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As the targets of chimeric antigen receptor (CAR)–T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a “size exclusion” model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.

BackgroundCell therapy has shown promise in the treatment of certain solid tumors, but its efficacy may be limited by inhibition of therapeutic T cells by the programmed cell death protein-1 (PD-1) receptor. Clinical trials are testing cell therapy in combination with PDCD1 disruption or PD-1-axis blockade. However, preclinical data to support these approaches and to guide the treatment design are lacking.MethodsMechanisms of tumor regression and interaction between cell therapy and PD-1 blockade were investigated in congenic murine tumor models based on targeting established, solid tumors with T-cell receptor T cells directed against tumor-restricted, non-self antigens (ie, tumor neoantigens).ResultsIn solid tumor models of cell therapy, PD-1 blockade mediated a reproducible but non-synergistic increase in tumor regression following adoptive T-cell transfer. Tumor regression was associated with increased tumor infiltration by endogenous T cells but not by transferred T cells. The effect was independent of PD-1 receptor expression by transferred T cells and was dependent on the endogenous T-cell repertoire and on tumor antigenicity. PD-1 blockade primarily induced cell state changes in endogenous tumor-antigen-specific T cells rather than transferred T cells.ConclusionsTogether, these findings support the concept that PD-1 blockade acts primarily through endogenous rather than transferred T cells to mediate a non-synergistic antitumor effect in solid tumor cell therapy. These findings have important implications for strategies to leverage PD-1 receptor disruption or blockade to enhance the efficacy of cell therapy.

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies. Electronic supplementary material The online version of this article (10.1186/s40425-019-0678-x) contains supplementary material, which is available to authorized users.

BackgroundRecurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alfa is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.MethodsWe conducted a phase II clinical trial evaluating bintrafusp alfa in adults with RRP. Papilloma samples before and after treatment with bintrafusp alfa were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.ResultsDual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject’s own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.ConclusionsIntact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.Trial registration numbersNCT03707587 and NCT02859454.

Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making. Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome. Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2–3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy. Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type–specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856–62. ©2017 AACR.

Targeting nonviral antigens in viral-driven cancer Adoptive cell transfer harnesses a patient's own T cells to destroy cancer. The strategy can successfully treat epithelial tumors driven by human papillomavirus (HPV), but it remains unclear why only some patients respond. Stevanović et al. examined the antitumor T cell response associated with HPV + cervical cancers that underwent complete regression. Unexpectedly, reactive T cells were not directed against virally associated antigens, but rather against cancer germline antigens or neoantigens not previously recognized by the immune system. These findings counter the widely held belief that T cell responses against viral antigens are responsible for therapeutic effects in HPV-driven cancers. Science , this issue p. 200

Background Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV)-driven disorder that causes substantial morbidity and can lead to fatal distal airway obstruction and post-obstructive pneumonias. Patients require frequent surgical debridement of disease, and no approved systemic adjuvant therapies exist. Methods A phase II study was conducted to investigate the clinical activity and safety of programmed death-ligand 1 (PD-L1) blockade with avelumab in patients with RRP. Results Twelve patients were treated. All patients with laryngeal RRP displayed improvement in disease burden, and 5 of 9 (56%) displayed partial responses. None of 4 patients with pulmonary RRP displayed a response. Using each patient’s surgical history as their own control, patients required fewer surgical interventions after avelumab treatment (p = 0.008). A subset of partial responders developed HPV-specific reactivity in papilloma-infiltrating T-cells that correlated with reduced HPV viral load and an increased Tissue Inflammation Signature. Conclusions Avelumab demonstrated safety and clinical activity in patients with laryngeal RRP. Further study of immune checkpoint blockade for RRP, possibly with longer treatment duration or in combination with other immunotherapies aimed at activating antiviral immunity, is warranted. Trial registration NCT, number NCT02859454, registered August 9, 2016. Electronic supplementary material The online version of this article (10.1186/s40425-019-0603-3) contains supplementary material, which is available to authorized users.

BackgroundAs heterogeneous tumors develop in the face of intact immunity, tumor cells harboring genomic or expression defects that favor evasion from T-cell detection or elimination are selected. For patients with such tumors, T cell-based immunotherapy alone infrequently results in durable tumor control.MethodsHere, we developed experimental models to study mechanisms of T-cell escape and demonstrated that resistance to T-cell killing can be overcome by the addition of natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) targeting programmed death ligand-1 (PD-L1).ResultsIn engineered models of tumor heterogeneity, PD-L1 CAR-engineered NK cells (PD-L1 t-haNKs) prevented the clonal selection of T cell-resistant tumor cells observed with T-cell treatment alone in multiple models. Treatment of heterogenous cancer cell populations with T cells resulted in interferon gamma (IFN-γ) release and subsequent upregulation of PD-L1 on tumor cells that escaped T-cell killing through defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs in vitro and in vivo.ConclusionsThese results describe the underlying mechanisms governing synergistic antitumor activity between T cell-based immunotherapy that results in IFN-γ production, upregulation of PD-L1 on T-cell escape cells, and the use of PD-L1 CAR-engineered NK cells to target and eliminate resistant tumor cell populations.

BackgroundBintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-βRII (a TGF-β ‘trap’) fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa.MethodsIn these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety.ResultsAs of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred.ConclusionBintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers.

BackgroundInterleukin-12 (IL-12) is a potent, proinflammatory cytokine that holds promise for cancer immunotherapy, but its clinical use has been limited by its toxicity. To minimize systemic exposure and potential toxicity while maintaining the beneficial effects of IL-12, we developed a novel IL-12-based therapeutic system that combines tumor-specific T-cell-mediated delivery of IL-12 with membrane-restricted IL-12 localization and inducible IL-12 expression.MethodsTherapeutic T cells targeting a tumor antigen were genetically engineered to express membrane-anchored IL-12 (aIL-12). Expression, function, and shedding of the aIL-12 molecule was assessed in vitro. Tumor treatment efficacy was assessed in vivo with T cell receptor (TCR) transgenic murine tumor models and a tumor xenograft model. Key outcomes were change in tumor size, circulating levels of IL-12 and other cytokines, and survival. Toxicity was assessed via change in body weight. Tumor growth curve measurements were compared using repeated-measures two-way analyses of variance.ResultsRetroviral gene transfer resulted in cell membrane expression of aIL-12 by transduced T cells. In each of two transgenic murine tumor models, tumor-specific T cells constitutively expressing aIL-12 demonstrated increased antitumor efficacy, low circulating IL-12 and interferon-γ, and no weight loss. Expression of aIL-12 via aNFAT-inducible promoter resulted in coordinate expression of aIL-12 with T cell activation. In an OT-I TCR transgenic murine tumor model, theNFAT-inducible aIL-12 molecule improved tumor treatment and did not result in detectable levels of IL-12 in serum or in weight loss. In a human tumor xenograft model, theNFAT-inducible aIL-12 molecule improved antitumor responses by human T cells coexpressing a tumor-specific engineered TCR. Serum IL-12 levels were undetectable with theNFAT-inducible construct in both models.ConclusionExpression of aIL-12 by tumor-targeting therapeutic T cells demonstrated low systemic exposure and improved efficacy. This treatment strategy may have broad applications to cellular therapy with tumor-infiltrating lymphocytes, chimeric antigen receptor T cells, and TCR T cells.

Tumor cell–intrinsic factors, unrelated to antigenicity, control immune cell infiltration and sensitivity to immunotherapy in a mouse model.

The search continues for a therapeutic niche for interleukin-15 in human cancer treatment.

Purpose: The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV+ tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV+ tumor cells. Experimental Design: T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV+ epithelial tumor cells. Results: We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01–restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01–restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16+ cervical, and head and neck cancer cell lines. Conclusions: These findings demonstrate that HPV-16+ tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16+ malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. Clin Cancer Res; 21(19); 4431–9. ©2015 AACR.

Adoptive T cell therapy can target and kill widespread malignant cells thereby inducing durable clinical responses in melanoma and selected other malignances. However, many commonly targeted tumor antigens are also expressed by healthy tissues, and T cells do not distinguish between benign and malignant tissues if both express the target antigen. As such, autoimmune toxicity from T-cell-mediated destruction of normal tissue has limited the development and adoption of this otherwise promising type of cancer therapy. A review of the unique biology of T-cell therapy and of recent clinical experience compels a reassessment of target antigens that traditionally have been viewed from the perspective of weaker immunotherapeutic modalities. In selecting target antigens for adoptive T-cell therapy, expression by tumors and not by essential healthy tissues is of paramount importance. The risk of autoimmune adverse events can be further mitigated by generating antigen receptors using strategies that reduce the chance of cross-reactivity against epitopes in unintended targets. In general, a circumspect approach to target selection and thoughtful preclinical and clinical studies are pivotal to the ongoing advancement of these promising treatments.

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3+ T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8+ cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ∼50 × 109 T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3+CD8+ cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I–mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I–mediated up-regulation of CD137 (4-1BB) expression on CD8+ cells suggested that 0–3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

Purpose: Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γc) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. Experimental Design: To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (TCM), effector memory (TEM), and stem cell memory (TSCM) subsets on the strength of tumor regression as well as the dose and type of clinically available γc cytokines, including IL-2, IL-7, IL-15, and IL-21. Results: We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γc cytokine only moderately correlated with response. Conclusion: Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γc cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo. Clin Cancer Res; 17(16); 5343–52. ©2011 AACR.

Background: Adoptive T cell therapy with gene-engineered T cells is an emerging cancer treatment strategy that has been applied successfully to the treatment of hematological cancers. We conducted a clinical trial to test proof of principle for this type of treatment in an epithelial cancer. Patients with human papillomavirus (HPV) 16-associated cancers were treated with gene-engineered T cells targeting HPV16 E7. Methods: The clinical trial was a phase I study with a 3 + 3 design and three dose levels (DL) of gene-engineered T cells (DL1: 1 x 109, DL2: 1 x 1010, DL3: 1 x 1011). Patients had metastatic HPV-16+ cancers from any primary tumor site. Treatment consisted of a one-time infusion of autologous T cells that were gene-engineered to express an HLA-A*02:01-restricted T-cell receptor (TCR) that targets HPV-16 E7 (E7 T cells). A lymphocyte-depleting conditioning regimen was administered before treatment. E7 T cell infusion was followed by high-dose systemic aldesleukin. Results: Twelve patients were treated (DL1, n=3; DL2, n=3; DL3, n=6). The age range was 31 to 59 years. The site of the primary cancer was vulva (n=1), head and neck (n=4), uterine cervix (n=5), and anus (n=2). Each patient had multiple metastases and had previously received 3 to 7 anti-cancer agents. The conditioning regimen consisted of cyclophosphamide 30 mg/Kg (n=6) or 60 mg/Kg (n=6) iv daily for 2 days overlapping with fludarabine 25 mg/m2 iv daily for 5 days. The E7 TCR was expressed by 90-99% of the infused T cells for each patient. E7 T cell cross-reactivity against healthy tissues was not identified. Cytokine-release syndrome was not observed. A single patient, at DL3, experience dose-limiting toxicity. Four patients experienced confirmed responses, and two patients experienced unconfirmed responses (i.e. met criteria for response at only one assessment) (Figure 1). Confirmed responses occurred in patients with cervical cancer, oropharyngeal cancer, vulvar cancer, and anal cancer. The duration of responses was 3 months (ongoing), 4 months, 8 months, and 9 months, respectively. These patients had previously received 7, 4, 7 and 3 prior anti-cancer agents, respectively. Three patients with confirmed responses had previously received PD-1 or PD-L1 checkpoint blockade. Four patients whose cancer progressed after E7 T cells received PD-1 or PD-L1 checkpoint blockade; none responded. Conclusions: Tumor regression can occur following treatment of an epithelial cancer with gene-engineered T cells. These findings support continued study of E7 T cells and possibly other types of gene-engineered T cells in epithelial cancers. Disclosures Adhikary: Kite Pharma: Employment. Schweitzer:Kite Pharma: Employment. Astrow:Kite Pharma: Employment. Hinrichs:Kite Pharma: Research Funding; NIH: Patents & Royalties: NIH patents related to cell therapy.

Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express human leukocyte antigen (HLA) class I antigen–restricted, melanoma antigen–reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non–MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight melanoma-associated antigen (HMW-MAA), which is highly expressed on more than 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA–specific CARs containing an antigen recognition domain based on variations of the HMW-MAA–specific monoclonal antibody 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3ζ activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA–specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA–expressing cell lines. Furthermore, the receptor functioned in both CD4+ and CD8+ cells, was non–MHC restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA–specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies. Cancer Res; 70(8); 3027–33. ©2010 AACR.

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-γ (IFN-γ) production, whereas depletion of interleukin (IL)–17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer–based immunotherapy of human malignancies.

The differentiation state of CD8+ T cells has emerged as a crucial determinant of their ability to respond to tumor and infection. Signals from T-cell receptors, co-stimulatory molecules and cytokine receptors direct the differentiation process. These signals ‘program’ sustained and heritable gene expression patterns that govern progressive differentiation and lineage commitment. The epigenetic mechanisms by which T cells are programmed are just beginning to be elucidated. Understanding the mechanisms that control CD8+ T-cell differentiation is important in the development of novel immunotherapy strategies.

Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor γ chain (γc), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-γ production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R−/− mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.

3009 Background: Engineered T-cell therapy has shown promise in B-cell malignancies and melanoma, but clinical investigation in epithelial cancers has been limited. Methods: We conducted a phase I/II clinical trial of T cells genetically engineered to express a T-cell receptor that targets an HLA-A*02:01-restricted epitope of E6 (E6 TCR T Cells) for patients with metastatic HPV-16+ carcinoma. The cell dose was escalated in cohorts of single patients (1 x 109, 1 x 1010, and 1-2 x 1011cells). Patients received a nonmyeloablative conditioning regimen of cyclophosphamide and fludarabine, a single infusion of E6 TCR T Cells, and systemic high-dose aldesleukin. Results: Twelve patients were treated, 9 at the highest cell dose, plus one retreatment. The cancer types were 6 cervical, 4 anal, 1 oropharyngeal, and 1 vaginal. No dose-limiting toxicity, autoimmune adverse events, or cytokine storm were observed. Two patients with anal cancer treated at the highest cell dose experienced partial tumor responses lasting 6 and 3 months after treatment. The patient with a 6-month response had complete regression of one tumor and partial regression of two tumors that were resected upon progression; she has no evidence of disease 22 months after treatment. T-cell receptor gene transfer efficiency was 45 and 51% in the responding patients, and 47-76% (median 61%) in the non-responding patients. Responding patients showed robust levels of E6 TCR T cell memory (30 and 46% of circulating T cells 1-month after treatment). Non-responding patients showed wide-ranging levels of E6 TCR T cell memory (range 4-53%, median 29%). Expression of programmed cell death protein 1 (PD-1) by circulating E6 TCR T Cells 1-month after treatment was low in all patients ( < 5%). The patient with a 6-month response had 7% E6 TCR T Cells in a resected tumor 10 months after treatment, 25% of which expressed PD-1. A patient with no response had no detectable E6 TCR T Cells in a resected tumor 3 months after treatment. Conclusions: E6 TCR T-cell therapy was safe at doses up to 2 x 1011 cells. Regression of metastatic HPV+ carcinoma occurred in two patients following treatment, suggesting that TCR T-cell therapy can mediate epithelial cancer regression. Clinical trial information: NCT02280811.

T Cells for Epithelial Tumors Malignant tumors harbor genetic alterations. Recently, adoptive T cell therapies have taken advantage of this: T cells specific for mutations in tumors are infused into patients to generate an antitumor immune response. Although therapeutic benefit has been seen for melanomas, effectiveness against more common epithelial tumors is unclear. Using whole-exome sequencing, Tran et al. (p. 641 ) identified tumor-infiltrating CD4+ T cells specific for a mutated antigen expressed by a tumor from a patient with metastatic cholangiocarcinoma. Infusion of this patient with an expanded-population, mutation-specific T cell resulted in tumor regression and stabilization of disease.

Adoptive T-cell therapy (ACT) is a potent and flexible cancer treatment modality that can induce complete, durable regression of certain human malignancies. Long-term follow-up of patients receiving tumor-infiltrating lymphocytes (TILs) for metastatic melanoma reveals a substantial subset that experienced complete, lasting tumor regression - and may be cured. Increasing evidence points to mutated gene products as the primary immunological targets of TILs from melanomas. Recent technological advances permit rapid identification of the neoepitopes resulting from these somatic gene mutations and of T cells with reactivity against these targets. Isolation and adoptive transfer of these T cells may improve TIL therapy for melanoma and permit its broader application to non-melanoma tumors. Extension of ACT to other malignancies may also be possible through antigen receptor gene engineering. Tumor regression has been observed following transfer of T cells engineered to express chimeric antigen receptors against CD19 in B-cell malignancies or a T-cell receptor against NY-ESO-1 in synovial cell sarcoma and melanoma. Herein, we review recent clinical trials of TILs and antigen receptor gene therapy for advanced cancers. We discuss lessons from this experience and consider how they might be applied to realize the full curative potential of ACT.

Adoptive T-cell therapies for cancer can induce tumor responses in patients with metastatic melanoma, synovial sarcoma, and B-cell malignancies. However, no cellular therapy has demonstrated activity in a common epithelial tumor. Adoptive transfer of tumor infiltrating lymphocytes (TIL) has high response rates and can induce complete and durable tumor regression in metastatic melanoma. We sought to determine the objective tumor response rate of TIL therapy for human papillomavirus (HPV)-associated malignancies. These common epithelial cancers occur at varied sites but universally express the immunogenic E6 and E7 viral oncoproteins making them rational targets for T cell-based therapeutic approaches. Herein we report the results of treatment of the first eight subjects (seven evaluable) from the cohort of patients with cervical cancer (which is necessarily associated with high-risk HPV infection) treated with TIL therapy. Tumors were HPV genotyped and TIL were generated from individual tumor fragment cultures then tested for reactivity against autologous dendritic cells loaded with pools of overlapping peptides spanning type-specific HPV E6 or E7 proteins. Cultures were selected for rapid expansion and administration to patients based on oncoprotein reactivity, T-cell growth rate, and CD8+ T cell frequency. Patients received non-myeloablative lymphoconditioning chemotherapy before cell infusion and high-dose bolus IL-2 after cell infusion. TIL from 6/8 patients displayed reactivity against E6 or E7. Objective tumor responses by RECIST criteria were observed in 3/7 evaluable patients; two have complete and ongoing regression of measurable disease at three and nine months after treatment. Adverse events were the expected combined toxicities of the preparative chemotherapy regimen and interleukin-2, and were consistent with those of TIL therapy for metastatic melanoma. No autoimmunity was observed. These early clinical trial results indicate that tumor responses can occur following administration of TIL for metastatic cervical cancer.

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.

Cluster of differentiation (CD)8+ T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8+ T cells for patient treatments is controversial and understudied. We investigated human CD8+ T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or “exhaustion,” maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.

Effector cells derived from central memory CD8 + T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1 − phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8 + T cells may allow superior efficacy upon adoptive transfer.

Interleukin-17 (IL-17)–secreting CD8+ T cells have been described, but they have not been thoroughly studied and they do not have a known role in cancer immunotherapy. We skewed CD8+ T cells to secrete IL-17 through priming in Th17-polarizing conditions. IL-17–producing CD8+ T cells demonstrated reduced expression of Eomes and diminished cytolytic differentiation in vitro. However, after adoptive transfer, these cells converted to interferon-γ–producing effector cells and mediated regression of large, established tumors. This improved antitumor immunity was associated with increased expression of IL-7R-alpha, decreased expression of killer cell lectin-like receptor G1, and enhanced persistence of the transferred cells. This report is the first description of a cancer therapy with IL-17–secreting CD8+ T cells. These findings have implications for the improvement of CD8+ T cell–based adoptive immunotherapy.

Graft-versus-tumor effects can be achieved after allogeneic bone marrow transplantation in patients with malignancies of the kidney or hematopoietic system but are often accompanied by severe graft-versus-host-disease (GVHD). We sought to maximize graft-versus-tumor while minimizing GVHD using tumor-specific allogeneic effector T cells rather than open-repertoire T cells. We transferred allogeneic CD8+ pmel-1 or CD4+ TRP-1 T cells specific for the melanoma-associated antigens, glycoprotein 100 (gp100) and tyrosinase-related protein-1 (TRP-1), respectively, into B16-melanoma–bearing mice. Mice receiving a preparative regimen of nonmyeloablating (5 Gy) total body irradiation experienced the rapid rejection of tumor-specific allogeneic lymphocytes with no impact on tumor growth. However, when mice were given more intense total body irradiation conditioning regimens combined with autologous bone marrow transplantation, adoptively transferred allogeneic tumor-specific T lymphocytes persisted at detectable levels for several weeks and mediated significant regression of large, vascularized tumors. We found that the risk of GVHD was low when tumor-specific T cells were transferred and significant toxicity was observed only when substantial numbers of open repertoire allogeneic naive T cells were mixed with the tumor-specific lymphocytes. Taken together, these data indicate that the use of tumor-specific allogeneic CD8+ T cells or CD4+ can result in significant antitumor effects in the absence of measurable GVHD.

Nonmutated tissue differentiation antigens expressed by tumors are attractive targets for cancer immunotherapy, but the consequences of a highly effective antitumor immune response on self-tissue have not been fully characterized. We found that the infusion ofex vivoexpanded adoptively transferred melanoma/melanocyte-specific CD8+T cells that mediated robust tumor killing also induced autoimmune destruction of melanocytes in the eye. This severe autoimmunity was associated with the up-regulation of MHC class I molecules in the eye and high levels of IFN-γ derived from both adoptively transferred CD8+T cells and host cells. Furthermore, ocular autoimmunity required the presence of the IFN-γ receptor on target tissues. Data compiled from >200 eyes and tumors in 10 independently performed experiments revealed a highly significant correlation (P< 0.0001) between the efficacy of tumor immunotherapy and the severity of ocular autoimmunity. Administration of high doses of steroids locally mitigated ocular autoimmunity without impairing the antitumor effect. These findings have particular importance for immunotherapies directed against self-antigens and highlight the need for targeting unique tumor antigens not expressed in critical tissues.

IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8+ T cells, but their distinct effects on antigen-driven differentiation of naive CD8+ T cells into effector CD8+ T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8+ T cells into granzyme B- and CD44-expressing effector CD8+ T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2–mediated acquisition of a cytolytic CD8+ T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8+ T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.

Lymphodepletion with chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells is a critical advancement in the treatment of patients with melanoma. More than 50% of patients that are refractory to other treatments experience an objective or curative response with this approach. Emerging data indicate that the key mechanisms underlying how TBI augments the functions of adoptively transferred T cells include (a) the depletion of regulatory T cells (Treg) and myeloid-derived suppressor cells that limit the function and proliferation of adoptively transferred cells; (b) the removal of immune cells that act as “sinks” for homeostatic cytokines, whose levels increase after lymphodepletion; and (c) the activation of the innate immune system via Toll-like receptor 4 signaling, which is engaged by microbial lipopolysaccharide that translocated across the radiation-injured gut. Here, we review these mechanisms and focus on the effect of Toll-like receptor agonists in adoptive immunotherapy. We also discuss alternate regimens to chemotherapy or TBI, which might be used to safely treat patients with advanced disease and promote tumor regression.

From the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland. Summary Glucocorticoids are potent immunosuppressive drugs that are generally withheld from cancer patients receiving immunotherapy. We sought to test the hypothesis that glucocorticoids might interfere with the function of cells after adoptive transfer. We gave dexamethasone, a potent synthetic glucocorticoid, to B16 melanoma–bearing mice receiving the adoptive cell transfer (ACT) of pmel-1 T-cell receptor transgenic CD8 + cells. Dexamethasone caused a profound lymphodepletion but, surprisingly, did not alter the antitumor efficacy of ACT-based regimens whether given before, during, or after ACT. Although dexamethasone radically decreased the number of native CD8 + splenocytes in recipient mice, it did not affect the numbers of CD8 + pmel-1 cells derived from ACT in these mice. In vitro proliferation assays revealed acute inhibition of naive pmel-1 CD8 + cells by dexamethasone without significant effect on activated cells. In vitro interferon (IFN)-γ release from activated pmel-1 CD8 + cells showed partial inhibition by dexamethasone, but this effect was relatively minor when compared with the near-complete inhibition of naive cells. Thus, glucocorticoids had a profound inhibitory effect on naive CD8 + T cells but had little impact on the proliferation and function of activated CD8 + pmel-1 T cells. Finally, because glucocorticoids had no effect on tumor regression in this model, it may be possible to use glucocorticoids in some patients receiving ACT-based immunotherapy regimens.

Immunotherapy using adoptive cell transfer is a promising approach that can result in the regression of bulky, invasive cancer in some patients. However, currently available therapies remain less successful than desired. To study the mechanisms of action and possible improvements in cell-transfer therapies, we use a murine model system with analogous components to the treatment of patients. T cell receptor transgenic CD8+ T cells (pmel-1) specifically recognizing the melanocyte differentiation antigen gp100 are adoptively transferred into lympho-depleted mice bearing large, established, 14-day subcutaneous B16 melanoma (0.5–1 cm in diameter) on the day of treatment. Adoptive cell transfer in combination with interleukin interleukin-2 or interleukin-15 cytokine administration and vaccination using an altered form of the target antigen, gp100, can result in the complete and durable regression of large tumor burdens. Complete responders frequently develop autoimmunity with vitiligo at the former tumor site that often spreads to involve the whole coat. These findings have important implications for the design of immunotherapy trials in humans.

Introduction: Adoptively transferred antigen-specific CD8+ T-cells can specifically produce IFN-gamma in response to tumor stimulation. We hypothesized that IFN-gamma production solely by adoptively transferred T-cells was required for anti-tumor efficacy. Methods: A murine model with analogous components to the treatment of patients was established. CD8+ T-cell receptor gp100-specific transgenic mice (Pmel-1) were crossed with IFN-gamma knockout mice (IFN-gamma−/−). Pmel-1 and Pmel-1IFN-gamma−/− cells were analyzed in vitro. In vivo function was assessed by adoptive transfer of Pmel-1 or Pmel-1IFN-gamma−/− cells into wild-type C57BL/6 or IFN-gamma−/− mice bearing large, established subcutaneous B16 melanoma (> 50mm 2 ). Cell transfer was undertaken in combination with active immunization with fowlpox virus encoding human gp100 (rFPVhgp100) plus interleukin-2 (IL-2). Tumor size (rank-sum test) and survival (Kaplan-Meier) were analyzed. Results: Pmel-1IFN-gamma−/− cells did not produce IFN-gamma, in vitro; yet, Pmel-1 and Pmel-1IFN-gamma−/− cells were able to produce GM-CSF, TNF-alpha, MIP-1alpha, and RANTES. Adoptive transfer of Pmel-1+rFPVhgp100+IL-2 led to the regression of large tumor burden in both wild-type and IFN-gamma−/− hosts. Pmel-1IFN-gamma−/− T-lymphocytes were less efficacious in a wild-type host. However, when Pmel-1IFN-gamma−/− cells were employed into IFN-gamma−/− hosts, therapeutic efficacy was abrogated with tumor growth similar to no treatment controls. Conclusions: These data suggest that IFN-gamma is essential for treatment of large, established melanoma, but this cytokine is not necessarily produced solely by adoptively transferred antigen specific T-lymphocytes and may be provided by the host's cells. These findings have important implications for the design of immunotherapy trials in humans.

Abstract 468 We have recently demonstrated that Th17-polarized TCR transgenic CD4+ T cells specific for TRP-1 melanoma antigen are superior to Th1-polarized cells in mediating effective anti-tumor responses against advanced disease after adoptive transfer. The therapeutic activity of Th17-skewed cells is critically dependent on their ability to secrete IFN-γ, suggesting that the Th17 subset might evolve in vivo. However, the developmental program of Th17-polarized cells in vivo remains substantially un- elucidated. We developed a novel TCR-transduction technique that enabled us to rapidly confer specificity for a cognate antigen upon any population of T cells, regardless of its genetic background, its previous polarization history or its state of differentiation. Using adoptive transfers into tumor-bearing hosts, we were able to study the functionality of these genetically-engineered T cells in vivo. In vitro, CD4+ T cells cultured in type 17 conditions acquired end-effector phenotype (CD62Llow, CD45RBlow), but proliferated slower than cells grown in type 1 condition. Thus, we hypothesized that Th17-polarized cells might represent a less mature, more central-memory like subset. This notion was supported by their ability to secrete high quantities of IL-2 and higher expression of IL-7 receptor. In contrast, Th1-polarized cells upon in vitro re-stimulation upregulated PRDM1 that encodes BLIMP1, a molecule associated with the end-effector senescent phenotype. Moreover, Th1-skewed cells overexpressed caspase 3 and were prone to activation-induced cell death as measured by annexin V assay, while type 17 cells were resistant to apoptosis, and robustly expanded in secondary cultures. Using the TCR gene transfer technique we tested the treatment outcomes when Th17-polarized cells deficient for IL-17A were used. In contrast to wild-type (WT)-derived Th17 cells that effectively eradicated established tumors, we observed significant impairment of treatment with IL-17A-deficent cells. Similarly, we observed reduction in treatment efficacy when CCR6-deficient Th17 cells were transferred. CCR6 is a receptor for CCL20, a chemokine highly induced Th17 cells and thought to contribute to the trafficking of those cells to the site of inflammation. In both cases however, the addition of exogenous vaccination and IL-2 significantly improved treatment efficacy. Thus, we concluded that Th17-associated factors play the role in the anti-cancer activity of type 17 cells. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-skewed cells derived from IFN-γ-deficient CD4+ cells as well as from t-bet-deficient mice, which are not able to develop type 1 responses. In contrast to WT-derived Th17 effectors, IFN-γ-deficient cells did not show any anti-tumor activity, while t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the capacity to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Overall, here we demonstrate that TCR gene engineered Th17-polarized cells can efficiently treat advanced tumor. The high activity of in vitro-generated anti-tumor Th17 cells relies on the contribution of type 17-associated characteristics, including both the secretion of inflammatory factors IL-17A and CCL20, as well as the superior capacity to survive and expand upon the secondary stimulation. Importantly however, type 1-defining t-bet-mediated plasticity in the lineage commitment is required for the full therapeutic effect, underscoring the dualistic nature of Th17-skewed cells. Disclosures: No relevant conflicts of interest to declare.

Adoptive cell transfer (ACT) of tumor-specific T lymphocytes is a powerful strategy for targeted therapy of cancer, but most of the research in this area has been focused on cytotoxic CD8+ T cells, which directly lyse MHC class I expressing targets. We have recently demonstrated that CD4+ TCR transgenic Th cells specific for self/tumor antigen tyrosinase-related protein 1 (TRP-1) have the ability to reject large established B16 murine melanoma in a model closely mimicking advanced human disease. Moreover, we showed that Th17-polarized cells were more effective in mediating complete tumor rejection than Th1-skewed cells that were capable of producing high quantities of interferon γ (IFN-γ). Interestingly, while Th1 and Th17 populations varied significantly in their phenotype, cytokines profiles, persistence and proliferation patterns in vivo, the Th17 anti-tumor function was critically dependent on the ability of the transferred cells to secrete IFN-γ. This suggests that the Th17 population might gradually acquire Th1-like properties in vivo, and that transcription factors regulating Th17 differentiation (ROR- γt) as well as IFN-γ production and Th1 polarization (t-bet) might be crucial for the effective rejection of the tumor. In order to emulate clinically relevant gene-therapy scenario we inserted TRP-1 TCR into open-repertoire CD4+ T cells from wild-type donors using a retroviral vector. Prior to transduction Th cells were stimulated under neutral (Th0) and polarizing Th1 and Th17 conditions. The majority of transduced cells expressed the Vβ14 chain, released appropriate polarization-defining cytokines upon specific antigenic stimulation in vitro and caused development of massive autoimmune vitiligo upon adoptive cell transfer into wild-type and Rag1−l/&minus mice. Gene-modified cells were readily detectable in Rag1−/− animals by flow-cytometry for more than 4 month after transfer. No off-target GVHD-like toxicities resulting from potential miss-pairing of endogenous and inserted TCR chains were observed. Th0 or Th1 and Th17-polarized TCR-transduced cells were all capable of treating mice bearing large (50–100mm2) B16 tumors, but complete cures with long-term survival occurred more robustly in animals treated with Th17-polarized effectors. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-polarized cells derived from t-bet-deficient donors, which are not able to develop Th1-type responses, most importantly, not capable of producing IFN-γ. In contrast to WT-derived Th17 effectors used as a control, t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the ability to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Currently, the clinical effectiveness of the ACT therapy might be hampered by the lack of high-avidity autologous effectors recognizing self/tumor antigens due to the central tolerance mechanisms. Here we demonstrate that the mature effector Th cells can be genetically engineered to express TCR recognizing MHC class II self/tumor antigen and those cells mediate powerful anti-cancer effect in vivo in a realistic model. While tumor-specific Th17-skewed CD4+ T cells are most effective in this setting, t-bet-mediated plasticity in lineage commitment is required for the full therapeutic effect.

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.

IL-2 is a critical T cell growth factor in vitro, but predominantly mediates tolerance in vivo. IL-2 is mainly produced by CD4+ Th cells, but the role of Th cell-derived IL-2 in vivo is controversial. We demonstrate that during immunity to a tumor/self-Ag, the predominant role of Th cell-derived IL-2 was to maintain IL-2Rα (CD25) on CD4+ T regulatory cells (Treg), which resulted in their maintenance of the Treg cell lineage factor, Forkhead/winged helix transcription factor (Foxp3), and tolerance. However, in the absence of Treg cells, Th cell-derived IL-2 maintained effector T cells and caused autoimmunity. IL-2R signaling was indispensable for Treg cell homeostasis and efficient suppressor function in vivo, but, surprisingly, was not required for their generation, because IL-2−/− and CD25−/− mice both contained Foxp3+ T cells in the periphery. IL-2R signaling was also important for CD8+ T cell immunity, because CD25−/− tumor-reactive CD8+ T cells failed to affect established tumors. Conversely, IL-2R signaling was not required for Th cell function. Lastly, administration of anti-IL-2 plus exogenous IL-15 to tumor-bearing mice enhanced the adoptive immunotherapy of cancer. Therefore, Th cell-derived IL-2 paradoxically controls both tolerance and immunity to a tumor/self-Ag in vivo.

Meeting abstracts Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to