Small molecule modulators of MHC class II antigen presentation: Mechanistic insights and implications for therapeutic application

Antigen presenting cells express MHC class II molecules bound to peptide fragments and are responsible for activating CD4⁺ T cells that then broadly influence many branches of the immune response. A growing interest in developing strategies to therapeutically influence the peptides to which naïve CD4⁺ T cells are exposed has led to the hunt for small molecules that modulate peptide presentation through the MHC class II pathway. Over the past decade a number of small molecules have been discovered that show surprising diversity in both structure and putative mechanisms. This review discusses how these small molecules were identified and compares the mechanisms by which they may act with what is known about the endogenous peptide exchanger, HLA-DM.     

An analysis of the role of skin Langerhans cells (LC) in the cytoplasmic processing of HIV-1 peptides after “Peplotion” transepidermal transfer and HLA class I presentation to CD8+ CTLs—An approach to immunization of humans

Skin Langerhans cells (LC) are antigen-presenting cells capable of expressing MHC class I and class II molecules on the plasma membrane. This molecular activity was reviewed to combine the knowledge of peptide presentation by MHC and HLA class I and class II molecules to prime CD8⁺ cytotoxic T cells (CTLs) and CD4⁺ T helper cells, respectively. The possible utilization of the skin dendritic cells for the development of antiviral CTLs and antibodies by synthetic peptides modeled according to the motifs of peptides that naturally interact with the peptide binding grooves of the various HLA haplotypes is discussed and evaluated. It may be possible that the introduction of synthetic viral peptides with motifs to fit the HLA class I haplotypes of a human population to the skin dendritic cells will prime selectively the cellular or the humoral immune responses. This approach may provide a new vaccination technique that applies synthetic virus peptides as vaccines for the immunization of humans. The neuropeptide CGRP interacts with LC and modulates antigen presentation.     

Hsp90–peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that mice immunized with a molecular chaperone-based vaccine derived from tumors became immune to further vaccination and that both CD8⁺ and CD4⁺ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90-conjugated peptides by APC into the MHC class II pathway for presentation to CD4⁺ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4⁺ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway.     

Small molecule modulators of MHC class II antigen presentation: mechanistic insights and implications for therapeutic application

Antigen presenting cells express MHC class II molecules bound to peptide fragments and are responsible for activating CD4(+) T cells that then broadly influence many branches of the immune response. A growing interest in developing strategies to therapeutically influence the peptides to which na?ve CD4(+) T cells are exposed has led to the hunt for small molecules that modulate peptide presentation through the MHC class II pathway. Over the past decade a number of small molecules have been discovered that show surprising diversity in both structure and putative mechanisms. This review discusses how these small molecules were identified and compares the mechanisms by which they may act with what is known about the endogenous peptide exchanger, HLA-DM.     

Post-translational modifications such as citrullination are excellent targets for cancer therapy

Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4⁺ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress. Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4⁺ T cell responses that would recognize these siPTM’s produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

The functional role of class II-associated invariant chain peptide (CLIP) in its ability to variably modulate immune responses

During the process of class II MHC assembly and cell surface expression, the class II-associated invariant chain peptide (CLIP) is removed from the peptide-binding groove of MHC, a task mediated by H-2M. This allows binding and presentation of peptide epitopes. We have previously shown that exogenously added CLIP interferes with this process and down-regulates the cell surface expression of class II molecules. In this study, we explored the effect of exogenously added CLIP on antigen-specific immune responses. In vivo studies with CLIP and various peptide and protein antigens with different affinities for I-A(d) molecules demonstrated that CLIP variably affects the T cell-mediated immune responses. Immunization with CLIP along with the antigen induced a shift from a T(h)1- to T(h)2-like response as determined by the cytokine profile and antibody isotype. These results suggest that the presence of exogenous CLIP can significantly influence the presentation of antigen by class II MHC molecules to CD4 T cells and thereby modulate immune responses. Exogenously added CLIP rapidly localized into the subcellular compartment of antigen-presenting cells where MHC class II molecules are present. We suggest that exogenous CLIP reduces the loading of peptides on the class II molecules, thus down-regulating MHC-peptide complexes on the cell surface. Alternatively, CLIP may bind to cell surface class II molecules and this complex is rapidly internalized resulting in reduced cell surface MHC class II expression. The reduced level of MHC-peptide complexes favors the activation of T(h)2 cells over T(h)1 cells. These results have implications in the regulation of immune responses, particularly the prevention of certain autoimmune diseases where T(h)1-type responses are pathogenic and T(h)2-type responses are protective.     

Accessory molecules in the assembly of major histocompatibility complex class I/peptide complexes: how essential are they for CD8+ T‐cell immune responses?

Summary: Assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum is a highly coordinated process that results in abundant class I/peptide complexes at the cell surface for recognition by CD8⁺ T cells and natural killer cells. During the assembly process, a number of chaperones and accessory molecules, such as transporter associated with antigen processing, tapasin, ER60, and calreticulin, assist newly synthesized class I molecules to facilitate loading of antigenic peptides and to optimize the repertoire of surface class I/peptide complexes. This review focuses on the relative importance of these accessory molecules for CD8⁺ T‐cell responses in vivo and discusses reasons that may help explain why some CD8⁺ T‐cell responses develop normally in mice deficient in components of class I assembly, despite impaired antigen presentation.     

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T‐cell recognition

While Burkitt lymphoma (BL) has a well‐known defect in HLA class I‐mediated antigen presentation, the exact role of BL‐associated HLA class II in generating a poor CD4⁺ T‐cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4⁺ T cells via the HLA class II pathway. This defect in CD4⁺ T‐cell recognition was not associated with low levels of co‐stimulatory molecules on BL cells, as addition of external co‐stimulation failed to elicit CD4⁺ T‐cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL‐associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II‐mediated antigen presentation and CD4⁺ T‐cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase‐like molecule that enhances class II‐mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II‐mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.     

Antigen processing and immune regulation in the response to tumours

The MHC class I and II antigen processing and presentation pathways display peptides to circulating CD8⁺ cytotoxic and CD4⁺ helper T cells respectively to enable pathogens and transformed cells to be identified. Once detected, T cells become activated and either directly kill the infected / transformed cells (CD8⁺ cytotoxic T lymphocytes) or orchestrate the activation of the adaptive immune response (CD4⁺ T cells). The immune surveillance of transformed/tumour cells drives alteration of the antigen processing and presentation pathways to evade detection and hence the immune response. Evasion of the immune response is a significant event tumour development and considered one of the hallmarks of cancer. To avoid immune recognition, tumours employ a multitude of strategies with most resulting in a down‐regulation of the MHC class I expression at the cell surface, significantly impairing the ability of CD8⁺ cytotoxic T lymphocytes to recognize the tumour. Alteration of the expression of key players in antigen processing not only affects MHC class I expression but also significantly alters the repertoire of peptides being presented. These modified peptide repertoires may serve to further reduce the presentation of tumour‐specific/associated antigenic epitopes to aid immune evasion and tumour progression. Here we review the modifications to the antigen processing and presentation pathway in tumours and how it affects the anti‐tumour immune response, considering the role of tumour‐infiltrating cell populations and highlighting possible future therapeutic targets.     

MHC class II-deficient mice allow functional human CD4+ T-cell development

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4⁺ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein–Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4⁺ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4⁺ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.     

Selectivity of the major histocompatibility complex class II presentation pathway of cortical thymic epithelial cell lines

Major histocompatibility complex (MHC) restriction of the immune response is established during positive selection of T cells in the thymus. This occurs mainly through interactions of T cell receptor of developing thymocytes with MHC/peptide ligands on cortical thymic epithelial cells (TEC). An ongoing controversy concerns the origin and the role of peptides involved in the positive selection of thymocytes. Evidence provided here shows that processing of MHC class II complexes in cortical TEC differs from that of medullary TEC. Removal of the invariant chain associated with MHC class II complexes was rapid and complete in medullary TEC which present peptides from both exogenous and cytosolic origin. In cortical TEC, a large fraction of class II dimers remained associated with a 10–12-kDa fragment of invariant chain (Ii). Incomplete removal of Ii correlated with the inability of cortical TEC to present peptides from exogenous origin. However, presentation of peptides from cytosolic proteins by cortical TEC remained possible. Thus, most peptides from exogenous proteins may be excluded from participating in positive selection of CD4⁺ T cells by a mechanism limiting Ii breakdown.     

LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4⁺ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4⁺ T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.     

Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2A^k MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact A^k αβ dimers of biosynthetically labeled Ii⁺ cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: A^k complexes were only found in the Ii⁺ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known A^k-binding peptides bound stably to A^k molecules on Ii^? cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of A^k: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.     

Composition of the HLA‐DR‐associated human thymus peptidome

Major histocompatibility complex class II (MHC‐II) molecules bind to and display antigenic peptides on the surface of antigen‐presenting cells (APCs). In the absence of infection, MHC‐II molecules on APCs present self‐peptides and interact with CD4⁺ T cells to maintain tolerance and homeostasis. In the thymus, self‐peptides bind to MHC‐II molecules expressed by defined populations of APCs specialised for the different steps of T‐cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC‐II‐associated peptides from five fresh human thymus samples. The data show a diverse self‐peptide repertoire, mostly consisting of predicted MHC‐II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA‐II‐bound peptidome and provide insight into how this peptidome is generated and how it drives T‐cell repertoire formation.     

Thymus‐specific serine protease regulates positive selection of a subset of CD4+ thymocytes

Thymus‐specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self‐peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4⁺ thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP‐deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II‐restricted TCR transgenes (Marilyn and OT‐II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT‐II CD4⁺ T cells. In contrast, selection of CD8⁺ T cells expressing an MHC class I‐restricted TCR transgene (OT‐I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4⁺ T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self‐peptides bound to MHC class II complexes.     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

Antigen‐loaded ER microsomes from APC induce potent immune responses against viral infection

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER‐enriched microsomal membranes (microsomes) isolated from DC contain more peptide‐receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide–MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide‐loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide‐pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide–MHC complexes armed microsomes from DC can be an important alternative to DC‐based vaccines for protection from viral infection.     

Need for cellular and humoral immune responses in bovines to ensure protection from foot-and-mouth disease virus (FMDV)—A point of view

The published studies on immunization of experimental animals, cattle, and sheep with synthetic peptides containing the antigenic domains in FMDV structural protein VP1 were analyzed. The results obtained with various FMDV synthetic peptides designed to stimulate the humoral immune response in bovines were compared to the current knowledge on MHC class I and class II, and the properties of the peptide binding grooves in each of them. X-ray crystallography of MHC class I proteins provided the three-dimensional structure of the peptide binding groove and led to the isolation and identification of self and viral peptides that naturally associate with the peptide binding grooves of both types of MHC and HLA molecules. The available knowledge of the amino acid motifs in MHC and HLA class I-bound viral peptides priming the CD8⁺ cytotoxic T cell responses must be coupled with the understanding of the three-dimensional structure of BoLA class I. This would aid in the development of an experimental approach to induce bovine anti-FMDV CD8⁺ cytotoxic cells to complement the humoral immune response to FMDV, which is currently achieved by a killed virus vaccine and, at the experimental level, by a peptide vaccine. Stimulation of both cellular and humoral immune responses against FMDV in cattle may reduce the risk of disease and virus shedding.