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T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Following solid organ or hematopoietic cell transplantation, refractory opportunistic viral reactivations are a significant cause of morbidity and mortality but can effectively be controlled by virus-specific T-cell transfer. Among effective and safe strategies is the use of "third-party" (neither from the transplant donor nor recipient) virus-specific T cells that can be manufactured from healthy donors and used as "off-the-shelf" therapies. Leukoreduction system chambers (LRSCs), recovered after routine plateletpheresis, were evaluated as a potential source of peripheral blood mononuclear cells (PBMCs) for the manufacturing of clinical-scale virus-specific T cell.Study Design and Methods: PBMCs from the same donors obtained either from LRSCs or peripheral blood were compared, focusing on T-cell function and phenotype as well as the potential to generate cytomegalovirus (CMV)-specific T-cell lines from both CMV seropositive and seronegative donors.Results: PBMCs from both sources were comparable except for a transient downregulation of CD62L expression on freshly extracted PBMCs from LRSCs. Both nonspecific stimulation using anti-CD3/CD28 antibodies and CMV peptides revealed that LRSCs or blood T cells were equivalent in terms of expansion, differentiation, and function. Moreover, PBMCs from LRSCs can be used to generate autologous monocyte-derived dendritic cells to prime and expand CMV-specific T cells from seronegative donors.Conclusion: LRSCs are a reliable source of PBMCs for the generation of virus-specific T cells for immunotherapy. These findings have implications for the development of third-party therapeutic T-cell products from well-characterized blood product donors. [ABSTRACT FROM AUTHOR]

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo . This is associated with p16 INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16 INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16 INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Janelle, Neault, Lebel, De Sousa, Boulet, Durrieu, Carli, Muzac, Lemieux, Labrecque, Melichar, Mallette and Delisle.)

The adoptive transfer of ex vivo -expanded T cells is a promising approach to treat several malignancies. Several lines of evidence support that the infusion of T cells with early memory features, capable of expanding and persisting after transfer, are associated with better outcomes. We report herein that exposure to exogenous TGFβ during human T-cell stimulation ex vivo leads to the accumulation of early/central memory (Tcm) cells. Exposure to TGFβ suppressed the expression of BLIMP-1, a key orchestrator of effector T-cell differentiation, and led to the upregulation of the memory-associated transcription factor ID3. Accordingly, this was associated with an early memory transcriptional signature in both CD4 + and CD8 + T-cell subsets. The T cells stimulated in the presence of TGFβ expanded normally, and displayed polyfunctional features and no suppressive activity. The adoptive transfer of ex vivo -stimulated T cells into immunodeficient mice confirmed that TGFβ-conditioned cells had an enhanced capacity to persist and mediate xenogeneic graft-versus-host disease, as predicted by their early T-cell memory phenotype. Chimeric antigen receptor-expressing T cells generated in the presence of exogenous TGFβ were cytotoxic and more effective at controlling tumor growth in immunodeficient animals. This work unveils a new role for TGFβ in memory T-cell differentiation and indicates that TGFβ signaling may be harnessed to program Tcm differentiation in the context of ex vivo T-cell stimulation for adoptive immunotherapy in humans., (©2019 American Association for Cancer Research.)