Upregulation Of HLA Class Research Articles

Abstract 5540: Single cell multiomic analysis of human brain metastasis reveals conserved dormancy population

Abstract This study explores the complex realm of brain metastasis, pivotal in crafting effective anti-tumor strategies. Leveraging single-cell multi-omics sequencing, we profiled the transcriptomes and chromatin accessibility landscapes of tumor cells and their surrounding microenvironment in human brain metastases that originated from across a spectrum of primary cancers. Our investigations have identified distinct, yet uniformly present, tumor cell subpopulations, each defined by unique gene expression patterns, chromatin accessibility, and the interactions between tumor and stromal cells. Notably, one such subpopulation exhibits a dormancy signature, characterized by enhanced interferon response, oxidative phosphorylation, and upregulated HLA class molecules, alongside alterations in circadian rhythms. Our trajectory analysis charts a course from these dormant cells to rapidly proliferating ones, with notable shifts in circadian rhythm genes, underscoring their potential role in brain metastasis. This aligns with recent findings that disruptions in circadian rhythms can modify stem cell characteristics in glioblastomas. This insight led us to focus on a specific circadian rhythm gene, highly active in the dormant cells and located on the HER2 amplicon—an area linked to high brain metastasis relapse rates in patients. We've developed inducible models for both overexpression and knockdown to scrutinize this gene's impact in her2 positive breast cancer cell lines, examining gene expression and proliferation in vitro. Complementing this, we are conducting spatial transcriptomic analysis to evaluate the microenvironmental regulation of tumor dormancy in human brain metastases. This multi-omics approach reveals a nuanced regulatory landscape of brain metastasis, highlighting a novel and often overlooked factor in metastasis progression and brain tumor relapse: the interplay between circadian rhythms and tumor dormancy. This discovery not only deepens our understanding of brain metastases but also opens potential new avenues for therapeutic interventions. Citation Format: Remi Klotz, Yiru Wang, Anu Sunkara, Frank Attenelo, Min Yu. Single cell multiomic analysis of human brain metastasis reveals conserved dormancy population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5540.

Cancer cell immunity-related protein co-expression networks are associated with early-stage solid-predominant lung adenocarcinoma.

Solid-predominant lung adenocarcinoma (SPA), which is one of the high-risk subtypes with poor prognosis and unsatisfactory response to chemotherapy and targeted therapy in lung adenocarcinoma, remains molecular profile unclarified. Weighted correlation network analysis (WGCNA) was used for data mining, especially for studying biological networks based on pairwise correlations between variables. This study aimed to identify disease-related protein co-expression networks associated with early-stage SPA. We assessed cancerous cells laser-microdissected from formalin-fixed paraffin-embedded (FFPE) tissues of a SPA group (n = 5), referencing a low-risk subtype, a lepidic predominant subtype group (LPA) (n = 4), and another high-risk subtype, micropapillary predominant subtype (MPA) group (n = 3) and performed mass spectrometry-based proteomic analysis. Disease-related co-expression networks associated with the SPA subtype were identified by WGCNA and their upstream regulators and causal networks were predicted by Ingenuity Pathway Analysis. Among the forty WGCNA network modules identified, two network modules were found to be associated significantly with the SPA subtype. Canonical enriched pathways were highly associated with cellular growth, proliferation, and immune response. Upregulated HLA class I molecules HLA-G and HLA-B implicated high mutation burden and T cell activation in the SPA subtype. Upstream analysis implicated the involvement of highly activated oncogenic regulators, MYC, MLXIPL, MYCN, the redox master regulator NFE2L2, and the highly inhibited LARP1, leading to oncogenic IRES-dependent translation, and also regulators of the adaptive immune response, including highly activated IFNG, TCRD, CD3-TCR, CD8A, CD8B, CD3, CD80/CD86, and highly inhibited LILRB2. Interestingly, the immune checkpoint molecule HLA-G, which is the counterpart of LILRB2, was highly expressed characteristically in the SPA subtype and might be associated with antitumor immunity. Our findings provide a disease molecular profile based on protein co-expression networks identified for the high-risk solid predominant adenocarcinoma, which will help develop future therapeutic strategies.

Sensitizing cancer cells to immune checkpoint inhibitors by microbiota-mediated upregulation of HLA class I.

Recent data have shown that gut microbiota has a major impact on the clinical response to immune checkpoint inhibitors (ICIs) in the context of solid tumors. ICI-based therapy acts by unlocking cognate cytotoxic T lymphocyte (CTL) effector responses, and increased sensitivity to ICIs is due to an enhancement of patients' tumor antigen (TA)-specific CTL responses. Cancer clearance by TA-specific CTL requires expression of relevant TAs on cancer cells' HLA class I molecules, and reduced HLA class I expression is a common mechanism used by cancer cells to evade the immune system. Here, we show that metabolites released by bacteria, in particular, phytosphingosine, can upregulate HLA class I expression on cancer cells, sensitizing them to TA-specific CTL lysis invitro and invivo, in combination with immunotherapy. This effect is mediated by postbiotic-induced upregulation of NLRC5 in response to upstream MYD88-NF-κB activation, thus significantly controlling tumor growth.

Brachyury-targeted immunotherapy combined with gemcitabine against head and neck cancer.

Brachyury is a transcription factor belonging to the T-box gene family and is involved in the posterior formation of the mesoderm and differentiation of chordates. As the overexpression of Brachyury is a poor prognostic factor in a variety of cancers, the establishment of Brachyury-targeted therapy would be beneficial for the treatment of aggressive tumors. Because transcription factors are difficult to treat with a therapeutic antibody, peptide vaccines are a feasible approach for targeting Brachyury. In this study, we identified Brachyury-derived epitopes that elicit antigen-specific and tumor-reactive CD4+ T cells that directly kill tumors. T cells recognizing Brachyury epitopes were present in patients with head and neck squamous cell carcinoma. Next, we focused on gemcitabine (GEM) as an immunoadjuvant to augment the efficacy of antitumor responses by T cells. Interestingly, GEM upregulated HLA class I and HLA-DR expression in tumor, followed by the upregulation of anti-tumor T cell responses. As tumoral PD-L1 expression was also augmented by GEM, PD-1/PD-L1 blockade and GEM synergistically enhanced the tumor-reactivity of Brachyury-reactive T cells. The synergy between the PD-1/PD-L1 blockade and GEM was also confirmed in a mouse model of head and neck squamous cell carcinoma. These results suggest that the combined treatment of Brachyury peptide with GEM and immune checkpoint blockade could be a promising immunotherapy against head and neck cancer.

Pan-Cancer HLA Gene-Mediated Tumor Immunogenicity and Immune Evasion.

Taken together, we highlight the prognostic potential of HLA genes in immunotherapies and suggest that higher tumor immunogenicity mediated by HLA expression may sometimes lead to tumor escape under strong selective pressure.

HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia/lymphoma.

Human T cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and induces chronic, persistent infection in infected individuals, with some developing adult T cell leukemia/lymphoma (ATL). HTLV-1 alters cellular differentiation, activation, and survival; however, it is unknown whether and how these changes contribute to the malignant transformation of infected cells. In this study, we used single-cell RNA-sequencing and T cell receptor-sequencing to investigate the differentiation and HTLV-1-mediated transformation of T cells. We analyzed 87,742 PBMCs from 12 infected and 3 uninfected individuals. Using multiple independent bioinformatics methods, we demonstrated the seamless transition of naive T cells into activated T cells, whereby HTLV-1-infected cells in an activated state further transformed into ATL cells, which are characterized as clonally expanded, highly activated T cells. Notably, the greater the activation state of ATL cells, the more they acquire Treg signatures. Intriguingly, the expression of HLA class II genes in HTLV-1-infected cells was uniquely induced by the viral protein Tax and further upregulated in ATL cells. Functional assays revealed that HTLV-1-infected cells upregulated HLA class II molecules and acted as tolerogenic antigen-presenting cells to induce anergy of antigen-specific T cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at the single-cell level.

A Novel Strategy for Off-the-Shelf T Cell Therapy Which Evades Allogeneic T Cell and NK Cell Rejection

Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of “off-the-shelf” T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a “missing-self” signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed.Methods. Here, we developed an immunologically stealth “off-the-shelf” T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo.Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an “off-the-shelf” CAR or TCR-T cell solution for a large proportion of the population.Conclusions. We have successfully developed a differentiated “off-the-shelf” approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. DisclosuresZhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

Synergistic Role of Leukemic and Non-Leukemic Immune Repertoires in CD8+ T-Cell Large Granular Lymphocytic Leukemia As Identified By Single-Cell Transcriptomics

Background: T-cell large granular lymphocytic leukemia (T-LGLL), a rare lymphoproliferative disorder of mature T cells, is characterized by the accumulation of activated effector T cells leading to a clonally restricted T-cell receptor (TCR) repertoire. Chronic antigen stimulation together with activating somatic STAT3 mutations have been proposed to lead to clonal expansion of leukemic cells. However, no holistic research has been done to show how leukemic and non-leukemic cells liaise to sustain abnormal immune reactivity in T-LGLL.Methods: We investigated the transcriptome and TCR repertoire in T-LGLL using: 1) single-cell RNA and TCR (scRNA+TCRαβ) sequencing from CD45+ sorted blood cells (T-LGLL n=11, healthy n=6), 2) TCRβ sequencing from blood mononuclear cells (T-LGLL n=48, healthy n=823), 3) bulk RNA sequencing (T-LGLL n=15, healthy n=5), 4) plasma cytokine profiling (T-LGLL n=9, healthy n=9), and 5) flow cytometry validations (T-LGLL n=6, healthy n=6) (Figure)Results: ScRNA+TCRαβ-seq data revealed that in healthy controls, hyperexpanded CD8+ T-cell clones (at least 10 cells with identical TCRs) preferentially had an effector memory phenotype, whereas in T-LGLL, the hyperexpanded clonotypes represented a more cytotoxic (increased expression of GZMB, PRF1, KLRB1) and exhausted (LAG3 and TIGIT) phenotype. Using flow cytometry, we confirmed that upon anti-CD3/CD28/CD49 antibody stimulation, T-LGLL clones (CD8+CD57+) expressed higher levels of cytotoxic proteins (GZMA /GZMB , PRF1) but were deficient in degranulation responses and cytokine secretion as measured by expression of CD107a/b and TNFα/IFNγ, respectively.Focused re-clustering of the extracted T-LGLL clones from the scRNA+TCRαβ-seq data revealed considerable heterogeneity among the T-LGLL clones and partly separated the mutated (mt) STAT3 and wild type (wt) STAT3 clones. STAT3wt clones upregulated T-cell activation and TCR signaling pathways, with a higher cytotoxicity and lower exhaustion score as compared to STAT3mt clones. This was validated with bulk RNA-seq data.To understand the antigen specificities of the T-LGLL clones, we combined previously profiled T-LGLL TCRs with our data to form the largest described dataset of 200 T-LGLL clones from 170 patients. Notably, T-LGLL clones were found to be private to each patient. Furthermore, the analysis by GLIPH2 algorithm grouping TCRs did not reveal detectable structural similarities, suggesting the absence of a unifying antigen in T-LGLL. However, in 67% of T-LGLL patients, the TCRs of leukemic clones shared amino acid level similarities with the rest of the non-leukemic TCR repertoire suggesting that the clonal and non-clonal immune repertoires are connected via common target antigens.To analyze the non-clonal immune repertoire in T-LGLL in detail, we compared our data to other published scRNAseq data from solid tumors (n=4) and hematologic cancers (n=8) and healthy controls (n=6). The analysis revealed that in T-LGLL also the non-leukemic CD8+ and CD4+ T cells were more mature, cytotoxic, and clonally restricted. When compared to healthy controls and other cancer patients, in non-leukemic T-LGLL the most upregulated pathway was IFNγ response.Finally, most of the upregulated cytokines in T-LGLL (e.g., CCL2/3/7, CXCL10/11, IL15RA) were secreted predominantly by monocytes and dendritic cells, which also had upregulated HLA class II expression and enhanced scavenging potential in T-LGLL patients. Ligand-receptor analysis with CellPhoneDB revealed that the number of predicted cell-cell interactions was significantly higher in T-LGLL as compared to reactive T-cell clones in healthy controls. The most co-stimulatory interactions (e.g., CD2-CD58, TNFSF14-TNFRSF14) occurred between the IFNγ secreting T-LGLL clones and the pro-inflammatory cytokine secreting monocytes.Conclusions: Our study shows a synergistic interplay between the leukemic and non-leukemic immune cell repertoires in T-LGLL, where an aberrant antigen-driven immune response including hyperexpanded CD8+ T-LGLL cells, non-leukemic CD8+ cells, CD4+ cells, and monocytes contribute to the persistence of the T-LGLL clones. Our results provide a rationale to prioritize therapies that target the entire immune repertoire and not only the T-LGLL clones in patients with T-LGLL. [Display omitted] DisclosuresLoughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Maciejewski: Alexion: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Bristol Myers Squibb/Celgene: Consultancy. Mustjoki: Novartis: Research Funding; BMS: Research Funding; Janpix: Research Funding; Pfizer: Research Funding.

OAB-019: CRISPR screens with single-cell transcriptome readout reveal potential mechanisms of response to natural killer cell treatment in multiple myeloma

<h3>Background</h3> Natural killer (NK) cell-based therapy is a promising approach to improve treatment responses in multiple myeloma (MM), which remains incurable. However, genetic determinants and mechanisms of sensitivity or resistance to NK cells in MM are incompletely understood. Here, we investigated the transcriptional impact of genes identified as regulators of sensitivity to NK cells in genome-wide CRISPR screens and integrated the findings with patient-derived data. <h3>Methods</h3> To systematically dissect the mechanisms by which select genes identified in our previous genome-wide studies influence MM cell response to NK cells, we performed pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform. Based on genome-wide screen ranking and biological relevance, 31 genes were selected. Pools of MM1.S and LP1 expressing sgRNAs targeting select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection and assignment to cells, followed by differential gene-expression analysis and patient data correlation. <h3>Results</h3> The analysis of the transcriptomic responses in MM cells upon NK cell exposure revealed that engagement by NK cells resulted in activation of IFNy-JAK-STAT signaling and upregulation of HLA class I genes, including the inhibitory NKG2A ligand HLA-E, as well as HLA class II. We then evaluated the impact of each sgRNA perturbation on gene expression in each condition, compared to the untreated control. Silencing of IFNy signaling mediators (JAK1, JAK2, STAT1, IFNGR2) prevented the induction of the IFNy response upon NK cell exposure, providing a mechanism for the observed sensitization to NK cells via reduced expression of HLA I genes. Disruption of TRAF2, NFKBIA, or NFKBIB, which also sensitized MM1.S to NK cells, induced NF-kB signaling. The induced NF-kB signature included increased expression of the death receptor FAS, pivotal for susceptibility to NK cells in the MM1.S CRISPR screen. We then correlated the findings with patient data from the CoMMpass dataset. Mutations in the NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes also identified experimentally by CROP-seq. Also, NFKBIA mutations were linked to reduced HLA-E expression in both MM patients and CROP-seq, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. Interestingly, patients with NLRC5 mutations had lower HLA-E expression consistent with CROP-seq data, indicating that although rare, NLRC5 mutations may predispose MM cells to higher NK-sensitivity. <h3>Conclusions</h3> Our data shed light on mechanisms of response to NK cells in MM and identify potential therapeutic targets for combination treatment. Furthermore, patient data correlation highlights subgroups that might have an increased susceptibility to NK cell treatment, highlighting the potential of such studies in the identification of predictive biomarkers.

Upregulation of HLA class I and antiviral immune responses in graves’ disease

Upregulation of HLA class I and antiviral immune responses in graves’ disease

Upregulation of HLA-Class I and II in Placentas Diagnosed with Villitis of Unknown Etiology.

The placenta utilizes many mechanisms to protect the haploidentical fetus from recognition by the maternal immune system. However, in cases of villitis of unknown etiology (VUE), maternal lymphocytes gain access into the placenta, causing significant health risks for the fetus. Evidence suggests that VUE is a rejection response between the mother and the haploidentical fetus. Therefore, we profiled human leukocyte antigen (HLA), an important predictor of transplant rejection, in VUE using placental tissue from ten patients with VUE and ten gestational age matched controls. Placentas were stained using novel multiplexed immunofluorescence (MxIF) to investigate morphology and HLA classes I and II. Gene expression was evaluated by microarray, and where available, tissue typing of mother/baby pairs was completed to determine HLA type. MxIF demonstrated strong CD8+ T cell infiltration and HLA class I staining both the distal and stem villi of VUE placentas. Compared to controls, VUE cases had significantly higher expression of HLA class II mRNA and pathway analysis demonstrated that 40% of the differentially expressed genes in VUE are related to tissue rejection. The data suggest that VUE resembles a rejection response between the mother and the fetus. It remains unknown what initiates immune recognition and why some mothers appear to be at higher risk for developing this condition than others. Understanding this etiology will be critical for developing effective interventions or prevention strategies during pregnancy.

Gemcitabine enhances cytotoxic activity of effector T-lymphocytes against chemo-resistant cholangiocarcinoma cells

Cholangiocarcinoma (CCA) can resist chemotherapy resulting in treatment failure. Gemcitabine, a chemotherapeutic drug, can sensitize cancer cells to become susceptible to cytotoxic T-lymphocytes (CTLs). We, therefore, hypothesized that a combination of gemcitabine and CTLs would be more effective for CCA treatment than individual therapy. To test this hypothesis, we conducted an in vitro study using gemcitabine combined with CTLs to treat gemcitabine-resistant CCA (KKU-213) cells. KKU-213 cells were pretreated with gemcitabine and tested for killing by CTLs activated by dendritic cells that were prepared by three different methods, including: (i) monocyte-derived dendritic cells pulsed with cancer cell lysate (Mo-DC + Lys), (ii) self-differentiated dendritic cells pulsed with cancer-cell lysate (SD-DC + Lys), and (iii) SD-DC presenting tumor-associated antigen PRKAR1A (SD-DC-PR). KKU-213 cells pretreated with gemcitabine were killed by CTLs activated by either SD-DC + Lys or SD-DC-PR more efficiently than those activated by Mo-DC + Lys. Furthermore, KKU-213 cells pretreated with a low dose (2 µM) of gemcitabine significantly enhanced the cytotoxic activity of CTLs activated by either SD-DC + Lys or SD-DC-PR at all evaluated effector (E) to target cell (T) ratios. At an E:T ratio of 5:1, KKU-213 cells pretreated with gemcitabine enhanced the cytotoxic activity of CLTs by approximately 2.5-fold (greater than 50% cell death) compared to untreated condition. The upregulation of HLA class I upon pretreatment of KKU-213 cells with gemcitabine may suggest a mechanism that leads to alteration of the antigen presentation process to promote CTL functions. These findings support the concept of combination therapy for overcoming chemo-resistant CCA.

Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment

BackgroundPatient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment.MethodsFour microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS.ResultsWe identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment.ConclusionsOnly 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response.

Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

CIITA-related block of HLA class II expression, upregulation of HLA class I, and heterogeneous expression of immune checkpoints in hepatocarcinomas: implications for new therapeutic approaches

ABSTRACTHepatocellular carcinoma (HCC) is the second cause of death for cancer worldwide, justifying the urgent need for novel therapeutic approaches. Immunotherapeutic strategies based on triggering and/or rescuing tumor antigen-specific T cells may be promising particularly if combined together. As preliminary step toward this goal, we have investigated the expression of antigen presenting molecules (HLA class I and class II) and immune checkpoints (PD-1 and PD-L1) in 43 HCC samples from distinct patients and in HCC cell lines. While normal hepatocytes did not express HLA class I and II, HCC cells strongly upregulated HLA class I while remaining negative for HLA class II. The absence of HLA class II expression in HCC cell lines correlated with lack of expression of the HLA class II transactivator, CIITA, which could not be rescued even after interferon-gamma treatment. This was due to high methylation levels of interferon-gamma-sensitive CIITA promoter IV strongly suggesting a biologically relevant developmental silencing of HLA-II expression in liver cell lineage. HCC tumor tissues showed a variable degree of leukocyte infiltration. Infiltrating lymphocytes expressed PD-1, while PD-L1 was expressed in cells with monocyte-macrophage morphology mostly localized at the tumor margin, but not in tumor cells. De novo expression of HLA class I, instrumental for presenting tumor antigens to cytotoxic T lymphocytes, and the correct characterization of the cells expressing checkpoint inhibitors in the tumor tissue should be the ground for setting novel strategies of combined approaches of immunotherapy in HCC based on tumor peptide vaccines and anti-checkpoint inhibitor antibodies.

HLA Class I Antigen Expression in Conjunctival Melanoma Is Not Associated With PD-L1/PD-1 Status.

Antitumor T cells need expression of HLA class I molecules but can be inhibited by ligands such as programmed death ligand 1 (PD-L1). We determined expression and regulation of these molecules in human conjunctival melanoma (CM) samples, cell lines, and murine xenografts. Immunofluorescence staining was performed to examine the expression of HLA-A, HLA-B/C, and β-2-microglobulin (B2M) in 23 primary CM samples. HLA class I expression was compared with clinicopathologic characteristics, the presence of tumor-infiltrating leukocytes, and PD-L1/PD-1 status. The effect of interferon γ (IFN-γ) on HLA class I expression was tested on three CM cell lines using quantitative PCR and flow cytometry. Furthermore, HLA class I expression was determined in CM cell line-derived murine xenografts. One third of tumors had positive HLA-A, HLA-B/C, and B2M expression. A positive expression was especially seen in thin and epibulbar tumors but was not associated with recurrences. HLA class I expression was correlated with M2 macrophage density and tended to associate with CD8+ T-cell density but was independent of PD-L1 or PD-1 expression. IFN-γ upregulated HLA class I expression and genes involved in HLA transcription and transportation on CM cell lines. Murine xenografts showed a comparable HLA class I expression as their respective cell lines. Our data indicate that subsets of CM have positive HLA class I expression, and HLA class I and PD-L1/PD-1 are expressed independently. When one considers immunotherapy, one should also analyze HLA class I expression, whose downregulation can limit the efficacy of T cell-mediated therapies.

IL-27 mediates HLA class I up-regulation, which can be inhibited by the IL-6 pathway, in HLA-deficient Small Cell Lung Cancer cells

BackgroundRecently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families.MethodsThe human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student’s T test was used for the statistical analysis of experimental replicates.ResultsIL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation.ConclusionsIn conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ.

Upregulation of HLA Class I Expression on Tumor Cells by the Anti-EGFR Antibody Nimotuzumab

Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor – T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.

20 IFN[formula omitted]-induced PD-L1 expression is JAK2 but not JAK1 dependent and its inhibition enhances NK-cetuximab mediated ADCC of HNSCC cells

Programmed death ligand 1 (PD-L1) is an immunosuppressive molecule expressed by many cancer types, including a large proportion of head and neck cancers (HNC), and ligation of its receptor, programmed death 1 (PD-1), induces exhaustion of effector T cells. It has been shown that interferon gamma (IFN γ ) induces PD-L1 expression in many cancer types including glioblastoma, melanoma, lung and kidney cancer. Importantly, the stimuli and mechanism for PD-L1 upregulation in HNC cells are not well characterized. IFN γ signals through Janus Kinase 1/2 (JAK1/2) heterodimer complex and mediates signal transducer and activator of transcription 1 (STAT1) phosphorylation, leading to type I cytokine expression, upregulation of antigen presentation, and tumor cell recognition by cytolytic T lymphocytes (CTL). We investigated basal PD-L1 expression and the mechanism by which IFN γ signaling upregulates PD-L1 in HNC cells including dependence on JAK/STAT pathway. We observed that IFN γ signaling increased PD-L1 expression in a JAK2 but not JAK1 dependent fashion. In addition, interferon alpha (IFN α ), which signals via JAK1/TYK2 did not upregulate PD-L1 expression while still upregulated HLA class I. Specific JAK2 inhibition downregulated NK cell-derived IFN γ induced PD-L1 expression and enhanced cetuximab mediated ADCC. Our data suggest a crucial role for JAK2/STAT1 in IFN γ mediated PD-L1 upregulation. JAK2 inhibition provides a promising strategy to increase tumor cell lysis through maintaining HLA class I while suppressing tumor cell expressed PD-L1 in combination with anti-EGFR cetuximab therapy.

Specific PML Isoforms and SATB1 Affect HLA Expression In Classical Hodgkin Lymphoma

IntroductionHodgkin Reed-Sternberg (HRS) cells are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I and class II are expressed in most EBV+ cHL cases, with an even enhanced expression in some cases. The mechanisms underlying enhanced or lost HLA expression are unknown. Promyelocytic leukemia protein (PML) is the main component of nuclear bodies (NBs) and it organizes the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. The PML transcript can be processed into seven different isoforms. Downregulation of PML isoform III or V results in a reduced HLA-A and HLA-G expression in Jurkat T cells, whereas PML enhances HLA class II expression by upregulating the class II transactivator (CIITA). Special AT-rich region binding protein 1 (SATB1) has been shown to be associated with PML-NBs in the HLA region. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G and HLA-H. AimTo determine if specific PML isoforms and SATB1 regulate HLA expression in cHL cell lines. MethodsWe stained HLA class I, HLA class II, PML-NBs and SATB1 in 54 EBV+ cHL cases and in 27 EBV- cHL cases. We analyzed HLA class I, HLA class II, PML isoforms I to V and SATB1 mRNA expression levels by qRT-PCR in cHL cell lines (n=6). Next, we inhibited SATB1 and PML expression in the cHL cell line L1236 using shRNA constructs. The effects of the shRNA constructs on HLA protein expression levels were assessed by Western blot and flow cytometry. ResultsWe observed a normal HLA class I membrane expression in 40% of EBV+ and 20% of EBV- cases. There was a stronger than normal HLA class I protein expression in approximately 40% of EBV+ cHL and not in EBV- cHL patients. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01), whereas the percentage of SATB1 positive cells was inversely correlated with the level of HLA class I expression in EBV+ cHL cases (p<0.05). We observed normal HLA class II expression in 50% of EBV+ and 50% of EBV- cases. A stronger than normal HLA class II expression was observed in 4 EBV+ and 1 EBV- cHL patients. The number of PML-NBs and the percentage of SATB1 positive cells were not correlated with classical HLA class II expression in EBV+, EBV- or the total cHL patient group.We further investigate the association of PML, SATB1 and HLA class I and II in six cell lines. At the mRNA level, significant positive correlations were found between PML-III and HLA-C and between PML-III and β2M. Borderline significant positive correlations were found between PML-V and CIITA and between PML-V and HLA-DPB1. Suggestive, though not significant, correlations were observed of various PML isoforms and SATB1 for several HLA genes. The limited number of cell lines, precluded more definitive conclusions for the other comparisons.To further study the role of specific PML isoforms and SATB1 in regulation of HLA gene expression, we inhibited PML-III, PML-V or SATB1 in L1236 by shRNA-based knockdown. The PML-III shRNA induced a ∼80% reduction at the PML-III mRNA level, but did not affect membranous HLA class I expression and possibly affected HLA class II expression levels in L1236. The shRNA for PML-V was not effective and needs to be optimized and experiments with a pan-PML shRNA are ongoing. The SATB1 shRNA1 induced a ∼75% at the SATB1 mRNA level and a ∼80% reduction at the protein level, while SATB1 shRNA2 showed a ∼75% reduction and a ∼25% reduction, respectively. Inhibition of SATB1 by shRNA1 significantly upregulated HLA class II expression (P=0.02) and no significant effect on HLA class I expression. Inhibition of SATB1 by shRNA2 showed no significant effect on HLA class I and II expression. ConclusionPML and SATB1 protein levels are associated with HLA class I in HRS cells. Various negative associations of SATB1 with HLA genes at the mRNA level support a possible regulatory effect. This was indeed observed upon inhibition of SATB1 in L1236 that induced enhanced HLA-DR/DP/DQ expression. The PML regulation appears to be complex with different PML isoforms showing different associations with specific HLA genes. Disclosures:No relevant conflicts of interest to declare.