Amelioration of acute graft-versus-host disease by NKG2A engagement on donor T cells

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic bone marrow transplantation, which is caused by donor T cells specific for host alloantigens. In a murine model, we found that donor T cells expressed a natural killer cell inhibitory receptor, CD94/NKG2A, during the course of aGVHD. Administration of an anti-NKG2A mAb markedly inhibited the expansion of donor T cells and ameliorated the aGVHD pathologies. These results suggested that the CD94/NKG2A inhibitory receptor expressed on host-reactive donor T cells can be a novel target for the amelioration of aGVHD.     

T cells, murine chronic graft-versus-host disease and autoimmunity

The chronic graft-versus-host disease (cGVHD) in mice is characterized by the production of autoantibodies and immunopathology characteristic of systemic lupus erythematosus (lupus). The basic pathogenesis involves the cognate recognition of foreign MHC class II of host B cells by alloreactive CD4 T cells from the donor. CD4 T cells of the host are also necessary for the full maturation of host B cells before the transfer of donor T cells. CD8 T cells play critical roles as well. Donor CD8 T cells that are highly cytotoxic can ablate or prevent the lupus syndrome, in part by killing recipient B cells. Host CD8 T cells can reciprocally downregulate donor CD8 T cells, and thus prevent them from suppressing the autoimmune process. Thus, when the donor inoculum contains both CD4 T cells and CD8 T cells, the resultant syndrome depends on the balance of activities of these various cell populations. For example, in one cGVHD model (DBA/2(C57BL/6xDBA/2)F1, the disease is more severe in females, as it is in several of the spontaneous mouse models of lupus, as well as in human disease. The mechanism of this female skewing of disease appears to depend on the relative inability of CD8 cells of the female host to downregulate the donor CD4 T cells that drive the autoantibody response. In general, then, the abnormal CD4 T cell help and the modulating roles of CD8 T cells seen in cGVHD parallel the participation of T cells in genetic lupus in mice and human lupus, although these spontaneous syndromes are presumably not driven by overt alloreactivity.     

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells

We have recently found that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to prevent graft-versus-host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM-BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM-BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV-DLI]). GvHD in recipients treated with allogeneic IBM-BMT plus IBM-DLI was far milder than in those treated with allogeneic IBM-BMT plus IV-DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4(+)CD25(+) and CD4(+)Foxp3(+) cells was significantly higher in recipients treated with IBM-BMT plus IBM-DLI than in those treated with IBM-BMT plus IV-DLI. Donor-derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM-BMT plus IBM-DLI, whereas Th1 cells were dominant in recipients treated with IBM-BMT plus IV-DLI. Furthermore, the production of transforming growth factor-beta and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM-DLI. Thus, IBM-BMT plus IBM-DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD. Disclosure of potential conflicts of interest is found at the end of this article.     

Activation-associated phenotype of CD3 T cells in acute graft-versus-host disease

During the effector phase of graft-versus-host disease (GvHD) response, donor T cells play an essential role and they are believed to change the expression of activation and co-stimulatory markers associated with functional alloreactivity. We analysed the expression of CD25, CD69, HLA-DR, CD154 and CD134 on CD4+ and CD8+ T cells by flow cytometry during acute GvHD (aGvHD) in 24 patients receiving human leucocyte antigen (HLA)-identical stem cell transplants. Expression of these molecules in nine patients with stages I-IV aGvHD was compared with 15 patients without aGvHD (n = 15). Serial analysis showed that peripheral blood of aGvHD patients presented a significant increase of CD4+ CD25+ cells (P < 0.03), CD4+ CD69+ (P < 0.04) and CD4+ CD134+ cells (P < 0.01). Additionally, there was a significant increase in CD8+ cells expressing CD134 (P = 0.007) and CD154 (P = 0.02). After resolution of aGvHD, the increased expression of these molecules returned to values comparable to patients without aGvHD. Only two of the 15 patients without clinical signs of aGvHD presented activated T cells that could not be attributed to development of aGvHD. In summary, our data show that multiple activation molecules are preferentially up-regulated on CD4+ and CD8+ T cells from patients with aGvHD. These patients had a significant increase in the expression of the co-stimulatory molecules CD134 and CD154.     

FTY720 enhances the activation-induced apoptosis of donor T cells and modulates graft-versus-host disease

FTY720 is a novel immunosuppressant that improves the outcomes after solid organ and bone marrow transplantation (BMT) due to the sequestration of T cells into LN. We tested the hypothesis that the sequestration of donor T cells in LN by FTY720 would enhance their interaction with host APC, thus causing a greater degree of activation-induced apoptosis of alloreactive T cells, and thereby resulting in a reduction of graft-vs.-host disease (GVHD). The short-term administration of FTY720 improved the recipient survival after allogeneic BMT. FTY720 treatment facilitated a rapid contraction of the donor T cell pool in association with an increased degree of apoptosis of donor T cells. The donor T cell reactivity to host alloantigens was diminished in host's LN and adoptive transfer of donor T cells isolated from LN of FTY720-treated recipients of allogeneic BMT induced less severe GVHD in secondary recipients than the transfer from controls. Caspase-dependent apoptosis was involved in this mechanism because FTY720-induced protection was abrogated when a pan-caspase inhibitor was administered. These findings thus demonstrate the presence of a novel mechanism by which FTY720 modulates the allogeneic T cell responses: namely, by the induction of activation-induced apoptosis of alloreactive T cells in LN.     

Regulatory T cells in graft-versus-host disease

Alloreactive T cells present in a bone marrow transplant are responsible for graft-vs-host disease, but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-vs-leukemia effect. The subpopulation of CD4⁺CD25⁺ immunoregulatory T cells was first identified based on its crucial role in the control of autoimmune processes, but they also play a role in alloreactive responses. Moreover, these cells could be used to develop innovative strategies in the field of transplantation and particularly to prevent graft-vs-host disease. Indeed, high numbers of CD4⁺CD25⁺ immunoregulatory T cells can modulate graft-vs-host disease if administered at the same time as allogeneic hematopoietic stem cell transplantation in mice. This review discusses various important issues regarding the possible use of CD4⁺CD25⁺ immunoregulatory T cells to modulate alloreactivity in hematopoietic stem cell transplantation.     

The role of danger signals and ectonucleotidases in acute graft-versus-host disease

Allogeneic hematopoietic cell transplantation (allo-HCT) represents the only curative treatment approach for many patients with benign or malignant diseases of the hematopoietic system. However, post-transplant morbidity and mortality are significantly increased by the development of acute graft-versus-host disease (GvHD). While alloreactive T cells act as the main cellular mediator of the GvH reaction, recent evidence suggests a critical role of the innate immune system in the early stages of GvHD initiation. Danger-associated molecular patterns released from the intracellular space as well as from the extracellular matrix activate antigen-presenting cells and set pro-inflammatory pathways in motion. This review gives an overview about danger signals representing therapeutic targets with a clinical perspective with a particular focus on extracellular nucleotides and ectonucleotidases.     

Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution.

Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host-reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2(b)] --> AKR/J [H-2(k)]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell-depleted bone marrow plus thymidine kinase-positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline-treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell-depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline-treated animals had persistent skewing in most Vbeta families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4(+) T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.     

Normal IgG protects against acute graft-versus-host disease by targeting CD4(+)CD134(+) donor alloreactive T cells

Intravenous immunoglobulin (IVIg) was shown to decrease the severity of acute graft-versus-host disease (aGVHD) in recipients of allogeneic bone marrow transplants. To investigate the mechanisms involved in the protective effect of IVIg, we have used the parent-into-F1 model in which parental lymphocytes are transferred into semi-syngeneic non-irradiated F1 rats. Here we report that IVIg, as well as F(ab')(2) fragments of IVIg, protected (Lewis x Brown-Norway) F1 rats against aGVHD induced by a single injection of Lewis lymphocytes. IVIg was given as five consecutive daily injections, starting on the day preceding that of the transfer of Lewis cells. Protection was associated with a decreased ability of lymphocytes to spontaneously proliferate and to produce NO and IFN-gamma, in the absence of an increased production of IL-10. We further demonstrate that protection was associated with a decrease in CD4(+) T cells bearing the activation marker CD134 in vivo, and with an enhanced apoptosis of activated CD4(+) T cells by IVIg, in vitro. Our observations suggest that the prevention of aGVHD by IVIg in this model is mediated by the induction of apoptosis of activated alloreactive CD4(+)CD134(+) donor T cells. The results further emphasize the role of normal immunoglobulin in modulating alloantigen immune responsiveness.     

Host T cells affect donor T cell engraftment and graft-versus-host disease after reduced-intensity hematopoietic stem cell transplantation.

Mixed chimerism in the T cell compartment (MCT) after reduced-intensity stem cell transplantation (RIST) may influence immune repopulation with alloreactive donor T cells. We examined effects of host T cell numbers on donor T cell engraftment and recovery and on acute graft-versus-host disease (aGVHD) in a relatively homogeneous patient population with respect to residual host T cells through quantified immune depletion prior to RIST and to donor T cells by setting the allograft T cell dose of 1x10(5) CD3+ cells/kg. In this setting, 2 patterns of early donor T cell engraftment could be distinguished by day +42: (1) early and complete donor chimerism in the T cell compartment (FDCT) and (2) persistent MCT. FDCT was associated with lower residual host CD8+ T cell counts prior to transplant and aGVHD. With persistent MCT, subsequent development of aGVHD could be predicted by the direction of change in T cell donor chimerism after donor lymphocyte infusion, and no aGVHD occurred until FDCT was established. MCT did not affect recovery of donor T cell counts. These observations suggest that the relative number and alloreactivity of donor and host T cells are more important than the absolute allograft T cell dose in determining donor engraftment and aGVHD after RIST.     

Involvement of both donor cytotoxic T lymphocytes and host NK1.1+ T cells in the thymic atrophy of mice suffering from acute graft-versus-host disease.

It has been reported that a dramatic decrease in the number of thymocytes (thymic atrophy) in mice suffering from acute graft-versus-host disease (GVHD) is ascribed to glucocorticoids. In this study, we examined the possibility that cellular immune responses may thus be involved in thymic atrophy. In contrast to chronic GVHD mice, acute GVHD (C57BL/6 X DBA/2) F1 (BDF1) hybrid mice, which were injected intravenously with both spleen and lymph node cells from C57BL/6 mice, showed a dramatic decrease in the number of CD4 CD8 double-positive thymocytes 2 or 3 weeks after the induction of GVHD. A flow cytometric analysis revealed the donor-derived T cells with either CD4 or CD8 molecules to infiltrate the thymus of the mice undergoing acute GVHD for 10 days. In a cytolytic assay, such thymus-containing cells exhibited a cytolytic activity specific to the host cells. In addition, anti-H-2d cytolytic T cells showed a high level of cytolytic activity against BDF1 (H-2bXd) thymocytes, whereas they also showed a low level of cytolytic activity against C57BL/6 (H-2b) thymocytes, thus suggesting that the thymus-infiltrating donor-derived T cells killed the host thymocytes through both anti-H-2d-specific and non-specific mechanisms. Interestingly, a flow cytometric analysis revealed both the percentage and the absolute cell number of host-derived NK1.1+ CD3+ cells to increase in the thymus of mice suffering from acute GVHD for 10 days. In addition, they also showed the cytolytic activity against YAC-1 cells and the mRNA expression of interleukin-12 (IL-12) in the thymus to be also significantly augmented on day 7 after the induction of acute GVHD. Collectively, our results indicate that the cellular immune responses such as donor cytotoxic T lymphocytes and host NK1.1+ T cells are therefore involved in the thymic atrophy of mice suffering from acute GVHD.     

Hydroxychloroquine for the prevention of acute graft-versus-host disease after unrelated donor transplantation.

Hydroxychloroquine (HCQ) is an immunosuppressive agent that interferes with antigen presentation and with activity against graft-versus-host disease (GVHD). In a phase II trial assessing the GVHD prophylactic effects of HCQ, 51 consecutive unrelated donor transplant recipients received HCQ in addition to cyclosporin A, methylprednisolone, and methotrexate. HCQ was initiated on pretransplantation day -21 at 800 mg/d and continued until day +100 after transplantation. HCQ was extremely well tolerated and was not associated with side effects. Pharmacokinetic analyses demonstrated large inter- and intrapatient variability. The addition of HCQ did not affect posttransplantation immune recovery. Grade II to IV acute GVHD was observed in 56% of patients, and grade III and IV GVHD was observed in 17%. Day +100 mortality was 22%. When compared with a matched cohort of patients reported to the International Bone Marrow Transplant Registry, patients receiving HCQ had comparable cumulative incidences of grade II to IV acute GVHD. However, lower incidences of grades III and IV GVHD and better GVHD-free survival were observed in HCQ-treated patients (P =.01). We conclude that prophylactic HCQ is well tolerated and associated with a low incidence of severe acute GVHD. An ongoing placebo-controlled randomized trial will further determine what role HCQ plays in preventing GVHD after allografting.     

Characterization of skin-infiltrating T lymphocytes during acute graft-versus-host disease

A panel of 34 clones was established from a cell line derived from the skin biopsy of a patient (genotype: A1, A2, B7, B8, DR3, DR6) undergoing acute graft-versus-host disease after semiallogeneic bone marrow transplantation with his mother's bone marrow (genotype: A1, A1, B7, B8, DR3, DR6). The T-cell line obtained presented the following phenotype: CD3+, CD4+, CD8-, CD16-, WT31+, T-cell receptor delta 1-, 4B4+, 2H4-, CD25+, DR+. This CD4+ T-cell line was poorly cytotoxic against the target cells tested, including the mother's phytohemagglutinin blasts as a negative control (autologous T cells), the father's phytohemagglutinin blasts bearing the mismatch haplotype, K562, U937, SVK14 (a keratinocyte cell line), and a panel of B-lymphoblastoid cell lines bearing HLA-A2, the known mismatch antigen. All but 1 of the 34 clones obtained were of CD4+ phenotype, and none was CD16+. Only the sole CD8+ clone showed significant cytotoxicity against the father's phytohemagglutinin blast; however, this cytotoxic activity was associated with the highest score for nonspecific killing against both K562 and U937. This work demonstrates the feasibility of obtaining a large panel of clones from a graft-versus-host disease target organ to constitute the basic cellular material for in vitro study of the graft-versus-host process.     

Critical role of endogenous Mtv in acute lethal graft-versus-host disease

Little is known about the etiology of the graft-versus-host disease (GVHD) occuring after transplantation of lymphoid cells incompatible for minor histocompatibility antigens (mHAg). Here, the potential role of host endogenous mouse mammary tumor virus (Mtv)-encoded superantigens (SAg) in the development of lethal GVHD was investigated. In a combination of H-2^d compatible mice, the presence of Mtv-7 and, to a lesser extent, of Mtv-1, -6, -13 in the host genome, highly increases the rate and severity of GVHD. Kinetic analyses of TCR Vβ gene expression in recipient mice consistently indicate a dramatic but transient infiltration of GVHD target organs by Mtv-SAg-specific T cells. This suggests that SAg encoded by endogenous Mtv, by activating large T cell subpopulations, would help the response to mHAg and thus play a critical role in the initiation or aggravation of GVHD.     

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM-BMT) greatly facilitates the engraftment of donor-derived cells, and IBM-BMT can attenuate graft-versus-host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.-BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM-BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex-disparate donors (BALB/c) were injected directly into the BM cavity (IBM-DLI) or injected intravenously (i.v.-DLI) along with IBM-BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM-BMT + i.v.-DLI showed severe graft-versus-host disease (GVHD), in contrast to those treated with IBM-BMT + IBM-DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM-BMT + IBM-DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM-BMT + i.v.-DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor-beta and hepatocyte growth factor in cultured BMSCs from IBM-BMT + IBM-DLI increased significantly when compared with those from the recipients of IBM-BMT + i.v.-DLI. Thus, the prevention of GVHD observed in the recipients of IBM-BMT + IBM-DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).     

The role of CD4+CD25hi regulatory T cells in the physiopathogeny of graft-versus-host disease

Donor T cells present within hematopoietic stem cell transplants promote engraftment, contribute to T-cell reconstitution and provide an antileukemic effect. However, they are also responsible for the life-threatening graft-versus-host disease. The subpopulation of CD4+CD25hi regulatory T cells, initially identified as crucial players in the regulation of autoimmune processes, might also play a role in the control of alloreactivity. Experimental studies in mice indicate that donor regulatory T cells indeed control alloreactive responses and reduce graft-versus-host disease. Recent clinical reports also suggest that higher numbers of CD4+CD25hi cells within the transplant or in the blood of grafted patients might be associated with reduced graft-versus-host disease. Hence, these cells are attractive immunoregulatory candidates to prevent graft-versus-host disease in humans.     

Mesenchymal stem cells for graft-versus-host disease: close encounters with T cells

Mesenchymal stem cells (MSC) have recently received centre stage attention because of their potent immunosuppressive effect which has also been successfully exploited in the clinical setting to treat graft-versus-host disease (GVHD); however, the path to clinical efficacy is hindered by the limited understanding of how MSC work and how best to use their potential. In this issue of the European Journal of Immunology, it is shown, using an animal model, that MSC can treat GVHD only if administered in the presence of active disease and that this requirement is strictly related to the presence of IFN-gamma. Here we summarise the knowledge regarding MSC mediated tolerance and the evidence supporting the notion that MSC must be 'licensed' to exert their effects. We also propose the idea that the instrumental effect of IFN-gamma activity on MSC-mediated immunomodulation relies upon IFN-gamma's ability to gather and retain suppressive and effector cells in the same anatomical compartment.     

The pathophysiology of acute graft-versus-host disease

Despite improvements in allogeneic stem cell transplantation, acute graft-versus-host disease (GVHD) remains a significant problem after transplantation, and it is still a major cause of post-transplant mortality. Disease progression is characterized by the differentiation of alloreactive T cells to effector cells leading to tissue damage, recruitment of additional inflammatory cell populations and further cytokine dysregulation. To make the complex process of acute GVHD more explicit, the pathophysiology of acute GVHD is often divided into three different phases. This review summarizes the mechanisms involved in the three phases of acute GVHD.