Veena Kapoor

Polarization of Tumor-Associated Neutrophil Phenotype by TGF-β: “N1” versus “N2” TAN

TGF-beta blockade significantly slows tumor growth through many mechanisms, including activation of CD8(+) T cells and macrophages. Here, we show that TGF-beta blockade also increases neutrophil-attracting chemokines, resulting in an influx of CD11b(+)/Ly6G(+) tumor-associated neutrophils (TANs) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of proinflammatory cytokines. Accordingly, following TGF-beta blockade, depletion of these neutrophils significantly blunts antitumor effects of treatment and reduces CD8(+) T cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8(+) T cells intratumorally. Together, these data suggest that TGF-beta within the tumor microenvironment induces a population of TAN with a protumor phenotype. TGF-beta blockade results in the recruitment and activation of TANs with an antitumor phenotype.

Gemcitabine Selectively Eliminates Splenic Gr-1+/CD11b+ Myeloid Suppressor Cells in Tumor-Bearing Animals and Enhances Antitumor Immune Activity

Purpose: Myeloid suppressor (Gr-1+/CD11b+) cells accumulate in the spleens of tumor-bearing mice where they contribute to immunosuppression by inhibiting the function of CD8+ T cells and by promoting tumor angiogenesis. Elimination of these myeloid suppressor cells may thus significantly improve antitumor responses and enhance effects of cancer immunotherapy, although to date few practical options exist. Experimental Design: The effect of the chemotherapy drug gemcitabine on the number of (Gr-1+/CD11b+) cells in the spleens of animals bearing large tumors derived from five cancer lines grown in both C57Bl/6 and BALB/c mice was analyzed. Suppressive activity of splenocytes from gemcitabine-treated and control animals was measured in natural killer (NK) cell lysis and Winn assays. The impact of myeloid suppressor cell activity was determined in an immunogene therapy model using an adenovirus expressing IFN-β. Results: This study shows that the chemotherapeutic drug gemcitabine, given at a dose similar to the equivalent dose used in patients, was able to dramatically and specifically reduce the number of myeloid suppressor cells found in the spleens of animals bearing large tumors with no significant reductions in CD4+ T cells, CD8+ T cells, NK cells, macrophages, or B cells. The loss of myeloid suppressor cells was accompanied by an increase in the antitumor activity of CD8+ T cells and activated NK cells. Combining gemcitabine with cytokine immunogene therapy using IFN-β markedly enhanced antitumor efficacy. Conclusions: These results suggest that gemcitabine may be a practical strategy for the reduction of myeloid suppressor cells and should be evaluated in conjunction with a variety of immunotherapy approaches.

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (Treg) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking Treg cells develop severe autoimmune disease, and depletion of Treg cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of Treg cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2–responsive Treg cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain Treg cells, the question arises as to the effects of IL-2 therapy on them. We monitored Treg cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4+CD25hi cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the Treg cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent Treg cells in normal hosts, and IL-2–induced Treg cell expansion was further augmented by lymphopenia. On a per-cell basis, Treg cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to Treg cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4+CD25+ Treg cell homeostasis.

Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T cells Expressing a Mesothelin-Specific Chimeric Antibody Receptor

Purpose: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects. Experimental Design: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors. Results: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity. Conclusions: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo. Clin Cancer Res; 17(14); 4719–30. ©2011 AACR.

The PDL1-PD1 axis converts human TH1 cells into regulatory T cells.

The inhibitory ligand PDL1 transforms immune cells from attackers into regulators. PDL1: Restoring the Peace With great power comes great responsibility. In superhero lore, special powers don’t separate the saviors from the evil villains they fight; instead, what matters is how the person behind the mask uses those powers. Immune cells are the superheroes of the body—they fight off infection and patrol the body for cancer. However, sometimes, even protective cells “go bad,” causing autoimmunity or graft-versus-host disease after transplant. Amarnath et al. now show that an inhibitory protein called programmed death ligand 1 (PDL1) can regulate renegade immune cells by converting immune response–promoting T helper type 1 (TH1) cells to regulatory T (Treg) cells—agents that selectively suppress activation of the immune system. TH1 cells secrete proinflammatory cytokines and are critical for the immune response to infection and cancer cells. In contrast to other subsets of TH cells, researchers believed TH1 cells to be relatively stable. However, PDL1 caused human TH1 cells to convert to Treg cells both in vitro and in vivo. These TH1-derived Treg cells inhibited graft-versus-host disease in mice after transplant. Moreover, inhibiting Treg differentiation by blocking the PDL1 receptor PD1 or pharmacologically inhibiting SHP1 and SHP2, which are signaling molecules that act downstream of PD1 activation, restored graft-versus-host disease in mice. These data provide the basis for future therapies: Because PDL1 is highly expressed on many cancers, inhibiting this pathway may restore T cell–mediated cancer surveillance; alternately, accentuating signaling through this pathway may prevent autoimmunity or graft-versus-host disease. With this knowledge, scientists and doctors may be able to ensure that T cells are the superheroes they are meant to be. Immune surveillance by T helper type 1 (TH1) cells is not only critical for the host response to tumors and infection, but also contributes to autoimmunity and graft-versus-host disease (GVHD) after transplantation. The inhibitory molecule programmed death ligand 1 (PDL1) has been shown to anergize human TH1 cells, but other mechanisms of PDL1-mediated TH1 inhibition such as the conversion of TH1 cells to a regulatory phenotype have not been well characterized. We hypothesized that PDL1 may cause TH1 cells to manifest differentiation plasticity. Conventional T cells or irradiated K562 myeloid tumor cells overexpressing PDL1 converted TBET+ TH1 cells into FOXP3+ regulatory T (Treg) cells in vivo, thereby preventing human-into-mouse xenogeneic GVHD (xGVHD). Either blocking PD1 expression on TH1 cells by small interfering RNA targeting or abrogation of PD1 signaling by SHP1/2 pharmacologic inhibition stabilized TH1 cell differentiation during PDL1 challenge and restored the capacity of TH1 cells to mediate lethal xGVHD. PD1 signaling therefore induces human TH1 cells to manifest in vivo plasticity, resulting in a Treg phenotype that severely impairs cell-mediated immunity. Converting human TH1 cells to a regulatory phenotype with PD1 signaling provides a potential way to block GVHD after transplantation. Moreover, because this conversion can be prevented by blocking PD1 expression or pharmacologically inhibiting SHP1/2, this pathway provides a new therapeutic direction for enhancing T cell immunity to cancer and infection.

Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils

The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.

Multifactorial T-cell Hypofunction That Is Reversible Can Limit the Efficacy of Chimeric Antigen Receptor–Transduced Human T cells in Solid Tumors

Purpose: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens. Experimental Design: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4–1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways. Results: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4). Conclusions: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell–based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function. Clin Cancer Res; 20(16); 4262–73. ©2014 AACR.

A Phase I Clinical Trial of Single-Dose Intrapleural IFN-β Gene Transfer for Malignant Pleural Mesothelioma and Metastatic Pleural Effusions: High Rate of Antitumor Immune Responses

Purpose: This phase 1 dose escalation study evaluated the safety and feasibility of single-dose intrapleural IFN-β gene transfer using an adenoviral vector (Ad.IFN-β) in patients with malignant pleural mesothelioma (MPM) and metastatic pleural effusions (MPE). Experimental Design: Ad.IFN-β was administered through an indwelling pleural catheter in doses ranging from 9 × 1011 to 3 × 1012 viral particles (vp) in two cohorts of patients with MPM (7 patients) and MPE (3 patients). Subjects were evaluated for (a) toxicity, (b) gene transfer, (c) humoral, cellular, and cytokine-mediated immune responses, and (d) tumor responses via 18-fluorodeoxyglucose-positron emission tomography scans and chest computed tomography scans. Results: Intrapleural Ad.IFN-β was generally well tolerated with transient lymphopenia as the most common side effect. The maximally tolerated dose achieved was 9 × 1011 vp secondary to idiosyncratic dose-limiting toxicities (hypoxia and liver function abnormalities) in two patients treated at 3 × 1012 vp. The presence of the vector did not elicit a marked cellular infiltrate in the pleural space. Intrapleural levels of cytokines were highly variable at baseline and after response to gene transfer. Gene transfer was documented in 7 of the 10 patients by demonstration of IFN-β message or protein. Antitumor immune responses were elicited in 7 of the 10 patients and included the detection of cytotoxic T cells (1 patient), activation of circulating natural killer cells (2 patients), and humoral responses to known (Simian virus 40 large T antigen and mesothelin) and unknown tumor antigens (7 patients). Four of 10 patients showed meaningful clinical responses defined as disease stability and/or regression on 18-fluorodeoxyglucose-positron emission tomography and computed tomography scans at day 60 after vector infusion. Conclusions: Intrapleural instillation of Ad.IFN-β is a potentially useful approach for the generation of antitumor immune responses in MPM and MPE patients and should be investigated further for overall clinical efficacy.

4-1BB Is Superior to CD28 Costimulation for Generating CD8+ Cytotoxic Lymphocytes for Adoptive Immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.

NAFLD causes selective CD4 + T lymphocyte loss and promotes hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4+ but not CD8+ T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4+ T lymphocytes have greater mitochondrial mass than CD8+ T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4+ T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4+ T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.