Engraftment of severe combined immune deficient mice receiving allogeneic bone marrow via In utero or postnatal transfer

Although in utero transplantation (IUT) has been shown to be effective in treating human severe combined immune deficiency (SCID), the relative merit of IUT as compared with postnatal bone marrow transplantation (BMT) for SCID is unknown. Therefore, comparative studies were undertaken in mice to determine the engraftment outcome in these two settings. Because T-cell depletion (TCD) reduces graft-versus-host disease (GVHD) severity but compromises alloengraftment, studies were performed with TCD or non-TCD BM and GVHD risk was assessed using a tissue scoring system and by the adoptive transfer of splenocytes from engrafted mice into secondary recipients. Non-SCID recipients received pre-BMT irradiation to simulate those circumstances in which conditioning is required for alloengraftment. IUT recipients of non-TCD and especially TCD BM cells in general had higher levels of donor T-cell and myeloid peripheral blood (PB) engraftment than nonconditioned SCID recipients. Increased TCD or non-TCD BM cell numbers in adult SCID recipients resulted in similar levels of PB engraftment as IUT recipients. However, under these conditions, mean GVHD scores were higher than in IUT recipients. The majority of adoptive transfer recipients of splenocytes from IUT recipients were GVHD-free, consistent with the in vitro evidence of tolerance to host alloantigens. Total body irradiation (TBI)-treated mice that had the highest engraftment had evidence of thymic damage as denoted by a higher proportion of thymic and splenic T cells with a memory phenotype as compared with IUT recipients. IUT mice had vigorous thymic reconstitution by 3 weeks of age. Our data indicate that IUT has a number of advantages as compared with postnatal BMT. Future studies examining the fine specificity of immunoreconstitution in IUT versus postnatal BMT are indicated.     

Improving the outcome of bone marrow transplantation by using CD52 monoclonal antibodies to prevent graft-versus-host disease and graft rejection

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic bone marrow transplantation, but can be avoided by removing T lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 monoclonal antibodies to eliminate T cells from both donor marrow and recipient to prevent both GVHD and rejection. Seventy patients receiving HLA-identical sibling transplants for acute myelogenous leukemia (AML) in first remission were studied. An IgM (CAMPATH-1M) was used for in vitro depletion of the graft and an IgG (CAMPATH-1G) for in vivo depletion of the recipient before graft infusion. No posttransplant immunosuppression was given. Results were compared with two control groups: (1) 50 patients who received bone marrow depleted with CAMPATH-1M, but no CAMPATH-1G in vivo; and (2) 459 patients reported to the International Bone Marrow Transplant Registry (IBMTR) who received nondepleted grafts and conventional GVHD prophylaxis with cyclosporin A (CyA) and methotrexate (MTX). The incidence of acute GVHD was 4% in the treatment group compared with 35% in the CyA/MTX group (P <.001). Chronic GVHD was also exceptionally low in the treatment group (3% v 36%; P <.001). The problem of graft rejection, which had been frequent in the historic CAMPATH-1M group (31%), was largely overcome in the treatment group (6%). Thus, transplant-related mortality of the treatment group (15% at 5 years) was lower than for the CyA/MTX group (26%; P =.04). There was little difference in the risk of leukemia relapse between the treatment group (30% at 5 years) and the CyA/MTX group (29%). Survival of the treatment group at 6 months was better than the CyA/MTX group (92% v 78%), although at 5 years the difference was not significant (62% v 58%) and neither was the difference in leukemia-free survival (60% v 52%). We conclude that T-cell depletion is a useful strategy to prevent GVHD, provided that measures are taken to ensure engraftment. Using CAMPATH-1G to deplete residual host lymphocytes is a simple and practical method to do this. At least in AML, the beneficial reduction in GVHD can be achieved without an increased risk of relapse.     

Ways to feel comfortable: teaching aids to promote children''s comfort

The plant lectin, soybean agglutinin (SBA), has been widely used to separate heterogeneous populations of cells. In the field of bone marrow transplantation, SBA has been used for partial depletion of T cells from bone marrow allografts to reduce graft-vs.-host disease. SBA's high affinity for many different tumor cells has also indicated its use as a tumor purging agent for autologous bone marrow transplants. We have compared two methods of cell separation using either soluble SBA agglutination, or SBA covalently attached to an activated polystyrene surface. The nonbinding SBA-cell populations generated by these two procedures were very similar in terms of cell recovery, light scatter properties, and phenotypic profile. Notably, both SBA- fractions were enriched in cells with the known progenitor markers, CD34, CD33, and HLA-DR, and were relatively depleted of SBA binding cells. In addition, the activity of each SBA- cell population was measured in vitro in short-term progenitor assays. Here, both SBA- populations were significantly enriched for CFU-GM. When device-separated SBA- cell populations were seeded into long-term bone marrow culture, they produced both increased progenitor activity and cell proliferation compared to unseparated BMMCs. The polystyrene technology described here could reduce or eliminate many of the drawbacks of soluble SBA agglutination, making SBA cell separation a viable and convenient technique for clinical application.     

Outcomes of recipients of both bone marrow and solid organ transplants. A review

In this review we examine the clinical outcomes of patients who have received both bone marrow transplantation (BMT) and solid organ transplantation (SOT) and discuss the possible immunologic consequences of the dual transplants. We collected cases through a comprehensive literature search (MEDLINE database, English literature only) covering the years 1990 through 1997 and correspondence with the International Bone Marrow Transplant Registry. Our study selected case reports of patients who have undergone both bone marrow and solid organ transplants; cases in which bone marrow transplantation was undertaken as an adjunct ot induce or augment donor-specific tolerance in a recipient to the transplanted organ were excluded. Clinical characteristics included patient's demographic information, underlying disorders for each transplant, source of donor organ or tissue, time between transplants, and immunosuppressive regimens used to prevent graft-versus-host disease (GVHD) or graft rejection. Clinical outcomes included patient survival, complications of transplantation, and donor-specific tolerance that was experienced in many cases. Twenty-one cases of SOT after BMT and 7 cases of BMT after SOT were reviewed. Solid organ transplantations included lung, liver, cardiac, and kidney for a variety of BMT-related complications including GVHD, hepatic veno-occlusive disease, chronic renal failure, end-stage pulmonary disease, and severe cardiomyopathy. Bone marrow transplants were performed following SOT for aplastic anemia and hematologic malignancies. Clinical outcomes for patients who received both BMT and SOT were variable and depended on transplant indication and degree of histocompatibility. Prior bone marrow transplantation may tolerize for a subsequent organ transplant from the same donor. Conversely, severe GVHD may follow BMT from human leukocyte antigen (HLA)-matched donors following SOT. The favorable survival in this high-risk group of patients may represent a literature review bias (that is, an undetermined number of unsuccessful cases may not have been reported). Nonetheless, dual transplantation is clearly feasible in selected cases.     

Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHC-mismatched donor marrow grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57-597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H-2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.     

Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.     

A comparison of the results of KTP and CO2 laser stapedotomy

Allogenic hematopoietic stem cell transplantation is associated with a severe complication induced by the T-cells present in the graft: graft-vs-host disease (GVHD). While effectively preventing GVHD, ex vivo T-lymphocyte depletion of the graft unfortunately increases graft rejection and reduces the graft-vs-leukemia (GVL) effect. The ex vivo transfer to the herpes simplex thymidine kinase (HS-tk) suicide gene into T-cells before their infusion with the hematopoietic stem cells should allow for selective in vivo depletion of these T-cells with ganciclovir (GCV) if subsequent GVHD was to occur. In patients not experiencing GVHD, and therefore at a higher risk of relapse, one could preserve the beneficial effects of the donor T-cells on tumor control. Lastly, the early presence of donor T-cells in all patients should contribute to successful engraftment. We have demonstrated that retroviral-mediated transfer of HS-tk and Neomycine resistance genes in T-lymphocytes, followed by G418 selection, results in T-cells specifically inhibited by GCV with no bystander effect. In a phase I study, escalating amounts of HS-tk expressing T-cells will be infused in conjunction with a T-cell depleted marrow graft to allogenic HLA identical recipients. Toxicity, survival, alloreactivity and GCV-sensitivity of the gene-modified cells will be monitored. If successful, such an approach could significantly contribute to expanding the use of alloreactivity as a treatment modality.     

Identification of a graft versus host disease-associated human minor histocompatibility antigen

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.     

Donor helper T-cell frequencies as predictors of acute graft-versus-host disease in bone marrow transplantation between HLA-identical siblings

BACKGROUND: Despite the current level of sophistication of molecular typing for class I and class II alleles, a significant proportion (20-40%) of recipients of HLA-identical sibling marrow develop severe, acute graft-versus-host disease (GVHD) after bone marrow transplantation. It has been suggested that the frequency of patient-specific helper T lymphocyte precursors (HTLp) detected in the HLA-identical sibling donor correlates with the incidence and severity of acute GVHD after transplantation. METHODS: This study group consisted of 42 patients who all received bone marrow from HLA-identical sibling donors from January 1990 to December 1996. Using a limiting dilution analysis, donor HTLp frequencies were determined on samples collected before transplantation. The HTLp assay used the cytotoxic T-cell line, CTLL-2, which proliferates in the presence of interleukin-2. The reliability and reproducibility of this assay was established by using cryopreserved batches of CTLL-2 cells of known sensitivity. RESULTS: The recipient-directed HTLp frequencies detected in the donor before transplantation were correlated with the incidence and severity of acute GVHD experienced by the recipient after transplantation. Statistical analysis revealed an extremely significant correlation between donor precursor frequencies and the development of acute GVHD in the patient after transplantation (P<0.0001). CONCLUSIONS: This study suggests that together with molecular typing the HTLp frequency should be considered when selecting the most suitable sibling donor for bone marrow transplantation.     

CD34 positive blood cells for allogeneic progenitor and stem cell transplantation

The transplantation of allogeneic peripheral blood progenitor cells (PBPC) provides complete and sustained hematopoietic and lymphopoietic engraftment. In healthy donors, large amounts of PBPC can be mobilized with hematopoietic growth factors. However, the high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic PBPC to an elevated risk of acute and chronic graft-versus-host disease. Thus, the use of appropriate T-cell reduction, but not depletion might reduce this risk. The hazards of graft rejection and a higher relapse rate can be avoided by maintaining a portion of the T-cells in the graft. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1000-fold reduction of T-cells. These purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation and can be used in the following instances: 1. As hematopoietic stem and progenitor cell transplantation instead of bone marrow cells, from HLA-identical family donors; 2. for increasing the stem cell numbers from HLA-mismatched or three HLA-loci different family donors in order to reduce the incidence of rejection but without increasing the T-cell number; 3. boosting of poor marrow graft function with stem cells from the same family donors; 4. transplantation from volunteer matched unrelated donors; 5. split transplantation of CD34+ and T-cells; 6. addition of ex vivo expanded CD34+ cells to blood cell or bone marrow transplantation; 7. generation of antigen specific immune effector cells and antigen presenting cells for cell therapy.     

In vivo infusion of anti-LFA-1 and anti-CD2 antibodies prevents graft failure after HLA partially incompatible bone marrow transplantation in children with high risk acute lymphoblastic leukaemia

Bone marrow transplantation (BMT) from matched sibling donors is the therapy of choice for children with high-risk acute lymphoblastic leukaemia in children. It is however not available to more than two-thirds of patients who lack a matched donor. Here, we review the outcome of 28 patients with high-risk ALL who were transplanted in France with alternative marrow sources such as HLA-phenoidentical unrelated volunteers and HLA-partially incompatible relatives. For these patients, we tested the possibility to prevent T-depleted marrow graft rejection by infusing in vivo two monoclonal antibodies directed against adhesion receptors i.e., LFA-1 and CD2. Two previous multicenter trials in children transplanted with partially incompatible bone marrow for inborn errors of metabolism showed their efficacy in this setting. Twenty eight patients were enrolled in this study and followed for a median of 4.4 years. Bone marrow engraftment occurred in 81% of the evaluable patients. Post-transplantation leukaemic relapse was the most frequent cause of death in this group of patients, and occurred in 39% of patients. The second most frequent complication was infectious disease, while an EBV-induced B-lymphocyte proliferative disorder occurred in four patients. In conclusion, T-cell-depletion combined with infusion of anti-LFA-1 and anti-CD2 antibodies is efficient in preventing graft failure and GVHD in this group of children with high-risk leukaemia undergoing partially incompatible BMT. The overall DFS is not improved in contrast to what has been previously observed in patients with immunodeficiencies transplanted with a similar rejection prophylaxis. Other approaches are therefore needed aiming either at preserving donor T-cell mediated immunity or accelerating immune reconstitution.     

In vitro sensitivity of post-bone marrow transplantation CFU-GM and BFU-E to TNF-alpha and IFN-gamma

Graft failure remains one of the limitations of successful marrow transplantation. T cell-depleted (TCD) bone marrow transplantation (BMT) is reported to have a higher incidence of graft failure than unmodified (UM) BMT. In most cases of secondary graft failure, no cellular immune mechanism has been identified and etiology remains unclear. In an effort to delineate a cytokine-mediated mechanism of secondary graft failure, we investigated colony-forming unit-granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) growth and pattern of inhibition by tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) in the early posttransplant period (day 28). Gradient-separated bone marrow mononuclear cells (BMMNC) from 38 recipients of TCD BMT, 15 recipients of UM BMT, and 23 normal donors (NLD) were plated in cultures of semisolid, serum-containing medium with the addition of stem cell factor (SCF), erythropoietin (Epo), and granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Three to seven times more CFU-GM and BFU-E colonies were cultures from NLD BM-derived BMMNC than from BMMNC of recipients of TCD or UM BMT (p = 0.0001). There was no difference in colony number between recipients of UM and TCD BMT on day 28 posttransplant, however. Under G-CSF culture conditions, CFU-GM colonies from recipients of UM and TCD BMT were more susceptible (p < or = 0.05) to suppression by IFN-gamma at concentrations of 1 and 100 U/mL than NLD BMMNC-derived colonies. No other difference in IFN-gamma inhibition was detected among the three groups. Under G-CSF and GM-CSF culture conditions, maximal inhibition was obtained at TNF-alpha concentrations > 10 ng/mL. Although early posttransplant BMMNC was more sensitive to inhibition than NLD-derived BMMNC, overall, no difference in colony growth or percent of inhibition induced by TNF-alpha or IFN-gamma was observed between recipients of unmodified and T. cell-depleted transplants. In this series, two recipients of TCD BM and one recipient of UM BMT developed graft failure; no distinct pattern of colony growth or colony inhibition was evident for those patients. The optimized in vitro conditions and specific cytokines used in this study do not indicate any quantitative or qualitative differences in the hematopoietic progenitors present in recipients of unmodified and T cell-depleted bone marrow early posttransplant to explain an increased risk of graft failure following a T cell-depleted BMT compared to an unmodified BMT.     

The kidney triggers graft-versus-host disease in experimental combined transplantation of kidney and stem cell-enriched peripheral leukocytes

As a preclinical step to human studies with combined stem cell-enriched peripheral leukocytes and organ transplantation from the same donor, a series of studies in rats was undertaken. These studies indicated that Lewis rats infused intravenously with major histocompatibility complex-incompatible (from Brown-Norway rats), stem cell-enriched peripheral leukocyte preparation alone never developed graft-versus-host disease (GHVD). However, GVHD invariably manifested in all animals a few days after the kidney was transplanted in rats that had been previously primed with stem cell-enriched peripheral leukocytes from the same kidney donor strain. GVHD was prevented by substituting the crude preparation of stem cell-enriched peripheral leukocytes with a purified preparation that was almost completely free of T lymphocytes. However, in these latter experiments all rats rejected their kidney graft within 10 days from the surgery. In rats previously given the crude stem cell-enriched peripheral leukocyte preparation, perioperative administration of the fusion protein CTLA4lg also prevented GVHD and prolonged kidney graft survival up to 106 to 175 days. By contrast, animals with kidney transplants, which were given CTLA4lg without stem cells, rejected their grafts within 35 days. All together, these findings may possibly contribute to the creation of rationally designed strategies of combining organ and bone marrow from the same donor to enhance mixed chimerism and prolong survival after organ transplantation.     

Murine gamma/delta-expressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens

T cells with antidonor specificities have been isolated from human recipients experiencing graft rejection after allogeneic bone marrow transplantation (BMT). Partial T-cell depletion of unrelated BM grafts with an anti- T-cell receptor (TCR) monoclonal antibody (MoAb) directed against the TCR alpha/beta heterodimer have shown that the incidence of graft-versus-host disease is low and that the incidence of durable engraftment is high. These studies suggest either that the number of residual TCR alpha/beta+ cells was sufficient to permit alloengraftment or that the preservation of cells other than TCR alpha/beta+ cells was beneficial for engraftment. With respect to the latter, one such candidate cell is the TCR gamma/delta+ T cell. Because no studies have specifically examined whether TCR gamma/delta+ cells might be capable of eliminating BM-derived hematopoietic cells, we established a new graft rejection model system in which transgenic (Tg) H-2d mice (termed G8), known to express gamma/delta heterodimers on high proportion of peripheral T cells, were used as BMT recipients. These Tg TCR gamma/delta+ cells respond vigorously to target cells that express the nonclassical major histocompatibility complex (MHC) class lb region gene products encoded in H-2T region of H-2T(b)+ strains. G8 Tg mice were used as recipients for C57BL/6 (B6: H-2(b); H-2T(b)) T-cell-depleted (TCD) donor BM. We show that G8 Tg (H-2(d), H-2T(d)) mice are potent mediators of B6 BM graft rejection and that the rejection process was inhibited by anti-TCR gamma/delta MoAbs. In contrast, BM from a B6 congenic strain that expresses the H-2T(a) allele, B6.A-Tl(a)/BoyEg, was readily accepted, suggesting that H-2T antigens on repopulating donor BM cells are the targets of host graft rejecting T cells that express the TCR gamma/delta heterodimer. PB chimerism studies were performed at > or = 1.5 months post-BMT using TCD BM from severe combined immunodeficient allogeneic donors, which is highly susceptible to rejection by the host. The addition of donor G8 TCR gamma/delta+ cells to TCD donor BM was shown to significantly increase alloengraftment in B6 recipients. These results show that (1) host TCR gamma/delta+ cells can reject repopulating donor cells, presumably by responding to nonclassical MHC class lb gene products expressed on BM-derived hematopoietic progenitor cells; and (2) donor TCR gamma/delta+ cells can facilitate the alloengraftment of rigorously TCD donor BM.     

Interleukin-12 prevents severe acute graft-versus-host disease (GVHD) and GVHD-associated immune dysfunction in a fully major histocompatibility complex haplotype-mismatched murine bone marrow transplantation model

BACKGROUND: We have recently reported that interleukin (IL)-12 prevents acute graft-versus-host disease (GVHD)-induced mortality in a full major histocompatibility complex- plus multiple minor antigen-mismatched A/J-->B10 bone marrow transplantation (BMT) model. Because most patients have access to a haploidentical, one haplotype-mismatched donor, we have now investigated the protective effect of IL-12 against GVHD and GVHD-associated immune dysfunction in a haploidentical CBD2F1 (H2kxd) --> B6D2F1 (H2bxd) strain combination. METHODS: GVHD was induced by injecting CBD2F1 marrow and spleen cells into lethally irradiated B6D2F1 mice. RESULTS: In untreated control mice, GVHD resulted in 87% mortality by day 8 after BMT, with no survivors beyond day 17. Treatment with a single injection of IL-12 on the day of BMT led to 87% long-term survival, with no significant weight loss, diarrhea or GVHD skin changes. The majority of T cells recovering in these mice showed the CD62L+, CD44low, CD45RBhigh naive phenotype. These T cells showed specific tolerance to both host and donor histocompatibility antigens, but normal anti-third party (H2s) alloresponses in vitro. B-cell proliferative responses to lipopolysaccharide were also normal in IL-12-protected mice. Moreover, normal negative selection of thymocytes bearing T cell receptors with Vbeta that recognize endogenous superantigens was observed among CD4+CD8- thymocytes, indicating a lack of GVHD-associated thymic selection abnormalities in IL-12-protected allogeneic BMT recipients. CONCLUSIONS: IL-12 provides permanent protection against an otherwise severe, rapidly lethal GVHD, with no clinical manifestations of chronic GVHD, immunosuppression or autoimmune features, in a full major histocompatibilty complex haplotype-mismatched murine BMT model.     

Prevention of marrow graft rejection without induction of graft-versus-host disease by a cytotoxic T-cell clone that recognizes recipient alloantigens

In allogeneic marrow transplantation, donor T cells that recognize recipient alloantigens prevent rejection but also cause graft-versus-host disease (GVHD). To evaluate whether the ability to prevent marrow graft rejection could be dissociated from the ability to cause GVHD, we generated a panel of four different CD8 cytotoxic T-lymphocyte clones specific for H2(d) alloantigens. Three of the clones caused no overt toxicity when as many as 20 x 10(6) cells were infused intravenously into irradiated H2(d)-positive recipients, and one clone caused acute lethal toxicity within 1 to 3 days after transferring 10 x 10(6) cells into H2(d)-positive recipients. One clone that did not cause toxicity was able to prevent rejection of (C57BL/6J x C3H/HeJ)F1 marrow in 800 cGy-irradiated (BALB/cJ x C57BL/6J)F1 recipients without causing GVHD. Large numbers of cells and exogenously administered interleukin-2 were required to prevent rejection. These results with different CD8 clones suggest that GVHD and prevention of rejection could be separable effects mediated by distinct populations of donor T cells that recognize recipient alloantigens.     

Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vbeta6(+) and Vbeta3(+)) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-gamma (IFN-gamma) to host antigens in vitro. These in vitro responses correlated with increased IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.     

Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier

We have investigated the in vivo administration of nonmitogenic anti-CD3F(ab')2 fragments for the prevention of lethal graft-vs-host disease (GVHD) in irradiated recipients of fully allogeneic bone marrow cells plus splenocyte (BMS) inocula. Recipients of anti-CD3F(ab')2 fragments administered for 1 mo post-bone marrow transplantation (BMT) had 100% survival without clinical or histopathological evidence of GVHD. Controls given saline injections succumbed by 39 days post-BMT. Similar results were obtained in groups of recipient mice given BMS in which T cells were depleted by in vitro anti-Thy-1.2 plus C' treatment. Further studies were undertaken to define mechanistic differences in the two approaches. Using Ly-5 congenic sources of donor bone marrow and spleen, we determined that anti-CD3F(ab')2 fragments induced TCR modulation and T cell depletion. Mature splenic-derived CD4+ cells were depleted to a greater extent than CD8+ cells. Early post-BMT, recipients receiving injections with control saline had the highest number of CD4+ and CD8+ cells (which may cause GVHD) followed by recipients of anti-CD3F(ab')2 fragments, with the fewest CD8+ cells observed in the anti-Thy-1.2 + C' treated group. CD3+CD4-CD8- cells (which may suppress GVHD generation) were present in higher numbers early post-BMT in recipients given anti-CD3F(ab')2 fragments as compared to recipients given anti-Thy-1.2 + C'-treated BMS. In long term survivors, a mononuclear T cell containing infiltrate without evidence of destruction was observed in sites of GVHD (lung and liver), consistent with a "Quilty" effect, which was not observed in either of the other two groups. Although survivors were tolerant of donor skin grafts and rejected third party grafts, recipients given anti-CD3F(ab')2 fragments but not anti-Thy-1.2 + C'-treated BMS had vigorous anti-host proliferative responses. These results demonstrate that although in vitro anti-Thy-1.2 + C' treatment of BMS (which is highly depletionary) and in vivo administration of anti-CD3F(ab')2 fragments (which is modulatory and less depletionary) are both effective strategies for GVHD, the cellular events involved in achieving GVHD prevention are indeed different.     

Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease

Allogeneic bone marrow transplantation is limited by the availability of suitable marrow donors and risk of graft-versus-host disease (GVHD) and opportunistic infection. In an attempt to ameliorate these limitations, umbilical cord blood has been postulated as an alternative source of allogeneic haemopoietic stem cells for transplantation. From September, 1994, umbilical cord blood from sibling donors has been used to reconstitute haemapoiesis in 44 children with acquired or congenital lympho-haemapoietic disorders, neuroblastoma, or metabolic diseases. Patients who had HLA-identical and HLA-1 antigen disparate grafts, had a probability of engraftment at 50 days after transplantation of 85%. No patient had late graft failure. The probability of grade II-IV GVHD at 100 days was 3% and the probability of chronic GVHD at one year was 6%. With a median follow-up of 1.6 years, the probability of survival for recipients of HLA-identical or HLA-1 antigen disparate grafts is 72%. We conclude that umbilical cord blood is a sufficient source of transplantable haemopoietic stem cells for children with HLA-identical or HLA-1 antigen disparate sibling donors with very low risk of acute or extensive chronic GVHD. The feasibility of umbilical-cord-blood transplantation with HLA-2 and HLA-3 antigen disparate sibling donors remains to be determined.     

Genetic recombination (+) RNA viruses

Graft-versus-host disease (GVHD) remains the principal complication limiting the wider application of allogeneic bone marrow transplantation (BMT). Advances in basic immunology during the last decade have demonstrated how interactions between immunologically competent cells are governed by cytokines, and much recent research has focused on the roles of these mediators in the pathogenesis of acute GVHD. This article reviews current evidence that dysregulated cytokine production can be considered a cascade of sequential monocyte and T-cell activation that is responsible for many of the manifestations of acute GVHD. We suggest that cytokine dysregulation can be conceptualized in three phases. Phase 1 is initiated by the conditioning of the host, which induces inflammatory processes in recipient tissues. Donor T-cell activation by host alloantigens and subsequent cytokine secretion in phase 2 is facilitated by the consequences of phase 1. The T-cell-derived cytokines of phase 2 activate distal inflammatory mediators, which, in synergy with T- and NK-cell-mediated cytotoxicity, produce the systemic morbidity of GVHD-associated immunosuppression in phase 3. Data from both experimental and clinical studies involving cytokines and their blockade in the prevention or treatment of GVHD are reviewed.