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.     

Tracking ex vivo-expanded CD4+CD25+ and CD8+CD25+ regulatory T cells after infusion to prevent donor lymphocyte infusion-induced lethal acute graft-versus-host disease.

Donor bone marrow (BM)-derived CD4+ CD25+ regulatory T cells, maturing in the host thymus, are critical in inhibiting graft-versus-host disease (GVHD) after donor lymphocyte infusion (DLI) in murine BM chimeras. Data presented here demonstrate that fresh CD25+ cells isolated from donor-type mice can be expanded ex vivo by a variety of methods. Ex vivo-expanded CD4+ CD25+ and CD8+ CD25+ cells were potent suppressors of donor response to host alloantigens in mixed lymphocyte reaction assays. Both fresh and ex vivo-expanded CD4+ CD25+ cells persisted long-term in vivo and effectively prevented DLI-induced GVHD in CD25-/- BM chimeras. Importantly, co-infused CD4+ CD25+ cells with DLI cells migrated to peripheral lymphoid organs and survived long-term in DLI-treated CD25-/- chimeras, but not in DLI-treated CD25+/+ chimeras, indicating homeostatic control of CD25+ cells and an available niche required for their long-term persistence. Furthermore, maintenance of CD25 expression seemed necessary for suppressive function, because only the CD25+ cell fraction, but not the CD25- fraction isolated after adoptive transfer, was suppressive in vitro. Ex vivo-expanded CD8+ CD25+ cells weakly prevented GVHD, apparently because of a rapid disappearance of these cells after adoptive transfer. Taken together, these data suggest that the therapeutic use of ex vivo-expanded CD4+ CD25+ cells may be a feasible, nontoxic modality for controlling GVHD in the clinic. Because of strict homeostatic control, an available niche may be required for long-term persistence of infused regulatory T cells.     

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.     

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.     

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).     

Effect of NK cells on GVHD in H-2 haploidentical bone marrow transplantation in mice

Objective: To study the effect of natural killer (NK) cells on graft-versus-host disease (GVHD) after H-2 haploidentical bone marrow transplantation (BMT) in mice. Methods :Murine model of H-2 haploidentical BMT was established by using Balb/c (H-2d) mouse as recipient, and Balb/c(H-2d)×C57BL/6 (H-2b) (H-2d/b) mouse as donor. Lethally irradiated Balb/c (H-2d) mice were transplanted with the bone marrow cells from Balb/c(H-2d)×C57BL/6(H-2b) (H-2d/b) mice containing donor spleen cells and/or NK cells. GVHD and survival rates were studied by observation of clinical manifestations and pathological changes. Results:In the group of bone marrow +spleen cells, GVHD was induced in 90% mice; but in the group plus with low amount of NK cells,GVHD was induced in 20% mice; and in the group transplanted with high amount of NK cells, GVHD was induced only in 10% mice. Compared to the group transplanted only with BM plus spleen cells, the incidences of GVHD in the latter two groups decreased significantly (P＜0.01) and the survival rates at different periods of 15, 30, 45 and 60 days increased obviously (P＜0.01 ). Conclusion: In mouse H-2 haploidentical BMT, alloreactive NK cells can reduce the incidence of GVHD and increase the survival rate.     

不同途径的供者淋巴细胞输注对移植物抗宿主病的影响

目的:观察髓腔内供者淋巴细胞输注(IBM-DLI)对异基因小鼠外周造血干细胞移植(allo-PBSCT)后移植物抗宿主病(GVHD)的影响.方法:雌性C57BL/6小鼠为受鼠,接受全身照射(TBI)预处理后,输注雄性BALB/c小鼠来源的经rhG-CSF动员后的外周造血干细胞,分别经尾静脉(IV)和髓腔内进行DLI,建立异基因GVHD模型,观察移植后小鼠的生存状态和GVHD发生情况,应用流式细胞仪检测受鼠体内嵌合体形成和CD4~+CD25~+调节性T细胞(Tregs)比例,酶联免疫吸附实验(ELISA)检测白细胞介素4(IL-4)、γ干扰素(IFN-γ)水平.结果:IBM-DLI组的受鼠GVHD发生比例和严重程度较IV-DLI组明显降低(P＜0.01);移植后第7天各组受鼠骨髓中供鼠来源的细胞比例均在95%以上;与IV-DLI组比较,脾细胞中Tregs比例在IBM-DLI组明显升高(P＜0.01),IBM-DLI组IL-4分泌增多,IFN-γ分泌减少 (P＜0.01).结论:与IV-DLI相比,IBM-DLI有利于减轻GVHD的发生,其机制可能与受鼠体内Tregs细胞比例增高以及Th细胞向Th2细胞分化有关.     

Randomized trial of CD8+ T-cell depletion in the prevention of graft-versus-host disease associated with donor lymphocyte infusion.

The application of DLI is limited by the potential development of GVHD. Results of single-arm trials suggest that CD8+ depletion of DLI may reduce the incidence of GVHD while still inducing pathologic and cytogenetic remissions. To test the impact of CD8 depletion on GVHD, we initiated a randomized trial comparing outcome among patients receiving unselected donor lymphocytes or CD8+-depleted cells. DLI was administered to patients with disease in remission to prevent relapse 6 months after T-cell-depleted allogeneic BMT. CD8 depletion was performed with monoclonal antibody and rabbit complement. Donor lymphocytes obtained from the original donor were infused fresh without cryopreservation. Infusions were adjusted so that all patients received 1.0 x 10(7) CD4+ cells/kg. Patients randomized to CD8 depletion received a median of 0.7 x 10(5) versus 32.0 x 10(5) CD8+ cells/kg in the unmanipulated cohort. Six (67%) of 9 patients receiving unselected DLI developed acute GVHD compared with 0 (0%) of 9 recipients of CD8-depleted DLI (P = .009). In the unselected group, 2 patients died while the disease was in remission, and 3 patients had relapses. In the CD8-depleted cohort, there were no toxic deaths and only 1 relapse. Measures of immunologic reconstitution by T-cell receptor excision circle analysis and T-cell receptor spectratyping demonstrated similar patterns of T-cell recovery in both the CD8-depleted and the unselected cohorts. Both groups converted from mixed to full donor hematopoietic chimerism after DLI. Our results indicate that CD8 depletion reduces the incidence of GVHD associated with DLI without adversely affecting conversion to donor hematopoiesis or immunologic recovery.     

Effects of intrabone marrow-bone marrow transplantation plus adult thymus transplantation on survival of mice bearing leukemia

We recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor is effective in mice bearing solid tumors. In the current study, we examined the effects of this strategy on the survival of mice with leukemia. One week after intravenous injection of 1×10(6) leukemic cells (EL-4, H-2(b)) into 8-week-old B6 (H-2(b)) mice, the mice were 8 Gy irradiated and transplanted with 1×10(7) bone marrow cells (BMCs) from 8-week-old BALB/c mice (H-2(d)) by IBM-BMT with or without donor lymphocyte infusion (DLI) or ATT. All the mice without treatment died within 70 days after injection of EL-4. About 40% of those treated with IBM-BMT alone died within 100 days due to tumor relapse. In contrast, those treated with IBM-BMT+DLI or ATT showed the longest survival rate without relapse of leukemia. In addition, the former showed less graft versus host disease (GVHD) than the latter. The mice treated with IBM-BMT+ATT also showed an intermediate percentage of effector memory (EM) and central memory (CM) cells between those treated with BMT alone and those treated with IBM-BMT+DLI. The numbers and functions of T cells increased in those treated with IBM-BMT+ATT with interleukin-2 and interferon-γ production. These results suggest that IBM-BMT+ATT is effective in the treatment of leukemia with strong graft versus leukemia without increased risk of GVHD.     

The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation.

The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment.     

Recipient-Derived Allo-iTregs Induced by Donor DCs Effectively Inhibit the Proliferation of Donor T Cells and Reduce GVHD

To compare the potency of recipient-derived, antigen-specific regulatory T cells induced by different dendritic cells (DCs; iTregs) and freshly isolated natural regulatory T cells (nTregs) in preventing mouse graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). CD4⁺ T cells from recipient BALB/c mice were stimulated with DCs from recipient BALB/c (syn-DCs), donor B6 (allo-DCs), and third-party C3H (third-party-DCs) mice to induce different iTregs. In parallel, nTregs were isolated from spleen cells of recipient BALB/c (syn-nTregs) and donor B6 (allo-nTregs) mice using magnetic-activated cell sorting. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the suppressive ability of these various regulatory T cells (Tregs). Both the iTregs and nTregs were transfused to GVHD mice on Days 0, 1, 3, and 5. Body weight, GVHD score, and survival time were monitored. Peripheral Tregs were subsequently examined on Days 7, 14, 21, and 28 after BMT, while chimerism was evaluated on Days 14 and 60. Histopathology of colon, liver, and spleen were also performed. DCs markedly induced CD25⁺ and Foxp3⁺ expression on CD4⁺ T cells. The allo-DC-induced Tregs (allo-iTregs) suppressed the proliferation of alloreactive T cells better than the other iTregs/nTregs in MLR assays (P < 0.05). Meanwhile, transfusion of the allo-iTregs reduced the severity of GVHD (P < 0.05), increased survival time compared with the GVHD group (P < 0.05), and enhanced the chimerism proportion. On Day 28 after BMT, the allo-iTregs group had the highest frequency of peripheral Tregs (P < 0.05). Recipient-derived allo-iTregs induced by donor DCs included predominant clones that specifically recognized donor antigens. These allo-iTregs not only prevented GVHD by suppressing the proliferation of donor-alloreactive T cells, but also promoted engraftment, and prolonged the survival of GVHD mice. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:825–836, 2019. © 2018 Wiley Periodicals, Inc.     

Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen.

Mixed hematopoietic chimerism can be induced in mice receiving allogeneic bone marrow transplantation (BMT) after nonmyeloablative host conditioning with depletion T cells with of anti-T cell monoclonal antibodies (mAbs), low-dose (3 Gy) total-body irradiation (TBI), and local thymic irradiation (7 Gy). These mice are specifically tolerant to donor and host antigens. When nontolerant donor T cells are given to chimeras several months after BMT, full donor-type chimerism develops, but graft-vs.-host disease (GVHD) does not occur. The induction of such lymphohematopoietic GVH reactions without GVHD could provide an approach to separating graft-vs.-leukemia (GVL) from GVHD in patients with hematologic malignancies. To make the nonmyeloablative conditioning regimen described above more cytoreductive for such malignancies, we have now modified it by replacing TBI with cyclophosphamide (CP). Treatment with anti-CD4 and anti-CD8 mAbs on day -5, 200 mg/kg CP on day -1, and 7 Gy thymic irradiation on day 0 was only slightly myelosuppressive and allowed fully major histocompatibility complex (MHC)-mismatched (with or without multiple minor antigen disparities) allogeneic bone marrow to engraft and establish long-term mixed chimerism in 40 to 82% of recipients in three different strain combinations. The administration of nontolerant donor spleen cells at 5 weeks or at 5, 8, and 11 weeks posttransplant was capable of eliminating host hematopoietic cells, leading to full or nearly full donor chimerism in six of six and two of four chimeric animals in two different strain combinations. No clinical evidence of GVHD was observed in any recipients of these donor leukocyte infusions (DLI). These studies demonstrate that induction of mixed chimerism with nonmyeloablative conditioning followed at appropriate times by DLI might allow lymphohematopoietic GVH reactions, and hence GVL effects, to eliminate chronic hematologic malignancies without causing clinically significant GVHD.     

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.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells.

Our previous studies in mice showed that the nonmyeloablative conditioning regimen of fractionated irradiation of the lymphoid tissues (total lymphoid irradiation; TLI) and depletive anti-T-cell antibodies (anti-thymocyte serum) markedly increased the percentage of regulatory DX5+ and natural killer 1.1+ T cells in the mouse spleen, and prevented acute lethal graft-versus-host disease (GVHD) in BALB/c mice (H-2(d)) following the transplantation of bone marrow (BM) and peripheral blood mononuclear cells (PBMC) from C57BL/6 (H-2(b)) donors. The object of the current study was to determine whether the TLI and anti-thymocyte serum regimen protected natural killer T-cell deficient CD1(-/-) BALB/c mice against GVHD after BM and PBMC transplantation from C57BL/6 donors, and whether a similar conditioning regimen of TLI and anti-thymocyte globulin (ATG) can prevent GVHD in Lewis rat (RT1(l)) hosts after BM and PBMC transplantation from ACI rat (RT1(a)) donors. The experimental results in mice showed that, although wild-type BALB/c hosts are protected in association with a marked increase in CD1- reactive T cells expressing the invariant TCR identified with a CD1 tetramer reagent; CD1(-/-) BALB/c hosts are not. Studies of chimeric donor cells in mice protected from GVHD showed donor T-cell polarization to a Th2 cytokine pattern. Results in rats showed that approximately 1000 fold more donor PBMC cells were required to induce a similar incidence of lethal GVHD in TLI and ATG conditioned hosts as compared with hosts conditioned with single-dose total-body irradiation or total-body irradiation and ATG. Surviving TLI and ATG conditioned rat hosts were complete chimeras. In conclusion, the TLI and ATG/anti-thymocyte serum conditioning regimen protects against GVHD in rats and mice, and regulatory natural killer T cells are required for protection.     

Adult thymus transplantation with allogeneic intra-bone marrow-bone marrow transplantation from same donor induces high thymopoiesis, mild graft-versus-host reaction and strong graft-versus-tumour effects

Although allogeneic bone marrow transplantation (BMT) plus donor lymphocyte infusion (DLI) is performed for solid tumours to enhance graft-versus-tumour (GVT) effects, a graft-versus-host reaction (GVHR) is also elicited. We carried out intra-bone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor to supply alloreactive T cells continually. Normal mice treated with IBM-BMT + ATT survived for a long time with high donor-derived thymopoiesis and mild GVHR. The percentage of CD4(+) FoxP3(+) regulatory T cells in the spleen of the mice treated with IBM-BMT + ATT was lower than in normal B6 mice or mice treated with IBM-BMT alone, but higher than in mice treated with IBM-BMT + DLI; the mice treated with IBM-BMT + DLI showed severe GVHR. In tumour-bearing mice, tumour growth was more strongly inhibited by IBM-BMT + ATT than by IBM-BMT alone. Mice treated with IBM-BMT + a high dose of DLI also showed tumour regression comparable to that of mice treated with IBM-BMT + ATT but died early of GVHD. By contrast, mice treated with IBM-BMT + a low dose of DLI showed longer survival but less tumour regression than the mice treated with IBM-BMT + ATT. Histologically, significant numbers of CD8(+) T cells were found to have infiltrated the tumour in the mice treated with IBM-BMT + ATT. The number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL)-positive apoptotic tumour cells also significantly increased in the mice treated with IBM-BMT + ATT. Allogeneic IBM-BMT + ATT thus can induce high thymopoiesis, preserving strong GVT effects without severe GVHR.     

Enhancement of allogeneic hematopoietic stem cell engraftment and prevention of GVHD by intra-bone marrow bone marrow transplantation plus donor lymphocyte infusion

We examined the effect of intra-bone marrow (IBM)-bone marrow transplantation (BMT) in conjunction with donor lymphocyte infusion (DLI) on the engraftment of allogeneic bone marrow cells (BMCs) in mice. Recipients that had received 6 Gy of radiation completely rejected donor BMCs, even when IBM-BMT was carried out. However, when BMCs were IBM injected and donor peripheral blood mononuclear cells (PBMNCs) were simultaneously injected intravenously (DLI), donor cell engraftment was observed 7 days after BMT and complete donor chimerism continued thereafter. It is of interest that the cells of recipient origin did not recover, and that the hematolymphoid cells, including progenitor cells (Lin-/c-kit+ cells) in the recipients, were fully reconstituted with cells of donor origin. The cells in the PBMNCs responsible for the donor BMC engraftment were CD8+. Recipients that had received 6 Gy of radiation, IBM-BMT, and DLI showed only a slight loss of body weight, due to radiation side effects, and had no macroscopic or microscopic symptoms of graft-versus-host disease. These findings suggest that IBM-BMT in conjunction with DLI will be a valuable strategy for allogeneic BMT in humans.     

Chloroquine prevention of murine MHC-disparate acute graft-versus-host disease correlates with inhibition of splenic response to CpG oligodeoxynucleotides and alterations in T-cell cytokine production.

The 4-aminoquinolines, chloroquine and hydroxychloroquine, are established, with a 52% response rate, as therapy for human steroid-refractory GVHD after BMT. Chloroquine affects numerous mechanisms that play a role in GVHD, including inhibition of major histocompatibility complex (MHC) class II antigen presentation, cytokine production, and antigen-presenting cell activation by bacterially derived CpG oligodeoxynucleotides (ODNs). Using an MHC-disparate murine model, we evaluated the effect of chloroquine treatment on the development of acute GVHD. We assessed the effect of chloroquine on the immunostimulatory responses induced by CpG ODNs after BMT. We also evaluated the impact of chloroquine on cytokine-producing populations known to affect GVHD, including CD4+ and CD8+ T-cell and CD3(+)/NK1.1(+) natural killer T-cell (NKT cell) populations. Twelve (86%) of 14 mice receiving phosphate-buffered saline solution (PBS) developed lethal GVHD; only 4 (29%) of 14 mice receiving chloroquine 20 mg/kg 3 times per week developed lethal GVHD (P < .01). Chloroquine significantly suppressed CpG ODN-induced splenic proliferation and interleukin 6 (IL-6) production associated with GVHD. Chloroquine suppressed CD8+ T-cell production of IL-2 and IL-4 associated with GVHD in this model and maintained an early expansion (day 7) of splenic NKT cells. These results indicate that the 4-aminoquinolines are effective in therapy for or prevention of acute GVHD secondary to MHC disparities. Chloroquine actions may include inhibition of CpG ODN augmentation of GVHD. Other mechanisms involved may include suppression of CD8+ T-cell production of IL-2 and IL-4 and an increase in NKT cells associated with GVHD inhibition by chloroquine.     

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.     

Combined CD4+ Donor Lymphocyte Infusion and Low-Dose Recombinant IL-2 Expand FOXP3+ Regulatory T Cells following Allogeneic Hematopoietic Stem Cell Transplantation

CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4⁺ lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4⁺DLI alone (P = .001). FOXP3 expression correlated with absolute CD4⁺CD25⁺ cell counts. Moreover, expanded CD4⁺CD25⁺ T cells displayed normal suppressive function and treatment with CD4⁺DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4⁺ cellular therapy may provide a mechanism to expand Treg in vivo.