J Immunother : first published as 10.1136/jitc-2020-SITC2020.0838 on 10 December 2020. Downloaded from Abstracts cell induction was positively correlated with DASA values, 837 INTERLEUKIN-10 DRIVES THE DEVELOPMENT OF T while cross-reactivity of induced T cells was inversely REGULATORY TYPE 1 (TR1) CELLS AND IS A TARGET correlated. FOR IMMUNOTHERAPY Conclusions Our results indicate that dissimilarity is key for 1Yanchun Ma*, 1Vera Bauer, 1Tanja Riedel, 1Thomas Hofer, 2Martin Roecken, both T-cell induction and discrimination from self. DASA may 1Ralph Mocikat. 1Helmholtz-Zentrum Muenchen, Munich, Germany; 2Eberhard-Karls- help predict immunogenic non-anchor type neoantigens inducing Universitaet Tuebingen, Tuebingen, Germany specific T-cell response from a variety of cancer mutation pools. Background In recent years, immunotherapy has become a http://dx.doi.org/10.1136/jitc-2020-SITC2020.0835 common tool of cancer treatment. In order to define thera- peutic targets, it is necessary to understand mechanisms of tumor-induced immunosuppression. In malignant B-cell lym- 836 RELEASING THE RESTRAINTS OF Vg9Vd2 T-CELLS IN phoma, the effects of the anti-inflammatory cytokine interleu- kin-10 (IL-10) remain poorly understood. Methods To investigate the role of IL-10 in a tumor microen- Laura Ridgley*, Angus Dalgleish, Mark Bodman-Smith. St George’s University of London, vironment, we used l-MYC-transgenic mice that spontaneously London, UK develop B-cell . The experiments were performed either in vivo or in vitro and the cell samples were then ana- Background Vg9Vd2 T-cells are a subset of cells with a crucial lyzed by flow cytometry. role in immunosurveillance which can be activated and Results In MYC tumors, CD4+Foxp3- effector T cells main- expanded by multiple means to stimulate effector responses, tained the expression of interferon-g (IFN-g), yet became often exploited in cancer immunotherapy. Little is known exhausted. Within this population we found a cell fraction of about the expression of checkpoint molecules on this cell pop- unknown origin coexpressing IFN-g and IL-10 that increased ulation and whether the ligation of these molecules can regu- during disease progression. These cells turned out to be T reg- late their activity. The aim of this study was to assess the ulatory type 1 (Tr1) cells, which are known to be immunosup- expression of activatory and inhibitory markers on Vg9Vd2T- pressive. When exposing homogeneous IFN-g-producing T cells to assess potential avenues of regulation to target with helper type 1 (Th1) cells to a MYC tumor milieu in vitro, immunotherapy. part of these cells started to express both, IFN-g and IL-10, Methods PBMCs were isolated from healthy donors and the and showed an increased level of programmed cell death pro- expression of activatory and inhibitory receptors was assessed tein 1 (PD-1). Notably, these changes diminished when an IL- on Vg9Vd2 T-cells by flow cytometry at baseline, following 10 neutralizing monoclonal antibody (mAb) was added to the 24 hours activation and 14 days expansion using zoledronic coculture, indicating that IL-10 is necessary for the Tr1 devel- acid (ZA) and Bacillus Calmette-Guerin (BCG), both with IL- opment and is involved in the upregulation of PD-1. In line 2. Activation and expansion of Vd2 cells was assessed by with these results, we treated l-MYC mice with anti-IL-10 expression of CD69 and by frequency of Vd2 cells, respec- mAb. This therapy not only led to significantly prolonged sur- tively. Production of effector molecules was also assessed fol- vival but also decreased expression of PD-1 on effector T cells lowing coculture with various tumour cell targets. The effect and increased proliferation of cytotoxic T cells. of blockade on Vg9Vd2 T-cells was also Conclusions In summary, these results showed the importance assessed. of IL-10 for the tumor immune escape in lymphoma. IL-10 Results Vg9Vd2 T-cells constitutively expressed high levels of induced a conversion from Th1 to Tr1 cells and elevated lev- NK-associated activatory markers NKG2D and DNAM1 els of PD-1. Both effects were diminished after IL-10 ablation. http://jitc.bmj.com/ which remained high following stimulation with ZA and Thus, targeting IL-10 might be a promising new approach of BCG. Vg9Vd2 T-cells expressed variable levels of checkpoint immunotherapy. inhibitor molecules at baseline with high levels of BTLA, Ethics Approval All animal studies were approved by Regier- KLRG1 and NKG2A and intermediate levels of PD1, TIGIT ung von Oberbayern, approval number 55.2-1-54. and VISTA. Expression of checkpoint receptors were modu- lated following activation and expansion with ZA and BCG http://dx.doi.org/10.1136/jitc-2020-SITC2020.0837

with decreased expression of BTLA and upregulation of on October 2, 2021 by guest. Protected copyright. numerous markers including PD1, TIGIT, TIM3, LAG3 and VISTA. Expression of these markers is further modulated 838 PHENOTYPIC AND FUNCTIONAL SIGNATURES OF upon coculture with tumour cell lines with changes reflecting PERIPHERAL AND TUMOR-RESIDENT gd T CELLS ARE activation of these cells with Vg9Vd2 T-cells expressing INFORMATIVE FOR OUTCOME OF CHECKPOINT inhibitory receptors PD1 and NKG2A producing the highest BLOCKADE IN level of TNF. Conclusions Our data reveals unique characteristics of Vd2in Graham Pawelec*, Kilian Wistuba-Hamprecht, Kilian Wistuba-Hamprecht. University of terms of their expression of immune checkpoints, which pro- Tübingen, Tubingen, Germany vide a mechanism which may be utilised by tumour cells to Background Immune checkpoint blockade (ICB) set a mile- subvert Vg9Vd2 T-cell cytotoxicity. Our work suggests differ- stone in cancer immunotherapy, but still only a fraction of ent profiles of immune checkpoints dependent on the method patients responds. Thus, there is an urgent need for bio- of stimulation. This highlights importance of expansion markers predicting outcome, and also for understanding the method in the function of Vg9Vd2 T-cells. Furthermore, this responsible mechanisms. gd T cells constitute a numerically work suggests important candidates for blockade by immune minor subset of 1-10% of the peripheral compartment checkpoint therapy in order to increase the successful use of in healthy people and have a major role in defense against Vg9Vd2 T-cells in cancer immunotherapy. multiple microbial and non-microbial challenges. Unlike the http://dx.doi.org/10.1136/jitc-2020-SITC2020.0836 majority of T cells, gd T cells bind their ligands in an MHC-

A500 J Immunother Cancer 2020;8(Suppl 3):A1–A559 J Immunother Cancer: first published as 10.1136/jitc-2020-SITC2020.0838 on 10 December 2020. Downloaded from Abstracts independent manner. We previously studied gd T cells, that with a 5-year survival of only 50% even when treated with also express checkpoint molecules, in patients in the pre- radical cystectomy. Immune checkpoint inhibitors have shown checkpoint blockade era and thereafter, and identified correla- promising results for treatment of bladder cancer; however, tions between subset frequencies of these unconventional T only around 30% of patients have a therapeutic effect and cells and patients‘ overall survival (OS). Here, we present a novel therapies are thus required. With the aim of pinpointing detailed phenotyping and functional investigation of tumor-res- novel targets for T-cell based therapy, we have performed ident as well as peripheral gd T cells. transcriptomic profiling of specific T cell populations in MIBC Methods Phenotyping was performed in stage IV melanoma and NMIBC, as well as in control bladder tissue. patients before and under PD-1+/-CTLA-4 blockade using as Methods Muscle-invasive (n=7) as well as non-muscle invasive basis our published OMIP-20 protocol.1 Cytokine expression (n=13) bladder tumor biopsies were obtained from untreated patterns and proliferative capacities were determined as patients and control bladder tissue (n=7). Upon digestion, described according to our established protocols.2 Primary cells were stained with an antibody panel to enable sorting of flow cytometry data analysis was performed using FlowJo CD8+ cytotoxic T-cells (CD8T), CD4+ T-helper cells (Th) (BD) and correlations with clinical meta data were determined and regulatory T-cells (Treg) using fluorescence activated cell using Prism (GraphPad) and SPSS (IBM). sorting. RNA was extracted and subject to sequencing. Differ- Results We found previously that low frequencies of peripheral ential expression analysis was performed, using DESeq2 Vd1 gd T cells were associated with prolonged OS. Here, we ( with padj investigated functional aspects and abundance of gd T cells Results Principal component analysis demonstrated that CD8T, within the tumor as well as in the blood. The peripheral Vd1 unlike Th and Tregs, cluster according to the invasiveness of but not the Vd2 differentiation signature revealed significantly the disease. Accordingly, many genes were significantly differ- lower proportions of naive and effector cells as well as an entially expressed between CD8T in MIBC and NMIBC com- accumulation of late differentiated cells in patients with high pared to control, and also between CD8T in MIBC compared Vd1 frequencies. The cytokine expression pattern (IFNg, TNF to NMIBC. Several genes associated with CD8 T-cell exhaus- and IL-17) and the degranulation marker CD107a were differ- tion were significantly upregulated in MIBC compared to both ent in patients with high versus low peripheral Vd1 frequen- NMIBC and control. Further, GSEA results indicated biologi- cies. The proliferative capabilities of Vd1 cells in melanoma cal differences of the CD8T compartment between different were limited in comparison to healthy subjects. Both Vd1 and tumor stages. Vd2 cells were found in tumor tissues, and these analyses are Conclusions The profiles of CD8 T-cells were ongoing, including analyses of replicative senescence through significantly different in NMIBC, MIBC and control. The CD57 expression. transcriptional profiles give clues on biological differences and Conclusions Our data provide novel insights into the role of disease progression and can be relevant for development of gd T cells in cancer rejection. The previously found negative novel treatment strategies. correlation of Vd1 T cells with OS is likely due to an accu- Ethics Approval The study was approved by the Regional mulation of mal-functioning, probably exhausted Vd1 T cells Ethics Committee (EPN - Regionala Etikprövningsnämnden i in patients with poor outcome of ICB. Thus, we suggest that Lund), approval number 2017/34. Vd1 T cells are promising candidates for future exploitation in Consent Written informed consent was obtained from all novel ICB-approaches. patients included in the study. Ethics Approval This study was approved by K. Wistuba-Ham- http://dx.doi.org/10.1136/jitc-2020-SITC2020.0839 precht´s Ethics Committee (approval nos. 490/2014BO1 and

792/2016BO2). http://jitc.bmj.com/

REFERENCES 840 PRESENCE AND DISTRIBUTION OF 1. Wistuba-Hamprecht K, Pawelec G, Derhovanessian E. OMIP-020: Phenotypic char- acterization of human gammadelta T-cells by multicolor flow cytometry. Cytometry IMMUNOSUPPRESSIVE PEPTIDE P3028 IN RELATION TO A 2014;85:522–524. doi:10.1002/cyto.a.22470 IMMUNE PHENOTYPE OF TONSILLAR CANCER 2. Beucke N, et al. Pitfalls in the characterization of circulating and tissue-resident 1Sabine Swoboda*, 2Rebecca Rosberg, 3David Gomez Jimenez, 1David Askmyr, human gammadelta T cells. J Leukoc Biol 2020. doi:10.1002/JLB.5MA1219-296R 3 1 4 1

Malin Lindstedt, Lennart Greiff, Leif Håkansson. Skåne University Hospital, Lund, on October 2, 2021 by guest. Protected copyright. http://dx.doi.org/10.1136/jitc-2020-SITC2020.0838 Sweden; 2Canimguide Therapeutics AB, Lund, Sweden; 3Lund University, Lund, Sweden; 4Canimguide Therapeutic AB, Lund, Sweden

Background A cancer lesion may avoid detection by the 839 TRANSCRIPTOMIC PROFILING OF T-CELL POPULATIONS immune system through a variety of immunosupressive mecha- IN NON-MUSCLE INVASIVE AND MUSCLE INVASIVE nisms involving myeloid-derived suppressor cells and regula- BLADDER CANCER tory T-cells. One such mechanism recently discovered is the : 1 1 1 1 2 immuno suppressive effect of a specific peptide, i.e., P3028, Viktor Sincic*, Milad Abolhalaj, Henrik Lilljebjörn, Alar Aab, Karin Hagerbrand, produced through degradation of albumin. In this study, 3Peter Ellmark, 1Thoas Fioretos, 1Carl Borrebaeck, 4Fredrik Liedberg, 1Kristina Lundberg. involving biopsies obtained from patients with tonsillar cancer 1Lund University, LUND, Sweden; 2Alligator Bioscience AB, Lund, Sweden; 3Alligator Bioscience AB. Lund University, Lund, Sweden; 4Skåne University Hospital, Lund University, (TC), P3028 is assessed in relation to overall immune pheno- Lund, Sweden type, as indicated by presence and distribution of CD8+ T- cells as well as to other specific immune cells. Background Bladder cancer is categorized as non-muscle inva- Methods Immunohistochemistry was performed on fresh frozen sive (NMIBC) or muscle invasive (MIBC). NIMBC makes up biopsies. CD8+ T-cells were used to classify the cancer lesions around 70% of the cases and although it is less aggressive, into immune phenotypes: ‘inflamed’ (lympho:cytes infiltrating the recurrence rate is 50-70%, thus requiring extensive moni- cancer cell areas), ‘immune excluded’ (lymphocytes in surround- toring. Additionally, there is a risk of progression into MIBC ing stroma, but few within cancer cell areas) and ‘desert’ (few

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