Regulatory T-cell depletion synergizes with gp96-mediated cellular responses and antitumor activity

Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6?J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.     

FOXP3~+调节性T细胞

调节性T细胞是一类可以调节其他多种免疫细胞功能的T细胞亚型,其正常生理功能对体内免疫稳态维持必不可少。调节性T细胞功能失调与人类多种重大疾病,如自身免疫性疾病、感染性疾病、过敏性疾病、恶性肿瘤、移植排斥的发生、发展及治疗都密切相关。调节性T细胞可分为多种亚型,其中最重要也是目前研究最多的为表达叉头状家族转录因子FOXP3的天然调节性T细胞及诱导调节性T细胞。深入研究FOXP3+调节性T细胞的发育及功能的分子及细胞免疫学机制,将为重大免疫性疾病的临床治疗提供创新性线索。     

Human Antigen-Specific Regulatory T Cells Generated by T Cell Receptor Gene Transfer

Background

Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.

Methodology/Principal Findings

To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging.

Conclusions/Significance

These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.     

Superantigen-induced proliferation of human CD4+CD25- T cells is followed by a switch to a functional regulatory phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki's disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25(+)FOXP3(+)) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3(-) T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non-Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non-Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25(+)FOXP3(+) cells arose from CD25(-) T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25(+)FOXP3(+) T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

Immune checkpoint blockade impairs immunosuppressive mechanisms of regulatory T cells in B-cell lymphoma

In malignant disease, CD4⁺Foxp3⁺ regulatory T cells (Tregs) hamper antitumor immune responses and may provide a target for immunotherapy. Although immune checkpoint blockade (ICB) has become an established therapy for several cancer entities including lymphoma, its mechanisms have not been entirely uncovered. Using endogenously arising λ-MYC-transgenic mouse B-cell lymphomas, which can effectively be suppressed by either Treg ablation or ICB, we investigated which mechanisms are used by Tregs to suppress antitumor responses and how ICB affects these pathways. During tumor development, Tregs up-regulated Foxp3, CD25, CTLA-4 and IL-10, which correlated with enhanced immunosuppressive functions. Thus, in contrast to other tumors, Tregs did not become dysfunctional despite chronic stimulation in the tumor microenvironment and progressive up-regulation of PD-1. Immunosuppression was mediated by direct contacts between Tregs and effector T cells and by IL-10. When λ-MYC mice were treated with ICB antibodies, Tregs revealed a less profound up-regulation of Foxp3, CD25 and IL-10 and a decreased suppressive capacity. This may be due to the shift towards a pro-inflammatory milieu fostered by ICB. In summary, an ICB-induced interference with Treg-dependent immunosuppression may contribute to the success of ICB.     

Rapid reconstitution of regulatory T-cell subsets is associated with reduced rates of acute graft-versus-host disease and absence of viremia after cord blood transplantation in children with reduced-intensity conditioning using alemtuzumab

Forkhead box P3 (FOXP3)+ regulatory T cell (Treg) reconstitution after unrelated donor umbilical cord blood transplantation in chemotherapy-naïve children is incompletely characterized. We studied 21 children with nonmalignant diseases receiving an identical alemtuzumab-containing regimen. We hypothesized that Treg recovery may be perturbed in patients not only by acute graft-versus-host disease (aGVHD) but also by viremia. Tregs and their memory and naïve subsets were serially monitored for proliferation and apoptosis along with conventional T cells (Tcon). A “reconstitution index” (RI) was calculated relative to pretransplantation values for each parameter. At 3 months post-UCBT, the RI of Tregs was faster compared with other immune components tested and was most rapid in patients free of aGVHD and viremia. There were significantly fewer Tregs in patients experiencing grade I–II aGVHD and/or viremia, leading to an imbalance between Tregs-Tcon ratios. Central and effector memory Tregs were most affected at this time point when they dominated in the circulation. Impaired Treg proliferation without increased apoptosis accounted for the reduced Treg-Tcon ratio. In patients affected with grade II aGVHD and viremia, the overall reduction in circulating Treg pool were associated with a more oligoclonal T-cell receptor β repertoire. Taken together, aGVHD and viremia can lead to defective Treg expansion homeostasis.     

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3⁺CD4⁺CD25⁺CD127^low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.     

PD-1+ immune cell infiltration inversely correlates with survival of operable breast cancer patients

The programmed death-1 (PD-1) molecule is mainly expressed on functionally “exhausted” CD8⁺ T cells, dampening the host antitumor immune response. We evaluated the ratio between effective and regulatory T cells (Tregs) and PD-1 expression as a prognostic factor for operable breast cancer patients. A series of 218 newly diagnosed invasive breast cancer patients who had undergone primary surgery at Ruijin Hospital were identified. The influence of CD8⁺ cytotoxic T lymphocytes, FOXP3⁺ (Treg cell marker), and PD-1⁺ immune cell counts on prognosis was analyzed utilizing immunohistochemistry. Both PD-1⁺ immune cells and FOXP3⁺ Tregs counts were significantly associated with unfavorable prognostic factors. In bivariate, but not multivariate analysis, high tumor infiltrating PD-1⁺ cell counts correlated with significantly shorter patient survival. Our results suggest a prognostic value of the PD-1⁺ immune cell population in such breast cancer patients. Targeting the PD-1 pathway may be a feasible approach to treating patients with breast cancer.     

热激蛋白gp96免疫学功能及在主动免疫治疗肿瘤和传染病中的应用

热激蛋白gp96可特异性结合来源于肿瘤和病毒的抗原肽,与抗原呈递细胞表面CD91等受体作用进入胞内,并在内质网中将结合的抗原通过抗原呈递链呈递给MHCI类分子,激活特异性T细胞。同时,与细胞表面Toll样受体(TLR)TLR2、TLR4等相互作用,激活天然免疫。近期研究发现调节性T细胞(Treg)对gp96免疫功能有显著抑制作用,随着对影响gp96免疫功能的免疫抑制机制的深入了解,以及利用汉逊酵母表达有免疫活性的全长gp96蛋白体系的建立,gp96将在治疗肿瘤及传染性疾病中发挥更大的作用。     

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8⁺ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8⁺ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8⁺ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8⁺ T cells, which led to higher ratios of CD8⁺/Treg and CD8⁺/CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8⁺ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.     

Neuropilin 1: function and therapeutic potential in cancer

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that acts as a co-receptor for a number of extracellular ligands including class III/IV semaphorins, certain isoforms of vascular endothelial growth factor and transforming growth factor beta. An exact understanding of the role of NRP1 in the immune system has been obscured by the differences in NRP1 expression observed between mice and humans. In mice, NRP1 is selectively expressed on thymic-derived Tregs and greatly enhances immunosuppressive function. In humans, NRP1 is expressed on plasmacytoid dendritic cells (pDCs) where it aids in priming immune responses and on a subset of T regulatory cells (Tregs) isolated from secondary lymph nodes. Preliminary studies that show NRP1 expression on T cells confers enhanced immunosuppressive activity. However, the mechanism by which this activity is mediated remains unclear. NRP1 expression has also been identified on activated T cells and Tregs isolated from inflammatory microenvironments, suggesting NRP1 might represent a novel T cell activation marker. Of clinical interest, NRP1 may enhance Treg tumour infiltration and a decrease in NRP1+ Tregs correlates with successful chemotherapy, suggesting a specific role for NRP1 in cancer pathology. As a therapeutic target, NRP1 allows simultaneous targeting of NRP1-expressing tumour vasculature, NRP1+ Tregs and pDCs. With the development of anti-NRP1 monoclonal antibodies and cell-penetrating peptides, NRP1 represents a promising new target for cancer therapies. This paper reviews current knowledge on the role and function of NRP1 in Tregs and pDCs, both in physiological and cancer settings, as well as its potential as a therapeutic target in cancer.     

Defining target antigens for CD25+ FOXP3 + IFN-gamma- regulatory T cells in chronic hepatitis C virus infection

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.     

Induction of CD4(+)CD25(+)FOXP3(+) regulatory T cells during human hookworm infection modulates antigen-mediated lymphocyte proliferation

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4(+)CD25(+)FOXP3(+) regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4(+)CD25(+)FOXP3(+) T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people.     

CD4+CD25high T cells are enriched in the tumor and peripheral blood of prostate cancer patients

In this study, we investigated whether CD4+CD25high regulatory T cells (Treg) are increased in the tumor tissue and peripheral blood of early-stage prostate cancer patients undergoing prostatectomy. We show that the prevalence of CD4+CD25high T cells inside the prostate was significantly higher in the tumor compared with benign tissue from the same prostate. Furthermore, the frequency of CD4+CD25high T cells in peripheral blood was significantly higher in prostate cancer patients compared with normal donors. A proportion of the CD4+CD25high T cells was also shown to be glucocorticoid-induced TNF receptor, ICOS, and FOXP3 positive. Moreover, CD4+CD25+ T cells from blood and supernatants from cultured prostate tumor tissue samples exhibited immunosuppressive function in vitro. Furthermore, supernatants from cultured prostate tissue samples and prostate cancer ascites fluid induced migration of CD4+CD25+ T cells and were shown to contain the regulatory T cell chemokine CCL22 by ELISA. Our findings indicate that Tregs are an important cellular component of early-stage prostate tumors, and thus new therapeutic strategies aimed at inhibition or depletion of Tregs may improve prostate cancer immunotherapy.     

Expansion of effector memory regulatory T cells represents a novel prognostic factor in lower risk myelodysplastic syndrome

Myelodysplastic syndromes are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia transformation. Studies of FOXP3(+) regulatory T cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared with age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4(+)FOXP3(+)CD25(+)CD127(low)CD45RA(-)CD27(-) Tregs (effector memory Tregs [Treg(EM)]) was significantly associated with anemia (p = 0.046), reduced hemoglobin (p = 0.038), and blast counts ≥5% (p = 0.006). In healthy donors, this Treg(EM) population constitutes only 2% of all Tregs (one to six Tregs per microliter) in peripheral blood but, when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 y (range 2.7-4.9 y) from sample acquisition, increased numbers of Treg(EM) cells proved to have independent prognostic importance in survival estimates, suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FOXP3(+) T cells as a whole. Based on multivariate analyses, Treg(EM) impacted survival independently from myeloblast characteristics, cytopenias, karyotype, and comorbidities. Based on these findings, Treg(EM) cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in myelodysplastic syndromes.     

Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function

Numerous reports have demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including type 1 diabetes, are deficient in their ability to control autologous proinflammatory responses when compared with nondiseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development. Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with type 1 diabetes on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished IL-2 responsiveness in Ag-experienced CD4(+) T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FOXP3 expression by Tregs and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene affect immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.