Copy Neutral LOH Affecting the Entire Chromosome 6 is a Frequent Mechanism of HLA Class I Alterations in Cancer

Total or partial loss of HLA class I antigens reduce the recognition of specific tumor peptides by cytotoxic T lymphocytes favoring cancer immune escape during natural tumor evolution. These alterations can be caused by genomic defects, such as loss of heterozygosity at chromosomes 6 and 15 (LOH-6 and LOH-15), where HLA class I genes are located. There is growing evidence indicating that LOH in HLA contributes to the immune selection of HLA loss variants and influences the resistance to immunotherapy. Nevertheless, the incidence and the mechanism of this chromosomal aberration involving HLA genes has not been systematically assessed in different types of tumors and often remains underestimated. Here, we used SNP arrays to investigate the incidence and patterns of LOH-6 and LOH-15 in a number of human cancer cell lines and tissues of different histological types. We observed that LOH in HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 was observed more often and was associated with homozygous HLA genotype and partial HLA loss of expression. We also discuss the immunologic and clinical implications of LOH in HLA on tumor clonal expansion and association with the cancer recurrence after treatment.

SARS-CoV-2 escape from cytotoxic T cells during long-term COVID-19

Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties, but the mode of selection underlying this process remains unclear. While escape from humoral immunity certainly plays a role in intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin’s lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented binding of known immunogenic SARS-CoV-2 HLA class I antigens, suggesting that virus immunoediting is largely driven by cytotoxic CD8 T cell clones. The two changes with the strongest effect, nsp3:T504A and nsp3:T504P, were experimentally assessed in a cytotoxic assay of the patient's CD8 T cells. Both these changes were associated with immune escape, with a stronger effect observed for nsp3:T504P, the change which ultimately got fixed. Together, these results suggest that CD8 T cell escape may be an underappreciated contributor to SARS-CoV-2 evolution in humans.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

T Cell Positive B Cell Negative Flow Cytometry Crossmatch (FCXM): Frequency, HLA-Locus Specificity, and Mechanisms Among 3073 Clinical FCXM Tests

Background. A T cell positive and B cell negative (T+B-) flow cytometry crossmatch (FCXM) result remains a conundrum since HLA-class I antigens are expressed on both T and B cells. We investigated the frequency, HLA specificity of the antibodies and mechanisms for the T+B- FCXM result. Methods. We analyzed 3073 clinical FCXM tests performed in an American Society of Histocompatibility and Immunogenetics accredited histocompatibility laboratory. The sera associated with the T+B- FCXM were also tested for donor HLA IgG antibodies using LABScreen single antigen assays. Results. Among the 3073 FCXM tests, 1963 were T-B-, 811 were T-B+, 274 were T+B+, and 25 were T+B-. IgG antibodies directed at donor HLA-A, B, or Cw locus determined antigens (DSA) were identified in all 25 sera and the summed mean fluorescence intensity (MFI) of DSA ranged from 212 to 53,187. Correlational analyses identified a significant association between the summed MFI of class I DSA, and the median channel fluorescence (MCF) of T cells treated with the recipient serum (Spearman rank correlation, rs=0.34, P=0.05) but not with the MCF of B cells (rs=0.23, P=0.24). We identified that differential binding of anti-HLA antibodies to T cells and B cells and the B cell channel shift threshold used to classify a B cell FCXM are potential contributors to a T+B- FCXM result. Conclusions. Our analysis of 3073 FCXM, in addition to demonstrating that HLA antibodies directed at HLA-A, B or Cw locus are associated with a T+B- result, identified mechanisms for the surprising T+B- FCXM result.

Differential immunopeptidome analysis revealed cancer specific amino acid usage of HLA class-I antigens and novel neoantigens of colorectal cancer

Knowing the nature of human leukocyte antigen (HLA) peptides, also called as immunopeptides, is indispensable to realize the cancer precision medicine such as cancer vaccination and the better prediction of efficacy for immunocheckpoint inhibitor (ICI) treatment. Direct interrogation of immunopeptides by mass spectrometry (MS) is of great use for that purpose but the reality in analyses of scarce tissue samples still confronts technical challenges. To elucidate characteristics of HLA class-I immunopeptides specifically presented on colorectal cancer (CRC), the optimized immunopeptide isolation method and differential ion mobility mass spectrometry (DIM-MS) with whole exome sequencing (WES)-based personalized database were established and subjected to differential analysis of tumor or normal tissues of CRC patients. From pilot experiments using 108 cells of colon cancer cell line HCT116, total 9,249 unique immunopeptides, including total 11 neoantigens, were identified. Next, approximately 40 mg of tumor or normal regions of CRC tissues were collected from 17 patients and analyzed by our personalized immunopeptidomic technology. As the result, 44,785 unique immunopeptides were profiled, in which 2 neoantigens carrying the mutation KRAS-G12V or CPPED1-R228Q were identified. Interestingly, specific amino acid usage of C-terminus trimming of immunopeptides was found in tumor-exclusive immunopeptides. Thus, our personalized immunopeptidome analysis significantly expands presented antigen knowledgebase and allows direct determination of neoantigens from scarce tissue specimens. This advanced immunopeptidomics holds promise to new outlook of research in immunopeptides both from basic and clinical aspects.

Peculiarities of distribution of HLA class I antigens in patients with lihen planus

The article presents data on the distribution characteristics of the HLA class I system antigens in patients with lichen planus. Aim. To study the patterns of distribution of HLA class I antigens in the general group. To establish the presence of an association of the disease with antigens of the HLA class I. Material and methods. Laboratory analysis of the distribution of HLA class I antigens was carried out in 60 patients with various forms of lichen planus who consider themselves Russian on the basis of linguistic and ethnicity. The duration of the disease is 120 years. Results. When analyzing the typing results in the general group of patients; a tendency to negative association of HLA-A11 and HLA-B7. It was found that the frequency of haplotype combinations A1-B8; A2-B27; A2-B40; A3-B35 significantly exceeded that of healthy people. Analysis of the frequency of phenotypic combinations revealed a significant increase in A3-A19 and B12-B35. Conclusion. Haplotype and phenotypic combinations of HLA A1-B8; A2-B27; A2-B40; A3-B35; A3-A19; B12-B35 are provoking factors in the development of various forms of the disease. The presence of these genetic traits in the individual increases the risk of developing lichen planus by 311 times. In turn; the HLA-A11 and B7 antigens play a protective role.

HDAC Inhibition Increases HLA Class I Expression in Uveal Melanoma

The treatment of uveal melanoma (UM) metastases or adjuvant treatment may imply immunological approaches or chemotherapy. It is to date unknown how epigenetic modifiers affect the expression of immunologically relevant targets, such as the HLA Class I antigens, in UM. We investigated the expression of HDACs and the histone methyl transferase EZH2 in a set of 64 UMs, using an Illumina HT12V4 array, and determined whether a histone deacetylase (HDAC) inhibitor and EZH2 inhibitor modified the expression of HLA Class I on three UM cell lines. Several HDACs (HDAC1, HDAC3, HDAC4, and HDAC8) showed an increased expression in high-risk UM, and were correlated with an increased HLA expression. HDAC11 had the opposite expression pattern. While in vitro tests showed that Tazemetostat did not influence cell growth, Quisinostat decreased cell survival. In the three tested cell lines, Quisinostat increased HLA Class I expression at the protein and mRNA level, while Tazemetostat did not have an effect on the cell surface HLA Class I levels. Combination therapy mostly followed the Quisinostat results. Our findings indicate that epigenetic drugs (in this case an HDAC inhibitor) may influence the expression of immunologically relevant cell surface molecules in UM, demonstrating that these drugs potentially influence immunotherapy.

Successful Clearance of Donor Specific Anti-HLA Antibodies after Desensitization with Rituximab, Velcade and Plasma-Exchange Followed By a Haploidentical Stem Cell Transplantion with Post-Transplantation Cyclophosphamide

Background: T cell - replete haploidentical stem cell transplantation (haploSCT) is increasingly performed and has expanded donor pool and transplant option for many patients (pt). However, the presence of recipient antibodies against donor HLA antigens (DSA) have been reported to be associated with engraftment failure and may limit the access to transplantation as the only lifesafing treatment modality. Guidelines for detection and treatment of DSA have been recently published but no standardized desensitization schedule exists so far. We report here the results of our institutional desensitization procedure initially developed for highly immunized patients with severe sickle cell disease undergoing haploSCT and then further adapted to all pt with DSA undergoing a mismatched allogeneic SCT. Methods: From March 2014 to July 2019, 20 pt had detectable DSA and did perform desensitization before undergoing a haploSCT. The DSA level was determined by using the LUMINEX technique (One Lamda, Inc). In case of positivity, the single antigen test was done to identify each class I and II HLA antibody-specificity. The values were expressed in mean fluorescence intensity (MFI). DSA were considered positive if the MFI value was ≥ 1000. The desensitization treatment included Rituximab 375 mg/m2, Velcade 1.3 mg/m2 and Plasma-Exchange followed by intravenous polyvalent immunoglobulins (Figure). DSA level controls were done routinely during desensitization, before starting the conditioning regimen and the day before graft injection. Pt who did not decrease DSA levels were not considered for haploSCT. Results: Median age was 61 years (range, 22-73). Diagnosis was acute myeloid leukemia in 6 pt, myelodysplastic / myeloproliferative syndrome in 10 pt, chronic lymphocytic leukemia in 1 pt and severe sickle cell disease in 3 pt. All donors were first-degree family members. Half of the pt were women [donor-recipient sex match: F/F=4; M/F=6; F/M=5; M/M=5pt]. The median MFI value before desensitization was 4700 (range, 1000-16000). Most pt (17) had DSAs against HLA class I antigens. Seventeen pt successfully decreased DSA levels to a median MFI value of 500 (range, 500-1200) and could proceed to haploSCT. They all engrafted and no DSA rebound was observed. One pt relapsed during the desensitization procedure and 2 pt with high DSAs above 10000 did not respond with remaining MFI of 16000 and 9500, respectively, and did not undergo haploSCT. One pt with CMML experienced primary graft failure for relapse and one pt with CLL did not engraft for unknown cause. Conclusions: The presence of DSA under 10000 should not be a barrier to transplantation. Our report shows that the hereby described desensitization schedule effectively cleared DSA allowing engraftment after haploSCT. Further studies are needed to determine the role of specificity and strength of DSA in order to better predict the likelihood of successful desensitization. Disclosures Harbi: Sanofi: Honoraria. Blaise:Jazz Pharmaceuticals: Honoraria.

844 Immunomodulatory activity of epigenetic drugs combinations in mesothelioma: laying the ground for new immunotherapeutic strategies

BackgroundGrowing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells.MethodsFour mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5.ResultsGuadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line.The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone.Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54).ConclusionsCombination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.

Potential Role of HLA Class I Antigens in the Glycolytic Metabolism and Motility of Melanoma Cells

Besides playing a crucial role in immune surveillance, human leukocyte antigens (HLA) possess numerous non-immune functions involved in cell communication. In the present study, screening of a panel of HLA class I- and HLA class II-specific monoclonal antibodies (mAbs) for their effects on the metabolism of human melanoma cells showed for the first time that the HLA-B,C-specific mAb B1.23.2 reduced the expression level of key glycolytic enzymes, but did not affect that of mitochondrial respiration effectors. As a result, the metabolism of melanoma cells shifted from a Warburg metabolism to a more oxidative phosphorylation. In addition, the HLA-B,C-specific mAb B1.23.2 downregulated the expression of glutamine transporter and glutaminase enzyme participating in the reduction of tricarboxylic acid cycle. The HLA-B,C-specific mAb B1.23.2-mediated reduction in energy production was associated with a reduction of melanoma cell motility. On the whole, the described results suggest that HLA class I antigens, and in particular the gene products of HLA-B and C loci play a role in the motility of melanoma cells by regulating their metabolism.