Nonclassical MHC Research Articles

Impaired endocytosis and accumulation in early endosomal compartments defines herpes simplex virus–mediated disruption of the nonclassical MHC class I–related molecule MR1

Presentation of metabolites by the Major Histocompatibility Complex Class-I-related protein 1 (MR1) molecule to Mucosal-Associated Invariant T (MAIT) cells is impaired during herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while pre-existing ligand-bound mature MR1 is surprisingly upregulated by HSV-1. Using flow cytometry, immunoblotting and high throughput fluorescence microscopy we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection, and that internalised molecules accumulate in EEA1-labelled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1 mediated downregulation of immature molecules, however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimise the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by MAIT cells.

Unveiling a novel NKG2A inhibitor based on monoclonal antibody to HLAE-peptide complex.

e14677 Background: HLAE is a non-classical MHC class I molecule complexed with the VMAPRTLFL nonapeptide, derived from the leader sequence of HLAG (HLAEpHLAG). HLAEpHLAG is overexpressed across a wide type of tumors prominently on Multiple Myeloma (MM). HLAEpHLAG complex is the ligand to CD94-NKG2A in the immune system which negatively regulates the activation of natural killer (NK) cells as well as NKG2A positive T cells. Harnessing of the HLAE-NKG2A axis by the cancer cells leads to evasion from the immune system attack resulting in cancer progression. Currently, several Immune Checkpoint Inhibitors (ICIs) target the NKG2A receptor which has a low specificity to cancer and high toxicity to the patient. Engineering a new ICI that targets the HLAEpHLAG complex can potentially yield improved outcomes in cancer therapy. Methods: Using the hybridoma system, a new mouse monoclonal antibody (mAb) called 4D7 that targets the HLAEpHLAG complex was developed. mAb 4D7 affinity was assessed in vitro with recombinant proteins and a panel of cell lines that expressed zero to high levels of HLAE. The functional impacts of the mAb 4D7 was examined in a system where various MM cell lines were separately co-cultured with primary NK (pNK) cells. pNK were isolated from the peripheral blood of 5 healthy donors, and their activity was examined in the presence of mAb 4D7 by CD107a degranulation assay. Also, the functional impacts of the mAb 4D7 on allogenic pNK and MM blast cells, isolated from the bone marrow of 6 MM patients were assessed. Results: mAb 4D7 was determined to be an IgG-kappa isotype showing a high specific binding to recombinant HLAEpHLAG. Cell lines with overexpression of membranal HLAEpHLAG: RPMI8226, U266, 721.221 HLAE, and 721.221 HLAG showed positive staining with the mAb 4D7. While cell lines with low to zero expression of HLA-E like 721.221 WT showed low or negative staining with the mAb 4D7. The degranulation of NKG2A+ pNK subset cells against MM target cells increased in presence of mAb 4D7. However, adding mAb 4D7 to NKG2A -pNK subset cells co-cultured with MM target cells did not affect the pNK activity. Similar results were obtained when the target and effector cells were isolated from the bone marrow of active treatment-naive MM patients. Conclusions: MAb 4D7 demonstrates a remarkably high affinity for binding to the HLAEpHLAG complex, ensuring the safety of its application. The ability of 4D7 mAb to effectively block the HLAEpHLAG complex and counteract the inhibition signal in NK cells. mAb 4D7 is a promising monoclonal antibody with substantial potential to serve as an ICI for clinical use in MM patients.

Multiple Isomers of Photolumazine V Bind MR1 and Differentially Activate MAIT Cells.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR β-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Sulfated bile acid is a host-derived ligand for MAIT cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function.

The effects of primary and secondary bacterial exposure on the seahorse (Hippocampus erectus) immune response

Evolutionary adaptations in the Syngnathidae teleost family (seahorses, pipefish and seadragons) culminated in an array of spectacular morphologies, key immune gene losses, and the enigmatic male pregnancy. In seahorses, genome modifications associated with immunoglobulins, complement, and major histocompatibility complex (MHC II) pathway components raise questions concerning their immunological efficiency and the evolution of compensatory measures that may act in their place. In this investigation heat-killed bacteria (Vibrio aestuarianus and Tenacibaculum maritimum) were used in a two-phased experiment to assess the immune response dynamics of Hippocampus erectus. Gill transcriptomes from double and single-exposed individuals were analysed in order to determine the differentially expressed genes contributing to immune system responses towards immune priming. Double-exposed individuals exhibited a greater adaptive immune response when compared with single-exposed individuals, while single-exposed individuals, particularly with V. aestuarianus replicates, associated more with the innate branch of the immune system. T. maritimum double-exposed replicates exhibited the strongest immune reaction, likely due to their immunological naivety towards the bacterium, while there are also potential signs of innate trained immunity. MHC II upregulated expression was identified in selected V. aestuarianus-exposed seahorses, in the absence of other pathway constituents suggesting a possible alternative or non-classical MHC II immune function in seahorses. Gene Ontology (GO) enrichment analysis highlighted prominent angiogenesis activity following secondary exposure, which could be linked to an adaptive immune process in seahorses. This investigation highlights the prominent role of T-cell mediated adaptive immune responses in seahorses when exposed to sequential foreign bacteria exposures. If classical MHC II pathway function has been lost, innate trained immunity in syngnathids could be a potential compensatory mechanism.

HLA-DQ8 Supports Development of Insulitis Mediated by Insulin-Reactive Human TCR-Transgenic T Cells in Nonobese Diabetic Mice.

In an effort to improve HLA-"humanized" mouse models for type 1 diabetes (T1D) therapy development, we previously generated directly in the NOD strain CRISPR/Cas9-mediated deletions of various combinations of murine MHC genes. These new models improved upon previously available platforms by retaining β2-microglobulin functionality in FcRn and nonclassical MHC class I formation. As proof of concept, we generated H2-Db/H2-Kd double knockout NOD mice expressing human HLA-A*0201 or HLA-B*3906 class I variants that both supported autoreactive diabetogenic CD8+ T cell responses. In this follow-up work, we now describe the creation of 10 new NOD-based mouse models expressing various combinations of HLA genes with and without chimeric transgenic human TCRs reactive to proinsulin/insulin. The new TCR-transgenic models develop differing levels of insulitis mediated by HLA-DQ8-restricted insulin-reactive T cells. Additionally, these transgenic T cells can transfer insulitis to newly developed NSG mice lacking classical murine MHC molecules, but expressing HLA-DQ8. These new models can be used to test potential therapeutics for a possible capacity to reduce islet infiltration or change the phenotype of T cells expressing type 1 diabetes patient-derived β cell autoantigen-specific TCRs.

Single-Cell RNA Sequencing Reveals the Interplay between Circulating CD4 + T Cells, B Cells and Cancer-Associated Monocytes in Classic Hodgkin Lymphoma Treated with PD-1 Blockade

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4 + T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA and scT-cell receptor (TCR) sequencing to characterize the peripheral immune response to PD-1 blockade and define non-CD8 + T-cell dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL who were treated with PD-1 blockade on the CheckMate 205 clinical trial (at cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed (ND), previously untreated cHL and 13 healthy donors. We first analyzed circulating CD4 + T cells given their likely importance in the response to PD-1 blockade in cHL. We found that patients with R/R cHL had markedly lower baseline TCR diversity than healthy donors across the entire CD4 + naïve/central memory (CM) T-cell space. Additionally, patients with the most favorable responses to PD-1 blockade had significantly increased CD4 + naïve/CM TCR diversity at baseline (C1D1) and more abundant naïve/CM subsets on treatment (C4D1). These response-related differences likely reflect a continued capacity to mount CD4 + T-cell responses to neoantigens. We also identified several circulating CD4 + T-cell populations that were significantly more abundant in patients with cHL than in healthy donors, including follicular helper-like T cells, IFN-stimulated cytotoxic T cells and actively proliferating CTLA4 + LAG3 + T cells. Compared to healthy donors, all evaluated patients with cHL had significantly reduced numbers of classically defined circulating NK cells and an expanded population of IFN-stimulated NK cells with likely reduced cytotoxic potential. In addition to marked deregulation of the NK cell compartment, all patients with cHL had decreased numbers of circulating B cells at all stages of differentiation. We used the scRNA sequencing data to reconstruct individual B-cell receptor (BCR) sequences with the TRUST4 algorithm and found that the numbers of total and unique BCRs, as well as chao1 diversity, were significantly decreased in patients with ND cHL in comparison to healthy donors. Additionally, patients with the most favorable responses to PD-1 blockade had significantly higher baseline BCR diversity and circulating B-cell numbers. Altogether, these data underscore the likely importance of B cell-mediated immune responses in cHL, either directly or via Fc-binding of innate effectors. Finally, the most abundant circulating CD3 - population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumor-promoting cytokines/chemokines, as well as PD-L1 and SIRPa. These inflamed IL1β + monocytes were virtually absent from the blood of healthy donors. Using RNAscope, we also detected macrophages with similar features in the intact cHL tumor microenvironment, in the immediate proximity of malignant HRS cells. To assess the broader significance of the inflamed IL1β + monocytes, we interrogated a single-cell compendium of monocytes and macrophages from multiple solid tumors and identified a population of monocytes/ macrophages with a similar transcriptional signature. These results suggest that similarly programmed inflammatory monocytes/macrophages are detectable in multiple cancers. We next assessed the clinical significance of circulating inflamed IL1β + monocytes in patients with R/R cHL who were treated with PD-1 blockade. Patients who did not respond to PD-1 blockade expressed significantly higher levels of the inflamed IL1β + monocyte transcriptional signature which led to the development of a predictive assay. We then identified a comparable circulating monocyte population with the same transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor, renal cell carcinoma, which underscored the broad significance of these findings. Taken together, our findings highlight the likely complementary role of CD4 + T cells, B cells and cancer-associated monocytes in the response to PD-1 blockade in cHL.

A prominent role of non-classical MHC class I L lineage genes in salmon antiviral immunity

A prominent role of non-classical MHC class I L lineage genes in salmon antiviral immunity

Proteome analysis, bioinformatic prediction and experimental evidence revealed immune response down-regulation function for serum-starved human fibroblasts

Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study, for the first time, we compared the proteomes of serum-starved human skin fibroblasts and peripheral blood mononuclear cells (PBMCs) using Nano-LC-ESI-tandem mass spectrometry. This analysis contributes to a better understanding of the underlying molecular mechanisms of chronic inflammation and cancer, which are intrinsically accompanied by growth factor deficiency.The proteomes of starved fibroblasts and PBMCs consisted of 307 and 294 proteins, respectively, which are involved in lymphocyte migration, complement activation, inflammation, acute phase response, and immune regulation. Starved fibroblasts predominantly produced extracellular matrix-related proteins such as collagen/collagenase, while PBMCs produced focal adhesion-related proteins like beta-parvin and vinculin which are involved in lymphocyte migration. PBMCs produced a more diverse set of inflammatory molecules like heat shock proteins, while fibroblasts produced human leukocytes antigen-G and -E that are known as main immunomodulatory molecules. Fifty-four proteins were commonly found in both proteomes, including serum albumin, amyloid-beta, heat shock cognate 71 kDa, and complement C3. GeneMANIA bioinformatic tool predicted 418 functions for PBMCs, including reactive oxygen species metabolic processes and 241 functions for starved fibroblasts such as antigen processing and presentation including non-classical MHC -Ib pathway, and negative regulation of the immune response. Protein-protein interactions network analysis indicated the immunosuppressive function for starved fibroblasts-derived human leucocytes antigen-G and –E. Moreover, in an in vitro model of allogeneic transplantation, the immunosuppressive activity of starved fibroblasts was experimentally documented. ConclusionUnder serum starvation-induced metabolic stress, both PBMCs and fibroblasts produced molecules like heat shock proteins and amyloid-beta, which can have pathogenic roles in auto-inflammatory diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, aging, and cancer. However, starved fibroblasts showed immunosuppressive activity in an in vitro model of allogeneic transplantation, suggesting their potential to modify such adverse reactions by down-regulating the immune system.

The non-classical major histocompatibility complex II protein SLA-DM is crucial for African swine fever virus replication

African swine fever virus (ASFV) is a lethal animal pathogen that enters its host cells through endocytosis. So far, host factors specifically required for ASFV replication have been barely identified. In this study a genome-wide CRISPR/Cas9 knockout screen in porcine cells indicated that the genes RFXANK, RFXAP, SLA-DMA, SLA-DMB, and CIITA are important for productive ASFV infection. The proteins encoded by these genes belong to the major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II (SLA II). RFXAP and CIITA are MHC II-specific transcription factors, whereas SLA-DMA/B are subunits of the non-classical MHC II molecule SLA-DM. Targeted knockout of either of these genes led to severe replication defects of different ASFV isolates, reflected by substantially reduced plating efficiency, cell-to-cell spread, progeny virus titers and viral DNA replication. Transgene-based reconstitution of SLA-DMA/B fully restored the replication capacity demonstrating that SLA-DM, which resides in late endosomes, plays a crucial role during early steps of ASFV infection.

Recognition of Listeria monocytogenes infection by natural killer cells: Towards a complete picture by experimental studies in rats.

The study of cellular immune responses in animal disease models demands detailed knowledge of development, function, and regulation of immune cells, including natural killer (NK) cells. Listeria monocytogenes (LM) bacterium has been explored in a large area of research fields, including the host pathogen interaction. Although the importance role of NK cells in controlling the first phase of LM burden has been investigated, the interaction between NK cells and infected cells in details are far from being comprehended. From in vivo and in vitro experiments, we can drive several important pieces of knowledge that hopefully contribute to illuminating the intercommunication between LM-infected cells and NK cells. Experimental studies performed in rats revealed that certain NK cell ligands are influenced in LM-infected cells. These ligands include both classical- and non-classical MHC class I molecules and C-type lectin related (Clr) molecules that are ligands for Ly49- and NKR-P1 receptors respectively. Interaction between these receptors:ligands during LM infection, demonstrated stimulation of rat NK cells. Hence, these studies provided additional knowledge to the mechanisms NK cells utilise to recognise and respond to LM infection outlined in the current review.

Mutation hot spots in MICA/MICB extracellular domains

MICA and MICB are non-classical MHC molecules that indicate cellular stress. They act as ligands for NKG2D receptors found on NK cells, thereby triggering a cytotoxic response against damaged, infected, or transformed cells. The production of soluble forms of MICA/MICB occurs via the cleavage of their extracellular domains (ECDs). The expression of MICA/MICB molecules in tumor sections or the levels of their soluble forms in blood have potential as diagnostic tools for cancer. They can predict important clinical outcomes for cancer patients, such as overall and recurrence-free survival. However, their extensive molecular polymorphism complicates the development of monoclonal antibodies (mAbs) for diagnostic use. Therefore, the diagnostic value of mAb-based assays may vary depending on the frequencies of allelic variants in local human populations. We examined the ECD amino acid sequences of more than 280 MICA and 50 MICB allelic variants. Additionally, we identified 172 and 58 single nucleotide polymorphisms (SNPs) located in the coding regions of the respective genes and resulting in amino acid replacements. The most frequent amino acid replacements (> 10%) in the ECD occur at 11 and 4 sites of MICA and MICB, respectively. We found that the frequencies of SNPs in the identified hot spots strongly correlate with each other in different human populations, despite the diversity of allelic variant frequencies. The functional role of only one site is known. The replacement of valine with methionine at position 152 enhances the affinity of MICA to NKG2D receptor. As the hot spots are dispersed throughout the entire ECD sequences, they may play a role other than modulating affinity with the NKG2D receptor interaction. We recommend that Ag sets used to validate anti-MICA/MICB mAbs meet two criteria. First, they should include both MICA and MICB alleles, as these genes have highly similar sequences. Second, the alleles should cover the variability observed in the identified hot spots.

All-Atom Simulations Reveal the Intricacies of Signal Transduction upon Binding of the HLA-E Ligand to the Transmembrane Inhibitory CD94/NKG2A Receptor.

Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.

HLA-DO promotes Treg differentiation by altering antigen presentation

Abstract HLA-DO in human (H2-O in mice) (DO) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. DO forms a stable complex with HLA-DM and requires DM for trafficking to MIIC. Using biochemical experiments, our lab has previously discovered that DO has differential effects on editing peptides of different sequences. DO enhances binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both thymic negative selection, and peripheral activation of CD4 T cells. Using single-cell RNA-sequencing (scRNA-seq) of peripheral CD4 T cells from unimmunized DO knock-out (KO) mice we found an increased frequency of “activated” CD4 T cells, in support of our biochemical model. This increased frequency corresponds with a decrease in “naïve” CD4 T cells in both scRNA-seq and FACs analysis. Interestingly, scRNA-seq analysis confirmed our previous findings of elevated regulatory T cells (Treg) levels in DO-KO mice. Genetic analysis showed that unimmunized DO-KO Tregs also have a more activated phenotype. These findings suggests that absence of DO generates a more stimulatory in vivo cellular environment for CD4 T cells, likely due to altered antigen presentation. Research supported by grants from NIH (R01 AI130210, R01 AI121174 and R37 AI060040)

Targeting senescent dermal fibroblasts responsible for hyperactive melanocytes in melasma

Targeting senescent dermal fibroblasts responsible for hyperactive melanocytes in melasma

Abstract 3467: GRWD5769: A first-in-class inhibitor of ERAP1, generating novel cancer antigens to drive de novo anti-tumor T cell responses

Abstract Immune checkpoint inhibitors (ICI) have changed the cancer treatment paradigm, yet significant unmet need remains. Clinical data shows that increasing antigen presentation diversity, genomic instability, tumor mutational burden and HLA diversity, as well as preventing permanent exhaustion of pre-existing cytotoxic T cells, caused by chronic cancer antigen stimulation, are all factors that improve clinical response to ICI. Additionally, increasing presentation of cancer-specific target peptides on cancer cells will enhance T cell receptor (TCR)-mediated therapeutics such as ImmTACs or TCR-Engineered T cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides loaded into classical and non-classical MHC Class I. In cancer, knockout or inhibition of ERAP1 changes a proportion of the antigen repertoire, generating and upregulating cancer antigens. This leads to the activation of de novo anti-tumor T cell responses, causing tumor growth inhibition and bypassing T cell exhaustion. We report the preclinical development, characterization and mechanistic analysis of the first-in-class ERAP1 inhibitor, GRWD5769. Extensive analysis of the immunopeptidomes of diverse cancer cell lines robustly showed that GRWD5769 modulates the cancer-related antigen repertoire across genotypes and cancer-type backgrounds. Novel or upregulated neoantigens generated by ERAP1 inhibition are conserved across cancer cell types and genetic backgrounds. ERAP1 inhibition in combination with an ICI (anti-PD1 mAb) diversified the TCR repertoire, upregulated prognostic immune gene markers in the tumor, including markers for recently activated (thus non-exhausted) T cells, and drove infiltration of T cells into syngeneic mouse model tumors. This combination is efficacious across syngeneic models, including different mouse strains and the effects of ERAP1 inhibition on the T cell response correlate with efficacy. Further, ex vivo human T cell co-cultures with cancer cell lines demonstrate that novel and upregulated neoantigens generated by ERAP1 inhibition drive tumor cell killing. In addition, these studies showed enhanced tumor cell killing when an ImmTAC was used in combination with ERAP1 inhibition to enhance presentation of the cancer-specific target peptide. In conclusion, the first-in-class, ERAP1 inhibitor clinical candidate, GRWD5769, drives novel anti-tumor T cell responses through neoantigen creation and circumventing T cell exhaustion. GRWD5769 has demonstrated a good safety profile in GLP toxicology studies and robust proof of mechanism and proof of principle biomarkers have been developed to provide a clear path to establish the activity and efficacy of GRWD5769 in patients in 2023. Citation Format: Andrew Leishman, Wayne Paes, Emma Sparrow, Ana Ribeiro, Michael Cundell, Milos Aleksic, Michael P. Pinggera, Jessica Sette, Kate Anderton, Nicola Ternette, Juliet Morgan, Jason J. Shiers, Martin Quibell, Peter I. Joyce. GRWD5769: A first-in-class inhibitor of ERAP1, generating novel cancer antigens to drive de novo anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3467.

Cover Story: Eur. J. Immunol. 4'23

European Journal of ImmunologyVolume 53, Issue 4 2370041 Cover PictureFree Access Cover Story: Eur. J. Immunol. 4'23 First published: 10 April 2023 https://doi.org/10.1002/eji.202370041AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Our cover image features immunostaining of classical and non-classical MHC class I molecules on HPV6/11 induced respiratory papilloma epithelia. HLA-A, B, and C are absent on these epithelial cells but are expressed in the papilloma stroma. However, non-classical MHC class I molecules, HLA-E and -G, are strongly expressed on papilloma epithelial cells and stroma. The image are from Israr et al. This is the strategy used by HPV6/11 infection to block NK activation and reduce HPV6/11 peptide presentation to T-cells. The authors also show that papilloma cells express increased amounts of PGE2 that directly inhibits NK cell function by signaling through the NK cell PGE2 receptors EP2 and EP4. The original image was digitally modified for the cover. Volume53, Issue4April 20232370041 RelatedInformation

Inhibition of LILRB2 by a Novel Blocking Antibody Designed to Reprogram Immunosuppressive Macrophages to Drive T-Cell Activation in Tumors.

Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.

Atomistic basis for the differential load response by the γδ and the chimeric Vγδ-Cαβ T-cell receptors.

αβ and γδ T cells perform vital functions in adaptive and transitional immunity, respectively, and they are both equipped with antigen-sensing T cell receptors. Prototypical recognition of sparse antigenic peptide-loaded major histocompatibility complex molecule (pMHC) by the αβ T-cell receptor (TCRαβ) is assisted by load, through catch bond formation. On the other hand, the contrasting abundance of γδ T cell receptor (TCRγδ) ligands suggests that the recognition may involve a mechanism that is less sensitive to load, as demonstrated in our recent studies. To elucidate structural basis for the load response, we performed a total of 12 µs all-atom molecular dynamics simulations of the 9C2 TCRγδ complexed with the nonclassical MHC molecule CD1d holding a sulfatide lipid antigen JLS (PDB 4LHU). Simulations were performed either without load or with 4.4 to 27.8 pN loads. Contrary to the case of TCRαβ, we did not find any noticeable stabilization of the TCRγδ-CD1d-JLS interface with applied load, supporting an absence of catch bond. We also studied a chimeric Vγδ-Cαβ TCR where the constant domains of PDB 4LHU were replaced with those of N15 TCRαβ (PDB 1NFD). Comparisons between the two systems in terms of interfacial contact dynamics and domain motion reveal how the constant domains can allosterically modulate the interface with the ligand in a load-dependent manner.

MHC Class II Presentation in Autoimmunity.

Antigen presentation by major histocompatibility complex class II (MHC-II) molecules is crucial for eliciting an efficient immune response by CD4+ T cells and maintaining self-antigen tolerance. Some MHC-II alleles are known to be positively or negatively associated with the risk of the development of different autoimmune diseases (ADs), including those characterized by the emergence of autoreactive T cells. Apparently, the MHC-II presentation of self-antigens contributes to the autoimmune T cell response, initiated through a breakdown of central tolerance to self-antigens in the thymus. The appearance of autoreactive T cell might be the result of (i) the unusual interaction between T cell receptors (TCRs) and self-antigens presented on MHC-II; (ii) the posttranslational modifications (PTMs) of self-antigens; (iii) direct loading of the self-antigen to classical MHC-II without additional nonclassical MHC assistance; (iv) the proinflammatory environment effect on MHC-II expression and antigen presentation; and (v) molecular mimicry between foreign and self-antigens. The peculiarities of the processes involved in the MHC-II-mediated presentation may have crucial importance in the elucidation of the mechanisms of triggering and developing ADs as well as for clarification on the protective effect of MHC-II alleles that are negatively associated with ADs.