T regulatory type 1 cells in squamous cell carcinoma of the head and neck: mechanisms of suppression and expansion in advanced disease

PURPOSE: Regulatory T cells play a major role in tumor escape from immunosurveillance. T regulatory cells type 1 (Tr1), a subset of regulatory T cells present in the tumor and peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC), mediate immune suppression and might contribute to tumor progression. EXPERIMENTAL DESIGN: CD4+CD25-T cells were isolated from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) of 26 HNSCC patients and 10 normal controls. The Tr1 cell phenotype was determined before and after culture in the presence of interleukin (IL)-2, IL-10, and IL-15, each at 10 to 20 IU/mL. Suppression was measured in carboxyfluorescein diacetate succinimidyl ester-based proliferation assays with or without neutralizing anti-IL-10 or anti-transforming growth factor-beta1 (TGF-beta1) monoclonal antibodies in Transwell systems. ELISA was used to define the Tr1 cytokine profile. RESULTS: Tr1 cells originate from CD4(+)CD25(-) precursors present in TIL and PBMC of HNSCC patients. Cytokine-driven ex vivo expansion of Tr1 precursors yielded CD4+CD25-Foxp3lowCD132+IL-10+TGF-beta1+ populations that mediated higher suppression than Tr1 cells of normal controls (P < 0.0001). Tr1 cells suppressed proliferation of autologous responders via IL-10 and TGF-beta1 secretion. Expression of these cytokines was higher in TIL-derived than PBMC-derived Tr1 cells (P < 0.0001). The Tr1 cell frequency and suppressor function were significantly higher in patients presenting with advanced than early disease stages and in patients "cured" by oncologic therapies than in those with active disease. CONCLUSIONS: In HNSCC, Tr1 cell generation is promoted at the tumor site. Tr1 cells use TGF-beta and IL-10 to mediate suppression. They expand during disease progression and also following cancer therapy in patients with no evident disease.     

Abnormal interleukin 10Ralpha expression contributes to the maintenance of elevated cyclooxygenase-2 in non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) cells are known to constitutively overexpress cyclooxygenase (COX)-2. Tumor COX-2-dependent production of PGE(2) triggers the synthesis of lymphocyte and macrophage interleukin (IL)-10 that, in turn, is known to potently suppress COX-2 in normal cells. Thus, we investigated the capacity of IL-10 to down-regulate COX-2 expression in NSCLC cells. Western blotting and ELISA analyses revealed that IL-10 did not affect COX-2 expression and subsequent PGE(2) production in NSCLC cells. Although normal human bronchial epithelial cells expressed both intracellular and membrane IL-10Ralpha, NSCLC cells only expressed intracellular but not cell surface membrane IL-10Ralpha. Unresponsiveness of COX-2 to IL-10 is due to the deficiency of IL-10Ralpha on the surface of NSCLC cells. Our findings highlight a novel mechanism contributing to maintenance of elevated COX-2 and PGE(2) in the lung tumor environment.     

Functional and phenotypic characteristics of CD4+CD25highFoxp3+ Treg clones obtained from peripheral blood of patients with cancer

Circulating human CD4(+)CD25(high)Foxp3(+) T cell populations (Treg) may contain activated CD4(+)CD25(+) T cells interfering with Treg evaluation. To gain insights into the phenotypic and functional characteristics of Treg in patients with cancer, we have analyzed CD4(+)CD25(high) populations at the clonal level. Single-cell sorted (SCS) CD4(+)CD25(high) T cells obtained from PBMC of normal controls (NC) or patients with squamous cell carcinoma of the head and neck (HNSCC) were plated at 1 cell/well in 96 well plates and expanded with anti-CD3/anti-CD28 Abs and 1,000 IU IL-2/mL in the presence or absence of rapamycin (1 nM). All generated clones were evaluated for the phenotype by flow cyometry and suppressor function in CFSE-based proliferation assays. Clones had heterogeneous CD25 expression levels. Cloning efficiency of CD4(+)CD25(high) T cells was low. CD25(high) clones expressed CTLA-4, Foxp3, CD62L, but little GITR and suppressed proliferation of autologous CD4(+)CD25(-) responder cells. Clones of activated CD4(+)CD25(interm./low) cells expressed intermediate to high levels of GITR and HLA-DR and did not suppress proliferation of responder cells. The number, suppressor phenotype and function of CD25(high) Treg clones were significantly enhanced in HNSCC patients relative to NC (p 相似文献   

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies.     

Tumor-specific CD4(+) suppressor T-cell clone capable of inhibiting rejection of syngeneic sarcoma in A/J mice

Elimination of CD4(+) T cells by anti-CD4 antibody caused regression of a methylcholanthrene-induced S713a sarcoma growing in syngeneic A/J mice, and the tumor regression was essentially required for CD8(+) T cells. A CD4(+) T-cell clone, designated T595B1, was established to elucidate the characteristics of CD4(+) suppressor T cells. T595B1 expressed CD3, T-cell receptor (TCR)beta, TCR-Vbeta2, CD4, CD25, CD45RB, CD44, LFA-1, and ICAM-1 molecules on its cell surface and showed MHC class II I-E(k)-restricted tumor antigen-specific proliferation. T595B1 cells specifically suppressed in vitro CTL induction of S713a in a dose-dependent manner. Furthermore, culture supernatant of T595B1 cells also suppressed in vitro CTL induction, but its suppressive activity was not specific. Cytokine analyses revealed that T595B1 cells secreted IL-4, IL-5, IL-6, and IL-10 but not IFN-gamma, IL-2, TNF, or TGFbeta, indicating that this clone belongs to the so-called T helper 2 (Th2) type. However, the suppressive activity of the culture supernatant to the in vitro CTL induction was not abrogated by any neutralizing antibody to IL-4, IL-5, IL-6, IL-10, or TGF-beta. Repeated adoptive transfer of T595B1 cells into syngeneic immune mice entirely impaired their capacity to reject S713a sarcoma, resulting in progressive tumor growth in these mice.     

Prevention of both direct and cross-priming of antitumor CD8+ T-cell responses following overproduction of prostaglandin E2 by tumor cells in vivo

Defects in antitumor immune responses have been associated with increased release of prostaglandin E(2) (PGE(2)) as a result of overexpression of cyclooxygenase (COX)-2 by tumors. In this report, we examine the effects of PGE(2) on antitumor CD8(+) T-cell responses generated both by cross-presenting dendritic cells and by direct priming by tumor cells. Our data show that PGE(2) inhibits dendritic cell maturation, resulting in the abortive activation of naive CD8(+) T cells, and is dependent on interleukin-10 production by dendritic cells. Interaction of tumor cells with na?ve CD8(+) T cells in the presence of PGE(2) in vitro results in the induction of CD8(+) CD28(-) T cells, which fail to proliferate or exhibit effector function. In vivo, overexpression of COX-2 by tumor cells results in a decrease in number of tumor-infiltrating dendritic cells and confers the ability of tumor cells to metastasize to the tumor draining lymph nodes.     

CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor

Expression of CD200, the gene encoding the ligand for the inhibitory immune receptor CD200R, is an independent prognostic factor for various forms of leukemia predicting worse overall survival of the patients. The enhanced expression of CD200 on the tumors implies that anti-tumor responses can be enhanced by blockage of the CD200-CD200R interaction. Indeed, antibody-mediated blockade of the CD200-CD200R inhibitory axis is currently evaluated in clinical tests to boost immune responses against CD200-expressing tumors. Here, we show that mice lacking CD200, the exclusive ligand for CD200R, are resistant to chemical skin carcinogenesis. Importantly, CD200R controls tumor outgrowth independently of CD200 expression by the tumor cells themselves. Furthermore, Cd200(-/-) mice do not become tolerant to intranasally administered antigens, suggesting that tumor rejection is normally suppressed through CD200-induced immune tolerance. Decreased tumor outgrowth is accompanied by increased expression of the proinflammatory cytokines interleukin (IL)-1β and IL-6 by the lymph node (LN) dendritic cells. During carcinogenesis, skin-draining LNs of Cd200(-/-) mice contain increased numbers of IL-17-producing FoxP3(+) cells, which preferentially home to the tumors. Thus, the CD200-CD200R axis induces tolerance to external and tumor antigens and influences the T-regulatory/Th17 cell ratio. We demonstrate for the first time that the absence of CD200R signaling inhibits outgrowth of an endogenous tumor irrespective of CD200 expression by the tumor cells. This important paradigm shift leads to a much broader applicability of CD200-blockade in the treatment of tumors.     

Interleukin-12-responding asialoGM1+CD8+ central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells

While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2. Although ASGM1(+)CD8(+) T cells showed no significant responses to IL-12 alone or IL-2 alone, pulse incubation of ASGM1(+)CD8(+) T cells with IL-12 at an earlier time (0-12 h), and subsequently with IL-2 at a later time (12-24 h), caused the same levels of proliferation, killer cell generation and IFN-gamma production as when they were incubated simultaneously with IL-12 plus IL-2 for 24 h. Thus, ASGM1(+)CD8(+) T cells appeared to respond to IL-12 directly to acquire IL-2 responsiveness and differentiate into IFN-gamma-producing killer T cells. Indeed, freshly isolated ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, expressed higher levels of IL-12R beta2 mRNA. The fact that IL-12 administration in vivo caused the generation of ASGM1(+)CD8(+) killer T cells in an IFN-gamma-dependent manner further indicated a physiological significance of ASGM1(+)CD8(+) central memory-type T cells in IL-12-induced immunoregulation for the therapy of tumors and infectious diseases.     

UV irradiation of immunized mice induces type 1 regulatory T cells that suppress tumor antigen specific cytotoxic T lymphocyte responses

We previously showed that exposure to UV radiation after immunization suppresses Th1 and Th2 immune responses, leading to impaired Ab and allo-immune responses, but the impact of UV radiation after immunization on anti-tumor immune responses mediated by tumor-specific CD8(+) T cell responses remains less clear. Furthermore, the exact phenotypic and functional characteristics of regulatory T cell population responsible for the UV-induced immunosuppression still remain elusive. Using the MBL-2 lymphoma cell line engineered to express OVA as a surrogate tumor Ag, here we demonstrate that UV irradiation after tumor Ag-immunization suppresses the anti-tumor immune response in a manner dependent on the immunizing Ag. This suppression was mediated by interleukin (IL)-10 released from CD4(+) CD25(+) T cells, by which impaired the induction of cytotoxic T lymphocytes (CTL) able to kill Ag-expressing tumor cells. In addition, we generated a panel of T cell clones from UV-irradiated and non-irradiated mice, and all of the clones derived from UV-irradiated mice had a Tr1-type regulatory T cell phenotype with expression of IL-10 and c-Maf, but not Foxp3. These Tr1-type regulatory T cell clones suppressed tumor rejection in vivo as well as Th cell activation in vitro in an IL-10 dependent manner. Given that suppression of Ag-specific CTL responses can be induced in Ag-sensitized mice by UV irradiation, our results may imply that exposure to UV radiation during premalignant stage induces tumor-Ag specific Tr1 cells that mediate tumor-Ag specific immune suppression resulting in the promotion of tumor progression.     

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8(+) memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A(+) tumors with adoptively transferred T cells possessing a transgenic Valpha8(+) T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 microg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8(+) Valpha8(+) P1CTL compared with mice receiving the CD8(+) Valpha8(+) P1CTL and PBS vehicle (26.3 versus 5.1% P < 10(-5)). Animals exhibiting complete tumor regression had a significant population of CD8(+) Valpha8(+) P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.     

Tumor-infiltrating dendritic cell subsets of progressive or regressive tumors induce suppressive or protective immune responses

Tumor-infiltrating dendritic cells (TID) have an ambivalent role in regulation of tumor regression or growth. However, their precise natures and molecular mechanisms have not been elucidated. In this study, we studied TIDs recruited in progressive P815 and regressive P198 tumors of the same origin. Our data showed that P815 tumors contained CD4+ 8+ and CD4- 8- TID815 subsets, whereas P198 tumors contained CD4+ 8+ and CD4+ 8- TID198 subsets. They similarly stimulate allogeneic T cell proliferation and have nitric oxide-mediated cytotoxicity to tumor cells with an exception of CD4- 8- TID815 with less efficiency. The newly identified fourth CD4+ 8+ TID815 or TID198 subset and the CD4+ 8- TID198 all express high levels of IFN-gamma and interleukin (IL)-6, whereas CD4- 8- TID815 secrete a marked level of transforming growth factor-beta. Vaccination of mice with P815 tumor lysate-pulsed CD4+ 8+ TID815 or TID198 and CD4+ 8- TID198 induced IFN-gamma-secreting Th1 and effective CTL responses leading to protective immunity against P815 tumor, whereas CD4- 8- TID815 stimulated IL-10-expressing Tr1 responses leading to immune suppression. Transfer of CD4+ Tr1 cells obtained from CD4- 8- TID815-immunized wild-type, but not IL-10(-/-) mice, into CD4+ 8+ TID815 immunized mice abolished otherwise inevitable development of antitumor immunity. Taken together, our findings provide an important insight into immunologic alterations in progressive and regressive tumors and an implication for dendritic cell-based approaches in the design of cancer vaccines.     

The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck.

Objective: Immune escape is a characteristic feature of head and neck squamous cell carcinoma (HNSCC). Regulatory T cells (Treg) might contribute to HNSCC progression by suppressing antitumor immunity, and their attributes in patients are of special interest. Methods: Multicolor flow cytometry was used to study the frequency and phenotype of Treg in peripheral blood lymphocytes of 35 patients with HNSCC and 15 normal controls (NC). CD4(+)CD25(high) T cells were purified by fluorescence-activated cell sorting and tested for regulatory function by coculture with carboxyfluorescein diacetate succinimidylester-labeled autologous CD4(+)CD25(-) responder cells. RESULTS: The percentages of circulating CD4(+)CD25(+) T cells were increased in HNSCC patients (5 +/- 3%) versus NC (2 +/- 1.5%). In patients, this cell subset largely contained CD4(+)CD25(high)Foxp3(+) T cells and only few CD25(low/interm) cells. In addition, the frequency of Treg positive for CD62L, CTLA-4, Fas, FasL, and Foxp3 was greater in the circulation of patients than in NC (P < 0.0001). In HNSCC patients, Treg mediated significantly higher suppression (78 +/- 7%) compared with Treg in NC (12 +/- 4%) with P < 0.0001. Surprisingly, higher Treg frequency (P < 0.0059) and levels of suppression (P < 0.0001) were observed in patients with no evident disease (NED) than in untreated patients with active disease (AD). CONCLUSIONS: The frequency of T cells with suppressor phenotype and function (Treg) was significantly greater in HNSCC patients who were NED after oncologic therapy relative to those with AD. This finding suggests that oncologic therapy favors expansion of Treg.     

Determination of a CD4+CD25−FoxP3+ T cells subset in tumor-draining lymph nodes of colorectal cancer secreting IL-2 and IFN-γ

CD4⁺CD25⁻FoxP3⁺ cells are a newly recognized subset of T cells which was first reported in autoimmune diseases. In our previous study, this subset was detected in tumor-draining lymph nodes (TDLNs) of patients with breast cancer. As little is known about their function in TDLNs of cancer patients, in this study, their frequency as well as their ability to produce interleukin (IL)-2, IL-10, or interferon (IFN)-γ were investigated in TDLNs of colorectal cancer (CRC) patients. Mononuclear cells were isolated from lymph nodes of 13 patients with CRC using Ficoll-Hypaque gradient centrifugation. Cells were stimulated in vitro and stained with CD25, CD4, FoxP3, IFN-γ, IL-10, and IL-2 or isotype matched antibodies and subjected to flow cytometry. The frequency of CD4⁺CD25⁻FoxP3⁺CD127^dim/− cells was significantly lower than CD4⁺CD25⁺FoxP3⁺CD127^dim/− population in TDLNs of CRC patients. The percentage of CD127^dim/− cells and also the MFI of FoxP3 expression was significantly lower in CD4⁺CD25⁻FoxP3⁺ in comparison with CD4⁺CD25⁺FoxP3⁺ population. Moreover, CD4⁺CD25⁻FoxP3⁺ cells contained higher percentages of IL-2- and IFN-γ-producing cells than CD4⁺CD25⁺FoxP3⁺ subpopulation. But, no difference was seen between two subsets in terms of IL-10 production. CD4⁺CD25⁻FoxP3⁺ cells in TDLNs of CRC patients had lower suppressive and higher effector properties in comparison with CD4⁺CD25⁺FoxP3⁺ conventional regulatory T cells.

     

Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host

The accumulation of myeloid suppressor cells (MSCs) is associated with immune suppression in tumor-bearing mice and in cancer patients. The suppressive activity of MSC correlates with the expression of the myeloid markers Gr-1, CD115 (macrophage colony-stimulating factor receptor), and F4/80. Gr-1(+)CD115(+) MSCs, in addition to being able to suppress T-cell proliferation in vitro, can induce the development of Foxp3(+) T regulatory cells (Treg) in vivo, which are anergic and suppressive. Furthermore, the secretion of interleukin (IL)-10 and transforming growth factor-beta by Gr-1(+)CD115(+) MSCs was induced and enhanced, respectively, on IFN-gamma stimulation. The development of Treg requires antigen-associated activation of tumor-specific T cells, depends on the presence of IFN-gamma and IL-10, and is independent of the nitric oxide-mediated suppressive mechanism by MSC. Our data provide evidence that Gr-1(+)CD115(+) MSC can mediate the development of Treg in tumor-bearing mice and show a novel immune suppressive mechanism by which MSCs can suppress antitumor responses.     

食管癌组织中共刺激分子与白介素-10 mRNA的表达

目的研究食管癌组织中共刺激分子CD80、CD86 mRNA的表达以及与TGF-β1、IL-10 mRNA表达水平的关系,探讨食管癌组织中树突细胞免疫功能降低及食管癌发生免疫逃逸的原因。方法应用逆转录聚合酶链反应(PT-PCR)技术,检测62例食管癌患者癌组织中CD80、CD86及TGF-β1、IL-10 mRNA的表达。以16例正常食管黏膜组织作为对照。结果食管癌组织中,CD80、CD86 mRNA的表达分别为0．631±0．212和0．530±0．210,均低于正常食管黏膜组织(P值分别为0．038和0．0002);Ⅰ、Ⅱ期患者高于Ⅲ、Ⅳ期患者(CD80:P=0．029;CD86:P=0．045);高中分化者高于低分化者(CD80:P=0．046;CD86:P=0．044)。食管癌组织中,TGF-β1、IL-10的mRNA表达分别为1．273±0．086和1．182±0．073,均高于正常食管黏膜组织(P值均为0．0001),且分别与CD80、CD86的mRNA表达呈负相关(P=0．0001)。结论食管癌组织中,CD80、CD86 mRNA的表达降低与TGF-β1、IL-10 mRNA的高表达呈负相关,癌组织内TGF-β1、IL-10 mRNA的表达增强是导致CD80、CD86 mRNA表达降低的重要影响因素,可能是引起DC功能缺陷和食管癌细胞发生免疫逃逸的原因之一。