Project Summary Adoptive cell transfer (ACT) therapies using tumor infiltrating T lymphocytes (TIL) for cancer patients have failed to fulfill their full potential, mostly due to the transfer of exhausted, short-lived CD8+ T cells. Recently, we discovered that T cell fitness can be improved by activating B cells in a TIL culture with the Toll-like receptor 9 (TLR) agonist CpG. Understanding and manipulating the TLR pathways that sustain T cell persistence will potentially unlock durable responses to tumors. Further, there is little research on how B and CD8+ T cells interact to promote T cell tumor immunity. In this proposal we seek to elucidate the mechanism by which T cells generated from a CpG-treated culture, are able to abolish established tumors in vivo without the need of IL-2 or vaccine administration. Our preliminary data shows B cells treated with CpG have overt CD40 (costimulatory protein) and concomitantly CD8+ T cells express CD40L. We found that at this time CD8+ T cells acquire a T follicular memory-like (TFML) phenotype denoted by CXCR5, ICOS, and PD-1 expression. We posit that CD40 signaling in B cells post CpG-treatment is responsible for the development of CD8+ TFML cells and potentiated antitumor activity. CD8+ T cells generated from CpG-treated cultures gain a unique phenotype denoted by high expression of the IL-2 cytokine receptor IL-2R and dim expression of CD39 My co-mentor Dr. Mark Rubinstein recently published the importance of the IL-2R in promoting engraftment of T cells in an ACT model. Conversely, CD39 which is only minimally expressed on CpG-generated T cells compared to vehicle treated T cells, is a marker associated with “terminally exhausted” CD8+ T cells in viral and cancer models. Thus, I seek to study the mechanisms underlying how CpG induces this potent cell product and the roles of IL-2R and CD39 in achieving robust antitumor responses. Overall, I hypothesize that TLR9 agonist CpG activates B cells in an antitumor T cell culture, promoting T cells to be imprinted with a TFML phenotype via CD40 signaling in B cells. I will examine this idea in Aim 1. Moreover, I posit that IL-2R and CD39, inversely regulated by TLR9 signaling, are responsible for augmenting the antitumor properties of adoptively transferred CD8+ T cells. This concept will be analyzed in Aim 2. Uncovering how B cells can be activated by CpG to become potent antigen presenting cells to CD8+ T cells in Aim 1 will establish a new paradigm for cancer therapy with Toll-like receptor agonist CpG. Also, findings from these studies will provide insight into the generation of T cells with strong immunological memory to tumors and will have important clinical implications for patients with cancer. Overall, the proposed research is expected to demonstrate that activation of the TLR9 pathway with CpG may sufficiently induce durable immunity against the growth and recurrence of advanced tumors.