Major Histocompatibility Complex Class I Molecules Research Articles

The immunopeptidomes of two transmissible cancers and their host have a common, dominant peptide motif.

SummaryTransmissible cancers are malignant cells that can spread between individuals of a population, akin to both a parasite and a mobile graft. The survival of the Tasmanian devil, the largest remaining marsupial carnivore, is threatened by the remarkable emergence of two independent lineages of transmissible cancer, devil facial tumour (DFT) 1 and devil facial tumour 2 (DFT2). To aid the development of a vaccine and to interrogate how histocompatibility barriers can be overcome, we analysed the peptides bound to major histocompatibility complex class I (MHC‐I) molecules from Tasmanian devil cells and representative cell lines of each transmissible cancer. Here, we show that DFT1 + IFN‐γ and DFT2 cell lines express a restricted repertoire of MHC‐I allotypes compared with fibroblast cells, potentially reducing the breadth of peptide presentation. Comparison of the peptidomes from DFT1 + IFNγ, DFT2 and host fibroblast cells demonstrates a dominant motif, despite differences in MHC‐I allotypes between the cell lines, with preference for a hydrophobic leucine residue at position 3 and position Ω of peptides. DFT1 and DFT2 both present peptides derived from neural proteins, which reflects a shared cellular origin that could be exploited for vaccine design. These results suggest that polymorphisms in MHC‐I molecules between tumours and host can be ‘hidden’ by a common peptide motif, providing the potential for permissive passage of infectious cells and demonstrating complexity in mammalian histocompatibility barriers.

Development of a Metastasis-Related Immune Prognostic Model of Metastatic Colorectal Cancer and Its Usefulness to Immunotherapy.

Background: Post-surgical recurrence of the metastatic colorectal cancer (mCRC) remains a challenge, even with adjuvant therapy. Moreover, patients show variable outcomes. Here, we set to identify gene models based on the perspectives of intrinsic cell activities and extrinsic immune microenvironment to predict the recurrence of mCRC and guide the adjuvant therapy.Methods: An RNA-based gene expression analysis of CRC samples (total = 998, including mCRCs = 344, non-mCRCs = 654) was performed. A metastasis-evaluation model (MEM) for mCRCs was developed using the Cox survival model based on the prognostic differentially expressed genes between mCRCs and non-mCRCs. This model separated the mCRC samples into high- and low-recurrence risk clusters that were tested using machine learning to predict recurrence. Further, an immune prognostic model (IPM) was built using the COX survival model with the prognostic differentially expressed immune-related genes between the two MEM risk clusters. The ability of MEM and IPM to predict prognosis was analyzed and validated. Moreover, the IPM was utilized to evaluate its relationship with the immune microenvironment and response to immuno-/chemotherapy. Finally, the dysregulation cause of IPM three genes was analyzed in bioinformatics.Results: A high post-operative recurrence risk was observed owing to the downregulation of the immune response, which was influenced by MEM genes (BAMBI, F13A1, LCN2) and their related IPM genes (SLIT2, CDKN2A, CLU). The MEM and IPM were developed and validated through mCRC samples to differentiate between low- and high-recurrence risk in a real-world cohort. The functional enrichment analysis suggested pathways related to immune response and immune system diseases as the major functional pathways related to the IPM genes. The IPM high-risk group (IPM-high) showed higher fractions of regulatory T cells (Tregs) and smaller fractions of resting memory CD4+ T cells than the IPM-low group. Moreover, the stroma and immune cells in the IPM-high samples were scant. Further, the IPM-high group showed downregulation of MHC class II molecules. Additionally, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and GDSC analysis suggested the IPM-low as a promising responder to anti-CTLA-4 therapy and the common FDA-targeted drugs, while the IPM-high was non-responsive to these treatments. However, treatment using anti-CDKN2A agents, along with the activation of major histocompatibility complex (MHC) class-II response might sensitize this refractory mCRC subgroup. The dysfunction of MEIS1 might be the reason for the dysregulation of IPM genes.Conclusions: The IPM could identify subgroups of mCRC with a distinct risk of recurrence and stratify the patients sensitive to immuno-/chemotherapy. Further, for the first time, our study highlights the importance of MHC class-II molecules in the treatment of mCRCs using immunotherapy.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Reverse Signaling by MHC-I Molecules in Immune and Non-Immune Cell Types.

Major histocompatibility complex (MHC) molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells, for example dendritic cells (DCs) and T cells, or immune cells and their targets, such as T cells and virus-infected or tumor cells. However, much less appreciated is the fact that MHC molecules can also act as signaling receptors. In this process, here referred to as reverse MHC class I (MHC-I) signaling, ligation of MHC molecules can lead to signal-transduction and cell regulatory effects in the antigen presenting cell. In the case of MHC-I, reverse signaling can have several outcomes, including apoptosis, migration, induced or reduced proliferation and cytotoxicity towards target cells. Here, we provide an overview of studies showing the signaling pathways and cell outcomes upon MHC-I stimulation in various immune and non-immune cells. Signaling molecules like RAC-alpha serine/threonine-protein kinase (Akt1), extracellular signal-regulated kinases 1/2 (ERK1/2), and nuclear factor-κB (NF-κB) were common signaling molecules activated upon MHC-I ligation in multiple cell types. For endothelial and smooth muscle cells, the in vivo relevance of reverse MHC-I signaling has been established, namely in the context of adverse effects after tissue transplantation. For other cell types, the role of reverse MHC-I signaling is less clear, since aspects like the in vivo relevance, natural MHC-I ligands and the extended downstream pathways are not fully known.The existing evidence, however, suggests that reverse MHC-I signaling is involved in the regulation of the defense against bacterial and viral infections and against malignancies. Thereby, reverse MHC-I signaling is a potential target for therapies against viral and bacterial infections, cancer immunotherapies and management of organ transplantation outcomes.

Ligand Design for Specific MHC Class I Molecules on the Cell Surface.

We have validated that ligand peptides designed from antigen peptides could be used for targeting specific major histocompatibility complex class I (MHC-I) molecules on the cell surface. To design the ligand peptides, we used reported antigen peptides for each MHC-I molecule with high binding affinity. From the crystal structure of the peptide/MHC-I complexes, we determined a modifiable residue in the antigen peptides and replaced this residue with a lysine with an ε-amine group modified with functional molecules. The designed ligand peptides successfully bound to cells expressing the corresponding MHC-I molecules via exchange of peptides bound to MHC-I. We demonstrated that the peptide ligands could be used to transport a protein or a liposome to cells expressing the corresponding MHC-I. This strategy may be useful for targeted delivery to cells overexpressing MHC-I, which have been observed in autoimmune diseases.

MiR34a Regulates Neuronal MHC Class I Molecules and Promotes Primary Hippocampal Neuron Dendritic Growth and Branching.

In the immune system, Major Histocompatibility Complex class I (MHC-I) molecules are located on the surface of most nucleated cells in vertebrates where they mediate immune responses. Accumulating evidence indicates that MHC-I molecules are also expressed in the central nervous system (CNS) where they play important roles that are significantly different from their immune functions. Classical MHC-I molecules are temporally and spatially expressed in the developing and adult CNS, where they participate in the synaptic formation, remodeling and plasticity. Therefore, clarifying the regulation of MHC-I expression is necessary to develop an accurate understanding of its function in the CNS. Here, we show that microRNA 34a (miR34a), a brain enriched noncoding RNA, is temporally expressed in developing hippocampal neurons, and its expression is significantly increased after MHC-I protein abundance is decreased in the hippocampus. Computational algorithms identify putative miR34a target sites in the 3′UTR of MHC-I mRNA, and here we demonstrate direct targeting of miR34a to MHC-I mRNA using a dual-luciferase reporter assay system. MiR34a targeting can decrease constitutive MHC-I expression in both Neuro-2a neuroblastoma cells and primary hippocampal neurons. Finally, miR34a mediated reduction of MHC-I results in increased dendritic growth and branching in cultured hippocampal neurons. Taken together, our findings identify miR34a as a novel regulator of MHC-I for shaping neural morphology in developing hippocampal neurons.

Atomistic structure and dynamics of the human MHC-I peptide-loading complex

The major histocompatibility complex class-I (MHC-I) peptide-loading complex (PLC) is a cornerstone of the human adaptive immune system, being responsible for processing antigens that allow killer T cells to distinguish between healthy and compromised cells. Based on a recent low-resolution cryo-electron microscopy (cryo-EM) structure of this large membrane-bound protein complex, we report an atomistic model of the PLC and study its conformational dynamics on the multimicrosecond time scale using all-atom molecular dynamics (MD) simulations in an explicit lipid bilayer and water environment (1.6 million atoms in total). The PLC has a layered structure, with two editing modules forming a flexible protein belt surrounding a stable, catalytically active core. Tapasin plays a central role in the PLC, stabilizing the MHC-I binding groove in a conformation reminiscent of antigen-loaded MHC-I. The MHC-I-linked glycan steers a tapasin loop involved in peptide editing toward the binding groove. Tapasin conformational dynamics are also affected by calreticulin through a conformational selection mechanism that facilitates MHC-I recruitment into the complex.

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

The class II peptide editor, H2-M, affects the development and repertoire of B-1 cells

Abstract Non-classical major histocompatibility complex class-II (MHCII) protein H2-M edits peptides bound to conventional MHCII (pMHCII) in favor of stable pMHCII complexes. Although the impact of H2-M deficiency on pMHCII expression and T cell activation is well-studied, information on how this protein affects the development and phenotypic profile of APCs is lacking. We found that the absence of H2-M in C57BL/6 (B6) mice, resulted in a loss of surface expression and peptide-cargo of MHCII molecules in B cells across lymphoid organs. Lower pMHCII expression reduced MHCII association with BCR, affecting the integrated BCR signaling. Importantly, in H2-M deficient B6 mice, compared to the wildtype mice, frequency and abundance of B-1 cells, but not conventional B-2 cells, was reduced in the spleen and peritoneum. This H2-M mediated effect on the B-1 cell population was only evident in the B6 background (I-Ab), but not in BALB/c (I-Ad/I-Ed), indicating an MHCII haplotype-dependent phenomenon. A decrease in B-1 cell number also was evident in immature B-1 cells, emphasizing that H2-M deficiency affects B-1 cell development. In H2-M KO mice compared to WT B6, B-1 cells display a significantly lower self-renewal capacity and a higher rate of apoptosis. Furthermore, H2-M deficiency alters the B-1 BCR repertoire, selecting for B-1 cells specific for highly abundant epitopes, but not for low-frequency epitopes. Collectively, these data identify the impact of H2-M/MHCII interaction as a regulator of BCR signaling that influences the selection, maturation, and maintenance of mature B-1 cell clones in the periphery.

Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I

Immune evasion is a major obstacle for cancer treatment. Common mechanisms include impaired antigen presentation through mutations or loss of heterozygosity (LOH) of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1–3. However, in pancreatic ductal adenocarcinoma (PDAC), a malignancy refractory to most therapies including ICB4, mutations causing MHC-I loss are rarely found5 despite the frequent downregulation of MHC-I expression6–8. Here we find that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation through an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced MHC-I cell surface expression and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, autophagy inhibition restores surface MHC-I levels, leading to improved antigen presentation, enhanced anti-tumour T cell response and reduced tumour growth in syngeneic hosts. Accordingly, anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface MHC-I expression. Autophagy inhibition, either genetically or pharmacologically with Chloroquine (CQ), synergizes with dual ICB (anti-PD1 and anti-CTLA4), and leads to an enhanced anti-tumour immune response. Our findings uncover a role for enhanced autophagy/lysosome function in immune evasion through selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB as a therapeutic strategy against PDAC.

Abstract B68: Pre-existing immune memory to cancer-associated phosphopeptides in healthy donors

Abstract Identifying appropriate antigens is essential to developing effective cancer vaccines. Protein phosphorylation plays a major role in signal transduction pathways that are dysregulated and contribute to the malignant phenotype of cancer cells. Our lab has shown that phosphopeptides derived from these proteins can be presented by major histocompatibility complex Class-I molecules and represent a new class of cancer-associated neoantigens. We previously reported that healthy donors without evident prior cancer had unusually robust CD8 T-cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides, suggesting that they might be due to pre-existing phosphopeptide specific memory T cells. To test this, we evaluated CD8+CD45RO+ memory T cells purified from the blood of healthy donors. These cells showed robust responses to 2-25% of HLA-A2+ and 0-23% of HLA-B7+ cancer-associated phosphopeptides. This demonstrates that healthy individuals with no evident cancer history have pre-existing immune memory to some cancer-associated phosphopeptides. We identified 3 “immunodominant” HLA-A2 associated phosphopeptides, in that memory responses were evident to them in the majority of analyzed donors. Such immunodominance suggests that these phosphopeptides result from signaling processes that occur commonly in transformation- or viral infection-related processes. In contrast, immunodominant phosphopeptides were not evident in the context of HLA-B7. While 2 donors showed pre-existing immune memory to a large number of shared targets, other donors showed memory to only small numbers of nonoverlapping phosphopeptides. This suggests that the mechanisms driving expression of these phosphopeptide antigens among healthy donors are much less common. We also found that responses to some phosphopeptides could be detected directly ex vivo. In some cases, the phosphopeptide targets of these ex vivo responses did not stimulate responses after 2 weeks in culture. These results suggest that some phosphopeptides in some donors may be the targets of ongoing immune responses composed of effector or effector memory cells that do not persist long-term in culture. These results provide evidence of regular, ongoing immune surveillance in healthy individuals to cancer-associated phosphopeptides. Citation Format: Amanda M. Lulu, Kara L. Cummings, Victor H. Engelhard. Pre-existing immune memory to cancer-associated phosphopeptides in healthy donors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B68.

Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef.

The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation.IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

Allergy-A New Role for T Cell Superantigens of Staphylococcus aureus?

Staphylococcus aureus superantigens (SAgs) are among the most potent T cell mitogens known. They stimulate large fractions of T cells by cross-linking their T cell receptor with major histocompatibility complex class-II molecules on antigen presenting cells, resulting in T cell proliferation and massive cytokine release. To date, 26 different SAgs have been described in the species S. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcal enterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoning and toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. In addition, there is growing evidence that SAgs are involved in allergic diseases. This review provides an overview on recent epidemiological data on the involvement of S. aureus SAgs and anti-SAg-IgE in allergy, demonstrating that being sensitized to SEs—in contrast to inhalant allergens—is associated with a severe disease course in patients with chronic airway inflammation. The mechanisms by which SAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, we discuss known and hypothetical pathways by which SAgs can drive an atopic disease.

HLA-DM catalytically enhances peptide dissociation by sensing peptide–MHC class II interactions throughout the peptide-binding cleft

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Abstract PR05: Autophagy facilitates immune evasion of pancreatic cancer through downregulation of MHC class I molecules

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy refractory to most therapies, including immune checkpoint blockade (ICB). Autophagy is a highly conserved self-degradation process that recycles cellular components. PDAC requires high levels of autophagy/lysosome function to meet their metabolic demand under nutrient-deprived conditions; however, little is known about substrates that are preferentially targeted and subsequent biologic impacts of their loss on PDAC pathogenesis. Here we found that cell surface expression of major histocompatibility complex class I (MHC-I) molecules, a critical component of antigen-presentation to CD8+ cytotoxic T lymphocytes (CTLs), is downregulated in PDAC cells though an autophagy-dependent pathway. Autophagy inhibition in PDAC cells restored surface MHC-I levels and improved antigen presentation, leading to enhanced antitumor T-cell response in syngeneic mouse models of PDAC, which was further enhanced by combination with ICB. Our findings indicate that elevated autophagy/lysosome function facilitates immune evasion of PDAC cells through selective targeting of MHC-I molecules and provide a rationale for the combination of autophagy inhibition and ICB as a therapeutic strategy against PDAC. This abstract is also being presented as Poster B61. Citation Format: Keisuke Yamamoto, Anthony Venida, Doug E. Biancur, Miwako Yamamoto-Kakiuchi, Albert P. Sohn, Rushika M. Perera, Alec C Kimmelman. Autophagy facilitates immune evasion of pancreatic cancer through downregulation of MHC class I molecules [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR05.

Substantial Influence of ERAP2 on the HLA-B*40:02 Peptidome: Implications for HLA-B*27-Negative Ankylosing Spondylitis

HLA-B*40:02 is one of a few major histocompatibility complex class I (MHC-I) molecules associated with ankylosing spondylitis (AS) independently of HLA-B*27. The endoplasmic reticulum aminopeptidase 2 (ERAP2), an enzyme that process MHC-I ligands and preferentially trims N-terminal basic residues, is also a risk factor for this disease. Like HLA-B*27 and other AS-associated MHC-I molecules, HLA-B*40:02 binds a relatively high percentage of peptides with ERAP2-susceptible residues. In this study, the effects of ERAP2 depletion on the HLA-B*40:02 peptidome were analyzed. ERAP2 protein expression was knocked out by CRISPR in the transfectant cell line C1R-B*40:02, and the differences between the peptidomes from the wild-type and ERAP2-KO cells were determined by label-free quantitative comparisons. The qualitative changes dependent on ERAP2 affected about 5% of the peptidome, but quantitative changes in peptide amounts were much more substantial, reflecting a significant influence of this enzyme on the generation/destruction balance of HLA-B*40:02 ligands. As in HLA-B*27, a major effect was on the frequencies of N-terminal residues. In this position, basic and small residues were increased, and aliphatic/aromatic ones decreased in the ERAP2 knockout. Other peptide positions were also affected. Because most of the non-B*27 MHC-I molecules associated with AS risk bind a relatively high percentage of peptides with N-terminal basic residues, we hypothesize that the non-epistatic association of ERAP2 with AS might be related to the processing of peptides with these residues, thus affecting the peptidomes of AS-associated MHC-I molecules.

MHC class I dysfunction of glioma stem cells escapes from CTL-mediated immune response via activation of Wnt/β-catenin signaling pathway.

Glioma stem cells (GSCs) decrease T cells cognition and evade systemic immunosurveillance via downregulations or defects of major histocompatibility complex class I (MHC-I) molecule and antigen-processing machinery (APM) components. Improvement of tumor surface antigens of GSCs may be effective strategy to trigger an adaptive immune response and activate cytotoxic T cells (CTLs) to eliminate glioma. In this study, our data indicated that downregulations of MHC-I and APM components expressions were associated with Wnt pathway activation in GSCs. Histone deacetylases (HDAC) inhibition improved MHC-I and APM components expressions, which could be partly reverted by Wnt pathway activation. Blocking CTLs-mediated killing decreased the anti-tumor effect of tumor lysate vaccine. The enhancement of T cells immune response resulting from HDAC inhibition was dependent on CTLs cognition on tumor antigens presented by upregulated MHC-I molecule in GSCs. These data suggest that suppression of stemness pathway may be effective for GSCs-based immunotherapy against immune-escaped tumors.

Elevated neoantigen levels in tumors with somatic mutations in the HLA-A, HLA-B, HLA-C and B2M genes

BackgroundThe major histocompatibility complex class I (MHC-I) molecule is a protein complex that displays intracellular peptides to T cells, allowing the immune system to recognize and destroy infected or cancerous cells. MHC-I is composed of a highly polymorphic HLA-encoded alpha chain that binds the peptide and a Beta-2-microglobulin (B2M) protein that acts as a stabilizing scaffold. HLA mutations have been implicated as a mechanism of immune evasion during tumorigenesis, and B2M is considered a tumor suppressor gene. However, the implications of somatic HLA and B2M mutations have not been fully explored in the context of antigen presentation via the MHC-I molecule during tumor development. To understand the effect that B2M and HLA MHC-I molecule mutations have on mutagenesis, we analyzed the accumulation of mutations in patients from The Cancer Genome Atlas according to their MHC-I molecule mutation status.ResultsSomatic B2M and HLA mutations in microsatellite stable tumors were associated with higher overall mutation burden and a larger fraction of HLA-binding neoantigens when compared to B2M and HLA wild type tumors. B2M and HLA mutations were highly enriched in patients with microsatellite instability. B2M mutations tended to occur relatively early during patients’ respective tumor development, whereas HLA mutations were either early or late events. In addition, B2M and HLA mutated patients had higher levels of immune infiltration by natural killer and CD8+ T cells and higher levels of cytotoxicity.ConclusionsOur findings add to a growing body of evidence that somatic B2M and HLA mutations are a mechanism of immune evasion by demonstrating that such mutations are associated with a higher load of neoantigens that should be presented via MHC-I.

Abstract 4030: Case classification with tumor antigen presenting and TGF-β signaling biomarkers to predict anti-PD-1 outcome in GI tract tumors using automated quantitative fluorescence multiplex IHC

Abstract Introduction: Anti-PD-1/L1 immune checkpoint blockade results in tumor stabilization or shrinkage in only 15-40% of patients. Predictive biomarkers are crucial in identifying responsive patients while excluding others from the toxicities of immunotherapies. Major histocompatibility complex class I (MHC-I) downregulation is one of the most frequent mechanisms of tumor escape from the host’s immune system, but little attention has been devoted to MHC-I expression in studies of the PD-1/L1 blockade. Recently, Tauriello and Mariathasan revealed stromal transforming growth factor (TGF)-β signaling in CD8+ T lymphocytes exclusion as a key determinant of resistance to PD-1/L1 blockade in colorectal and urothelial carcinomas. We present here a multiplex panel IHC of MHC-I, β2-microglobulin (B2M), CD14, TGF-β receptor 2 (TGFBR2), and pan-cytokeratin (panCK) in tumor micro-environment, and their predictive values to anti-PD-1 treatment. Methods: With the multiplex panel, 51 pre-pembrolizumab treatment patient specimens were stained, including pancreatic, colorectal and cholangiocarcinoma (33 non-responders: 17 PD, 13 SD, 3 NE; 18 responders: 14 PR, 4 CR). Pathologists annotated tumor areas on whole slide scans. HALO High-Plex FL module was used for image analysis. Epithelial tumor (panCK+) and stroma (panCK-) were masked with HALO’s random forest classifier. Spatial location, count, intensity, and percent abundance of each marker were identified. 43 features were designed based on the rationale of hypothesized biological significance. MATLAB was used for feature selection, ranking, and prediction of responses to anti-PD1 treatment. Results: There was a trend of higher MHCI expression on tumor cells in the responders than non-responders to pembrolizumab treatment. Heterogeneous MHCI expression of tumor cells, and fraction of TGFBR2+ CD14+ cells in stroma were the top features ranked by Relieff k-nearest neighbor (k=30) for the prediction of the response to pembrolizumab treatment. Using Quadratic Discriminant Analysis (QDA) with five-fold cross-validation, the prediction accuracy was 76.5%. Independent validation was not performed due to small sample size. Conclusions: Deep immune characterization of tumor microenvironments using high dimensional feature spaces derived from multiplex IHC staining may provide insightful directions on finding and validating predictive markers for various immunotherapy regiments (ex. PD-1/L1 blockade; dual TGF-β and PD-1/L1 blockade; combination of PD-1/L1 blockade with other treatments that enhance MHC-I molecules on tumor cells). Acknowledgement: We thank Nick Cummins, Jorge Lozano, and John Hurley for their technical assistance. Citation Format: Xiangxue Wang, Shizen Moh, Antony Hubbard, José L. Muñoz-Rodríguez, Mehrnoush Khojasteh, Jim Martin, Qingfeng Zhu, Robert Anders, Luis Diaz, Lidija Pestic-Dragovich, Lei Tang, Wenjun Zhang. Case classification with tumor antigen presenting and TGF-β signaling biomarkers to predict anti-PD-1 outcome in GI tract tumors using automated quantitative fluorescence multiplex IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4030.

Peptide editing and MHC binding mechanisms of Tapasin and TAP binding protein related, TAPBPR

Abstract Cell surface major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity to microbes and tumors by presenting intracellular peptides for recognition by CD8+ T cells. Since the repertoire of bound peptides is crucial to MHC-I stability as well as to T cell and NK cell recognition, the molecular mechanism by which peptides are selectively loaded onto MHC-I molecules is of considerable interest. A key role is played by the well-characterized chaperone tapasin as indicated by reduced cell surface MHC-I levels and altered peptide repertoires in tapasin deficient cell lines and mice. Despite extensive studies of the functional role of tapasin in shaping peptide repertoires, direct structural information on its interaction with MHC-I is lacking. Following the recent biochemical and crystallographic studies of the interaction of the tapasin homolog, TAPBPR, with MHC-I, we have engineered soluble versions of tapasin for comparison with TAPBPR. Using surface plasmon resonance assays of purified, untethered proteins, we observe that tapasin binds directly to an MHC-I molecule loaded with truncated peptides but with lower apparent affinity than TAPBPR. Similarly, using fluorescence polarization, we find that tapasin facilitates peptide exchange onto MHC-I with a lower efficiency than TAPBPR. These experiments permit direct comparisons of tapasin/MHC-I and TAPBPR/MHC-I interactions and help elucidate the functional relationship of these two distinct chaperones in shaping the MHC-I peptide repertoire.