Chimeric antigen receptor (CAR)–T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses.

Activation of naive CD8 + T cells with antigen in the absence of skewing cytokines triggers their differentiation into effector CTL, which induces death of target cells. We show that CD8 + T cells activated in the presence of the cytokines IL-6 or IL-21 plus TGF-b similar to CD4 + T cells, develop into IL-17-producing (Tc17) cells. These cells display greatly suppressed cytotoxic function along with low levels of the CTL markers: T-box transcription factor Eomesodermin, granzyme B and IFN-c. Instead, these cells express hallmark molecules of Th17 program including retinoic acid receptor-related orphan receptor (ROR)ct, RORa, IL-21 and IL-23R. The expression of the type 17 master regulator RORct is causally linked to Tc17 generation, because its overexpression stimulates production of IL-17 in the presence of IL-6 or IL-21. Both, upregulation of the type 17 program as well as suppression of CTL differentiation are STAT3 dependent. Furthermore, Tc17 cells producing IL-17 but not granzyme B are also detectable in EAE, a mouse model for multiple sclerosis. Our data point to the existence of mutually exclusive CTL and Tc17 developmental pathways in vitro and in vivo.Key words: CTL . EAE . RORgt . STAT3 . Tc17Supporting Information available online Introduction CTL are important effector cells in the immune response to intracellular pathogens and tumors. They differentiate from naive CD8 + T cells following activation by antigen in the absence of skewing cytokines, and during this process they acquire the ability to destroy their targets by releasing cytotoxic molecules such as perforin and granzymes, from granules into the immunological synapse. In addition, CTL secrete cytokines, mostly IFN-g and TNF-a, which function to induce or augment inflammation [1][2][3].Two T-box transcription factors, Eomesodermin (Eomes) and T-bet, are important for the development of effector and memory CTL [4][5][6]. Studies using deletion, overexpression or dominant negative analogs of these factors have suggested that both of them are involved in the regulation of expression of granzyme B and perforin [4,5,7]. Consistent with these data, CD8 + T cells with combined deletion of the Eomes and Tbx21 (encoding T-bet) genes differentiate into cells with highly impaired cytotoxic activity and IFN-g production [8]. Instead, these cells produce Th17 type cytokines and express the IL-23 receptor (IL-23R) as well as the transcription factor retinoic acid receptor-related orphan receptor (ROR)gt, both of 1716Frontline which are characteristic for the type 17 differentiation program [8]. Thus, the phenotype of CD8 + T cells deficient for both Eomes and T-bet is reminiscent of the newly described Th17-cell subset.Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22, which are highly pro-inflammatory and induce severe autoimmunity, e.g. during EAE, the mouse model for multiple sclerosis [9]. The differentiation of these cells requires TGF-b in combination with 11]. Two additional cytokines, IL-21 and IL-23, are also critically involved in the diffe...

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The NF-jB/Rel family member c-Rel was described to be required for the development of T H 1 responses. However, the role of c-Rel in the differentiation of T H 17 and regulatory CD41