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CD8 T cells are the main effectors of the adaptive immune system for T helper (Th) 1 or cytotoxic type of responses. In several types of cancer, CD8 lymphocyte infiltrates correlate with good prognosis. CD8 T cells can destroy directly transformed cells and secrete proinflammatory cytokines as IFN-γ and TNF-α, favoring the recruitment and activity of other effectors of antitumoral immunity such as M1 macrophages and NK cells. Hence, in the past few years the potential for the use of adaptive immune system as a base for immunotherapies has expanded enormously and it has focused in CD8 T cells activity. Chimeric antigen receptor (CAR)-T cell technology is one of those striking findings with remarkable successes in the treatment of refractory and/or relapse (r/r) B cell acute lymphoblastic leukemia (B-ALL). The duration of the event-free survival and the rates of overall survival overpass the previously existent treatments prompting to its proposal as therapy for less advanced stages of disease. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTPN2, enhance proinflammatory type I interferon signaling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling. Very recently other groups have linked PTPN2 deficiency has with enhanced CD8 antitumoral responses. Unpublished observations from our laboratory using PTPN1/PTPN2 dual specific small molecule inhibitors suggested a role for both phosphatases in the enhancement of CD8 T cell cytotoxic activity. Following this lead, we decided to develop a genetic model to study the contribution of each enzyme to the CD8 T cell ability to respond to antigenic cues. Indeed, we found that a combined total or partial reduction of these genes increased the cytotoxic activity and IFN-γ secretion of CD8 T cells, underscoring synergistic but non-redundant mechanisms of action. The reduced activity of these phosphatases correlated with an increase in the tonic signals mediated by the JAK-STAT axis, particularly STAT 3 and STAT5a. As consequence, cells are sensitized to respond to type 1 and type 2 interferons. Transcriptomic analysis of these enhanced effector cells correlated their functional state with an increased expression, at transcript and protein level, several CD8 relevant transcription factors including of IRF4, Myb and the Basic leucine zipper transcriptional factor ATF-like 3 (BatF3). Henceforth, our results suggest that small molecule inhibitors with dual specificity for PTP1B and TC-PTP are a powerful therapeutic tool for potentiating CTL cytotoxic responses and CTL based immunotherapies as CAR-T cells. Citation Format: Luis Alberto Perez-Quintero, Yevgen Zolotarov, Zuzet Martinez-Cardoba, Chu Han Feng, Alexandre Poirier, Kelly Anne Pike, Jean-Sebastien Delisle, Michel L. Tremblay. Concomitant reduction of PTPN1/PTPN2 activity synergistically enhanced antitumoral effector function of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG16.

Mounting a T helper 1 (Th1) type of response is required for the successful priming of antigen specific CD8 T-cells which ultimately lead to effective antitumoral responses. Several cellular components of the immune system participate of the Th1 decision-making process. They include innate cells sensing the transformed cells, mainly dendritic cells (DCs), and the further involvement of antigen specific CD4 T-cells promoting direct activation of CD8 T-cells through cross-presentation (1). The correct stimulation of DCs by type I interferons and molecular pattern sensors as STING is required to drive their differentiation to conventional DC 1 (cDC1) which through the secretion of large amounts of IL-12 and the expression of CD40 skew the CD4 T-cells differentiation to Th1 (2). However, this system is sensitive to modifications of the tumor cytokine microenvironmen,t leading to failure. Adoptive cell transfer (ACT) cancer immunotherapies are promising treatments for advanced malignancies. These therapies attempt to supply key cellular actors missing and induce proper antitumoral immunity. They include mainly the in vitro modification of autologous cells, from DCs loaded with tumor antigens to the expression of chimeric antigen receptor (CAR) on T and NK cells. Constant development of these technologies has given successful results as clinical trials have already showed up to 90 % of complete remission in relapsed/refractory B cell acute lymphoid leukemia (B-ALL) (3), treatment now approved for commercial use. However, there is still lack of consistency in the responses obtained from different individuals and in different trials. Previously our laboratory has already shown that modulation of the JAK-STAT inhibitory protein tyrosine phosphatases (PTPs), PTPN1 and PTP-N2, enhance proinflammatory type I interferon signalling while decreases the effects of immunosuppressive cytokines acting through STAT3 signaling (4, 5). Specifically, we have demonstrated that partial inhibition of these phosphatases in immune cells enhances the secretion of IL-12p70 by DCs[6] and increases the cytotoxic activity of antigen specific CD8 T-cells [Pérez-Quintero LA, Tremblay ML, unpublished results]. Moreover, the partial inhibition of these phosphatases in CD8 T-cells enhance the acquisition of a central memory (Tcm) phenotype, which has been found to be associated with better remission in CAR-T-cells therapies. Nevertheless, alternative methods to enrich this phenotype, as cytokine cocktails, have proven expensive and ineffective. Having this in mind, we propose here the use of small-molecule inhibitors specific for PTPN1 and PTPN2 as a simple and cost-effective method to enhance antitumoral responses. By treating ex vivo the cellular products in animal models for DCs and CD8 ACT therapies we show, as a proof of concept, a marked improvement on the reduction of tumor burden and remission, with the late goal of translating these findings into a clinical setup. References: 1. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment. Nature Immunology 2013;14:1014. 2. Theisen D,Murphy K. The role of cDC1s in vivo: CD8 T-cell priming through cross-presentation [version 1]. 2017;6. 3. Oluwole OO,Davila ML. At The Bedside: Clinical review of chimeric antigen receptor (CAR) T-cell therapy for B cell malignancies. Journal of Leukocyte Biology 2016. 4. Pike KA, et al. Protein tyrosine phosphatase 1B is a regulator of the interleukin-10–induced transcriptional program in macrophages. Science Signaling 2014;7(324): ra43-ra43. 5. Pike KA, et al. TC-PTP regulates the IL-7 transcriptional response during murine early T-cell development. Scientific Reports 2017;7(1):13275. 6. Penafuerte C, et al. Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling. OncoImmunology 2017;6(6):e1321185. Citation Format: Luis Alberto Perez-Quintero, Kelly Anne Pike, Penafuerte Claudia, Michel Tremblay. Enhancing antitumoral activity of cell-based immunotherapies by modulating the JAK-STAT axis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B035.