Chimeric antigen receptor T-cell therapy for solid tumors

Chimeric antigen receptor (CAR) T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias). This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment. Chimeric antigen receptor (CAR) T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias). This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment. Infusion of T cells directed against specific antigens has demonstrated promise in HIV and cancer therapy. Along with immune checkpoint blockade,1Topalian SL Drake CG Pardoll DM Immune checkpoint blockade: a common denominator approach to cancer therapy.Cancer Cell. 2015; 27: 450-461Abstract Full Text Full Text PDF PubMed Scopus (2616) Google Scholar this approach is triggering a paradigm shift in cancer immunotherapy. Perhaps the most exciting of these approaches has been the use of T cells that have been genetically modified to express chimeric antigen receptor (CAR) genes. CARs are comprised of an extracellular single-chain variable fragment (scFv), which serves as the targeting moiety, a transmembrane spacer, and intracellular signaling/activation domain(s) (Figure 1). The CAR constructs are transfected into T cells, using plasmid transfection, mRNA or via viral vector transduction, to direct them toward tumor-associated antigens (TAAs). CAR structure has evolved significantly from the initial composition involving only the CD3ζ signaling domain, dubbed a "first-generation CAR." Since then, in an effort to augment T-cell persistence and proliferation, costimulatory endodomains were added, giving rise to second- (e.g., CD3ζ plus 41BB- or CD28-signaling domains) and third-generation (e.g., CD3ζ plus 41BB- and CD28-signaling domains) CARs. CARs have also been constructed in the context of human leukocyte antigen targeting intracellular molecules.2Willemsen RA Debets R Hart E Hoogenboom HR Bolhuis RL Chames P A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes.Gene Ther. 2001; 8: 1601-1608Crossref PubMed Scopus (60) Google Scholar The adoptive transfer of CAR T cells has demonstrated remarkable success in treating blood-borne tumors; prominently, the use of CD19 CARs in leukemias,3Gill S Maus MV Porter DL Chimeric antigen receptor T cell therapy: 25 years in the making.Blood Rev. 2015; (Epub ahead of print).PubMed Google Scholar and indications in patients with lymphoma and myeloma are being explored. A growing number of clinical trials have focused on solid tumors, targeting surface proteins including carcinoembryonic antigen (CEA), the diganglioside GD2, mesothelin, interleukin 13 receptor α (IL13Rα), human epidermal growth factor receptor 2 (HER2), fibroblast activation protein (FAP), and L1 cell adhesion molecule (L1CAM) (reviewed in Gill et al.3Gill S Maus MV Porter DL Chimeric antigen receptor T cell therapy: 25 years in the making.Blood Rev. 2015; (Epub ahead of print).PubMed Google Scholar and Fousek et al.4Fousek K Ahmed N The evolution of T-cell therapies for solid malignancies.Clin Cancer Res. 2015; 21: 3384-3392Crossref PubMed Scopus (68) Google Scholar). Unfortunately, the clinical results have been much less encouraging. To date, the two most positive trials reported have used GD2 CARs to target neuroblastoma (3 of 11 patients with complete remissions),5Louis CU Savoldo B Dotti G Pule M Yvon E Myers GD et al.Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.Blood. 2011; 118: 6050-6056Crossref PubMed Scopus (818) Google Scholar and HER2 CARs for sarcoma (4 of 17 patients showing stable disease).6Ahmed N Brawley VS Hegde M Robertson C Ghazi A Gerken C et al.Human epidermal growth factor receptor 2 (HER2)-specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.J Clin Oncol. 2015; 33: 1688-1696Crossref PubMed Scopus (652) Google Scholar The reason for this is not yet known, but is likely multifactorial. The solid tumor landscape presents unique barriers that are absent in hematological malignancies, and these barriers, either by themselves or in combination with various tumor- and/or host cell-borne factors eventually neutralize CAR activity. Unlike the "liquid tumor" environment of blood malignancies, CAR T cells must successfully traffic to solid tumor sites in spite of potential T-cell chemokine receptor-/tumor-derived chemokine mismatches and successfully infiltrate the stromal elements of solid tumors in order to elicit TAA-specific cytotoxicity, regardless of antigen loss or heterogeneity. Even after successful trafficking and infiltration, T cells must surmount challenges conferred by: (i) an environment characterized by oxidative stress, nutritional depletion, acidic pH, and hypoxia; (ii) the presence of suppressive soluble factors and cytokines; (iii) suppressive immune cells (regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM) or neutrophils (TAN); and (iv) T-cell-intrinsic negative regulatory mechanisms (e.g., upregulation of cytoplasmic and surface inhibitory receptors) and overexpression of inhibitory molecules. Lastly, the CAR T cells, themselves, may be problematic given their potential immunogenicity and toxicity. In this mini-review, we discuss some of the key immunosuppressive barriers and other negative elements within solid tumors that ultimately neutralize the function of antitumor T cells, and CAR T cells in particular (Figure 2). The first step in adoptive T-cell therapy is choosing an optimal TAA for CAR T-cell targeting. Ideally, the TAA is highly expressed on the surface of all tumor cells but not on important normal tissues (or at least lowly expressed). However, unlike the success story of CD19 CARs in leukemias, where CD19 is consistently expressed on tumors and only on "dispensable" B cells, specific target antigens on solid tumors have been more difficult to identify. So far, roughly 30 solid tumor antigens are being evaluated for CAR T-cell therapy; these include neoantigens (for instance, mutated sequences), oncofetal or developmental antigens, tumor-selective antigens (i.e., enriched expression of antigens on neoplastic cells, but low basal expression on normal cells), and endogenous tumor-specific antigens; a recent list of CAR targets that are currently being evaluated in clinical trials is available.3Gill S Maus MV Porter DL Chimeric antigen receptor T cell therapy: 25 years in the making.Blood Rev. 2015; (Epub ahead of print).PubMed Google Scholar It should be noted that the scFv avidity to TAA may also be important, and immunoediting and subsequent removal of the most immunogenic epitopes may lead to tumor escape.7Restifo NP Dudley ME Rosenberg SA Adoptive immunotherapy for cancer: harnessing the T cell response.Nat Rev Immunol. 2012; 12: 269-281Crossref PubMed Scopus (1213) Google Scholar Neoantigens on the surface of solid tumors represent an especially attractive target for CAR therapy as their expression is restricted to tumor cells. It is now recognized that most neoantigens are likely to due to tumor-specific mutations and are thus highly individualized (and hence not practical for CAR therapy). However, several neoepitopes that are more generalized have been identified. CAR T cells targeting the mutated EGF receptor is an illustration of this approach; EGFR variant 3 (EGFRvIII) is only expressed on malignant tumor cells (mostly glioblastomas). EGFRvIII CARs have shown promise in treating animal models of glioblastomas8Johnson LA Scholler J Ohkuri T Kosaka A Patel PR McGettigan SE et al.Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.Sci Transl Med. 2015; 7: 275ra22Crossref PubMed Scopus (333) Google Scholar,9Morgan RA Johnson LA Davis JL Zheng Z Woolard KD Reap EA et al.Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.Hum Gene Ther. 2012; 23: 1043-1053Crossref PubMed Scopus (244) Google Scholar and clinical trials testing the efficacy of EGFRvIII CAR in patients with glioblastomas are underway (NCT02209376, NCT01454596). Abnormal glycosylation of extracellular glycoprotein MUC1 is also seen in a large variety of cancers; MUC-1-targeting CAR T cells against MUC1-overexpressing breast cancer xenografts were shown to significantly delay tumor progression.10Wilkie S Picco G Foster J Davies DM Julien S Cooper L et al.Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor.J Immunol. 2008; 180: 4901-4909Crossref PubMed Scopus (252) Google Scholar A similar success was reported for CAR T cells targeting MUC16, which is overexpressed in many ovarian carcinomas.11Chekmasova AA Rao TD Nikhamin Y Park KJ Levine DA Spriggs DR et al.Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice following adoptive transfer of T cells genetically targeted to the MUC16 antigen.Clin Cancer Res. 2010; 16: 3594-3606Crossref PubMed Scopus (106) Google Scholar CEA is an example of an antigen expressed during developmental growth, but restricted in normal adult tissues and in transformed cells. Evidence of tumor eradication by CEA-CAR T cells in mice has been reported.12Chmielewski M Hahn O Rappl G Nowak M Schmidt-Wolf IH Hombach AA et al.T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice.Gastroenterology. 2012; 143: 1095-1107Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar A recent phase 1 trial using CEA-redirected transgenic T-cell receptor (TCR) T cells, however, showed that while one out of three metastatic colon cancer patients demonstrated an objective response to the therapy as exhibited by regression of metastasis to the lungs and liver, all three patients had to endure transient colitis.13Parkhurst MR Yang JC Langan RC Dudley ME Nathan DA Feldman SA et al.T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.Mol Ther. 2011; 19: 620-626Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar Cancers arising from virus transformation may express viral products that are attractive targets for therapy since these products are not displayed on normal tissues; for instance, human papillomavirus (HPV)-transformed ovarian cancers.14Kenter GG Welters MJ Valentijn AR Lowik MJ Berends-van der Meer DM Vloon AP et al.Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia.N Engl J Med. 2009; 361: 1838-1847Crossref PubMed Scopus (833) Google Scholar Tumor-selective (versus tumor-specific) antigens include targets that are overexpressed on transformed cells but expressed at low levels on normal tissues. This includes mesothelin, a glycoprotein whose overexpression in mesothelioma, ovarian, and pancreatic carcinomas, and low expression on peritoneal, pleural, and pericardial surfaces, has made it an attractive target for CAR therapy.15Morello A Sadelain M Adusumilli PS Mesothelin-targeted CARs: driving T cells to solid tumors.Cancer Discov. 2016; 6: 133-146Crossref PubMed Scopus (289) Google Scholar,16Pastan I Hassan R Discovery of mesothelin and exploiting it as a target for immunotherapy.Cancer Res. 2014; 74: 2907-2912Crossref PubMed Scopus (165) Google Scholar Two mesothelin-specific CARs have been reported; one based on the SS1 antibody17Chowdhury PS Viner JL Beers R Pastan I Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity.Proc Natl Acad Sci USA. 1998; 95: 669-674Crossref PubMed Scopus (181) Google Scholar—a mouse anti-human scFv which is currently being evaluated in a clinical trial at the University of Pennsylvania (NCT02159716), and another designated P4, a fully human scFv.18Lanitis E Poussin M Hagemann IS Coukos G Sandaltzopoulos R Scholler N et al.Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor.Mol Ther. 2012; 20: 633-643Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar A fully human scFv targeting mesothelin was recently described by another group, and is currently being tested in a clinical trial (NCT02414269).19Adusumilli PS Cherkassky L Villena-Vargas J Colovos C Servais E Plotkin J et al.Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity.Sci Transl Med. 2014; 6: 261ra151Crossref PubMed Scopus (345) Google Scholar Treatment using T cells electroporated with the mRNA encoding SS1-CAR, while promising, raised concerns about potential immunogenicity-related toxicity (see below). The notion of targeting multiple antigens (for instance, the expression of 2 scFvs,20Wilkie S van Schalkwyk MC Hobbs S Davies DM van der Stegen SJ Pereira AC et al.Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling.J Clin Immunol. 2012; 32: 1059-1070Crossref PubMed Scopus (340) Google Scholar) or non-tumor cell–related antigens (i.e., a CAR-targeting the stromally-expressed FAP,21Wang LC Lo A Scholler J Sun J Majumdar RS Kapoor V et al.Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.Cancer Immunol Res. 2014; 2: 154-166Crossref PubMed Scopus (333) Google Scholar or the VEGF receptor on tumor endothelium has also been explored.22Chinnasamy D Tran E Yu Z Morgan RA Restifo NP Rosenberg SA Simultaneous targeting of tumor antigens and the tumor vasculature using T lymphocyte transfer synergize to induce regression of established tumors in mice.Cancer Res. 2013; 73: 3371-3380Crossref PubMed Scopus (75) Google Scholar It should be noted that for any of the targets chosen, the scFv avidity to TAA may also be important. Another issue is that immunoediting and subsequent removal of the most immunogenic epitopes may lead to tumor escape.7Restifo NP Dudley ME Rosenberg SA Adoptive immunotherapy for cancer: harnessing the T cell response.Nat Rev Immunol. 2012; 12: 269-281Crossref PubMed Scopus (1213) Google Scholar Once a CAR that targets appropriate tumor antigen is generated and infused into a patient, an immediate obstacle is the ability of these CAR T cells to successfully target and infiltrate the solid tumor. This process is dependent on the appropriate expression of adhesion receptors on both T cells and the tumor endothelium, and a "match" between the chemokine receptors on the CAR (primarily CXCR3 and CCR5) and the chemokines secreted by the tumors. Unfortunately, there is often a chemokine/chemokine receptor "mismatch", with tumors producing very small amounts of, for instance, CXCR3 ligands, resulting in inefficient targeting of CXCR3high CD8+ effectors to tumor sites.23Harlin H Meng Y Peterson AC Zha Y Tretiakova M Slingluff C et al.Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment.Cancer Res. 2009; 69: 3077-3085Crossref PubMed Scopus (748) Google Scholar One approach to overcome this problem is to design CAR T cells that coexpress "better-matched" chemokine receptors. For example, using mesothelioma tumors that make large amounts of CCL2, we and others demonstrated enhanced intratumoral migration of CAR T cells when they coexpressed the appropriate CCR2b transgene, thus leading to subsequent tumor eradication.24Moon EK Carpenito C Sun J Wang LC Kapoor V Predina J et al.Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor.Clin Cancer Res. 2011; 17: 4719-4730Crossref PubMed Scopus (371) Google Scholar Similarly, the use of GD2-CAR T cells coexpressing CCR2b exhibited improved trafficking and tumor control compared to GD2-CAR alone.25Craddock JA Lu A Bear A Pule M Brenner MK Rooney CM et al.Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b.J Immunother. 2010; 33: 780-788Crossref PubMed Scopus (366) Google Scholar We have also recently found that the genetic inhibition of protein kinase A activation in CAR T cells increased their ability to infiltrate tumors in vivo due to higher baseline expression of CXCR3.26Newick K O'Brien S Sun J Kapoor V Maceyko S Lo A et al.Augmentation of CAR T cell trafficking and antitumor efficact by blocking protein kinase A (PKA) localization.Cancer Immunol Res. 2016; (In press.)PubMed Google Scholar Due to poor trafficking after intravenous injection, local instillation of CARs is also being explored; there are a number of clinical trials that are evaluating the merits of site-specific (i.e., systemic versus regional versus intratumoral) administration of CAR T cells in solid tumors (NCT02498912, NCT02414269, NCT01818323). One potential limitation is that local instillation is often more technically challenging than simple intravenous administration. Another potential issue is that although site-specific injection of CAR T cells will likely result in higher T-cell levels locally, the ability of these CARs to exit the tumor, enter the blood and then traffic to other tumor sites (which presumably exist in advanced cancer patients) is uncertain. The ongoing studies will help to address these issues. Several groups have also demonstrated the successful use of oncolytic viruses armed with chemotactic chemokines in attempts to attract CAR T cells to tumor sites. Oncolytic viruses have been shown to successfully and specifically infect tumor cells, and lyse them. The use of oncolytic adenoviral vector expressing CCL5 and GD2-CAR T cells robustly controlled neuroblastoma progression in mice and improved CAR T-cell influx,27Nishio N Diaconu I Liu H Cerullo V Caruana I Hoyos V et al.Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors.Cancer Res. 2014; 74: 5195-5205Crossref PubMed Scopus (218) Google Scholar and similar observations were attained with the use of HER2-CAR T cells loaded with modified oncolytic viruses.28VanSeggelen H Hammill JA Dvorkin-Gheva A Tantalo DG Kwiecien JM Denisova GF et al.T cells engineered with chimeric antigen receptors targeting NKG2D ligands display lethal toxicity in mice.Mol Ther. 2015; 23: 1600-1610Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar The solid tumor microenvironment presents many problems for CAR T cells. There are purely physical/anatomical barriers, such as stroma that characterizes many types of cancers, and the associated high tissue pressure that prevents extravasation. Countering these barriers by reducing tumor fibroblast numbers using FAP-CAR T cells21Wang LC Lo A Scholler J Sun J Majumdar RS Kapoor V et al.Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity.Cancer Immunol Res. 2014; 2: 154-166Crossref PubMed Scopus (333) Google Scholar or by having the CARs secrete an enzyme that degrades matrix29Caruana I Savoldo B Hoyos V Weber G Liu H Kim ES et al.Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes.Nat Med. 2015; 21: 524-529Crossref PubMed Scopus (432) Google Scholar have both shown some success in augmenting CAR T-cell function in animal models. The metabolic landscape within the tumor microenvironment is markedly stressful and inhospitable toward T cells. Prominent hallmarks of the tumor microenvironment include hypoxia and nutrient starvation; under these conditions, elevated lactate generation (leading to acidosis) and the lack of glucose and other metabolites inhibit T-cell proliferation and cytokine production.30Jacobs SR Herman CE Maciver NJ Wofford JA Wieman HL Hammen JJ et al.Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways.J Immunol. 2008; 180: 4476-4486Crossref PubMed Scopus (553) Google Scholar,31Fischer K Hoffmann P Voelkl S Meidenbauer N Ammer J Edinger M et al.Inhibitory effect of tumor cell-derived lactic acid on human T cells.Blood. 2007; 109: 3812-3819Crossref PubMed Scopus (1084) Google Scholar The lack of nutrients (specifically amino acids such as tryptophan, arginine, and lysine) may also activate the integrated stress response, causing protein translation shutdown or autophagy responses in effector T cells as a means of survival in order to generate an intracellular source of nutrients.32Howie D Waldmann H Cobbold S Nutrient sensing via mTOR in T cells maintains a tolerogenic microenvironment.Front Immunol. 2014; 5: 409Crossref PubMed Scopus (43) Google Scholar For example, the amino acid tryptophan is essential for many biological functions though it cannot be synthesized, and hence must be obtained via dietary means. Tryptophan metabolism as catalyzed by tumor- and MDSC-expressed indolamine-2,3-dioxygenase leads to T-cell anergy and death, and Treg accumulation. In a solid tumor xenograft model of CD19-expressing tumor cells transduced with indolamine-2,3-dioxygenase, Ninomiya and colleagues showed the failure of adoptively transferred CD19 CAR T cells to control progression of indolamine-2,3-dioxygenase-expressing tumors.33Ninomiya S Narala N Huye L Yagyu S Savoldo B Dotti G et al.Tumor indoleamine 2,3-dioxygenase (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs.Blood. 2015; 125: 3905-3916Crossref PubMed Scopus (221) Google Scholar MDSC may also reduce the bioavailability of the key amino acid arginine (see below). Preliminary work has suggested that manipulation of key cellular regulators of protein synthesis (i.e., the mammalian target of rapamycin) might augment the efficacy of adoptively transferred cells.34Veliça P Zech M Henson S Holler A Manzo T Pike R et al.Genetic regulation of fate decisions in therapeutic T cells to enhance tumor protection and memory formation.Cancer Res. 2015; 75: 2641-2652Crossref PubMed Scopus (19) Google Scholar Sukumar and colleague also showed that inhibiting glycolysis promoted the formation of memory cells, and enhanced antitumor activity.35Sukumar M Liu J Ji Y Subramanian M Crompton JG Yu Z et al.Inhibiting glycolytic metabolism enhances CD8+ T cell memory and antitumor function.J Clin Invest. 2013; 123: 4479-4488Crossref PubMed Scopus (565) Google Scholar Many studies have reported the presence of immunosuppressive soluble factors in the sera, ascites fluid, and tissue extracts from cancer patients that could inhibit CAR T cells. Prostaglandin E2 (PGE2), a small molecule derivative of arachidonic acid produced by the inducible cyclooxygenase 2 (COX2) enzyme, is generated by both tumor cells and macrophages; many studies have reported PGE2-mediated inhibition of T-cell proliferation, suppression of CD4 help, and subversion of CD8 differentiation.36Goodwin JS Bankhurst AD Messner RP Suppression of human T-cell mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell.J Exp Med. 1977; 146: 1719-1734Crossref PubMed Scopus (641) Google Scholar Adenosine, a purine nucleoside seen at high levels during hypoxia, is another potent inhibitor of T-cell proliferation and activity. Both PGE2 and adenosine illicit their immunosuppressive effects via signaling through their own G-coupled receptors which activate protein kinase A in a cyclic AMP-dependent manner.37Su Y Huang X Raskovalova T Zacharia L Lokshin A Jackson E et al.Cooperation of adenosine and prostaglandin E2 (PGE2) in amplification of cAMP-PKA signaling and immunosuppression.Cancer Immunol Immunother. 2008; 57: 1611-1623Crossref PubMed Scopus (51) Google Scholar We recently demonstrated that genetic inhibition of protein kinase A activation in CAR T cells can enhance their antitumor efficacy.26Newick K O'Brien S Sun J Kapoor V Maceyko S Lo A et al.Augmentation of CAR T cell trafficking and antitumor efficact by blocking protein kinase A (PKA) localization.Cancer Immunol Res. 2016; (In press.)PubMed Google Scholar Cytokines, implicated in inflammatory responses at tumor sites, can be a double-edged sword, which may bolster or inhibit the antitumor response. One of the most important inhibitory tumor cytokines is TGFβ. In addition to its ability to promote epithelial-to-mesenchymal transition, enhance matrix production, promote metastasis, and skew the immune response toward a Th2 phenotype,38Oh SA Li MO TGF-ß: guardian of T cell function.J Immunol. 2013; 191: 3973-3979Crossref PubMed Scopus (190) Google Scholar TGFβ has direct negative effects on T-cell effector functions.39Massagué J TGFbeta in Cancer.Cell. 2008; 134: 215-230Abstract Full Text Full Text PDF PubMed Scopus (3040) Google Scholar A few approaches have been used to counteract this effect. We previously showed that systemic blockade of TGFβ was efficacious in augmenting adoptive T-cell therapy.40Wallace A Kapoor V Sun J Mrass P Weninger W Heitjan DF et al.Transforming growth factor-beta receptor blockade augments the effectiveness of adoptive T-cell therapy of established solid cancers.Clin Cancer Res. 2008; 14: 3966-3974Crossref PubMed Scopus (72) Google Scholar To counteract TGFβ effects specifically in T cells, CAR T cells expressing a dominant negative TGFβ receptor have been created. These CAR T cells were resistant to TGFβ suppression and demonstrated augmented efficacy in animal models.41Bollard CM Rössig C Calonge MJ Huls MH Wagner HJ Massague J et al.Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity.Blood. 2002; 99: 3179-3187Crossref PubMed Scopus (261) Google Scholar Other inhibitory cytokines include IL10 and IL4; although these have not been directly targeted by alterations in T cells, two groups have constructed chimeric IL4 receptors so that IL4 engagement resulted in signaling that mimicked that of IL2.42Leen AM Sukumaran S Watanabe N Mohammed S Keirnan J Yanagisawa R et al.Reversal of tumor immune inhibition using a chimeric cytokine receptor.Mol Ther. 2014; 22: 1211-1220Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar,43Wilkie S Burbridge SE Chiapero-Stanke L Pereira AC Cleary S van der Stegen SJ et al.Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4.J Biol Chem. 2010; 285: 25538-25544Crossref PubMed Scopus (132) Google Scholar One of these groups combined this with the use of CAR T cells targeting the tumor-associated antigen MUC1 and showed enhanced efficacy.43Wilkie S Burbridge SE Chiapero-Stanke L Pereira AC Cleary S van der Stegen SJ et al.Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4.J Biol Chem. 2010; 285: 25538-25544Crossref PubMed Scopus (132) Google Scholar Finally, it has also been possible to use introduce activating cytokines to improve the tumor microenvironment milieu to augment CAR function. A few groups have designed CARs or T cells that release the stimulatory cytokine IL12 upon TCR engagement.44Chmielewski M Hombach AA Abken H Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.Immunol Rev. 2014; 257: 83-90Crossref PubMed Scopus (227) Google Scholar Although the approach worked extremely well in animal models, a recent clinical trial in which the IL12 gene, driven by an NFAT promoter in adoptively transferred tumor-infiltrating lymphocytes (TILs) resulted in unacceptable toxicity.45Zhang L Morgan RA Beane JD Zheng Z Dudley ME Kassim SH et al.Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma.Clin Cancer Res. 2015; 21: 2278-2288Crossref PubMed Scopus (231) Google Scholar Finding ways to more tightly control IL12 release or the use of less toxic cytokines (i.e., type 1 interferons) might allow this strategy to proceed in the clinic. Within the tumor microenvironment, various suppressive surveilling immune cells, Tregs, MDSC, and TAM/TAN with the so called M2 and N2 phenotype are known to present a barrier against successful antitumor immunity. Although there is extensive literature describing the immunosuppressive nature of these cells, to date, their effects on CAR T-cell therapy has not been extensively examined. One technical factor to consider is that in order to study these cell-cell interactions, mouse CAR T cells must be injected into immunocompetent mice. Given the major differences between the behavior of mouse versus human CAR T cells (e.g