Immunomodulation after allogeneic bone marrow transplantation by CD4+CD25+ regulatory T cells

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation (BMT) caused by donor T cells. Immunosuppression mediated by CD4(+)CD25(+) regulatory T cells has been shown to ameliorate such pathogenic immune responses in animal models. Here, we summarize recent findings from experimental and clinical studies and propose a model for peripheral tolerance induction after BMT.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Linomide administration following bone marrow transplantation in mice

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.     

Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination

We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.     

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

Therapeutic potential of CD4+ CD25+ regulatory T cells in allogeneic transplantation

The subpopulation of CD4+ CD25+ immunoregulatory T cells constitutes less than of the entire CD4+ T-cell pool in mice and 2% in humans. These cells play a crucial role in the control of autoimmune processes. More recently, in vitro and in vivo data also indicate that CD4+ CD25+ immunoregulatory T cells can regulate alloreactivity. This renders them good candidates for innovative strategies in the field of transplantation. Inducing a state of immune tolerance with immunoregulatory T cells would alleviate the need for immunosuppression, and the occurrence of late allograft failure represents a major goal of transplantation immunology. Here we discuss how these naturally occurring CD4+ CD25+ immunoregulatory T cells can be used to modulate alloreactivity in hematopoietic stem cell and solid organ transplantation.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3ε⁺ and FC-CD3ε⁻ subpopulations in the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells. Here, we demonstrate that the induction of FoxP3⁺CD4⁺CD25⁺ regulatory T cells in coculture is mediated by not only the FC-CD3ε⁻ subset but also the FC-CD3ε⁺ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3⁺CD4⁺CD25⁺ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.     

Accumulation and local proliferation of antigen-specific CD4+ T cells in antigen-bearing tissue

Although activation and subsequent expansion of naive CD4(+) T cells within lymph nodes is well characterized, the fate of T effector cells activated within peripheral tissues during secondary reactions is poorly defined. Therefore, we studied the recruitment, proliferation and egress of antigen-specific Th1 effector cells in comparison with nonspecific Th1 cells throughout a delayed-type hypersensitivity reaction (DTH). Although we observed a high turnover of Th1 effector cells with unspecific high-rate recruitment and CCR7-dependent egress from the inflamed tissue in the early, acute DTH phase, a strong, selective accumulation of antigen-specific T cells occurred during the chronic, late DTH phase. This was mainly based on local proliferation of CD4(+) effector cells within the DTH tissue and concomitant retention. Considering the strong CCR7-dependent Th cell egress found in this model, the reduced CCR7 expression on antigen-specific T cells isolated from late-phase DTH tissue most likely contributes to the retention of these cells within the tissue. Thus, peripheral tissues can support not only the proliferation of CD8(+) T cells, as recently shown, but also that of CD4(+) T effector cells, forming a pool of tissue-resident T cells.     

TGF-beta expression by allogeneic bone marrow stromal cells ameliorates diabetes in NOD mice through modulating the distribution of CD4+ T cell subsets

BMSCs could promote the regeneration of islet beta-cell, but the status of BMSCs under diabetes is still unknown. Our study verified the effect of allogeneic BMSCs (ICR) transferred into NOD mice on blood glucose and CD4+ T cells subsets function. In vivo experiment, BMSCs could decrease blood glucose, weaken lymphocytes proliferation. In vitro experiment, the distribution of CD4+ T cell subsets was changed after co-culture with BMSCs, resulting in a greater frequency of Treg cells and reduced representation of Th17 cells. After TGF-beta blockade, CD4+ T cells differentiated along a route favoring development of Th17, but not Treg cells. Thus, NOD can be treated by BMSCs which changes the distribution of CD4+ T cells, increases the number of Treg cells, and inhibits the differentiation of Th17 cells. And the positive effects of allogeneic BMSCs in the treatment of NOD mice depend on the regulation of TGF-beta secreted by BMSCs.     

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4⁺ and CD8⁺ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4⁺ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8⁺ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4⁺ and CD8⁺ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4⁺ T cells and the maintenance of the suppressive activity of CD8⁺ T cells.     

CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25- and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.     

Interleukin 2 regulation following semi-allogeneic bone marrow transplantation in mice

Success of allogeneic and autologous bone marrow transplantation (BMT) is hampered by susceptibility to infection during the first two post-treatment years. Further, in treating malignant diseases, impaired anti-host reactivity for donor cells may contribute to a high rate of relapse. Both complications are a consequence of immune deficiency involving B and T lymphocytes. The present study evaluates several key parameters of the immunologic reconstitution mechanism in mice subjected to myeloablative total body irradiation following semi-allogeneic (parental) BMT. This resulted in a gradual reduction of splenic CD3, CD4 and CD8 cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15–20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT.     

Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II+ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite graft survival. In rejecting mice the T cell response was mediated by two T cell subsets: 1) CD4+ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2) IFN-gamma-producing CD8+ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8+ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4+ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8+ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal provided by professional APCs in the periphery.     

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4 + CD25 + T regulatory cells are able to limit the effectiveness of CD4 + T cells and are able to favor the maintenance and the progression of B. abortus infection.     

CD40L启动CD4^＋T细胞缺乏小鼠免疫反应的研究

随着HIV感染者及各类医疗措施导致的免疫受损者的增多,探讨一种适合免疫缺陷人群的预防机会感染的策略越来越受到重视。研究表明,CD4^＋T细胞是抵抗肺孢子菌等机会感染的最主要因素,但不是唯一的因素。其中CD40配体（CD40L）被认为是一种可以启动B细胞和CD8^＋T细胞反应的关键因子。为探讨CD4^＋L是否能在缺乏CD4^＋T细胞的小鼠体内启动免疫反应,本文研究了用卵白蛋白（OVA）作为模型抗原,联合应用CD40L引起的免疫反应。结果显示,同时应用OVA和CD40L,可使CD4^＋T细胞耗竭小鼠体内抗OVA IgG抗体和抗原特异性IFN明显增多,提示在CD4^＋T细胞缺乏的宿主体内,CIMOL可以启动B细胞和CD8^＋T细胞类免疫反应。该结果为抗肺孢子菌等机会性感染的免疫预防研究提供可贵的资料。     

Insights into CD4+ memory T cells following Leishmania infection

Recent publications by Zaph et al. have highlighted the distinct requirements for generating and maintaining different subpopulations of CD4(+) memory T cells after infection with Leishmania major in mice. These studies have advanced the understanding of the nature of long-lasting immunity to Leishmania and, when considered within the context of previous work on both murine and human leishmaniasis, will aid the design of effective vaccines.