IntroductionThe role of CD4 ϩ cells in antitumor immunity remains controversial and poorly understood. 1,2 They are known to mediate potent therapeutic effect in the setting of hematopoietic stem cell allotransplantation and donor lymphocyte infusion in hematologic malignancy, 3,4 but antigen-specific T helper (Th) cells have been studied to much lesser extent. A lack of clarity regarding CD4 ϩ cells is due, in no small part, to the complexity of their biology. CD4 ϩ T cells can differentiate into diverse subsets with specific phenotypes that can have self-reinforcing and opposing functions, but these T-cell subsets have not been comprehensively studied in tumor-bearing mice.Historically, CD4 ϩ T lymphocytes have been thought of as mere providers of stimuli to help the putatively more important CD8 ϩ effectors, which eliminate cancer by direct cytotoxicity. [5][6][7] There are several studies showing that CD4 ϩ T helper (Th) cells are capable of protecting the host against tumor challenge and even of mediating tumor regression on their own in the setting of either solid or hematopoietic disease. [8][9][10][11][12][13] Furthermore, protection was maintained against MHC class II-negative multiple myeloma model and involved cross-presentation by professional antigenpresenting cells (APCs) and activation of tumoricidal activity mediated by macrophages secreting IFN-␥. 14 A similar IFN-␥-dependent mechanism was involved in the rejection of MHC class II-negative tumor in severe combined immunodeficient (SCID) mice. 15 In some cases, the ability to reject antigen-expressing tumor by specific naive Th cells was thought to be substantially better than the ability of CD8 ϩ cells. 16 Classically, effector CD4 ϩ T cells have been categorized into T helper 1 (Th1) and T helper 2 (Th2) subsets. 17,18 Limited studies indicate that both subtypes elicit antitumor effects, 19-21 but the Th1-polarized cells, secreting IFN-␥ and capable of enhancing activity of cytotoxic CD8 ϩ lymphocytes, have traditionally been regarded as more efficient. [22][23][24][25] However, it is also clear that CD4 ϩ T regulatory cells (T regs ) can efficiently suppress the function of antitumor CD8 ϩ T cells. 5,[26][27][28] Recently, the novel Th17 lineage, generated in the presence of TGF-␤ and IL-6 and expanded under the influence of IL-23, [29][30][31] has been associated with responses against certain infections and implicated in the development of autoimmunity in animal models that had been previously linked to Th1-type responses (experimental autoimmune encephalitis, collagen-induced arthritis). 32,33 They also seem to play an important role in the pathogenesis of graft-versus-host disease (GVHD). 34,35 Th17 cells have been found in various tumors, including mycosis fungoides, Sézary syndrome, and prostate cancer. 36,37 Kryczek et al reported the presence of naturally occurring Th17 cells and T regs in the tumor microenvironment and tumor-draining lymph nodes in both human and mice tumors. 38 Proinflammatory cytokines including IL-17A, IL-6, and I...

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8 + T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8 + T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8 + T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.

IntroductionInfusion of tumor-reactive T cells is emerging as an effective treatment for Epstein-Barr virus-associated malignancies and for melanoma. 1-3 T cells with reactivity against tumor antigens can be generated through T-cell receptor (TCR) gene engineering, 4 expanded in vitro, and administered to patients. 5 This approach permits not only targeting of any antigen for which a TCR can be isolated but also selection and induction of T-cell subsets with optimal therapeutic potential. Identification of these subsets is a critical challenge in the advance of cancer immunotherapy. 6,7 Th17-lineage CD4 ϩ T cells, which express the transcription factor ROR-␥t and produce the proinflammatory cytokine interleukin-17 (IL-17), demonstrate enhanced antitumor immunity. 8,9 IL-17-producing CD8 ϩ T cells have been identified in mice and in humans, 10-13 and they can be generated through in vitro priming with Th17-polarizing cytokines. 14 However, a role for these cells in antitumor immunity has not been described. CD4 ϩ T cells can differentiate into lineages with diverse effector functions. In contrast, CD8 ϩ T cells, through the redundant expression of Eomes and T-bet, are fated to develop into cytolytic effector cells that produce IFN-␥ and express granzyme B and perforin. 11,15 It is not known if induction of IL-17 production in this cytolytic lineage would enhance its ability to mediate tumor destruction. Interestingly, the improved antitumor immunity observed with Th17 cells appears to be IL-17-independent, suggesting that it is not IL-17 secretion per se, but rather another characteristic conferred by Th17-polarization, that improves their function. 8 To study tumor therapy with type 17-skewed CD8 ϩ T cells, we used the pmel-1 model of adoptive immunotherapy of B16 melanoma. 16 This model reproduces the clinical challenge of targeting the shared tumor/self-antigen, gp100, in poorly immunogenic, vascularized B16 melanoma tumors. Pmel-1, gp100-specific, TCR-transgenic CD8 ϩ T cells that were primed and expanded before infusion were used to parallel human peripheral blood lymphocytes that are TCR transduced and expanded for clinical therapy. 5 Methods Mice and tumor linesPmel-1/Thy1.1 TCR-transgenic mice have been described previously. 16,17 They were housed in the National Institutes of Health (NIH) Clinical Research Center vivarium and maintained in compliance with the NIH Animal Care and Use Committee. C57BL/6 mice were purchased from The Jackson Laboratory. The B16F10 and MCA205 tumor lines were obtained from the National Cancer Institute (NCI) tumor repository. All animal studies were approved by the NCI Animal Care and Use Committee. In vitro assaysType 17 skewing was accomplished by priming pmel-1/Thy1.1 splenocytes with 1 M hgp100 25-33 in rmIL-6 (5 ng/mL) and recombinant human transforming growth factor-␤ (rhTGF-␤; 10 ng/mL; R&D Systems), and anti-IFN-␥ (10 g/mL; eBioscience). Beginning 2 days after priming, cell cultures were expanded with rhIL-2 (30 IU/mL; Novartis). Control cells were primed and expand...

T-cell-based immunotherapies can be effective in the treatment of large vascularized tumors, but they rely on adoptive transfer of substantial numbers (∼20 million) of tumor-specific T cells administered together with vaccination and high-dose interleukin (IL)-2. In this study, we report that ∼10,000 T cells gene-engineered to express a single-chain IL-12 molecule can be therapeutically effective against established tumors in the absence of exogenous IL-2 and vaccine. Although IL-12-engineered cells did not perist long-term in hosts, they exhibited enhanced functionality and were detected in higher numbers intratumorally along with increased numbers of endogenous natural killer and CD8 + T cells just before regression. Importantly, transferred T cells isolated from tumors stably overproduced supraphysiologic amounts of IL-12, and the therapeutic effect of IL-12 produced within the tumor microenvironment could not be mimicked with high doses of exogenously provided IL-12. Furthermore, antitumor effects could be recapitulated by engineering wild-type open-repertoire splenocytes to express both the single-chain IL-12 and a recombinant tumor-specific T-cell receptor (TCR), but only when individual cells expressed both the TCR and IL-12, indicating that arrested migration of T cells at the tumor site was required for their activities. Successful tumor eradication was dependent on a lymphodepleting preconditioning regimen that reduced the number of intratumoral CD4 + Foxp3 + T regulatory cells.Our findings reveal an approach to genetically modify T cells to reduce the cell number needed, eliminate the need for vaccines or systemic IL-2, and improve immunotherapy efficacy based on adoptive transfer of gene-engineered T cells.

BackgroundClinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.MethodsAntigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.ResultsTandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.ConclusionTandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0246-1) contains supplementary material, which is available to authorized users.

Depleting host immune elements with nonmyeloablative regimens prior to the adoptive transfer of tumor-specific CD8(+) T cells significantly enhances tumor treatment. In the current study, superior antitumor efficacy was achieved by further increasing the intensity of lymphodepletion to a level that required HSC transplantation. Surprisingly, the HSC transplant and not the increased lymphodepletion caused a robust expansion of adoptively transferred tumor-specific CD8(+) T cells. The HSC-driven cell expansion of effector, but not of naive, CD8(+) T cells was independent of in vivo restimulation by MHC class I-expressing APCs. Simultaneously, HSCs also facilitated the reconstitution of the host lymphoid compartment, including inhibitory elements, not merely via the production of progeny cells but by enhancing the expansion of cells that had survived lymphodepletion. Profound lymphodepletion, by myeloablation or by genetic means, focused the nonspecific HSC boost preferentially toward the transferred tumor-specific T cells, leading to successful tumor treatment. These findings indicate that CD8(+) T cell-mediated tumor responses can be efficiently driven by HSCs in the myeloablative setting and have substantial implications for the design of new antitumor immunotherapies.

Lymphodepletion prior to adoptive cell transfer (ACT)-based immunotherapies can enhance anti-tumor responses by augmenting innate immunity, by increasing access to homeostatic cytokines, and by depressing the numbers of regulatory T cells and myeloid-derived suppressor cells. Although it is clear that high-dose total body irradiation (HD-TBI) given together with hematopoietic stem cell (HSC) transplantation effectively enhances ACT, the relationship between the intensity of lymphodepletion and tumor treatment efficacy has not been systematically studied. Using the pmel-1 mouse model of self/tumor-reactive CD8+ T cells, we observed a strong correlation between the intensity of the conditioning regimen and the efficacy of ACT-based treatments using linear regression analysis. This was the case for preparative TBI administered either as a single dose (R2 = 0.97, p < 0.001) or in fractionated doses (R2 = 0.94, p < 0.001). Increased amounts of preparative TBI were directly correlated with progressively more favorable ratios of transferred tumor-reactive CD8+ T cells towards endogenous cells with the potential for inhibitory activity including: CD4+ cells (potentially T regulatory cells); Gr1+ cells (which are capable of functioning as myeloid-derived suppressor cells): and endogenous CD8+ and NK1.1+ cells (that can act as “sinks” for homeostatic cytokines in the post-ablative setting). With increasing ablation, we also observed elevated LPS levels in the sera and heightened levels of systemic inflammatory cytokines. Thus, increased intensity lymphodepletion triggers enhanced tumor treatment efficacy and the benefits of HD-TBI must be titrated against its risks.