While adoptive transfer of autologous tumor antigen-specific T cell immunotherapy can produce remarkable clinical efficacy, most patients do not achieve durable complete responses. We hypothesized that reducing susceptibility of T cells to activation-induced cell death (AICD), which increases during the rapid in vitro expansion of therapeutic T cells before their infusion, might improve the persistence of adoptively transferred cells. Our investigations revealed that repetitive stimulation of the T cell receptor (TCR) induced AICD, as a result of activating the DNA damage response pathway through ATM-mediated Ser15 phosphorylation of p53. Activation of this DNA damage response pathway also occurred upon antigen-specific restimulation in TCR-transduced TIL1383I T cells prepared for adoptive transfer to patients as part of a clinical trial. Notably, treatment with the antioxidant N-acetyl cysteine (NAC) significantly reduced upregulation of the DNA damage marker γH2AX, subsequent ATM activation and cell death. In the Pmel mouse model of melanoma, the presence of NAC during ex vivo T cell expansion improved the persistence of adoptively transferred cells, reduced tumor growth and increased survival. Taken together, our results offer a preclinical proof of concept for the addition of NAC to current therapeutic T cell expansion protocols, offering immediate potential to improve the quality and therapeutic efficacy of adoptive T cell therapeutics infused into patients.

Background:Ceramide is important for cellular signaling. Results: Increasing the expression of ceramide synthase 6 (CerS6) results in transcriptional activation of acid ceramidase independent of catalytic CerS6 activity. Conclusion: Modulation of a single member of the ceramide synthase family impacts on sphingolipid composition and ceramide metabolizing enzymes. Significance: Understanding how CerS impacts gene expression and signaling is important for the development of novel therapeutic approaches.

Persistence of effector cytotoxic T lymphocytes (CTLs) during an immunological response is critical for successfully controlling a viral infection or tumor growth. Various cytokines are known to play an important part in regulating the immune response. The IL-2 family of cytokines that includes IL-2 and IL-15 are known to function as growth and survival factors for antigen-experienced T cells. IL-2 and IL-15 possess similar properties, including the ability to induce T cell proliferation. Whereas long term IL-2 exposure has been shown to promote apoptosis and limit CD8+ memory T cell survival and proliferation, it is widely believed that IL-15 can inhibit apoptosis and helps maintain a memory CD8+ T-cell population. However, mechanisms for superior outcomes for IL-15 as compared to IL-2 are still under investigation. Our data shows that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules Glutathione reductase (GSR), Thioredoxin reductase 1 (TXNDR1), Peroxiredoxin (PRDX), Superoxide dismutase (SOD). An increased expression of cell-surface thiols, intracellular glutathione, and thioredoxins was also noted in IL-15 cultured T cells. Additionally, IL-15 cultured T cells also showed an increase in cytolytic effector molecules. Apart from increased level of Granzyme A and Granzyme B, IL-15 cultured T cells exhibit increased accumulation of reactive oxygen (ROS) and reactive nitrogen (RNS) species as compared to IL-2 cultured T cells. Overall, this study suggests that T cells cultured in IL-15 show increase persistence not only due to increased anti-apoptotic proteins but also due to increased anti-oxidant levels, which is further complimented by increased cytolytic effector functions.

Repetitive stimulation of T cell receptor (TCR) with cognate antigen results in robust proliferation and expansion of the T cells, and also imprints them with replicative senescence signatures. Our previous studies have shown that life-span and anti-tumor function of T cells can be enhanced by inhibiting reactive oxygen species (ROS) or intervening with ROS dependent JNK activation that leads to its activation induced cell death (AICD). Since tumor suppressor protein p53 is also a redox active transcription factor that regulates cellular ROS generation that triggers downstream factor mediating apoptosis, we determined if p53 levels could influence persistence and function of tumor reactive T cells. Using h3T TCR transgenic mice, with human tyrosinase epitope reactive T cells developed on p53 knock-out (KO) background, we determined its role in regulating anti-tumor T cell function. Our data shows that as compared to h3T cells, h3T-p53 KO T cells exhibited enhanced glycolytic commitment that correlated with increased proliferation, IFN-γ secretion, cytolytic capacity, expression of stemness gene signature and decreased TGF-β signaling. This increased effector function correlated to the improved control of subcutaneously established murine melanoma after adoptive transfer of p53-KO T cells. Pharmacological inhibition of human TCR transduced T cells using a combination of p53 inhibitors also potentiated the T cell effector function and improved persistence. Thus, our data highlights the key role of p53 in regulating the tumor reactive T cell response and that targeting this pathway could have potential translational significance in adoptive T cell therapy.

Sphingolipids regulate critical cellular processes including inflammation. Ceramide, which serves a central role in sphingolipid metabolism, is generated by six ceramide synthases (CerS) that differ in substrate specificity. CerS6 preferentially generates C16-ceramide and its mRNA is highly expressed in immune tissues. In this study we analyzed how deficiency of CerS6 impacts on the development of colitis using an adoptive transfer model. Adoptive transfer of CerS6-deficient splenocytes, which have significantly decreased levels of C16-ceramide, showed that CerS6-deficiency protected against the development of colitis. However, adoptively transferred cells isolated from the lamina propria of the large intestine from wild type or CerS6-deficient groups showed no differences in the percentages of immune-suppressive regulatory T cells, pro-inflammatory Th17 cells, or their ability to express IL-17. In vitro polarization of wild type or CerS6-deficient splenocytes also revealed no defects in the development of T cell subsets. Our data suggest that protection from colitis following adoptive transfer of CerS6-deficient splenocytes maybe related to their ability to migrate and proliferate in vivo rather than subset development or cytokine expression.