Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder the efficacy of adoptively transferred T cells. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces hetero-dimerization of these receptors and phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. In several previous reports, a dominant negative receptor (DNR) version of TGFBR2, that does not contain signaling domains, was shown to protect T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs.

Herein, we report the development of a novel TGF-β signal conversion platform that provides a T cell supportive signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of a T cell stimulating interleukin receptor. The signaling domains of interleukin receptors that were chosen for this platform are naturally activated via cytokine-induced hetero-dimerization and induce STAT transcription factor signaling pathways beneficial to effector T cell function and persistence. Based on the ability of lentiviral vectors to express large and complex gene expression cassettes, we constructed single vectors encoding both chimeric TGF-β receptors (CTBR) in the context of either a CAR or transgenic TCR. This required expression of four unique proteins from a single lentiviral vector when CTBR was combined with a transgenic TCR consisting of unique alpha and beta chains.

To demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on multiple functional attributes of CTBR expressing T cells, including gene expression, cytokine production, cytotoxicity, and metabolic function. As anticipated, the STAT signaling signature induced by a particular CTBR was predictive of the functional consequence of exposure to TGF-β. In the case of a CTBR based on IL-12 signaling domains (CTBR12) that strongly induced STAT4 phosphorylation, CTBR12 expressing T cells secreted significantly greater amounts of IFNγ than control T cells following activation in the presence of TGF-β. This result was consistent with IL-12-mediated STAT4 signaling, which induces IFNγ production in effector T cells. We next evaluated CTBR expressing T cells in a stringent serial re-stimulation assay in the presence of TGF-β but absence of exogenous IL-2 cytokine support. Antigen driven T cell expansion was severely limited by exposure to TGF-β in this assay, and either CAR or transgenic TCR expressing cells ultimately lost cytotoxic activity over time. T cells overexpressing the dominant negative TGF-β receptor failed to expand and clear tumor cells following multiple rounds of antigen-driven stimulation in the presence of TGF-β, recapitulating the impact of tumor-derived TGF-β on infiltrating lymphocyte function. On the other hand, CTBR expressing cells maintained their ability to expand and kill tumor targets in the presence of TGF-β.

Together, these data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into a STAT signal that supports T cell effector function. The specific impact on T cell function is dependent on the nature of the signaling domains utilized. This platform is relevant in the context of both CAR T cells and transgenic TCR T cells, and has the potential to produce superior T cell responses in the immunosuppressive tumor microenvironment.


Boyerinas: bluebird bio: Employment, Equity Ownership. Miller: bluebird bio: Employment, Equity Ownership. Murray: bluebird bio: Employment, Equity Ownership. Evans: bluebird bio: Employment, Equity Ownership. Parsons: bluebird bio: Employment, Equity Ownership. Seidl: bluebird bio: Employment, Equity Ownership. Friedman: bluebird bio: Employment, Equity Ownership. Morgan: bluebird bio: Employment, Equity Ownership, Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.