Interleukin-15 administration increases graft-versus-tumor activity in recipients of haploidentical hematopoietic SCT.

Utilizing a clinically relevant haploidentical (HI) murine transplant model, lethally irradiated B6D2F1 (H2K(b/d)) mice were transplanted with T cell-depleted (TCD) BM from B6CBAF1 (H2K(b/k)) mice. We found that administration of IL-15 significantly increases the numbers of CD8+ T and natural killer (NK) cells in spleen and BM after transplantion without GVHD. Graft-versus-tumor (GVT) potency of the graft was evaluated upon tumor challenge using P815 tumor cells (H2(d)). IL-15 administration without T-cell infusion did not result in any survival improvement. However, IL-15 in combination with very low-dose T-cell infusion (1 × 10(4)) significantly increased GVT activity and improved survival in recipients of HI hematopoietic SCT (HSCT). This effect was observed when IL-15 was given at a later time point, rather than immediately following transplantation. IL-15 administration also specifically increased slow-proliferative CD8+ T-cell proliferation and IFN-γ secretion in CD8+ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester)-labeled HI T-cell infusion, whereas there was no effect on CD4+ T-cell proliferation, suggesting the critical effect of IL-15 on CD8+ T-cell homeostasis in HI host. We conclude that IL-15 can be used for enhancing antileukemia effect of HI-HSCT, which requires presence of donor-derived T cells.

Haploidentical hematopoietic SCT increases graft-versus-tumor effect against renal cell carcinoma.

Allogeneic hematopoietic SCT (HSCT) has been shown to be an effective treatment option for advanced renal cell cancer (RCC). However, tumor resistance/relapse remains as the main post transplant issue. Therefore, enhancing graft-versus-tumor (GVT) activity without increasing GVHD is critical for improving the outcome of HSCT. We explored the GVT effect of haploidentical-SCT (haplo-SCT) against RCC in murine models. Lethally irradiated CB6F1 (H2K(b/d)) recipients were transplanted with T-cell-depleted BM cells from B6CBAF1 (H2K(b/k)) mice. Haplo-SCT combined with a low-dose haploidentical (HI) T-cell infusion (1 × 10(5)) successfully provided GVT activity without incurring GVHD. This effect elicited murine RCC growth control and consequently displayed a comparative survival advantage of haplo-SCT recipients when compared with MHC-matched (B6D2F1CB6F1) and parent-F1 (B6CB6F1) transplant recipients. Recipients of haplo-SCT had an increase in donor-derived splenic T-cell numbers, T-cell proliferation and IFN-γ-secreting donor-derived T-cells, a critical aspect for anti-tumor activity. The splenocytes from B6CBAF1 mice had a higher cytotoxicity against RENCA cells than the splenocytes from B6 and B6D2F1 donors after tumor challenge. These findings suggest that haplo-SCT might be an innovative immunotherapeutic platform for solid tumors, particularly for renal cell carcinoma.

Adenovirus hexon T-cell epitope is recognized by most adults and is restricted by HLA DP4, the most common class II allele.

The immunogenicity of adenovirus (Ad) vectors is enhanced by virus-specific memory immune responses present in most individuals as a result of past exposure to these ubiquitous pathogens. We previously identified the first human T-cell epitope from the major capsid protein hexon, H910-924, and found that it is highly conserved among different Ad serotypes. Memory/effector T-cell responses to H910-924 were detected in 14 of 18 (78%) healthy adults by an interferon-gamma ELISPOT assay. Hexon peptide-specific CD4 T-cell lines were generated from three HLA-typed donors and analyzed using a panel of HLA homozygous B-cell lines and monoclonal antibodies to HLA class II loci. These studies reveal that the hexon epitope is restricted by HLA DP4, a class II allele present in 75% of the population. Analysis of overlapping peptides and peptides with single residue mutations identified a HLA DP4-binding motif. Additionally, antibodies to the hexon peptide were detected in all donor sera by dot blot assay and ELISA. Therefore, most individuals exhibit both memory B- and T-cell responses to this highly conserved epitope on hexon, an obligate component of all Ad vectors, including 'gutted' vectors. These data suggest that current strategies for the use of Ad gene therapy vectors will not evade memory immune responses to Ad.

Amifostine and autologous hematopoietic stem cell support of escalating-dose melphalan: a phase I study.

This study was conducted to define a new maximum tolerated dose and the dose-limiting toxicity (DLT) of melphalan and autologous hematopoietic stem cell transplantation (AHSCT) when used with the cytoprotective agent amifostine. Fifty-eight patients with various types of malignancy who were ineligible for higher-priority AHSCT protocols were entered on a phase I study of escalating doses of melphalan beginning at 220 mg/m(2) and advancing by 20 mg/m(2) increments in planned cohorts of 4 to 8 patients until severe regimen-related toxicity (RRT) was encountered. In all patients, amifostine 740 mg/m(2) was given on 2 occasions before the first melphalan dose (ie, 24 hours before and again 15 minutes before). AHSCT was given 24 hours after the first melphalan dose. Melphalan was given in doses up to and including 300 mg/m(2). Hematologic depression was profound, although it was rapidly and equally reversible at all melphalan doses. Although mucosal RRT was substantial, it was not the DLT, and some patients given the highest melphalan doses (ie, 300 mg/m(2)) did not develop mucosal RRT. The DLT was not clearly defined. Cardiac toxicity in the form of atrial fibrillation occurred in 3 of 36 patients treated with melphalan doses >/=280 mg/m(2) and was deemed fatal in 1 patient given melphalan 300 mg/m(2). (Another patient with a known cardiomyopathy was given melphalan 220 mg/m(2) and died as a result of heart failure but did not have atrial fibrillation.) Another patient given melphalan 300 mg/m(2) died of hepatic necrosis. The maximum tolerated dose of melphalan in this setting was thus considered to be 280 mg/m(2), and 27 patients were given this dose without severe RRT. Moreover, 38 patients were evaluable for delayed toxicity related to RRT; none was noted. Tumor responses have been noted at all melphalan doses and in all diagnostic groups, and 21 patients are alive at median day +1121 (range, day +136 to day +1923), including 16 without evidence of disease progression at median day +1075 (range, day +509 to day +1638). Amifostine and AHSCT permit the safe use of melphalan 280 mg/m(2), an apparent increase over the dose of melphalan that can be safely administered with AHSCT but without amifostine. Further studies are needed not only to confirm these findings, but also to define the antitumor efficacy of this regimen. Finally, it may be possible to evaluate additional methods of further dose escalation of melphalan in this setting.

Activity of single-agent melphalan 220-300 mg/m2 with amifostine cytoprotection and autologous hematopoietic stem cell support in non-Hodgkin and Hodgkin lymphoma.

High-dose chemotherapy using melphalan (HDMEL) is an important component of many conditioning regimens that are given before autologous hematopoietic stem cell transplantation (AHSCT). In contrast to the situation in myeloma, and to a lesser degree acute leukemia, only a very limited published experience exists with the use of HDMEL conditioning as a single agent in doses requiring AHSCT for lymphoma, both Hodgkin lymphoma (HL) and especially non-Hodgkin lymphoma (NHL). Thus, we report results of treating 26 lymphoma patients (22 with NHL and four with HL) with HDMEL 220-300 mg/m(2) plus amifostine (AF) cytoprotection and AHSCT as part of a phase I-II trial. Median age was 51 years (range 24-62 years); NHL histology was varied, but was aggressive (including transformed from indolent) in 19 patients, indolent in two patients and mantle cell in one. All 26 patients had been extensively treated; 11 were refractory to the immediate prior therapy on protocol entry and two had undergone prior AHSCT. All were deemed ineligible for other, 'first-line' AHSCT regimens. Of these 26 patients, 22 survived to initial tumor evaluation on D +100. At this time, 13 were in complete remission, including four patients who were in second CR before HDMEL+AF+AHSCT. Responses occurred at all HDMEL doses. Currently, seven patients are alive, including five without progression, with a median follow-up in these latter patients of D +1163 (range D +824 to D +1630); one of these patients had a nonmyeloablative allograft as consolidation on D +106. Conversely, 14 patients relapsed or progressed, including five who had previously achieved CR with the AHSCT procedure. Two patients, both with HL, remain alive after progression; one is in CR following salvage radiotherapy. Six patients died due to nonrelapse causes, including two NHL patients who died while in CR. We conclude that HDMEL+AF+AHSCT has significant single-agent activity in relapsed or refractory NHL and HL. This experience may be used as a starting point for subsequent dose escalation of HDMEL (probably with AF) in established combination regimens.

Mucosal injury in patients undergoing hematopoietic progenitor cell transplantation: new approaches to prophylaxis and treatment.

Hematopoietic progenitor cell transplantation is often associated with severe mucosal toxicity. The need for parenteral analgesics and parenteral nutrition are evidence of the severity of the problem in individual patients. However, the increased risk for systemic infection related to bacteremia associated with the breakdown of mucosal barriers is a significant cause of morbidity and mortality as well. There is a multitude of grading scales, demonstrating the lack of consensus among clinicians in this area. Multiple agents have been used prophylactically and therapeutically to address mucositis. While efforts have been less successful in the past, the advent of newer agents including amifostine, keratinocyte growth factor, transforming growth factor beta and interleukin-11 provides hope that this toxicity will be significantly decreased in the near future.

Nonmyeloablative conditioning allows for more rapid T-cell repertoire reconstitution following allogeneic matched unrelated bone marrow transplantation compared to myeloablative approaches.

Nonmyeloablative pretransplantation conditioning regimens have resulted in durable engraftment of allogeneic hematopoietic stem cells. In contrast to conventional fully myeloablative approaches, nonmyeloablative regimens are associated with a marked reduction of morbidity and mortality in the early posttransplantation period. Consequently, such reduced-intensity transplantation approaches can be used in older and frailer patients who would not tolerate fully ablative regimens. However, it is currently unclear how this radically different transplantation strategy affects immunological reconstitution. To address this important issue, we used T-cell receptor Vbeta spectratype analysis to examine the distribution of complementarity-determining region 3 (CDR3)-size bands as a measure of the complexity of the redeveloping T-cell repertoire. For this study, we evaluated the T-cell repertoire of 9 patients receiving T-cell replete, matched unrelated donor transplants following fully ablative or nonmyeloablative conditioning regimens. All 4 of the myeloablative and 2 of the nonmyeloablative patients received bone marrow, whereas 3 other nonmyeloablative patients received peripheral blood stem cells. The results of the spectratype analysis demonstrated that the patients who received nonmyeloablative conditioning together with either bone marrow or peripheral blood stem cells exhibited more rapid reconstitution of T-cell repertoire complexity.

Effect of T cell subset dose on outcome of T cell-depleted bone marrow transplantation.

T cell depletion using the murine monoclonal antibody (moAb) T10B9 is unique in that the T cell receptor (TCR)gamma delta bearing subset is relatively spared compared to the TCR alpha beta + subset. We evaluated the probabilities of engraftment, acute and chronic graft-versus-host disease (GVHD), relapse, and survival in 273 recipients of marrow T cell depleted using T10B9. Sixty-two patients received marrow from an HLA-identical sibling, 54 patients received partially matched related donor marrow and 157 patients received unrelated donor marrow. Limiting dilution analysis (LDA) was used to estimate total clonable T cell dose in all patients and a modified LDA using moAb-coated immunomagnetic beads was used to estimate TCR alpha beta +, CD4+, and CD8+ T cells in a subset of patients. TCR gamma delta + cell dose was estimated by flow cytometry. Cox proportional hazards regression models were used to assess the impact of T cell subset dose/kg of body weight on outcome. We found a significant association of TCR gamma delta + T cell dose (P = 0.004), but not TCR alpha beta + T cell dose or total clonable T cell dose, with the probability of engraftment. TCR alpha beta +, CD4+, CD8+ and total clonable T cell dose were significantly associated (P < 0.001) with the risks of grade 2-4 acute GVHD in recipients of marrow from related donors but not in recipients of marrow from unrelated donors. Neither total clonable T cell dose nor any T cell subset dose was found to be significantly associated with chronic GVHD, relapse or survival. The results confirm preclinical studies showing TCR gamma delta + T cells promote engraftment. TCR gamma delta + T cells are not associated with an increased risk of acute GVHD while TCR alpha beta T cells are associated with acute GVHD but not engraftment in recipients of marrow grafts T cell depleted using T10B9. These findings support the hypothesis that T cell subsets differentially contribute to alloengraftment and GVHD.

Association of donor-derived host-reactive cytolytic and helper T cells with outcome following alternative donor T cell-depleted bone marrow transplantation.

Recipients of marrow from alternative donors (unrelated or HLA-mismatched related donors) have a higher incidence of post-transplant complications compared to recipients of marrow from HLA-identical siblings. HLA disparity undetected by routine typing techniques has been suggested as one cause for the increased complications observed. Limiting dilution analysis (LDA) of donor-derived, host-reactive T cell precursor frequency prior to transplant has been proposed as a surrogate indicator of underlying HLA disparity which might be used to predict transplant outcome and aid in donor selection. We compared results of LDA of host-reactive IL-2 producing helper T lymphocytes (HTLp) and/or cytolytic T lymphocytes (CTLp) in 77 alternative marrow donor/recipient pairs with transplant outcome using univariate and multivariate analysis. All donor grafts were depleted ex vivo of mature T cells. Median patient age was 15 years (1-53). Donor selection was based on serologic typing for HLA class I and high resolution oligotyping for HLA-DRB1-DRB5, and HLA-DQB1. HLA-A and HLA-B locus antigens were retrospectively defined by one dimensional isoelectric focusing (IEF). Cox proportional hazards regression models were used to assess the impact of frequency and estimated cell dose of CTLp and HTLp on outcome. The CTLp assay was most sensitive to HLA-A and HLA-B locus disparity detected by serology or IEF. The HTLp assay detected class I disparity but was most strongly reactive in the presence of HLA-DRB1 disparity. Univariate analysis indicated a significant association of CTLp frequency and dose with severe (grades 3-4) acute graft-versus-host disease (GVHD), and of CTLp dose with chronic GVHD. Both assays were associated with survival and neither assay was associated with relapse. After adjustment for other significant covariables including known HLA disparity, the association of CTLp with acute GVHD was lost, however, CTLp frequency and CTLp dose remained associated with survival and HTLp frequency was associated with chronic GVHD. These data support the hypothesis that post-BMT complications may be influenced not only by T cell dose but by the alloreactive potential of the cells infused. LDA of alloreactive potential was useful in detecting disparity and in predicting survival or chronic GVHD in recipients of alternative donor TCD marrow grafts.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

A new limiting dilution culture system for the detection of T cell subsets in T cell-depleted marrow grafts.

T cell depletion (TCD) has been achieved using techniques that cause the inactivation, lysis, or physical removal of T cells from the donor marrow. The clinical results of TCD reflect, in part, the degree of TCD achieved and the subsets that are removed. To better evaluate TCD using the monoclonal antibody (mAb) T10B9, we have performed a series of flow cytometry and mAb blocking studies and have developed a new limiting dilution assay (LDA) that allows the detection of T cell subsets that survive treatment. T cell growth was stimulated with PHA, rIL-2, and irradiated feeder PBMC in a total well volume of 20 microliters. Growth was scored by microscopic examination on days 14-16 of incubation. Immunomagnetic beads coated with mAb were added to the growing wells and incubated, then the plates were fixed to a template of samarium cobalt magnets before washing away nonadherent cells. Wells in which > 50 cells bound > or = 2 beads were scored as positive. Flow cytometry indicated that T10B9 recognized all T cells, but complement-mediated lysis spared a significant proportion of the TCR gamma delta + subset. The epitope recognized by T10B9 on TCR gamma delta + cells appears to be differentially expressed compared with TCR alpha beta + T cells based on antibody blocking studies. In contrast to antibodies to CD3 epsilon, T10B9 binds less well to TCR gamma delta + cells, possibly resulting in incomplete complement-mediated lysis of this subset. The relative sparing of TCR gamma delta + cells was found in marrow and peripheral blood. Subset LDA confirmed that the TCR gamma delta + cells detected by flow cytometry were capable of growth and further showed that OKT3 did not spare TCR gamma delta + cells. The subset LDA should prove useful in helping to assess the role of T cell subsets in clinical events post-TCD bone marrow transplantation.

Unrelated donor bone marrow transplantation to treat severe aplastic anaemia in children and young adults.

Alternative donor bone marrow transplantation (BMT) to treat severe aplastic anaemia (SAA) in children and young adults has been complicated by high rates of graft rejection and severe graft-versus-host disease (GVHD). We hypothesized that increased immunosuppression combined with T-cell depletion of the marrow graft would enable successful use of unrelated donor BMT in this disease. Preconditioning consisted of cytosine arabinoside, cyclophosphamide, and total body irradiation (TBI). T-cell depletion was with the anti-CD3 antibody T10B9. GVHD prophylaxis consisted of cyclosporine A. 28 previously transfused patients were transplanted. Nine donor/recipient pairs were HLA matched. As of 1 January 1996, 15/28 (54%) patients are alive, transfusion independent and well with a range of follow-up of 13 months to 8 years (median 2.75 years). Fatalities include all three patients with nonengraftment and all three patients with grade III/IV GVHD. Other fatalities were due to infections or therapy-related toxicity. The incidence >or= grade II acute GVHD was 28%. These data show that in children with SAA who have failed immunosuppression, unrelated donor BMT offers a reasonable hope of long-term survival.

Human CD8 transgene regulation of HLA recognition by murine T cells.

A series of human CD8 transgenic (hCD8 Tg) mice with differential expression in the thymus and periphery were produced to investigate CD8 coreceptor regulation of repertoire selection and T cell responses. Expression of hCD8 markedly enhanced responses to both HLA class I molecules and hybrid A2/Kb molecules providing functional evidence for a second interaction site, outside of the alpha 3 domain, which is essential for optimal coreceptor function. Peripheral T cell expression of hCD8 was sufficient to augment responsiveness to HLA class I, as hCD8 Tg mice which lacked thymic expression responded as well as mice expressing hCD8 in the thymus and periphery. Both murine CD8+ and CD4+ T cells expressing hCD8 transgenes exhibited markedly enhanced responses to foreign HLA class I, revealing the ability of T cell receptor repertoires selected on either murine class I or class II to recognize human class I major histocompatibility complex (MHC). In contrast to recognition of foreign class I, thymic expression of hCD8 transgenes was absolutely required to enhance recognition of antigenic peptide restricted by self-HLA class I. Thus, our studies revealed disparate requirements for CD8 coreceptor expression in the thymus for selection of a T cell repertoire responsive to foreign MHC and to antigenic peptides bound to self-MHC, providing a novel demonstration of positive selection that is dependent on human CD8.

Use of unrelated marrow grafts compensates for reduced graft-versus-leukemia reactivity after T-cell-depleted allogeneic marrow transplantation for chronic myelogenous leukemia.

The effect of donor/recipient histocompatibility on relapse in patients receiving T-cell-depleted (TCD) grafts for chronic myelogenous leukemia (CML) was evaluated. Specifically, we sought to determine whether TCD results in an attenuation of the graft-versus-leukemia (GVL) effect on recipients of unrelated marrow grafts similar to that observed in HLA-identical sibling marrow transplantations. This question was addressed by comparative analysis of the relapse rates in marrow grafts who otherwise received identical preparative regimens and graft-versus-host disease (GVHD) prophylaxis schedules (T-cell depletion with T10B9 monoclonal antibody and complement plus posttransplant cyclosporine) and by serial molecular analyses using the polymerase chain reaction (PCR) to detect the bcr/abl RNA transcript in patients transplanted with unrelated donor grafts. Patients transplanted with advanced disease (accelerated phase or blast crisis) had equally high relapse rates, regardless of whether they received HLA-identical sibling (56%;95% confidence interval [CI], 29% to 82%) or unrelated marrow grafts (8%; 95% CI, 0% to 28%) had a significantly lower incidence of relapse than did patients transplanted with HLA-identical marrow grafts (47%; 95% CI, 23% to 71%; P = .002). Because all patients were similarly treated, these data indicate that the lower relapse rate in these unrelated patients was caused by an augmented GVL effect that was most likely attributable to increased HLA disparity between donor and recipient. The probability of developing both acute and chronic GVHD was significantly increased in chronic-phase recipients of unrelated marrow grafts, suggesting that the enhanced GVL effect was at least partly GVHD-associated. The lack of such a finding in advanced disease patient receiving unrelated marrow grafts raises the possibility that clinically significant GVL effect after TCD marrow transplantation was limited and confined to patients with more indolent disease. Serial PCR analyses for the presence of the bcr/abl RNA transcript showed that the vast majority of patients transplanted in chronic phase with unrelated marrow grafts were persistently PCR-negative, indicating that the GVL effect was durable in these patients. Most of these patients were observed to become PCR negative within 1 to 2 months after transplantation, showing that early eradication of leukemia was possible with TCD marrow grafts. This study shows that the use of unrelated marrow grafts compensates for reduced GVL reactivity associated with TCD in patients transplanted for CML. Furthermore, these data indicate that, in selected patient populations with CML, TCD can be used to reduce GVHD without a commensurate compromise in the GVL effect.

Therapeutic plasma exchange does not appear to be effective in the management of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome following bone marrow transplantation.

Recognition of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) following BMT has increased in recent years. The pathogenesis and etiology may be related to endothelial cell damage secondary to irradiation and/or CsA. Optimal management of this condition remains unclear. Due to similarity between this syndrome and classical TTP, patients with TTP/HUS following BMT are commonly treated with therapeutic plasma exchange (TPE). We describe our experience with 9 such patients who were treated with TPE (8 cases) and immunoadsorption with a Staphylococcal Protein A column (1 case). The exchanges were done with fresh frozen plasma and/or cryoprecipitate-depleted frozen plasma. Out of 8 patients treated with TPE, 6 died within 2 months of TPE due to secondary infections, metabolic disturbances and progression of TTP/HUS. Of these 6 patients, 5 had no hematological response, while 1 had hematological improvement. Two patients are alive 4 and 3 years later, however, they had shown only minimal hematological response at the end of 28 and 20 TPE, respectively. Their renal function remains stable but severely reduced. The ninth patient who received Staphylococcal Protein A column treatment died within 5 days of treatment without hematological improvement. Thus, in contrast to its effectiveness in classical TTP, TPE does not appear to be as effective in the management of well established TTP/HUS following BMT.