Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells.

IL-17-producing antigen-specific human T cells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ T cells, the degree to which T cell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per T cell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Ex vivo blockade of PI3K gamma or delta signaling enhances the antitumor potency of adoptively transferred CD8+ T cells.

Adoptive T cell transfer therapy induces objective responses in patients with advanced malignancies. Despite these results, some individuals do not respond due to the generation of terminally differentiated T cells during the expansion protocol. As the gamma and delta catalytic subunits in the PI3K pathway are abundant in leukocytes and involved in cell activation, we posited that blocking both subunits ex vivo with the inhibitor IPI-145 would prevent their differentiation, thereby increasing antitumor activity in vivo. However, IPI-145 treatment generated a product with reduced antitumor activity. Instead, T cells inhibited of PI3Kγ (IPI-549) or PI3Kδ (CAL-101 or TGR-1202) alone were more potent in vivo. While T cells coinhibited of PI3Kγ and PI3Kδ were less differentiated, they were functionally impaired, indicated by reduced production of effector cytokines after antigenic re-encounter and decreased persistence in vivo. Human CAR T cells expanded with either a PI3Kγ or PI3Kδ inhibitor possessed a central memory phenotype compared to vehicle cohorts. We also found that PI3Kδ-inhibited CARs lysed human tumors in vitro more effectively than PI3Kγ-expanded or traditionally expanded CAR T cells. Our data imply that sole blockade of PI3Kγ or PI3Kδ generates T cells with remarkable antitumor properties, a discovery that has substantial clinical implications.

Distinct host preconditioning regimens differentially impact the antitumor potency of adoptively transferred Th17 cells.

BACKGROUND: How distinct methods of host preconditioning impact the efficacy of adoptively transferred antitumor T helper cells is unknown. METHODS: CD4+ T cells with a transgenic T-cell receptor that recognize tyrosinase-related peptide (TRP)-1 melanoma antigen were polarized to the T helper 17 (Th17) phenotype and then transferred into melanoma-bearing mice preconditioned with either total body irradiation or chemotherapy. RESULTS: We found that preconditioning mice with a non-myeloablative dose of total body irradiation (TBI of 5 Gy) was more effective than using an equivalently dosed non-myeloablative chemotherapy (cyclophosphamide (CTX) of 200 mg/kg) at augmenting therapeutic activity of antitumor TRP-1 Th17 cells. Antitumor Th17 cells engrafted better following preconditioning with TBI and regressed large established melanoma in all animals. Conversely, only half of mice survived long-term when preconditioned with CTX and infused with anti-melanoma Th17 cells. Interleukin (IL)-17 and interferon-γ, produced by the infused Th17 cells, were detected in animals given either TBI or CTX preconditioning. Interestingly, inflammatory cytokines (granulocyte colony stimulating factor, IL-6, monocyte chemoattractant protein-1, IL-5, and keratinocyte chemoattractant) were significantly elevated in the serum of mice preconditioned with TBI versus CTX after Th17 therapy. The addition of fludarabine (FLU, 200 mg/kg) to CTX (200 mg/kg) improved the antitumor response to the same degree mediated by TBI, whereas FLU alone with Th17 therapy was ineffective. CONCLUSIONS: Our results indicate, for the first time, that the antitumor response, persistence, and cytokine profiles resulting from Th17 therapy are impacted by the specific regimen of host preconditioning. This work is important for understanding mechanisms that promote long-lived responses by adoptive cellular therapy, particularly as CD4+ based T-cell therapies are now emerging in the clinic.

Progressively Enhancing Stemness of Adoptively Transferred T Cells with PI3Kδ Blockade Improves Metabolism and Antitumor Immunity.

Generating stem-like memory T cells (TSCM) is a potential strategy to improve adoptive immunotherapy. Elucidating optimal ways to modulate signaling pathways that enrich TSCM properties could identify approaches to achieve this goal. We discovered herein that blocking the PI3Kδ pathway pharmaceutically to varying degrees can generate T cells with increasingly heightened stemness properties, based on the progressive enrichment of the transcription factors Tcf1 and Lef1. T cells with enhanced stemness features exhibited metabolic plasticity, marked by improved mitochondrial function and glucose uptake after tumor recognition. Conversely, T cells with low or medium stemness were less metabolically dynamic, vulnerable to antigen-induced cell death, and expressed more inhibitory checkpoint receptors. Only T-cell receptor-specific or chimeric antigen receptor (CAR)-specific T cells with high stemness persisted in vivo and mounted protective immunity to tumors. Likewise, the strongest level of PI3Kδ blockade in vitro generated human tumor-infiltrating lymphocytes and CAR T cells with elevated stemness properties, in turn bolstering their capacity to regress human solid tumors. The stemness level of T cells in vitro was important, ultimately impacting their efficacy in mice bearing three distinct solid tumors. Lef1 and Tcf1 sustained antitumor protection by donor high CD8+ TSCM or CD4+ Th17SCM, as deletion of either one compromised the therapeutic efficacy. Collectively, these findings highlight the importance of strategic modulation of PI3Kδ signaling in T cells to induce stemness and lasting protective responses to solid tumors. SIGNIFICANCE: Elevating T-cell stemness by progressively blocking PI3Kδ signaling during ex vivo manufacturing of adoptive cell therapies alters metabolic and functional properties to enhance antitumor immunity dependent on Tcf1 and Lef1.

Distinct host preconditioning regimens differentially impact the antitumor potency of adoptively transferred Th17 cells.

Background: Mechanisms by which distinct methods of host preconditioning impact the efficacy of adoptively transferred antitumor T helper cells is unknown. Methods: CD4 + T cells with a transgenic TCR that recognize TRP-1 melanoma antigen were polarized to the T helper 17 (Th17) phenotype and then transferred into melanoma-bearing mice preconditioned with either total body irradiation or chemotherapy. Results: We found that preconditioning mice with a non-myeloablative dose of total body irradiation (TBI of 5 Gy) was more effective than using an equivalently dosed non-myeloablative chemotherapy (CTX at 200 mg/kg) at augmenting therapeutic activity of anti-tumor TRP-1 Th17 cells. Anti-tumor Th17 cells engrafted better following preconditioning with TBI and regressed large established melanoma in all animals. Conversely, only half of mice survived long-term when preconditioned with CTX and infused with anti-melanoma Th17 cells. IL-17 and IFN-g produced by the infused Th17 cells, were detected in animals given either TBI or CTX preconditioning. Interestingly, inflammatory cytokines (G-CSF, IL-6, MCP-1, IL-5, and KC) were significantly elevated in the serum of mice preconditioned with TBI versus CTX after Th17 therapy. Conclusions: Our results indicate, for the first time, that the antitumor response, persistence, and cytokine profiles resulting from Th17 therapy are impacted by the specific regimen of host preconditioning. This work is important for understanding mechanisms that promote long-lived responses by ACT, particularly as CD4 + based T cell therapies are now emerging in the clinic.

The degree of T cell stemness differentially impacts the potency of adoptive cancer immunotherapy in a Lef-1 and Tcf-1 dependent manner.

Generating stem memory T cells (T SCM ) is a key goal for improving cancer immunotherapy. Yet, the optimal way to modulate signaling pathways that enrich T SCM properties remains elusive. Here, we discovered that the degree to which the PI3Kδ pathway is blocked pharmaceutically can generate T cells with differential levels of stemness properties. This observation was based on the progressive enrichment of transcriptional factors of stemness (Tcf-1 and Lef-1). Additional investigation revealed that T cells with high stemness features had enhanced metabolic plasticity, marked by heightened mitochondrial function and glucose uptake. Conversely, T cells with low or medium features of stemness expressed more inhibitory checkpoint receptors (Tim-3, CD39) and were vulnerable to antigen-induced cell death. Only TCR-antigen specific T cells with high stemness persisted following adoptive transfer in vivo and mounted protective immunity to melanoma tumors. Likewise, the strongest level of PI3Kδ blockade in vitro generated human tumor infiltrating lymphocytes (TILs) and CAR T cells with heightened stemness properties, in turn bolstering their capacity to regress human mesothelioma tumors. We find that the level of stemness T cells possess in vitro differentially impacts their potency upon transfer in three tumor models. Mechanistically, both Lef-1 and Tcf-1 sustain anti-tumor protection by high T SCM , as deletion of either one compromised cellular therapy. Collectively, these findings highlight the therapeutic potential of carefully modulating PI3Kδ signaling in T cells to confer high stemness and mediate protective responses to solid tumors.

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity.

BACKGROUND: Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity. METHODS: In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS: Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS: Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Fundamentals of T Cell Metabolism and Strategies to Enhance Cancer Immunotherapy.

Emerging reports show that metabolic pathways can be targeted to enhance T cell-mediated immunity to tumors. Yet, tumors consume key metabolites in the host to survive, thus robbing T cells of these nutrients to function and thrive. T cells are often deprived of basic building blocks for energy in the tumor, including glucose and amino acids needed to proliferate or produce cytotoxic molecules against tumors. Immunosuppressive molecules in the host further compromise the lytic capacity of T cells. Moreover, checkpoint receptors inhibit T cell responses by impairing their bioenergetic potential within tumors. In this review, we discuss the fundamental metabolic pathways involved in T cell activation, differentiation and response against tumors. We then address ways to target metabolic pathways to improve the next generation of immunotherapies for cancer patients.

Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16F10 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TIL are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and human oral cavity cancer TIL, evaluating relationships between inhibitory receptor expression and function. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.

IL6 Fuels Durable Memory for Th17 Cell-Mediated Responses to Tumors.

The accessibility of adoptive T-cell transfer therapies (ACT) is hindered by the cost and time required for product development. Here we describe a streamlined ACT protocol using Th17 cells expanded only 4 days ex vivo. While shortening expansion compromised cell yield, this method licensed Th17 cells to eradicate large tumors to a greater extent than cells expanded longer term. Day 4 Th17 cells engrafted, induced release of multiple cytokines including IL6, IL17, MCP-1, and GM-CSF in the tumor-bearing host, and persisted as memory cells. IL6 was a critical component for efficacy of these therapies via its promotion of long-term immunity and resistance to tumor relapse. Mechanistically, IL6 diminished engraftment of FoxP3+ donor T cells, corresponding with robust tumor infiltration by donor effector over regulatory cells for the Day 4 Th17 cell product relative to cell products expanded longer durations ex vivo. Collectively, this work describes a method to rapidly generate therapeutic T-cell products for ACT and implicates IL6 in promoting durable immunity of Th17 cells against large, established solid tumors. SIGNIFICANCE: An abbreviated, 4-day ex vivo expansion method licenses Th17 cells to confer long-lived immunity against solid malignancies via induction of systemic IL6 in the host.See related commentary by Fiering and Ho, p. 3795.

Fueling Cancer Immunotherapy With Common Gamma Chain Cytokines.

Adoptive T cell transfer therapy (ACT) using tumor infiltrating lymphocytes or lymphocytes redirected with antigen receptors (CAR or TCR) has revolutionized the field of cancer immunotherapy. Although CAR T cell therapy mediates robust responses in patients with hematological malignancies, this approach has been less effective for treating patients with solid tumors. Additionally, toxicities post T cell infusion highlight the need for safer ACT protocols. Current protocols traditionally expand T lymphocytes isolated from patient tumors or from peripheral blood to large magnitudes in the presence of high dose IL-2 prior to infusion. Unfortunately, this expansion protocol differentiates T cells to a full effector or terminal phenotype in vitro, consequently reducing their long-term survival and antitumor effectiveness in vivo. Post-infusion, T cells face further obstacles limiting their persistence and function within the suppressive tumor microenvironment. Therapeutic manipulation of T cells with common γ chain cytokines, which are critical growth factors for T cells, may be the key to bypass such immunological hurdles. Herein, we discuss the primary functions of the common γ chain cytokines impacting T cell survival and memory and then elaborate on how these distinct cytokines have been used to augment T cell-based cancer immunotherapy.

Sex Differences in Using Systemic Inflammatory Markers to Prognosticate Patients with Head and Neck Squamous Cell Carcinoma.

Background: Remarkable discrepancy exists in outcomes between men and women for multiple malignancies. We sought to expose sex differences in using platelet count and neutrophil-to-lymphocyte ratio (NLR) to predict overall survival for select cancer types with focus on head and neck squamous cell carcinoma (HNSCC).Methods: Peripheral blood samples from 9,365 patients seen in a tertiary teaching hospital with nine different primary tumors were retrospectively examined. HNSCC RNA-sequencing data from The Cancer Genome Atlas were analyzed by two computational means [Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE)] to extend our observations to the tumor microenvironment.Results: For HNSCC, platelet count was more predictive of overall survival for males [log-rank test: HR = 1.809; 95% confidence interval (CI), 1.461-2.239 vs. HR = 1.287; 95% CI, 0.8901-1.861], whereas NLR was more predictive for females (HR = 2.627; 95% CI, 1.716-4.02 vs. HR = 1.261; 95% CI, 0.998-1.593). For females, lymphocyte count was more associated with survival than neutrophil count (multivariate Cox regression: P = 0.0015 vs. P = 0.7476). Both CIBERSORT (P = 0.0061) and ESTIMATE (P = 0.022) revealed greater immune infiltration in females. High tumor infiltration by T lymphocytes was more strikingly associated with survival in females (HR = 0.20, P = 0.0281) than in males (HR = 0.49, P = 0.0147).Conclusions: This is the first study to comprehensively demonstrate sex bias in the clinical utility of platelet, granulocyte, and lymphocyte counts as biomarkers to prognosticate HNSCC patients.Impact: This work emphasizes the necessity to consider sex in appraising inflammatory markers for cancer risk stratification. Cancer Epidemiol Biomarkers Prev; 27(10); 1176-85. ©2018 AACR.

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma.

Ewing sarcoma cells depend on the EWS-FLI1 fusion transcription factor for cell survival. Using an assay of EWS-FLI1 activity and genome-wide RNAi screening, we have identified proteins required for the processing of the EWS-FLI1 pre-mRNA. We show that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require the RNA-binding protein HNRNPH1 to express in-frame EWS-FLI1. We also demonstrate the sensitivity of EWS-FLI1 fusion transcripts to the loss of function of the U2 snRNP component, SF3B1. Disrupted splicing of the EWS-FLI1 transcript alters EWS-FLI1 protein expression and EWS-FLI1-driven expression. Our results show that the processing of the EWS-FLI1 fusion RNA is a potentially targetable vulnerability in Ewing sarcoma cells.