In absence of co-stimulation, our cells displayed an exhausted phenotype, characterized by the upregulation of PD-1/TIM3 and diminished cytolytic function. suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. Methods First (1G – CD3) and second generation (2G – 41BB.CD3) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CARICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by Mmp16 PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 – activation + signal 2 – co-stimulation) and 4/7ICR (signal 3 – cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. Conclusions Our findings demonstrate the feasibility of targeting breast cancer using transgenic T cells equipped to thrive in the suppressive tumor milieu and highlight the importance of providing transgenic T cells with signals that recapitulate physiologic TCR signaling C [activation (signal 1), co-stimulation (signal 2) and cytokine support (signal 3)] – to promote in vivo persistence and memory formation. Electronic supplementary material The online version of this article (10.1186/s40425-018-0347-5) contains supplementary material, which is available to authorized users. Keywords: Chimeric antigen receptor, Genetic engineering, Inverted cytokine receptor, T cell therapy, Breast cancer Background Breast cancer is the most prevalent malignant disease of women in the developed world and remains one of the leading causes of death; in 2017 an estimated 252,710 new cases of invasive breast cancer were diagnosed in women . Although early detection and advances in conventional chemo-, radio-, and antibody-based therapies have substantially increased cure rates (99% 5-year survival in patients with localized disease), the 5-year survival of those with distant metastases is only 27%, highlighting the need for novel therapies . The adoptive transfer of T cells modified to express tumor-targeted chimeric antigen receptors (CARs) has proven to be effective for the treatment of a range of refractory hematologic malignancies including ALL, B-CLL, and lymphoma and holds promise for the treatment of solid tumors [2C6]. However, extension of this approach to metastatic breast cancer requires both the identification of an appropriate antigen to target Tenapanor and consideration of additional genetic strategies to protect these cells from the suppressive tumor microenvironment (TME). Indeed, the breast cancer TME is infiltrated by regulatory T cells [7, 8], myeloid-derived suppressor cells (MDSCs) [9, 10], and rich in inhibitory/Th2-polarized cytokines such as IL4 [11C13], that promote tumor survival [14C17], migration and invasion [18, 19], and directly inhibit Th1-polarized effector T cells [20, 21]. We now explore the feasibility of targeting metastatic breast cancer using T cells modified with a CAR targeting the tumor associated antigen (TAA) mucin1 (MUC1), whose overexpression in underglycosylated form has been associated with tumor invasiveness and metastatic potential [22C28]. Further, to ensure that our CAR T cells remain operative in Tenapanor the tumor microenvironment, we co-express an inverted cytokine receptor (ICR) encoding the cytokine-binding Tenapanor portion of the IL4 receptor exodomain linked to the immunostimulatory IL7 receptor signaling endodomain (4/7ICR) [29, 30]. We demonstrate the potent, selective, and sustained anti-tumor activity of these dual transgenic T cells in the IL4-rich breast cancer microenvironment and highlight the importance of transgenically delivering a combination of signals that recapitulate physiological T cell signaling (activation, co-stimulation, and cytokine support) to ensure durable benefit. Methods Donor and cell lines Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers after informed consent on protocols approved by the Baylor College of Medicine Institutional Review Board. The cell lines MDA MB 468, MCF-7,.