Dávila, Eduardo

Abstract The recent FDA approval of the first T-cell receptor–engineered (TCR) T-cell cancer therapy has solidified the treatment as a viable therapeutic strategy. However, only a fraction of patients who received this treatment achieved substantial and long-lasting responses, highlighting the need to understand the molecular underpinnings of these responses and develop more effective strategies to improve this therapy. Previous research has demonstrated that the activation of myeloid differentiation primary response 88 (MyD88) signaling in tumor-specific T cells functions as a robust costimulatory signaling pathway that promotes effective antitumor T-cell responses. In this study, we developed a strategy for improving TCR T-cell therapies by creating a synthetic TCR in which the intracellular domain of CD3ζ and a modified MyD88 are appended to the β-chain of a tumor-reactive TCR. The β:CD3ζ:MyD88 TCR activated MyD88 signaling in a tumor antigen/TCR-specific manner, and β:CD3ζ:MyD88 TCR T cells exhibited enhanced functionality and a reduced propensity to become exhausted. Furthermore, MyD88 signaling promoted T-cell persistence and expansion in vitro. In vivo, tumor-infiltrating β:CD3ζ:MyD88 TCR T cells skewed the immunosuppressive tumor microenvironment toward a less immunosuppressive state. Finally, β:CD3ζ:MyD88 TCR T cells substantially delayed tumor growth kinetics in a melanoma model. These findings introduce an approach for improving TCR-dependent T-cell responses by directly appending signaling domains to the full-length TCR and exemplify the utility of activating MyD88 signaling in tumor-specific T cells. Significance: A chimeric β:CD3ζ:MyD88 TCR reduces exhaustion and improves antitumor T-cell responses, overcoming the limited efficacy of current TCR-engineered T-cell cancer therapies.

Abstract There is a dire need to understand the cellular signals that can potentiate T-cell responses to weakly immunogenic tumor antigens (TAg) and to tumors expressing suboptimal levels of these antigens. We examined the anti-tumor activity and survival of TLR2-MyD88-stimulated tumor-specific CD8 T-cells derived from melanoma patients and TAg-specific T-cell receptor transgenic pmel mice. TLR2 engagement on pmel CD8 T-cells, but not on TLR2-/-pmel or MyD88-/-pmel T-cells, reduced the activation threshold to a subdominant TAg, resulting in increased production of effector molecules and cytotoxicity. Wild-type or MyD88-/- mice treated with pmel T-cells and TLR2 ligand, but not TLR2-/-pmel or MyD88-/-pmel T-cells, showed significant tumor regression of an established melanoma tumor. Over-expressing TLR2 in pmel T-cells eradicated established tumors and four-times fewer cells were needed to generate anti-tumor responses. The enhanced anti-tumor activity was associated with improved survival and increased effector function of TLR2-MyD88-stimulated T-cells. Activating TLR-MyD88 signals in patient-derived T-cells reduced the activation threshold to several weakly immunogenic TAgs, resulting in increased cytokine production, expansion and cytotoxicity. These data highlight the physiological importance of activating TLR-MyD88 signals within tumor-reactive T-lymphocytes for enhancing their longevity and augmenting effector function against suboptimal levels of weakly-immunogenic antigens.

Abstract The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.

Many of the mechanisms that govern T-cell homeostasis remain obscure. Here we report that repeated administration of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG-ODN) into mice induces a systemic antigen-independent expansion of naive and memory T cells in a full T-cell compartment. Expansion of T cells was observed on both CD4+ and CD8+ T-cell subsets and was produced not by inducing the proliferation of the cells but by preventing their death. The antiapoptotic effects of CpG-ODN on T cells were observed against activation-induced death and growth factor withdrawal–mediated death. The ability of CpG-ODN to protect T cells from these forms of death was associated with the up-regulation of antiapoptotic gene products including c-FLIP, bcl-xL, and, to some extent, bcl-2. The effect of CpG-ODN on naive and memory T cells required the expression of CD28 and was not dependent on the presence of B lymphocytes, suggesting that other antigen-presenting cells that respond to CpG-ODN, such as dendritic cells, may provide antiapoptotic signals to T cells in an antigen-independent but CD28/B7-dependent fashion. The present findings suggest that CpG-ODN can disrupt normal T-cell homeostasis not by acting as a mitogen but by preventing T-cell death that normally takes place as a mechanism to maintain steady-state levels of T cells. These findings support a potential means to expeditiously replenish and maintain the peripheral lymphocyte population after severe immunodepletion such as that which occurs in HIV-infected individuals and individuals undergoing cytoablative therapies.