1028 IL-12 and IL-27 upregulate CD39 expression on CD8⁺ T-cells and differentially affect CD39⁺CD8⁺ T-cell effector function

<h3>Background</h3> Tumor-specific CD8⁺ T-cells play a critical role in tumor control, as demonstrated by the success of immune checkpoint inhibitors and adoptive cell therapy. Studies of mice and human tumor-infiltrating lymphocytes (TILs) demonstrate that tumor-specific CD8⁺ TILs can be defined by CD39 expression on their surface. CD39 is commonly considered an immunosuppressive enzyme, as it depletes ATP. However, CD39 is also associated with mitigating activation-induced cell death and mediating leukocyte trafficking. It is, however, unclear whether CD39 expression on tumor-specific T-cells can be regulated by putative anti-tumor factors such as pro-inflammatory cytokines similar to the phenotype and anti-tumor properties of CD8⁺ TILs. IL-12 and IL-27 have established roles in promoting effector T-cell differentiation, expansion, and cytotoxic activity. Both cytokines are implicated in the upregulation of CD39 by T regulatory cells, suggesting they may regulate CD39 expression in other cell types, including tumor-specific CD8⁺ T-cells. We hypothesize that IL-12 and IL-27 induce CD39 upregulation on CD8⁺ T- cells, modulating their anti-tumor properties. <h3>Methods</h3> An engineered immunogenic, syngeneic neuroblastoma (neuro-2a) mouse model was used for <i>in vitro</i> and <i>in vivo</i> experiments. CD8⁺ T-cells or bulk splenocytes isolated from naïve or neuro-2a vaccinated mice were stimulated <i>in vitro</i> using anti-CD3/CD28 Dynabeads and IL-2 ± IL-12 or IL-27. Flow cytometry was used to determine the phenotype of CD8⁺ T-cells and assess effector activity. Additionally, we inhibited IL-12 activity <i>in vivo</i> to study the effect on CD8⁺ TIL expression of CD39. An isotype control antibody was administered to a separate group to act as a control. <h3>Results</h3> Our <i>in vitro</i> results demonstrate that CD8⁺ T-cells stimulated in the presence of IL-12 or IL-27 had higher expression of CD39 compared to stimulated controls. In addition, we found a higher frequency of CD39⁺CD8⁺ T-cells expressing IFNγ and CD107a than CD39^- counterparts. Finally, blocking IL-12 activity <i>in vivo</i> reduced CD39⁺CD8⁺ TIL frequency compared to the isotype control group. <h3>Conclusions</h3> Our results establish that IL-12 and IL-27 induce CD39 expression on CD8⁺ T-cells and blocking IL-12 reduced CD39⁺CD8⁺ TIL frequency. Furthermore, CD39 expression is associated with improved effector CD8⁺ T-cell function. Future experiments will assess the functionality of CD39⁺CD8⁺ T-cells using <i>ex vivo</i> cytotoxicity assays. Data generated in this study will provide novel information on the mechanism of CD39 induction and its effect on CD8⁺ T-cell function, which can be exploited to improve future cancer therapies.

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