Thymocyte/splenocyte-derived CD4+CD25+Treg stimulated by anti-CD200R2 derived dendritic cells suppress mixed leukocyte cultures and skin graft rejection

BACKGROUND: CD200 delivers immunoregulatory signals following engagement of its receptor, CD200R. A family of CD200Rs (CD200R1-4) has been described. Spleen expresses cell surface CD200R1, while bone marrow shows predominantly expression of cell surface CD200R2/R3. We showed that dendritic cell precursors (DCp) cultured with anti-CD200R2/3 develop the capacity to induce CD4(+)CD25(+) regulatory T cells (Treg) from peripheral lymphocytes. We now characterize DCs involved in induction of antigen-specific Treg from thymocytes or peripheral T cells, and the properties of Treg cells maintained in long-term culture. METHODS: Bone marrow DCp (C3H or BL/6 origin) were cultured for 8 days with GMCSF, IL-4 and anti-CD200R2, or with CD200Fc and a previously described peptide inhibitor of CD200R1 to allow preferential engagement of non-CD200R1 receptors by CD200. Mixed leukocyte cultures (MLCs) were initiated with allogeneic responder lymphocytes/thymocytes (BL/6 or C3H) and mitomycin-c treated DCs to induce Treg. Treg cells were maintained by reculture with DCs derived in the same manner and IL-2, cloned at limiting dilution, and tested for their ability to suppress MLCs and skin graft rejection in vivo. RESULTS: Foxp3(+) CD4(+)CD25(+) Treg were derived from 60-hr thymocyte and splenocyte T cell cultures using both DC populations. Cloned C3H Treg (Foxp3(+)) suppressed both C3H anti-BL/6 reactivity in a fresh MLC and rejection of BL/6 skin allografts in C3H recipients; the converse was true for BL/6 Treg. CONCLUSIONS: We conclude that CD200 triggering of bone-marrow DCs in the absence of CD200R1 engagement induces CD4(+)CD25(+) Treg, and these cloned antigen-specific Treg may have clinical utility.     

Induction of tolerance-inducing antigen-presenting cells in bone marrow cultures in vitro using monoclonal antibodies to CD200R

CD200 to CD200R interactions produce immunoregulation. We investigated whether the expression of CD200R on dendritic cell (DC) precursors affects their developmental fate. C57BL/6 bone marrow (BM) cells were cultured in vitro in the presence of (interleukin-4 + granulocyte-macrophage colony-stimulating activity) to generate allostimulatory DCs, which were in turn used to induce cytotoxic T-lymphocyte and cytokine production after culture with C3H responder spleen cells. Some marrow cultures included anti-CD200R antibodies. The inclusion of monoclonal antibodies in different isoforms of CD200R in the BM culture led to a generation of cells (tolerogenic DCs) that were unable to produce allostimulation in vitro with responder cells. Cells taken from these latter mixed leukocyte cultures (MLCs) now contained CD4(+)CD25(+) cells able to inhibit the antigen-specific MLC response of fresh C3H responder cells to stimulation with C57BL/6 cells, but not stimulation with BALB/c cells. Tolerogenic DCs, infused in vivo into mice receiving C57BL/6 skin grafts, produced antigen-specific decreased rejection of BL/6 allografts, not BALB/c allografts, compared with mice receiving control DCs (generated from BM in the absence of anti-CD200R). The induction of CD4(+)CD25(+) suppressor cells in MLCs using tolerogenic DCs from the initial BM cultures could be overcome by using limiting numbers of tolerogenic DCs and an excess of allostimulatory DCs derived from BM cultures maintained in the absence of anti-CD200R. These data indicate that anti-CD200R biases stem cells in BM toward the development of suppressive antigen-presenting cells, which can induce CD4(+)CD25(+) regulatory T cells. Tolerogenic DCs have the potential to modify graft acceptance in vivo.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

阻断共刺激通路诱导产生免疫无能状态

目的 探讨同时阻断CD40／CDl54和B7／CD28共刺激通路能否诱导产生免疫无能状态以及无能状态的逆转条件。方法 以C3H小鼠脾细胞为刺激细胞，BALB／c小鼠脾细胞为反应细胞，C57BL／6J小鼠脾细胞为第三方细胞，在体外双向混合淋巴细胞培养中加入不同浓度的抗CDl54和抗CD80单克隆抗体，诱导产生无能细胞。在无能细胞中分别加入经7射线照射后的C3H小鼠或C57BL／6J小鼠脾细胞，或者直接加入不同浓度重组小鼠白细胞介素2(nmIL-2)，或者同时加入经7射线照射后的C3H小鼠细胞和不同浓度的mnIL-2刺激，观察无能状态的逆转情况。结果 联合应用抗CDl54和抗CD80单克隆抗体能显著抑制体外双向混合淋巴细胞培养反应的细胞增殖；第三方刺激细胞能够逆转无能细胞的无能状态；单纯加入nmIL-2或C3H小鼠细胞再次刺激不能逆转无能状态，而只有同时加入C3H小鼠细胞和nmIL-2刺激才能逆转无能细胞的无能状态。结论 同时阻断CD40／CDl54和B7／CD28通路能诱导产生抗原特异性的免疫无能状态，而且只有同时给予抗原和外源性IL-2再次刺激才能逆转这种无能状态。     

The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice

CD4(+)CD25(+)Regulatory T (Treg) cells are crucial for negatively regulating immune responses. Rapamycin (rapa) is an immunosuppressive agent which is widely used for preventing acute graft rejection in patients and has been used to induce operational tolerance in mouse models. The aim of the present study was to determine the effect of rapa on CD4(+)CD25(+)Foxp3(+)Treg cells in a mouse model. After C57BL/6 mice were intraperitoneally given 1.5 mg/kg/day of rapa for 14 days, the percentages, cell numbers, phenotype and function of CD4(+)CD25(+)Treg cells were determined by flow cytometry as well as the in vitro and in vivo functional assays. The cell numbers of CD4(+) and CD4(+)CD25(+)Treg cell subsets were markedly decreased in rapa-treated mice as reported. However, rapa significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in spleens and thymi of mice (P<0.01) respectively. Furthermore, splenic CD4(+)CD25(+)Treg cells in rapa-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro and in vivo. Thus, rapa could significantly enhance the percentages of CD4(+)CD25(+)Foxp3(+)Treg cells in the thymus and the periphery while keeping these cells functional, indicating that CD4(+)CD25(+)Treg cells are more resistant to rapa than other CD4(+)T cells. The different effects of rapa on CD4(+)CD25(+)Treg and T effector cells make rapa to be a favorable choice for inducing immune tolerance to self-, allo-, or xeno-antigens.     

Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection.

INTRODUCTION: This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice. METHODS: "Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses. RESULTS: The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and >100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts. CONCLUSIONS: These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.     

Mice lacking CD200R1 show absence of suppression of lipopolysaccharide-induced tumor necrosis factor-alpha and mixed leukocyte culture responses by CD200

BACKGROUND: CD200:CD200R interactions deliver immunoregulatory signals. A family of CD200Rs (CD200R1-5) has been described, and engagement of CD200R1 by its ligand CD200 suppresses LPS-induced macrophage cytokine production, decreases alloimmune responses in vivo and in vitro, and suppresses collagen-induced arthritis. METHODS: We generated C57BL/6 mice lacking the genomic exons encoding the extracellular domains of the CD200R1 molecule using transformation of ES cells and explored cell subtypes and immune responses in these mice. RESULTS: Myeloid cells/splenocytes from CD200R1(-/-) mice were not stained in FACS by anti-CD200R1 mAb. Stimulation of splenic tumor necrosis factor-alpha production by lipopolysaccharide was enhanced relative to control (+/+) mice and was not suppressed by addition of exogenous CD200Fc. Modulation of alloreactivity in mixed leukocyte cultures by CD200Fc depended upon CD200R1+ stimulatory cells, although maximal immunoregulation by CD200Fc occurred only when CD200R1+ T responder cells also were used. CD200Fc failed to suppress graft rejection in CD200R1(-/-) mice. CONCLUSION: CD200:CD200R1 plays an immunoregulatory role in vivo.     

Depleting anti-CD4 monoclonal antibody (GK1.5) treatment: influence on regulatory CD4+CD25+Foxp3+ T cells in mice

BACKGROUND: CD4(+)CD25(+) regulatory T (Treg) cells are often essential for the maintenance of immunologic self-tolerance and transplant tolerance in some cases. The effects of depleting anti-CD4 monoclonal antibody (GK1.5), which was used in transplant tolerance induction, on CD4(+)CD25(+) Treg cells have not been investigated. METHODS: Three weeks after BALB/c mice were injected with GK1.5 or phosphate-buffered saline, the levels, phenotype and immunosuppressive function of CD4(+)CD25(+) Treg cells in these mice were detected. RESULTS: The numbers of CD4 and CD4(+)CD25(+) Treg cells in the periphery were markedly decreased in GK1.5-treated mice. However, GK1.5 treatment significantly enhanced the ratios of CD4(+)CD25(+) T cells or CD4(+)CD25(+)Foxp3 T cells to CD4(+) T cells in the periphery (P<0.01). Compared with the control mice, more CD4(+)CD25(+) T cells in GK1.5-treated mice showed CD45RB and CD62L phenotype. Furthermore, enriched CD4(+)CD25(+) Treg cells in GK1.5-treated mice show immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as those from the control mice in vitro. CONCLUSIONS: GK1.5 could significantly enhance the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in the periphery while keeping these cells functional, indicating that GK1.5 might affect the potential induction of immune tolerance by different influences on CD4(+)CD25(+)Treg cells and CD4(+)CD25(-) T cells in periphery.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Discrete monoclonal antibodies define functionally important epitopes in the CD200 molecule responsible for immunosuppression function

BACKGROUND: Both murine and human CD200 fusion proteins (CD200Fc) act as immunosuppressants after engagement of cell-bound receptors (CD200R). Anti-CD200 monoclonal antibodies (mAbs) augment activity in mixed leukocyte cultures (MLCs) (increased cytotoxic T lymphocyte/cytokine production) after neutralization of endogenous CD200 activity. Previous studies documented critical regions in the N-terminal domains of both CD200 and CD200R1 for ligand:receptor binding and defined a number of synthetic CD200 and CD200R peptides that antagonize that interaction. METHODS: We used a panel of mAbs to mouse and human CD200Fc to compare the rank activities of antibodies for binding (flow cytometric analysis [FACS] or enzyme-linked immunoadsorbent assay [ELISA]) to CD200 with their abilities to augment immune reactivity in MLCs. RESULTS: Only mAbs defining epitopes in the N-terminal domain could augment MLC reactivity (or block immunosuppression by soluble CD200Fc), whereas mAbs targeting C-domain epitopes, although reactive in ELISA or FACS (targeting cell surface CD200), were inactive in MLCs. CONCLUSION: In addition to defining the importance of N-terminal epitopes for CD200 function, rank comparison of mAbs for FACS staining of CD200 expressed on various cell types indicates heterogeneity in expressed CD200.     

Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival

BACKGROUND: The expression of costimulatory molecules on antigen-presenting cells is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODNs) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendritic cells (DCs). MATERIALS AND METHODS: DCs were propagated from C57BL/10 (B10; H2b) bone marrow cells in granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, and transduced with anti-CD80 or anti-CD86 antisense ODNs (base-mismatched ODNs as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DCs was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. RESULTS: ODNs were effectively incorporated by DCs, which were enhanced by the presence of lipofectamine. Antisense ODNs targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DCs and inhibited their capacity to elicit the proliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2k) spleen T cells. This was associated with decreased IL-2, but elevated IL-4 production, and an increase in T-cell apoptosis. In contrast with the administration of control DCs into C3H recipients that exacerbated rejection of B10 cardiac allografts, injection of DCs transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. CONCLUSION: Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective approach to modify DCs that renders them tolerogenic by inducing T-cell hyporesponsiveness and apoptosis. This may lead to the development of new therapeutic strategies in transplantation.     

Prolongation of survival following depletion of CD4+CD25+ regulatory T cells in mice with experimental brain tumors

OBJECT: Regulatory CD4+CD25+ T cells have been shown to play an important role in the regulation of the immune response. Whereas the presence of these cells has been associated with immune suppression, the lack of regulatory T (Treg) cells has been shown to induce autoimmunity. The purpose of this study was to define the role of Treg cells in tumors of the central nervous system (CNS). METHODS: The authors implanted syngeneic GL261 tumor cells in the brains or flanks of C57BL/6 mice. The resulting tumors were later removed at specific time points, and the presence of tumor-infiltrating lymphocytes was analyzed by performing flow cytometry for the presence of Treg cells. In a separate experiment, mice with GL261 tumors were treated with injections of anti-CD25 monoclonal antibody (mAb) to determine whether depletion of Treg cells may have an impact on the length of survival in mice with brain tumors. Tumor-infiltrating lymphocytes isolated from mice with GL261 tumors were found to have a significant increase in the presence of Treg cells compared with control lymphocytes (p < 0.05). Moreover, Treg cells isolated in murine brain tumors expressed FoxP3, CTLA-4, and CD62L. Mice treated with anti-CD25 mAb lived significantly longer than tumor-bearing control animals (p < 0.05). An analysis of brains in surviving animals showed a depletion of CD4+CD25+ T cells. CONCLUSIONS: The results of this study indicate that CD4+CD25+ Treg cells play an important role in suppressing the immune response to CNS tumors. These Treg cells may therefore represent a potentially novel target for immunotherapy of malignant gliomas.     

Dendritic Cells and B Cells Cooperate in the Generation of CD4CD25FOXP3 Allogeneic T Cells

Background

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) play an essential role in immune tolerance, suppressing responses against self-antigens. Additionally, Treg play an important role in maintaining immunosuppression to alloantigens as well as to other antigens. It is well known that in the gut, a subset of dendritic cells produces retinoic acid (RA), which together with transforming growth factor (TGF-β) is able to differentiate naïve T cells into Treg. The aim of this study was to establish the role of antigen-presenting cells (APC) in the differentiation of allogeneic Tregs under the effect of RA and TGF-β.

Methods

Splenic CD4⁺CD25⁻ naïve T cells from C57BL/6 mice were co-cultured with splenic CD11c-enriched APC from Balb/c mice in the presence of TGF-β, RA, and interleukin (IL-2). After 6 days of culture, cells were analyzed for the expression of Foxp3 by flow cytometry. Additionally, we investigated the role of B cells and dendritic cells (DCs) and their stimulatory capacity in the generation of Tregs.

Results

Our results showed that co-culture of naive T cells with the appropriate level of stimulation by APC in the presence of TGF-β, RA, and IL-2 provided a new powerful approach to generate allogeneic Treg cells. We demonstrated that although B cells and DCs can generate Tregs by themselves, a mixure of both APC improved their capacity to efficiently generate Tregs. Also, we observed that although the addition of IL-2 to the cultures was not crucial to generate Tregs, it was required to optimize their expansion and cell survival.     

Costimulation blockade-induced cardiac allograft tolerance: inhibition of T cell expansion and accumulation of intragraft cD4(+)Foxp3(+) T cells

BACKGROUND: Previous studies have demonstrated that anti-CD40L or anti-B7 requires the presence of CD4(+)CD25(+) regulatory T cells (Treg) to induce antigen specific hyporesponsiveness. Other tolerance strategies involving Treg have shown a dependency on interleukin (IL)-10. The objective of this study was to investigate the role of CD4(+)CD25(+) Treg and IL-10 when treating transplant recipients with cytotoxic T lymphocyte-associated antigen (CTLA)-4 immunoglobulin (Ig), anti-CD40L, and anti-lymphocyte function-associated antigen (LFA)-1. METHODS: Recombinase activating gene-deficient (Rag1(-/-) mice were transplanted with BALB/c hearts and adoptively transferred with IL-10(-/-) CD4(+) T cells, CD4(+)CD25(-) T cells or CD4(+)CD25(-)CD103(-) T cells and treated with costimulation blockade. Intragraft T cells from C57BL/6 recipients were analyzed for the expression of the Foxp3 protein after tolerance induction. RESULTS: Mice reconstituted with IL-10(-/-) CD4(+) T cells, CD4(+)CD25(-) T cells or CD4(+)CD25(-) CD103(-) T cells and treated with costimulation blockade accepted allografts permanently. Analysis of cells from recipient mice adoptively transferred with CD4(+)CD25(-) T cells contained a population of CD4(low)CD25(+) T cells 100 days after transplantation. Costimulation blockade partially prevented the homeostatic proliferation of CD4(+)CD25(-)CD103(-) T cells in Rag-1(-/-) recipients. Accepted allografts contained an elevated number of CD4(+)Foxp3(+) T cells. CONCLUSIONS: These results indicate that T-cell derived IL-10 is not essential for induction of graft acceptance in mice treated with costimulation blockade, but that treatment limits T-cell expansion in the recipients. The results further indicate that tolerance is maintained by intragraft CD4(+)Foxp3(+) T cells.     

阻断ICOS/B7h信号的供体特异性输血对移植受体CD4+CD25+调节性T细胞的影响

目的 观察阻断ICOS/B7h信号的供体特异性输血(DST)对异基因小鼠心脏移植术后体内CD4+CD25+调节性T细胞(Treg)的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组:供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组:移植当天给予受体鼠(C57BL/6)尾静脉注射5×106 ICOS-Fc靶定的供体(BALB/C)脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4+CD25+Treg的亚群比例,利用逆转录-聚合酶链反应(RT-PCR)检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4+CD25+Treg对CD4+CD25-效应T细胞(Teff)的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[(84.38±29.14)d比(7.00±0.76)d,P＜0.01].各组中,治疗组受体外周血中CD4+CD25+Treg亚群比例显著上调[(15.60±5.69)%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4+CD25+Treg能够更高效地抑制CD4+CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4+CD25+Treg在耐受的形成与维持中均起着重要作用.     

Dendritic Cells Armed With Anti-CD3 mAbs Reduce Pulmonary Metastases, Prolong Survival, and Engender Antitumor Effector Cells Demonstrable by Adoptive Transfer

Background: Dendritic cells (DCs) pulsed with tumor cells or peptides are effective antitumor agents in a number of tumor models. In light of our earlier demonstration that T-cell signaling via the CD3 proteins induces cytolytic activity and constrains tumor progression, we equipped DCs pulsed with tumor cells with anti-CD3 mAbs and tested their antitumor efficacy in a murine renal cell cancer pulmonary metastasis model.Methods: We investigated the antitumor efficacy of DCs pulsed with whole irradiated tumor cells (DC/R) or DCs pulsed with irradiated tumor cells and armed with anti-CD3 mAbs (DC/R/anti-CD3 mAbs). Experimental end points included the number of pulmonary metastases and survival of tumor-inoculated mice.Results: Our studies demonstrate that arming tumor-pulsed DCs with anti-CD3 mAbs results in a superior outcome compared to that from tumor-pulsed DCs alone in terms of reduction in the number of pulmonary metastases and survival times. Furthermore, adoptive transfer experiments revealed that the splenocytes from DC/R/anti-CD3 mAbs-treated mice are superior to splenocytes from DC/R-treated mice in reducing renal cancer pulmonary metastases in severe combined immunodeficient (SCID) beige mice.Conclusion: Our data suggest that the therapeutic efficacy of DCs pulsed with tumor cells can be augmented by arming them with anti-CD3 mAbs. DC-based treatment regimens that currently are being pursued in clinical trials might be improved by equipping such cells with anti-CD3 mAbs.     

乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞水平的检测及意义

目的：探讨乳腺癌患者外周血CD4+CD25+Foxp3+调节性T细胞（Treg）水平检测的意义。 方法：流式细胞术检测74例乳腺癌患者与30例健康对照者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞百分比,分析CD4+CD25+Foxp3+Treg细胞水平与乳腺癌患者临床病理特征及相关免疫组化指标的关系。 结果：乳腺癌患者外周血CD4+CD25+Foxp3+Treg占CD4+T细胞的百分比高于健康对照者[（9.15± 2.24）% vs.（2.29±1.36）%],差异有统计学意义（P<0.05）。统计分析显示,乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移、pTNM分期以及HER-2、pS2、nm23的表达有关（均P<0.05）,而与肿瘤大小、病理类型以及雌激素受体（ER）、孕激素受体（PR）、p53、Ki-67表达无关（均P>0.05）。进一步相关性分析显示,CD4+CD25+Foxp3+Treg细胞水平与肿瘤组织学分级、淋巴结转移数、pTNM分期、HER-2的表达呈正相关（r=0.583,r=0.333,r=0.919,r=0.604,均P<0.05）而与pS2、nm23表达呈负相关（r=-0.229,r=-0.401,均P<0.05）。 结论：乳腺癌患者外周血CD4+CD25+Foxp3+Treg细胞水平升高,并与与乳腺癌的进展、转移密切相关,对其检测可能有助于患者预后及治疗效果的评估。