Mesenchymal cells recruit and regulate T regulatory cells

OBJECTIVE: Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. MATERIALS AND METHODS: Human MSCs and different T cell populations (CD3(+), CD3(+)/CD45RA(+), CD3(+)/CD45RO(+), CD4(+)/CD25(+), CD4(+)/CD25(+)/CD45RO(+), CD4(+)/CD25(+)/CD45RA(+)) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. RESULTS: We demonstrate MSC recruit Tregs from a fraction of CD3(+) and from immunoselected CD3(+)/CD45RA(+) and CD3(+)/CD45RO(+) fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4(+)/CD25(bright)/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25(+), CD4/CD25(+)/CD45RA(+), and CD4/CD25(+)/CD45RO(+)) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. CONCLUSIONS: In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.     

Phosphoantigen-activated V gamma 2V delta 2 T cells antagonize IL-2-induced CD4+CD25+Foxp3+ T regulatory cells in mycobacterial infection

Although Foxp3(+) T regulatory cells (Tregs) are well documented for their ability to suppress various immune cells, T-cell subsets capable of counteracting Tregs have not been demonstrated. Here, we assessed phosphoantigen-activated Vgamma2Vdelta2 T cells for the ability to interplay with Tregs in the context of mycobacterial infection. A short-term IL-2 treatment regimen induced marked expansion of CD4(+)CD25(+)Foxp3(+) T cells and subsequent suppression of mycobacterium-driven increases in numbers of Vgamma2Vdelta2 T cells. Surprisingly, activation of Vgamma2Vdelta2 T cells by adding phosphoantigen Picostim to the IL-2 treatment regimen down-regulated IL-2-induced expansion of CD4(+)CD25(+)Foxp3(+) T cells. Consistently, in vitro activation of Vgamma2Vdelta2 T cells by phosphoantigen plus IL-2 down-regulated IL-2-induced expansion of CD4(+)CD25(+)Foxp3(+) T cells. Interestingly, anti-IFN-gamma-neutralizing antibody, not anti-TGF-beta or anti-IL-4, reduced the ability of activated Vgamma2Vdelta2 T cells to down-regulate Tregs, suggesting that autocrine IFN-gamma and its network contributed to Vgamma2Vdelta2 T cells' antagonizing effects. Furthermore, activation of Vgamma2Vdelta2 T cells by Picostim plus IL-2 treatment appeared to reverse Treg-driven suppression of immune responses of phosphoantigen-specific IFNgamma(+) or perforin(+) Vgamma2Vdelta2 T cells and PPD-specific IFNgamma(+)alphabeta T cells. Thus, phos-phoantigen activation of Vgamma2Vdelta2 T cells antagonizes IL-2-induced expansion of Tregs and subsequent suppression of Ag-specific antimicrobial T-cell responses in mycobacterial infection.     

Expression of CD30 in patients with acute graft-versus-host disease

Acute GVHD (aGVHD) remains a major source of morbidity after allogeneic hematopoietic cell transplantation. CD30 is a cell-surface protein expressed on certain activated T cells. We analyzed CD30 expression on peripheral blood T-cell subsets and soluble CD30 levels in 26 patients at the time of presentation of aGVHD, before the initiation of treatment, compared with 27 patients after hematopoietic cell transplantation without aGVHD (NONE). Analysis by flow cytometry showed that patients with aGVHD had a greater percentage of CD30 expressing CD8(+) T cells with the difference especially pronounced in the central memory subset (CD8(+)CD45RO(+)CD62L(+)): GVHD median 12.4% (range, 0.8%-33.4%) versus NONE 2.1% (0.7%, 17.5%), P < .001. There were similar levels of CD30 expression in naive T cells, CD4(+) T cells, and regulatory (CD4(+)CD127(low)CD25(+)) T cells. Plasma levels of soluble CD30 were significantly greater in patients with GVHD: median 61.7 ng/mL (range, 9.8-357.1 ng/mL) versus 17.4 (range, 3.7-142.4 ng/mL) in NONE (P < .001). Immunohistochemical analysis of affected intestinal tissue showed many CD30(+) infiltrating lymphocytes present. These results suggest that CD30 expression on CD8(+) T-cell subsets or plasma levels of soluble CD30 may be a potential biomarker for aGVHD. CD30 may also represent a target for novel therapeutic approaches for aGVHD.     

CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.     

CD4+CD25+ T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen-specific inhibitors of rejection with different phenotypes, suggesting separate pathways of activation by Th1 and Th2 responses

CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.     

Severe immune dysregulation affects CD4(+)CD25(hi)FoxP3(+) regulatory T cells in HIV-infected patients with low-level CD4 T-cell repopulation despite suppressive highly active antiretroviral therapy

We hypothesized that CD4(+)CD25(hi)FoxP3(+) regulatory T cells (Tregs) could be involved in the high immune activation existing in patients with low-level CD4 T-cell repopulation under suppressive high active antiretroviral therapy (hereafter, "LLR patients"). Sixteen LLR patients, 18 human immunodeficiency virus (HIV)-infected controls (hereafter, "HIV controls"), and 16 healthy subjects were included. The frequency of CD4(+)CD25(hi)FoxP3(+) and HIV-specific Treg suppressive function were assessed. Relationships between Treg and CD4/CD8 activation (HLA-DR/CD38) and the frequency of naive CD4 T-cells were assessed. Low-level patients showed a higher Treg frequency but reduced HIV-specific immunosuppressive functions than HIV controls. Whereas in healthy subjects a strong negative correlation between Tregs and activated CD8 T cells emerged (r?=?-0.75, P?相似文献   

Preferential attachment of HIV particles to activated and CD45RO+CD4+ T cells.

We have studied the binding of biotinylated HIV particles to various cell lines and peripheral blood mononuclear cells (PBMCs). Viruses were harvested from cultures of cell surface-biotinylated cells productively infected with HIV-IIIB. Labeled HIV particles bound to and infected CD4(+) cell lines and PBMCs. The interaction between gp120 and CD4 contributed in part to HIV binding to CD4(+) cells. However, HIV binding was for the most part independent of CD4 expression and sensitive to polyanion inhibition. Polyanion-sensitive interactions involved heparan sulfate in cell lines but not in primary T cells. Interestingly, HIV binding to primary cells was heterogeneous and targeted discrete subsets of CD4(+) and CD4(-) cells. The CD4(+) T cell subset that displayed high HIV-binding capacity contained mostly CD4(+)CD45RO(+) cells, whereas the subset showing undetectable HIV binding contained higher proportions of CD4(+)CD45RO(-) cells. Consistently, purified CD4(+)CD45RO(-) cells or purified CD4(+) T cells with low virus-binding capacity showed lower HIV entry and delayed HIV replication when compared with purified CD4(+)CD45RO(+) or purified CD4(+) T cells with high virus-binding capacity, respectively. Our data suggest that the binding of HIV to cell surface-expressed CD4 might be inefficient in a subset of CD4(+) T cells and that increased binding of HIV to activated and CD4(+)CD45RO(+) cells may contribute to the higher susceptibility of these cells to HIV infection.     

Long-term effects of intermittent interleukin 2 therapy in patients with HIV infection: characterization of a novel subset of CD4(+)/CD25(+) T cells

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV(+)) patients receiving highly active antiretroviral therapy (HAART), and HIV(+) patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4(+)/CD25(+) T cells. Increased CD25 expression, especially in CD4(+) T cells but also in CD8(+) T cells, without increases in expression of the beta and gamma chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4(+) T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4(+)/CD25(+) T cells correlated positively with both the total and naive CD4(+) T-cell counts. A discrete population of CD45 double intermediate RA(+)/RO(+) CD4(+) cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4(+) count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4(+)/CD25(+) and CD4(+)/CD25(-) cells from IL-2-treated HIV(+) patients proliferated in response to mitogens, specific antigens, and T-cell-receptor-mediated stimuli. Thus, intermittent administration of IL-2 in HIV(+) patients leads to preferential expansion of a unique subset of CD4(+) T cells that may represent a critical population in T-cell homeostasis.     

Regulatory potential and control of Foxp3 expression in newborn CD4+ T cells

Thymectomy at day 3 after birth leads to autoimmune disease in some genetic backgrounds. Disease is thought to be caused by the lack/paucity of regulatory T cells. We show that 3-day-old mice already contain a significant compartment of Foxp3-expressing CD25(+)CD4(+) splenocytes. Whereas, in adult spleen, the subsets of regulatory T cells (CD25(+) and/or CD103(+)) express high amounts of Foxp3 mRNA, in 3-day-old mice, both thymic and splenic CD25(+)CD4(+) T cell subsets express lower amounts of Foxp3 mRNA, and CD103(+) cells are barely detected. In adult day 3-thymectomized mice, the CD25(+)CD4(+) T cell subset is overrepresented (most of the cells being CD103(+)) and expresses high amounts of Foxp3 mRNA, independent of the development of autoimmune gastritis. These cells control inflammatory bowel disease and the homeostatic expansion of lymphocytes. This study demonstrates that the peripheral immune system of newborn mice is endowed of a remarkable regulatory potential, which develops considerably in the absence of thymic supply.     

The immunosuppressive drug FK778 induces regulatory activity in stimulated human CD4+ CD25- T cells

The induction of transplantation tolerance involves a T-cell-mediated process of immune regulation. In clinical transplantation, the use of immunosuppressive drugs that promote or facilitate this process would be highly desirable. Here, we investigated the tolerance-promoting potential of the immunosuppressive drug FK778, currently under development for clinical therapy. Using a human allogeneic in vitro model we showed that, upon T-cell receptor (TCR) triggering, FK778 induced a regulatory phenotype in CD4+ CD25- T cells. Purified CD4+ CD25- T cells primed in the presence of FK778 showed hyporesponsiveness upon restimulation with alloantigen in the absence of the drug. This anergic state was reversible by exogenous interleukin-2 (IL-2) and was induced independent of naturally occurring CD4+ CD25+ regulatory T cells. Pyrimidine restriction was a crucial requirement for the de novo induction of regulatory activity by FK778. The FK778-induced anergic cells showed suppressor activity in a cell-cell contact-dependent manner; were CD25(high), CD45RO+, CD27-, and CD62L-; and expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and FoxP3. The cells revealed delayed p27(kip1) degradation and enhanced phosphorylation of STAT3. In conclusion, the new drug FK778 shows tolerizing potential through the induction of a regulatory T-cell subset in CD4+ CD25- T cells.     

Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+ CD25+ FoxP3+ T cells

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3(+) regulatory T-cell (T(REG)) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for T(REG) homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on T(REG) homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4(+)CD25(+)FoxP3(+) T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25(+)FoxP3(+) T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4(+)FoxP3(+) T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4(+)FoxP3(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.     

Increased CD4~+CD45RA~-FoxP3~(low) cells alter the balance between Treg and Th17 cells in colitis mice

AIM To investigate the role of regulatory T cell(Treg) subsets in the balance between Treg and T helper 17(Th17) cells in various tissues from mice with dextran sulfate sodium-induced colitis.METHODS T r e g c e l l s, T r e g c e l l s u b s e t s, T h 1 7 c e l l s, a n d CD4+CD25+FoxP 3+IL-17+ cells from the lamina propria of colon(LPC) and other ulcerative colitis(UC) mouse tissues were evaluated by flow cytometry. Forkhead box protein 3(FoxP 3), interleukin 17A(IL-17A), and RORC m RNA levels were assessed by real-time PCR, while interleukin-10(IL-10) and IL-17 A levels were detected with a Cytometric Beads Array.RESULTS In peripheral blood monocytes(PBMC), mesenteric lymphnode(MLN), lamina propria of jejunum(LPJ) and LPC from UC mice, Treg cell numbers were increased(P 0.05), and FoxP 3 and IL-10 mR NA levels were decreased. Th17 cell numbers were also increased in PBMC and LPC, as were IL-17 A levels in PBMC, LPJ, and serum. The number of FrI subset cells(CD4+CD45RA+FoxP 3low) was increased in the spleen, MLN, LPJ, and LPC. FrI I subset cells(CD4+CD45RA-Fox P3high) were decreased among PBMC, MLN, LPJ, and LPC, but the number of Fr III cells(CD4+CD45RA-FoxP 3low) and CD4+CD25+FoxP 3+IL-17A+ cells was increased. Fox P3 m RNA levels in CD4+CD45RA-Fox P3 low cells decreased in PBMC, MLN, LPJ, and LPC in UC mice, while IL-17 A and RORC mR NA increased. In UC mice the distribution of Treg, Th17 cells, CD4+CD45RA-FoxP 3high, and CD4+CD45RA-FoxP 3low cells was higher in LPC relative to other tissues.CONCLUSION Increased numbers of CD4+CD45RA-FoxP 3low cells may cause an imbalance between Treg and Th17 cells that is mainly localized to the LPC rather than secondary lymphoid tissues.     

Low percentages of regulatory T cells in common variable immunodeficiency (CVID) patients with autoimmune diseases and its association with increased numbers of CD4+CD45RO+ T and CD21low B cells

BackgroundCommon variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies defined by marked reductions in serum IgG, IgA and/or IgM levels and recurrent bacterial infections. Some patients are associated with defects in T cells and regulatory T cells (Tregs), resulting in recurrent viral infections and early-onset autoimmune disease.MethodsWe analyzed whether there is an association between Tregs cells (CD4+CD25+CD127^low and CD4+CD25+FoxP3+); memory T cells (CD4+CD45RO+); memory B cells (CD19+CD27-IgD-); and CD21^low B cells (CD19+CD38^lowCD21^low); as well as autoimmune manifestations in 36 patients with CVID (25 women and 11 men, mean age 24 years), all by flow cytometry.ResultsFourteen patients presented with autoimmune diseases (AI) (39%), including 11 with autoimmune thrombocytopenia (ITP) (31%); two with vitiligo (6%); one with systemic lupus erythematosus (LES) (3%); and one with multiple sclerosis (MS) (3%). CVID patients with AI had a reduced proportion of Tregs (both CD4+CD25+CD127^low and FoxP3+ cells) compared with healthy controls. CVID patients with AI had expanded CD21^low B cell populations compared with patients who did not have AI. A correlation between increased CD4+CD45RO T cell populations and reduced Tregs was also observed.ConclusionsOur results showed that 39% of patients with CVID had AI and reduced Tregs populations. Research in this area might provide noteworthy data to better understand immune dysfunction and dysregulation related to CVID.     

CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are potent suppressors of the adaptive immune system, but their effects on innate immune cells are less well known. Here we demonstrate a previously uncharacterized function of Tregs, namely their ability to steer monocyte differentiation toward alternatively activated macrophages (AAM). AAM are cells with strong antiinflammatory potential involved in immune regulation, tissue remodeling, parasite killing, and tumor promotion. We show that, after coculture with Tregs, monocytes/macrophages display typical features of AAM, including up-regulated expression of CD206 (macrophage mannose receptor) and CD163 (hemoglobin scavenger receptor), an increased production of CCL18, and an enhanced phagocytic capacity. In addition, the monocytes/macrophages have reduced expression of HLA-DR and a strongly reduced capacity to respond to LPS in terms of proinflammatory mediator production (IL-1beta, IL-6, IL-8, MIP-1alpha, TNF-alpha), NFkappaB activation, and tyrosine phosphorylation. Mechanistic studies reveal that CD4(+)CD25(+)CD127(low)Foxp3(+) Tregs produce IL-10, IL-4, and IL-13 and that these cytokines are the critical factors involved in the suppression of the proinflammatory cytokine response. In contrast, the Treg-mediated induction of CD206 is entirely cytokine-independent, whereas the up-regulation of CD163, CCL18, and phagocytosis are (partly) dependent on IL-10 but not on IL-4/IL-13. Together these data demonstrate a previously unrecognized function of CD4(+)CD25(+)Foxp3(+) Tregs, namely their ability to induce alternative activation of monocytes/macrophages. Moreover, the data suggest that the Treg-mediated induction of AAM partly involves a novel, cytokine-independent pathway.     

Thymic retention of CD4+CD25+FoxP3+ T regulatory cells is associated with their peripheral deficiency and thrombocytopenia in a murine model of immune thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. Several reports have observed that ITP is associated with a peripheral deficiency of tolerance-inducing CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). Using a murine model of ITP, we analyzed Tregs in the spleen and thymus. CD61 knockout mice were immunized against wild-type (CD61(+)) platelets, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice. Compared with SCID mice receiving naive splenocytes, within 2 weeks after transfer, the ITP SCID mice became thrombocytopenic (< 200 × 10(9) platelets/L) and had increased serum anti-CD61 antibodies. The quantity of thymic Tregs by 2 weeks after transfer was significantly elevated, whereas Tregs in the spleens were significantly reduced. Treatment of the ITP mice with 2 g/kg intravenous immunoglobulin raised the platelet counts, reduced antibody production, and normalized the thymic and splenic Treg populations. Compared with thymocytes from ITP mice treated with intravenous immunoglobulin, thymocytes from untreated ITP mice delayed the onset of ITP when administered before engraftment with immune splenocytes. These results suggest that ITP in mice is associated with a peripheral Treg deficiency because of thymic retention and therapy normalizes the Tregs.     

Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4

Regulatory T cells (Treg) comprise multiple subsets and are important in controlling immunity and inflammation. However, the induction and mode of action of the various distinct Treg subsets remain ill defined, particularly in humans. Here, we describe a human CD8+ lymphocyte activation gene-3 (LAG-3)+CD25+FoxP3+ Treg subset, which suppresses T cells partly through the secretion of CC chemokine ligand 4 (CCL4), which can inhibit T cell activation by interfering with T cell receptor signaling. CD8+ Tregs are expanded by antigen in in vivo-primed donors, and can be detected in pathogen-infected human tissue. This CD8+LAG-3+CD25+FoxP3+CCL4+ Treg subset thus may play a role in immunoregulation in humans, including infectious diseases.