Prevention of graft-versus-host disease by anti IL-7Ralpha antibody

Graft-versus-host disease (GVHD) continues to be a serious complication that limits the success of allogeneic bone marrow transplantation (BMT). Using IL-7-deficient murine models, we have previously shown that IL-7 is necessary for the pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by antibody-mediated blockade of IL-7 receptor alpha (IL-7Ralpha) signaling. C57/BL6 (H2K(b)) recipient mice were lethally irradiated and underwent cotransplantation with T-cell-depleted (TCD) BM and lymph node (LN) cells from allogeneic BALB/c (H2K(d)) donor mice. Following transplantation, the allogeneic BMT recipients were injected weekly with either anti-IL-7Ralpha antibody (100 mug per mouse per week) or PBS for 4 weeks. Anti-IL-7Ralpha antibody treatment significantly decreased GVHD-related morbidity and mortality compared with placebo (30% to 80%). IL-7Ralpha blockade resulted in the reduction of donor CD4(+) or CD8(+) T cells in the periphery by day 30 after transplantation. Paradoxically, the inhibition of GVHD by anti-IL-7Ralpha antibody treatment resulted in improved long-term thymic and immune function. Blockade of IL-7R by anti-IL-7Ralpha antibody resulted in elimination of alloreactive T cells, prevention of GVHD, and improvement of donor T-cell reconstitution.     

Hepatocyte growth factor preserves graft-versus-leukemia effect and T-cell reconstitution after marrow transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT). When GVHD is controlled by T-cell-depleted grafts or immunosuppressants, BM transplant recipients often suffer from an increased rate of leukemic relapse and impaired reconstitution of immunity. Using a mouse BMT model, we investigated the effects of hepatocyte growth factor (HGF) gene transfection on the severity of GVHD, the graft-versus-leukemia effect, and the reconstitution of T cells after BMT. After HGF gene transfer, acute GVHD was reduced, while mature donor T-cell responses to host antigens were preserved, resulting in a significant improvement of leukemia-free survival. HGF gene transfer promoted regeneration of bone marrow-derived T cells and the responsiveness of these cells to alloantigens. Furthermore, HGF preserved the thymocyte phenotype and thymic stromal architecture in mice with GVHD. This suggested that HGF exerts a potent protective effect on the thymus, which in turn promotes reconstitution of bone marrow-derived T cells after allogeneic BMT. These results indicate that HGF gene transfection can reduce acute GVHD preserving the graft-versus-leukemia effect, while promoting thymic-dependent T-cell reconstitution after allogeneic BMT.     

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.     

Rapidly proliferating CD44hi peripheral T cells undergo apoptosis and delay posttransplantation T-cell reconstitution after allogeneic bone marrow transplantation

Delayed T-cell recovery is an important complication of allogeneic bone marrow transplantation (BMT). We demonstrate in murine models that donor BM-derived T cells display increased apoptosis in recipients of allogeneic BMT with or without GVHD. Although this apoptosis was associated with a loss of Bcl-2 and Bcl-X(L) expression, allogeneic recipients of donor BM deficient in Fas-, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Bax-, or BM-overexpressing Bcl-2 or Akt showed no decrease in apoptosis of peripheral donor-derived T cells. CD44 expression was associated with an increased percentage of BM-derived apoptotic CD4(+) and CD8(+) T cells. Transplantation of RAG-2-eGFP-transgenic BM revealed that proliferating eGFP(lo)CD44(hi) donor BM-derived mature T cells were more likely to undergo to apoptosis than nondivided eGFP(hi)CD44(lo) recent thymic emigrants in the periphery. Finally, experiments using carboxyfluorescein succinimidyl ester-labeled T cells adoptively transferred into irradiated syngeneic hosts revealed that rapid spontaneous proliferation (as opposed to slow homeostatic proliferation) and acquisition of a CD44(hi) phenotype was associated with increased apoptosis in T cells. We conclude that apoptosis of newly generated donor-derived peripheral T cells after an allogeneic BMT contributes to delayed T-cell reconstitution and is associated with CD44 expression and rapid spontaneous proliferation by donor BM-derived T cells.     

Differential use of Fas ligand and perforin cytotoxic pathways by donor T cells in graft-versus-host disease and graft-versus-leukemia effect

In allogeneic bone marrow transplantation (BMT) donor T cells are primarily responsible for antihost activity, resulting in graft-versus-host disease (GVHD), and for antileukemia activity, resulting in the graft-versus-leukemia (GVL) effect. The relative contributions of the Fas ligand (FasL) and perforin cytotoxic pathways in GVHD and GVL activity were studied by using FasL-defective or perforin-deficient donor T cells in murine parent --> F1 models for allogeneic bone marrow transplantation. It was found that FasL-defective B6.gld donor T cells display diminished GVHD activity but have intact GVL activity. In contrast, perforin-deficient B6.pfp(-/-) donor T cells have intact GVHD activity but display diminished GVL activity. Splenic T cells from recipients of B6.gld or B6.pfp(-/-) T cells had identical proliferative and cytokine responses to host antigens; however, splenic T cells from recipients of B6.pfp(-/-) T cells had no cytolytic activity against leukemia cells in a cytotoxicity assay. In experiments with selected CD4(+) or CD8(+) donor T cells, the FasL pathway was important for GVHD activity by both CD4(+) and CD8(+) T cells, whereas the perforin pathway was required for CD8-mediated GVL activity. These data demonstrate in a murine model for allogeneic bone marrow transplantation that donor T cells mediate GVHD activity primarily through the FasL effector pathway and GVL activity through the perforin pathway. This suggests that donor T cells make differential use of cytolytic pathways and that the specific blockade of one cytotoxic pathway may be used to prevent GVHD without interfering with GVL activity.     

Absence of regulatory T-cell control of TH1 and TH17 cells is responsible for the autoimmune-mediated pathology in chronic graft-versus-host disease

Graft-versus-host disease (GVHD) remains the major complication after allogeneic bone marrow transplantation (BMT). The process whereby acute GVHD mediated by alloreactive donor T cells transitions into chronic GVHD, which is characterized by prominent features of auto-immunity, has long been unresolved. In this study, we demonstrate that GVHD-associated autoimmunity and, by extension, chronic GVHD is attributable to the progressive loss of CD4(+)CD25(+)Foxp3(+) regulatory T cells during the course of acute GVHD. This leads to the expansion of donor-derived CD4(+) T cells with T(H)1 and T(H)17 cytokine phenotypes that release proinflammatory cytokines and cause autoimmune-mediated pathological damage. These T cells are present early after transplantation, indicating that the pathophysiological events that lead to chronic GVHD are set in motion during the acute phase of GVHD. We conclude that the absence of CD4(+)CD25(+) regulatory T cells coupled with unregulated T(H)1 and T(H)17 cells leads to the development of autoimmunity and that donor-derived T(H)1 and T(H)17 cells serve as the nexus between acute and chronic GVHD.     

Cellular and cytokine effectors of acute graft versus host disease

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT) and Older BMT recipients are at greater risk for acute graft-versus-host-disease. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older recipients. GVHD mortality and morbidity, as well as pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4+ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2FI BM chimeras created with either old or young BM. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. We also evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD in mouse models. In bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results suggest new strategies for the prevention and treatment of this toxic complication of BMT.     

Recovery from established graft-vs-host disease achieved by bone marrow transplantation from a third-party allogeneic donor

OBJECTIVE: We investigated whether established graft-vs-host disease (GVHD) could be successfully treated by a second allogeneic bone marrow transplantation (BMT) through elimination of first donor-derived lymphocytes responsible for GVHD. MATERIALS AND METHODS: In a murine GVHD model of BDF1 (H-2(b/d))-->B6C3F1(H-2(b/k)), GVHD mice underwent a second BMT using a graft (1 x 10(7) bone marrow and 3 x 10(7) spleen cells) from a major histocompatibility complex (MHC) antigen haploidentically mismatched (to host and also to first donor) mouse strain, B6B10F1(H-2(b/s)), following low-dose total body irradiation (TBI) 2 to 3 weeks after the first BMT. RESULTS: Results demonstrated that severe GVHD could be successfully and stably treated by a second allogeneic BMT. For successful treatment of GVHD, rapid achievement of full second-donor T-cell chimerism was required. Furthermore, we showed that mice with GVHD could easily accept MHC haploidentically mismatched second-donor hematopoietic cells even after minimal conditioning (2-4 Gy TBI) because they were in a profoundly immunosuppressed state, and that the mice were relatively resistant to new development of GVHD by second-donor grafts. Furthermore, the timing of the second BMT, the intensity of conditioning treatment (GVHD mice are very sensitive), and donor selection were also found to be important for obtaining successful outcomes. Increased regulatory T cells and reduction of interferon-gamma levels may be involved in tolerance induction. CONCLUSIONS: We demonstrated that established GVHD in a murine GVHD model could be successfully treated by a second BMT from a third-party allogeneic donor.     

Cellular and cytokine effectors of acute graft versus host disease

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT) and Older BMT recipients are at greater risk for acute graft-versus-host-disease. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older recipients. GVHD mortality and morbidity, as well as pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4⁺ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. We also evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD in mouse models. In bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results suggest new strategies for the prevention and treatment of this toxic complication of BMT.     

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.     

Irradiated donor leukocytes promote engraftment of allogeneic bone marrow in major histocompatibility complex mismatched recipients without causing graft-versus-host disease.

Graft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL --> (SJL x C57BL6) F1, C57BL6 --> B10.RIII, and C57BL6 --> B10.BR mouse donor --> recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 x 10(6) to 75 x 10(6) irradiated donor splenocytes administered in multiple injections from day -1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFkappaB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.     

Interleukin 18 preserves a perforin-dependent graft-versus-leukemia effect after allogeneic bone marrow transplantation

We have recently shown that early administration of interleukin 18 (IL-18) after bone marrow transplantation (BMT) attenuates acute graft-versus-host disease (GVHD) in a lethally irradiated parent into F1 (B6-->B6D2F1) BMT model. In this study, we investigated whether IL-18 can maintain graft-versus-leukemia (GVL) effect in this context. B6D2F1 mice received transplants of T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either syngeneic (H2(b/d)) or allogeneic B6 (H2(b)) donors. Recipient mice were treated with recombinant murine IL-18 or the control diluent. Initial studies demonstrated that IL-18 treatment did not affect the proliferative responses or the cytolytic effector functions of T cells after BMT. In subsequent experiments, animals also received host-type P815 mastocytoma cells at the time of BMT. All syngeneic BM transplant recipients died from leukemia by day 18. The allogeneic BM transplant recipients effectively rejected their leukemia regardless of treatment and IL-18 significantly reduced GVHD-related mortality. Examination of the cytotoxic mechanisms with perforin-deficient donor T cells demonstrated that perforin is critical for the GVL effect. Taken together these data demonstrate that IL-18 can attenuate acute GVHD without impairing the in vitro cytolytic function or the in vivo GVL activity after allogeneic BMT.     

Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versus-host disease

Prolonged immunodeficiency after allogeneic bone marrow transplantation (BMT) causes significant morbidity and mortality from infection. This study examined in murine models the effects of interleukin-7 (IL-7) given to young and middle-aged (9-month-old) recipients of major histocompatibility complex (MHC)-matched or -mismatched allogeneic BMT. Although administration of IL-7 from day 0 to 14 after syngeneic BMT promoted lymphoid reconstitution, this regimen was ineffective after allogeneic BMT. However, IL-7 administration from day 14 (or 21) to 27 after allogeneic BMT accelerated restoration of the major lymphoid cell populations even in middle-aged recipients. This regimen significantly expanded donor-derived thymocytes and peripheral T cells, B-lineage cells in bone marrow and spleen, splenic natural killer (NK) cells, NK T cells, and monocytes and macrophages. Interestingly, although recipients treated with IL-7 had significant increases in CD4(+) and CD8(+) memory T-cell populations, increases in naive T cells were less profound. Most notable, however, were the observations that IL-7 treatment did not exacerbate graft-versus-host disease (GVHD) in recipients of an MHC-matched BMT, and would ameliorate GVHD in recipients of a MHC-mismatched BMT. Nonetheless, graft-versus-leukemia (GVL) activity (measured against 32Dp210 leukemia) remained intact. Although activated and memory CD4(+) and CD8(+) T cells normally express high levels of IL-7 receptor (IL-7R, CD127), activated and memory alloreactive donor-derived T cells from recipients of allogeneic BMT expressed little IL-7R. This might explain the failure of IL-7 administration to exacerbate GVHD. In conclusion, posttransplant IL-7 administration to recipients of an allogeneic BMT enhances lymphoid reconstitution without aggravating GVHD while preserving GVL.     

Lymphopenia-induced proliferation of donor T cells reduces their capacity for causing acute graft-versus-host disease

OBJECTIVE: T cells that undergo lymphopenia-induced proliferation (LIP) are characterized by greater effector and anti-tumor function than na?ve T cells. But the ability of these T cells in causing graft-versus-host disease (GVHD) is not known. METHODS: We tested the hypothesis that donor T cells that had undergone LIP would cause more severe GVHD than na?ve T cells by utilizing well-characterized murine experimental models of allogeneic bone marrow transplantation (BMT). RESULTS: Contrary to our hypothesis, LIP of donor T cells under either noninflammatory or irradiated conditions caused significantly reduced GVHD as determined by survival, clinical, pathologic, and biochemical parameters than na?ve T cells. Compared to na?ve donor T cells, LIP T cells demonstrated reduced expansion in vivo and in vitro after allogeneic BMT. The reduction in GVHD mortality and severity was observed across multiple strains after allogeneic BMT. In vivo mechanistic studies by cell depletion demonstrated an increase in the CD44(hi) "memory" phenotype T cells and not the CD4(+)CD25(+) T cell subset to be critical for the reduction in GVHD. CONCLUSIONS: These data demonstrate that LIP of T cells regulates acute GVHD severity in contrast to their ability to cause increased allograft rejection, autoimmunity, or anti-tumor immunity.     

An absence of CCR5 on donor cells results in acceleration of acute graft-vs-host disease

OBJECTIVE: Chemokines have been postulated to play a role in the pathogenesis of graft-vs-host disease (GVHD) after allogeneic hematopoietic transplantation. Recent reports have indicated that the absence of donor expression of CCR5 on T cells ameliorates GVHD in models using no conditioning of the recipient. We therefore assessed the role of CCR5 on donor cells in models where intensive conditioning of the recipient occurs, thus more appropriately mirroring the clinical experience. METHODS: Lethally irradiated mice received allogeneic bone marrow transplants. Recipients were given full MHC-mismatched donor bone marrow and splenocytes from CCR5 knockout (KO) mice vs wild-type (WT) control donors. RESULTS: Recipients of CCR5 KO donor cells succumbed to acute GVHD at an accelerated rate compared to mice receiving WT cells. Donor CD8+ T cells expanded to a significantly greater extent in recipients of CCR5 KO vs WT control cells. T cells recovered from recipients of CCR5 KO cells produced more IFN-gamma and TNF-alpha and proliferated to a T-cell mitogen at a significantly greater level then T cells from recipients of WT cells, indicating that CCR5 plays a role in downregulating donor alloreactive CD8+ T-cell expansion. Histological assessment of the mice indicated pathological lesions in the kidneys and a greater degree of liver pathological changes in mice that received CCR5 KO donor grafts. CONCLUSIONS: These results indicate that the role of CCR5 in allogeneic bone marrow transplants and GVHD is more complex than initially thought. In a murine transplant model with intensive conditioning, the overall effect of absent CCR5 expression on donor cells results in greater GVHD and donor T-cell expansion.     

L-Selectin(hi) but not the L-selectin(lo) CD4+25+ T-regulatory cells are potent inhibitors of GVHD and BM graft rejection

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after bone marrow transplantation (BMT). CD4(+)CD25(+) immune regulatory T cells (Tregs), long recognized for their critical role in induction and maintenance of self-tolerance and prevention of autoimmunity, are also important in the regulation of immune responses in allogeneic bone marrow (BM) and solid organ transplantation. Published data indicate that ex vivo activated and expanded donor Tregs result in significant inhibition of lethal GVHD. This study provides a direct comparison of LSel(hi) and LSel(lo) Tregs for GVHD inhibition and for the promotion of allogeneic BM engraftment. Imaging of green fluorescent protein-positive effectors in GVHD control mice and LSel(hi) and LSel(lo) Treg-treated mice vividly illustrate the multisystemic nature of GVHD and the profound inhibition of GVHD by LSel(hi) Tregs. Data indicate that LSel(hi) Tregs interfere with the activation and expansion of GVHD effector T cells in secondary lymphoid organs early after BMT. Either donor- or host-type LSel(hi), but not LSel(lo), Tregs potently increased donor BM engraftment in sublethally irradiated mice, an event occurring independently of transforming growth factor beta signaling of host T cells. These data indicate that Treg cellular therapy warrants clinical consideration for the inhibition of GVHD and the promotion of alloengraftment.     

Successful liver allografts in mice by combination with allogeneic bone marrow transplantation.

Successful liver allografts were established by combination with allogeneic bone marrow transplantation. When liver tissue of BALB/c (H-2d) or C57BL/6J (H-2b) mice was minced and grafted under the kidney capsules of C3H/HeN (H-2k) mice, it was rejected. However, when C3H/HeN mice were irradiated and reconstituted with T-cell-depleted BALB/c or BALB/c nu/nu bone marrow cells, or with fetal liver cells of BALB/c mice, they accepted both donor (stem-cell)-type (BALB/c) and host (thymus)-type (C3H/HeN) liver tissue. Assays for both mixed-lymphocyte reaction and induction of cytotoxic T lymphocytes revealed that the newly developed T cells were tolerant of both donor (stem-cell)-type and host (thymus)-type major histocompatibility complex determinants. We propose that liver allografts combined with bone marrow transplantation should be considered as a viable therapy for patients with liver disease such as liver cirrhosis and hepatoma.     

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.     

Absence of clinical GVHD and the in vivo induction of regulatory T cells after transplantation of facilitating cells

Graft-versus-host disease (GVHD) and failure of engraftment limit clinical bone marrow transplantation (BMT) to patients with closely matched donors. Engraftment failure of purified allogeneic hematopoietic stem cells (HSCs) has been decreased in various BMT models by including donor BM-derived CD8(+)/alphabetagammadeltaTCR(-) facilitating cells (FCs) or CD8(+)/alphabetaTCR(+) T cells in the BM inoculum. To aggressively investigate the GVHD potential of these donor CD8(+) populations, a purified cell model of lethal GVHD was established in a murine semiallogeneic parent --> F(1) combination. Lethally irradiated recipients were reconstituted with purified donor HSCs alone or in combination with splenic T cells (T(SP)), BM-derived T cells (T(BM)), or the FC population. In marked contrast to the lethal GVHD present in recipients of HSCs plus T(SP) or CD8(+) T(BM), recipients of donor HSC+FC inocula did not exhibit significant clinical or histologic evidence of GVHD. Instead, HSC+FC recipients were characterized by increased splenocyte expression of transforming growth factor-beta (TGF-beta) and the induction of the regulatory T-cell genes CTLA4, GITR, and FoxP3. These findings suggest that the FCs, which express a unique FCp33-TCRbeta heterodimer in place of alphabetaTCR, permits HSC alloengraftment and prevents GVHD through the novel approach of regulatory T-cell induction in vivo.     

Adoptive immunotherapy of BCR-ABL-induced chronic myeloid leukemia-like myeloproliferative disease in a murine model

Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia(+) leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2(b)) mice with BCR-ABL-transduced Balb/c (H-2(d)) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL-induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4(+) and CD4(-) cells. BCR-ABL-induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)-mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2(d)) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8(+) cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia(+) B-lymphoblastic leukemia to DLI.