Newborn blood can engraft adult mice without inducing graft-versus-host disease across non H-2 antigens

Cells derived from human cord blood were recently used instead of bone marrow (BM) for transplantation. However, several questions concerning the potential of these cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce a graft-versus-host disease (GVHD) remain unanswered. Here we used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non H-2 antigens. Few mature T cells were found in NBB as in adult BM and both contain around 10% to 20% SCA-1+ pluripotent progenitor cells. Yet numerous immature CD4+CD8+ TCR alpha/beta low Thy-1high T cells were present in NBB. In contrast, adult peripheral blood (PB) exhibits a mature T-cell phenotype and contains few progenitor cells. The injection of blood pooled from one to three newborn mice resulted in the engraftment of 71% to 86% of F1 recipients, which survived more than 100 days without clinical signs of GVHD. The injection of BM leads to 100% survival whereas the injection of adult PB resulted in rapid mortality, both without signs of GVHD. In NBB-engrafted F1 surviving mice, T-cell reconstitution preceded B-cell reconstitution, and the degree of donor cell chimerism increased progressively with time. Additionally, 2 to 4 months after transplantation, T cells from these mice were tolerant to host non H-2 antigens but kept reactivity against third-party Ags. Host specific tolerance in NBB-engrafted F1 mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells were able to reconstitute the lymphohematopoietic system of lethally irradiated adult mice without inducing GVHD against incompatible non H-2 antigens. Thus, this experimental model in vivo may be relevant to the human cord blood transplantation.     

Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies

Unrelated cord blood transplantation (CBT) has now become more common, but as yet there have been only a few reports on its outcome compared with bone marrow transplantation (BMT), especially for adults. We studied the clinical outcomes of 113 adult patients with hematologic malignancies who received unrelated BM transplants (n = 45) or unrelated CB transplants (n = 68). We analyzed the hematopoietic recovery, rates of graft-versus-host disease (GVHD), risks of transplantation-related mortality (TRM) and relapse, and disease-free survival (DFS) using Cox proportional hazards models. The time from donor search to transplantation was significantly shorter among CB transplant recipients (median, 2 months) than BM transplant recipients (median, 11 months; P < .01). Multivariate analysis demonstrated slow neutrophil (P < .01) and platelet (P < .01) recoveries in CBT patients compared with BMT patients. Despite rapid tapering of immunosuppressants after transplantation and infrequent use of steroids to treat severe acute GVHD, there were no GVHD-related deaths among CB transplant recipients compared with 10 deaths of 24 among BM transplant recipients. Unrelated CBT showed better TRM and DFS results compared with BMT (P = .02 and P < .01, respectively), despite the higher human leukocyte antigen mismatching rate and lower number of infused cells. These data strongly suggest that CBT could be safely and effectively used for adult patients with hematologic malignancies.     

Cord blood transplantation provides better reconstitution of hematopoietic reservoir compared with bone marrow transplantation

Delayed hematopoietic recovery is the main factor precluding a wider use of cord blood (CB) transplants. We hypothesized that this delayed engraftment might not be related to an insufficient number of stem cells in the graft, but to an intrinsic difficulty of these cells to undergo differentiation. To test our hypothesis, 2 groups of children were compared; 12 received a CB transplant and 12 an adult bone marrow (BM) transplant. We studied neutrophil and platelet recovery and, at a median time of approximately 1 year after transplantation, the frequency of colony-forming cells (CFCs) and long-term culture initiating cells (LTC-ICs) in the BM of the 2 groups. Recipients of BM transplants received 1-log more cells and had significantly faster neutrophil and platelet recovery. Conversely, the frequency of committed and early progenitors was significantly higher in the BM of children given CB cells compared with BM transplant recipients (median count of CFC/2 x 10(4) BM mononuclear cells, 20 versus 11, P =.007; median count of LTC-IC/10(6) BM mononuclear cells, 8.2 versus 0.2 P =.001). CB, but not adult BM stem cells, can better restore the host hematopoietic progenitor cell reservoir; the delayed engraftment after CB transplantation may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.     

A Critical Role for CD48 Antigen in Regulating Alloengraftment and Lymphohematopoietic Recovery After Bone Marrow Transplantation

The binding of CD2, present on T cells, to its counterreceptor CD48facilitates adhesion, signaling, alloantigen-induced cytokine production, and cytotoxic T-lymphocyte responses. Becausethese T-cell functions have been implicated in graft-versus-hostdisease (GVHD) pathogenesis, we have analyzed the effects of theCD2:CD48 pathway on GVHD mediated by CD4⁺ andCD8⁺ T cells infused into sublethally irradiatedrecipients. CD4⁺ T-cell-mediated, and to a lesserextent, CD8⁺ T-cell-mediated GVHD was inhibited byCD2 + 48 monoclonal antibody (MoAb) infusion. To assessthe effects of combined MoAb infusion on alloengraftment, two differentalloengraftment bone marrow transplantation (BMT) models were used. Inboth, MoAb infusion markedly inhibited alloengraftment andhematopoietic recovery post-BMT. To determine if the adverse effects onlymphohematopoiesis in the allogeneic BMT recipients were caused by animmune or nonimmune mechanism, studies were performed in congenic BMTrecipients to preclude an immune mechanism as the cause for delayedrecovery post-BMT. MoAb infusion resulted in impairedlymphohematopoietic recovery in congenic BMT recipients and markedlyreduced day 12 colony-forming unit-spleen formation in syngeneic BMTrecipients, consistent with a nonimmune mediated mechanism. Because thespleen is a site of early hematopoietic recovery post-BMT, studies were performed using adult splenectomized syngeneic BMT recipients. MoAbinfusion delayed recovery in both nonsplenectomized and splenectomized recipients post-BMT, indicating that the delayed hematopoietic recoverywas not the consequence of an abnormal homing pattern of hematopoieticprogenitors to the spleen early post-BMT. CD48 MoAb was necessaryand sufficient for the inhibition of GVHD lethality and delayedlymphohematopoietic effects of the combined MoAb regimen. CD48 MoAbwas found to induce a profound modulation of CD48 antigen expression onBM cells, suggesting that the CD48 antigen may have an importantfunction in hematopoiesis in the BM compartment. Taken together, thesedata provide evidence that the CD48 antigen plays a critical role inregulating hematopoiesis in post-BMT.     

Distinct graft-versus-leukemic stem cell effects of early or delayed donor leukocyte infusions in a mouse chronic myeloid leukemia model

Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.     

Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graft-versus-leukemia reactivity, and tolerance after bone marrow transplantation

Graft rejection, mixed chimerism, graft-versus-host disease (GVHD), leukemia relapse, and tolerance are interrelated manifestations of immunologic reactivity between donor and host cells that significantly affect survival after allogeneic bone marrow transplantation (BMT). In this report, a mouse model of BMT, in which the donor and host were compatible at the major histocompatibility complex (MHC), was used (1) to examine the interrelationship of pretransplant conditioning and T-cell content of donor BM with regard to lymphoid chimerism and GVHD and (2) to determine how these factors affected graft-versus-leukemia (GVL) reactivity and donor-host-tolerance. AKR (H-2k) host mice were administered optimal or suboptimal total body irradiation (TBI) as pretransplant conditioning followed by administration of BM cells from B10.BR (H-2k) donor mice with or without added spleen cells as a source of T lymphocytes. Transplanted mice were injected with a supralethal dose of AKR leukemia cells 20 and 45 days post-BMT to assess GVL reactivity in vivo. The pretransplant conditioning of the host and T-cell content of the donor marrow affected the extent of donor T-cell chimerism and the severity of GVH disease. GVL reactivity was dependent on transplantation of mature donor T cells and occurred only in complete chimeras. Transplantation of T-cell-deficient BM resulted in the persistence of host T cells, ie, incomplete donor T-cell chimerism, even when lethal TBI was used. Mixed chimerism was associated with a lack of GVL reactivity, despite the fact that similar numbers of donor T cells were present in the spleens of mixed and complete chimeras. In this model, moderate numbers of donor T cells facilitated complete donor T-cell engraftment, caused only mild GVHD, and provided a significant GVL effect without preventing the subsequent development of tolerance after conditioning with suboptimal TBI. In contrast, severe, often lethal, GVHD developed when the dose of TBI was increased, whereas tolerance and no GVH/GVL reactivity developed when the T-cell content of the marrow was decreased.     

Beta2 integrins separate graft-versus-host disease and graft-versus-leukemia effects

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.     

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.     

Two cytotoxic pathways of natural killer cells in human cord blood: implications in cord blood transplantation

Using K562 cells as a target we investigated cord blood (CB)–natural killer (NK) cytolytic pathways. The cytotoxicity of fresh CB-NK cells was significantly lower than that of peripheral blood mononuclear cells (PB MNCs). When CB was incubated with IL-2, the level of CB-NK cytotoxicity was increased and boosted to the level observed in PB-NK cells. Fresh CB-NK cells induced apoptosis in target cells. Activated CB cells induced apoptosis and necrosis in target cells, at the same level as PB MNCs. CB stem cell transplantation may also induce graft-versus-host disease (GVHD)/graft-versus-leukaemia (GVL), similar to bone marrow transplantation.     

Graft engineering using ex vivo methods to limit GVHD: fludarabine treatment generates superior GVL effects in allogeneic BMT

OBJECTIVE: To compare the abilities of different ex vivo methods of treating donor lymphocytes to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia (GVL) activity in murine models of allogeneic bone marrow transplantation. METHODS: Donor/recipient pairs included MHC fully mismatched, MHC haplomismatched, and MiHA mismatched strain combinations. T cell-depleted BM (TCD-BM) was transplanted in combination with untreated, fludarabine-treated, 7.5-Gy gamma-irradiated, or psoralen/UVA (PUVA)-treated splenocytes. GVL activity was studied by adding a lethal number of recipient-type lymphoma cells. Posttransplant survival was determined, and GVHD and GVL activity were assessed by clinical and pathological scoring. Hematopoietic chimerism and donor T-cell expansion were analyzed by flow cytometry of peripheral blood samples at 30 and 60 days posttransplant. RESULTS: Allogeneic splenocytes treated with fludarabine, 7.5 Gy gamma-irradiation, or PUVA had significantly diminished GVHD activity, and all treated donor T cells facilitated engraftment by low-dose TCD-BM. Allogeneic splenocytes treated with fludarabine and, to a lesser extent, PUVA retained GVL activity and contributed more to donor T-cell chimerism compared to gamma-irradiated donor splenocytes. CONCLUSION: Among ex vivo methods of treating donor T cells to limit their proliferative capacity, fludarabine exposure had the greatest differential ability to inhibit the GVHD activity of allogeneic lymphocytes, while preserving their GVL activity and ability to engraft recipients. Thus, ex vivo treatment with fludarabine was superior to gamma-irradiation or PUVA in separating GVL from GVHD activity in murine models of allogeneic bone marrow transplantation.     

Expanded donor natural killer cell and IL-2, IL-15 treatment efficacy in allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD), leukemia relapse, and immune deficiency remain the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor natural killer cells (NK) and cytokines have good potential in GVHD prevention and immune reconstitution enhancement. Improved survival after allo-HSCT therefore requires effective prophylaxis to reduce GVHD and strategy to mediate graft-versus-leukemia (GVL) effect. We studied the administration of expanded donor NK cell infusion and interleukin-2 (IL-2) and IL-15 mixture treatment in a murine allo-HSCT model for its effects on GVHD, immune reconstitution and leukemia relapse. In the GVHD model, recipient mice were reconstituted with bone marrow (BM) cells and splenocytes via vein. In the leukemia model, recipient mice were inoculated with EL9611 leukemia cells via vein 8 d prior to transplant. NK cell infusion mice group had lower clinical GVHD scores and suffered less severe GVHD-associated weight loss than control mice group. 90% of control mice died of leukemia relapse within 52 d post-transplant. NK cell infusion and IL-2 and IL-15 treatment recipient mice had improved survival compared with control mice. NK cell infusion and IL-2 and IL-15 treatment recipient mice had also accelerated lymphoid immune reconstitution compared with control mice group. Expanded donor NK cell infusion and IL-2 and IL-15 treatment could promote lymphoid immune reconstitution, mitigate GVHD, and reduce leukemia relapse in allo-HSCT recipients. The findings may have important significance for complication prevention in clinical allo-HSCT.     

Delayed infusion of immunocompetent donor cells after bone marrow transplantation breaks graft-host tolerance allows for persistent antileukemic reactivity without severe graft-versus-host disease

The development of graft-host tolerance after bone marrow transplantation (BMT) is crucial to avoid the problems of graft-versus- host disease (GVHD) and graft rejection. GVHD can be eliminated by depleting mature donor T cells from the BM inoculum, thereby facilitating the development of graft-host tolerance. However, T-cell depletion often results in an increased incidence of graft rejection and an increased frequency of leukemia relapse. Thus, although graft- host tolerance is a desirable outcome, it can pose a significant threat to leukemia-bearing hosts. Using a major histocompatability complex (MHC)-matched allogeneic model of BMT (B10.BR into AKR), we found that irradiated recipients given donor BM alone displayed mixed T-cell chimerism and did not develop GVHD. Graft-host tolerance developed by 8 weeks after BMT in these chimeras, and they were susceptible to low- dose leukemia challenge. When sufficient numbers of donor spleen cells, as a source of T-cells, were added to the BM graft, AKR hosts developed severe and lethal GVHD. Antihost reactive donor T cells persisted in chimeras undergoing GVHD, indicating that graft-host tolerance did not develop. When administration of the spleen cells was delayed for 7 to 21 days after BMT, there was significantly less mortality because of GVHD. Day 21 was the optimal time for infusion of cells without development of GVHD. Graft-host tolerance was broken by the delayed infusion of donor cells, as indicated by the persistence of antihost- reactive donor T cells in these chimeras in T-cell receptor cross- linking and mixed lymphocyte reaction assays. Importantly, the persistence of antihost-reactive donor T cells correlated with along- term antileukemic effect that was still present at 100 days after transplant. Multiple infusions of immunocompetent donor cells could be administered without increasing the risk for GVHD if delayed until 21 days post-BMT. Delayed infusions of donor spleen cells also resulted in a long-term antileukemic effect in the absence of GVHD in an MHC- haplotype-mismatched model of BMT (SJL into [SJL x AKR]F1). Although delayed infusion of normal donor cells did not induce GVHD, spleen cells from donors previously sensitized to host alloantigens induced GVHD when infused 21 days after BMT. Thus, the ability of previously activated cells to induce GVHD was not inhibited in the same manner as naive cells. Results from limiting dilution analysis assays indicated that alloactivated interleukin-2-secreting CD4+ T cells were preferentially inhibited over cytolytic T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-2 in bone marrow transplantation: preclinical studies.

Interleukin-2 (IL-2) promotes the generation and proliferation of killer cells in the peripheral blood and bone marrow (BM) both in vitro and in vivo. When employed in a syngeneic bone marrow transplantation (BMT) setting and followed by IL-2 therapy, murine BM cells activated with IL-2 in vitro (ABM) demonstrate potent graft-versus-leukemia (GVL) and anticytomegalovirus effects. ABM cells retain the capacity to reconstitute the hemopoietic system both in normal and leukemic mice. This therapy does not cause graft-versus-host disease (GVHD). Human ABM cells carry out purging of leukemia without loss of progenitor cell activity in vitro. The purging ability of ABM can be augmented by interleukin-1, interferon, and tumor necrosis factor. IL-2 therapy stimulates the veto suppressor cell activity of T cell-depleted BM, and has reduced GVHD and permitted engraftment of mismatched allogeneic BM in murine models. Future studies should determine the optimum treatment schedules with IL-2 for improving the GVL effect in autologous BMT, and for abolishing GVHD in allogeneic BMT settings.     

Interleukin-10 administration decreases survival in murine recipients of major histocompatibility complex disparate donor bone marrow grafts

Studies in mice and humans have indicated that the predominance of interleukin-4 (IL-4)- and IL-10-producing T-helper type 2 (Th2) cells may serve to downregulate acute graft-versus-host disease (GVHD) reactions, whereas IL-2-producing Th1 cells have been implicated in facilitating acute GVHD. We explored the possibility that the in vivo infusion of IL-10 would inhibit acute GVHD induced by fully allogeneic donor grafts. Unexpectedly, IL-10 infusions resulted in a dose- dependent increase in GVHD-induced mortality. The acceleration of lethal GVHD by IL-10 occurred in irradiated recipients of T-cell- depleted bone marrow (BM) plus 5, 15, or 25 x 10(6) splenocytes but did not influence the post-BM transplantation (post-BMT) survival rate of recipients of BM without splenocytes, suggesting that the IL-10 effects were not due to toxicity. Antimurine IL-10-neutralizing monoclonal antibody injections, administered to diminish endogenous IL-10, reduced GVHD-associated mortality and improved the clinical appearance of the recipients. For BM graft rejection studies, IL-10 was infused into sublethally irradiated recipients of anti-Thy 1.2 + C' T-cell-depleted, fully allogeneic BM grafts. In a short-term (day 7) in vivo assay, IL- 10 infusions significantly inhibited allogeneic (but not syngeneic) BM proliferation in vivo, indicative of increased graft rejection. In long- term chimerism experiments, IL-10 infusions caused a significant increase in early post-BMT mortality caused by a profound anemia typically associated with graft rejection and aplasia. A slightly higher irradiation dose (650 cGy v 600 cGy) eliminated the anemia but did not reverse the graft rejection process associated with IL-10 administration. We conclude that the in vivo infusion of exogenous IL- 10 in recipients of fully allogeneic donor grafts results in accelerated GVHD and graft rejection in the strain combinations tested to date.     

Allogeneic cell-mediated immunotherapy of leukemia with immune donor lymphocytes to upregulate antitumor effects and downregulate antihost responses

Donor lymphocyte infusion mediates most effective graft- versus-leukemia (GVL) effects following induction of host-versus-graft tolerance by transplantation of donor stem cells. This study was designed to maximize GVL effects across both major (MHC) and minor (mHgs) histocompatibility barriers in recipients inoculated with murine B-cell leukemia (BCL1), using specifically immune donor lymphocytes. GVL effects were induced with donor spleen cells from mice immunized across MHC or mHgs barriers with BCL/1 cells or normal BALB/c spleen cells. Our data suggest that spleen cells from donor mice immunized against murine B-cell leukemia of BALB/c origin, or to a lesser extent against normal host alloantigens, induce better therapeutic GVL effects with less great-versus-host disease (GVHD) across both mHgs and MHC. The cytokine profile of effector cells inducing predominantly GVL effects with reduced GVHD across MHC and mHg barriers consisted preferentially of upregulated IFN-gamma, IL-2, IL-10 and IL-12 in donors, implying a Th-1 to Th-2 cytokine shift. We hypothesize that immunotherapy with immune donor lymphocytes sensitized in vivo or in vitro with allogeneic tumor cells or normal host cells together with allogeneic BMT may provide an effective approach for amplifying GVL effects, while reducing procedure-related morbidity and mortality due to uncontrolled GVHD.     

Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provide a strong graft-versus-leukaemia effect in mice

Allogeneic lymphocytes administered with an unmanipulated bone marrow transplant provide a strong antileukaemic effect, the so-called graft-versus-leukaemia (GVL) effect. On the other hand, T-cell-mediated graft-versus-host-disease (GVHD) observed after transplantation of unmanipulated BM graft causes substantial morbidity and mortality. The aim of the present study was to determine the antileukaemic potential of enriched IL-2 activated NK cells administered 2 h after BMT. Balb/c (H-2^d) mice were given a dose of A20 (H-2^d, B-cell leukaemia) cells 2 d prior to lethal total body irradiation (TBI) and transplantation of either syngeneic or allogeneic anti-Thy1.2 (CD90) depleted bone marrow cells. Either syngeneic (Balb/c, H-2^d) or allogeneic (C57BL/6, H-2^b) enriched and IL-2 (200 U/ml for 24 h) activated NK cells were given 2 h after BMT. Injection of A20 leukaemia into normal Balb/c recipients led to death after a median of 14 d. A lethal dose of TBI followed by either syngeneic or allogeneic Thy1.2-depleted BMT resulted in a modest antileukaemic effect. The adoptive transfer of syngeneic enriched and IL-2 preincubated NK cells given at time of BMT exerted a significantly better GVL effect. However, the infusion of allogeneic enriched NK cells resulted in a stronger GVL effect. These results clearly demonstrate that allogeneic NK cells are superior to syngeneic NK cells in their potential to eradicate residual leukaemia cells after BMT without mediating clinical overt GVHD. This experimental setting may offer a strategy for treatment of haematological malignancies in a phase of minimal residual disease.     

Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.     

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.     

Cellular therapy: exploiting NK cell alloreactivity in transplantation

Allogeneic hematopoietic transplantation relies on T-cell alloreactions for engraftment and the graft-versus-leukemia (GVL) effect. In human leukocyte antigen (HLA) haplotype-mismatched transplants, extensive T-cell depletion of the graft is essential to prevent GVHD. This raises the question of whether mismatched transplants exert any GVL effect and whether it will ever be possible to reduce the intensity of preparative regimens. Because natural killer (NK) cells are negatively regulated by MHC class I-specific inhibitory receptors, mismatched transplants may trigger NK-cell alloreactivity. HLA class I disparities driving NK-cell alloreactions in the GVH direction mediate strong GVL effects, produce higher engraftment rates, and do not cause GVHD. In murine MHC-mismatched transplant models with no donor T-cell reactivity against the recipient, the pre-transplant infusion of donor-vs-recipient alloreactive NK cells conditioned the recipients to bone marrow transplantation without GVHD. NK-cell alloreactivity may be a unique therapeutic tool for tolerance induction and clearance of leukemia in hematopoietic transplantation.