BackgroundClassical human leukocyte antigen (HLA) class I molecules are expressed by most somatic cells and present peptides to cytotoxic T cells. The HLA-genotype of an individual contains up to six different HLA-I molecules and defines the repertoire of peptides that can be presented to cytotoxic T cells. Homozygosity for one or more HLA-loci could translate in a smaller repertoire of tumour neoantigens possibly presented to cytotoxic T cells in an individual and potentially predispose such individuals with a disadvantage to fight a nascent tumour.Material and MethodsHigh-resolution HLA-genotyping from germline normal DNA of 80 esophago-gastric adenocarcinoma (EGA) patients was performed with the NGS method by Illumina. Whole exome sequencing (WES) was performed on tumor tissue and normal peripheral blood cells (n=39). The data were processed, and non-synonymous mutations were called. The amount of potential high-affinity binders derived from 10 cancer testis antigens (CTAs) frequently expressed in EGA and non-synonymous mutations obtained from WES data were determined using an in-silico approach for MHC-binding (IEDB.org). RNA-extraction and gene expression profiling were performed using the NanoString technology.ResultsWe compared the frequency of HLA homozygosity in EGA patients to an HLA-matched reference population derived from a large cohort of bone marrow donors (n=7.615 out of 615.017 donors). We demonstrate that EGA patients are more likely to be homozygous for at least one HLA-I gene than the control population. In EGA patients, 35% of HLA-A, -B, and -C alleles were homozygous in comparison with 19% of HLA alleles among the HLA-matched general population. This difference corresponded to an odds ratio (OR) for homozygosity of 2.282 (95% confidence interval (CI) 1.442-3.615, p<0.001). The odds ratios for homozygosity at HLA-A (OR=1.885, CI=1.111-3.236, p<0.05), HLA-B (OR=3.045, CI=1.346-6.499, p<0.05) and HLA-C (OR=2.170, CI=1.445-3.579, p<0.05) were significantly different. We then aimed to estimate the influence of HLA-homozygosity in the context of tumour immune surveillance. Predictions by IEDB analysis resource tool indeed showed a reduced repertoire of high and moderate-affinity MHC-binders (both CTA-derived and mutation-derived peptides) in the homozygous cohort. Our findings demonstrate a reduced amount of potentially immunogenic peptides in EGA patients with HLA-homozygosity for at least one locus, which may result in impaired cancer immunosurveillance. In line with this observation, we also found increased levels of CTA expression in homozygous compared to heterozygous patients. After artificial modification of the genotype of homozygous patients to a heterozygous genotype, the set of predicted good-binding peptides was comparable to the heterozygous cohort.ConclusionOur results highlight the effect of HLA-I homozygosity on the immunopeptidome as important prerequisite of anti-tumor immunity. The high frequency of genomic HLA-I homozygosity observed in the EGA cohort may reflect an increased cancer risk for these patients. Together with previous reports demonstrating reduced survival after checkpoint therapy, our study suggests consideration of germ-line HLA-homozygosity for the design and interpretation of immunotherapeutic trials.Disclosure InformationM.A. Garcia-Marquez: None. M. Thelen: None. E. Bauer: None. K. Wennhold: None. J. Lehmann: None. D. Keller: None. B. Gathof: None. L. Maas: None. J. George: None. C. Bruns: None. A. Quaas: None. M. von Bergwelt-Baildon: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. M. Peifer: None. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS.
were analysed using the CEL-seq2 platform. Interactions between tumour and immune cells were predicted using an unbiased ligand-receptor interaction analysis. Proteins secreted by tumour cells were analysed by performing mass spectrometry on conditioned medium from patient derived organoid cultures. The conditioned medium was concentrated using a 3kDa Millipore filter and prepared for LC-MS. Mass spectrometry data were acquired in data-dependent acquisition mode. For determining suppression, healthy donor PBMCs were stimulated with anti-CD28/anti-CD3 Dynabeads in the presence of recombinant proteins or concentrated conditioned medium from neuroblastoma organoids. In vitro killing assays were performed with GFP and luciferase transduced organoids and healthy donor PBMCs. Results Analysis of scRNA-seq of 25 neuroblastoma tumours showed a negative correlation between MIF and MDK expression and the cytotoxicity of NK cells, CD8 and gd-T cells within the tumour. These findings could be confirmed with a previously published SEQC bulk-RNAseq dataset containing 498 patients. Next to that, a higher expression of MIF and MDK correlated with poor survival in the same dataset. In the secretome from cultured neuroblastoma organoids, we have used mass spectrometry to identify MIF and MDK amongst the top 100 most abundant proteins from a total of ~1200 proteins. In vitro, we showed that rMIF and rMDK have a suppressive effect on the activation of T cells and the amount of cytotoxic factors such as granzyme B and Perforin are produced by the T cells. This confirms our hypothesis that MIF and MDK negatively influence the cytotoxicity of T cells. Conclusions Using two different unbiased analyses -scRNA-seq analysis of tumours and mass spectrometry of neuroblastoma organoid secretome-, we have identified MIF and MDK as immunosuppressive factor in neuroblastoma. Currently, we are testing several MIF and MDK inhibitors to test if T cell mediated killing of neuroblastoma can be increased if the immunosuppressive MIF and MDK are blocked.
BackgroundSecondary lymphoid organs (SLO) are involved in induction and enhancement of anti-tumor immune responses on different tumor entities. Recent evidence suggests that anti-tumor immune responses may also be induced or enhanced in the tumor microenvironment in so called tertiary lymphoid structures (TLS). It is assumed that TLS represent a hotspot for T cell priming, B cell activation, and differentiation, leading to cellular and humoral anti-tumor immune response.MethodsFFPE-slides of 120 primary pancreatic ductal adenocarcinoma (PDAC) patients were immunohistochemically (IHC) stained for CD20, CD3, CD8 and HLA-ABC to analyze spatial distribution of tumor-infiltrating lymphocytes. 5-color immunofluorescence staining was performed to further investigate structural components of TLS in comparison to lymphoid follicles in SLOs. Microscope-based laser microdissection and Nanostring-base RNA expression analysis were used to compare gene expression in PDAC, TLS, SLOs and normal pancreatic tissue.ResultsTLS were frequently detected in PDAC and were mainly localized along the invasive tumor margin. In less than 10% of the cases TLS were infiltrating the tumors. Interestingly, 20% of the patients had no TLS. Results of TLS will be correlated with clinical parameters, Immunoscore and immune escape mechanisms. 5-color Immunofluorescence staining revealed similar organization and function of TLS and SLO. Finally, gene expression analyzed by Nanostring revealed largely overlapping expression patterns in TLS and SLO.ConclusionsThe results clearly demonstrate close similarities between SLO and TLS in terms of composition, distribution and gene expression Patterns.Disclosure InformationM. Thelen: None. M.A. García-Márquez: None. T. Nestler: None. S. Wagener-Ryczek: None. J. Lehmann: None. E. Staib: None. F. Popp: None. F. Gebauer: None. P. Lohneis: None. M. Odenthal: None. S. Merkelbach-Bruse: None. C. Bruns: None. K. Wennhold: None. M. von Bergwelt-Baildon: None. H.A. Schlößer: None.
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