antigen presentation on class i mhc proteins

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MHC Class I, Class II, Antigen Processing, And Presentation

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Major Histocompatibility Class I (MHC Class I)

This is the first class of the MHC molecule that encodes the glycoproteins that are expressed on the surface of nearly all nucleated cells.
Their major function is to present antigen-processed peptides to the T-cytotoxic cells by the cytosolic pathway.
In humans, the MHC class I protein is encoded by the HLA-A, -B, and -C genes.
This class of the MHC class I is made up of two chains i.e a transmembrane glycoprotein with a molecular weight of 45,000, which is noncovalently associated with a non–MHC-encoded polypeptide with molecular weight of 12,000 that is known as β2-microglobulin.
Class I molecules are to be found on virtually all nucleated cells in the body except on cells in the retina and brain.

Structure of MHC Class I

MHC Class I molecules in both human and mouse consist of two polypeptide chains that dramatically differ in size.
The larger (α) chain has a molecular weight of 44 kDa in humans and 47 kDa in the mouse, and is encoded by an MHC Class I gene.
The smaller chain, called β-2 microglobulin, has a molecular weight of 12 kDa in both species, and is encoded by a nonpolymorphic gene that is mapped outside of the MHC complex.
There are no known differences in the structure of the human MHC Class I antigen a chains encoded by the HLA-A locus compared to those encoded by the HLA-B or the HLA-C loci, or in the structure of the murine MHC Class I antigen a chains encoded by the H-2K locus compared to those encoded by the H-2D or H-2L loci.

Regardless of which of these loci codes it, the α chain can be subdivided into the following regions or domains:

the peptide-binding domain;
the immunoglobulin-like domain;
the transmembrane domain; and
the cytoplasmic domain.
The peptide-binding domain is the most N-terminal; it is the only region of the molecule where allelic differences in the amino acid sequence can be localized.
As seen from its name, the peptide-binding domain of the molecule includes the site to which antigenic peptides bind.
It makes much sense to have this site exactly where the allelic differences are, because different MHC alleles accommodate peptides better or worse, thus influencing on the magnitude of the T-cell response.
X-ray crystallography showed that the peptide-binding site in the MHC Class I molecules looks like a cleft that has a ‘‘floor’’ and two ‘‘walls’’ formed by spiral shaped portions of the alpha chain, called alpha 1 and alpha 2.
Since the ‘‘floor’’ of the peptide-accommodating cleft is closed, only relatively small peptides, consisting of 9 to 11 amino acid residues, can be ‘‘stuffed’’ there.
The immunoglobulin-like domain is structurally conserved, and resembles a domain of an antibody C-region.
It contains the binding site for the T-cell accessory molecule CD8.
The transmembrane and the cytoplasmic domains ensure that the alpha chain spans the membrane and is properly expressed by the cell.
The β-2-microglobulin chain is also vitally important for the proper expression of the alpha chain.
There are some mutant lymphoid cell lines (notably Daudi) that do not express MHC Class I molecules because of the defect in the β-2-microglobulin gene.

Major Histocompatibility Class II (MHC Class II)

The class II MHC genes encode glycoproteins expressed primarily on antigen-presenting cells (macrophages, dendritic cells, and B cells), where they present processed antigenic peptides to TH cells.
The class II proteins are encoded by the HLA-D region and the HLA-D regions have three families, DP-, DQ-, and DR-encoded molecules.
This class retains control of immune responsiveness and the different allelic forms of these genes confer differences in the ability to mount an immune response against a given antigen.
The HLA-D locus-encoded proteins are made up of two noncovalently associates transmembrane glycoproteins with a molecular weight of 33,000 and 29,000 respectively.
They have a restricted tissue distribution and they are chiefly found on macrophages, dendritic cells, B-cells, and other antigen-presenting cells. They are also expressed on other cells such as endothelial cells and/or epithelial cells induced by IFN-γ.

Structure of MHC Class II

Class II MHC molecules in both humans and mice consist of two polypeptide chains that have a similar, albeit not identical size.
One of them is called alpha (α) and the other beta (β).
The molecular weight of the α chain is 32–34 kDa, and of the β chain 29–32 kDa.
A separate gene controls each of the chains.
Thus, the murine I-A locus actually consists of the Iα and Iβ genes, the human HLA-DR locus of the HLA-DRα and HLA-DRβ, etc. Both the α and the β genes are polymorphic.
The β genes of some of the MHC Class II loci can be tandemly duplicated, so, instead of one gene per homologous chromosome, a cell can have two or three.
Because of that, one cell can simultaneously express more than two allelic products of each of the MHC Class II loci.
For example, a cell can express allelic products of its HLA-DR molecule that can be identified as HLADRα1– HLA-DRβ1; HLA-DRα2 – HLA-DRβ2; HLA-DRα1 – HLA-DRβ2; HLA-DRα2 – HLA-DRβ1; etc.
Overall, one cell can simultaneously express as many as 20 different MHC Class II gene products because of this tandem duplication phenomenon.

The structure of the α and the β chains of the MHC Class II molecules resembles that of the alpha chain of the MHC Class I molecules in that the former can be also divided into the peptide-binding, the immunoglobulin-like, the transmembrane, and the cytoplasmic domains.

One important difference, however, is that the peptide-binding cleft in Class II molecules is formed by both alpha and beta chains.

Although positioned close to each other in space, the spirals of the alpha and the beta chains that form the cleft are not physically bound to each other.

Because of that, the ‘‘floor’’ of the peptide-accommodating cleft in Class II MHC molecules is ‘‘open,’’ or ‘‘has a hole’’ in it.

That allows MHC Class II molecules to accommodate peptides that are larger than those that fit MHC Class I molecules.

The immunoglobulin-like domain of the MHC Class II molecules contains the binding site for a T-cell accessory molecule, CD4.

This site cannot bind the above-mentioned CD8 molecule.

Major Histocompatibility Class III (MHC Class III)

Class III MHC genes encode for various secreted proteins that have immune functions, including the component of the complement system and molecules that are involved in inflammation such as cytokines.

Antigen Processing and Presentation

The recognition of protein antigens by T-lymphocytes required that the antigens be processed by Antigen-presenting Cells, then displayed within the cleft of the MHC molecules on the membrane of the cell.
This involves the degradation of the protein antigens into peptides, a process known as antigen processing.
When the antigen has been processed and degraded into peptides, it then associates with MHC molecules within the cell cytoplasm forming a peptide-MHC complex. This complex is then transported to the membrane, where it is displayed by a process of antigen presentation.
The MHC Class I and class II MHC molecules associated with peptides that have been processed in different intracellular compartments.
The Class I MHC molecules bind peptides derived from endogenous antigens that have been processed within the cytoplasm of the cell such as tumor proteins, bacterial proteins, or viral proteins, or cellular proteins, and processed within the c ytosolic pathway .
Class II MHC molecules bind peptides derived from exogenous antigens that are internalized by phagocytosis or endocytosis and processed within the endocytic pathway .

A. Cytosolic pathway: Endogenous antigen

This is the pathway that processes and presents the endogenous antigen using the Class I MHC molecules.
The antigen proteins are degraded intracellularly to short peptides by a cytosolic proteolytic system that is present in all cells. These proteins targeted for proteolysis have a small protein known as ubiquitin attached to them.
The ubiquitin-protein conjugate then gets degraded by a multifunctional protease complex known as a proteasome.
Each proteasome is a large (26S), cylindrical particle that consists of four rings of protein subunits and a central channel of 10–50 Å diameter.
The proteasome can cleave peptide bonds between 2-3 different amino acid combinations in an ATP-dependent process.
Degradation of the ubiquitin-protein complex takes place in the central hollow of the proteasome.
The peptides are then transported from the cytosol to the rough endoplasmic reticulum. This is enabled by the transporter protein, designated TAP (transporter associated with antigen processing) is a membrane-spanning heterodimer consisting of two proteins: TAP1 and TAP2 .
The TAP1 and TAP2 proteins each have a domain projecting into the lumen of the Rough endoplasmic reticulum (RER), and an ATP-binding domain that extends into the cytosol.
Both TAP1 and TAP2 belong to the family of ATP-binding cassette proteins found in the membranes of many cells, including bacteria.
They mediate ATP-dependent transport of amino acids, sugars, ions, and peptides.
the peptides that are generated in the cytosol by the proteasome, are translocated into the Rough Endoplasmic Reticulum (RER) by TAP proteins by a process that utilizes hydrolyzed ATP. TAP proteins have a high affinity for peptides sizes of 8-10 amino acids, the optimum length for class I MHC binding.
Additionally, TAP proteins favor peptides with hydrophobic or basic carboxyl-terminal amino acids, which is the preferred anchor residue for class I MHC molecule, and therefore, TAP is optimized to transport peptides that will interact with class I MHC molecules.
Next, the peptides that are assembled with class I MHC are aided by chaperone molecules that facilitate the folding of polypeptides.
The alpha and beta-2-microglobulin components of the class I MHC molecules are synthesized on the polysomes along the rough endoplasmic reticulum. These components are assembled into a stable class I MHC molecules complex that can exit the RER requiring the presence of a peptide in the binding groove of the class molecule.
The first chaperone involved is known as calnexin, which is a resident membrane protein of the endoplasmic reticulum. Calnexin associates with the class I α chain and promotes its folding. When the Beta-2-microglobulin binds to the α chain, the calnexin is released, and the class I molecule associates with the chaperone calreticulin and with tapasin.
Tapasin is a TAP-associated protein that brings the TAP transporter into proximity with the class I molecule and allows it to acquire an antigenic peptide. The physical association of the α chain-beta-2-microglobulin heterodimer with the TAP protein promotes peptide capture by the class I molecule before the peptides are exposed to the RER.
The peptides not bound by class I molecules are rapidly degraded.
After binding, the class I molecule displays increased stability and can dissociate from calreticulin and tapasin, exit from the RER, and proceed to the cell surface via the Golgi.
An additional chaperone protein, ERp57, associates with calnexin and calreticulin complexes. The precise role of this resident endoplasmic reticulum protein in the class I peptide assembly and loading process has not yet been defined, but it is thought to contribute to the formation of disulfide bonds during the maturation of class I chains.

B. Endocytic Pathway: Exogenous antigen

Antigen-presenting cells can internalize antigen by phagocytosis, endocytosis, or both. Macrophages internalize antigen by both phagocytosis and endocytosis. Most of the other APCs are poorly phagocytic and can only internalize the antigen by pinocytosis or endocytosis, whereas most other APCs are not phagocytic and therefore they internalize the exogenous antigen only by endocytosis of by pinocytosis. B-cells which are also APCs internalizes the antigen effectively by receptor-mediated endocytosis using antigen-specific membrane antibody receptors.

When the exogenous antigen is internalized, it is degraded into peptides in the compartments of the endocytic processing pathway.
The breaking down of antigens into peptides takes 1-3 hours to transverse the endocytic pathway and appear at the cell surface in the form of a peptide-class II MHC complex.
In this pathway, three acidic compartments: early endosome (pH 6.0-6.5), late endosome or endolysosomes (pH 5.0-6.0); and lysosomes (pH 4.5-5.0). The internalized antigen moves from the early to late endosomes and later to the lysosomes where they encounter the hydrolytic enzyme, with a decreasing pH in each compartment.
the lysosomes have a unique collection of 40 acid-dependent hydrolases including proteases, nucleases, glycosidases, lipases, phospholipases, and phosphatases. Within the compartments of the endocytic pathway, the antigen is degraded into oligopeptide made up of 13-18 residues, that bind to class II MHC molecule. The hydrolytic enzymes are active in low Ph, they inhibit antigen processing chemical agents that may increase the compartment pH and that of protease inhibitors.
Movement of the peptides from one compartment to the next has been associated with small transport vesicles.
After getting to the final compartments, they return to the cell periphery fusing with the plasma membrane, enabling the recycling of surface receptors.
The antigen-presenting cells express both MHCI and MHC II molecules, therefore to prevent binding of MHC II to the same set of antigenic peptides as those of class I MHC, some mechanisms must exist to prevent this.
When the MHC II has been synthesized within the RER, three pairs of class II chains associate with a preassembled trimer of a protein known as an invariant chain (Ii, CD74). The trimeric protein interacts with the peptide-binding cleft of the class II MHC molecules, preventing any endogenously derived peptides from binding to the cleft while the MHC class II remains within the RER.
The invariant chain is also involved in the folding of class II MHC and its chains, the exit from the RER, and routing it to the endocytic processing pathway from the trans-Golgi network into the endocytic vesicles.
Secondly, the peptides assemble with class II MHC molecule by displacing CLIP (Class-II associated invariant chain peptide). Most of class II MHC-invariant chain complexes are transported from the RER where they are formed through the Golgi complex and trans-Golgi network, and then through the endocytic pathway, moving from early endosomes to late endosomes then finally to the lysosomes.
This increases the proteolytic activity from each compartment to the next.
This causes the degradation of the invariant chain gradually, leaving a short fragment of the invariant chain known as the CLIP (Class II-associated invariant chain peptide) that remains bound to the class II molecule after the invariant chain has been cleaved with the endosomal compartment.
CLIP occupies the peptide-binding groove of the class II MHC molecule, preventing premature binding of the antigenic peptide. HLA-DM molecule catalyzes the exchange of CLIP with the antigenic peptides. It is found in mammalian cells, mice, and rabbits. HLA-DM is neoclassical and nonpolymorphic.
When the HLA-DM and class II CLIP complex react, it facilitates the exchange of CLIP for another peptide but in the presence of HLA-DO, it can bind to HLA-DM reducing the efficiency of the exchange reaction.
The HLA-DO which has a similar structure as that of HLA-DM helps to modulate the function of HLA-DM, however, the function is obscure.

Presentation of Non-peptide antigens

Nonpeptide antigens are also recognized by the immune system, these are antigens that are derived from infectious agents such as Mycobacterium tuberculosis.
These antigens are recognized by T-cell Receptors known as δγ-TCR (T-cell receptor are dimers of αβ and δγ) which are derived from glycolipid of bacterial pathogens such as Mycobacterium tuberculosis.
These nonprotein antigens are presented by members of the CD1 family of nonclassical class I molecules.
The CD1 family of molecules associates with β2-microglobulin and it has its structure similar to that of MHC I molecules. It has 5 genes that encode for human CD1 molecules (CD1A-E, encoding the gene products CD1a-d, no E has been identified yet. These genes are located on the chromosomes and not on MHC I.
They are classified into two groups based on sequence homology. Group 1 includes CD1A, B, C, and E; CD1D is in group 2. All mammalian species have CD1 genes, although the number varies. Rodents have only group 2 CD1 genes, whereas rabbits, like humans, have five genes, including both group 1 and 2 types.
The sequence identity of CD1 with classical class I molecules is considerably lower than the identity of the class I molecules with each other. CD1D1 as compared to class I MHC shows that the antigen-binding groove of Cd1d1 is deeper and more voluminous than that of class I MHC molecule.

Clinical Significance of Antigen processing and presentation

Sometimes the antigen-presenting cells (APCs) can deliver self-antigens which cause autoimmune diseases. When the self-antigens are presented to the T-cells, it initiates an immune reaction against our own tissues, causing autoimmune disorders such as Graves Disease, rheumatoid arthritis.
In Graves’ disease, TSHR (Thyroid-stimulating hormone receptors) acts as the self-antigen, which is presented to T-cells activating B-cells which produce autoantibodies against TSHRs in the thyroid. This leads to the activation of TSHRs causing hyperthyroidism and leading to goiter.
Immunology by Kuby, 5th Edition
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Faith Mokobi

BiteSized Immunology: Systems & Processes

Antigen Processing and Presentation

In order to be capable of engaging the key elements of adaptive immunity (specificity, memory, diversity, self/nonself discrimination), antigens have to be processed and presented to immune cells. Antigen presentation is mediated by MHC class I molecules , and the class II molecules found on the surface of antigen-presenting cells (APCs) and certain other cells.

MHC class I and class II molecules are similar in function: they deliver short peptides to the cell surface allowing these peptides to be recognised by CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The difference is that the peptides originate from different sources – endogenous, or intracellular , for MHC class I; and exogenous, or extracellular for MHC class II. There is also so called cross-presentation in which exogenous antigens can be presented by MHC class I molecules. Endogenous antigens can also be presented by MHC class II when they are degraded through autophagy.

MHC class I presentation

MHC class I molecules are expressed by all nucleated cells. MHC class I molecules are assembled in the endoplasmic reticulum (ER) and consist of two types of chain – a polymorphic heavy chain and a chain called β2-microglobulin. The heavy chain is stabilised by the chaperone calnexin , prior to association with the β2-microglobulin. Without peptides, these molecules are stabilised by chaperone proteins : calreticulin, Erp57, protein disulfide isomerase (PDI) and tapasin. The complex of TAP, tapasin, MHC class I, ERp57 and calreticulin is called the peptide-loading complex (PLC). Tapasin interacts with the transport protein TAP (transporter associated with antigen presentation) which translocates peptides from the cytoplasm into the ER. Prior to entering the ER, peptides are derived from the degradation of proteins, which can be of viral- or self origin. Degradation of proteins is mediated by cytosolic- and nuclear proteasomes, and the resulting peptides are translocated into the ER by means of TAP. TAP translocates peptides of 8 –16 amino acids and they may require additional trimming in the ER before binding to MHC class I molecules. This is possibly due to the presence of ER aminopeptidase (ERAAP) associated with antigen processing.

It should be noted that 30–70% of proteins are immediately degraded after synthesis (they are called DRiPs – defective ribosomal products, and they are the result of defective transcription or translation). This process allows viral peptides to be presented very quickly – for example, influenza virus can be recognised by T cells approximately 1.5 hours post-infection. When peptides bind to MHC class I molecules, the chaperones are released and peptide–MHC class I complexes leave the ER for presentation at the cell surface. In some cases, peptides fail to associate with MHC class I and they have to be returned to the cytosol for degradation. Some MHC class I molecules never bind peptides and they are also degraded by the ER-associated protein degradation (ERAD) system.

There are different proteasomes that generate peptides for MHC class-I presentation: 26S proteasome , which is expressed by most cells; the immunoproteasome, which is expressed by many immune cells; and the thymic-specific proteasome expressed by thymic epithelial cells.

Antigen presentation

On the surface of a single cell, MHC class I molecules provide a readout of the expression level of up to 10,000 proteins. This array is interpreted by cytotoxic T lymphocytes and Natural Killer cells, allowing them to monitor the events inside the cell and detect infection and tumorigenesis.

MHC class I complexes at the cell surface may dissociate as time passes and the heavy chain can be internalised. When MHC class I molecules are internalised into the endosome, they enter the MHC class-II presentation pathway. Some of the MHC class I molecules can be recycled and present endosomal peptides as a part of a process which is called cross-presentation .

The usual process of antigen presentation through the MHC I molecule is based on an interaction between the T-cell receptor and a peptide bound to the MHC class I molecule. There is also an interaction between the CD8+ molecule on the surface of the T cell and non-peptide binding regions on the MHC class I molecule. Thus, peptide presented in complex with MHC class I can only be recognised by CD8+ T cells. This interaction is a part of so-called ‘three-signal activation model’, and actually represents the first signal. The next signal is the interaction between CD80/86 on the APC and CD28 on the surface of the T cell, followed by a third signal – the production of cytokines by the APC which fully activates the T cell to provide a specific response.

MHC class I polymorphism

Human MHC class I molecules are encoded by a series of genes – HLA-A, HLA-B and HLA-C (HLA stands for ‘Human Leukocyte Antigen’, which is the human equivalent of MHC molecules found in most vertebrates). These genes are highly polymorphic, which means that each individual has his/her own HLA allele set. The consequences of these polymorphisms are differential susceptibilities to infection and autoimmune diseases that may result from the high diversity of peptides that can bind to MHC class I in different individuals. Also, MHC class I polymorphisms make it virtually impossible to have a perfect tissue match between donor and recipient, and thus are responsible for graft rejection.

MHC class II presentation

MHC class II molecules are expressed by APCs, such as dendritic cells (DC), macrophages and B cells (and, under IFNγ stimuli, by mesenchymal stromal cells, fibroblasts and endothelial cells, as well as by epithelial cells and enteric glial cells). MHC class II molecules bind to peptides that are derived from proteins degraded in the endocytic pathway. MHC class II complexes consists of α- and β-chains that are assembled in the ER and are stabilised by invariant chain (Ii). The complex of MHC class II and Ii is transported through the Golgi into a compartment which is termed the MHC class II compartment (MIIC). Due to acidic pH, proteases cathepsin S and cathepsin L are activated and digest Ii, leaving a residual class II-associated Ii peptide (CLIP) in the peptide-binding groove of the MHC class II. Later, the CLIP is exchanged for an antigenic peptide derived from a protein degraded in the endosomal pathway. This process requires the chaperone HLA-DM, and, in the case of B cells, the HLA-DO molecule. MHC class II molecules loaded with foreign peptide are then transported to the cell membrane to present their cargo to CD4+ T cells. Thereafter, the process of antigen presentation by means of MHC class II molecules basically follows the same pattern as for MHC class I presentation.

As opposed to MHC class I, MHC class II molecules do not dissociate at the plasma membrane. The mechanisms that control MHC class II degradation have not been established yet, but MHC class II molecules can be ubiquitinised and then internalised in an endocytic pathway.

MHC class II polymorphism

Like the MHC class I heavy chain, human MHC class II molecules are encoded by three polymorphic genes: HLA-DR, HLA-DQ and HLA-DP. Different MHC class II alleles can be used as genetic markers for several autoimmune diseases, possibly owing to the peptides that they present.

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Physiology, mhc class i.

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Last Update: September 26, 2022 .

Introduction

Major histocompatibility complex (MHC) class I is a diverse set of cell surface receptors expressed on all nucleated cells in the body and platelets. MHC is also known as human leukocyte antigen (HLA), of which there are 3 subtypes: HLA-A, HLA-B, and HLA-C. These molecules play a vital role in the immune system recognizing self from nonself, presenting foreign antigens to other immune cells. HLA class I alleles are extremely polymorphic among the world population, which presents issues relating to human tissue transplants. [1] [2]

Issues of Concern

MHC class I cross-reactivity is often the mediator in transplant reactions. A host T-cell may bind the MHC molecule on the donor's grafted tissue and recognize the graft as nonself. Subsequently, it mounts an immune attack on the graft via a cascade of immune cell activation. This type of reaction is labeled a T-cell–mediated reaction. T-cell–mediated reactions are responsible for acute transfusion reactions, with symptoms arising in days to weeks after transplant. The host may have also become sensitized via a previous transplant, blood transfusion, or pregnancy. The sensitization process resulting in anti–HLA antibodies is called alloimmunization and is poorly understood. Anti–HLA antibodies are responsible for hyperacute transfusion reactions, which arise minutes to hours after transfusion. [1] [3] [1]

Before transplanting any hemopoietic precursors, such as stem cells or solid tissue transplants of kidneys or livers, a pretransplant crossmatch test is necessary to evaluate the reactivity of the recipient's HLA antibodies against the donor's HLA proteins. [4] HLA class I proteins are highly immunogenic. If the HLA types do not match, there can be a hyperacute reaction in which the recipient's immune system recognizes the graft as foreign and signals the body's immune system to destroy the allograft.

Another concern is the association of specific HLA alleles leading to a genetic predisposition to developing some disease conditions. Although it is often unclear exactly how these HLA class I subtypes have implications in the pathogenesis of the disease, their presence may aid in diagnosing particular diseases. Further research is needed to discover how these alleles contribute to disease and any therapies that could prevent their action.

Cellular Level

MHC class I molecules are protein structures consisting of three alpha domains and a beta-two macroglobulin domain. The HLA class I gene is located on the chromosome, while beta-2 macroglobulin encoding is on chromosome 15. [5] The HLA class I alleles are codominant expressed, and inheritance is via simple Mendelian inheritance patterns. [6]

The alpha-1 and -2 domains are the binding cleft for various peptides, which are then presented to a T-cell receptor. One end of the alpha domain also serves as the binding site for an inhibitory receptor in natural killer (NK) cells. The beta-2 macroglobulin acts to stabilize the peptide binding. [5] The binding cleft of MHC class I tyrosine residues flank me and create closed ends that limit the peptide size; it can bind to around 8 to 10 amino acids. These amino acids are of cytosolic origin. Self or foreign cytosolic proteins are degraded via the proteasome and transported into the lumen of the endoplasmic reticulum. There, the peptides are loaded onto an MHC class I via a chaperone protein named tapasin. The peptide-bound MHC class I is then transported to the cell’s plasma membrane, presenting the peptide to CD8+ T-cell receptors. [7]

HLA class I functions as part of the adaptive immune system and plays a vital role in recognizing self from nonself and providing protection against viruses and tumors. In a healthy person, HLA class I binds degraded cytosolic self-proteins and then transports these fragments to the cell membrane, where they present to CD8+ T lymphocytes. When the presented peptide is of foreign origin, such as one derived from a virus-infected cell, the CD8+ T cell eliminates the infected cell. MHC class I–presenting self-proteins also serve as an inhibitory signal to NK cells; this prevents NK killing from healthy cells.

Related Testing

HLAs are identifiable via multiple detection methods.

Molecular Testing

Sequence-specific primer polymerase chain reaction

Sequence-specific primer polymerase chain reaction (PCR) uses various primers complementary to specific HLA DNA sequences. The DNA is plated into a multiwell plate with different primers. If the DNA extracted from that cell is complementary to the primer, it is amplified, and the product can be run on a gel via electrophoresis. The band can be identified as a primer and matched to known candidate HLA alleles. [8]

Sequence-specific oligonucleotide probes

One way to detect the high polymorphism seen in these genes is via PCR paired with sequence-specific oligonucleotides. This method involves amplifying the gene via PCR and then probing DNA with a fluorescent tag. The HLA type is determined using known HLA alleles as a reference, and that gene may undergo sequencing. [8] [9] [8]

Direct DNA sequencing

Another technique is to use Sanger sequencing or next-gen sequencing to sequence the entire gene of a specific HLA variant after amplification via PCR. Once the sequence is known, it can be compared to previously published HLA alleles. [8]

Serological Testing

Serological testing generally uses a recipient's lymphocytes obtained from their sera and incubated with anti–sera-containing antibodies against various HLA class 1 subtypes. The solution is then incubated with rabbit sera, providing a source of complement; a dye is also added to identify dead cells. This assay progresses serially with different HLA antibodies put into each well of a tray. Eliminating those wells with a positive result allows for determining the HLA type. This method provides a relatively quick and easy way to determine general subtypes of HLA's present. Still, it does not offer an in-depth analysis of the true molecular identity of the HLAs. This method is less common today because of its inability to detect small changes in HLA types that may make an immunological difference and cause a transfusion reaction. [8] [10] [8]

Antibody Testing

Cytotoxic cell-based antibody testing can also effectively measure the recipient's risk of having a positive crossmatch. In this method, 30 to 40 donor cell lymphocytes are mixed with dye and complemented with the recipient's serum. Suppose the recipient's serum contains high enough HLA-specific antibodies against a particular donor. In that case, the lymphocyte complement is activated, the cell will die, the dye will be taken up, and that well in the plate will be visually identifiable as a positive result. This test yields a value known as a percentage panel reactive antibody. Its result measures the recipient's risk of a positive crossmatch in a similar population of donors. This test does lack the ability to take race and different HLA frequencies in a population into account, weakening its value. [8]

Clinical Significance

Transplant rejection remains one of the most clinically relevant issues surrounding the HLA class I molecule. Identifying the donor and recipient’s HLA status is imperative to prevent transplantation reactions. Continuing education on the different serological, molecular, and cellular tests available to clinicians and their proper use, and healthcare professionals must learn how to interpret them.

Spondylosis Ankylosis

Research has shown a strong correlation between HLA-B27 and ankylosing spondylitis. Among those with the HLA-B27 allele, 5% to 6% develop ankylosing spondylitis. [11] Ankylosing spondylitis is a seronegative spondyloarthropathy that leads to progressive spine and sacroiliac joint stiffness. This stiffness results from improper bone deposition, leading to fused vertebrae. The exact role of HLA-B27 in AS is still unknown. [12]

Behcet Disease

Research has found the HLA-B51 allele to be the greatest risk factor for developing Behcet disease. Behcet disease is an autoinflammatory condition resulting in normally immune-privileged sites such as the brain, eye, and joints infiltrating neutrophils. The link between the pathogenesis of Behcet disease and HLA B51 is still unclear. More research is needed to ascertain the true connection between the HLA-B21 allele and Behcet disease. Researchers postulate that it could be due to antigen presentation to CD8+ cells or HLA-B51 ’s interaction with NK receptor KIR3DL1. [13]

Psoriasis, an autoimmune condition, is highly linked to specific HLA class I alleles, including HLA-A01 , HLA-A02 , HLA-B13 , HLA-B17 , HLA-B39 , HLA-B57 , HLA-Cw06 , and HLA-Cw07. The role of HLA in psoriasis is currently unknown. [14]

Birdshot Chorioretinopathy

Birdshot chorioretinopathy, a form of posterior uveitis, correlates with HLA-A29 , with 85% to 97.5% of those diagnosed carrying the allele. [15]

HLA-B27 , HLA-B51 , HLA-C06 , and HLA-B5701 all confer protection against HIV infection. The reason appears to be that the peptides presented by these alleles are structurally resistant to mutation. Another idea is that those CD8-positive T cells that interact with these specific alleles have higher functionality in riding the body of HIV-infected cells. [15]

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Review Article
Published: 11 November 2011

Towards a systems understanding of MHC class I and MHC class II antigen presentation

Jacques Neefjes 1 ,
Marlieke L. M. Jongsma 1 ,
Petra Paul 1 &
Oddmund Bakke 2

Nature Reviews Immunology volume 11 , pages 823–836 ( 2011 ) Cite this article

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Antigen presentation
Antigen processing and presentation
MHC class I
MHC class II
Signal transduction

MHC class I and MHC class II molecules have long been intensively studied, and this has resulted in a global view of these processes. New technologies, including genome-wide small interfering RNA screens and systems biology approaches, have identified numerous additional pathways that control antigen presentation by MHC molecules.

MHC molecules are polymorphic, and the biology of the various alleles differs, such that they can potentially have different consequences with regards to the relevant immune responses. This point is best-defined for MHC class I molecules. In this Review, we bring this into context with the current understanding of the general MHC class I antigen presentation process.

The biology of MHC molecules touches almost all areas in the field of cell biology. Various new findings from the area of cell biology have consequences for MHC class I and MHC class II antigen presentation.

The immune system is a relatively late addition in our progress through evolution, and many immune-specific molecules exist. Unique functions of some of these, including the immunoproteasome in interferon-induced damage clearance, have been recently uncovered.

Through a combination of small interfering RNA screens, microarrays and cell biological approaches, novel pathways that control MHC class II expression and transport in dendritic cells have been defined. The systems biology of MHC molecules will yield more surprises.

A dynamic field of research has many unsolved issues. A survey of the views of almost 50 group leaders in the field of antigen presentation has provided democratic opinions on the variety of unsettled topics within the field.

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.

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antigen presentation on class i mhc proteins

A guide to antigen processing and presentation

High-throughput discovery of MHC class I- and II-restricted T cell epitopes using synthetic cellular circuits

MR1 antigen presentation to MAIT cells and other MR1-restricted T cells

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Acknowledgements

We thank our colleagues for their input in the controversial items section and I. Berlin, S. van Kasteren, O. Landsverk and A. Lammerts van Beuren-Brandt for critical reading. We apologize to our colleagues for not citing every relevant paper owing to length limitations. This work was supported by European Research Council (ERC) and Netherlands Organization for Scientific Research (NWO) grants to J.N. and an NWO visiting grant to O.B.

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Neefjes, J., Jongsma, M., Paul, P. et al. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 11 , 823–836 (2011). https://doi.org/10.1038/nri3084

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Understanding MHC Class I Presentation of Viral Antigens by Human Dendritic Cells as a Basis for Rational Design of Therapeutic Vaccines

Nadine van montfoort, evelyn van der aa, andrea m woltman.

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Edited by: Marianne Boes, University Medical Centre Utrecht, Netherlands

Reviewed by: Kristen J. Radford, Mater Medical Research Institute, Australia; Laurence C. Eisenlohr, Thomas Jefferson University, USA

*Correspondence: Andrea M. Woltman, Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, Room Na-1006, P.O. Box 2040, Rotterdam 3000 CA, Netherlands e-mail: [email protected]

This article was submitted to Antigen Presenting Cell Biology, a section of the journal Frontiers in Immunology.

Received 2014 Jan 31; Accepted 2014 Apr 7; Prepublished 2014 Mar 3; Collection date 2014.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Effective viral clearance requires the induction of virus-specific CD8 + cytotoxic T lymphocytes (CTL). Since dendritic cells (DC) have a central role in initiating and shaping virus-specific CTL responses, it is important to understand how DC initiate virus-specific CTL responses. Some viruses can directly infect DC, which theoretically allow direct presentation of viral antigens to CTL, but many viruses target other cells than DC and thus the host depends on the cross-presentation of viral antigens by DC to activate virus-specific CTL.

Research in mouse models has highly enhanced our understanding of the mechanisms underlying cross-presentation and the dendritic cells (DC) subsets involved, however, these results cannot be readily translated toward the role of human DC in MHC class I-antigen presentation of human viruses. Here, we summarize the insights gained in the past 20 years on MHC class I presentation of viral antigen by human DC and add to the current debate on the capacities of different human DC subsets herein. Furthermore, possible sources of viral antigens and essential DC characteristics for effective induction of virus-specific CTL are evaluated.

We conclude that cross-presentation is not only an efficient mechanism exploited by DC to initiate immunity to viruses that do not infect DC but also to viruses that do infect DC, because cross-presentation has many conceptual advantages and bypasses direct immune modulatory effects of the virus on its infected target cells.

Since knowledge on the mechanism of viral antigen presentation and the preferred DC subsets is crucial for rational vaccine design, the obtained insights are very instrumental for the development of effective anti-viral immunotherapy.

Keywords: virus, human dendritic cell, cross-presentation, CTL priming, MHC class I-antigen presentation, viral immunity, immunotherapy, virus–host interaction

Role of Dendritic Cells in the Induction of Anti-Viral Immunity

Immune responses to viral infections are a complex interplay between the virus, target cells, and cells of the immune system. Effective viral clearance requires the induction of virus-specific CD8 + cytotoxic T lymphocytes (CTL), which have the capacity to eradicate the virus by direct and indirect mechanisms ( 1 ). DC, a low frequent population of white blood cells play a central role in the induction of virus-specific CTL, since they are the most potent antigen presenting cells and unique for their capacity to activate naïve T cells ( 2 ). DC are located at strategic positions at sites of pathogen entry, where they continuously sample the environment for invading pathogens. Capturing antigens in combination with encountering danger signals from pathogens induces maturation of DC and their migration to secondary lymphoid organs where they can activate naïve T cells. Activation of naïve CD8 + T cells and polarization toward effective CTL requires presentation of MHC class I–peptide complexes (signal 1) together with co-stimulation (signal 2) and the presence of cytokines (signal 3) such as IL-12 ( 3 ) and IFNα ( 4 ).

Dendritic cells comprise a family of different subsets, diverging in ontogeny, localization, and phenotype. Each DC subset has its own specialized immune functions with regard to the functional interactions with all kind of immune cells, including T cells, B cells, and NK cells, due to differential expression of receptors and intrinsic differences in their ability to produce different cytokines and other membrane-bound and soluble immune modulatory molecules ( 5 ). Human DC subsets present in blood, peripheral, and lymphoid tissues can be classified in two main categories: plasmacytoid DC (pDC) and myeloid DC (mDC), which can be further divided into BDCA1 + (CD1c + ) and BDCA3 + (CD141 + ) DC ( 6 ). pDC are specialized in the production of high amounts of anti-viral type I interferon (IFN; IFNα/β) upon activation ( 7 ), whereas BDCA1 + DC are known for their high production of IL-12 and their ability to induce T cell responses ( 5 ). BDCA3 + DC, on the other hand, can produce high levels of type III IFN (IFNλ) ( 8 ), which possess direct anti-viral activity, and induce Th-1 responses ( 9 ). In the skin, two additional mDC subsets have been characterized, epidermal Langerhans cells (LC) and dermal interstitial DC (intDC) ( 10 ). Since DC represent a very rare population in the human body that hampers isolation of sufficient numbers, in vitro -generated DC differentiated from monocytes ( 11 ) or hematopoietic progenitor cells ( 12 ) are frequently used for functional studies on human DC.

The notion that DC compared to other antigen presenting cells stand out in their capacity to induce strong virus-specific CTL goes back more than 20 years, when it was reported that human blood-derived DC exposed to HIV-1 or influenza virus could induce proliferation of autologous CTL ( 13 , 14 ). At that time, it was not known whether the efficacy of DC reflected specialized antigen presentation pathways or that other factors were responsible for the efficacy of DC in virus-specific CTL cell induction. At least it was noted that only low numbers of DC were sufficient to induce influenza-specific T cells ( 14 ).

Now we know that DC, in addition to their broad expression of pattern-recognition receptors (PRR) and excellent T cell stimulatory capacities, harbor unique specialized antigen presentation pathways, that are of major importance for their central role in the induction of virus-specific immunity; DC can efficiently facilitate MHC class I presentation of endogenously synthesized antigens, a process that is active in all nucleated cells, but also facilitate MHC class I presentation of antigen engulfed from exogenous sources, a process called cross-presentation ( 15 ). DC are very efficient in capturing exogenous antigen, because they express a diverse repertoire of receptors and exploit various mechanisms to engulf antigens, including endocytosis, phagocytosis, and pinocytosis. The cross-presentation capacity of DC may be crucial for the induction of virus-specific CTL during infections with viruses that do not infect DC.

Seminal mouse studies have demonstrated the importance of cross-presentation for the generation of virus-specific CTL responses ( 16 – 18 ). In addition, mouse studies have provided important insights into the cell-biological mechanisms underlying cross-presentation by DC ( 19 , 20 ). However, composition of the human DC compartment and susceptibility to viruses differ largely between mice and men. In addition, the mechanism of cross-presentation by human DC is less well-understood. Therefore, research on MHC class I presentation of viral antigens by human DC is of great importance to understand the induction of virus-specific CTL in humans.

The study into antigen presentation of viruses by subsets of human DC ex vivo has been facing several technical challenges, which has hampered the understanding of this process for many viruses. However, some recent technical advancements have become available that empowered this research. For example, the possibility to more efficiently isolate human DC subsets from peripheral blood and other organs and the development of a new generation of protocols to generate human DC subsets in vitro ( 21 , 22 ), as was previously shown for BDCA1 + monocyte-derived DC (moDC) ( 11 ) and CD34 + HPC-derived intDC and LC, that resemble mDC found in mucosal tissues including skin ( 12 , 23 ). These technical advancements have revived the scientific interest in the interactions between viruses and different human DC subsets. Since 2010, a significant body of literature has been published on presentation of viral antigens by different human DC subsets that facilitated this review, which is based for a large part on studies using human DC.

In the present review, the different mechanisms employed by human DC to facilitate MHC class I presentation of viral antigens are discussed. For this purpose, possible sources of viral antigens, essential DC characteristics for optimal MHC class I presentation of viral antigens, and host factors important for virus-specific CTL induction are defined. Furthermore, the roles of the various human DC subsets of human DC in these processes are evaluated. Since knowledge on mechanisms of virus-specific CTL induction by human DC subset is crucial for rational vaccine design, recommendations for development of effective anti-viral immune therapies will be provided based on the insights obtained in this review.

Sources of Viral Antigen for MHC Class I Presentation by DC

Virus-infected DC can use endogenously synthesized viral proteins as antigens for presentation in MHC class I, whereas non-infected DC need to actively engulf exogenous viral antigens for cross-presentation. Here, we discuss possible sources of viral antigen obtained from different viruses for MHC class I presentation by human DC.

Human moDC are permissive for quite a number of viruses including measles virus (MV), human cytomegalovirus (HCMV), influenza A virus (IAV), human T-cell lymphotropic virus type 1 (HTLV-1), dengue virus (DV), vaccinia virus (VV), respiratory syncytial virus (RSV), herpes simplex virus (HSV), and human metapneumovirus (hMPV) ( 24 – 36 ). Although moDC can take up HIV-1, they are largely refractory to HIV-1 productive infection ( 37 ), whereas, productive infection of peripheral blood-derived BDCA1 + DC and pDC has been demonstrated ( 38 ). In addition to moDC, RSV also infects BDCA1 + and BDCA3 + mDC ( 39 ) and IAV infects BDCA1 + mDC, but not pDC ( 40 ). LC are permissive for MV, but only after maturation ( 25 ). Although LC can take up HIV-1, they are not permissive for HIV-1 replication and transmission, but rather prevent it by degradation ( 41 ). Permissiveness to infection indicates that these viruses not only enter human DC, they also induce a certain level of protein neo-synthesis in DC that ranges from restricted synthesis of early viral proteins ( 33 ) to extensive synthesis of multiple viral proteins and secretion of viral progeny ( 26 ). Intracellular synthesis of viral antigens by DC suggests that these infected DC may facilitate direct presentation of viral antigens in MHC class I and activation of virus-specific cytotoxic T cells (CTL). MHC class I presentation of viral antigens has been reported for DC infected with IAV, MV, HTLV-1, and HCMV, albeit sometimes with low efficiency ( 14 , 25 , 27 , 31 , 42 ).

Nevertheless, it has been demonstrated in several independent studies, involving IAV, HIV-1, and MV, that the efficiency of MHC class I-antigen presentation of replication-incompetent virus was at least comparable to replication-competent virus ( 25 , 40 , 43 – 46 ). These heat-or UV-treated replication-incompetent viruses have lost the capacity to induce synthesis of viral proteins, but still efficiently enter DC to act as exogenous sources of viral antigen. It was estimated that MHC class I presentation of replication-incompetent IAV by BDCA1 + mDC was 300 times more efficient than MHC class I presentation of replication-competent IAV ( 40 ). These studies clearly show that, at least for the viruses studied, endogenous synthesis of viral antigens is not required for MHC class I presentation and that cross-presentation is an efficient mechanism to facilitate MHC class I presentation of viral antigens.

Thus, cross-presentation is not only an efficient mechanism exploited by DC to initiate immunity to viruses that do not infect DC but also contributes to initiation of anti-viral immunity to viruses that do infect DC. In fact, cross-presentation seems a clever way to bypass direct immune modulatory effects of the virus on its infected target cells. For instance, interference with MHC class I presentation is commonly used by herpes viruses to evade immunity [reviewed by Ref. ( 47 )] and is also exploited by IAV, as was elegantly shown by comparing CMV-specific CTL proliferation by CMV-antigen loaded IAV-infected and uninfected BDCA1 + mDC ( 40 ). In addition, early during HIV infection, part of the DC compartment is depleted, which may contribute to decreased activation of adaptive immunity ( 48 ). Virus-induced cell death is also reported for RSV ( 34 , 39 ) and VV ( 33 ).

In addition to replication-incompetent viral particles, other sources of exogenous viral antigens for cross-presentation by human DC include virus-like particles (VLP), viral proteins, and virus-infected cells (Figure 1 ). VLP morphologically and immunologically resemble infectious viral particles because they contain the natural viral envelop proteins, however, they are not infectious, because they do not contain the viral genome. Although some VLP naturally occur in vivo , they are often man-made, being used as safe representatives of viral particles to study virus–host interactions ( 49 ) or in the context of vaccine research ( 50 , 51 ). VLP can be efficient sources of exogenous viral antigen for cross-presentation by DC, as was demonstrated for hepatitis C virus (HCV) VLP ( 49 ), human papilloma virus 16 (HPV16) VLP ( 50 ), and VLP composed of the coat protein of papaya mosaic virus (PapMV) ( 51 ).

Overview of different pathways underlying MHC class I presentation of viral antigens by human DC . Although direct MHC class I class I presentation may contribute to virus-specific CTL induction (dashed arrow), cross-presentation is an effective mechanism for MHC class I presentation of viruses that do not infect DC but also for those viruses that do infect DC. Sources of viral antigen that can be efficiently cross-presented by human DC include viral proteins, (infectious) viral particles, VLP, and virus-infected cells, also referred to as cell-associated Ag. Endocytic receptors including CLR, FcR and other receptors (Table 1 ) play an important role in the uptake of Ag for cross-presentation. Cross-presentation can be enhanced by opsonization. Two main pathways for cross-presentation have been described that are also relevant for cross-presentation of viruses by human DC and are characterized by differences in the mechanism of protein degradation and differences in kinetics (black arrows). The slower cytosolic pathway, that relies on proteasomal degradation in the cytosol, is important for cross-presentation of viral particles, infected cells, and opsonized viral proteins (A) . The relatively fast vacuolar pathway is independent of proteasomal degradation and is important for cross-presentation of VLP (B) . Alternatively, DC can obtain viral peptides or MHC class I-peptide complexes by interaction with virus-infected cells. EE, early endosome; LE, late endosome; PR, proteasome.

Recombinant proteins such as HCV-derived NS3 ( 52 ), HIV-1-derived Nef ( 53 ), HCMV-derived pp65 ( 9 , 54 ), and hepatitis B virus (HBV)-derived hepatitis B surface antigen (HBsAg) ( 55 , 56 ) are sources of exogenous antigens that are often used to study the mechanism of cross-presentation by DC. Nevertheless, the efficiency of cross-presentation of these recombinant proteins is relatively low compared to other sources of viral antigens. Moreover, with the exception of HBsAg, which is secreted by human hepatocytes and can be measured in peripheral blood, most proteins are not naturally occurring as soluble proteins in vivo but are only present in/associated with infected cells.

Cell-associated antigen, i.e., antigen associated to or present in infected target cells, represents another important source of viral antigens that can be encountered by DC. Albert and colleagues contributed the first evidence of this by showing that uptake of apoptotic IAV-infected monocytes by moDC leads to efficient activation of influenza-specific CTL ( 57 ). After this study, a compelling number of studies have confirmed that virus-infected target cells can be efficient antigen sources for cross-presentation in many infections. For instance, VV-infected monocytes ( 45 , 58 ), HTLV-1 infected CD4 + T cells ( 31 ), MV-infected B cell lines ( 25 ), HCMV-infected fibroblasts ( 27 , 59 ), and EBV-transformed B cells ( 60 , 61 ) are reported as efficient sources of viral antigens for cross-presentation by human DC. The latter study nicely illustrated the high efficiency of this mechanism by demonstrating activation of EBV-specific CTL by DC cross-presenting EBV latency antigens that were expressed at low levels in EBV-transformed B cells ( 61 ).

In the above-mentioned studies, apoptotic or necrotic virus-containing cells or cell remnants were used as sources of cell-associated antigens for cross-presentation. Transfer of viral peptides from infected cells to DC could represent an alternative efficient mechanism underlying cross-presentation of cell-associated viral antigens. Two different mechanisms facilitating peptide exchange between cells have been described, including transfer of antigenic peptides via intercellular communication channels, called gap junctions ( 62 ), and direct transfer of MHC class I/peptide complexes from infected cells to DC, named cross-dressing ( 63 , 64 ). The relevance of these pathways in presentation of viral antigens by human DC and induction of virus-specific T-cell immunity should be further evaluated.

In summary, for efficient viral antigen presentation to CD8 + T cells, DC can acquire viral antigens from various sources. Although direct presentation of endogenously generated antigen by virus-infected DC has been reported for some viruses, evidence to support an important role for this mechanism in the induction of virus-specific CTL is lacking. In contrast, there is compelling evidence that cross-presentation of exogenously acquired viral antigen is highly efficient and provides an excellent way for the host to bypass evasion mechanisms that several viruses employ to prevent direct MHC class I presentation in infected target cells.

Endocytic Receptors Involved in Uptake of Viruses by DC

Being intracellular parasites, viruses use the host machinery for internalization, proliferation, and transmission. DC are attractive target cells for viral entry because they express numerous receptors at their cell surface and they migrate through the body, which facilitates viral dissemination. Viruses can enter DC via docking with their viral envelop to endocytic receptors expressed at the cell membrane ( 43 , 44 , 46 ). A commonly described receptor used by viruses to enter DC is DC-specific C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN/CD209). DC-SIGN is involved in the infection of moDC by DV ( 32 , 65 ), HCMV ( 28 ), HSV ( 66 ), MV ( 67 ), and IAV ( 68 ) and also in DC-mediated transmission of HIV-1 ( 69 ) and HTLV-1 ( 70 ) to CD4 + T cells. DC-SIGN is part of the large family of C-type lectin receptors (CLR), comprising Ca 2+ -dependent receptors that each have unique functions but share the recognition of carbohydrate structures present on micro-organisms ( 71 ). Other CLR family members involved in interaction with viruses include Langerin (CD207), involved in the interaction with MV and HIV-1 ( 25 , 41 ), DC immunoreceptor (DCIR) ( 72 ), proposed as an alternative receptor for HIV-1 promoting infection in cis and trans and macrophage mannose receptor (MMR/CD206), possibly involved in uptake of HBsAg by liver BDCA1 + DC ( 73 ). Also non-CLR can be involved in the interaction with viruses or VLP. DC-specific heparin sulfate proteoglycan Syndecan-3 cooperates together with DC-SIGN to facilitate infection of DC and transmission to CD4 + T cells ( 74 ) and is involved in the interaction with HPV VLP ( 75 ). Since expression of endocytic receptors varies widely between DC subsets (Table 1 ), the different subsets will likely have specialized roles in the interaction with different viruses, determined by the combination of receptors expressed on each DC subset.

Summary of receptors that are involved in DC–virus interaction on different DC subsets .

pDC, plasmacytoid DC; LC, Langerhans cell; intDC, interstitial DC; moDC, monocyte-derived DC; nf, information not found .

Are these CLRs only involved in supporting viruses to enter the host or did they evolve to support activation of the host’s immune system through antigen presentation? Langerin is an important receptor for interaction with pathogens in the skin and has been shown to support antigen presentation in MHC class II, but its role in MHC class I-mediated antigen presentation is under debate ( 25 ). Moris et al. showed that blocking of DC-SIGN partly reduced MHC class I presentation of internalized HIV-1 by DC, arguing in favor of a role of DC-SIGN in cross-presentation of HIV-1 ( 91 ). In contrast, Sabado et al. showed that blocking of DC-SIGN, DEC-205 (CD205), or MR did not reduce MHC class I presentation of HIV-1 antigens ( 46 ) whereas Tjomsland and colleagues showed that blockade of MR even promoted cross-presentation of HIV-1 by DC ( 92 ). Thus, the physiological role of DC-SIGN in cross-presentation of HIV-1 is thus far inconclusive, which may be explained by differences in experimental set-up such as the HIV-1 strain used. Antibody-mediated delivery of antigen to the CLRs MR, DEC-205 ( 82 ), DCIR ( 81 ), DC-SIGN ( 93 ), and CLEC9A ( 94 ) (Table 1 ) on human DCs facilitates efficient cross-presentation. These examples show that CLR can facilitate cross-presentation, however, the physiological role of these receptors in cross-presentation of viral antigens is still under debate.

Whereas CLR can directly recognize viral envelop antigens, complement receptors and Fc receptors (FcR) selectively recognize viral antigens that are opsonized with complement and immunoglobulins, respectively. Antigen immune complexes naturally exist and are formed when pre-existing antibodies bind to blood-borne antigens in the circulation, for example, during HCMV re-infection ( 85 ). Binding of immune complexes to Fcγ receptor (FcγR) on DC leads to efficient cross-presentation in MHC class I ( 85 ). Strikingly, the observation that FcR-dependent uptake of HBsAg can enhance activation of HBV-specific CTL was made years before the concept of cross-presentation by DC was recognized ( 95 ), indicating that opsonization of viral antigens may be important for generating virus-specific CTL. Similarly, opsonization of antigen by complement can efficiently enhance cross-presentation, as was recently demonstrated for HIV-1 by targeting HIV-1 particles to CR3 ( 92 ). In addition, although not classically referred to as opsonization, binding of high-density lipoprotein (HDL) to HCV VLP supported efficient Scavenger receptor B-mediated uptake and cross-presentation ( 96 ). A similar role for extracellular heat-shock proteins (HSP) has been proposed [reviewed by Ref. ( 97 )], mainly based on mouse studies in the field of cancer immunotherapy. However, the role of HSP in cross-presentation of viral antigens by human DC remains to be investigated.

Although these results indicate that several endocytic receptors may be involved in facilitating cross-presentation, their exact role needs to be determined. Especially recognition of viral antigens by opsonins seems to be an effective way of natural antigen targeting to DC for cross-presentation. Increased knowledge on the receptors used by viruses for infection on the one hand and the receptors that facilitate cross-presentation on the other hand may be of great value for therapeutic interventions.

Mechanisms Underlying Cross-Presentation

One of the intriguing aspects of cross-presentation is that processing of incoming antigen needs to be very efficient to compete with the vast amount of endogenous proteins for MHC class I binding. In addition, cross-presentation requires access of incoming antigen to the MHC class I pathway that is mechanistically separated from the uptake vesicles ( 98 ).

Dendritic cells harbor unique pathways to facilitate these logistic and mechanistic challenges underlying cross-presentation. Based on research of numerous groups, two main models have been put together for the mechanisms underlying cross-presentation of exogenous antigens, referred to as the “cytosolic” pathway and the “vacuolar” pathway [reviewed by Ref. ( 20 )]. These pathways are not mutually exclusive and may operate together in one cell ( 99 ). The most discriminative aspects between the two pathways are discussed below.

In the cytosolic pathway, antigens are degraded by the proteasome, a large enzyme complex situated in the cytosol that makes this pathway sensitive to inhibitors of proteasomal degradation. Alternatively, in the vacuolar pathway, both antigen degradation and MHC class I presentation occur in the endocytic compartment. Involvement of this pathway can be experimentally addressed by confirming resistance to inhibition of proteasomal degradation and sensitivity to inhibition of lysosomal proteolysis.

Lysosomal proteolysis has a detrimental role in the cytosolic cross-presentation pathway. It was experimentally demonstrated that limiting lysosomal proteolysis by chemically increasing the lysosomal pH favors cross-presentation of viral proteins HCV-derived NS3 and HIV-derived Nef by preventing complete degradation of potential MHC class I binding epitopes ( 53 ). Several different adaptations on the endocytic compartment, including a differential lysosomal protease activity, mechanisms to control the lysosomal pH, and antigen storage compartments, together endow DC to facilitate cross-presentation via the cytosolic pathway ( 100 – 102 ). Cross-presentation via the cytosolic pathway further requires export of internalized antigens from the endocytic compartment to the cytosol for proteasomal degradation, which is probably the rate-limiting step in this pathway, at least for protein antigen. Many enveloped viruses can enter the cytoplasm as part of their infection strategy that requires fusion of the viral envelope with the endosomal membrane to release the viral genome into the cytoplasm. This endosomal fusion capacity probably underlies the efficiency of cross-presentation of viral particles, at least for those particles that are able to enter the cytoplasm of DC. The mechanism of cytosolic delivery for other viral antigens and viruses that do not undergo endosomal fusion in human DC is largely unknown. Candidate proteins that may be involved in cytosolic delivery include HSP and p97 and sec61, which belong to the endoplasmic reticulum-associated protein degradation (ERAD) machinery ( 20 ), however, the role of these molecules in human DC is poorly studied.

Interestingly, the cytosolic and vacuolar pathway has totally different kinetics, which can be used to determine which pathway is involved ( 103 ). Whereas cross-presentation via the vacuolar pathway is fast and can be detected after 20 min ( 104 ), cross-presentation via the cytosolic pathway is much slower and formation of MHC class I–peptide complexes via this pathway may take at least 8 h ( 100 ), probably because it relies on MHC class I neo-synthesis ( 20 ). In contrast, MHC class I loading in the vacuolar pathway occurs in the endocytic compartment and depends on recycling of MHC class I molecules that are constitutively internalized by a highly regulated process ( 105 ).

Viral Road to Cross-Presentation

The cytosolic and the vacuolar pathways were largely established based on model antigens and mouse studies. It is important to assess if these models are applicable to cross-presentation of viral antigens by human DC.

As discussed above, viral particles use receptors expressed on the plasma membrane to enter DC and uptake of viruses often involves endocytosis. After receptor-mediated endocytosis, the cargo is transported through the endocytic compartment, a highly regulated network of vesicles with different characteristics and functions ( 103 ). An important function of the endocytic system is to sort internalized receptors and cargo to different locations for either degradation or recycling. Viruses use the endocytic system to exert their fusion capacity, however, at the same time DC use it to obtain viral antigen for cross-presentation. For example, when IAV reaches late endosomes, the low pH enforces conformational change, leading to hemagglutinin-mediated fusion of the endosomal and viral membranes and release of the viral RNA and proteins into the cytoplasm ( 106 ). IAV is efficiently cross-presented, at least when its fusogenic activity is intact ( 43 , 107 ). The fusion dependence was also observed for HIV; cross-presentation of HIV-1 was completely absent when fusion-incompetent HIV-1 mutants were used or fusion was inhibited chemically ( 44 , 46 ). Cross-presentation of HIV-1 viral particles is sensitive to proteasome inhibitors, but enhanced by inhibition of lysosomal proteolysis ( 46 ). Taken together, the above-mentioned work suggests a role for the cytosolic pathway in cross-presentation of fusion-competent viral particles, at least by mDC. Interestingly, cross-presentation of IAV by pDC is not sensitive to proteasome inhibitors, but is sensitive to inhibition of endosomal processing. Together with fast MHC class I presentation, this study suggests a role for the vacuolar pathway for cross-presentation of IAV by pDC.

Evidence from different studies involving IAV-infected monocytes ( 108 ), HCMV-infected fibroblasts ( 27 ), and EBV-transformed B cells ( 61 ) suggests that cross-presentation of cell-associated antigen involves uptake by receptor-mediated phagocytosis and that antigen processing is dependent on the proteasome, but also sensitive to inhibition of lysosomal proteolysis ( 109 ). Cross-presentation of Ag–Ig immune complexes also requires both proteasomal and endosomal antigen processing ( 85 ). Taken together, these data indicate that although cross-presentation of both cell-associated antigen and Ag–Ig immune complexes require proteasomal degradation, they may need some degree of lysosomal proteolysis to facilitate translocation of antigens from lysosomes to cytoplasm. Since these sources of viral antigen do not have intrinsic fusogenic capacity, they rely on functional specializations of DC to export Ag of the endocytic compartment to the cytosol ( 103 ).

Interestingly, several lines of evidence suggest that VLP follow a different pathway for cross-presentation. Cross-presentation of PapMV VLP, HCV VLP, and HBV VLP was not affected by proteasome inhibitors but sensitive to reagents that inhibit lysosomal proteolysis ( 51 , 96 , 110 ). Furthermore, it was shown that cross-presentation of HBV VLP by both mouse DC ( 110 ) and human DC (our own unpublished observations) is fast and TAP-independent. Together, these studies suggest that cross-presentation of VLP occurs via the vacuolar pathway.

The differences in cross-presentation pathways between fusion-competent viruses and VLP suggest that different vesicles within the endocytic compartment are involved. Chatterjee et al. showed that antigen targeting via MR or DEC-205 both lead to cross-presentation via different compartments ( 82 ). Evidence for a process of sorting comes from an elegant study by Lakadamyali et al., where it was shown that after endocytosis, IAV is sorted into a population of dynamic endosomes that rapidly becomes more acidic, which is necessary for the virus to enter the cytoplasm ( 111 ). In contrast, an alternative non-viral ligand, transferrin is sorted into a different population of static endosomes that facilitate recycling of antigen and receptors to the cell surface.

Antigen targeting to DC-SIGN can result in trafficking to different cellular compartments, as was shown for HCV envelop protein and Lewis X uptake via DC-SIGN ( 112 ). In addition, antibody-mediated antigen targeting to the neck region of DC-SIGN was dramatically more efficient with regard to cross-presentation of the targeted antigen compared to targeting to the carbohydrate-binding domain, and these differences were related to different endocytic trafficking ( 93 ). Taken together, these studies suggest that endocytic sorting is important for the fate of antigens and that sorting occurs at the receptor level. The nature of the sorting signal and the role of endocytic receptors and their adaptor molecules in this process remains to be further elucidated. However, an indication that poly-ubiquitination may be involved in sorting and antigen translocation comes from a mouse study involving the MMR ( 113 ).

We conclude that both the cytosolic and the vacuolar pathways are applicable to cross-presentation of viral antigen by human DC, depending on the type of viral antigen that is encountered by DC (Figure 1 ). The studies discussed above suggest that VLP preferentially traffic via the vacuolar pathway for cross-presentation, whereas protein antigen, fusion-competent viral particles, cell-associated antigen, and Ig-opsonized antigen preferentially traffic via the cytosolic pathway for cross-presentation, except in pDC that may preferentially facilitate the vacuolar pathway. Since the above-mentioned studies together suggest that antigen is sorted into pathways with different efficiency of cross-presentation at the receptor level, it is of high importance to gain more knowledge on the receptors used for internalization of viral antigens and their exact role in the sorting of Ag to different pathways in order to fully understand the cross-presentation of viral antigens. Currently, besides VLP, no other viral antigens were found that utilize the vacuolar cross-presentation pathway in human mDC, thus the physiological role of this pathway remains to be further understood. However, since this pathway is highly efficient, as was demonstrated in pDC ( 114 ), further understanding of the mechanisms underlying the vacuolar pathway may be of interest for therapeutic purposes.

DC Maturation as a Critical Factor for CTL Induction

Antigen presentation in MHC class I can lead to CTL priming or tolerance, depending on the context in which DC encounter the antigen ( 15 ). Sensing of danger signals by PRR on DC (Table 1 ) induce DC maturation, a differentiation process initiated after innate immune recognition that regulates key functions involved in CTL induction, including migration, antigen presentation, co-stimulation, and production of cytokines. Co-stimulation lowers the threshold for antigen recognition by the T-cell receptor and is important for proliferation, survival, effector function, and memory formation of T cells. Changes in antigen presentation after DC maturation include upregulation of MHC class I molecules ( 42 ), enhanced proteasomal activity ( 115 ), and reduced lysosomal antigen degradation ( 116 ) due to lower expression of lysosomal proteases ( 107 ). It is well-accepted that matured human DC have an enhanced capacity to activate virus-specific CTL ( 25 , 42 , 56 , 60 , 117 , 118 ). Importantly, however, the experimental stimuli used for induction of DC maturation are often not representative for the type of danger signals that are encountered by DC during viral infection in vivo .

Which danger signals can be naturally encountered by PRR on DC during viral infection? Viruses can display danger signals of various nature including viral nucleic acids, replication intermediates, carbohydrate structures, and proteins on the envelop, that can be sensed by PRR on DC (Table 1 ). IAV and RSV, both ssRNA viruses, induce maturation of different human DC subsets including moDC, BDCA1 + mDC, and pDC ( 34 , 39 , 42 , 119 , 120 ). Also VLP have been shown to induce DC maturation ( 49 , 50 , 75 ), which is not dependent on TLR but may be mediated by a recently identified innate recognition mechanism ( 121 ). In addition to virus-derived danger signals, virus-induced danger signals produced by the host in response to viral infection can induce DC maturation. Examples of such virus-induced host-derived maturation signals include cytokines such as IFNα/β and TNFα secreted by virus-infected cells ( 122 ) and damage-associated molecular patterns (DAMP) released by damaged or dying cells ( 123 ). During interaction of DC with cell-associated Ag, DC can encounter both virus-derived danger signals and host-derived maturation signals ( 27 , 124 , 125 ) or host cell-derived DAMP, such as TLR4 ligand high-mobility group box 1 (HMGB1) ( 126 ) or CLEC9A ligand F-actin ( 127 ).

The induction of DC maturation by virus-derived and virus-induced stimuli suggests that these factors also enhance CTL priming, however, direct experimental evidence on the contribution of virus-induced DC maturation on CTL induction by human DC is limited. IAV-infection of DC is associated with strong DC maturation and efficient antigen-specific CTL proliferation ( 42 , 117 ). Similarly, TLR agonist poly I:C that mimics viral double-stranded RNA (dsRNA) is a strong inducer of DC maturation and effectively enhances cross-presentation of recombinant viral antigen by several subsets of human DC ( 9 , 56 , 128 , 129 ). Also TLR7/8 agonists have been shown to enhance DC-induced CTL expansion and effector function in vitro ( 81 ). In contrast, cross-presentation of cell-associated antigen was inhibited when polyI:C or IAV were present in the captured dead cells, suggesting that virus-derived danger signals may also have a detrimental effect on cross-presentation, which may be specific for cross-presentation of cell-associated antigen ( 130 ). IFNα, a widely studied representative of virus-induced signals, can exert multiple effects on human DC that promote CTL cross-priming [reviewed by Ref. ( 4 )]. For example, moDC differentiated in the presence of IFNα, so called IFNα-DC, have superior cross-presentation capacity compared to classical moDC ( 52 , 131 ). In conclusion, although it is widely accepted that virus-derived and virus-induced stimulatory signals are required for effective cross-priming of virus-specific CTL, it has been difficult to experimentally address this hypothesis in the currently used in vitro models. Challenges include the low precursor frequency of naïve virus-specific CD8 + T cells and dissection of the separate contributions of DC maturation and antigen presentation to CTL induction.

Interference with DC maturation and thereby subverting the development of effective CTL induction is an important mechanism of immune evasion used by many viruses. Examples of viruses that interfere with DC maturation are MV ( 132 ), VV, via the production of cytokine receptor homologs ( 33 ), HSV, via destabilization of host mRNA ( 35 , 133 ) and HCMV, which prevents upregulation of co-stimulatory molecules and production of cytokines ( 134 ) and induces TGFβ production by its target cells ( 124 ). Furthermore, DC isolated from patients with chronic HIV, HBV, and HCV infections showed functional impairments in the capacity to produce IL-12 or induce T-cell activation, which may be a direct effect of the virus on DC and thereby the cause of the failing adaptive immune response, but could also be the consequence of the chronic infection ( 135 , 136 ).

The connection between innate immune recognition of viruses by human DC and the induction of virus-specific CTL is an important subject for further study. In addition, the PRR and pathways underlying recognition of viruses by DC and the mechanisms by which viruses circumvent these pathways needs to be further explored. Novel molecular techniques such as the ability to knock down PRR in human DC will empower this research, which is important for the development of therapeutic interventions.

DC Subsets Involved in Cross-Presentation of Viral Antigen

Before 2010, the large majority of studies on cross-presentation of viral antigen by human DC were performed with in vitro -generated moDC, however, more recently a number of groups have succeeded in obtaining sufficient numbers of DC from blood or other organs to assess the ability and mechanism of cross-presentation of viral antigens by different human DC subsets.

BDCA3 + DC were initially recognized as a subset with superior cross-presentation capacity compared to other human DC subsets ( 9 , 21 , 89 , 137 ). Comparison of transcriptional profiles revealed that BDCA3 + DC represent the human equivalent of murine CD8α + and CD103 + DC ( 56 , 138 ), which have a superior intrinsic cross-presentation capacity compared to other DC subsets ( 139 ). In parallel, selective expression of CLEC9A ( 84 ), a receptor that senses dead cells ( 140 ) and facilitates cross-presentation by mouse ( 141 ) and human DC ( 94 ), suggested that human BDCA3 + DC would excell in cross-presentation of dead cell material. Superior capacity to cross-present cell-associated antigen by BDCA3 + DC was demonstrated by several independent studies ( 9 , 21 , 89 , 102 , 137 ), however, not observed in all studies ( 118 ). Although BDCA3 + DC are highly capable of cross-presenting cell-associated antigen, cross-presentation of cell-associated antigen has also been demonstrated for BDCA1 + DC ( 102 ), pDC ( 89 , 118 ), and moDC ( 31 , 57 ). Also for other types of antigen, cross-presentation is not restricted to the BDCA3 + DC subset. Cross-presentation of protein antigen was shown for peripheral blood and tissue-derived BDCA1 + DC ( 9 , 128 ), BDCA-2 + pDC ( 102 , 128 ), and BDCA3 + DC ( 9 , 56 , 102 , 128 , 137 ), as well as for in vitro -generated CD34 + -derived DC ( 102 ) and moDC, as discussed above. Although BDCA3 + DC are highly capable of cross-presenting cell-associated antigen, cross-presentation of cell-associated antigen has also been demonstrated for BDCA1 + DC ( 102 ), pDC ( 89 , 118 ), and moDC ( 54 ).

Both BDCA3 + and BDCA1 + DC share the specialized machinery that is associated with efficient cross-presentation capacity, i.e., high phagosomal pH, production of ROS within endocytic compartments, and efficient transfer of exogenous antigens into the cytosol ( 102 ). Both subsets have a similar efficiency of endogenous MHC class I presentation after transfection, a similar efficiency of cross-presentation of heat-inactivated IAV that can egress to the cytosol at low pH and a similar efficiency of cross-presentation of antigen that is selectively delivered to early endosomes ( 107 ). Nevertheless, BDCA3 + DC were superior compared to BDCA1 + DC at cross-presentation of antigen that was artificially targeted to lysosomes by using antigen conjugated to DEC-205 targeting antibodies ( 107 ). This suggests that although both DC subsets can efficiently cross-present Ag delivered to early endosomes, BDCA3 + DC may exhibit a specialized machinery to transfer Ag from late endosomes and lysosomes to the cytosol. This DC characteristic might explain the superior capacity to cross-present IgG-opsonized antigen targeted to FcγR that could not be attributed to superior FcγR expression and/or antigen uptake in these cells ( 85 ).

Plasmacytoid DC contribute to anti-viral immune responses by producing large amounts of IFNα/β, however, their role as professional antigen presenting cell in the initiation of virus-specific T-cell responses was initially questioned based on controversial results in mice ( 86 ). Direct comparison of intrinsic characteristics that can influence cross-presenting capacity, such as phagosomal pH and ROS production, between pDC and BDCA1 + and BDCA3 + mDC was hampered due to inconclusive data for pDC ( 102 ). However, pDC express a broad repertoire of antigen-uptake receptors on their cell surface such as FcR and CLR BDCA-2, DEC-205, DCIR that can facilitate the uptake and cross-presentation of viral antigens ( 116 ) (Table 1 ). In addition, pDC can efficiently transfer exogenous Ag into the cytosol suggesting that they may be capable of cross-presenting antigen via the cytosolic pathway ( 102 ). Numerous functional studies showed that human pDC can cross-present recombinant protein antigens, long peptide antigens, IAV-derived antigens, and cell-associated antigens ( 88 , 118 , 119 , 142 ). In addition, it was also demonstrated that pDC can efficiently cross-present viral antigen via the vacuolar pathway, which may be facilitated by MHC class I storage in recycling endosomes ( 114 ). Taken together, we conclude that human pDC can efficiently facilitate cross-presentation of a wide range of viral antigens. Direct comparison of cross-presentation efficiency between human pDC and mDC was thus far inconclusive, with one study showing a higher efficiency of cross-presentation by pDC ( 114 ), another study showing superior MHC class I-restricted IAV presentation by BDCA1 + mDC ( 40 ) and three studies concluding that pDC and BDCA1 + or BDCA3 + mDC have similar cross-presentation efficiencies ( 118 , 119 , 142 ).

Although blood DC required DC maturation for efficient cross-presentation, skin or lymph node DC can cross-present under steady state conditions, which might be due to a more mature/activated status of these tissue DC compared to circulating DC ( 56 , 102 , 143 ). In addition to BDCA1 + and BDCA3 + DC, skin contains Langerin + LC and dermal intDC, often referred to as CD14 + DC. Comparison of CD14 + DC to other skin DC subsets indicated that this subset showed the least cross-presenting capacity among skin subsets ( 10 , 56 ), which may be related to the finding that these cells express immunoglobulin-like transcript receptors that antagonize CTL development ( 144 ). Cross-presentation capacity of LC cells is under debate and may vary upon the source of LC and type of antigen used in experiments. Cross-presentation of recombinant protein antigen by in vitro -generated LC has been demonstrated in several independent studies ( 10 , 102 , 145 ), however, cross-presentation of replication-incompetent MV and MV-infected cells by skin-derived LC was absent ( 25 ). Sine LC are potentially interesting vaccine target cells, because of their presence at mucosal sites such as skin and higher respiratory tract ( 25 ), further studies on the cross-presentation capacity of primary LC are required.

We conclude that essential mechanisms of cross-presentation are present among most human DC subsets, with the exception of CD14 + DC. Superiority of cross-presentation among DC subsets can be attributed to the repertoire of uptake receptors and adaptations in the endocytic compartment and may vary depending on the type of antigen.

Technical Limitations and Novel Approaches

Although several technical advancements have potentiated the study of MHC class I-antigen presentation by human DC, several important questions remain to be addressed.

One of the current technical challenges is to measure antigen presentation at the level of DC. The purest read-out would be to measure MHC class I-antigen complexes at the surface of DC (signal 1 only), however, tools are lacking ( 20 ). The best current available method to quantify MHC class I-antigen presentation is a read-out involving activation or in vitro induction of virus-specific T cells. However, it should be taken into account that activation of virus-specific T cells results from a combination of TCR ligation by MHC class I–peptide complexes (signal 1) and other stimuli provided by DC such as cytokines and co-stimulation (signal 2 and 3).

The study of induction of human CD8 + T cells by DC is also hampered by the extreme low frequency of naïve virus-specific T cells in peripheral blood. As discussed above, MHC class I presentation by human DC has been most frequently studied for IAV, HIV-1, and CMV. For these viruses, it has been possible to obtain sufficient numbers of “memory” T cells from peripheral blood and use T-cell expansion and IFNγ production as read-outs for antigen presentation in an autologous setting ( 13 , 14 , 54 ). Virus-specific T-cell clones to other viruses can be obtained by several rounds of antigen-specific expansion in vitro . However, performance of such in vitro -generated clones in cross-presentation studies is complicated due to their limited life span and the allogenic bias present in experiments because DC and T cells are not from the same donor. A novel promising approach for the study of cross-presentation of viruses by human DC is the use of T-cell receptor transfer to generate autologous virus-specific T cells ( 146 , 147 ). Such T cells are evaluated in the context of immunotherapy of patients but may also be exploited as tools to monitor antigen presentation by DC.

Recommendations and Considerations for Development of Therapeutic Vaccine Strategies

Chronic viral infections such as HIV, HBV, and HCV are a big health burden and affect 100 millions of patients worldwide. Viral persistence is associated with a failure of the patient’s immune response to eradicate the virus ( 136 ). In addition to chronic persistent infections, reactivation of latent infections including HCMV, EBV, and HPV is a major threat for immune compromised patients. In addition, a high proportion of these chronic and latent infections including HIV, HBV, HCV, EBV, HPV, and HTLV is related to the development of malignancies later in life ( 148 ). Immunotherapy represents an attractive therapeutic intervention to combat such infections and prevent virus-related malignancies by using the body’s own defense mechanisms. To accomplish this, immunotherapy is directed to improve virus-specific immunity and eradicate the virus but also generate protective memory responses to prevent re-infections. Moreover, immunotherapy should overcome T-cell exhaustion and anergy, often observed in patients with chronic infections ( 148 ).

Insights into the mechanisms underlying effective priming of virus-specific CTL by human DC are instrumental for the development of effective virus-specific immunotherapy. We identified cross-presentation as a crucial mechanism for the induction of virus-specific CTL and embrace the concept to utilize the effective cross-presentation mechanisms naturally present in DC for immunotherapy. In line with this concept, antibody-mediated antigen targeting to endocytic receptors is an emerging approach employed by numerous groups to target antigen to DC for cross-presentation. Endocytic receptors that efficiently facilitate cross-presentation by human DC include FcγRIIA, CLEC9A, DEC-205, and DCIR ( 81 , 85 , 94 , 116 , 149 ). An advantage of antigen targeting to specific receptors is the possibility to select receptors that are uniquely expressed by distinct subsets of DC (Table 1 ), such as proposed for XCR1 ( 150 ) or CLEC9A ( 94 ). Selective targeting to DC prevents antigen consumption by irrelevant cells, which may lead to reduced availability of antigen to DC and improper T-cell activation.

As discussed previously, DC maturation is crucial for virus-specific CTL induction. Although the endocytic receptors are very potent in internalizing antigen, their role in promoting DC maturation is less clear. Therefore, the combination of antigen targeting with adjuvants is an important field of study. FcγR have been shown to facilitate both efficient antigen uptake and DC maturation, however, it was recently shown that FcγR-dependent DC maturation in human DC is less strong than was previously observed in mice DC ( 85 , 151 ). Other interesting approaches that combine antigen targeting to DC and DC maturation in one cargo include TLR-ligand–peptide conjugates ( 152 ) and nanoparticles that contain both antigen and adjuvant ( 116 ).

Since DC comprise a heterogeneous family of subsets that differ in location, frequency, receptor expression, and functional specializations, it is important to design a therapeutic vaccine with the desired DC subset in mind. Based on accumulated evidence from in vitro studies on antigen presentation by human DC subsets, we conclude that most human DC subsets have the basic capacity to cross-present, as long as the antigen is efficiently targeted to an endocytic compartment that favors cross-presentation. Nevertheless, DC subsets do have unique functional characteristics, such as type of cytokine production, which can have high impact on the type of immune response induced. Moreover, DC subsets express different PRR (Table 1 ) and only adjuvants for a selected number of TLRs are currently available at clinical grade.

In addition to antigen targeting to DC in vivo , recruiting of DC precursors may represent an attractive immunotherapeutic approach, as was recently proposed for monocytes, which can contain a natural reservoir of HBsAg that can be presented in MHC class I upon differentiation of these monocytes to moDC ( 153 ).

Concluding Remarks and Future Perspectives

Based on two decades of research into MHC class I-restricted presentation of viral antigen by human DC, we conclude that cross-presentation of viral antigens is a highly efficient mechanism for defense against viruses. Furthermore, cross-presentation of viral antigens seems not only pivotal for defense against viruses that do not infect DC, but also for those that infect DC, as demonstrated by in vitro studies using replication-incompetent IAV, HIV-1, and MV. Since these viruses represent a selection of all viruses that can productively infect human DC, the contribution of direct presentation by human DC infected with other viruses cannot be completely ruled out. Nevertheless, as discussed in this review, cross-presentation has many conceptual advances compared to direct presentation by infected DC.

So far, knowledge on the presentation of viral antigens by human DC is mainly derived from in vitro studies. Whether these studies faithfully represent the in vivo situation is of course difficult to predict. Several caveats from these in vitro studies include the use of in vitro -generated DC, which may behave differently than their in vivo counterparts, the use of laboratory adapted virus strains, and pseudo-typed viruses, which may have tropisms that may not represent the in vivo situation, and the use of recombinant viral proteins and TLR ligands that are not fully representative for antigens or danger signals that can be encountered in vivo . Nevertheless, taking these limitations into account, together these studies have given us an important understanding of the mechanisms underlying MHC class I presentation of viral antigens by human DC. This knowledge is an important basis for the rational design of therapeutic vaccines for chronic viral infections.

Interesting venues for further research include identification of DC receptors involved in viral infection and initiation of immune response, elucidation of the molecular signals underlying sorting of viral antigen to endocytic compartments that favor cross-presentation and the role of virus-derived danger signals and virus-induced maturation stimuli in cross-presentation and CTL priming.

A more detailed knowledge of these key factors in virus–host interaction will further empower the design of novel therapeutics for infectious diseases.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This study was supported by a VIDI grant (project 91712329) from the Netherlands Organisation for Scientific Research (NWO) to Andrea M. Woltman.

Abbreviations

DC, dendritic cell; CTL, cytotoxic T lymphocyte; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; moDC, monocyte-derived dendritic cell; LC, Langerhans cell; PRR, pattern-recognition receptor; VLP, virus-like particle; CLR, C-type lectin receptor; FcR, Fc receptor; TLR, Toll-like receptor.

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Major histocompatibility complex (mhc) class i and mhc class ii proteins: conformational plasticity in antigen presentation.

$\r\nMarek Wieczorek&#x;$

1 Protein Biochemistry, Institute for Biochemistry, Freie Universität Berlin, Berlin, Germany
2 Computational Molecular Biology Group, Institute for Mathematics, Berlin, Germany

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity.

Introduction

Major histocompatibility complex (MHC) class I and class II proteins play a pivotal role in the adaptive branch of the immune system. Both classes of proteins share the task of presenting peptides on the cell surface for recognition by T cells. Immunogenic peptide–MHC class I (pMHCI) complexes are presented on nucleated cells and are recognized by cytotoxic CD8+ T cells. The presentation of pMHCII by antigen-presenting cells [e.g., dendritic cells (DCs), macrophages, or B cells], on the other hand, can activate CD4+ T cells, leading to the coordination and regulation of effector cells. In all cases, it is a clonotypic T cell receptor that interacts with a given pMHC complex, potentially leading to sustained cell:cell contact formation and T cell activation.

Major histocompatibility complex class I and class II share an overall similar fold. The binding platform is composed of two domains, originating from a single heavy α-chain (HC) in the case of MHC class I and from two chains in the case of MHC class II (α-chain and β-chain) (Figure 1 A). The two domains evolved to form a slightly curved β-sheet as a base and two α-helices on top, which are far enough apart to accommodate a peptide chain in-between. Two membrane-proximal immunoglobulin (Ig) domains support the peptide-binding unit. One Ig domain is present in each chain of MHC class II, while the second Ig-type domain of MHC class I is provided by non-covalent association of the invariant light chain beta-2 microglobulin (β 2 m) with the HC. Transmembrane helices anchor the HC of MHC class I and both chains of MHC class II in the membrane (Figure 1 A).

Figure 1. Structural characteristics of major histocompatibility complex (MHC) class I and MHC class II proteins and their compartment-dependent loading with processed peptides. (A) Domain topology of a pMHC class I and pMHC class II complex. (B) Structure of HLA-A68 in complex with an HIV-derived peptide (PDB: 4HWZ, left) and HLA-DR1 in complex with a hemagglutinin-derived peptide (1DLH, right). Indicated are the supposed interaction sites of MHC class I with tapasin and of MHC class II with DM as dashed gray lines. The peptide is shown in yellow with its N and C-terminus marked and relevant pockets are labeled green (C) Simplified illustration of MHC class I (left) and II (right) processing and peptide-editing pathways. CLIP, class II-associated invariant chain peptide; Caln., calnexin; Calr., calreticulin; ER, endoplasmic reticulum; PLC, peptide loading complex.

The groove in-between the two helices accommodates peptides based on (i) the formation of a set of conserved hydrogen bonds between the side-chains of the MHC molecule and the backbone of the peptide and (ii) the occupation of defined pockets by peptide side chains (anchor residues P2 or P5/6 and PΩ in MHC class I and P1, P4, P6, and P9 in MHC class II) ( 1 – 4 ). The type of interactions of individual peptide side-chains with the MHC depend on the geometry, charge distribution, and hydrophobicity of the binding groove. Predicting the affinity of these distinct MHC–antigen interactions for individual allotypes has been a long-standing goal in the community. While good progress has been made in developing and optimizing bioinformatic algorithms to estimate peptide binding to MHC proteins, these in silico predictions, however, still yield false positives ( 5 , 6 ), and often fail in predicting immunodominance. We argue that understanding the relevance of transient or energetically excited protein conformations that are visited during the equilibrium fluctuations of the molecular structure is important for making good predictions.

In MHC class I, the binding groove is closed at both ends by conserved tyrosine residues leading to a size restriction of the bound peptides to usually 8–10 residues with its C-terminal end docking into the F-pocket ( 7 – 9 ). In contrast, MHC class II proteins usually accommodate peptides of 13–25 residues in length in their open binding groove, with the peptide N-terminus usually extruding from the P1 pocket ( 10 ). It has been reported that the interactions at the F pocket region in MHC class I and the P1 region (including the P2 site) in MHC class II appear to have a dominant effect on the presentation of stable pMHC complexes and on the immunodominance of certain peptidic epitopes ( 11 – 16 ). Interestingly, these pockets are located at opposite ends of the binding groove of the respective MHC class I and MHC class II structures (Figure 1 B).

The most polymorphic human MHC class I and class II proteins (human leukocyte antigens, HLAs) are each expressed from three gene regions (MHC class I: HLA-A, -B, -C; MHC class II: HLA-DR, -DP, -DQ), which are all highly polymorphic. This allelic variation mainly affects the nature and composition of the peptide-binding groove and thus modulates the peptide repertoire that is presented on the surface by MHC class I or MHC class II proteins for CD8+ or CD4+ T cell recognition, respectively. A good match of the peptide and the MHC binding groove is an important, but certainly not the sole determinant of its presentation. In fact, the formation of a pMHC complex depends on its peptide-loading pathway, in which the selection of peptides is influenced by several factors, such as antigen availability, protease activity, or the availability of chaperones. In addition, for each MHC class, a “catalyst” is available to enhance peptide exchange for certain peptides: tapasin for MHC class I and HLA-DM for MHC class II. These molecules edit the presented peptide repertoire and bias the exchange reaction toward the presentation of thermodynamically stable complexes. Tapasin and HLA-DM thus act similar to typical enzymes by reducing the energy barrier for peptide exchange. However, in the case of HLA-DM and tapasin, no covalent bonds are formed or cleaved during the exchange reaction.

The MHC class I HC folds and assembles with β 2 m in the lumen of the endoplasmic reticulum (ER). The partially folded heterodimer is then incorporated into the peptide-loading complex (PLC) for peptide binding and exchange. In the PLC, tapasin is a protein that catalyzes, together with other chaperones, the loading of high-affinity peptides derived from proteolysis of endogenously expressed proteins (Figure 1 C, left panel) ( 17 , 18 ). In the absence of tapasin, some class I allotypes (such as HLA-B*44:02) are retained in the ER (tapasin-dependent), whereas other class I proteins (tapasin-independent, such as HLA-B*44:05 and HLA-B*27:09) can bind peptides and travel to the cell surface ( 19 – 22 ). There is no crystal structure of the MHC class I/tapasin complex, but several structural models and mutational studies suggested that tapasin binds two regions in the HC of MHC class I, a loop in the α 3 domain (residues 222–229), and a region of the α 2 domain (residues 128–137) adjacent to the F-pocket (Figure 1 B) ( 18 , 21 , 23 – 29 ).

Major histocompatibility complex class II proteins fold in the ER in complex with a protein called invariant chain (Ii) ( 30 ) and are then transported to late endosomal compartments (also coined MHC class II compartment, MIIC). There, Ii is cleaved by cathepsin proteases and a short fragment remains bound to the peptide-binding groove of MHC class II proteins, termed class II-associated invariant chain peptide (CLIP). This placeholder peptide is then normally exchanged against higher affinity peptides, which are derived from proteolytically degraded proteins available in endocytic compartments (Figure 1 C, right panel). HLA-DM accelerates peptide exchange, with different allelic variants being more or less susceptible to catalysis. HLA-DM has a highly similar structural fold compared to classical MHC class II proteins, but its closed-up binding groove prevents peptide binding. Crystal structures of HLA-DM in complex with the MHC class II protein HLA-DR1 ( 31 ) and in complex with the competitive inhibitor HLA-DO ( 32 ) revealed that HLA-DM mainly contacts the α 1 -domain of MHC class II proteins close to the P1 pocket and additionally the membrane-proximal β2-domain, in line with previous mutational analyses (Figure 1 B) ( 13 , 33 – 35 ).

Despite the structural differences between tapasin and HLA-DM as well as their presumably opposite sites of interaction with regard to the orientation of the binding groove, a similar mode of action has been suggested, hinting at a possible convergent evolution of the two exchange catalysts (Figure 1 B) ( 36 ). A common feature seems to be that both catalysts target regions in the vicinity of those pockets in the peptide-binding groove that are of great relevance for the stability of the respective pMHC complex ( 11 , 13 , 15 , 31 , 37 ). Furthermore, in both cases, the binding of a high-affinity peptide is able to release the interaction with tapasin/DM ( 13 , 26 , 38 , 39 ) and allows for transport of stable pMHC complexes to the cell surface.

While the general hallmarks of antigen processing and editing have been established, the discussion is now moving toward the dynamics of the system, both at the cellular and molecular level. The mechanistic questions relate to a description of how exactly peptides are selected for presentation and how tapasin and HLA-DM catalyze this reaction in an allele-specific manner.

Structural Variations in MHC Complexes

Many allelic variants of MHC class I and MHC class II bound to individual peptide antigens display different biochemical features, but surprisingly, their “ground-state,” i.e., thermodynamically most stable conformations reported by the many available pMHC X-ray structures are very similar. In contrast, increasing experimental and computational evidence of wild type (WT) and mutant MHC complexes over the past years incontestably revealed that changes in conformational dynamics in MHC proteins have to accompany peptide loading and exchange ( 22 , 40 – 46 ).

To highlight possible dynamic regions within ground-state crystal structures of human MHC class I and class II proteins bound to a peptide, we performed a global B-factor analysis of all available X-ray crystal structures of human MHC complexes in the absence of any other binding partner. In each structure, we normalized the B-factor values of each alpha carbon (CA) atom to the global mean. Then the variance of all the normalized B-factor values for each CA atom, in 297 human pMHC class I and 41 human pMHC class II structures, was calculated and depicted with a blue to red color spectrum, respectively (heat map on structures of HLA-A*0201 and HLA-DR1, Figures 2 A,B). Overall in the binding groove of class I (class II), the α-helix in the α2 (β1) domain displays higher B-factor variation values than the α-helix in the α1 (α1) domain and the β-strands from both domains. Among the pMHCI structures, although B-factor variation values in the N-terminus-proximal helical segments indicate the existence of a certain degree of dynamics, the α2-helical region around the peptide C-terminus displays the highest variation in B-factors (Figure 2 A). Among the class II structures, high B-factor variation values are found especially in β-strands 2 and 4 of the α-chain, the 3 10 helical region and almost the entire β-chain α-helix (Figure 2 B). Our analysis is corroborated by a previous comparison of 91 different pMHC class II crystal structures ( 47 ). In this analysis, conformational heterogeneities were observed in three regions: the 3 10 -helical region (α45–54), the kink region in the β1-helix (β62–71), and the β2-domain (β105–112).

Figure 2. Global B-factor analysis of X-ray crystal structures of MHC class I and MHC class II . Shown is the variance of the normalized residual B factor values of CA atoms (A) derived from 297 human pMHC class I structures is plotted as blue to red spectrum on a HLA-A*0201/peptide complex (PDB: 5HHN) and (B) from 41 human pMHC class II structures is plotted on a DR1/peptide complex (PDB: 4X5W).

It is known that, to some extent, structural variations can be introduced by variable peptide-binding modes. In this context, peptides longer than 8–10 residues have been reported to bind to the MHC class I binding groove ( 48 – 52 ). To accommodate the increase in length, the peptides have to bulge out, leaving the central residues (between p2 and pΩ) exposed to solvent. This is usually achieved by a kink in the backbone in the middle part of the peptide. Recently, two crystal structures of HLA-A2 bound to15-mer peptides have been solved ( 53 ). The two peptides follow a binding mode similar to that of the canonical peptides with two anchor residues in the B and F pockets. While one of the peptides showed a mobile central conformation similar to another reported long peptide ( 48 , 54 ), the other peptide adopted an unusual rigid β-hairpin secondary structure. Furthermore, although the binding of the N- and C-termini at both ends of the binding groove is conserved in almost all the MHC class I complexes, some exceptions have been reported. For example, in the F pocket of HLA-A2, the C terminal residue of the peptide extends by ~1 Å leading to a significant rearrangement of the pocket with only one of the standard hydrogen bonds (at Thr143) preserved ( 55 ). Another example is seen in the HLA-B35 protein, the short N-terminus of the 8-mer peptide does not reach the A pocket. Instead, the hydrogen bonds between the amino group of P1 residue and residue 45 of MHC class I are mediated by a water molecule ( 56 ).

Since, in the case of MHC class II proteins, the peptide ligand within the binding groove usually adopts a pseudosymmetrical PPII helix-like conformation, bidirectional binding is theoretically possible. An interesting case represents a crystal structure of a DR1/CLIP complex, in which the peptide binds in a very unusual, inverted orientation (C-terminus close to the P1 pocket). The driving force for this peptide inversion is the formation of three additional H-bonds of P1-close residues and the backbone of the peptide’s C-terminus ( 57 , 58 ). Biological and biochemical evidence for the existence of other pMHC class II isomers have been described in the context of autoimmunity ( 59 ). Since MHC class II proteins have open binding grooves, peptides can protrude outwards and even bind in different registers. In this regard, an insulin B chain-derived peptide (InsB 9–23 ) was suggested to induce type 1 diabetes (T1D) in a thermodynamically less favored, low-affinity binding register ( 60 , 61 ).

Apart from variable peptide binding, catalyst binding can induce more significant conformational variation, as seen in DM-bound DR. By designing a P1-anchor-free pMHC class II complex, Pos et al. could increase the affinity for the pMHC class II to DM and solve the crystal structure for the covalently tethered DR1/HA/DM complex ( 31 ). In this MHC class II/DM complex, the interaction interface is primarily composed by the α-subunits of DM and DR1 (~65% of the entire interaction surface, Figure 1 B). DM binding to DR stabilizes a rearranged conformation in the vicinity of the P1 pocket of DR1 resulting from the absence of critical peptide-MHC class II interactions in this region. The extended region in the DR1α-chain (α52–55) and the 3 10 -helix adopt an α-helical fold (Figure 3 A). Compared to other parts of the pMHC class II structure, it was shown that this site indeed represents a conformationally labile region ( 35 , 46 ). In addition, the C-terminal part of the β1-α-helix (β86–91) becomes slightly less structured (Figure 3 B).

Figure 3. Conformational rearrangements upon DM binding and structural variations in type 1 diabetes-susceptible DQ complexes. (A) Structural rearrangement in the α1-S4 strand and 3 10 -helical region seen in DR1 when bound to DM (limon cartoon) compared to DR1 unbound DM (red). (B) DM-induced rearrangements in the P1-pocket and the surrounding helical segments. PDBs used in (A,B) 1DLH and 4FQX. (C) Overlay of DQ2/ag (PDB: 1S9V), DQ6/hyp 1–13 (PDB:1UVQ) and DQ8/InsB 9–23 (PDB: 1JK8) showing the structural variations of the 3 10 helix and the P1-proximal β1-helix. Interdomain communication as exemplarily indicated by the hydrogen bond between αR52 and βE86/βT89 in the DQ8 allele variant is thought to increase the stability of these regions and was previously discussed to be linked to a lowered DM-susceptibility ( 62 , 63 ). ag, αI-gliadin; hyp, hypocretin peptide 1–13; InsB, insulin B chain 9–23. (D) Structural alignment of DR1/CLIP (PDB: 3QXA) and DR1-αF54C/CLIP (PDB: 3QXD), a mutant that shows an altered conformation in the 3 10 helix and an increased DM susceptibility.

A feature, which is also present in DM-bound DO, is the intermolecular H-bonds between two conserved residues (DRα W43 and DMα N125). The formation of these critical bonds (as well as other interactions) likely depends on the “flipping out” movement of αW43 from the P1 pocket of DR1 toward DMα (Figure 3 A). This movement of DR1 αW43 was suggested to be triggered by partial dissociation of the peptide’s N-terminus or by transient destabilization of contacts of the peptide N-terminus when bound to MHC class II ( 13 , 64 ). As a consequence, the P1 site is stabilized by a repositioning of two phenylalanine sidechains (DRα F51 and DRβ F89), thereby compensating for the loss of peptide anchors and αW43 from this region (Figure 3 B). Incoming peptides have to compete with these repositioned Phe residues for the P1/P2 site in order to be selected for display. Interestingly, structural characteristics of T1D-conferring HLA-DQ alleles were indeed discussed to be linked to decreased DM-sensitivity ( 62 ). The analysis of several DQ variants indicated structural differences of the T1D-risk variants DQ2 and DQ8 when compared to DQ1, DQ 6, or DR variants. The decrease in DM-susceptibility of these two proteins was explained by a stabilization of the 3 10 helical region ( 63 ) (Figure 3 C). However, the exact relationship between structural variations in the 3 10 helix and DM-susceptibility are not clear, as highly DM-susceptible DR complexes can display a different conformational mode, compared to DM-susceptible DQ alleles (Figure 3 D).

The observed changes lead to the question as to how much structural plasticity in these regions preexists in the peptide-loaded form and provide a prerequisite for catalyzed as well as for spontaneous peptide exchange. For example, do pMHC complexes sample conformations observed in simulations of empty proteins or in complex with the catalyst? How would allelic variation affect the distribution of MHC proteins within the conformational space and thereby influence the presented peptide repertoire? Could variation in protein plasticity also account for the association of specific MHC alleles with immune diseases? Since the polymorphic peptide-binding groove of MHC proteins defines its affinity for a certain peptide, substitutions of even a single amino acid may lead to significantly different affinities for individual peptides. In general, the critical factors defining whether a peptide is presented or not are determined at the different levels of antigen processing and presentation such as uptake route, amount, and folding state of the antigenic protein, amenability to proteolytic degradation, and catalysis of the complex, etc. However, at the molecular level, it has been shown that certain polymorphisms shape individual pockets in the peptide-binding groove to optimally present an autoimmunogenic self-peptide ( 65 – 67 ). In other cases, the functional impact of disease-associated polymorphisms remained enigmatic and suggests that dynamics might account for the observed differences.

Dynamics of Peptide-Free MHC Proteins

While simulations and experimental studies vary in the features ascribed to peptide-free MHC proteins, they certainly agree in attributing a substantial degree of dynamics to the peptide-binding groove. Thus, binding of peptides to MHC proteins is of utmost importance for the stabilization of the known MHC fold ( 40 , 68 ). The lack of a crystal or an NMR structure of peptide-free MHC protein hinders an accurate description of the structural changes upon peptide binding and this is probably due to the ensemble character of the peptide-free conformers. This ensemble character, however, has been probed by computational techniques, as discussed in the following paragraphs.

MHC Class I

Most of the conformational dynamics information on the peptide-free class I have been revealed by molecular dynamics (MD) simulations. In such simulations, peptide-free class I protein is modeled from the crystal structure by deleting the atoms of the bound peptide. In the absence of peptide, an increased conformational flexibility of the F pocket region was observed for several allelic variants (HLA-A*02:01, HLA-B*44:02, HLA-B*44:05, HLA-B*27:05, HLA-B*27:09, H-2D b , and H-2K b ) ( 9 , 15 , 21 , 22 , 69 – 71 ). Longer simulations of chicken and human class I allotypes showed increased global motion in the peptide-free form when compared to the peptide-bound proteins ( 72 , 73 ). By combining molecular docking and MD simulations, a conformational transition of the 3 10 helical segment of H-2L d between the peptide-bound and peptide-free class I was observed. Thus, a conformational reorganization close to the A and B pockets upon peptide binding was proposed ( 74 ).

Experimentally, circular dichorism (CD) was used to measure the thermal denaturation temperature (T m ) of the peptide-free HLA-B*07:02 (B7/β2m). By increasing the temperature, a gradual loss of the structure-specific signal of B7/β 2 m in the CD spectrum of peptide-free class I was detected, indicating a more heterogeneous conformational population. Furthermore, in the absence of peptide, the binding groove of B7/β 2 m was more sensitive to enzymatic proteolysis when compared to the peptide-bound form ( 40 ). Saini et al. studied the unfolding of H-2K b by measuring the intrinsic tryptophan florescence. The results pointed to a folding intermediate of peptide-free class I proteins that are more structured than a molten globule ( 75 ). This was in line with a previous study arguing for a native-like conformation of in vitro refolded empty murine class I proteins ( 76 ).

Structural studies using NMR indicated a loss of the binding-groove fold in the peptide-free form of a HLA-C allotype. In particular, NMR spectra of these peptide-free MHC class I protein show the loss of selected methionine NH resonances of the β-sheet floor of the peptide-binding groove, which indicated unfolding or conformational exchange of this part of the protein ( 77 ). This is consistent with previous reports indicating that especially the binding groove is undergoing conformational exchange in the absence of the bound peptide ( 40 , 78 ).

A work that focused on complexes with a partly filled MHC class I binding groove pointed to a requirement of the stabilization of the F pocket region. By using a refolded H-2D b in the presence of a pentamer peptide (NYPAL), which binds to the C to F pockets in the binding groove, a X-ray crystal structure of the pMHCI complex could be solved ( 79 ). Thus, peptide/class I interactions at the F pocket region seem to be sufficient to keep class I in a folded state. In agreement, short dipeptides mimicking the peptide C-terminus of high-affinity ligands support the folding of HLA-A*0201, displaying high peptide-receptivity ( 14 ). To stabilize peptide-free class I in a folded form independently from the peptide, the Springer group created a novel variant by introducing a disulfide bond to restrain the high flexibility of the F pocket region. The disulfide mutant showed an increased peptide and β 2 m affinity and bypassed the cellular quality control ( 80 ).

MHC Class II

Physiologically, the question if peptide-free MHC class II proteins play a role in adaptive immunity is posed by studies indicating that unloaded MHC class II proteins are abundantly present on the surface of immature DCs. There, they are able to bind ligands from the extracellular milieu and activate T cells ( 81 , 82 ). Two isomers of peptide-unloaded MHC proteins seem to exist, each displaying different kinetic properties ( 83 ). While the peptide-receptive empty isomer of DR1 binds peptide rapidly, the conversion to the non-receptive isomer within less than 5 min dramatically reduces the peptide-binding capacity of human DR ( 41 , 84 , 85 ). Studies using circular dichroism and size exclusion chromatography predicted a conformational change of peptide-free MHC class II upon peptide binding, and an increase in the overall stability ( 44 , 68 , 86 ). Peptide-free MHC class II proteins thus show a lower degree of helicity and an increased hydrodynamic radius compared to peptide-loaded MHC class II.

Carven and Stern studied ligand-induced conformational changes by selective chemical side-chain modification of peptide-free DR1 followed by mass spectroscopy analysis ( 87 ). The results of this study were inconsistent with a partly unfolded state of DR1 in the absence of ligand, but rather indicated a more localized conformational change induced upon peptide binding. However, empty MHC class II proteins harbor the hallmarks of partially unstructured peptide-binding domains when studied spectroscopically. NMR-spectra of the α-chain of such peptide-free MHC class II proteins barely display any signals for residues corresponding to the folded peptide-binding groove, indicating that the binding groove undergoes conformational exchange ( 46 ). Taken together, it appears likely that the empty binding groove dynamically samples different native-like conformations. Interestingly, similar to MHC class I, certain small molecules and dipeptides increase peptide-receptivity of peptide-free MHC class II proteins, presumably by preventing a “closure” of the binding groove ( 88 , 89 ). In similar to the F pocket of MHCI, the predicted site of interaction is the most dominant pocket of the binding groove (P1).

In a MD simulation study combined with peptide-binding assays, it was shown that a well-conserved residue, βN82, which also contributes disproportionally to pMHCII stability ( 12 , 90 ), is participating in the control of peptide receptivity ( 91 ). The authors suggested that the “non-receptive” state of peptide-free DR1 is induced by a molecular lock through the formation of a hydrogen-bond between DRβ N82A and DRα Q9. The observed narrowing of parts of the binding groove-flanking α-helices likely represents the trigger for such a clamped conformation. Using MD simulations, another investigation suggested a movement of the α51–59 region into the P1–P4 site of the binding groove in the empty state. In this state, the α51–59 region adopted a ligand-like conformation. In addition, an increased flexibility of the β2 domain as well as the β50–70 helical region were observed ( 92 ). A higher flexibility of the β58–69 helical region was also seen in MD simulations of the HLA-DR3 protein upon in silico peptide removal ( 93 ) (Table 1 ). Interestingly, this helical segment is also recognized by monoclonal antibodies designed to bind to the peptide-free conformation ( 81 , 94 ).

Table 1. Computational studies on MHC class I and II dynamics .

Finally, it has to be noted that the timescale of the experimental descriptions of empty MHC molecules differs vastly from the theoretical studies. While the latter describe the initial events of conformational changes accompanying peptide removal, the experimental investigations observe the properties of the empty MHC species at or near equilibrium.

Dynamic Features of Peptide-Bound MHC Complexes

While the study of empty MHC proteins is of theoretical and conceptual interest, nature has engineered the antigen-presenting system in a way that prevents the accumulation of isolated, non-peptide-bound MHC molecules. Endogenous peptides derived from the proteasome in case of class I or from the invariant chain in the case of class II first bind and eventually are replaced by antigenic peptide. This inherently dynamic process is enabled by intrinsic features of the MHC molecules and several studies suggest that pMHC complexes sample different and transient conformations dependent on the bound peptide and the allelic variant under investigation ( 46 , 64 , 71 , 72 , 95 , 100 , 106 , 113 , 114 ).

Changes in conformational dynamics in MHC class I are heterogeneously distributed along its peptide-binding groove, as suggested by both computational and experimental studies. For example, MD simulations showed a subtype-dependent conformational flexibility of the F pocket region. Residues 114 and 116 of the HC, at the bottom of the F pocket, and residues 74 and 77 from the α 1 -helix, engaging the peptide’s C-terminus, show an altered mobility in different MHC class I allotypes ( 9 , 22 , 70 , 71 , 96 , 115 , 116 ). Consistently, it was shown that the dynamics of the MHC class I binding groove was most profoundly affected by C-terminal residues of the peptide ( 15 ). In longer MD simulations, in addition to varying protein plasticity in the F pocket region, an enhanced sampling of conformations in the α 3 -domain upon peptide binding was observed ( 72 , 73 ) (Table 1 ).

Experimental observations at the atomistic level, derived from NMR-based relaxation-dispersion experiments, have elucidated the peptide dependency of minor states on the stability of pMHCI complexes. Conformational fluctuations of different HLA-B*35:01 complexes were localized to the peptide-binding groove, including residues of the B, E, and F-pocket, but not in the IgG-like domains ( 113 ). Interestingly, the presence of minor conformations in pMHCI complexes (ranging from approximately >1 to 4.5%) could be positively correlated to the thermostability and surface presentation of the pMHCI complex under investigation, implying that a minor conformation considerably contributes to pMHCI stability. Similar, investigations of HLA-A*02:01 loaded with different peptides by HD exchange/MS and fluorescence anisotropy revealed that fluctuations within the binding groove depend on the ligand bound to MHC class I ( 112 ). Despite these ligand-sensitive changes in dynamics, the α 2 -helix showed a general higher flexibility than the α 1 -helix. The authors concluded that the observed variations in dynamics throughout the peptide-binding site could influence receptor engagement, entropic penalties during receptor binding, and the population of binding-competent states (see also Table 2 ).

Table 2. Experimental studies on major histocompatibility complex (MHC) class I and II dynamics .

Another study also showed that β 2 m seems to sense allelic as well as peptide-induced conformational variations and accommodates to them, showing a high degree of plasticity within the inter-domain interface with the HC domains ( 110 ). NMR-chemical-shift changes of complexes were most pronounced in the region close to the F-pocket. By comparing the dynamics in the ns–ms timescale of HC-bound and free β 2 m, Hee et al. demonstrated that most residues gain rigidity upon HC binding ( 111 ). Nevertheless, three sites (region around His31, site around Asp53 and Lys58, and region around Ser88) remained flexible in the mature complex. Interestingly, His31 and Ser88 are located underneath the F-pocket, which stability is known to be important for tapasin function. Moreover, Lys58 and Ser88 are also known to interact with several other proteins, including the natural killer cell receptors Ly49A CD8 and LIR1 ( 117 – 119 ). Conformational sampling of these regions could thus be critical for the interaction with these receptors.

A similarly heterogeneous picture in regards to altered binding groove dynamics can be observed for MHCII: by dissecting the thermodynamics of peptide MHC class II interactions, Ferrante et al. conclusively demonstrated how dynamics of pMHCII complexes are linked to peptide affinity and DM-susceptibility ( 106 ). Using different biophysical techniques and MD simulations, the authors showed that peptide binding events can be driven by a considerable proportion of conformational entropy (if enthalpic interactions are less favored). MD simulations suggest that peptide-dependent conformational fluctuations involve alterations of α-chain residues DR1α-43–54 and β-chain residues 63–68 and 79–90. Wieczorek et al. performed H/D-exchange measurements in combination with NMR spectroscopy to obtain residue-specific experimental information about the stability of individual secondary structure elements. Several regions undergoing conformational fluctuation even in highly stable pMHCII complexes were thus revealed. These fluctuations are confirmed by extensive (~100 μs) MD simulations and Markov model analyses that reveal transient conformations with obvious relevance for the peptide-exchange pathway (see below for details) ( 46 ). In particular, the highest lability was seen in DRα 46–62, in parts of β-strands s2–s4 sitting underneath the N-terminal part of the α 1 helix, β65–93 and several loops connecting the β-strands of the peptide-binding site. Interestingly, this experimental piece of evidence is also in line with the global B-factor analysis presented here (Figure 2 B); a certain degree of dynamics thus seems to already be encoded even in the context of a high affinity pMHC complex. Earlier on, it was shown by HD-exchange/MS measurements that conserved peptide–MHC class II contacts (H–bonds) are strong at the P1 pocket-proximal site of the peptide (especially position βN82) in highly stable immunodominant complexes, which is in agreement with previous biochemical studies ( 12 , 35 , 90 ). However, local destabilization induced by a point mutation in the DR1 complex and the use of a different ligand (DR1-αF54C/CLIP) strongly enhanced fluctuations of the peptide and especially weakened contacts around the P1 site ( 35 ). This implies that weakening interactions by substitutions in the peptide or MHC (allelic variation) would have an influence on conformational fluctuations that correlate with DM-susceptibility.

MHC Dynamics During Peptide Exchange

While the studies described in the previous section unambiguously demonstrate the dynamic features of pMHC complexes, the question arises naturally in how far these properties translate into peptide exchange. For pMHCII complexes experimental progress has been made in identifying intermediate or transient conformations of pMHCII with regard to catalyzed or intrinsic peptide exchange ( 13 , 31 , 32 , 35 , 46 ). For pMHCI molecules the atomistic description of structural changes during peptide exchange mostly relies on MD simulation studies, supported by mutational analysis and circumstantial biophysical evidence ( 14 , 22 , 37 , 73 ).

Within cells, tapasin is a key protein that mediates the binding of high-affinity peptides to most class I proteins. To date, there is no crystal structure of the tapasin/MHC class I complex. Based on mutational studies, however, two regions have been shown to be essential for tapasin interaction with the HC of MHC class I: a loop in the α 3 domain (residues 222–229) and a part of the α 2 domain (residues 128–137) ( 17 , 23 , 25 , 120 , 121 ). Two major functions have been proposed for tapasin: (i) a chaperone-like stabilization of empty class I proteins ( 20 – 22 , 24 , 122 ) and (ii) a peptide-editing function through peptide-exchange catalysis ( 26 , 123 , 124 ). Several computational models have been published to describe the mechanism of action of tapasin on MHC class I ( 28 , 29 , 121 ). Most researchers agree on the importance of the F-pocket region for peptide exchange ( 14 , 21 , 37 , 80 , 125 ). However, the association of F-pocket dynamics and the peptide-exchange mechanism remain a matter of debate. So far, dynamics in the F-pocket region in the presence of peptide have not revealed any significant conformational exchange phenomena in most MD simulations (Table 1 ). Recently, longer MD simulations (microsecond timescale) of tapasin/pMHCI complexes indicated that binding of tapasin to HLA-B*44:02 accelerates the dissociation of low-affinity peptides ( 29 , 37 ). Using a computational systems model ( 73 , 126 ), it was shown that peptide exchange seems to depend on the opening and closing rate of the binding groove in the presence of peptide. According to this study, the pMHCI opening rate is peptide-dependent, but pMHCI closing is allele-dependent. Consequently, a low-affinity peptide complex would display fast opening rates, but only if the MHC allele variant has an F-pocket signature (more plasticity) that allows for fast closing in the presence of a high-affinity peptide (as B44:05), it would lead to efficient peptide exchange in the absence of catalyst. Allele variants with a rigid F-pocket conformation (as B44:02) in contrast depend on tapasin to sample the necessary conformational states to close the binding groove quickly.

It has to be considered that as a default, tapasin is present in the cell and that it may also provide the necessary function as a chaperone to prevent the collapse of empty MHC class I molecules into a non-receptive state ( 29 , 127 ) as it is experimentally measured in tapasin-deficient cells ( 21 , 22 ). However, in the absence of a crystal structure of the tapasin/MHC class I complex, it is difficult to rationalize the dynamics with regard to tapasin binding and exchange.

Two seminal studies made it unambiguously clear that HLA-DM recognizes complexes showing a P1-destabilized conformation ( 13 , 31 ). However, since DM-susceptible structures rarely show any of the changes present in the DM-bound structure (e.g., folding of α-46–55 into an α-helix and unfolding of C-terminal β-helix), the question of the conformational prerequisites for DM binding arises. As mentioned previously, an important study indeed demonstrated that HLA-DM attacks pMHC class II complexes at a site of conformational lability, the 3 10 -helical region ( 35 ). This segment, together with a neighboring unstructured segment (α52–55) folds into an α-helix when bound to DM ( 31 ). Interestingly, increased fluctuations of this region could be observed by other computational and experimental studies, implying the existence of higher conformational entropy within this region ( 46 , 106 ). Noteworthy, the α-helix in the region β86–β91 opens up to a certain degree in the DM-bound structure of DR1. However, a considerable influence of P1-remote sites on conformational dynamics and DM-dependence was recently demonstrated ( 64 ). In this study, alteration of P9-pocket/peptide interactions influenced dynamics of the pMHCII, likely in regions relevant for DM binding. The authors concluded that the key determinants for HLA-DM recognition are conformational dynamics present in HLA-DR1. Similarly, and as already mentioned above, Ferrante et al. explained the relationship between entropic penalties and DM binding in a thermodynamic context ( 106 ). According to their experimental and computational results, higher conformational entropy of pMHCII complexes correlates with DM susceptibility.

A recent study by our group in the MHC class II field explored internal motions of pMHC class II molecules along the conformational peptide-exchange pathway in a more conceptual model ( 46 ). Using NMR/HDX (hydrogen deuterium exchange) and MD simulations of over 200 μs in total, followed by Markov State model (MSM) analysis ( 128 – 132 ), we have identified transient conformations relevant for the DM-catalyzed and non-catalyzed (spontaneous) peptide-editing process ( 46 ). In agreement with the general view, the catalyzed pathway depends on the particular destabilization of the region surrounding the P1 pocket, sharing in part features of MHC class II bound to DM. More specifically, it has been suggested for MHC class II that pMHC complexes have to sample P1-pocket-destabilized conformations to allow for HLA-DM binding ( 13 ).

The non-catalyzed pathway, however, was correlated to the ground state of the pMHCII complex and, therefore, is directly correlated with thermodynamic stability. Indeed, it was shown that, removal of two hydrogen bonds between β82N of the MHC class II and the backbone of the peptide in the mutant DR1βN82A drastically reduces stability and, at the same time, dramatically enhances non-catalyzed peptide exchange. Nevertheless, binding to HLA-DM is also enhanced for the βN82A mutant, leading to the somewhat paradoxical finding that an MHCII molecule might bind tightly to a catalyst that it does not need for exchanging peptide. This can be best conceptualized when assuming that the βN82A mutant of HLA-DR1 in addition to increased spontaneous exchange more frequently samples a rare conformation along the pathway of catalyzed exchange. MD simulations in conjunction with MSM analysis indeed show that an excited state structurally correlated with features of the HLA-DM bound conformation. This excited state was seen to be significantly more frequently sampled in the mutant compared to WT HLA-DR1.

However, a similar intermediate state can be defined for the very stable WT protein, where peptide release from the pockets was not mandatory for the observation of the early intermediates. Thus, if the pMHCII forms a stable complex, the peptide editing depends on the population of rare conformations that can be selected by the catalyst DM for binding. This study demonstrated the critical importance of residues 80–93 of the β1 helix for catalyzed exchange, suggesting that β1 helical unfolding is critical for the rearrangement of this segment as it is observed in the DR1-DM structure ( 31 ). As the study shows, mutations in the β1 helix (e.g., βE87P) designed to specifically destabilize the C-terminal part of the β1 helix without disrupting H-bonds to the peptide, are able to over-proportionally shift the dynamics toward the HLA-DM-dependent pathway ( 46 ).

In conclusion, this model helps to reconcile discrepancies in the hypothesized correlations of peptide affinity, pMHC stability, DM susceptibility, and catalytic effect ( 133 ).

Conclusion and Outstanding Questions

Major histocompatibility complex proteins are encoded by oligogenic and highly polymorphic genes and most polymorphisms map to the regions important for peptide binding. The pMHC complexes display various degrees of flexibility along the binding groove, and these dynamic features seem to correlate with the propensity for peptide exchange. Of interest is the fact that tapasin and DM both bind their MHC targets in regions of enhanced dynamics. Short, destabilized helical segments together with their adjacent structural elements seem to represent the requirements for transient binding of the respective catalyst. The degree of these local flexibilities can be correlated to a higher dependence of a particular pMHC complex on tapasin or DM. Polymorphic substitutions might not only change the binding preference for certain ligands but also the overall stability and dynamics of the corresponding allelic variants. In turn, this will affect the conformational ensemble recognized by the peptide editors and in principle should be able to explain why certain alleles seem to possess a generally lower taspasin or DM dependence. In this way, MHC molecules may become a paradigmatic example of how differences in the dynamic landscape of protein complexes translate into distinct functional outcomes of physiological relevance.

How far have we come and what has to be done to achieve this goal? Figures 4 and 5 summarize the findings described in this review and also emphasize the most daunting questions in the field that need to be answered in order to formulate a unifying concept of antigen exchange. What seems to be clear is that both type of MHC molecules can exchange peptide along two distinct pathways, with the ratio of spontaneous versus catalyzed exchange certainly being different for the allelic protein variants and pMHC complex. While dynamics often correlates with thermodynamic stability, it has yet to be seen which type of motions are critical for catalysis and which structural elements are indispensable for these transitions to occur. For MHC class II molecules, the structure of the MHCII–DM complexes provides a cornerstone ( 31 ), and the early intermediates (μs-ms timescale) toward the DM-bound form could be defined ( 35 , 46 ) (Figure 5 ). However, there are no structural insights about the replacement of DM by incoming peptide, thus requiring experimental and simulation strategies to follow the fate of DM-prebound MHCII molecules. In the case of MHCI, a tapasin-class I complex structure is required in order to provide a reliable framework for further experimental and theoretical studies. Similarly, characterization of empty MHC molecules will certainly aid in defining the dynamic modes that are explored by the peptide-binding domains. Since it has been shown that empty MHC molecules can be rescued by the chaperoning function of the exchange catalysts ( 134 , 135 ) and thus the dynamics that occur upon peptide exchange are likely to show features of the empty state. It seems, therefore, highly desirable to compare the two systems on time scales down to a few milliseconds.

Figure 4. Thermodynamic model for peptide exchange of major histocompatibility complex (MHC) class I . Peptide–MHC class I (pMHCI) complexes can follow two mechanistic pathways for peptide exchange starting from pMHCI ground state (state 1). In the tapasin-catalyzed pathway, tapasin modulates conformational changes in the α2-1 helix (red) of the F pocket region (pink) and the α3 domain (not shown) that accelerate the kinetics of peptide dissociation (state 2) and the loading of a high-affinity peptide (3). More intermediates states (between state 1 and state 3) need to be identified by computational studies and/or NMR and X-ray crystallography. In the non-catalyzed pathway, the peptide dissociates from the sub-optimally-loaded intermediate state (state 1′). The resulting empty MHC molecule shows subtype-dependent dynamics (especially at the F pocket region, pink) and thus can exist in a stable peptide-receptive form (state 2′) or in an unstable form (state 2″) that is chaperoned by tapasin for peptide binding. The structures used in states 1 and state 3 were modified from PDB: 1UXS (shown in white). The models used in states 1′, 2, 2′, and 2″ represent suggested states by computational and experimental studies (shown in limon).

Figure 5. Thermodynamic model for peptide editing of major histocompatibility complex class II . pMHCII complexes can follow two mechanistic pathways for peptide exchange. The DM-catalyzed route requires multi-step transitions starting from the pMHCII ground state (1). This includes initially an out-flip movement of αW43 or the destabilization of β80–93 region via spontaneous conformational sampling of rare conformations (state 2′). DM would preferably select for conformations that are sampled on longer timescales and which show both, an out-flip movement of αW43 and a destabilized β80–93 region. Binding of DM to the energetically excited intermediate (which shows in part features of the DM-bound state) would then induce further rearrangements in the 3 10 -helical region (state 3′) and thereby accelerate peptide-release. Binding of peptides which can displace the stabilizing interactions complete the peptide exchange process (state 4). Spontaneous (non-catalyzed) peptide exchange depends on the intrinsic stability of the pMHCII complex and does not rely on the sampling of rare conformations (state 2). Binding of a new peptide would likely require dissociation of the bound peptide, leading to the empty state (state 3) which rapidly converts into the non-receptive state (state 3b) but can also be chaperone by DM (state 3c) in order to allow for high-affinity peptide binding (state 4). Structures used in state 1, 3′, and 4 were derived from PDB: 4QXA, 4FQX, and 1DLH, respectively. Cartoons shown in 2, 2′, 3, and 3b were derived from molecular dynamic simulations ( 46 , 91 ).

For both MHC classes, more sophisticated NMR experiments capitalizing on selective amino acid side-chain labeling protocols are probably required and methods relying on CEST or relaxation dispersion should be able to yield more direct information on the anticipated intermediate states ( 136 , 137 ). So far, in-depth NMR experiments are restricted to certain stable pMHC complexes and the investigation of other alleles have been hampered by the in-availability of other variants such as the disease-relevant DQ alleles. There is a need to expand the experimental basis of dynamically investigated pMHC complexes in order to test the predictions made on the basis of the dynamic features of just a few alleles. Solutions are most likely to come from protein engineering approaches in combination with the use of different expression systems. The increasing importance of MD simulations arises from the fact that micro-to-milli-second simulations in combination with Markov State Modeling will become more of a standard in the field. This is essential, because the critical intermediates of antigen exchange seem to be populated at this time scale. Once we are able to conceptualize conformational peptide exchange, we will be in the position to better predict MHC peptide occupancies in the context of cellular editing mechanisms and we will understand and be able to manipulate the action of small molecules or biological macromolecules that modulate peptide exchange.

Author Contributions

MW, EA, and CF designed and wrote the manuscript. JS, MÁ-B, SS, and FN critically read, discussed, and edited the content prior to submission.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

CF is thankful for funding by the DFG (SFB958, SFB854, TR186 and SPP1623).

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Keywords: antigen presentation, major histocompatibility complex, HLA, protein dynamics, peptide exchange, tapasin, HLA-DM, adaptive immunity

Citation: Wieczorek M, Abualrous ET, Sticht J, Álvaro-Benito M, Stolzenberg S, Noé F and Freund C (2017) Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation. Front. Immunol. 8:292. doi: 10.3389/fimmu.2017.00292

Received: 13 December 2016; Accepted: 28 February 2017; Published: 17 March 2017

Reviewed by:

Copyright: © 2017 Wieczorek, Abualrous, Sticht, Álvaro-Benito, Stolzenberg, Noé and Freund. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Christian Freund, christian.freund@fu-berlin.de

† These authors have contributed equally to this work.

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Antigen Processing and Presentation. In order to be capable of engaging the key elements of adaptive immunity (specificity, memory, diversity, self/nonself discrimination), antigens have to be processed and presented to immune cells. Antigen presentation is mediated by MHC class I molecules, and the class II molecules found on the surface of ...
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The Major Histocompatibility complex (MHC) system known as the human leukocyte antigen (HLA) in humans is located on the short arm of chromosome 6 (6p21.3) and contains the most polymorphic gene cluster of the entire human genome. Furthermore, the HLA consists of three regions which have been designated as class I, class II, and class III based on the structure and function of gene products ...
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Major histocompatibility complex (MHC) class I is a diverse set of cell surface receptors expressed on all nucleated cells in the body and platelets. MHC is also known as human leukocyte antigen (HLA), of which there are 3 subtypes: HLA-A, HLA-B, and HLA-C. These molecules play a vital role in the immune system recognizing self from nonself, presenting foreign antigens to other immune cells ...
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The purest read-out would be to measure MHC class I-antigen complexes at the surface of DC (signal 1 only), however, tools are lacking . The best current available method to quantify MHC class I-antigen presentation is a read-out involving activation or in vitro induction of virus-specific T cells. However, it should be taken into account that ...
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MHC Class I, Class II, Antigen Processing, And Presentation

Major Histocompatibility Class I (MHC Class I)

Structure of MHC Class I

Major Histocompatibility Class II (MHC Class II)

Structure of MHC Class II

Major Histocompatibility Class III (MHC Class III)

Antigen Processing and Presentation

A. Cytosolic pathway: Endogenous antigen

B. Endocytic Pathway: Exogenous antigen

Presentation of Non-peptide antigens

Clinical Significance of Antigen processing and presentation

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BiteSized Immunology: Systems & Processes

Antigen Processing and Presentation

MHC class I presentation

Antigen presentation

MHC class I polymorphism

MHC class II presentation

MHC class II polymorphism

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Introduction

Structural Variations in MHC Complexes

Dynamics of Peptide-Free MHC Proteins

MHC Class I

MHC Class II

Dynamic Features of Peptide-Bound MHC Complexes

MHC Dynamics During Peptide Exchange

Conclusion and Outstanding Questions

Author Contributions

Conflict of Interest Statement

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