The expansion of immunosuppressive cells represents a cardinal strategy deployed by tumors to escape from detection and elimination by the immune system. Regulatory T lymphocytes (Treg) and ...myeloid-derived suppressor cells (MDSC), major components of these inhibitory cellular networks, have drawn intense scrutiny in recent years. In patients with cancer and in animal tumor models, these suppressor cells accumulate in the tumor microenvironment, secondary lymphoid tissues, and in the blood. Equipped with the ability to suppress innate and adaptive anticancer immunity, these cells also foster disease development by promoting tumor neoangiogenesis and by enhancing cancer metastasis. They therefore represent major impediments for anticancer therapies, particularly for immune-based interventions. Recent work has provided evidence that beyond their direct cytotoxic or cytostatic effects on cancer cells, several conventional chemotherapeutic drugs and agents used in targeted therapies can promote the elimination or inactivation of suppressive Tregs or MDSCs, resulting in enhanced antitumor immunity. We analyze findings pertinent to this concept, discuss the possible molecular bases underlying the selective targeting of these immunosuppressive cells by antineoplastic agents, and consider current challenges and future prospects related to the integration of these molecules into more efficient anticancer chemoimmunotherapeutic strategies.
A patient with recurrent multifocal glioblastoma received chimeric antigen receptor (CAR)-engineered T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2). Multiple ...infusions of CAR T cells were administered over 220 days through two intracranial delivery routes - infusions into the resected tumor cavity followed by infusions into the ventricular system. Intracranial infusions of IL13Rα2-targeted CAR T cells were not associated with any toxic effects of grade 3 or higher. After CAR T-cell treatment, regression of all intracranial and spinal tumors was observed, along with corresponding increases in levels of cytokines and immune cells in the cerebrospinal fluid. This clinical response continued for 7.5 months after the initiation of CAR T-cell therapy. (Funded by Gateway for Cancer Research and others; ClinicalTrials.gov number, NCT02208362 .).
Stimulation of toll-like receptor-9 (TLR9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. Because ...TLR9 is located intracellularly, we hypothesized that methods that enhance its internalization may also potentiate its immunostimulatory response. The goal of this study was to evaluate carbon nanotubes (CNT) as a CpG delivery vehicle in brain tumor models.
Functionalized single-walled CNTs were conjugated with CpG (CNT-CpG) and evaluated in vitro and in mice bearing intracranial GL261 gliomas. Flow cytometry was used to assess CNT-CpG uptake and antiglioma immune response. Tumor growth was measured by bioluminescent imaging, histology, and animal survival.
CNT-CpG was nontoxic and enhanced CpG uptake both in vitro and intracranial gliomas. CNT-mediated CpG delivery also potentiated proinflammatory cytokine production by primary monocytes. Interestingly, a single intracranial injection of low-dose CNT-CpG (but not free CpG or blank CNT) eradicated intracranial GL261 gliomas in half of tumor-bearing mice. Moreover, surviving animals exhibited durable tumor-free remission (>3 months), and were protected from intracranial tumor rechallenge, demonstrating induction of long-term antitumor immunity.
These findings suggest that CNTs can potentiate CpG immunopotency by enhancing its delivery into tumor-associated inflammatory cells.
Microglia (MG) and macrophages (MPs) represent a significant component of the inflammatory response to gliomas. When activated, MG/MP release a variety of pro-inflammatory cytokines, however, they ...also secrete anti-inflammatory factors that limit their cytotoxic function. The balance between pro and anti-inflammatory functions dictates their antitumor activity. To evaluate potential variations in MG and MP function in gliomas, we isolated these cells (and other Gr1+ cells) from intracranial GL261 murine gliomas by FACS and evaluated their gene expression profiles by microarray analysis. As expected, arginase 1 (Arg1, M2 marker) was highly expressed by tumor-associated Gr1+, MG and MP. However, in contrast to MP and Gr1+ cells that expressed Arg1 shortly after tumor trafficking, Arg1 expression in MG was delayed and occurred in larger tumors. Interestingly, depletion of MPs in tumors did not prevent MG polarization, suggesting direct influence of tumor-specific factors on MG Arg1 upregulation. Finally, Arg1 expression was confirmed in human GBM samples, but most Arg1+ cells were neutrophils and not MPs. These findings confirm variations in tumor MG and MP polarization states and its dependency on tumor microenvironmental factors.
While the role of T helper 17 lymphocytes (Th17) in the pathogenesis of autoimmune diseases and in infectious immunity has been relatively well defined, the impact of these cells and their associated ...cytokines on cancer development is still under debate. Although multiple reports have indicated that Th17 can promote anticancer immunity, others have argued that these cells may exhibit tumor-promoting properties. This dichotomy in the function of Th17 lymphocytes in cancer may be related to the versatile nature of these cells, being capable of differentiating into either proinflammatory Th1 or suppressive FoxP3-expressing Treg cells or hybrid T cell subsets depending on the underlying environmental conditions. In the current review, we examine the role of Th17 lymphocytes and Th17-associated cytokines in cancer and discuss how factors that control their final lineage commitment decision may influence the balance between their tumor-promoting versus tumor-suppressing properties.
BackgroundChimeric antigen receptor-(CAR) T cell therapy is seeing encouraging progress in solid tumors, however, factors such as the suppressive tumor microenvironment (TME) hinder its ...effectiveness.1 We recently completed a phase I trial of IL13Rα2-targeted CAR-T cells for recurrent glioblastomas (GBM) and other high-grade gliomas, the largest clinical study completed to date (NCT02208362).2–5 Samples from this trial were evaluated for TGFβ, an immunosuppressive cytokine highly upregulated in GBM.MethodsTGFβ levels were measured using ELISA. Syngeneic mouse studies were used to evaluate the impact of TGFβ blockade on CAR-T therapy. Flow cytometry was used to characterize the TME and CRISPR/Cas technology was utilized to knockout TGFβR2.ResultsWe measured TGFβ levels in 781 patient samples collected from the tumor site (tumor fluid; TF) or cerebral spinal fluid (CSF) before and after CAR-T administration. Compared to CSF, TF exhibited a higher concentration of TGFβ indicating a more immunosuppressive environment in tumor bed. We also found that post CAR-T therapy TGFβ levels decreased in the TF, but not the CSF. Furthermore, higher TGFβ levels in TF correlated with poor prognosis and lack of response to CAR-T therapy. These results indicate that targeting the TGFβ pathway may enhance CAR-T therapy against GBM. Using our syngeneic glioma model, we evaluated the importance of inhibiting the TGFβ pathway. We demonstrated that pretreatment with a TGFβR1 inhibitor (LY3200882) significantly augmented the efficacy of CAR-T therapy and improved overall survival of mice bearing large established tumors. TME characterization revealed upregulation of a migratory phenotype for both CAR and endogenous T cells. These results indicate that targeting TGFβ in the TME augments CAR-T cell function by potentially enhancing both endogenous and CAR T cell migration and trafficking into the tumor. Next, we assessed the impact of blocking TGFβ-signaling on human CAR-T cells. Targeting the TGFβ pathway (using TGFβR1 inhibitor) enhanced CAR-T cell antitumor activity and proliferation following multiple tumor-rechallenges in vitro. Gene expression analyses revealed upregulation of genes associated with cytotoxic and memory-stem-like phenotypes and down-regulation of genes associated with exhaustion and inhibitory pathways. Based on these results, we next evaluated TGFβ-resistant CAR-T cells in vivo and demonstrated that blocking TGFβ-signaling through TGFβR2 knockout augmented the efficacy of CAR-T cells in a large immunosuppressive GBM tumor model in syngeneic mice.ConclusionsCollectively, our results indicate that inhibiting the TGFβ pathway either in TME or CAR T cells is essential for enhancing CAR-T cell efficacy in GBM.AcknowledgementsThis work was supported in part by Mustang Bio., Inc, Cancer Center Support Grant P30 CA33572, and The Kenneth T. and Eileen L. Norris Foundation. We also thank Lilly for providing the TGFbR1 inhibitor (LY3200882) for our studies.ReferencesYap T, et al. First-In-Human Phase I Study of a Next-Generation, Oral, TGFb Receptor 1 Inhibitor, LY3200882, in Patients with Advanced Cancer. Clin Cancer Res 2021;27(24):6666–76Brown CE, et al. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med, 2016;375(26):2561–9.Alizadeh D, et al. IFNg Is Critical for CAR T Cell-Mediated Myeloid Activation and Induction of Endogenous Immunity. Cancer Discov. 2021;11(9):2248–65.Tang N, et al. TGF-beta inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight 2020;5(4).Han J, et al. TGF-beta signaling and its targeting for glioma treatment. Am J Cancer Res, 2015;5(3):945–55.Ethics ApprovalAll studies related to analyzing patient samples or healthy donors were obtained with informed written consent (whenever necessary) after protocols were approved by the City of Hope IRB and IND committee. All animal experiments were performed using protocols approved by the City of Hope IACUC.
MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor-immune evasion. The mechanisms by which tumors promote the expansion and/or function of these ...suppressive cells and the cross-talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor-induced gr-MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF-β1-mediated generation of CD4(+)CD25(+)FoxP3(+) iTregs. Furthermore, gr-MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4(+) cells by gr-MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD-1 and PD-L1 signaling, or COX-2. These findings thus indicate that gr-MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.
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