CLIP Peptide Research Articles

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide–MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

Scientific policy session: European GrantAntigen-delivery through invariant chain boosts CD8 and CD4 T cell immunity

Eradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and -II. Finally, we verified that Ii-expressing dendritic cells could prime CD4+ and CD8+ T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.

MHC class II-restricted-presentationMHC class II and DM molecules in chickens: different yet again

The chicken MHC is simple and compact, with strong genetic associations for resistance to infectious disease, due at least in part to high expression of a single classical class I gene, BF2. The closely-linked TAP and tapasin genes are highly polymorphic, and co-evolve with the dominantly-expressed BF2 gene, due to the rarity of recombination between genes of this system. The classical class II system has some similarities to the class I system, with two class II B (BLB), one DMA and two DMB genes, all of which are closely-linked and polymorphic. Like β2-microglobulin, the class II A gene is monomorphic and located distant to the MHC. Only BLB2 is well-expressed in spleen and blood. The objective is to determine to what extent the chicken class II system follows the same principles as the class I system. RNA levels were determined by RT-PCR and RT-qPCR, proteins were detected by Western blot, flow cytometry and tissue section staining, and genes were knocked out (KO) by CRISPR-Cas9. RNAs for BLB2 and DMB2 are widely expressed, while BLB1 and DMB1 are only strongly expressed in gut tissues. Tissue staining shows very high DMB1 (and BLB1) staining in intestinal epithelial cells (IECs), while sorted IECs show similar levels for DMB1 and DMB2 by RT-PCR, but only DMB1 by western blot. Both BLB1 and BLB2 class II molecules mostly bear CLIP peptides in DMA KO cells, but some alleles are exquisitely DM-dependent and others less so. Both BLB1 and BLB2 are affected in DMB1 and DMB2 KO cells. The presence of high level DMB1 (and BLB1) expression in IECs but not elsewhere is unprecedented for class II molecules, and the exact ways in which the different DM loci and alleles affect the two class II molecules remain interesting questions still to answer.

Dysfunction of Dendritic Cells in Tumor-Draining Lymph Nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes (TDLN) and to identify the mechanisms that lead to defective tumor antigen presentation. We transplanted tumors into the flank of mice and analyzed DCs from TDLN and contralateral non-draining lymph nodes (NDLN). Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN either in vivo or ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake by DCs from TDLN. Interestingly, DCs in TDLN also showed poor removal of the Invariant-chain CLIP peptide from the MHC-II peptide binding groove. This is likely to be due to a functional defect of the peptide-editor H2-M. Using Invariant-chain mutant mice and H2-M knock-out mice we confirmed that defective peptide exchange from MHC-II molecules hampered the anti-tumor responses and lead to enhanced tumor growth. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis could allow us to “correct” these DC defects, thereby allowing CD4 T cell priming to provide the necessary help for antitumor CD8 T cells.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo or ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.

HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY

Human leukocyte antigen (HLA) molecules play critical roles in our adaptive immune system by signaling a cell's health status to the immune system, through presentation of small peptides. Understanding HLA biology is important because of its prominent role in autoimmune diseases and cancer immunotherapy. Although both the HLA class I and class II antigen processing and presentation pathways have been studied extensively, the fundamental rules in HLA class II antigen presentation still remain less understood. To clarify the mechanistic and adaptive differences between the HLA systems, we challenged a B lymphoblastic cell line (JY), widely used as model system in studying antigen presentation, with a high temperature treatment to mimic a “fever-like state”, representing one of the most common physiological responses to infection. In the absence of real invading pathogenic peptides to present, we could focus on delineating the intrinsic HLA pathway adaptations in response to high temperature in this particular cell line. Following a three-pronged approach, we performed quantitative analyses of the proteome, the HLA class I ligandome, as well as the HLA class II ligandome. The data reveals that elevated temperature may already prepare these cells for an immune-like response through increased HLA class II presentation capacity and specific release of, from the invariant chain originating, CLIP peptides. Interestingly, at high temperature, prominent changes in the composition of the CLIP repertoire were observed, with enrichment of peptides containing C-terminal extensions beyond the CLIP-core region. Collectively, these illustrate intriguing temperature sensitive adaptations in this B cell line.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II molecules) and CD8 cytotoxic T cells (through MHC-I molecules). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo and ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLN was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.

A Missing Switch in Peptide Exchange for MHC Class II Molecules.

Antigen processing and loading of peptides onto MHC class II molecules is a multistep process that involves vesicular transport of the MHCII molecules along the secretory pathway, where they eventually merge with antigen-containing endocytic vesicles or phagosomes (1). It is within these late endosomal or lysosomal compartments that protein antigens become degraded by proteases, most prominently by cathepsins, and where catalyzed peptide exchange by HLA-DM fulfills its role in the efficient replacement of the invariant chain-derived peptide CLIP by high-affinity pathogen- or host cell-derived peptides. Protease action may be limited by protein antigen abundance and redox conditions, while HLA-DM is regulated at several stages, including by expression levels, pH, or the co-expression of the competitive inhibitor HLA-DO. HLA-DM activity leads to significant changes in the immunopeptidome of antigen-presenting cells, thereby tailoring T cell responses and often shifting antigenicity toward high-affinity immunodominant epitopes (2). Control of DM activity by DO has been described to be of prime importance in thymic epithelial cells, in a subset of dendritic cells, and in B cells when entering the germinal centers for affinity maturation and class switching (3, 4). In all of these cases, the switch from a broader, self-peptide (CLIP) dominated immunopeptidome to a more focused repertoire is necessitated by the requirement for more stringent antigen presentation, often preceding more intense T cell reactivity and proliferation. Here, we review data on this cellular switch in the functionality of antigen presentation and propose that it is promoted by an as yet poorly understood molecular switch. Acknowledging that general biophysical parameters such as pH and redox are important for antigen processing in general, an elusive DM-DO switch is postulated that would allow rapid and strong shifts in immunopeptidomes. We capitalize on theoretical considerations to back our opinion that a regulatable switch would have the advantage of allowing for a rapid and possibly signal-dependent change in the peptide selection process, as might be required in the context of rapidly changing immunological conditions.

Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning

B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.

Antigen-delivery through invariant chain (CD74) boosts CD8 and CD4 T cell immunity

ABSTRACTEradication of tumors by the immune system relies on the efficient activation of a T-cell response. For many years, the main focus of cancer immunotherapy has been on cytotoxic CD8 T-cell. However, stimulation of CD4 helper T cells is critical for the promotion and maintenance of immune memory, thus a good vaccine should evoke a two-dimensional T-cell response. The invariant chain (Ii) is required for the MHC class II heterodimer to be correctly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells (APC). We previously showed that by replacing the Ii CLIP peptide by an MHC-I cancer peptide, we could efficiently load MHC-I. This prompted us to test whether longer cancer peptides could be loaded on both MHC classes and whether such peptides could be accommodated in the CLIP region of Ii. We here present data showing that expanding the CLIP replacement size leads to T-cell activation. We demonstrate by using long peptides that APCs can present peptides from the same Ii molecule on both MHC-I and -II. In addition, we present evidence that antigen presentation after Ii-loading was superior to an ER-targeted minigene construct, suggesting that ER-localization was not sufficient to obtain efficient MHC-II loading. Finally, we verified that Ii-expressing dendritic cells could prime CD4+ and CD8+ T cells from a naïve population. Taken together our study demonstrates that CLIP peptide replaced Ii constructs fulfill some of the major requirements for an efficient vector for cancer vaccination.

Serotonin: its place today in sleep preparation, triggering or maintenance

Serotonin (5-HT) is involved in sleep in two different ways. First, when released during waking by the axonal nerve endings, it influences the synthesis of hypnogenic substances in specific brain targets. Such a synthesis might be in keeping with the waking qualitative aspects. As an example, the hypnogenic CLIP peptide (ACTH18-39) is synthesized when stressful events occur during wakefulness. Second, when released during sleep within the nucleus raphe dorsalis (nRD) by dendrites of 5-HT neurons, it contributes to 5-HT perikarya silencing through an auto-inhibitory process. Nitric oxide, co-synthesized with 5-HT, may act in synergy with this amine at both mentioned levels. Regarding the triggered hypnogenic substances, they induce sleep through acting on two components within the nRD: (1) the 5-HT component; its silencing is necessary to remove the gating effect exerted on phasic sleep events (ponto-geniculo-occipital, PGO, waves); (2) a substance P component; its silencing is necessary, at least, to alleviate the tonic influence exerted on somatic muscles. These two components may constitute the brain “sleep switch-on” mechanism allowing wake/sleep alternation. Pharmacological procedures influencing this switch may be determinant for treating insomniac patients. Serotonin appears thus to be involved in sleep preparation, triggering and maintenance.

Elucidating the role of HLA-DO/H2-O in modulating the diversity of the MHC Class II self peptide repertoire

Abstract The classical MHC Class II molecules HLA-DP, DQ and DR proteins bind antigenic peptides in the endosomes of APCs and present them to CD4+ T cells whereas HLA-DM and DO are the non-classical proteins. DM catalyzes the exchange of CLIP peptides with other endosomal peptides whereas DO acts as an inhibitor of DM activity. The clear biochemical effect of DO in inhibiting DM is reported but its effect on the peptide repertoire is still not clear. We used Quadrupole Orbitrap mass spectrometry for characterizing peptides presented by the MHC Class II protein DR1 from CRISPR/Cas9 deleted LG2 cells. We identified 6139 peptides in wild type cells (WT) but only 5227 peptides from DO−/− cells (DO-KO). The label free quantitation analysis of WT Vs DO-KO showed DM sensitive CLIP peptides were present in lower abundance in DO-KO, consistent with cell surface staining experiments. We analyzed peptide diversity in the eluted peptidome using diversity measures like Shannon’s Entropy, Simpson’s diversity and Berger-Parker Index. In each case the diversity was higher for the WT peptidome as compared to DO-KO. We screened several peptides for binding affinity and DM susceptibility by analyzing their relative abundances in WT and DO-KO. Peptides from group WT > DO-KO, WT only showed lower binding affinity and high DM sensitivity as compared to peptides in DO > WT, DO only. The same phenomenon was observed for peptides eluted from I-Ab purified from B cells and thymus of BL/6 as compared to H2-O−/− mice. These results indicate that DO/H2-O broadens the peptide repertoire whereas its absence restricts the repertoire resulting in presentation of fewer different epitopes, and highlights the role of DO in regulating the MHC-II peptide repertoire by modulating DM peptide editing.

Molecular Mapping Alzheimer's Disease: MALDI Imaging of Formalin-fixed, Paraffin-embedded Human Hippocampal Tissue.

A method for the molecular mapping of formalin-fixed, paraffin-embedded human hippocampal tissue affected by Alzheimer's disease (AD) is presented. This approach utilizes imaging mass spectrometry (IMS) with matrix-assisted laser desorption/ionization (MALDI). The usefulness of this technique in comparing diseased versus nor mal tissue at the molecular level while continuing to maintain topological and morphological integrity is evident in the preliminary findings. The critical correlation of the deparaffination, washing, matrix deposition, and analysis steps in handling the tissue sections and how these steps impact the successful mapping of human hippocampal tissue is clearly demonstrated. By use of this technique we have been able to identify several differences between the hippocampal AD tissue and the control hippocampal tissue. From the observed peptide clip masses we present preliminary identifications of the amyloid-beta peptides known to be prominent in the brains of those with AD. We have obtained high-resolution mass spectra and mass images with 100μm spatial resolution. Future experiments will couple this work with MALDI LIFT experiments to enable top down proteomics of fresh frozen tissue, which is not possible with paraffin-embedded tissues.

Laser-Induced In-Source Decay Applied to the Determination of Amyloid-Beta in Alzheimer's Brains.

A method for the analysis of amyloid-beta peptides in isolated plaques and intact tissue sections affected by Alzheimer's disease (AD) is presented. This method employs matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and the inherent laser-induced in-source decay (ISD) that occurs coupled with imaging mass spectrometry (IMS) to investigate the composition of these samples eliminating the need for other confirmational MS/MS techniques. These results demonstrate this technique's usefulness for the identification of amyloid-beta peptides in tissue and isolated senile plaques from AD patients using the reproducible fragmentation pattern demonstrated via the laser-induced ISD of synthetic amyloid-beta peptide clips (1-40, 1-42). Clear differences between the hippocampal AD tissue and the control hippocampal tissue regarding the presence of amyloid-beta have been identified. These are based on laser-induced ISD of standard amyloid-beta clips as controls as well as the analysis of isolated senile plaques as a confirmation before tissue analysis. Using the resulting observed peptide clip masses from the control data, we present mass spectrometry based identification of the amyloid-beta peptides in both isolated plaques and hippocampal regions of those patients diagnosed with AD.

Peptidomic analysis of type-1 diabetes associated HLA-DQ molecules and impact of HLA-DM editing (APP5P.108)

Abstract HLA-DM is critical in editing peptide repertoires presented by APC, thus influencing the selection/activation of CD4+ T cells. We previously reported that type-1 diabetes (T1D) associated HLA-DQ2, 8 & DQ2/8 molecules (DQs) are relatively resistant to DM editing of the CLIP peptides. In this study, we further studied DM sensitivity of T1D-associated DQs by peptidomic analysis. Compared to a T1D-protective DQ6, the percentages of shared peptides among T1D-associated DQs were significantly higher. There were greater numbers of nested peptide sets and long peptides (>20 aa) eluted from T1D-associated DQs. Predicted peptide binding motifs of DQ2, 8 & 2/8 shared charged anchor residues, while hydrophobic anchors were present in DQ6 peptides. Competition binding assays comparing the eluted peptides from DM sensitive, dependent, or resistant groups showed that the binding affinities of the DM sensitive peptides were significantly lower than that of CLIP1 peptide. The increased sDQ8-CLIP1 complex stability, with the intrinsic half-life of 58 h measured by fluorescence polarization assay, was 8 times longer than that of sDQ6-CLIP1 (7 h), further supporting the DM resistance of T1D associated DQs. Collectively, T1D associated DQs share distinct characteristics, including binding of peptides from known T1D auto-antigens. Our new data further support the hypothesis that DM resistance in T1D associated DQs affects the peptide repertoires, thus contributing to the pathogenesis of T1D.

OP0185 Endogenous B Cell-Targeted Antigen Expression Induces Tolerance to Collagen Type II Arthritis in Mice

BackgroundRheumatoid arthritis (RA) is a destructive autoimmune disease. Today’s treatment is associated with side effects and only suppresses the disease. A safe and effective therapy would be to re-establish tolerance,...

Comparative lipidomics reveals a global sampling of major cellular membrane lipids by human CD1 proteins (P5006)

Abstract Human CD1 proteins have differentially shaped grooves and present bacterial or self lipid antigens to T cells, but it is unknown whether the pool of endogenous lipids are differently sampled by each type of CD1 proteins. We used recently established lipidomic approach to analyze the molecules extracted from human CD1a, CD1b, CD1c, and CD1d proteins. In the study, we demonstrated that this highly sensitive and accurate lipidomic approach detected more than one thousand diverse molecules defined by accurate mass retention time values, which represented hundreds of lipid species associated with CD1 proteins. More than fifty percent of these molecules were shared by all four types of CD1 proteins and additional thirty percent shared by two or three CD1 isoforms. Further mass spectrometry analyses of collision-induced dissociation showed that the molecules eluted from all four types of CD1 proteins mainly are lipids from subcellular membranes, including phospholipids of conventional, ether-linked, and lyso forms and sphingolipids. Surprisingly, CD1 isoforms with bigger grooves preferred to lipids with shorter chains, which can be explained by small spacer or scaffold lipids that bind together with antigens. Whereas previous studies argued that one lipid molecule blocks CD1 grooves, analogous to MHC class II-associated CLIP peptide, we conclude that CD1 proteins broadly sample cellular lipids and two small lipids are able to be adapted into a big groove.

Ubiquitination of HLA‐DO by MARCH family E3 ligases

HLA-DO (DO) is a nonclassical MHC class II (MHCII) molecule that negatively regulates the ability of HLA-DM to catalyse the removal of invariant chain-derived CLIP peptides from classical MHCII molecules. Here, we show that DO is posttranslationally modified by ubiquitination. The location of the modified lysine residue is shared with all classical MHCII beta chains, suggesting a conserved function. Three membrane-associated RING-CH (MARCH1, 8 and 9) family E3 ligases that polyubiquitinate MHCII induce similar profiles of polyubiquitination on DOβ. All three MARCH proteins also influenced trafficking of DO indirectly by a mechanism that required the DOβ encoded di-leucine and tyrosine-based endocytosis motifs. This may be the result of MARCH-induced ubiquitination of components of the endocytic machinery. MARCH9 was by far the most efficient at inducing intracellular redistribution of DO but did not target molecules for lysosomal degradation. The specificity of MARCH9 for HLA-DQ and HLA-DO suggests a need for common regulation of these two MHC-encoded molecules.

Tollip-induced down-regulation of MARCH1

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA+ HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms.