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.     

The initial phase of graft-versus-host disease is associated with a decrease of CD4+CD25+ regulatory T cells in the peripheral blood of patients after allogeneic stem cell transplantation

The mechanisms that induce and control the alloimmune inflammation of graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (allo-SCT) are still incompletely understood. In the murine system, GvHD can be suppressed by CD4(+)CD25(+) regulatory T cells (TREG), which are generally involved in the suppression of inflammatory reactions. A disruption of the homeostasis between TREG and conventional T cells might therefore be associated with the inflammatory reactions of GvHD. We repetitively measured the frequency of TREG in the peripheral blood of 29 patients within the first 71-373 days after allo-SCT and correlated the results with the clinical course. We demonstrate that the initial phase of GvHD is associated with a significant reduction of TREG in the peripheral blood, while at later stages and during intensified immunosuppressive therapy, increased numbers of TREG appear. These results might indicate a pathogenic role for reduced numbers of TREG in the induction of human GvHD.     

Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells

Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4?CD25?Foxp3? regulatory T-cell (Treg) proliferation and function in vitro. We investigated whether the combination of RAPA plus IL-2 could impact acute GVHD induction after bone marrow transplantation (BMT). RAPA plus IL-2 resulted in improved survival and a reduction in acute GVHD lethality associated with an increased expansion of donor type CD4?Foxp3? Tregs and reduced CD4?CD25? conventional T cells (Tcons). RAPA plus IL-2, but not either drug alone, increased both expansion of donor natural Tregs and conversion of induced Tregs from donor CD25? Tcons while IL-2 alone increased conversion of Tregs from CD25? Tcon. RAPA plus IL-2 treatment resulted in less production of IFN-γ and TNF, cytokines known to be important in the initiation of acute GVHD. These studies indicate that the pharmacologic stimulation of T cells with IL-2 and the suppression of Tcon proliferation with RAPA result in a selective expansion of functional Tregs and suppression of acute GVHD.     

IL-13 production by donor T cells is prognostic of acute graft-versus-host disease following unrelated donor stem cell transplantation

Despite the success of human leukocyte antigen (HLA) typing in allogeneic stem cell transplantation (SCT) it is rare to find an unrelated donor that is perfectly matched, making identification of "permissive" mismatches of paramount importance. Here, we describe novel associations between donor T-cell cytokine production during donor-antipatient mixed lymphocyte reactions (MLRs) and acute graft-versus-host disease (aGVHD). The data reveal positive correlations between both Th1-type and Th2-type cytokine production and GVHD and the assay established could potentially represent a useful tool for identification of permissible unrelated SCT donors. Associations between interleukin 13 (IL-13) levels and aGVHD were by far the strongest predictor of a GVHD (P =.0002). All patients suffering severe (grade III) aGVHD following SCT had donors who produced very high pretransplantation IL-13 responses, while those developing little or no aGVHD (grades 0-I) produced no IL-13 at all. IL-13 levels were independent of all other cytokines measured as well as cytotoxic T-lymphocyte precursor (CTLp) frequencies. The cytokines IL-5, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) also predicted development of aGVHD (P <.05 for all 3), appearing to be coproduced in the assay and correlating with estimated CTLp frequencies. The data challenge the notion that aGVHD is purely a Th1-type cytokine-driven response, high-lighting a novel and highly significant link between the Th2-type cytokine IL-13 and aGVHD.     

Serum cytokine levels and acute graft-versus-host disease after HLA-identical hematopoietic stem cell transplantation

OBJECTIVE: The aim of this study was to investigate whether the serum levels of soluble interleukin-2R (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10, and transforming growth factor-beta(1) (TGF-beta(1)) were associated with the development of acute graft-vs-host disease (aGVHD). PATIENTS AND METHODS: Serum cytokine levels were sequentially measured by sandwich enzyme-linked immunosorbent assay in 13 patients who had received full-match allogeneic hematopoietic stem cell transplantation (HSCT). RESULTS: Serum sIL-2R and IL-10 levels from the 1st to the 15th week post transplantation were significantly higher in the group that developed aGVHD than in the group without aGVHD. sIL-2R levels increased in direct correlation to engraftment and at onset of aGVHD, whereas IL-10 levels increased transiently following HSCT. The mean TNF-alpha concentration in the first weeks after transplantation was augmented in the group that developed aGVHD. Furthermore, a decrease in TGF-beta(1) levels after engraftment was significantly associated with aGVHD. No correlation was found between aGVHD and the other cytokines. CONCLUSIONS: These results support the idea that a balance between cytokines derived from type 1 and type 2 T-helper cells may be important in the development and control of aGVHD. Although sIL-2R, TNF-alpha, IL-10, and TGF-beta(1) levels have been correlated with aGVHD, sIL-2R levels at engraftment may provide a better parameter for early detection of aGVHD after allogeneic HSCT.     

Critical role for CCR5 in the function of donor CD4+CD25+ regulatory T cells during acute graft-versus-host disease

CD4+CD25+ regulatory T cells (T(regs)) have been shown to inhibit graft-versus-host disease (GVHD) in murine models, and this suppression was mediated by T(regs) expressing the lymphoid homing molecule l-selectin. Here, we demonstrate that T(regs) lacking expression of the chemokine receptor CCR5 were far less effective in preventing lethality from GVHD. Survival of irradiated recipient animals given transplants supplemented with CCR5-/- T(regs) was significantly decreased, and GVHD scores were enhanced compared with animals receiving wild-type (WT) T(regs). CCR5-/- T(regs) were functional in suppressing T-cell proliferation in vitro and ex vivo. However, although the accumulation of T(regs) within lymphoid tissues during the first week after transplantation was not dependent on CCR5, the lack of function of CCR5-/- T(regs) correlated with impaired accumulation of these cells in the liver, lung, spleen, and mesenteric lymph node, more than one week after transplantation. These data are the first to definitively demonstrate a requirement for CCR5 in T(reg) function, and indicate that in addition to their previously defined role in inhibiting effector T-cell expansion in lymphoid tissues during GVHD, later recruitment of T(regs) to both lymphoid tissues and GVHD target organs is important in their ability to prolong survival after allogeneic bone marrow transplantation.     

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Background

A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care.

Design and Methods

Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease.

Results

Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO⁺ CD27⁻) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25^high Foxp3⁺) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease.

Conclusions

Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8+ T cells

Cytomegalovirus (CMV) infection causes significant morbidity and mortality in the setting of immunodeficiency, including the immune reconstitution phase following allogeneic stem cell transplantation (SCT). We assessed CMV-specific CD4(+) and CD8(+) T-cell responses in 87 HLA-A*0201-positive (A2+) and/or B*0702-positive (B7+) allogeneic stem cell transplant recipients using HLA-peptide tetramer staining and cytokine flow cytometry (CFC) to examine the association of CMV-specific immune reconstitution and CMV antigenemia following SCT. Strong CMV-specific T-cell responses recovered in most subjects (77 of 87, 88%) after SCT. Frequencies of CMV-specific CD8(+) T cells were significantly higher in those subjects who experienced early antigenemia relative to those who did not (2.2% vs 0.33%, P =.0002), as were frequencies of CMV-specific CD4(+) T cells (1.71% vs 0.75%, P =.002). Frequencies of CMV-specific CD8(+) T cells were also higher in subjects experiencing late antigenemia (2.4% vs 0.57%). When we combined tetramer staining and an assessment of cytokine production in a single assay, we found that individuals who experienced CMV antigenemia had lower tumor necrosis factor-alpha (TNF-alpha)-producing fractions of tetramer-staining CMV-specific CD8(+) T cells than subjects who did not (25% vs 65%, P =.015). Furthermore, individuals at high risk for CMV reactivation, including patients with acute graft-versus-host disease and those receiving steroids, had low fractions of cytokine-producing CMV-specific CD8(+) T cells (25% and 27%, respectively). These data suggest that the inability to control CMV reactivation following allogeneic SCT is due to the impaired function of antigen-specific CD8(+) T cells rather than an inability to recover sufficient numbers of CMV-specific T cells.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28⁻ suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28⁻ T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28⁻ T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Contribution of CD4+ and CD8+ T cells to graft-versus-host disease and graft-versus-leukemia reactivity after transplantation of MHC-compatible bone marrow.

A murine model of allogeneic bone marrow (BM) transplantation was used to determine the relative importance of CD4+ and CD8+ T cells in establishing donor T cell chimerism and in the development of graft-versus-host (GVH) and graft-versus-leukemia (GVL) reactivity. Mature donor T cells were essential for complete chimerism when host mice (AKR, H-2k) were conditioned with suboptimal irradiation (9 Gy = LD50). Transplantation of donor BM (B10.BR, H-2k) resulted in mixed chimerism, whereas mice given BM containing additional T cells developed into complete and stable chimeras. Depletion of T cell subsets was associated with an increase in the frequency of mixed chimerism. The incidence of lethal GVHD was dependent on the number of T cells added to the BM inoculum. Ex vivo depletion of CD4+ T cells eliminated GVH-associated mortality. Removal of CD8+ T cells had no effect on overall survival. In contrast to the GVH results, removal of either CD4+ or CD8+ T cells compromised GVL reactivity, indicating that an optimal GVL response required both CD4+ and CD8+ T cells. T cell-subset depletion did not interfere with the induction of donor-host tolerance in these chimeras and may have facilitated its development. The loss of GVH/GVL effector cells as a result of T cell depletion and the development of donor-host tolerance may act synergistically to prevent or suppress GVH and GVL reactivity.     

In vitro-expanded donor alloantigen-specific CD4+CD25+ regulatory T cells promote experimental transplantation tolerance

CD4+CD25+ regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25- effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro-generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.     

Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease

Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30-/-) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30-/- animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.     

Percentage of CD3+CD4-CD8-gammadeltaTCR- T cells is increased HIV disease.

HIV patients given highly active antiretroviral therapy (HAART) experience a rapid rise in alphabetaT cell numbers, but changes in gammadeltaT cell populations have not been described. Here we investigate the effects of immune reconstitution and immune restoration diseases (IRDs) on expression of a pan-gammadeltaT cell receptor (TCR) marker on double-negative (CD3(+)CD4(-)CD8(-)) T cells and T cells expressing CD4 or CD8. IRDs are inflammatory disorders associated with preexisting infections in patients who have achieved immune reconstitution after HAART. Proportions of CD3(+)CD4(-)CD8(-) T cells and total gammadeltaT cells were not affected by CD4(+) T cell counts, HAART, or a history of IRD, but levels of CD4(-)CD8(-)gammadeltaTCR(-) T cells were higher in patients with <15% CD4(+) T cells.