Guangyong Peng

Tumor Microenvironments Direct the Recruitment and Expansion of Human Th17 Cells

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases, their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients, but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study, we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers, suggesting that development of tumor-infiltrating CD4+ Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment, we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell–cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs, demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments, and provide new insights relevant for the development of novel cancer immunotherapeutic approaches.

Tumor-Infiltrating γδ T Lymphocytes Predict Clinical Outcome in Human Breast Cancer

Understanding and dissecting the role of different subsets of regulatory tumor-infiltrating lymphocytes (TILs) in the immunopathogenesis of individual cancer is a challenge for anti-tumor immunotherapy. High levels of γδ regulatory T cells have been discovered in breast TILs. However, the clinical relevance of these intratumoral γδ T cells is unknown. In this study, γδ T cell populations were analyzed by performing immunohistochemical staining in primary breast cancer tissues from patients with different stages of cancer progression. Retrospective multivariate analyses of the correlations between γδ T cell levels and other prognostic factors and clinical outcomes were completed. We found that γδ T cell infiltration and accumulation in breast tumor sites was a general feature in breast cancer patients. Intratumoral γδ T cell numbers were positively correlated with advanced tumor stages, HER2 expression status, and high lymph node metastasis but inversely correlated with relapse-free survival and overall survival of breast cancer patients. Multivariate and univariate analyses of tumor-infiltrating γδ T cells and other prognostic factors further suggested that intratumoral γδ T cells represented the most significant independent prognostic factor for assessing severity of breast cancer compared with the other known factors. Intratumoral γδ T cells were positively correlated with FOXP3+ cells and CD4+ T cells but negatively correlated with CD8+ T cells in breast cancer tissues. These findings suggest that intratumoral γδ T cells may serve as a valuable and independent prognostic biomarker, as well as a potential therapeutic target for human breast cancer.

Tumor-Derived γδ Regulatory T Cells Suppress Innate and Adaptive Immunity through the Induction of Immunosenescence

Fundamentally understanding the suppressive mechanisms used by different subsets of tumor-infiltrating regulatory T (Treg) cells is critical for the development of effective strategies for antitumor immunotherapy. γδ Treg cells have recently been identified in human diseases including cancer. However, the suppressive mechanisms and functional regulations of this new subset of unconventional Treg cells are largely unknown. In the current studies, we explored the suppressive mechanism(s) used by breast tumor-derived γδ Treg cells on innate and adaptive immunity. We found that γδ Treg cells induced immunosenescence in the targeted naive and effector T cells, as well as dendritic cells (DCs). Furthermore, senescent T cells and DCs induced by γδ Treg cells had altered phenotypes and impaired functions and developed potent suppressive activities, further amplifying the immunosuppression mediated by γδ Treg cells. In addition, we demonstrated that manipulation of TLR8 signaling in γδ Treg cells can block γδ Treg–induced conversion of T cells and DCs into senescent cells in vitro and in vivo. Our studies identify the novel suppressive mechanism mediated by tumor-derived γδ Treg cells on innate and adaptive immunity, which should be critical for the development of strong and innovative approaches to reverse the tumor-suppressive microenvironment and improve effects of immunotherapy.

Human tumor-infiltrating Th17 cells have the capacity to differentiate into IFN-γ+ and FOXP3+ T cells with potent suppressive function.

Accumulating evidence suggests that Th17 cells and Tregs may exhibit development plasticity and that CD4+ Tregs can differentiate into IL‐17‐producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor‐infiltrating T lymphocytes (TILs). We showed that Th17 clones generated from TILs can differentiate into IFN‐γ‐producing and FOXP3+ cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells. We further demonstrated that T‐cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage‐specific transcriptional factor and cytokine genes. In addition to expressing IFN‐γ and FOXP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Tregs. We further demonstrated that the Th17‐derived Tregs, unlike naturally occurring CD4+CD25+ Tregs, did not reconvert back into Th17 cells even under Th17‐biasing cytokine conditions. These results provide the critical evidence that human tumor‐infiltrating Th17 cells can differentiate into Tregs and indicate a substantial developmental plasticity of Th17 cells.

Human regulatory T cells induce T-lymphocyte senescence

Regulatory T (Treg) cells have broad suppressive activity on host immunity, but the fate and function of suppressed responder T cells remains largely unknown. In the present study, we report that human Treg cells can induce senescence in responder naive and effector T cells in vitro and in vivo. Senescent responder T cells induced by human Treg cells changed their phenotypes and cytokine profiles and had potent suppressive function. Furthermore, Treg-mediated molecular control of senescence in responder T cells was associated with selective modulation of p38 and ERK1/2 signaling and cell-cycle-regulatory molecules p16, p21, and p53. We further revealed that human Treg-induced senescence and suppressor function could be blocked by TLR8 signaling and/or by specific ERK1/2 and p38 inhibition in vitro and in vivo in animal models. The results of the present study identify a novel mechanism of human Treg cell suppression that induces targeted responder T-cell senescence and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression.

Specific Recruitment of γδ Regulatory T Cells in Human Breast Cancer

Understanding the role of different subtypes of tumor-infiltrating lymphocytes (TIL) in the immunosuppressive tumor microenvironment is essential for improving cancer treatment. Enriched γδ1 T-cell populations in TILs suppress T-cell responses and dendritic cell maturation in breast cancer, where their presence is correlated negatively with clinical outcomes. However, mechanism(s) that explain the increase in this class of regulatory T cells (γδ Treg) in patients with breast cancer have yet to be elucidated. In this study, we show that IP-10 secreted by breast cancer cells attracted γδ Tregs. Using neutralizing antibodies against chemokines secreted by breast cancer cells, we found that IP-10 was the only functional chemokine that causes γδ Tregs to migrate toward breast cancer cells. In a humanized NOD-scid IL-2Rγ(null) (NSG) mouse model, human breast cancer cells attracted γδ Tregs as revealed by a live cell imaging system. IP-10 neutralization in vivo inhibited migration and trafficking of γδ Tregs into breast tumor sites, enhancing tumor immunity mediated by tumor-specific T cells. Together, our studies show how γδ Tregs accumulate in breast tumors, providing a rationale for their immunologic targeting to relieve immunosuppression in the tumor microenvironment.

Granzyme A Produced by γ9δ2 T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

Human Herpesvirus 6 Suppresses T Cell Proliferation through Induction of Cell Cycle Arrest in Infected Cells in the G2/M Phase

ABSTRACT Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus that primarily infects immune cells and strongly suppresses the proliferation of infected cells. However, the mechanisms responsible for the regulation and suppression mediated by HHV-6 are still unknown. In this study, we examined the ability of HHV-6A to manipulate cell cycle progression in infected cells and explored the potential molecular mechanisms. We demonstrated that infection with HHV-6A imposed a growth-inhibitory effect on HSB-2 cells by inducing cell cycle arrest at the G2/M phase. We then showed that the activity of the Cdc2–cyclin B1 complex was significantly decreased in HHV-6A-infected HSB-2 cells. Furthermore, we found that inactivation of Cdc2–cyclin B1 in HHV-6A-infected cells occurred through the inhibitory Tyr15 phosphorylation resulting from elevated Wee1 expression and inactivated Cdc25C. The reduction of Cdc2–cyclin B1 activity in HHV-6-infected cells was also partly due to the increased expression of the cell cycle-regulatory molecule p21 in a p53-dependent manner. In addition, HHV-6A infection activated the DNA damage checkpoint kinases Chk2 and Chk1. Our data suggest that HHV-6A infection induces G2/M arrest in infected T cells via various molecular regulatory mechanisms. These results further demonstrate the potential mechanisms involved in immune suppression and modulation mediated by HHV-6 infection, and they provide new insights relevant to the development of novel vaccines and immunotherapeutic approaches.

Human Herpesvirus 6 Latent Infection in Patients With Glioma

The etiology of glioma remains unclear so far. Human herpesvirus 6 (HHV-6) might be associated with glioma, but there is no direct evidence to support this. High percentages of HHV-6 DNA and protein were detected in tissue from gliomas, compared with normal brain tissue. In addition, a strain of HHV-6A was isolated from the fluid specimens from glioma cysts. High levels of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α, and transforming growth factor β (TGF-β) were detected in the cyst fluid specimens from HHV-6-positive patients with glioma. Furthermore, HHV-6A infection promoted IL-6, IL-8, and TGF-β production in astrocyte cultures. Our studies strongly suggest the involvement of HHV-6 infection in the pathogenesis of glioma.

TLR8 signaling enhances tumor immunity by preventing tumor‐induced T‐cell senescence

Accumulating evidence suggests the immunosuppressive microenvironments created by malignant tumors represent a major obstacle for effective anti‐tumor immunity. A better understanding of the suppressive mechanisms mediated by tumor microenvironments and the development of strategies to reverse the immune suppression are major challenges for the success of tumor immunotherapy. Here, we report that human tumor cells can induce senescence in naïve/effector T cells, exhibiting potent suppressive function in vitro and in vivo. We further show that tumor‐derived endogenous cyclic adenosine monophosphate (cAMP) is responsible for the induction of T‐cell senescence. Importantly, activation of TLR8 signaling in tumor cells can block the induction and reverse the suppression of senescent naïve and tumor‐specific T cells in vitro and in vivo, resulting in enhanced anti‐tumor immunity. These studies identify a novel mechanism of human tumor‐mediated immune suppression and provide a new strategy to reverse tumor immunosuppressive effects for tumor immunotherapy.