Increased frequency of CD4(+)CD25(high) Treg cells inhibit BCG-specific induction of IFN-gamma by CD4(+) T cells from TB patients

Cell-mediated immunity plays a considerable role in the protection against Mycobacterium tuberculosis infection. The immune response to tuberculosis (TB) was dominated by both CD4(+) T cells with the T helper 1 type cytokines and CD8(+) T cells. Recent studies have suggested that the circumstances in which protective or tissue-damaging T cell responses to microbes are affected by the activity of Treg (CD4(+)CD25(high)) cells. In the present study, we demonstrated that the frequencies of CD4(+)CD25(+) and CD4(+)CD25(high) T cells in TB patients were significantly higher compared to normal individuals. These Treg cells expressed CTLA-4 and Foxp3 at protein level and displayed activation and memory phenotypes as assessed by flow cytometric analysis. The frequencies of CD4(+)CD25(high)CTLA-4(+) and CD4(+)CD25(high)Foxp3(+) T cells within the total CD4(+) T cell population were significantly increased in the blood of TB patients compared to healthy donors. Moreover, the expression of GITR on Treg cells was higher in TB patients than in normal donors. The phenotypic analysis demonstrated that CD4(+)CD25(high) Treg expressed higher levels of CD45RO and HLA-DR, and lower levels of CD45RA compared to CD4(+)CD25(low) and CD4(+)CD25(-) T cells. The addition of CD4(+)CD25(high) T cells back to cultures could significantly suppress the antigen-specific production of IFN-gamma induced by BCG-stimulated CD4(+)CD25(-) T cells, suggesting that Treg might play a key role in the control of cellular immune responses in TB infection.     

Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function

CD4(+)CD25(+)Foxp3(+) regulatory T cells (CD25(+) Treg cells) direct the maintenance of immunological self-tolerance by active suppression of autoaggressive T-cell populations. However, the molecules mediating the anergic state and regulatory function of CD25(+) Treg cells are still elusive. Using differential proteomics, we identified galectin-10, a member of the lectin family, as constitutively expressed in human CD25(+) Treg cells, while they are nearly absent in resting and activated CD4(+) T cells. These data were confirmed on the mRNA and protein levels. Single-cell staining and flow cytometry showed a strictly intracellular expression of galectin-10 in CD25(+) Treg cells. Specific inhibition of galectin-10 restored the proliferative capacity of CD25(+) Treg cells and abrogated their suppressive function. Notably, first identified here as expressed in human T lymphocytes, galectin-10 is essential for the functional properties of CD25(+) Treg cells.     

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.     

Effect of interleukin-15 on effector and regulatory function of anti-CD3/anti-CD28-stimulated CD4(+) T cells

Autologous transfer of anti-CD3/anti-CD28 (CD3/CD28)-activated CD4(+) T cells may benefit patients receiving autologous stem cell transplant with severe CD4 lymphopenia. Interleukin (IL)-15, an IL-2-like cytokine that promotes T cell survival may enhance immune reconstitution in conjunction with adoptive immunotherapy. We investigated the effect of IL-15 on effector and regulatory function of CD3/CD28-activated CD4(+) T cells. IL-15 upregulated CD45RO and CD25 whereas it down regulated CD62L expression of CD3/CD28-stimulated CD4(+) T cells. Both type 1 (IFN-gamma, tumor necrosis factor (TNF)-alpha) and type 2 (IL-5 and IL-10) production by CD3/CD28-activated CD4(+) T cells was further enhanced by IL-15. Co-culture experiments revealed that CD3/CD28-activated CD4(+) T cells down regulated proliferation of autologous peripheral blood lymphocytes (PBLs) and CD8(+) PBL subsets upon TCR ligation, a contact-dependent effect that was further enhanced by pretreatment with IL-15. Flow cytometric analysis of cell mixture with carboxyfluorescein diacetate succinimidyl ester and Annexin-V-PE staining revealed that CD3/CD28+IL-15-activated CD4(+) T cells showed increased apoptosis over CD4(+) T cells stimulated with CD3/CD28 alone. Taken together, pretreatment of CD3/CD28-activated CD4(+) T cells with IL-15 may increase regulatory function but may aggravate activation-induced apoptosis of CD3/CD28 CD4(+) T cells.     

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.     

CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells

Foxp3 expression was initially thought to be restricted to the CD4(+)CD25(+) regulatory T-cell population. However, recent studies suggest that forkhead box P3 (Foxp3) is expressed in CD4(+)CD25(-) T cells in aged mice. In the present study in B-cell non-Hodgkin lymphoma (NHL), we found that a subset of intratumoral but not peripheral blood CD4(+)CD25(-) T cells, comprising about 15% of intratumoral CD4(+) T cells, express Foxp3 and are capable of suppressing the proliferation of autologous infiltrating CD8(+) T cells. In vitro activation with OKT3/anti-CD28 antibody (Ab) or dendritic cells (DCs) induced Foxp3 expression in a subset of these CD4(+)CD25(-)Foxp3(-) T cells. We found that the presence of lymphoma B cells during activation augmented activation-induced Foxp3 expression in CD4(+)CD25(-) T cells. We also found that CD70(+) lymphoma B cells significantly contributed to the activation-induced Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Furthermore, the blockade of CD27-CD70 interaction by anti-CD70 Ab abrogated lymphoma B-cell-mediated induction of Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Taken together, these studies reveal a novel role for NHL B cells in the development of intratumoral regulatory T cells.     

CD49d provides access to "untouched" human Foxp3+ Treg free of contaminating effector cells

The adoptive transfer of regulatory Foxp3(+) T (Treg) cells has been shown in various animal models to prevent inflammatory immune and autoimmune diseases. Translation into therapeutic applications, however, is hindered by the lack of suitable techniques and markers. CD25, commonly used to isolate Treg cells from mice, has only limited value in humans as it is also present on proinflammatory CD4(+) effector cells. Here we show that clean populations of human Foxp3(+) Treg cells can be obtained with antibodies directed against CD49d. The marker is present on proinflammatory peripheral blood mononuclear cells but is absent on immune-suppressive Treg cells. Depletion with alpha-CD49d removes contaminating interferon-gamma (IFN-gamma)- and interleukin-17 (IL-17)-secreting cells from Treg preparations of CD4(+)CD25(high) cells. More importantly, in combination with alpha-CD127 it allows the isolation of "untouched" Foxp3(+) Treg (ie, cells that have not been targeted by an antibody during purification). The removal of CD49d(+)/CD127(+) cells leaves a population of Foxp3(+) Treg virtually free of contaminating CD25(+) effector cells. The cells can be expanded in vitro and are effective suppressors both in vitro and in vivo. Thus, CD49d provides access to highly pure populations of untouched Foxp3(+) Treg cells conferring maximal safety for future clinical applications.     

Apoptosis-induced decrease of intrathyroidal CD4(+)CD25(+) regulatory T cells in autoimmune thyroid diseases.

BACKGROUND: We previously showed that the proportion of CD4(+) T cells was lower and both the proportion and intensity of Fas expression on intrathyroidal CD4(+) T cells were higher in the thyroid than in the peripheral blood of patients with autoimmune thyroid disease (AITD). OBJECTIVE: To clarify whether the intrathyroidal CD4(+)CD25(+) regulatory T (Treg) cells are decreased by Fas-mediated apoptosis in patients with AITD. DESIGN: We examined intrathyroidal and peripheral lymphocytes in 20 patients with AITD (15 patients with Gravesa disease and five patients with Hashimotoas disease) and peripheral lymphocytes in 10 healthy volunteers by three-color flow cytometry. MAIN OUTCOME: The proportion of CD4(+)CD25(+) cells was lower in the thyroid of patients with AITD than in the peripheral blood of the same patients or the peripheral blood of the healthy subjects. The proportions of CD4(+)CD25(+)CD69() cells and Forkhead box P3 (Foxp3)(+)CD4(+)CD25(+) cells, which constitute more specific Treg subsets than CD4(+)CD25(+) cells, were also lower in the thyroid than in the peripheral blood of patients with AITD. The proportion of apoptotic cells was higher among intrathyroidal CD4(+) cells than among peripheral CD4(+) cells and higher among intrathyroidal CD4(+)CD25(+) cells than among intrathyroidal CD4(+)CD25() cells. CONCLUSION: These results indicate that intrathyroidal Treg cells are decreased in response to apoptosis in patients with AITD. This decrease in Treg cells may contribute to the incomplete regulation of autoreactive T cells in AITD.     

Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is still a major complication after allogeneic stem cell transplantation. In murine models, freshly isolated or ex vivo expanded CD4(+)CD25(high) regulatory T cells (Treg) are able to ameliorate GVHD while maintaining graft-versus-leukaemia reactions. However, in the human setting, prospective studies of this population and its interaction with activated non-regulatory CD134(+) (OX40) lymphocytes during post-transplant follow-up are lacking. In this study, we prospectively quantified CD4(+)CD25(high) and activated CD134(+) lymphocytes in 119 peripheral blood samples from 35 consecutive patients who underwent allogeneic bone marrow transplantation (BMT). Fifty-five samples obtained less than 100 d after allogeneic BMT, were not statistically different regarding CD4(+)CD25(high) Treg or CD134(+) lymphocytes compared with those obtained from patients with (n = 35) or without (n = 20) acute GVHD. Chronic GVHD was associated with a small, but not statistically significant, increase in the number of Treg (9.9 vs. 6.7 x 10(6)/L). However, the CD134/CD25(high) ratio was significantly higher during chronic GVHD (cGHVD) when compared with either patients without cGVHD (67.7 +/- 40.3 vs. 4.0 +/- 0.9, P < 0.01) or cGVHD after treatment (67.7 +/- 40.3 vs. 3.7 +/- 0.8, P < 0.01). Our findings suggest that the suppressive activity of CD4(+)CD25(high) Treg could be abrogated in vivo during cGVHD by CD134 expression in a much higher number of activated donor T lymphocytes. In addition to CD4(+)CD25(high)ex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.     

Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling

CD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25(+)Foxp3(+)) from naive CD4 T cells (CD25(-)Foxp3(-)) by T-cell receptor stimulation with additional transforming growth factorβ (TGFβ) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2-inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling.     

Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.     

Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells have been shown to inhibit adaptive responses by T cells. Natural killer (NK) cells represent an important component of innate immunity in both cancer and infectious disease states. We investigated whether CD4(+)CD25(+) Treg cells could affect NK cell function in vivo by using allogeneic (full H2-disparate) bone marrow (BM) transplantation and the model of hybrid resistance, in which parental marrow grafts are rejected solely by the NK cells of irradiated (BALB/c x C57BL/6) F(1) recipients. We demonstrate that the prior removal of host Treg cells, but not CD8(+) T cells, significantly enhanced NK cell-mediated BM rejection in both models. The inhibitory role of Treg cells on NK cells was confirmed in vivo with adoptive transfer studies in which transferred CD4(+)CD25(+) cells could abrogate NK cell-mediated hybrid resistance. Anti-TGF-beta mAb treatment also increased NK cell-mediated BM graft rejection, suggesting that the NK cell suppression is exerted through TGF-beta. Thus, CD4(+)CD25(+) Treg cells can potently inhibit NK cell function in vivo, and their depletion may have therapeutic ramifications for NK cell function in BM transplantation and cancer therapy.     

Suppressor activity and potency among regulatory T cells is discriminated by functionally active CD44

CD4CD25(+) regulatory T cells are fundamental to the maintenance of peripheral tolerance and have great therapeutic potential. However, efforts in this regard have been hampered by limiting cell numbers in vivo, an anergic phenotype in vitro, and a rudimentary understanding of the molecular basis for the functional state of CD4CD25(+) regulatory T cells (Treg cells). Here we show heterogeneity of suppressor activity among activated CD4CD25(+) Treg cells and that, within this population, the functionally active, hyaluronan-binding form of CD44 (CD44(act)) is strikingly correlated with superior suppressor activity. Within 16 hours after in vitro activation, CD44(act) can discriminate enhanced suppressive function in in vitro proliferation assays and in an in vivo bone marrow engraftment model. The expression of other surface markers and that of Foxp3 are similar irrespective of hyaluronan binding and associated degree of suppressor potency. Furthermore, CD44(act) is induced on resting CD4CD25(+) cells in vivo by allogeneic stimulation, with similar functional consequences. These results reveal a cell-surface marker that delineates functional activity within a population of activated CD4CD25(+) regulatory T cells, thereby providing a potential tool for identifying regulatory activity and enriching for maximal suppressor potency.     

Depletion of CD25^＋CD4^＋T cells （Tregs） enhances the H BV-specific CD8^＋ T cell response primed by DNA immunization

AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.     

Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells

CD4(+)CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance, and their adoptive transfer gives protection from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4(+)CD25+ Treg cells display phenotypic and functional characteristics similar to those of murine CD4(+)CD25+ Treg cells: namely, hyporesponsiveness to T-cell receptor (TCR) stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4(+)CD25+ Treg cells have been hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40 000-fold expansion of highly purified human CD4(+)CD25high T cells in vitro through the use of artificial antigen-presenting cells for repeated stimulation via CD3 and CD28 in the presence of high-dose interleukin 2 (IL-2). Expanded CD4(+)CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4(+)CD25high T cells, and maintained expression of the lymph node homing receptors L-selectin (CD62L) and CCR7. The ability to rapidly expand human CD4(+)CD25high Treg cells on a large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine.     

Proliferation and foxp3 expression in virus-specific memory CD8+ T lymphocytes

Foxp3 plays a critical role in development of CD4+ regulatory T lymphocytes (Tregs). It was originally proposed as a specific marker for Tregs, but recent studies have shown that Foxp3 can be expressed in proliferating CD8+ and CD4+ T lymphocytes. We further investigated the association between Foxp3 expression and proliferation of peripheral blood CD4+ and CD8+ T lymphocytes and focused on virus-specific memory CD8+ T lymphocytes. We found that resting peripheral blood bulk and cytomegalovirus- or HIV-1-specific CD8+ T lymphocytes do not normally express Foxp3. However, stimulation in vitro triggered these cells to express Foxp3 as well as CD25, and the addition of interleukin-2 possibly enhanced the expression of Foxp3. These data demonstrate that proliferation itself is sufficient to induce the Treg-like phenotype. Given that others have demonstrated Treg functional activity in such "induced Tregs," these results suggest that virus-specific CD8+ T lymphocytes have the capacity to acquire regulatory functions. Although the implications of Foxp3 expression in virus-specific CD8+ T lymphocytes in the immunologic control of persistent HIV-1 viremia remain to be determined, our results are consistent with Foxp3 expression playing an essential role in regulation of cell proliferation and functional outcomes for HIV-1-specific CD8+ T lymphocytes.