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.     

Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4⁺ T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4⁺ T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II–peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II–peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4⁺ T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II–peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide–MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.     

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope

Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection. However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized. In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope. A cytotoxic T lymphocyte response to this peptide could be demonstrated in M. bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation. When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified. Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC. The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d. These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection. Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.     

Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude

Cell-mediated immunity stems from the proliferation of naive T lymphocytes expressing T cell antigen receptors (TCRs) specific for foreign peptides bound to host major histocompatibility complex (MHC) molecules. Because of the tremendous diversity of the T cell repertoire, naive T cells specific for any one peptide:MHC complex (pMHC) are extremely rare. Thus, it is not known how many naive T cells of any given pMHC specificity exist in the body or how that number influences the immune response. By using soluble pMHC class II (pMHCII) tetramers and magnetic bead enrichment, we found that three different pMHCII-specific naive CD4(+) T cell populations vary in frequency from 20 to 200 cells per mouse. Moreover, naive population size predicted the size and TCR diversity of the primary CD4(+) T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides are stronger immunogens than others.     

Detection of low-frequency human antigen-specific CD4(+) T cells using MHC class II multimer bead sorting and immunoscope analysis

MHC class II tetramers are attractive tools to study antigen-specific CD4(+) T cell responses in various clinical situations in humans. HLA-DRA1*0101/DRB1*0401 MHC class II heterodimers were produced as empty molecules using the Drosophila melanogaster expression system. Peptide binding experiments revealed that these molecules could be loaded efficiently with appropriate MHC class II tumor epitopes. Interestingly, MHC class II tetramer staining was influenced by modifications in membrane lipid rafts, and could in itself induce activation changes of stained CD4(+) T cells at 37 degrees C. In order to increase the threshold of detection of poorly represented peripheral antigen-specific CD4(+) T cells, we combined cell sorting using MHC class II multimer beads together with TCR analysis using the immunoscope technology. This strategy greatly increased the sensitivity of detection of specific CD4(+) T cells to frequencies as low as 4 x 10(-6) among peripheral blood mononuclear cells. Such a combined approach may have promising applications in the immunomonitoring of patients under vaccination protocols to tightly follow induced antigen-specific CD4(+) T cells expressing previously identified TCR.     

A CD4 domain 1 CC' loop peptide analogue enhances engraftment in a murine model of bone marrow transplantation with sublethal conditioning.

Host CD4(+) T cells that survive sublethal or even lethal preconditioning regimens can participate in the process of hematopoietic stem cell graft rejection, particularly when the transplantations are performed across a major histocompatibility complex (MHC) class II barrier. To enhance donor marrow engraftment, we tested the efficacy of a small synthetic cyclic heptapeptide, 802-2 (CNSNQIC), which was designed to closely mimic the CD4 domain 1 CC' surface loop, theoretically involved in CD4/MHC class II complex oligomerization and subsequent CD4(+) T-cell activation. Previously, this peptide was found to have inhibitory activity in murine models for CD4(+) T cell-dependent graft-versus-host disease and skin allograft rejection. Herein, we used the MHC class II--disparate bm12 --> B6-CD45.1 sublethal irradiation transplantation model to test the possibility that the 802-2 peptide could enhance the engraftment of donor T cell-depleted bone marrow (ATBM). Sublethally irradiated B6-CD45.1 mice that received bm12 ATBM in combination with the 802-2 peptide demonstrated increased donor marrow cell engraftment as compared with mice that received ATBM alone; this suggests that the 802-2 peptide may be useful as an immunomodulating agent to overcome MHC class II mismatch barriers in hematopoietic stem cell transplantation.     

Role of MHC class II expressing CD4+ T cells in proteolipid protein(91-110)-induced EAE in HLA-DR3 transgenic mice

MHC class II molecules play a central role in the control of adaptive immune responses through selection of the CD4(+) T cell repertoire in the thymus and antigen presentation in the periphery. Inherited susceptibility to autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and IDDM are associated with particular MHC class II alleles. Advent of HLA transgenic mice has helped us in deciphering the role of particular HLA DR and DQ class II molecules in human autoimmune diseases. In mice, the expression of class II is restricted to professional antigen-presenting cells (APC). However, in humans, class II is also expressed on T cells, unlike murine T cells. We have developed new humanized HLA class II transgenic mice expressing class II molecules not only on APC but also on a subset of CD4(+) T cells. The expression of class II on CD4(+) T cells is inducible, and class II(+) CD4(+) T cells can present antigen in the absence of APC. Further, using EAE, a well-established animal model of MS, we tested the functional significance of these class II(+) CD4(+) T cells. DR3.AEo transgenic mice were susceptible to proteolipid protein(91-110)-induced EAE and showed CNS pathology accompanied by widespread inflammation and demyelination seen in human MS patients, suggesting a role for class II(+) CD4(+) T cells in the pathogenesis.     

CpG motifs of DNA vaccines induce the expression of chemokines and MHC class II molecules on myocytes

Determining how an immune response is initiated after in vivo transfection of myocytes with plasmids encoding foreign antigens is essential to understand the mechanisms of intramuscular (i. m.) genetic immunization. Since myocytes are facultative antigen-presenting cells lacking MHC class II and co-stimulatory molecules, it was assumed that their unique role upon DNA vaccination is to synthesize and secrete the protein encoded by the plasmid. Here we describe that i. m. injection of unmethylated CpG motifs induced the expression of chemokines (monocyte chemotactic protein-1) and MHC class II molecules on myocytes. Our results indicate that immunostimulatory DNA sequences (CpG motifs) of DNA vaccines augment synthesis of chemokine by myocytes with subsequent recruitment of inflammatory cells secreting IFN-gamma, a potent cytokine that up-regulates the expression of MHC class II molecules on myocytes. A myoblast cell line triple transfected with plasmids encoding MHC class II molecules and an immunodominant CD4 T cell epitope of influenza virus presented the endogenously synthesized peptide and activated specific T cells. These findings suggest that one mechanism for the immunogenicity of DNA vaccines consists in the presentation of peptides to CD4 T cells by in vivo plasmid-transfected myocytes.     

Suppression of major histocompatibility complex class II-associated invariant chain enhances the potency of an HIV gp120 DNA vaccine

Summary One function of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is to prevent MHC class II molecules from binding endogenously generated antigenic epitopes. Ii inhibition leads to MHC class II presentation of endogenous antigens by APC without interrupting MHC class I presentation. We present data that in vivo immunization of BALB/c mice with HIV gp120 cDNA plus an Ii suppressive construct significantly enhances the activation of both gp120-specific T helper (Th) cells and cytotoxic T lymphocytes (CTL). Our results support the concept that MHC class II-positive/Ii-negative (class II(+)/Ii(-)) antigen-presenting cells (APC) present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4(+) and CD8(+) T cells. Activated CD4(+) T cells locally strengthen the response of CD8(+) CTL, thus enhancing the potency of a DNA vaccine.     

Human duodenal epithelial cells constitutively express molecular components of antigen presentation but not costimulatory molecules

Constitutive expression of major histocompatibility complex (MHC) class II molecules by duodenal epithelial cells (EC) suggests that they can present antigen to CD4(+) T cells. However, other molecular components including invariant chain (Ii), HLA-DM, and costimulatory molecules CD80, CD86 and CD40, are required for efficient T-cell activation. We have investigated whether normal human duodenal EC possess these molecules and whether they can mediate MHC class II antigen presentation. EC were isolated from duodenal biopsies from patients in whom pathology was excluded. Freshly-isolated duodenal EC did not stimulate autologous T-cell proliferation against purified protein derivative of tuberculin. Flow cytometry and immunoblot analysis revealed that duodenal EC constitutively express HLA-DR, Ii, and HLA-DM. Surface MHC class II associated invariant chain peptide (CLIP) was not detectable, suggesting that HLA-DM functions normally in CLIP removal. Duodenal EC expressed SDS-stable HLA-DR alphabeta heterodimers, indicating that peptide binding had occurred. Surface expression of CD80, CD86 or CD40 was not detected although mRNA for these costimulatory molecules was present in all samples. These results suggest that nondiseased human duodenal EC can process and present antigen by the MHC class II pathway, but that they may induce anergy, rather than activation, of local T cells.     

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.     

Peptide dependency of alloreactive CD4+ T cell responses

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.     

Dimerization of soluble disulfide trap single-chain major histocompatibility complex class I molecules dependent on peptide binding affinity

Stable presentation of peptide epitope by major histocompatibility complex (MHC) class I molecules is a prerequisite for the efficient expansion of CD8(+) T cells. The construction of single-chain MHC class I molecules in which the peptide, β(2)-microglobulin, and MHC heavy chain are all joined together via flexible linkers increases peptide-MHC stability. We have expressed two T cell epitopes that may be useful in leukemia treatment as single-chain MHC class I molecules, aiming to develop a system for the expansion of antigen-specific CD8(+) T cells in vitro. Disulfide trap versions of these single-chain MHC molecules were also created to improve anchoring of the peptides in the MHC molecule. Unexpectedly, we observed that soluble disulfide trap single-chain molecules expressed in eukaryotic cells were prone to homodimerization, depending on the binding affinity of the peptide epitope. The dimers were remarkably stable and efficiently recognized by conformation-specific antibodies, suggesting that they consisted of largely correctly folded molecules. However, dimerization was not observed when the disulfide trap molecules were expressed as full-length, transmembrane-anchored molecules. Our results further emphasize the importance of peptide binding affinity for the efficient folding of MHC class I molecules.     

Mini-review: Presentation of pathogen-derived antigens in vivo

Most intracellular pathogens induce robust T cell responses upon infection of mammalian hosts. In most cases, these T cell responses are protective and result in pathogen clearance. It is therefore important to determine how T cells are primed and how they differentiate into cytokine-secreting and/or cytotoxic effector cells. In contrast to B cells, which recognize soluble Ag, CD8(+) and CD4(+) T cells react to Ag-derived peptides bound to MHC I or MHC II molecules, respectively. Therefore, elucidating the mechanisms by which pathogen-derived Ag become available for presentation is necessary to understand how pathogens trigger T cell responses in vivo. Although many excellent reviews have focused on the mechanisms involved in Ag processing, very few have pointed to the specificity of host-pathogen interactions. In this respect, it should be noticed that these interactions are very different from one pathogen to another, and may result in the involvement of different cells and molecules. Because of space limitations, we have decided to focus this review on two intracellular pathogens--vaccinia virus and Listeria monocytogenes. We have chosen these two pathogens because they both induce a strong CD8(+) T cell response and because they have been extensively studied by both microbiologists and immunologists.     

Antigen-independent adhesion of CD45RA (naive) and CD45RO (memory) CD4 T cells to B cells.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreasing the threshold for thymocyte activation biases CD4+ T cells toward a regulatory (CD4+CD25+) lineage

Thymus-derived CD4(+)CD25(+) regulatory T (T(r)) cells play a critical role in suppressing aberrant responses to self in vivo. The factors that influence a CD4(+) T cell's decision to commit to an immunoregulatory T(r) cell lineage are currently unknown. In the present study, we found that in mice, abundantly expressing a few or one peptide(s) bound to MHC class II molecules, a large portion of conventional CD4(+) T cells could be biased towards the commitment to a T(r) lineage by reducing the threshold required for thymocyte activation. This occurred in the presence of either an antisense glucocorticoid receptor transgene or a pharmacological inhibitor of glucocorticoid synthesis. These results demonstrate a novel in vivo pathway for the generation of T(r) cells, and raise the possibility that therapeutic enhancement of the T(r) cell repertoire through pharmacological manipulation of TCR signaling thresholds may provide a feasible means of ameliorating autoimmunity.