Abstract 1986: Differential T cell response within the tumor microenvironment observed across different indications
Authors: Basak Nandini P. , K Vasanth , Jaganathan Kowshik , K Gowri Shankar , A Manimaran , M Rajashekar , Ganesh MS , Prabha Amritha , BV Prakash , Prakash C Jaya , Govindan Sindhu , Malhotra Ritu , Kar Pradeep , M Oliyarasi , Suri Anirudh , Rao Rachita , Sankaran Satish 
Published: June 2022

Abstract
Cytotoxic T-cells (CTLs) in the Tumor immune Microenvironment (TiME) play an important role in mounting anti-tumor immune response. Immunotherapeutic (I/O) agents like checkpoint inhibitor aim to prevent anergy whilst T cell agonists co-stimulate CTLs to reinvigorate them and thus leading to tumor cytotoxicity. In this study we investigate the abundance and functionality of the intratumoral CTLs post stimulation, across different tumor indications, to understand the differential response across samples and indications to immune cell stimulation. These include Head and Neck Squamous cell carcinoma (HNSCC), Renal Cell Carcinoma (RCC), stomach adenocarcinoma (ca-Stomach) and Triple Negative Breast Cancer (TNBC). Using the Farcast࣪ TiME tumor histo-culture platform, that preserves both tumor and immune components in culture, we stimulated tumor explants with anti-CD3 (10ng/ml) + IL2 (100units/ml) for T-cell activation. Supernatants were collected pre-treatment and at 24-hour intervals for evaluation of cytokine release. At termination the explants were either fixed in formalin to generate paraffin blocks or dissociated into single cells for flowcytometry. 3-5 samples from each tumor indication were included in this study. Flowcytometry based immunophenotyping indicated presence of 48.6% ±5.4 live CD3+ T-cells and 20.6% ± 3.1 live CD3+CD8+ CTLs across all indications in the absence of any stimulation. HNSCC, RCC and ca-Stomach had similar proportion of CTLs, while TNBC had much lower proportions of T cell and CTLs. HNSCC and TNBC had a very low proportion of functional Granzyme-B+ CTLs (0.29%±0.08) whereas ca-Stomach and RCC had relatively higher proportion of these cells (6.9%±2.5). The proliferating Ki67+ CTLs sub-population was low across all indications (0.37%±0.2). Upon stimulation there was a significant increase (&gt;2.5-fold, p&lt;0.05) in the proliferating CTLs sub-population across all indications though the cytokine release pattern varied. HNSCC showed a consistent increase (7.4-fold) in functional CTLs in all samples accompanied by a significant increase in INFɣ, Granzyme B and Perforin secretion. RCC and TNBC showed minimal change in functional CTLs but showed a significant increase in secretion of these cytokines. In ca-Stomach there was an increase in INFɣ secretion, but not Granzyme B and Perforin. In summary, while CTLs were present in TiME, the proportions of activated and proliferating sub-populations differed across indications. The presence of active CTLs did not always translate to tumor cytotoxicity. This could be due to other pro-tumor cells and factors that offset the anti-tumor activity of T cells. The TiME platform thus provides a unique platform to study complex interactions between tumor and intratumoral immune cell populations that could be further probed to understand differential response in patients to immunotherapy mediated tumor cytotoxicity.
Citation Format: Nandini P. Basak, Vasanth K, Kowshik Jaganathan, Gowri Shankar K, Manimaran A, Rajashekar M, MS Ganesh, Amritha Prabha, Prakash BV, C Jaya Prakash, Sindhu Govindan, Ritu Malhotra, Pradeep Kar, Oliyarasi M, Anirudh Suri, Rachita Rao, Satish Sankaran. Differential T cell response within the tumor microenvironment observed across different indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1986.