异基因外周造血干细胞移植后CD4^＋ CD25^＋调节性T细胞与GVHD关系的研究

目的:研究异基因外周血干细胞移植(Allo-PBSCT)后CD4 CD25 调节性T细胞(CD4 CD25 Tregs)与急、慢性移植物抗宿主病(a/cGVHD)的关系。方法:采用流式细胞术动态监测50例血液病恶性肿瘤患者Allo-PBSCT过程中,在移植前、植入时、植入后 30d、 60d、 120d、 360d外周血CD4 CD25 Tregs在CD4 T细胞中的百分含量。结果:Allo-PBSCT过程中,CD4 CD25 Tregs的百分含量在植入时降到最低,然后逐渐回升, 180d最高, 360d回落到移植前水平。发生aGVHD患者外周血的CD4 CD25 Tregs百分含量与无aGVHD患者无显著性差异,发生cGVHD患者CD4 CD25 Tregs百分含量较无cGVHD患者显著性增加。结论:Allo-PBSCT术后发生aGVHD时,受者调控CD4 CD25 Tregs数量的功能尚未恢复正常,发生cGVHD时,CD4 CD25 Tregs的数量可以反馈性地增加,但由于多种原因,CD4 CD25 Tregs难以抑制cGVHD的发生。     

Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD

CD4+CD25+ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4+CD25+ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4+CD25- T cells. Here we examined the differential effect of CD62L+ and CD62L- subsets of CD4+CD25+ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4+CD25+ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4+CD25- T cells, only the CD62L+ subset of CD4+CD25+ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4+CD25+CD62L+ Treg cells were transferred compared with the CD62L- subset. Subsequently, CD4+CD25+CD62L+ Treg cells showed a significantly higher capacity than their CD62L- counterpart to inhibit the expansion of donor CD4+CD25- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.     

Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4+CD25high regulatory T cells

Chronic graft-versus-host disease (cGVHD) is characterized by a state of profound immunodeficiency in association with alloreactive and autoimmune phenomena. These observations indicate an impairment of immunologic tolerance that could involve both central and peripheral mechanisms. Defective thymic function may contribute to dysregulation of central tolerance, but few studies have addressed peripheral tolerance. Recently a population of CD4+CD25+ T cells (Treg cells) has been characterized, which controls immunologic reactivity in vivo and which on transfer can prevent experimental acute GVHD. We investigated the number and function of peripheral blood CD4+CD25high T cells in patients more than 100 days after allogeneic hematopoietic stem cell transplantation. Patients with cGVHD had markedly elevated numbers of CD4+CD25high T cells as compared to patients without GVHD. CD4+CD25high T cells derived from patients in both groups were of donor origin, lacked markers of recent activation, and expressed intracellular CD152. In contrast to controls, CD4+CD25high T cells derived from patients with cGVHD were characterized by lower surface CD62L expression. In vitro, CD4+CD25high T cells were hyporesponsive to polyclonal stimulation and suppressed the proliferation and cytokine synthesis of CD4+CD25- cells, an effect that was independent of interleukin 10. These results indicate that chronic graft-versus-host injury does not occur as a result of Treg cell deficiency.     

Regulatory dendritic cells protect against cutaneous chronic graft-versus-host disease mediated through CD4+CD25+Foxp3+ regulatory T cells

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity and mortality in allogeneic bone marrow transplantation (alloBMT). However, effective strategies for the treatment of cGVHD have not been established. In this study, we examined the therapeutic utility of modified dendritic cells (DCs) with a greater capacity to regulate immune responses than previously known tolerogenic DCs, regulatory DCs (DC(regs)), in the major histocompatibility complex-compatible, and multiple minor histocompatibility antigen-incompatible model of cGVHD in alloBMT. Treatment of the recipient mice after alloBMT with the recipient-type DC(regs) led to greater suppression of the incidence and severity of cutaneous cGVHD than rapamycin, whereas treatment with the recipient-type mature DCs promoted the pathogenesis. Analysis of the recipient mice suggested that the protective effect of the recipient-type DC(regs) involved the peripheral generation of alloreactive CD4(+)CD25(+)Foxp3(+)regulatory T (T(R)) cells from donor-derived CD4(+)CD25(-)Foxp3(-) T cells. Thus, immunotherapy with DC(regs) is a promising strategy for the treatment of cGVHD in alloBMT mediated through the induction of a dominant tolerance involving CD4(+)CD25(+)Foxp3(+) T(R) cells.     

Severe chronic graft-versus-host disease is characterized by a preponderance of CD4(+) effector memory cells relative to central memory cells

Donor alloreactive CD4(+) T cells are important to the pathogenesis of chronic graft-versus-host disease (cGVHD), but specific subsets of CD4(+) T cells responsible for GVHD have not been defined. We hypothesized that cGVHD might be associated with a preponderance of CD4(+) effector memory cells (CCR7(-)/CD62L(low), CD4(EM)). We analyzed CCR7 and CD62L expression on CD4(+) T cells from stem cell transplantation patients, who did or did not develop cGVHD, and healthy donors. Patients with cGVHD had a higher percentage of CD4(EM) cells (35.5% +/- 2.9%) than healthy donors (13.8% +/- 0.7%; P <.0001) or patients without cGVHD that received a transplant (21.7% +/- 2.1%; P <.01). Using corticosteroid dose as a surrogate marker for cGVHD severity, severe cGVHD was associated with a higher percentage of CD4(EM) cells. The proportion of CD4(EM) cells in corticosteroid-dependent patients with systemic lupus erythematosis or Wegener granulomatosis did not differ from patients without cGVHD that received a transplant. This finding implies that overrepresentation of CD4(EM) cells is a unique feature of cGVHD.     

Stimulation with 4-1BB (CD137) inhibits chronic graft-versus-host disease by inducing activation-induced cell death of donor CD4+ T cells

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, is a costimulator for activated T cells. Previous studies have established that treatment with agonistic anti-4-BB monoclonal antibody (3H3) is effective in reversing the progression of spontaneous systemic lupus erythematosus. Its therapeutic effect is mediated by suppression of autoantibody production. In this report, we show that a single injection of 3H3 blocks chronic graft-versus-host disease (cGVHD) in the parent-into-F1 model. In particular, donor CD4+ T cells are rapidly eliminated from host spleens by activation-induced cell death after 4-1BB triggering. Since donor CD4+ T cells are required for the development of cGVHD, and 3H3-mediated inhibition of autoantibody production occurs without donor CD8+ T cells, 3H3 blocks cGVHD by preventing alloreactive donor CD4+ T cells from activating host B cells. Importantly, 3H3 treatment can reverse the progression of advanced cGVHD. Our findings indicate that agonistic anti-4-1BB monoclonal antibody has potential as an immunotherapeutic agent for preventing and treating cGVHD.     

Low telomerase activity in CD4+ regulatory T cells in patients with severe chronic GVHD after hematopoietic stem cell transplantation

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the control of chronic graft-versus-host disease (cGVHD). In this study, we examined telomere length and telomerase activity of Treg and conventional CD4(+) T cells (Tcon) in 61 patients who survived more than 2 years after allogeneic hematopoietic stem cell transplantation. Cell proliferation and expression of Bcl-2 were also measured in each subset. Treg telomere length was shorter and Treg telomerase activity was increased compared with Tcon (P < .0001). After transplantation, Treg were also more highly proliferative than Tcon (P < .0001). Treg number, telomerase activity, and expression of Bcl-2 were each inversely associated with severity of cGVHD. These data indicate that activation of telomerase is not sufficient to prevent telomere shortening in highly proliferative Treg. However, telomerase activation is associated with increased Bcl-2 expression and higher Treg numbers in patients with no or mild cGVHD. In contrast, patients with moderate or severe cGVHD have fewer Treg with lower levels of telomerase activity and Bcl-2 expression. These results suggest that failure to activate Treg telomerase may restrict proliferative capacity and increase apoptotic susceptibility, resulting in the loss of peripheral tolerance and the development of cGVHD.     

Correlation of peripheral blood OX40+(CD134+) T cells with chronic graft-versus-host disease in patients who underwent allogeneic hematopoietic stem cell transplantation.

There is no reliable laboratory indicator of the onset of chronic graft-versus-host disease (cGVHD). This study looks at whether the expression of OX40, a member of the tumor necrosis factor receptor family, is related to the development of cGVHD in patients who underwent allogeneic hematopoietic stem cell transplantation. Peripheral blood mononuclear cells from 22 patients after day 100 were subjected to multicolor flow cytometry. The percentages of both OX40+CD4+ and OX40+CD8+ T cells were significantly higher in patients with cGVHD than those without (P <.0001 and P =.001, respectively). Serial analyses showed that OX40+CD4+ T cells elevated before the onset of cGVHD and closely correlated with the therapeutic response. The expression of CD25, CD69, and HLA-DR was partially detectable on OX40+ T cells. These results indicate that serial measurement of OX40+ T cells is useful for predicting the onset as well as the therapeutic response of cGVHD and raise a possibility that the OX40/gp34 system is involved in the pathogenesis of cGVHD.     

Donor CD4+ T and B cells in transplants induce chronic graft-versus-host disease with autoimmune manifestations

Chronic graft-vs-host disease (GVHD) is a major cause of morbidity and mortality of long-term survivors of allogeneic hemato-poietic cell transplantation (HCT). Chronic GVHD can have features of an autoimmune collagen vascular disease with clinical manifestations similar to autoimmune scleroderma and systemic lupus erythematosus (SLE). However, the pathogenesis of chronic GVHD is poorly understood. It is unclear how autoreactive T and B cells are generated in chronic GVHD recipients. We have recently developed a new chronic GVHD model by transplantation of donor DBA/2 (H-2d) spleen cells into major histocompatibility complex (MHC)-matched but minor antigen-mismatched sublethally irradiated BALB/c (H-2d) recipients as well as athymic BALB/c(nu/nu) and adult-thymectomized BALB/c recipients. Both euthymic and athymic BALB/c recipients developed high levels of serum IgG autoantibodies, sclerodermatous skin damage, and glomerulonephritis. Disease induction required both donor CD25-CD4+ T and B cells in transplants. In contrast, donor CD25+CD4+ T regulatory (Treg) cells prevented the disease induction. These results indicate that host thymus is not required for induction of chronic GVHD and that quiescent autoreactive T and B cells in transplants from nonautoimmune donors may be activated and expanded to cause chronic GVHD with autoimmune manifestations in allogeneic recipients, and donor Treg cells can suppress this process.     

Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ

The application of allogeneic stem cell transplantation (alloSCT) is limited by graft-versus-host disease (GVHD). GVHD can be divided into acute and chronic forms that likely have different requirements for initiation and pathogenesis mechanisms. In prior studies we demonstrated that residual host antigen-presenting cells (APCs) were required to initiate acute GVHD (aGVHD) mediated by CD8 T cells. In contrast, here we demonstrate that either donor or host APCs can initiate CD4-mediated GVHD in a model that has features of chronic GVHD (cGVHD). Both donor and host APCs must provide CD80/86-dependent costimulation to elicit maximal cGVHD, and there is no GVHD when both donor and host lack CD80/86. Finally, we were surprised to find that, although either donor or host APCs are sufficient to stimulate skin cGVHD, donor APCs play a dominant role in intestinal cGVHD. Both CD40 and CD80/86 are critical for donor APC function in intestinal cGVHD, but only CD80/86 is required for skin cGVHD. Thus, there are target-tissue-specific differences in APC requirements. These results identify differences in APC requirements between CD8-mediated aGVHD and CD4-mediated cGVHD. They further highlight donor APCs as additional targets for GVHD therapy.     

Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative treatment for both inherited and acquired diseases of the hematopoietic compartment; however, its wider use is limited by the frequent and severe outcome of graft-versus-host disease (GVHD). Unfortunately, efforts to reduce GVHD by removing donor T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic hematopoietic stem/progenitor cell engraftment without inducing GVHD could increase numbers of potential recipients while broadening the pool of acceptable donors. Although unfractionated CD4(+) T cells have not been shown to be an efficient facilitating population, CD4(+)CD25(+) regulatory cells (T-reg's) were examined for their capacity to support allogeneic hematopoietic engraftment. In a murine fully major histocompatibility complex (MHC)-mismatched BMT model, cotransplantation of donor B6 T-reg's into sublethally conditioned BALB/c recipients supported significantly greater lineage-committed and multipotential donor progenitors in recipient spleens 1 week after transplantation and significantly increased long-term multilineage donor chimerism. Donor engraftment occurred without GVHD-related weight loss or lethality and was associated with tolerance to donor and host antigens by in vitro and in vivo analyses. Donor CD4(+)CD25(+) T cells may therefore represent a potential alternative to unfractionated T cells for promotion of allogeneic engraftment in clinical hematopoietic cell transplantation.     

Recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation

Human Valpha24+ natural killer T (NKT) cells have an invariant T-cell receptor-alpha chain and are activated in a CD1d-restricted manner. Valpha24+ NKT cells are thought to regulate immune responses and to play important roles in the induction of allograft tolerance. In this report, we analyzed the recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation and its correlation with graft-versus-host disease (GVHD). Patients who received a dose-reduced conditioning regimen, antithymocyte globulin- or CAMPATH-1H-containing conditioning regimen were excluded. NKT cells were reconstituted within 1 month after transplantation in peripheral blood stem cell transplantation recipients, while their numbers remained low for more than 1 year in bone marrow transplantation (BMT) recipients. The number of Valpha24+ NKT cells in BMT recipients with acute GVHD was lower than that in patients without acute GVHD, and both the CD4+ and CD4- Valpha24+ NKT subsets were significantly reduced. With regard to chronic GVHD, BMT recipients with extensive GVHD had significantly fewer Valpha24+ NKT cells than other patients. Furthermore, the number of CD4+ Valpha24+ NKT cells was also significantly reduced in patients with chronic extensive GVHD. Our results raise the possibility that the number of Valpha24+ NKT cells could be related to the development of GVHD.     

CD4+CD25+ cell depletion from the normal CD4+ T cell pool prevents tolerance toward the intestinal flora and leads to chronic colitis in immunodeficient mice

BACKGROUND: CD4+CD25+ regulatory T cells have been shown to prevent immune-mediated colitis in mice; however, it is unclear whether the absence of CD4+CD25+ in the normal CD4+ T cell pool is responsible for the development of chronic colitis. Using the T cell-deficient Tgepsilon26 mouse model, we show that CD4+CD25- cells but not CD4+CD25+ cells induce a severe intestinal inflammation. Transfer of CD4+CD25+ cells, together with CD4+CD25- cells, ameliorated intestinal inflammation, and reconstitution with the whole mesenteric lymph node cell pool did not induce colitis in recipients. Transferred CD4+CD25- cells were found mainly in the mesenteric lymph nodes, where they showed an activated TH1-like phenotype. In the absence of regulatory CD4+CD25+ T cells, recipient CD4 cells secreted IFN-gamma in response to stimulation with intestinal bacterial antigen that was prevented in vivo and in vitro by regulatory CD4+CD25+ cells. These studies suggest that CD4+CD25- cells have a strong colitogenic effect in the Tgepsilon26 colitis model and that CD4+CD25+ cells may be the main regulators that prevent or downregulate the proinflammatory effect of colitogenic T cells in the Tgepsilon26 mouse model.     

IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.     

TGF-beta in allogeneic stem cell transplantation: friend or foe?

Donor treatment with granulocyte-colony-stimulating factor (G-CSF) attenuates the ability of donor T cells to induce acute graft-versus-host disease (aGVHD) but increases the severity of chronic GVHD (cGVHD). We investigated the role of the regulatory cytokine transforming growth factor beta (TGF-beta) in this paradox in well-established murine models of aGVHD and cGVHD wherein recipients undergo transplantation with splenocytes from donors treated with G-CSF. Neutralization of TGF-beta after stem-cell transplantation (SCT) significantly increased the severity of aGVHD, and the concurrent prevention of interleukin-10 (IL-10) production further exaggerated this effect. Early after SCT, donor T cells were the predominant source of TGF-beta and were able to attenuate aGVHD in a TGF-beta-dependent fashion. Although the neutralization of TGF-beta augmented the proliferation and expansion of donor T cells after SCT, it paradoxically impaired cellular cytotoxicity to host antigens and associated graft-versus-leukemia (GVL) effects. In cGVHD, neutralization of TGF-beta from day 14 after SCT attenuated histologic abnormalities, and CD11b+ mononuclear cells infiltrating sclerodermatous skin produced 50-fold more TGF-beta than corresponding T cells. Thus, though the production of TGF-beta by donor T cells early after transplantation attenuates aGVHD and is required for optimal GVL, the production of TGF-beta late after SCT is preferentially from mononuclear cells and mediates cGVHD. These data have important implications for the timing of therapeutic TGF-beta neutralization to prevent cGVHD after allogeneic SCT.     

Prevention and treatment of graft-versus-host disease by down-regulation of anti-host reactivity with veto cells of host origin.

Control of graft-versus-host disease (GVHD) post allogeneic spleen cell transplantation was partly achieved by administration of host-type lymphocytes. (C57BL/6 x BALB/c) F1 mice were conditioned by total body irradiation and transplanted with parental C57BL/6 spleen cells to induce severe GVHD. Infusion of mature host-type lymphocytes did not change the degree of chimerism but protected most of the recipients from lethal GVHD. Treatment of host-type blood cells with anti-CD8 antibodies resulted in loss of the GVHD protective effects mediated by host cells, whereas after treatment of the host with anti-CD4 antibodies protective host cells given 4 days after induction of GVHD resulted in successful rescue of all transplanted recipients from lethal GVHD. Administration of host blood cells could effectively protect against GVHD induced by donor spleen cells and low-dose rIL-2. GVHD protection resulted from down-regulation of alloreactive donor T cells and not by rejection of GVHD effector cells. Taken together, administration of host hematopoietic cells particularly host CD8+ T cells, may be considered for prevention or control of GVHD following allogeneic bone marrow transplantation, thus also explaining the increased resistance of mixed chimeras to GVHD.     

Graft-host tolerance in bone marrow transplant chimeras. Absence of graft-versus-host disease is associated with unresponsiveness to minor histocompatibility antigens expressed by all tissues

Because bone marrow (BM) transplantation is used with increasing frequency, it is important to elucidate the mechanisms involved in the establishment of tolerance to host minor histocompatibility antigens (MiHA) in recipients transplanted with T-cell-undepleted marrow grafts. We have previously shown that BM chimeras transplanted across MiHA barriers showed specific unresponsiveness to MiHA expressed on recipient-type concanavalin A blasts. Because expression of many MiHA is tissue-specific, we wanted to determine if chimera T lymphocytes would be tolerant to MiHA expressed by all host tissues and organs. To investigate this issue, we measured in vivo proliferation of lymphoid cells from normal C57BL/10 (B10) mice and (B10-->LP) chimeras in tissues and organs of lethally irradiated syngeneic and allogeneic recipients. Donor B10 cells were either untreated, or depleted with anti-Thy-1.2, anti-CD4, or anti-CD8 antibodies. Transplantation of B10 cells in LP recipients triggered an important T-cell-dependent 125I- dUrd uptake in several organs that involved both CD4+ and CD8+ cells. Using Thy-1-congeneic mice we showed that in long-term chimeras practically all CD4+ and CD8+ T lymphocytes were derived from hematopoietic progenitors and not from mature T cells present in the BM graft. When (B10-->LP) BM chimera cells were injected to secondary recipients, no proliferation was observed in any organ of LP hosts whereas normal proliferation was seen in H-2k allogeneic hosts. Thus, in these BM chimeras, tolerance encompasses MiHA expressed by all organs.     

T memory stem cells after allogeneic haematopoietic cell transplantation: unique long-term kinetics and influence of chronic graft-versus-host disease

T memory stem cells (TSCMs) are a subset of primitive T cells capable of both self-renewal and differentiation into all subsets of memory and effector T cells. Therefore, TSCMs may play a role in immune reconstitution and graft-versus-host disease (GVHD) in patients receiving allogeneic haematopoietic cell transplantation (HCT). We conducted a cross-sectional study to evaluate the proportions, absolute counts, phenotypes and functions of TSCMs in 152 adult patients without disease recurrence at least 12 months after undergoing HCT. CD4⁺ TSCMs were negatively correlated with number of months after transplantation in HCT patients that received cord blood transplantation, but not in patients that received bone marrow transplantation or peripheral blood stem cell transplantation. The proportions and absolute counts of CD4⁺ TSCMs and expression levels of inducible co-stimulator (ICOS) in CD8⁺ TSCMs were significantly higher in patients with mild and moderate/severe cGVHD compared to patients without cGVHD. These data suggested that, more than 12 months after allogeneic HCT, the kinetics of CD4⁺ TSCMs were dependent on the type of donor source, and further that CD4⁺ TSCMs and ICOS levels in CD8⁺ TSCMs were associated with cGVHD.