Graft-vs.-host and graft-vs.-leukemia reactions after delayed infusions of donor T-subsets.

Infusions of donor leukocytes have been given to allogeneic bone marrow recipients after transplant to treat leukemia relapse. Treatment with these delayed infusions of donor cells has been called delayed or donor leukocyte infusion (DLI). While graft-vs.-host disease (GVHD) has typically been less severe than expected after DLI, it still remains a significant risk factor. Recently, we used a full major histocompatibility complex (MHC)-mismatched model (C57BL/6 into AKR) to determine how increased immunogenetic disparity affects GVH and graft-vs.-leukemia (GVL) reactions after DLI. In contrast to an MHC-matched model (B10.BR into AKR), GVHD was still observed when MHC-mismatched donor T cells were infused 3 weeks posttransplant. Limiting dilution analysis was used to determine the frequency of alloreactive cytotoxic T lymphocytes (CTL) and interleukin (IL)-2-secreting T helper cells in the spleens of MHC-mismatched recipients 7 days after DLI treatment. GVHD correlated with elevated frequencies of alloreactive T-helper cells. One strategy for reducing the severity of GVHD after DLI is the selective administration of CD4 or CD8 T-subsets. Delayed infusion of purified T-subsets 3 weeks posttransplant resulted in significantly less GVHD than infusion of a mixture of the two subsets. No GVH-associated mortality was observed after DLI with purified donor CD4+ T cells. In GVL studies, MHC-mismatched CD8+ T cells were the most potent antitumor effectors against an acute T cell leukemia. The GVL effect of MHC-mismatched T-subsets was compared with that of MHC-matched subsets. When naive MHC-matched cells were given as DLI, depletion of either T-subset eliminated the GVL effect. CD8+ T cells from MHC-matched donors primed against host alloantigens, however, mediated a CD4 (T-helper)-independent GVL reaction. Together, these results suggest that administration of T-subsets can significantly reduce GVHD after DLI without loss of the beneficial GVL effect.     

Photochemical treatment of donor lymphocytes inhibited their ability to facilitate donor engraftment or increase donor chimerism after nonmyeloablative conditioning or establishment of mixed chimerism.

Donor T-cells can provide a graft-versus-leukemia effect and help to promote donor engraftment after allogeneic BMT; however, these benefits can be outweighed by the ability of the cells to induce life-threatening GVHD. Photochemical treatment (PCT) of T-cells with S-59 psoralen and long-wavelength UV-A light can inhibit their proliferative capacity and significantly decrease their ability to induce acute GVHD after allogeneic BMT. PCT donor T-cells have been shown to facilitate donor engraftment in a myeloablative BMT model. In this study, we examined whether donor T-cells subjected to PCT ex vivo could retain the ability to facilitate engraftment or increase donor chimerism after nonmyeloablative BMT or after establishment of mixed hematopoietic chimerism. In a transplantation model in which mice were conditioned for BMT with sublethal (600 cGy) TBI, an infusion of PCT donor T-cells was unable to facilitate engraftment of donor BM. A BMT model was used in which a mixture of allogeneic and syngeneic marrow cells was infused into lethally irradiated recipients for establishment of mixed hematopoietic chimerism. The goal was to determine whether PCT donor splenocytes could increase levels of donor chimerism. Recipients of splenocytes treated with UV-A light only (no S-59 psoralen) and given at the time of BMT or in a donor lymphocyte infusion (DLI) had significantly higher levels of donor chimerism than did recipients of BM only. Although PCT donor splenocytes given at the time of BMT modestly increased donor chimerism, PCT donor splenocytes given in a DLI did not increase donor chimerism. A nonmyeloablative BMT model was employed for determining whether DLI given relatively late after BMT could increase donor chimerism. Recipient mice were conditioned for BMT with a combination of low-dose TBI (50 or 100 cGy) and anti-CD154 (anti-CD40L) monoclonal antibody for achievement of low levels of mixed chimerism. When control mixed chimeras were given a DLI 71 days after BMT, donor chimerism was significantly increased. In contrast, PCT of the donor cells eliminated the ability of the cells to increase donor chimerism after infusion. Together results from these 3 distinct BMT models indicate that PCT of donor T-cells significantly inhibited the ability of the cells to facilitate donor engraftment after nonmyeloablative BMT or to increase donor chimerism in mixed hematopoietic chimeras when the cells were administered in a DLI.     

Induction of stable long-term mixed hematopoietic chimerism following nonmyeloablative conditioning with T cell-depleting antibodies, cyclophosphamide, and thymic irradiation leads to donor-specific in vitro and in vivo tolerance.

BACKGROUND: Successful transplantation of solid organs relies on long-term immunosuppression for the prevention of graft rejection. Donor-specific tolerance without the need for continuous immunosuppression can be observed after allogeneic BMT. However, its routine use for tolerance induction has been precluded so far by the high conditioning-related toxicity of standard BMT regimens. Our laboratory has recently established a cyclophosphamide (CTX) plus thymic irradiation (TI)-based nonmyeloablative conditioning protocol for the treatment of hematologic malignancies. We have recently described the successful clinical application of this approach for the induction of donor-specific tolerance in a patient receiving a living-related kidney transplant, which resulted in graft acceptance without long-term immunosuppression. The aim of this study was to evaluate the induction and maintenance of host-versus-graft tolerance following this CTX-plus-TI-based regimen in a mouse model. METHODS: Induction of mixed hematopoietic chimerism and development of donor-specific tolerance following the CTX-based nonmyeloablative conditioning regimen (200 mg/kg CTX, in vivo T-cell depletion [anti-CD4 monoclonal antibody (MoAb) GK1.5 and anti-CD8 MoAb 2.43], and 7 Gy TI) was studied in the fully major histocompatibility complex (MHC)-mismatched B10.A (H2a)-->B6 (H2b) strain combination. RESULTS: The conditioning regimen allowed allogeneic bone marrow engraftment and persistent (>30 weeks) mixed lymphohematopoietic chimerism in almost all recipients. TI was essential to allow engraftment and development of tolerance, which was evident in all lasting chimeras. Compared to animals receiving a similar TBI-based conditioning regimen, overall levels of chimerism were significantly lower in the CTX-plus-TI-conditioned animals. However, donor-specific tolerance in vitro and in vivo was evident in CTX-plus-TI-conditioned chimeras. Tolerance was associated with the presence of donor-type MHC class II+ cells in the thymus and deletion of donor-reactive cells, as determined by Mtv-8 and Mtv-9 superantigen-mediated deletion of Vbeta11+ and Vbeta5/1.2+ T cells. CONCLUSION: Engraftment, long-term chimerism, and induction of donor-specific tolerance can be achieved using a nonmyeloablative CTX-based conditioning regimen in fully MHC-mismatched BMT recipients without the induction of GVHD.     

Graft-versus-leukemia and graft-versus-host reactions after donor lymphocyte infusion are initiated by host-type antigen-presenting cells and regulated by regulatory T cells in early and long-term chimeras.

Regulatory T (T(reg)) cells and host antigen-presenting cells (APCs) have been implicated in graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect after donor lymphocyte infusion (DLI), but their relative contributions remain unclear in early versus long-term complete donor or mixed murine allogeneic hematopoietic stem cell (HSC) chimeras. We have previously demonstrated that donor HSC-derived Thy1(+) T(reg) cells, consisting primarily of CD4(+)CD25(+) cells, play an important role in the suppression of graft-versus-host (GVH) reactivity when DLI is given to complete donor chimeras 28 days after HSC transplantation. Data presented here demonstrate that protection against GVHD exerted by Thy1(+) T(reg) cells is less evident with time and eventually is not required in long-term complete donor chimeras because of an absence of host-type APCs to activate alloreactive T cells. Lethal GVHD was observed when Thy1(+) T(reg) cells were depleted from complete donor chimeras given by DLI at day 28, 35, or 42; however, T(reg) cell depletion and DLI at day 70 no longer induced GVHD-associated mortality. Moreover, the failure of DLI to induce GVHD with T(reg) depletion correlated with a loss of the DLI-induced GVL effect in long-term (day 100) complete donor chimeras. In contrast to the results from complete donor chimeras, GVL reactivity in day 100 mixed chimeras was robust after DLI. Loss of a DLI-induced GVL effect in long-term complete donor chimeras was attributed to the absence of host APCs because the infusion of exogenous host-type dendritic cells partially restored both DLI-induced GVL and GVH reactions in day 100 complete donor chimeras. The GVL and GVH reactions restored by infusion of host dendritic cells in day 100 complete donor chimeras were at least partially regulated by T(reg) cells because transient depletion of CD25(+) cells increased both the GVL effect and the severity of GVHD after DLI. Taken together, these data suggest that T(reg) cells can regulate DLI-induced GVL and GVH reactions in both early and long-term complete donor chimeras, and a state of mixed chimerism is superior to complete donor chimerism because host-type APCs facilitate a DLI-induced GVL effect without severe GVHD.     

Dose and timing of interleukin (IL)-12 and timing and type of total-body irradiation: effects on graft-vs.-host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients.

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.     

Donor natural killer (NK1.1+) cells do not play a role in the suppression of GVHD or in the mediation of GVL reactions after DLI.

Donor regulatory T cells (CD3+ alphabetaT-cell receptor [TCR]+) derived from the repopulating host thymus have been shown to be primarily responsible for suppression of GVHD following DLI therapy in murine BMT models. However, natural killer (NK) T cells also have regulatory properties, and a role for NK T cells in suppression of GVH reactivity has not been completely excluded. NK cells may also contribute to the graft-versus-leukemia (GVL) effect associated with DLI therapy. In this study, we used a murine BMT model (C57BL/6 into AKR) to study whether depletion of donor NK cells had any impact on the suppression of GVH reactivity after DLI or on the DLI-induced GVL effect against acute T-cell leukemia. Depletion of donor NK cells was accomplished in vivo by giving DLI-treated bone marrow chimeras multiple injections of anti-NK1.1 monoclonal antibody (MoAb). The chimeras treated with anti-NK1.1 MoAb had significantly fewer splenic NK1.1 cells than nontreated chimeras, and splenocytes from anti-NK1.1-treated mice were deficient in the ability to generate lymphokine-activated lytic activity. Results presented here showed that NK-cell depletion had no effect on the suppression of GVH reactivity after DLI. When DLI-treated chimeras were challenged with an acute T-cell leukemia, NK-cell depletion had no discernible effect on GVL reactivity. These preclinical data suggest that donor NK cells do not have a significant role in the suppression of GVHD after DLI or in the mediation of GVL reactivity induced by DLI.     

Separation of graft-vs.-tumor effects from graft-vs.-host disease in allogeneic hematopoietic cell transplantation

Allogeneic hematopoietic cell transplantation (HCT) is an increasingly widely used treatment modality in hematological malignancies. Alloreactivity mediated by donor T cells (and, in some settings, by donor natural killer cells) can produce durable immunologic control or eradication of residual malignancy after allogeneic HCT. However, graft-vs.-tumor (GVT) effects are variably effective and are often accompanied by deleterious alloreactivity against normal host tissue, manifesting as graft-vs.-host disease (GVHD). A major focus of current research in HCT is the separation of beneficial GVT effects from GVHD. Here we review a number of approaches currently under investigation to specifically augment GVT effects, including the identification of minor histocompatibility antigens (mHA), adoptive immunotherapy with tumor-specific or mHA-specific cytotoxic T lymphocytes, vaccination of the donor or recipient to stimulate tumor-specific immunity, and adoptive transfer of natural killer cells. In addition, we review strategies being investigated to specifically suppress GVHD while sparing GVT, including the manipulation and infusion of regulatory T cells, the use of novel pharmacologic and biologic agents, and the use of mesenchymal stem cells. Ultimately, advances in separation of GVT from GVHD will further enhance the potential of allogeneic HCT as a curative treatment for hematological malignancies.     

Successful allogeneic stem cell transplantation with nonmyeloablative conditioning in patients with relapsed hematologic malignancy following autologous stem cell transplantation.

The use of myeloablative preparative therapy and allogeneic stem cell transplantation (alloSCT) as salvage therapy for adult patients with relapsed hematologic malignancy after autologous stem cell transplantation (autoSCT) is generally unsuccessful due to very high treatment-related mortality rates. We evaluated the outcome of HLA-matched related donor alloSCT following nonmyeloablative preparative therapy in 13 patients (median age, 38 years) with relapsed hematologic malignancies (Hodgkin's disease, n = 4; Hodgkin's disease and advanced myelodysplastic syndrome, n = 1; non-Hodgkin's lymphoma, n = 6; multiple myeloma, n = 2) after initial autoSCT. Median time from autoSCT to alloSCT was 12 months (range, 3-24 months); 6 patients had chemotherapy-refractory disease following autoSCT, 6 were in untreated relapse, and 1 had a partial response from salvage chemotherapy. Preparative therapy consisted of cyclophosphamide, 150-200 mg/kg; peritransplantation anti-thymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal irradiation); and a very short course of cyclosporine as GVHD prophylaxis. All patients achieved initial mixed chimerism as defined by greater than 1% donor peripheral white blood cells. Seven patients, who had no evidence of GVHD, received prophylactic DLI beginning 5 to 6 weeks after transplantation for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-tumor effect. Six patients showed conversion to full donor chimerism and 1 lost the graft. Grade II or greater acute GVHD occurred in 9 patients. Seven patients achieved a complete response; 6 had no response. The median survival time of the 13 patients is currently 10 months (range, 3-39 months), with an overall survival probability at 2 years of 45% (95% confidence interval [CI], 19%-73%) and a disease-free survival probability at 2 years of 37.5% (95% CI, 12%-65%). Thus, this novel nonmyeloablative alloSCT strategy followed by prophylactic DLI was well tolerated and can result in durable disease-free survival among patients with advanced hematologic malignancies after a failed autoSCT. Further follow-up and evaluation of additional patients are required to conclusively establish the role of this strategy in the treatment of hematologic malignancies after an autologous transplantation.     

Unmanipulated HLA 2-3 antigen-mismatched (haploidentical) stem cell transplantation using nonmyeloablative conditioning.

The major problems in human leukocyte antigen (HLA)-mismatched stem cell transplantation (SCT) are graft failure and graft-versus-host disease (GVHD). Less-intensive regimens should be associated with a lower release of inflammatory cytokines and possibly less GVHD. The objective of this study was to investigate whether HLA-haploidentical SCT can be performed using nonmyeloablative conditioning and pharmacologic GVHD prophylaxis, including glucocorticoids. Using conditioning consisting of fludarabine, busulfan, and anti-T-lymphocyte globulin and GVHD prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg/day), 26 patients who had hematologic malignancies in an advanced stage or with a poor prognosis underwent transplantation using peripheral blood stem cells from an HLA-haploidentical donor (2-3 antigen mismatches in the graft-versus-host [GVH] direction) without T-cell depletion. All patients except for 1 achieved donor-type engraftment. Rapid hematologic engraftment was achieved (neutrophils > 0.5 x 10(9)/L on day 12 and platelets > 20 x 10(9)/L on day 12), with full donor chimerism achieved by day 14. Fifteen patients did not develop acute GVHD clinically, and only 5 patients developed grade II GVHD. The recovery of CD4+ T cells was delayed compared with that of CD8+ T cells. Sixteen of the 26 patients are alive in complete remission. Four died of transplantation-related causes, and 6 died of progressive disease. These data suggest that nonmyeloablative conditioning, GVHD prophylaxis consisting of tacrolimus and methylprednisolone, and early therapeutic intervention for the GVH reaction allow stable engraftment and effectively suppress GVHD in HLA 2-3 antigen-mismatched SCT.     

Graft-versus-host reactions and the effectiveness of donor lymphocyte infusions.

We retrospectively analyzed 83 consecutive recipients of donor lymphocyte infusions (DLI) after allogeneic transplantation for factors associated with disease response and graft-versus-host disease (GVHD). DLI was highly effective in relapsed chronic phase chronic myeloid leukemia (CML), with 71% of patients achieving durable complete remissions (CR). In relapsed acute myeloid leukemia, DLI led to durable CRs in 31% of patients; the rate was <20% in all other diseases. Achieving full donor chimerism and GVHD were predictive of CR. Grade II or higher acute or chronic GVHD occurred in 36 (43%) patients and contributed to death in 13 (16%). Even more patients, 33 (40%), died of their underlying malignancy, including 10 who developed active GVHD. In relapsed CML, most durable CRs occurred without clinically apparent GVHD, yet all responders achieved full donor chimerism, including 6 with coincident normal host hematopoiesis at the time of DLI. Thus, in CML, potent lymphohematopoietic graft-versus-host reactions occurred even in the absence of clinically apparent GVHD; this confirms the ability to dissociate these processes and argues against a leukemia-specific immunologic effect. DLI clearly has efficacy in the treatment of relapsed disease after allogeneic transplantation. However, with the exception of CML, most patients die of their underlying disease because of insufficient antitumor activity even with active GVHD.     

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation.

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 x 10(7) CD3(+) cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI >/=40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was /=grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism >/=40% or 相似文献   

Fludarabine,Campath, and Low-Dose Cyclophosphamide (FCClow) with or without TBI Conditioning Results in Excellent Transplant Outcomes in Children with Severe Aplastic Anemia

Various reduced-intensity conditioning regimens are in use for allogeneic hematopoietic cell transplant (HSCT) in patients with idiopathic severe aplastic anemia (SAA). We describe the use of fludarabine, Campath, and low-dose cyclophosphamide (FCC^low) conditioning in 15 children undergoing related or unrelated donor transplants. Total body irradiation (TBI) of 2 Gy was added for unrelated donor HSCT. At a median follow-up of 2.3 years, the failure-free survival was 100%, with low rates of infection and toxicity. There was no occurrence of grade III to IV acute graft-versus-host disease (GVHD). All patients had full donor myeloid chimerism post-HSCT, even with mixed chimerism in the T cell lineage. The absence of chronic GVHD and long-term stable mixed donor T cell chimerism confirms immune tolerance following FCC^low (± TBI) conditioned transplantation in children with SAA.     

Allogeneic cell therapy for patients who relapse after autologous stem cell transplantation.

Allogeneic donor leukocytes can be used after nonmyeloablative conditioning to exploit their graft-versus-tumor (GVT) activity in the setting of reduced conditioning-regimen toxicity. This approach may be particularly useful for patients who relapse after autologous stem cell transplantation (SCT). However, GVT activity, toxicity, and ability to establish mixed chimerism may differ in patients who were heavily pretreated prior to SCT compared with patients treated earlier in the course of their disease. We have performed a series of studies of nonmyeloablative allogeneic transplantation and present data on the subset of 14 patients treated for relapse after autologous SCT: 4 patients received no conditioning and unstimulated donor leukocyte infusions (DLI), 10 patients received conditioning with fludarabine and cyclophosphamide followed by unstimulated or granulocyte-colony-stimulating factor (G-CSF)-stimulated allogeneic peripheral blood stem cells (PBSCs), 4 patients received no graft-versus-host disease (GVHD) prophylaxis, and 10 patients received cyclosporine GVHD prophylaxis. All but 1 patient had sustained donor chimerism at least 30 days after allogeneic cell therapy (ACT), and 8 patients had more than 80% donor chimerism after ACT. Acute GVHD developed in 11 patients (grade III-IV, n = 6). Aplasia was more frequent in the patients receiving unstimulated PBSCs, despite the development of mixed chimerism. There were 6 complete responses and 4 partial responses; response was independent of conditioning and growth-factor stimulation of the donor graft. Five patients died of treatment-related causes and 4 patients died from progressive disease. Four patients remained alive 27 to 194 weeks (median, 66 weeks) after ACT. Prior autologous SCT may define a subset of patients at particularly high risk for GVHD and other toxicity after ACT. However, these data show that ACT with either DLI or G-CSF-stimulated blood cells results in direct GVT activity in some patients with Hodgkin's disease, myeloma, and non-Hodgkin's lymphoma, even after relapse from autologous SCT. Most patients developed donor chimerism with minimal conditioning. Alternative prophylactic regimens that control GVHD while maintaining GVT are needed to improve outcomes in these heavily pretreated patients.     

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.     

Induction of tolerance by mixed chimerism with nonmyeloblative host conditioning: the importance of overcoming intrathymic alloresistance.

A nonmyeloablative conditioning regimen, consisting of depleting doses of anti-CD4 and anti-CD8 monoclonal antibodies (MoAbs) given on days -6 and -1 and 3 Gy of whole body irradiation given on day 0, allows the engraftment of fully major histocompatibility complex (MHC)-mismatched allogeneic bone marrow and the induction of tolerance for the graft. If MoAbs are given on day -5 only, permanent chimerism and tolerance are not observed in most animals. The addition of thymic irradiation to the single MoAb treatment permits tolerance induction in these mice, suggesting that residual host thymocytes reject donor marrow in recipients of 1, but not 2, MoAb injections. In this study, both CD4+ and CD8+ thymocytes were found to be responsible for residual alloreactivity in mice receiving only 1 MoAb injection. Co-receptor coating and downmodulation on residual thymocytes occur to a greater extent in recipients of 2 MoAb injections than in recipients of a single MoAb injection. This downmodulation may play a role in the loss of alloreactivity. Our results suggest that a second MoAb injection inactivates mature, functional donor-alloreactive CD4+ and CD8+ host thymocytes.     

Ex vivo fludarabine exposure inhibits graft-versus-host activity of allogeneic T cells while preserving graft-versus-leukemia effects.

Allogeneic donor T cells in bone marrow transplantation (BMT) can contribute to beneficial graft-versus-leukemia (GVL) effects but can also cause detrimental graft-versus-host disease (GVHD). A successful method for the ex vivo treatment of donor T cells to limit their GVHD potential while retaining GVL activity would have broad clinical applications for patients undergoing allogeneic hematopoietic cell transplantation for malignant diseases. We hypothesized that donor lymphocyte infusions treated with fludarabine, an immunosuppressive nucleoside analog, would have reduced GVHD potential in a fully major histocompatibility complex-mismatched C57BL/6 --> B10.BR mouse BMT model. Recipients of fludarabine-treated donor lymphocyte infusions (F-DLI) had significantly reduced GVHD mortality, reduced histopathologic evidence of GVHD, and lower inflammatory serum cytokine levels than recipients of untreated DLI. Combined comparisons of GVHD incidence and donor-derived hematopoietic chimerism indicated that F-DLI had a therapeutic index superior to that of untreated DLI. Furthermore, adoptive immunotherapy of lymphoblastic lymphoma using F-DLI in the C57BL/6 --> B10.BR model demonstrated a broad therapeutic index with markedly reduced GVHD activity and preservation of GVL activity compared with untreated allogeneic T cells. Fludarabine exposure markedly reduced the CD4+CD44(low)-naive donor T-cell population within 48 hours of transplantation and altered the relative representation of cytokine-producing CD4+ T cells, consistent with T-helper type 2 polarization. However, proliferation of fludarabine-treated T cells in allogeneic recipient spleens was equivalent to that of untreated T cells. The results suggest that fludarabine reduces the GVHD potential of donor lymphocytes through effects on a CD4+CD44(low) T-cell population, with less effect on alloreactive T cells and CD4+CD44(high) memory T cells that are able to mediate GVL effects. Thus, F-DLI represents a novel method of immune modulation that may be useful to enhance immune reconstitution among allograft recipients with reduced risk of GVHD while retaining beneficial GVL effects.     

Opposing roles of interferon-gamma on CD4+ T cell-mediated graft-versus-host disease: effects of conditioning.

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.     

Protection from lethal graft-vs.-host disease by donor stem cell repopulation.

Graft-vs.-host reaction (GVHR) induced in non-irradiated F1 mice with DBA/2J parental spleen and lymph node (LN) cells usually does not lead to acute GVH disease (GVHD). This contrasts with the GVHR induced in other parent-F1 combinations involving both major histocompatibility complex (MHC) class I and class II differences between donor and host. Most signs of acute GVHD in non-irradiated F1 mice relate to immunodeficiency following destruction of the lymphohemopoietic system of the host, which leads to wasting and death due to infections. This sequence of events is prevented when donor lymphoid cells, originating from grafted stem cells, repopulate the destroyed lymphohemopoietic system of the host. To examine whether a "silent" repopulation of the F1 host by donor stem cells might underly the absence of clinical signs of acute GVHD when GVHR is induced with DBA/2J lymphoid cells, GVHR was induced with LN cells, which do not contain stem cells. Indeed, GVHR induced in (C57BL/10 x DBA/2J)F1 (BDF1) mice with 80 x 10(6) DBA/2J LN cells led to acute GVHD. Signs of acute GVHD such as wasting and death did not occur when donor stem cells, from an inoculum of DBA/2J spleen and LN cells, were allowed to repopulate the lymphohemopoietic system of the host. The effect of donor stem cells on clinical signs of acute GVHD was more apparent when (B10.D2 x DBA/2J)F1, instead of DBA/2J, lymphoid cells were used to induce GVHR. The detection of alloreactive anti-host cytotoxic T lymphocyte (CTL) activity during acute GVHD induced with DBA/2J donor lymphoid cells supports the hypothesis that such CTL contribute to the destruction of the host immune system in acute GVHD.     

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.     

In situ analyses of in ovo graft-vs.-host reaction induced by thymic nurse cell lymphocytes

In both mammalian and avian systems, thymic nurse cells (TNC) have been shown to harbor a heterogeneous population of T lymphocytes (TNC-L) some of which exhibit a postselectional phenotype. By tranplanting micromanipulated single chicken TNC onto the chorionallantoic membrane (CAM) of major histocompatibility complex (MHC)-disparate embryos, an experimental system which allows for the detection of lymphocytes with graft-vs.-host (GVH) reactivity, we demonstrate here that TNC enclose lymphocytes that can develop into both CD4⁺ single-positive (sp) and CD8⁺ sp, T cell receptor (TcR)αβ⁺, or TcRγδ⁺ cells. This finding was additionally confirmed by serial transfer of primary expanded alloreactive T cells onto the CAM of secondary hosts. All donor TNC-L expressed MHC class II molecules and the interleukin-2 receptor a chain in primary and secondary GVH reactions. Furthermore, we observed selective accumulation of CD8⁺ and TcRγδ⁺ host lymphocytes in the CAM upon the induction of a local GVH reaction, most probably as a consequence of the pathological alteration of the epithelium.