CLIP Peptide Research Articles

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA‐DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein-peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)-peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE-FL) can resolve multiple MHC-peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE-FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.

Electrostatic potential as a tool to understand interactions between malaria vaccine candidate peptides and MHC II molecules

One of the most important problems in vaccine development consists in understanding receptor–ligand interactions between Class II Major Histocompatibility Complex molecules (MHC II) and antigenic peptides involved in inducing an appropriate immune response. In this study, we used X-ray crystallography structural data provided by the HLA-DRβ1*0301–CLIP peptide interaction to compare native non-immunogenic and specifically-modified immunogenic peptides derived from the malarial SALSA protein, by analyzing molecular electrostatic potential surfaces on the most important regions of the peptide binding groove (Pockets 1, 4, 6 and 9). Important differences were found on the electrostatic potential induced by these peptides, particularly in MHC II conserved residues: Qα9, Sα53, Nα62, Nα69, Yβ30, Yβ60, Wβ61, Qβ70, Kβ71 and Vβ86, the same ones involved in establishing hydrogen bonds between Class II molecule-peptide and the recognition by T cell receptor, it correlating well with the change in their immunological properties. The results clearly suggest that modifications done on the electrostatic potential of these amino acids could favor the induction of different immune responses and therefore, their identification could allow modifying peptides a priori and in silico, so as to render them into immunogenic and protection-inducers and hence suitable components of a chemically-synthesized, multi-antigenic, minimal subunit based vaccine.

MultiRTA: A simple yet reliable method for predicting peptide binding affinities for multiple class II MHC allotypes

BackgroundThe binding of peptide fragments of antigens to class II MHC is a crucial step in initiating a helper T cell immune response. The identification of such peptide epitopes has potential applications in vaccine design and in better understanding autoimmune diseases and allergies. However, comprehensive experimental determination of peptide-MHC binding affinities is infeasible due to MHC diversity and the large number of possible peptide sequences. Computational methods trained on the limited experimental binding data can address this challenge. We present the MultiRTA method, an extension of our previous single-type RTA prediction method, which allows the prediction of peptide binding affinities for multiple MHC allotypes not used to train the model. Thus predictions can be made for many MHC allotypes for which experimental binding data is unavailable.ResultsWe fit MultiRTA models for both HLA-DR and HLA-DP using large experimental binding data sets. The performance in predicting binding affinities for novel MHC allotypes, not in the training set, was tested in two different ways. First, we performed leave-one-allele-out cross-validation, in which predictions are made for one allotype using a model fit to binding data for the remaining MHC allotypes. Comparison of the HLA-DR results with those of two other prediction methods applied to the same data sets showed that MultiRTA achieved performance comparable to NetMHCIIpan and better than the earlier TEPITOPE method. We also directly tested model transferability by making leave-one-allele-out predictions for additional experimentally characterized sets of overlapping peptide epitopes binding to multiple MHC allotypes. In addition, we determined the applicability of prediction methods like MultiRTA to other MHC allotypes by examining the degree of MHC variation accounted for in the training set. An examination of predictions for the promiscuous binding CLIP peptide revealed variations in binding affinity among alleles as well as potentially distinct binding registers for HLA-DR and HLA-DP. Finally, we analyzed the optimal MultiRTA parameters to discover the most important peptide residues for promiscuous and allele-specific binding to HLA-DR and HLA-DP allotypes.ConclusionsThe MultiRTA method yields competitive performance but with a significantly simpler and physically interpretable model compared with previous prediction methods. A MultiRTA prediction webserver is available at http://bordnerlab.org/MultiRTA.

Small molecule enhancers of peptide binding to MHC class II: Implications for DM function (130.40)

Abstract MHC II molecules present peptides derived from exogenous protein antigens. Antigens are delivered through the endosomal/lysosomal pathway to the MIIC which contains DM. Nascent MHC II molecules are directed to this same compartment through association with the invariant chain (Ii). During this process Ii is progressively cleaved leaving the placeholder peptide CLIP in the peptide binding groove of the newly translated MHC II molecule. In the MIIC, DM facilitates the exchange of CLIP peptide for high affinity peptides derived from internalized antigens before export of MHC II to the cell surface. Peptides bound to MHC II are generally very stable and can have half lives of days to weeks, while MHC II molecules that have lost peptide rapidly denature, preventing opportunistic binding of unsuitable peptides at the cell surface. These mechanisms greatly hinder peptide immunogenicity as unstructured peptide do not survive to intersect with the MIIC and there is a paucity of empty MHC II receptors on the cell surface to accept exogenously supplied peptides. Through high-throughput screening efforts we have found a number of small molecules that enhance peptide binding to MHC II. One of these small molecules is able to mimic the function of DM at the cell surface and greatly enhances peptide display in vivo. Modeling of putative small molecule interaction sites has led to the discovery of residues that are critical for DM function.

Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM

Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: A molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.

Posttranslational Regulation ofI-Edby Affinity for CLIP

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

CD4+ T cells specific for a model latency‐associated antigen fail to control a gammaherpesvirus in vivo

CD4(+) T cells play a major role in containing herpesvirus infections. However, their cellular targets remain poorly defined. In vitro CD4(+) T cells have been reported to kill B cells that harbor a latent gammaherpesvirus. We used the B cell-tropic murine gammaherpesvirus-68 (MHV-68) to test whether this also occurred in vivo. MHV-68 that expressed cytoplasmic ovalbumin (OVA) in tandem with its episome maintenance protein, ORF73, stimulated CD8(+) T cells specific for the H2-K(b)-restricted OVA epitope SIINFEKL and was rapidly eliminated from C57BL/6 (H2(b)) mice. However, the same virus failed to stimulate CD4(+) T cells specific for the I-A(d)/I-A(b)-restricted OVA(323-339) epitope. We overcame any barrier to the MHC class II-restricted presentation of an endogenous epitope by substituting OVA(323-339) for the CLIP peptide of the invariant chain (ORF73-IRES-Ii-OVA), again expressed in tandem with ORF73. This virus presented OVA(323-339) but showed little or no latency deficit in either BALB/c (H2(d)) or C57BL/6 mice. Latent antigen-specific CD4(+) T cells therefore either failed to recognize key virus-infected cell populations in vivo or lacked the effector functions required to control them.

Bridging phases in high-response piezoelectric materials

Proteolytic activity is important for normal functioning of an organism andmust be rigorously controlled due to its potential danger. Failure in biological control mechanisms of proteolytic activities may result in a disease. Emerging biological roles of lysosomal cysteine proteases are bringing them in the focus of drug discovery research. They are expressed in variety of organisms frombacteria to humans (for the group ofmammalian homologues the term cysteine cathepsins is used). Cysteine cathepsins are lysosomal proteases involved in processing and digestion. These enzymes have rather short active site cleft, comprising of only three well defined substrate binding subsites (S2, S1 and S1’) and rather broad binding areas (S4, S3, S2’, S3’), which makes them distinct from other protease classes such as serine and aspartic with six and eight substrate binding sites, respectively. There are eleven human enzymes currently known (cathepsins B, C, F, H, L, K, O, S, V, X andW). Exopeptidases (cathepsins B, C, H, X), in contrast to endopeptidases (such as cathepsins L, S, V and F), provide features, which facilitate binding of Nand Cterminal groups of substrates in the active site cleft. Besides clear preference for free chain termini in the case of exopeptidases, the substrate binding sites exhibit no strict specificity. Their subsite specificity is rather exclusive than limiting. Our study of their interactions with protein inhibitors addresses their roles in the endosomal pathway of the immune system response. During this process foreign proteins are degraded to peptides of appropriate length, which must be delivered at the moment when the last piece of invariant chain (CLIP peptide) leaves the binding groove of the MHC class II molecules andmakes it available for peptide binding and antigen presentation. P12

Structural biology of cysteine cathepsins

Proteolytic activity is important for normal functioning of an organism andmust be rigorously controlled due to its potential danger. Failure in biological control mechanisms of proteolytic activities may result in a disease. Emerging biological roles of lysosomal cysteine proteases are bringing them in the focus of drug discovery research. They are expressed in variety of organisms frombacteria to humans (for the group ofmammalian homologues the term cysteine cathepsins is used). Cysteine cathepsins are lysosomal proteases involved in processing and digestion. These enzymes have rather short active site cleft, comprising of only three well defined substrate binding subsites (S2, S1 and S1’) and rather broad binding areas (S4, S3, S2’, S3’), which makes them distinct from other protease classes such as serine and aspartic with six and eight substrate binding sites, respectively. There are eleven human enzymes currently known (cathepsins B, C, F, H, L, K, O, S, V, X andW). Exopeptidases (cathepsins B, C, H, X), in contrast to endopeptidases (such as cathepsins L, S, V and F), provide features, which facilitate binding of Nand Cterminal groups of substrates in the active site cleft. Besides clear preference for free chain termini in the case of exopeptidases, the substrate binding sites exhibit no strict specificity. Their subsite specificity is rather exclusive than limiting. Our study of their interactions with protein inhibitors addresses their roles in the endosomal pathway of the immune system response. During this process foreign proteins are degraded to peptides of appropriate length, which must be delivered at the moment when the last piece of invariant chain (CLIP peptide) leaves the binding groove of the MHC class II molecules andmakes it available for peptide binding and antigen presentation. P12

A theoretical analysis of HLA-DRβ1*0301–CLIP complex using the first three multipolar moments of the electrostatic field

Interactions between the HLA-DRβ1*0301 molecule and several occupying peptides obtained from computational substitutions made to the CLIP peptide are studied. The exploration was carried out using a vector composed of the first three terms of the multipolar expansion of the electrostatic field, namely, charge (q), dipole (d) and quadrupole (C). Comparisons between pocket–peptide interactions established that the binding pockets for this HLA molecule are ordered in terms of their importance for binding peptides, as follows: P1 >>> P4 > P6 > P7 > P9. A set of electrostatically distinct amino acids that determine interaction stability and specificity were identified for each pocket. The β74R residue was especially identified as being the key amino acid mediating the occupying peptide binding for pocket 4; this residue has been recently associated with Graves' disease.

Human Dendritic Cell Expression of HLA-DO Is Subset Specific and Regulated by Maturation

Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO. In this study, we report that, in fact, certain types of human primary DC express DO. These include Langerhans cells (LC) and some subtypes of circulating blood DC. Specifically, the majority of BDCA-3(+) DC, a small subset of uncertain function, are DO(+), while smaller proportions of CD11c(+), BDCA-1(+) (myeloid) DC, at most a minority of CD123(+)/BDCA-2(+) (plasmacytoid) DC, and no detectable CD16(+) (myeloid) DC, express DO. Immunohistochemistry of human tonsil sections demonstrates that tonsillar interdigitating DC are also DO(+). In a subset of immature LC with higher DO expression, an increased fraction of surface DR molecules carry CLIP peptides, indicating that DO functions as a DM inhibitor in these cells. LC expression of DO is down-regulated by maturation stimuli. DM levels also decrease under these conditions, but the DM:DO ratio generally increases. In the myeloid cell types tested, DO expression correlates with levels of DObeta, but not DOalpha, implying that modulation of DObeta regulates DO dimer abundance in these cells. The range of APC types shown to express DO suggests a broader role for DO in immune function than previously appreciated.

Upregulation of the CLIP self peptide on mature dendritic cells antagonizes T helper type 1 polarization.

Dendritic cells (DCs) initiate and regulate immunity against foreign and self antigens. Here we identified more than 200 individual major histocompatibility complex class II-associated peptides on human DCs and found that mature DCs selectively upregulated the self peptide CLIP. CLIP cosegregated together with foreign antigenic peptides in tetraspan microdomains on the surface and localized to DC-T cell synapses. The increased representation of CLIP-major histocompatibility complex class II complexes favored polarization of autologous naive T cells toward the nonpolarized and T helper type 2 (T(H)2) phenotype. There was also a considerably higher T(H)2/T(H)1 ratio in H2-DM-deficient mice, which have a CLIP(hi) phenotype, in contrast to wild-type, CLIP(lo) mice. Thus, the self peptide CLIP on DCs qualifies as an endogenous regulator in priming of T helper cells by antagonizing the polarization toward the T(H)1 phenotype.

Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers.

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Downmodulation of antigen presentation by H2-O in B cell lines and primary B lymphocytes.

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigen-presenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.

Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate β69

Berylliosis is a chronic granulomatous disorder caused by inhalation of Be dusts that is driven by the accumulation Be-specific CD4+ Th1-cells at disease sites. Susceptibility to berylliosis has been associated with the supratypic variant of HLA-DP gene coding for glutamate at position β69 (HLA-DPβGlu69). The aim of this study was to test the hypothesis that the HLA-DPβGlu69 residue plays a role in the interaction with Be. To this end, soluble HLA-DP2 molecule (carrying βGlu69) and its mutated form carrying lysine at position β69 (HLA-DP2Lys69) were produced in Drosophila melanogaster and then used in a Be binding assays. BeSO 4 (1–1000 μM) was used to compete for the binding of the biotinilated invariant chain-derived peptide CLIP (50 μM). BeSO 4 was capable of compete out biotin-CLIP binding from the HLA-DP2 (IC50%: 4.5 μM of BeSO 4 at pH 5.0 and 5.5 μM of BeSO 4 at pH 7.5), but not from the HLA-DP2Lys69 molecule (IC50%: 480 μM of BeSO 4 at pH 5.0 and 220 μM of BeSO 4 at pH 7.5). Moreover, the binding of NFLD.M60, a MoAb recognizing an epitope in the HLA-DP peptide binding region, to the HLA-DP2, but not to the HLA-DP2Lys69 soluble molecules was inhibited BeSO 4. NFLD.M60 binding to HLA-DP2, but not to HLA-DP2Lys69 stably transfected murine cells was also inhibited by Be both at pH 5.0 and at pH 7.5. The data indicate a direct interaction of Be with the HLA-DPGlu69 molecule, in the absence of antigen processing.

H2-Mβ1 and H2-Mβ2 Heterodimers Equally Promote CLIP Removal in I-Aq Molecules from Autoimmune-prone DBA/1 Mice

Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4(+) helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4(+) T cells. In contrast to the HLA-DM region in humans, the beta-chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta1 and Mbeta2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-A(q) alone or in the presence of H2-Malphabeta1 or H2-Malphabeta2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-A(q) molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-A(q) molecules. Moreover, I-A(q) cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-A(q) exhibits a high affinity for CLIP since virtually all I-A(q) molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.

Nonclassical MHC class II molecules.

Major histocompatibility complex (MHC) class II molecules are cell surface proteins that present peptides to CD4(+) T cells. In addition to these wellcharacterized molecules, two other class II-like proteins are produced from the class II region of the MHC, HLA-DM (DM) and HLA-DO (DO) (called H2-M, or H2-DM and H2-O in the mouse). The function of DM is well established; it promotes peptide loading of class II molecules in the endosomal/lysosomal system by catalyzing the release of CLIP peptides (derived from the class II-associated invariant chain) in exchange for more stably binding peptides. While DM is present in all class II- expressing antigen presenting cells, DO is expressed mainly in B cells. In this cell type the majority of DM molecules are not present as free heterodimers but are instead associated with DO in tight heterotetrameric complexes. The association with DM is essential for the intracellular transport of DO, and the two molecules remain associated in the endosomal system. DO can clearly modify the peptide exchange activity of DM both in vitro and in vivo, but the physiological relevance of this interaction is still only partly understood.

H-2M molecules, like MHC class II molecules, are targeted to parasitophorous vacuoles of Leishmania-infected macrophages and internalized by amastigotes of L. amazonensis and L. mexicana.

In their amastigote stage, Leishmania are obligatory intracellular parasites of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have properties similar to those described for the MHC class II compartments of antigen-presenting cells, sites where peptide-class II molecule complexes are formed before their expression at the cell surface. After infection with Leishmania amazonensis or L. mexicana, endocytosis and degradation of class II molecules by intracellular amastigotes have also been described, suggesting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have now studied the fate of the MHC molecule H-2M in mouse macrophages infected with Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-derived CLIP peptide from the peptide-binding groove of class II molecules and facilitates the binding of antigenic peptides. H-2M was detected in PV of mouse macrophages infected with various Leishmania species including L. amazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class II molecule complexes and especially of complexes with parasite peptides. The present data indicate, however, that if this process occurs, it does not lead to a clear increase of SDS-stable compact (alpha)(beta) dimers of class II. In PV that contained L. amazonensis or L. mexicana, both class II and H-2M molecules often colocalized at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and possibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania species and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infected macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN2 or Z-Phe-PheCHN2 or by incubation of the parasites with the same inhibitors before infection. These data thus support the idea that amastigotes of certain Leishmania species capture and degrade some of the molecules required for antigen presentation. To examine whether endocytosis of class II molecules by the parasites occurs through interactions with parasite components involving their peptide-binding groove, we made use of the fact that a large fraction of the class II molecules of H-2M(alpha) knock-out H-2(b) mice are occupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CLIP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H-2M-independent and (2) does not necessarily involve the peptide-binding pocket of these molecules. Altogether, these data are compatible with an endocytic mechanism based on general properties shared by classical and non-classical class II molecules.