11393 (to ZS and JK), Marie Curie 749010 (to DXB), NIH grants R01GM100114 (to DSL), P50GM085273 (to the New Mexico Spatiotemporal Modeling Center), and P30CA118100 (to the UNM Comprehensive Cancer Center). DF and AJRH acknowledge financial support from the NWO-funded Netherlands Proteomics Centre through the National Road Map for Large-scale Infrastructures program X-Omics (project 184.034.019). ES and LB are supported by grants from INSERM, CNRS, Université de Nantes, FRM (DEQ20170839118), and Ligue Contre le Cancer AO GO2019 (Côtes d'Armor, Association pour la Recherche contre le Cancer (PJA20191209404). This work was realized in the context of the LabEx IGO program, which is supported by the French National Research Agency Investissements d'Avenir.

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γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire–phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.

Background: Treatment response to chemoradiotherapy and immune checkpoint blockade is limited in gastroesophageal adenocarcinoma (GEA). This is likely caused by the fact that the majority of GEAs, belonging to the chromosomally instable subgroup (70%), are often immune excluded. We hypothesize that the immune excluded state is caused by secretion of immune suppressive factors by the tumor cells, to protect themselves from immune attack. By identifying and targeting these factors we aim to increase immune infiltration and increase treatment response. Method: To better understand the drivers of immune exclusion in GEA, we optimized methods to analyze the secretome of freshly dissociated GEA tumor biopsies (n=36) by plating single cells in a 96 well plate for 48 hours and performing targeted proteomics on the supernatant (50ul) using the Olink Target 96 ImmunoOncology panel. Using the same panel, we also analyzed the secretomes of patient-derived organoids (n=7). In parallel the immune infiltrate was characterized immediately in freshly dissociated biopsies with 14 color flow cytometry, and in FFPE material through 6 opal multicolor immunohistochemistry to determine the spatial organization of the immune infiltrate. Results: We identified 62 factors in the panel that are secreted by at least 25% of the biposies. Pro- and anti-inflammatory factors were found to correlate frequently. Using ranked scores for the pro- and anti-inflammatory factors we were able to identify 7 patients with a predominantly inflamed secretome and 29 with a suppressive secretome. The inflamed secretomes are characterized by IFN-gamma, CXCL9-10-11, the presence of granzymes and higher CD8 T cell levels identified by flow cytometry, whereas tumors with dominant anti-inflammatory profile had lower CD8 higher CD4 T cell rates. Notably, the anti-inflammatory secretomes are characterized by an overall lack of pro-inflammatory factors. Among the factors that are most frequently detected (>85% of patients) are galectin-9, IL-8, VEGFA, HO-1 and CAIX, all factors with potential immune suppressive properties. These are also detected in the secretomes of the organoids, indicating direct tumor mediated suppressive effect in the GEA TME. Conclusion: The analysis revealed that GEAs secrete immune suppressive factors and that the state of the TME as influenced by the secretome is reflected in the immune infiltrate composition. We found that tumors lacking a CD8 T cell infiltrate showed an absence of pro-inflammatory factors and had a secretome predominantly made up of anti-inflammatory factors that were also secreted by organoids. Moving forward we aim to further identify GEA associated suppressive factors by using a broader panel to analyze the secretome. Additionally, functional studies will determine whether targeting these immune suppressive factors will facilitate responses to immune checkpoint blockade Citation Format: Jasper Sanders, Micaela H. Harrasser, Tessa S. van Schooten, Emma N. Bos, Benthe H. Doeve, Maarten F. Bijlsma, Hanneke W. van Laarhoven, Donald L. van der Peet, Sarah Derks. Characterizing the secretome of freshly dissociated gastroesophageal tumor biopsies to identify immune suppressive factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2324.