Minimal observed impact of HLA genotype on hospitalization and severity of SARS-CoV-2 infection

HLA is a critical component of the viral antigen presentation pathway. We investigated the relationship between severity of SARS-CoV-2 disease and HLA type in 3,235 individuals with confirmed SARS-CoV-2 infection. We found only the DPB1 locus to be associated with the binary outcome of whether an individual developed any COVID-19 symptoms. The number of peptides predicted to bind to an HLA allele had no significant relationship with disease severity both when stratifying individuals by ancestry or age and in a pooled analysis. Age, BMI, asthma status, and autoimmune disorder status were predictive of severity across multiple age and individual ancestry stratificiations. Overall, at the population level, we found HLA type is significantly less predictive of COVID-19 disease severity than certain demographic factors and clinical comorbidities.

Targeting Cross-Presentation as a Route to Improve the Efficiency of Peptide-Based Cancer Vaccines

Cross-presenting dendritic cells (DC) offer an attractive target for vaccination due to their unique ability to process exogenous antigens for presentation on MHC class I molecules. Recent reports have established that these DC express unique surface receptors and play a critical role in the initiation of anti-tumor immunity, opening the way for the development of vaccination strategies specifically targeting these cells. This study investigated whether targeting cross-presenting DC by two complementary mechanisms could improve vaccine effectiveness, in both a viral setting and in a murine melanoma model. Our novel vaccine construct contained the XCL1 ligand, to target uptake to XCR1+ cross-presenting DC, and a cell penetrating peptide (CPP) with endosomal escape properties, to enhance antigen delivery into the cross-presentation pathway. Using a prime-boost regimen, we demonstrated robust expansion of antigen-specific T cells following vaccination with our CPP-linked peptide vaccine and protective immunity against HSV-1 skin infection, where vaccine epitopes were natively expressed by the virus. Additionally, our novel vaccination strategy slowed tumor outgrowth in a B16 murine melanoma model, compared to adjuvant only controls, suggesting antigen-specific anti-tumor immunity was generated following vaccination. These findings suggest that novel strategies to target the antigen cross-presentation pathway in DC may be beneficial for the generation of anti-tumor immunity.

Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

SARS-CoV-2 inhibits induction of the MHC class I pathway by targeting the STAT1-IRF1-NLRC5 axis

The MHC class I-mediated antigen presentation pathway plays a critical role in antiviral immunity. Here we show that the MHC class I pathway is targeted by SARS-CoV-2. Analysis of the gene expression profile from COVID-19 patients as well as SARS-CoV-2 infected epithelial cell lines reveals that the induction of the MHC class I pathway is inhibited by SARS-CoV-2 infection. We show that NLRC5, an MHC class I transactivator, is suppressed both transcriptionally and functionally by the SARS-CoV-2 ORF6 protein, providing a mechanistic link. SARS-CoV-2 ORF6 hampers type II interferon-mediated STAT1 signaling, resulting in diminished upregulation of NLRC5 and IRF1 gene expression. Moreover, SARS-CoV-2 ORF6 inhibits NLRC5 function via blocking karyopherin complex-dependent nuclear import of NLRC5. Collectively, our study uncovers an immune evasion mechanism of SARS-CoV-2 that targets the function of key MHC class I transcriptional regulators, STAT1-IRF1-NLRC5.

LC3 and NLRC5 Interaction Inhibits NLRC5-Mediated MHC Class I Antigen Presentation Pathway in Endometrial Cancer

Background: The major histocompatibility complex class I (MHC- I) transactivator, nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 5 (NLRC5), serves as a target for immune evasion in many cancers, including endometrial cancer (EC). An inhibition of autophagy can contribute to immunotherapy by assisting the MHC-I-mediated antigen presentation in cancer. However, the underlying mechanism for autophagy-regulated MHC-I in EC remains unclear. Our study aimed to investigate the effect of autophagy on NLRC5 and MHC-I-mediated antigen presentation, and to identify the potential mechanisms underlying this process in EC.Methods: We examined the levels of autophagy and MHC-I genes by performing transmission electron microscopy (TEM), RNA-seq sequencing, western blotting, and qRT-PCR. The t-test, F-test, Kaplan-Meier analysis, and Pearson’s correlation analysis were used for statistical evaluations of tissue microarrays. Immunofluorescence staining, co-immunoprecipitation (CO-IP), and glutathione S-transferase (GST) pull-down assay were performed. HEC-1A, AN3CA, and Ishikawa EC cells were transfected designed, and the role of LC3 and NLRC5 in MHC-I-mediated antigen presentation in EC was further evaluated in a xenotransplantation model of HEC-1A cell line. Results: Autophagy was upregulated in EC endometrium as compared to that in normal endometrium. MHC I and NLRC5 expressions were lower in EC endometrium than in normal endometrium. Autophagy played a negative role in the MHC-I genes expression in vitro. Furthermore, a negative correlation was found between LC3 and NLRC5 levels, and LC3 interacted with NLRC5 to inhibit NLRC5-mediated MHC-I antigen presentation pathway in vitro and in vivo. Conclusion: An upregulation of LC3 in EC patients may contribute to tumor immune escape by restricting the NLRC5-mediated MHC-I antigen presentation pathway, suggesting that inhibiting LC3 and promoting NLRC5 may be a promising immunotherapy strategy in the management of EC.

Low abundance members of the gut microbiome are potent drivers of immune cell education.

One of the main goals of microbiome research is to identify bacterial members that significantly affect host phenotypes and understand their contributions to disease pathogenesis. Studies identifying bacterial members that dictate host phenotype have focused mainly on the dominant members, and the role of low abundance microbes in determining host phenotypes and pathogenesis of diseases remains unexplored. In this study, we compared the gut bacterial community of mice with wide-ranging microbial exposure to determine if low abundance bacteria vary based on microbial exposure or remain consistent. We noted that similar to the high abundance bacterial community, a core community of low abundance bacteria made up a significant portion of the gut microbiome irrespective of microbial exposure. To determine the effect of low abundance bacteria on community structure and host gene expression, we devised a microbiome dilution strategy to delete out low abundance bacteria and engrafted the diluted microbiomes into germ-free mice. Our approach successfully excluded low abundance bacteria from small and large intestinal bacterial communities and induced global changes in microbial community structure and composition in the large intestine. Gene expression analysis of intestinal tissue revealed that loss of low abundance bacteria resulted in a drastic reduction in expression of multiple genes involved MHC class II antigen presentation pathway and T-cell cytokine production in the small intestine. The effect of low abundance bacteria on MHC class II expression was found specific to the intestinal epithelium at an early timepoint post-colonization and correlated with bacteria belonging to the family Erysipelotrichaceae. We conclude that low abundance bacteria have a significantly higher immuno-stimulatory effect compared to dominant bacteria and are thus potent drivers of early immune education in the gut. Therefore, characterizing the immune interaction of low abundance bacteria with the host will offer greater insight into the intestinal immune landscape and disease pathogenesis.

Histone Acetyltransferase (HAT) Activator, YF2, Modulates the p53:BCL6 Axis and Antigen Presentation in Diffuse Large B-Cell Lymphomas

A hallmark of DLBCL is epigenetic derangements characterized by monoallelic mutations in histone acetyltransferases (HATs); EP300 (p300) and CREBBP (CBP). The intact allele offers the opportunity for targeted therapies designed to overcome mutational dysregulation. We reported the discovery of YF2, a first-in-class HAT activator that demonstrates selective cytotoxicity in HAT-mutated DLBCL and induces HAT-mediated histone acetylation in vitro and in vivo. Here, we detail the mechanisms of action and the downstream effects of YF2 treatment. A unique feature of CBP/p300 is that it harbors a regulatory loop within its catalytic domain that undergoes autoacetylation which is critical for maintaining normal function. In order to determine if YF2 is able to induce the autoacetylation of p300/CBP, thereby increasing its catalytic activity, hypoacetylated CBP/p300 was combined with YF2 and Ac-CoA. YF2 demonstrated significant induction of CBP/p300 autoacetylation. To understand how YF2 interacts with HATs we analyzed the thermal stability, via thermal shift assay, of CBP/p300 subunits in the presence of YF2. We observed a T m shift when utilizing the full p300 (ΔT m = -2.9 oC)/CBP(ΔT m = -3.4 oC) catalytic core, which includes the catalytic, PHD/RING, and bromodomain. YF2 does not interact directly with the catalytic domain as there were no observed T m shift. YF2 significantly interacts with the bromodomain (ΔT m = -5.6 oC). In silico analysis has shown that the bromodomain has 3 TRP domains that are predicted to interact with small molecules. Next, we sought to determine how resistance to HDAC inhibitors (HDACi) and mutations/loss of HATs affects sensitivity to YF2. We first developed cell lines to be 10-fold resistant to HDACi romidepsin. When treated with YF2, resistant-SUDHL-6 was more sensitive to YF2 than the parental cell line (Resistant IC 50 = 2.2µM vs Parental IC 50 = 7.22µM). We found no change in YF2 sensitivity in the HAT wt OCI-Ly1 cell line. We performed CRISPR KO of EP300 in wt OCI-Ly7 cell line. A single cell clone with EP300 mutations was identified (OCI-Ly7-EP300 +/-). ICE analysis revealed that the percentage of indels was 12%. OCI-Ly7-EP300 +/- had lower p300 protein expression and were more sensitive to YF2 (IC 50 = 14.05µM) compared to wt (IC 50 = 23.7µM) when measured by Annexin V and CellTiter Glo assay. CBP/p300 is involved in the transcriptional activation of p53 through direct acetylation. YF2 induced both CBP (EC 50 = 15.47µM) and p300 (EC 50 = 6.05µM) mediated acetylation of p53 in cell free assays. As measured by RNA-Seq, YF2 altered multiple pathways regulated by CBP/p300 such as apoptosis and the p53 pathways. The p53 pathway was significantly upregulated in all cell lines. Validation of this pathway via qPCR, revealed p21, BAI1, ATM, FAS, FOS were upregulated in all cell lines. Additionally, YF2 induced G2/M arrest in a dose dependent manner when assessed via flow cytometry. We also observed modest increases in p21 and decrease CCND1 expression with YF2 treatment. BCL6, a transcriptional repressor linked to B-cell lymphomagenesis, is in part regulated through acetylation by CBP/p300. Mechanistically, CBP and the BLC6/SMRT/HDAC3 repressor complex co-occupy enhancers in the MHC Class II loci. Lack of functional CBP drives BCL6 mediated MHC repression resulting in reduced MHC gene expression and altered antigen presentation. In cell free assays, we YF2 induced p300 mediated BCL6 acetylation (IC 50 = 1.58 µM). We hypothesized HAT activation by YF2 could increase MHC expression in DLBCL. RNA-Seq analysis revealed YF2 led to upregulation of the interferon gamma pathway. Significantly, cell lines treated with YF2 showed increased MHC Class I and II expression when analyzed via flow cytometry. In summary, these findings demonstrate that YF2 interacts with the RING and bromodomains, leading to an allosteric change within the catalytic pocket to facilitate increased acetylation. In addition, YF2 leads to CBP/p300 autoacetylation, further enhancing enzymatic activity. We also demonstrated that YF2 is highly selective to DLBCL harboring HAT mutations and overcomes resistance to HDACi. Additionally, YF2 treatment modulates the p53:BCL6 axis, cell cycle progression, and antigen presentation pathway potentially restoring immune surveillance. These results support future clinical application of YF2 in HAT mutated lymphomas. Figure 1 Figure 1. Disclosures Amengual: Seagen: Consultancy; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau; Appia Pharmaceuticals: Research Funding.

Histone Hypoacetylation-Driven CD9 Repression Arrests Differentiation and Evades Immunosurveillance in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells accompanied by impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain poor, underscoring the need to decipher the underlying pathology and implement therapeutic interventions. Emerging evidence suggest myeloblasts could evolve machineries to evade T cell patrol and hinder immunotherapies. Here, we present a new mechanism driving immune escape in the context of pediatric AML, based on our discoveries of CD9 in hematopoietic stem cells (HSC; Leung et al, Blood, 2011) and acute lymphoblastic leukemia (ALL; Leung et al, Leukemia, 2020). We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of cell surface CD9 on blasts of pediatric AML patients (13.2%, n=81) was significantly lower than that of pediatric ALL patients (90.4%, n=181, P<0.001) or that on CD34+ HSC of normal bone marrow donors (48.4%, n=22, P=0.014). Among pediatric AML cases, the blasts of 32 patients (39.5%) were CD9+. The 5-year RFS rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018). Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P<0.01), Ki-67 expression (P=0.041) and colony formation (P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastic reduction of leukemic load in the bone marrow, spleen, blood and liver by 70.7-91.8% (P<0.05), a significantly prolonged survival duration (P<0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals transplanted with CD9- MV4-11 cells. A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) and samples (n=9) with a histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), restored activating histone acetylation marks (4.1-41.6-fold, P<0.05) and suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). Mechanistically, global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ patient samples. Moreover, single-cell transcriptomic analyses of total bone marrow cells from MV4-11-tranplanted mice detected a significant enrichment of differentially regulated genes functioning in the antigen processing and presentation pathway. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P<0.001) in PMA-mediated monocyte/macrophage-like AML differentiation cultures preceding the appearance of lineage markers CD14, CD36 and iCD68 as well as the antigen presentation molecule MHC-I. In the overexpression system, CD9 not only elevated the expression of monocytic markers, but also promoted basal and IFNγ-induced MHC-I/II expression (P<0.01) through the JAK/STAT axis. Inter-patient comparisons of bone marrow samples (n=27) revealed a higher MHC-I expression in CD9+ than CD9- AML (MFI: 61820 vs. 18601, P<0.001). Interestingly, tetraspanin CD9 physically bound to MHC-I/II and formed an immune complex as revealed by co-immunoprecipitation. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Syngeneic transplantation in immunocompetent mice, AML/T cell co-cultures, antigen-specific assays and panobinostat priming/T cell adoptive transfer are currently underway to fully dissect the role of CD9 in leukemia immunity. Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immunosurveillance in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy. Disclosures No relevant conflicts of interest to declare.

914 Loss of LKB1 is associated with resistance to IFN-gamma and T cell killing in non-small cell lung cancer

BackgroundKRAS-mutant non-small cell lung cancers (NSCLC) have exhibited unique response patterns to immunotherapy based on their co-occurring mutations. Patients harboring KRAS & STK11/LKB1 co-mutations (KL) have experienced shorter progression-free and overall survival compared to those with only KRAS mutations (K). Despite their limited responses, KL tumors exhibit a tumor mutational burden comparable to their K counterparts, suggesting the presence of additional mechanisms impairing antigen-specific responses. Accordingly, here we investigated the role of the MHC I antigen processing and presentation pathway in KL tumors.MethodsTCGA lung adenocarcinoma (LUAD) data were investigated for changes in expression of HLA molecules and chaperones involved in antigen processing and presentation. In mice, we performed single cell RNA sequencing of resected LKR13 K and KL tumors to evaluate changes in the tumor microenvironment and intrinsic differences in tumor antigen processing machinery. In vitro experiments were performed using the ovalbumin antigen to evaluate changes in antigen-specific T cell responses.ResultsExpression of HLA-A (p<0.0001), -B (p<0.0001), -C (p<0.0001), and beta2-microglobulin (B2M, p<0.0002) was downregulated in KL tumors from TCGA, as were expression of the TAP1 (p<0.001) and TAP2 (p<0.001) transporter associated with antigen processing subunits. LKR13 KL tumors exhibited similar patterns with lower H2-k1 (p<0.0001), H2-d1 (p<0.0001), B2m (p<0.0001), Tap1 (p<0.0001) and Tap2 (p<0.0001). As a result, LKR13 KL were resistant to recognition (p<0.005) and killing (56.9% K versus 7.8% KL) by OT-I T cells. Decreased expression of IFN-gamma-regulated genes such as PSMB8 (p<0.001), PSMB9 (p<0.0001), PSMB10 (p<001), CIITA (p<0.0001), NLRC5 (p<0.0001), IFNGR1 (p<0.0001), and IFNGR2 (p<0.0001) was also noted in KL tumors. Accordingly, KL tumors were unresponsive to exogenous IFN-gamma stimulation, maintaining repression of surface H2-Kb and resistance to T cell recognition (p<0.05) and killing (12.8% K versus 4% KL). Expression of T cell chemokines and receptors CXCR3 (p<0.0001), CXCL9 (p<0.0001), and CXCL10 (p<0.0001) was also repressed, potentially contributing to the lack of T cell infiltration in KL tumors.ConclusionsKRAS-mutant tumors harboring STK11/LKB1 alterations have an immunosuppressed phenotype and resistance to PD-1/PD-L1 inhibitors. Our findings provide evidence that these alterations are associated with markedly reduced antigen presentation and resistance to T cell killing, responsiveness to IFN-gamma stimulation, and impaired production of T cell chemokines, providing mechanistic insights into this immunosuppressed phenotype that could help guide the development of new therapeutic strategies for enhancing anti-tumor immunity.