High Risk Of Graft Rejection Research Articles

High-risk corneal allografts and why they lose their immune privilege

Corneal allografts are routinely performed without HLA typing or systemic immunosuppressive drugs. However, certain conditions create high risks for immune rejection. This review discusses recent insights into the mechanisms that rob the corneal allograft of its immune privilege. Studies in mice have revealed that stimuli that induce new blood vessel growth in the cornea also elicit proliferation of lymph vessels. Lymph vessels facilitate migration of antigen-presenting cells to regional lymph nodes in which they induce alloimmune responses. The presence of blood vessels in the corneal graft bed creates a unique chemokine milieu that stimulates recruitment of sensitized lymphocytes into the corneal allograft. Other data indicate that although corneal allograft survival is closely associated with Foxp3 expression in CD4+CD25+Foxp3+ T regulatory cells (Tregs), reduced expression of Foxp3 in Tregs creates a high risk for graft rejection. Recent evidence indicates that allergic diseases have a profound impact on the immune response and produce a dramatic increase in corneal allograft rejection. Understanding the underlying mechanisms that create 'high-risk' hosts may provide important therapeutic targets for restoring immune privilege of corneal allografts and enhancing their survival.

HLA-matched sibling transplantation with BM and CD34+-purified PBSCs in adult patients with high-risk severe aplastic anemia to overcome graft rejection without an increase in GVHD

The transplantation of a large number of stem cells can overcome graft rejection but with the increased risk of GVHD. In this study, we analyzed the outcome of 32 adult patients with acquired severe aplastic anemia (SAA) who were at a high risk for graft rejection, including multiple transfusions (median 147 units, range 20-680) and long disease duration (median 67 months, range 3-347), and who had received both BM and CD34(+)-purified PBSCs from an HLA-matched sibling donor to reduce graft rejection. T cells in PBSCs were depleted using a magnetic-activated cell sorting method (CliniMACS system). Conditioning regimens consisted largely of CY and antithymocyte globulin (ATG) with fludarabine (FLU) or procarbazine (PCB). With a median follow-up of 89 months, the 8-year probability of survival was 87.5%. Neutrophils and plts promptly recovered, and none of the patients developed graft failure. The cumulative incidences of acute and chronic GVHD were 9.4 and 18.0%, respectively. Sustained engraftment and excellent survival without an apparent increase in the rate of GVHD in high-risk patients using the current approach showed that high-dose SCT with both BM and CD34(+)-purified PBSCs may yield better outcomes in heavily transfused and/or allo-immunized patients with SAA.

Treosulfan-based conditioning regimen in a second matched unrelated peripheral blood stem cell transplantation for a pediatric patient with CGD and invasive aspergillosis, who experienced initial graft failure after RIC

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by a defect of phagocyte NADPH-oxidase and characterized by severe, recurrent bacterial and fungal infections. Invasive aspergillosis (IA) is the leading cause of mortality in patients with CGD. We report the case of a 3-year-old boy with CGD, who developed IA despite antifungal prophylaxis. His treatment consisted of a 10-month-long multi-drug antifungal therapy, together with surgery, but these did not cause any substantial clinical improvement. BMT in high-risk patients with CGD remains a challenge due to both, higher risk of graft rejection and inflammatory flare in the course of immune recovery. Our patient rejected the first matched unrelated donor (MUD) allograft after RIC regimen recommended by the EBMT Inborn Errors Working Party for high-risk patients. After treosulfan-based conditioning and second MUD peripheral blood stem cell transplantation both, full reconstitution of the granulocytic series and complete recovery from IA, were achieved.

HEMATOPOIETIC STEM CELL TRANSPLANTATION IN THALASSEMIA AND SICKLE CELL DISEASE. EXPERIENCE OF MEDITERRANEAN INSTITUTE OF HEMATOLOGY IN A MULTI-ETHNIC POPULATION.

Hematopoietic stem cell transplantation (HSCT) still remains the only definitive cure currently available for patients with thalassemia and sickle cell anemia. Results of transplant in thalassemia and in sickle cell anemia have steadily improved over the last two decades due to improvements in preventive strategies, and effective control of transplant-related complications. From 2004 through 2009, 145 consecutive patients with thalassemia and sickle cell anemia, ethnically heterogeneous from Mediterranean and Middle East countries, were given HSCT in the International Center for Transplantation in Thalassemia and Sickle Cella Anemia in Rome. This experience is characterized by two peculiarities: patients were ethnically very heterogeneous and the vast majority of these patients were not regularly transfesed/chelated and therefore were highly sensitized due to RBC transfusions without leukodepletion filters. Consequently, they could have a high risk of graft rejection as a result of sensitization to HLA antigens. The Rome experience of SCT in patients with thalassemia and sickle cell anemia confirmed the results obtained in Pesaro, and most importantly showed the reproducibility of these results in other centers.

PANEL REACTIVE ANTIBODY IN THALASSEMIC SERUM INHIBITS PROLIFERATION AND DIFFERENTIATION OF CORD BLOOD CD34+ CELLS IN VITRO

Thalassemic children are at a high risk of graft rejection in cord blood transplantation. To investigate this possible mechanism, the authors evaluated the effect of panel reactive antibody on the growth of CD34+ cells in vitro. On semisolid medium, CD34+ cells derived from cord blood were incubated with thalassemic serum, and colony-forming units were counted after 10 days of culture. After incubation with serum-specific panel reactive antibody, profound decreases were found in the numbers of CFU-GM, CFU-GEMM and BFU-E compared with controls. The results indicated that serum-specific panel reactive antibody might have an apparent inhibition effect on proliferation and differentiation of cord blood CD34+ cells.

The perspectives of treatment of liver insufficiency by stem cells

The insufficiency of liver functions remains one of the major causes of death. The liver transplantation is the most effective method for treating severe liver diseases. The shortage of donor organs and high risk of graft rejection are the main problems for liver transplantation. Stem cells and isolated hepatocytes are the alternative means for repopulating liver after various injuries instead of liver transplantation. This review analyses achievements in therapy of liver insufficiency by means of stem cells in model experiments on animals as well as in clinical practice and also perspectives of employment of stem cells for treatment of liver insufficiency.

Overcoming Graft Rejection in Adult Patients with High-Risk Severe Aplastic Anemia Using Supplemental CD34+-Enriched PBSC in Combination with BM.

The transplantation of a large number of stem cells can overcome the graft rejection with the pitfall of an increased incidence of graft-versus host disease (GVHD). We analyzed the clinical outcome of adult patients with severe aplastic anemia (SAA) at high risk for graft rejection who received a transplant with both bone marrow (BM) and CD34+-enriched peripheral blood stem cells (PBSC) to decrease the graft rejection. Between December 1999 and April 2006, 31 consecutive adult patients with SAA who were multi-transfused (more than 40 units of red blood cells ± platelets) and/or had a 3 years or longer period prior to transplant were investigated. The median age of patients was 39 years (range, 21–52) and 19 (61.3%) were women. The median interval from diagnosis to transplant was 62 months (range, 2–347). The median number of transfusions before transplant was 144 units (range, 20–680). Twenty-three (74.2%) patients received a prior immunosuppressive treatment and 3 (9.7%) prior allogeneic transplants. All patients received both BM and CD34+-enriched PBSC from sibling donors. PBSC were collected on days 5 and 6 of G-CSF (10μg/kg/day), and T cells were depleted using magnetic cell sorting (CliniMACS system, Miltenyi Biotec, Germany). Marrow harvesting was performed 48 hours after the last leukapheresis. Conditioning regimens consisted of cyclophosphamide/antithymocyte globulin + procarbazine (n=19) or fludarbine (n=9). Other 3 patients with a history of prior transplant received irradiatin based conditining regimens. GVHD prophylaxis consisted of cyclosporin and short-term methotrexate. Patients were transplanted with a median mononuclear cell dose of 1.35 (range, 0.43–4.05)×108/kg of body weight. The median CD34+ cell counts in the CD34+-enriched PBSC and unpurged BM were 7.99 (range 2.26–23.49)×106/kg and 4.41 (range, 1.34–15)×107/kg, respectively. With a median follow-up of 66 months, the 5-year probability of survival was 87.1%. The median time to neutrophils ≥0.5×109/L and platelet ≥20×109/L without platelet transfusions were 13 days (range, 10–32) and 16 days (range, 11–40), respectively. No patient developed graft failure. The cumulative incidence of acute GVHD over grade II and chronic GVHD were 9.7% and 16.1%, respectively. In conclusion, high rates of durable engraftment and excellent long-term survival were achieved by our approach which seems to play a role in overcoming the sensitization to histocompatibility antigens. Remarkably, the T-cell depleted PBSC apparently did not increase the incidence of GVHD. The high dose stem cell component therapy may be feasible for high-risk SAA adult patients.

Forced Expression of the “IY” Mutant Inosine Monophosphate Dehydrogenase II Results in Physiologically Significant Resistance to Mycophenolic Acid In Vitro.

Nonmyeloablative conditioning regimens followed by allogeneic hematopoietic cell transplantation (HSCT) rely on pre and post transplant immunosuppression to overcome host-versus-graft reactions and control graft-versus-host-disease (GVHD). Patients with less than 50% of donor T cells by day 14 after transplant have a higher risk of graft rejection and relapse than those with more than 50% donor chimerism (Blood 2004; 104(8):2254). However, with increasing donor T-cell chimerism the risk of acute GVHD increases. Immunosuppression may inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells. Genetically modifying T cells with a drug resistance gene is one way to confer protection from the effects of the immunosuppressive agents, retain donor T cell function, and increase donor T cell chimerism by day 14. Mycophenolate mofetil (MMF) has been used in the post transplant setting to decrease the severity of GVHD and reduce the risk of host vs. graft reaction leading to graft failure. The active metabolite of MMF, mycophenolic acid (MPA), inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH). The type II isoform of IMPDH is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells which lack the salvage pathway for guanosine synthesis. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthetic activity ex vivo (BBA 2002; 1594:27, JBC. 1997; 272:961, BBA. 1994; 1217:156). We have generated six mutants of IMPDH II based on in vitro data and x-ray crystallography of the MPA binding site and transduced them into a human T cell line (Jurkat), a canine large granular lymphocyte cell line (CLGL-90) and a fibroblast cell line (NIH3T3) using oncoretroviral transduction. The mutants include: S276A (Amut), S276F (Fmut), T333I + S351Y (IYmut), L30F + Q277R (FRmut), Q277R + A462T (RTmut), and S276Y (Ymut). We generated bicistronic (IRES containing) retroviral vectors that expressed either HA-tagged wild type human IMPDH II (WT) or an HA-tagged IMPDH mutant and EGFP. We show that forced expression of WT will increase the MPA IC50 3 fold (IC50=3uM) compared to non-transduced cells. Forced expression of only the IY mutant in all cell lines tested was able to transfer resistance to MPA in excess of WT (IC50>50uM). This is clinically relevant since MPA concentrations measured after transplant in patients on MMF are around 8uM (Blood. 2005; 106(13):4381). To show that resistance was related to the expression of the mutant enzyme we correlated GFP by FACS with HA-IMPDH II expression by western blot analysis, and correlated this with resistance to MPA in vitro. This was true regardless of selection in MPA or transduction and cell sorting for >90% GFP positive cells. Analysis of IMPDH activity in lysates of transduced cells is currently underway. This is the first report where forced expression only of the IY mutant provides a clear enhanced resistance to MPA in vitro compared to cells over expressing wild type IMPDH II. Currently, we are generating bicistronic maloney based oncoretroviral vectors which carry a suicide gene (CD34-TK) and the IMPDH-HA IY mutant which will allow us to pharmacologically modulate the level of donor T cells after allogeneic stem cell transplantation.

Reduced-Intensity Bone Marrow Transplantation for Heavily Transfused Patients with Severe Aplastic Anemia - Japanese Single Center Experience.

Background: Bone marrow transplantation (BMT) is a curative therapy for severe aplastic anemia (SAA). Outcome of BMT can be negatively affected when there is organ damage such as heart failure, liver dysfunction and diabetes mellitus after multiple transfusions in patients with long-standing SAA. Multiple transfusions also sensitize SAA patients with alloantigens and place them at high risk of graft rejection; it is an important problem after BMT for patients with multiply transfused SAA. We investigated the use of the combination of fludarabine monophosphate (FLU), cyclophosphamide (CY) and anti-thymocyte globulin (ATG) as the conditioning regimen in heavily transfused SAA patients.Methods: Four patients with heavily transfused SAA were treated with allogeneic BMT from unrelated and sibling donor. They were treated with a reduced-intensity conditioning regimen that included FLU (120 mg/m2), CY (1,200 mg/m2) and ATG (horse 40 mg/kg, rabbit 15 mg/kg).Results: The regimen was well tolerated, and engraftment rapidly occurred with some therapy-related toxicity encountered. Chimerism analysis on day 30 after transplant showed reconstitution with 100% donor cells in three patients. Among these three patients, two patients showed late graft rejection. At this time of reporting, only one patient is alive with complete donor chimerism.Conclusion: The combination of FLU/CY/ATG was well tolerated also in heavily transfused SAA patients: problems such as graft rejection remain.

Pivotal Role of Complement-Fixing HLA Alloantibodies in Presensitized Kidney Allograft Recipients

Recipient presensitization represents a major risk factor for kidney allograft loss. Complement fixation may be a critical attribute of deleterious alloantibodies. We investigated clinical impact of complement-fixing HLA presensitization employing [C4d]FlowPRA, a novel assay permitting selective detection of HLA panel reactive antibody (PRA)-triggered C4 complement split product deposition. A cohort of 338 kidney transplants was evaluated for presensitization applying [C4d]FlowPRA together with [IgG]FlowPRA and complement-dependent cytotoxicity (CDC)-PRA. Analysis of HLA class I alloreactivities revealed a high incidence of C4d-positive graft dysfunction in [IgG]FlowPRA(+)/[C4d]FlowPRA(+) and [IgG]FlowPRA(+)/[C4d]FlowPRA(-) recipients (23% and 22% vs. 3% in [IgG]FlowPRA(-) patients). Only patients with complement-fixing HLA class I immunization had inferior graft survival [75% (3 years) vs. 91% and 89%, respectively (p=0.036)]. Despite frequent finding of capillary C4d deposition (28%), complement-fixing HLA class II immunization was not associated with inferior survival rates. This may have been due to reduction of clinical effects by intense immunosuppression in presensitized patients. Evaluating CDC, 29% of CDC-PRA(+)/[C4d]FlowPRA(+) recipients had C4d-positive graft dysfunction. For these patients 3-year graft survival was worst, followed by CDC-PRA(+)/[C4d]FlowPRA(-) and CDC-PRA(-) patients (76% vs. 81% vs. 90%, p=0.014). Results highlight a strong impact of complement-fixing HLA presensitization. Discerning complement-activating abilities of HLA alloantibodies, [C4d]FlowPRA may help identify recipients at particularly high risk for graft rejection and loss.

Overcoming graft rejection in heavily transfused and allo‐immunised patients with bone marrow failure syndromes using fludarabine‐based haematopoietic cell transplantation

Allogeneic haematopoietic cell transplantation (HCT) can cure a variety of non-malignant haematological disorders. Although transplant outcomes for selected patients with severe aplastic anaemia (SAA) and paroxysmal nocturnal haemoglobinuria (PNH) have improved, older age, allo-immunisation from transfusions, prior immunosuppressive therapy and a prolonged time from diagnosis to transplantation are associated with worse outcome. Because of its potent immunosuppressive effects, we investigated a fludarabine-based non-myeloablative conditioning regimen in patients with transfusion-dependent non-malignant haematological disorders at increased risk for graft rejection with conventional transplant conditioning. Twenty-six patients with transfusion dependent/anti-thymocyte globulin (ATG)-refractory SAA, PNH or pure red cell aplasia underwent HCT from a human leucocyte antigen (HLA)-compatible relative. Transplant conditioning consisted of cyclophosphamide (120 mg/kg) and fludarabine (125 mg/m2) with or without ATG. Ciclosporine, alone or combined with mycophenolate mofetil or methotrexate, was used as graft-versus-host disease (GVHD) prophylaxis. All patients achieved durable engraftment and transfusion-independence. Twenty-four of 26 patients are alive at a median of 21 months following transplantation. Although a high cumulative incidence of acute (65% grades II-IV, 54% grades III-IV) and chronic GVHD (56%) was observed, only one patient died from transplant-related causes (cumulative incidence 7%). These data show that HCT following fludarabine-based non-myeloablative conditioning results in durable engraftment and excellent survival in SAA and PNH patients at high risk for graft rejection.

Bone Marrow Transplantation and Immunosuppressive Therapy in Children with Aplastic Anemia: Data from a Prospective Multinational Trial in Germany, Austria and Switzerland.

In children severe aplastic anemia is a rare, lifethreatening disease. Patients present with peripheral pancytopenia and hypoplastic bone marrow. The disease is sub-classified into a severe (PMNs >200/μl: SAA) and a very severe (PMNs <200/μl: vSAA) form. Children with vSAA are especially prone to die from infectious complications. Bone marrow transplantation (BMT) and immunosuppressive therapy (IST) are the two treatment options. However, most of the affected children lack a compatible sibling donor (MSD) and BMT from a matched unrelated donor (MUD) is still complicated by a high risk of graft rejection and graft-versus-host disease. A retrospective analysis based on the EBMT registry data showed in 1990 that in children with vSAA IST with anti-thymocyte-globulin (ATG) +/− androgens resulted in an inferior survival rate (37%) compared to SAA (56%), and BMT, even from MUD, was recommended for this group.We report the results of a prospective multicentre trial with a combined IST regimen of cyclosporin A (CSA) and ATG for treatment of SAA in children. By biological selection depending on the availability of a MSD, patients were assigned to either the BMT (n=67) or the IST group (n=146). 62 patients received BMT (except 5 patients whose parents refused) after a conditioning treatment with ATG and cyclophosphamide. 151 children (146 without a MSD, 5 with a MSD) were treated with combined IST with ATG (horse 0,75ml/kg BW for 8 days), CSA (5mg/kg BW, adjusted to blood levels), prednisolone (1–2mg/kg tapered until day 28) and in cases with PMN <500/μl with G-CSF (5μg/kg BW) in addition. Response to IST and survival was evaluated on day 112, at 6 months and every 6 months afterwards. Complete response (CR) was registered when haemoglobin reached normal values, platelets >100,000/μl and PMN >1,500/μl. The median follow-up at time of analysis was 50 months (range 0.85–116 months).97 vSAA patients received IST. In this formerly high-risk group the 5-year survival rate could be improved to 93% (95%-Confidence Interval (CI): 88%–98%). Surprisingly, SAA patients showed a significantly lower 5-year survival rate of 81% (95%-CI: 69%–93%%; p-value: 0.0266). Similarly, vSAA patients reached CR in 68% (95%-CI: 58% – 77%) of cases, compared to 45% in SAA patients (95%-CI: 31% – 59%, p-value: 0.009)(best response after one year). When treated with BMT, both the vSAA and the SAA patients reached comparable survival rates (vSAA 89%; 95%-CI: 80%–99% and SAA 96%; 95%-CI: 89%–100%).We conclude that in children without a MSD treatment with combined IST and G-CSF converts the former bad prognosis factor severe granulocytopenia into an indicator of better response and survival. Our findings strongly suggest that especially in children with vSAA the immune system plays the key role, and the per se healthy stem-cell compartment is able to compensate for the loss of stem cells as soon as the T-cell-mediated destruction is stopped. Therefore severe granulocytopenia may serve as a surrogate marker for an immune-mediated disease instead of a stem-cell disorder.

Fanconi Anemia: Contribution of Molecular Analyses to the Identification of Bone Marrow Graft Donors and the Study of Chimerism in Grafted Patients

We report on the effectiveness of molecular studies regarding Fanconi anemia (FA) for a better selection of bone marrow graft donors and for post-transplant follow up. Ten unrelated FA patients and their families were analyzed by microsatellite markers. In 9 cases, the cytogenetic investigation of potential human leukocyte antigen (HLA)-identical related donors was normal, and the molecular analyses confirmed that they were also either normal or heterozygous carriers. For 1 patient, cytogenetic analysis of an HLA-identical sibling donor yielded ambiguous results with a relatively high number of chromosomal breakages using cross-linking agents. However, genotyping of this potential donor demonstrated his heterozygous state. Nine patients have received allogeneic bone marrow transplantation from HLA-matched related donors. Microsatellite analysis showed complete chimerism (CC) in all cases. The median follow up was 54 months (range 8-144 months). One patient out of 9 with CC rejected her graft without prior detection of a transitional mixed chimerism. Among these patients, 1 died 25 months after the transplantation of a chronic graft-versus-host-disease (GVHD). We conclude that, when the cytogenetic studies are not conclusive, molecular analyses are crucial to distinguish heterozygous carriers from asymptomatic FA Tunisian patients. Molecular analyses also allowed the evaluation of hematopoietic chimerism after allogeneic bone marrow transplantation and might be of value to identify patients with a high risk for graft rejection.

Successful allogeneic hematopoietic stem cell transplantation using triple agent immunosuppression in severe aplastic anemia patients.

Graft rejection in patients with severe aplastic anemia (SAA) following allogeneic hematopoietic stem cell transplantation (HSCT) is strongly associated with a large number of prior transfusions and with prolonged disease duration before transplant. We retrospectively analyzed the outcomes and the factor affecting these multitransfused SAA patients, who had received triple agent immunosuppression and high doses of stem cells to overcome rejection. In total, 113 patients with SAA who had a median 16 months (range 1-216) of disease duration were transplanted using HLA-matched sibling donors after conditioning with cyclophosphamide (CY), procarbazine (PCB), and ATG. Graft failure occurred in 16 of the eligible 113 patients, and with a median follow-up of 30 months (range, 1-80), probability of overall rejection was 15%. Specifically, the multitransfused patients who received high doses of stem cells with T-cell depletion showed the lowest rejection rate, 5.6%, compared with 30.3% in multitransfused patients with bone marrow stem cells alone (P=0.0310). Disease duration (P=0.0338) and the number of infused CD34+cells (P=0.0101) were associated with a high risk of graft rejection on multivariate analysis. ABO mismatch and the number of CD34+ cells were significant factors in the incidence of acute graft-versus-host-disease (GVHD). The incidence of chronic GVHD among patients with sustained engraftment was 13/109 (11.9%). With the same follow-up period, probability of disease-free survival for the entire group of patients at 6 years was 89% and the only factor associated with long-term survival was rejection (P=0.0241). These results suggest that allogeneic HSCT conditioned with triple agent immunosuppression, and specifically with high-dose stem cell return is probably an effective treatment for successful engraftment in SAA patients with a high risk of rejection.

Systemic cyclosporin A in high risk penetrating keratoplasties: a case-control study

AIMTo examine the efficacy of systemic cyclosporin A (CSA) in preventing rejection and graft failure in high risk keratoplasty (PK).METHODSA retrospective case-control study with 49 patients in both the CSA...

Durable engraftment of major histocompatibility complex–incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide

AbstractTreatment of leukemia by myeloablative conditioning and transplantation of major histocompatibility complex (MHC)–mismatched stem cells is generally avoided because of the high risk of graft rejection or lethal graft-versus-host disease (GVHD). This study shows that MHC-incompatible cells can engraft stably after nonmyeloablative conditioning with immunosuppressive chemotherapy and low-dose total body irradiation (TBI). Long-term mixed hematopoietic chimerism, clonal deletion of donor-reactive T cells, and bidirectional cytotoxic T-cell tolerance were achieved by transplanting MHC-mismatched marrow cells into recipients conditioned with pretransplantation fludarabine or cyclophosphamide (Cy), 50 to 200 cGy TBI on day −1, and Cy 200 mg/kg intraperitoneally on day 3. In this model, long-term donor chimerism was proportional to the dose of TBI or donor marrow cells. Pretransplantation fludarabine and posttransplantation Cy were both required for alloengraftment, but the drugs had additional effects. For example, fludarabine sensitized host stem cells to the toxicity of TBI, because animals conditioned with both agents had higher chimerism than animals conditioned with TBI alone (P < .05). Also, posttransplantation Cy attenuated lethal and nonlethal GVH reactions, because F1 recipients of host-reactive, parental spleen cells survived longer (P < .05) and had lower donor cell chimerism (P < .01) if they received posttransplantation Cy than if they did not. Finally, delayed infusions of donor lymphocytes into mixed chimeras prolonged survival after leukemia challenge (P < .0001) without causing lethal GVHD. These results indicate that stable engraftment of MHC-incompatible cells can be induced after fludarabine-based, nonmyeloablative conditioning and that it serves as a platform for adoptive immunotherapy with donor lymphocyte infusions.

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients.

Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.

Total body irradiation: current indications

The choice of dose and fractionation for total body irradiation is made difficult by the large number of considerations to be taken into account. The outcome of bone marrow transplantation after total body irradiation can be understood in terms of tumour cell killing, engraftment, and normal tissue damage, each of these endpoints being influenced by irradiation-, disease-, transplant-, and patient-related factors. Interpretation of clinical data is further hampered by the overwhelming influence of logistic constraints, the small numbers of randomised studies, and the concomitant variations in total dose and fraction size or dose rate. So far, three cautious conclusions can be drawn in order to tentatively adapt the total body irradiation schedule to clinically-relevant situations. Firstly, the organs at risk for normal tissue damage (lung, liver, lens, kidney) are protected by delivering small doses per fraction at low dose rate. This suggests that, when toxicity is at stake (e.g., in children), fractionated irradiation should be preferred, provided that interfraction intervals are long enough. Secondly, fractionated irradiation should be avoided in case of T-cell depleted transplant, given the high risk of graft rejection in this setting. An alternative would be to increase total (or fractional) dose of fractionated total body irradiation, but this approach is likely to induce more normal tissue toxicity. Thirdly, clinical data have shown higher relapse rates in chronic myeloid leukaemia after fractionated or low dose rate total body irradiation, suggesting that fractionated irradiation should not be recommended, unless total (or fractional) dose is increased. Total body irradiation-containing regimens, primarily cyclophosphamide/total body irradiation, are either equivalent to or better than the chemotherapy-only regimens, primarily busulfan/cyclophosphamide. Busulfan/cyclophosphamide certainly represents a reasonable alternative, especially in patients who may not be eligible for total body irradiation because of prior irradiation to critical organs.

Bone Marrow Transplantation From Genetically HLA-Nonidentical Donors in Children With Fatal Inherited Disorders Excluding Severe Combined Immunodeficiencies: Use of Two Monoclonal Antibodies to Prevent Graft Rejection

For children with life-threatening inborn errors of metabolism without a matched related bone marrow donor, transplantation from an HLA genetically nonidentical donor is the only therapeutic option. To reduce the high risk of graft rejection in this setting without increasing the conditioning regimen, a protocol based on the infusion of an antiadhesion antibody directed against the CD11a (leukocyte function-associated antigen 1 [LFA-1]) molecule was performed by the European Bone Marrow Transplantation-European Society for Immunodeficiency group with promising results. To optimize engraftment, and thereby survival, further, the additional blockade of a second important leukocyte adhesion and signalization pathway mediated by the CD2 and LFA-3 interaction was attempted in a multicenter protocol conducted by the European Bone Marrow Transplantation-European Society for Immunodeficiency group. Results of this study (ie, engraftment and survival) were compared with a historical control group that received the anti-LFA-1 antibody alone. Factors that may have affected engraftment and survival were also considered in this study. Forty-four children with inborn errors, including inherited immunodeficiencies (excluding severe combined immunodeficiencies), Chédiak-Higashi syndrome, familial hemophagocytic lymphohistiocytosis, and malignant osteopetrosis, received bone marrow from HLA-nonidentical related donors or from HLA-identical unrelated donors at 13 European centers between August 1990 and June 1993. Bone marrow was depleted of T cells by use of either erythrocyte (E) rosetting or monoclonal antibodies (MoAbs) to prevent graft-versus-host disease. The conditioning regimen consisted of busulfan and cyclophosphamide for all patients plus etoposide for patients with osteopetrosis, familial hemophagocytic lymphohistiocytosis, and Chédiak-Higashi syndrome. Infusions of MoAbs specific for the CD11a and the CD2 molecules were started 4 and 3 days, respectively, before and continued through the first 10 and 11 days, respectively, after bone marrow transplantation (a total of 14 injections). The overall sustained engraftment rate was 69.8%, with full chimerism in 80.6% of patients and no late graft rejection with the use of two MoAbs versus 65.7% and 58.1%, respectively, in the control group, in which only one MoAb was infused. The overall actuarial survival rate with a functional graft was 40.9%, with a mean follow-up of 39.3 months with two MoAbs versus 37.8% with one. The engraftment rate was significantly influenced by the T-cell depletion method, with better results for recipients of E rosette- depleted marrow (78.6% vs 20% for Campath 1-M plus complement-depleted marrows). Graft-versus-host disease and the kinetics of immune reconstitution were similar in both groups. The overall engraftment rate and overall survival rate with engraftment in patients treated with anti-LFA-1 and anti-CD2 were similar to those in patients treated with anti-LFA-1 antibody alone. However, although the number of patients is too small to draw definitive conclusions, results from the combined use of the two MoAbs indicates a trend toward better engraftment and survival after infusion of E rosette-depleted marrow. Further improvement in survival would demand additional strategies to hasten immunologic recovery.

Outcome of Unrelated Donor Bone Marrow Transplantation in 40 Children With Hurler Syndrome

Long-term survival and improved neuropsychological function have occurred in selected children with Hurler syndrome (MPS I H) after successful engraftment with genotypically matched sibling bone marrow transplantation (BMT). However, because few children have HLA-identical siblings, the feasibility of unrelated donor (URD) BMT as a vehicle for adoptive enzyme therapy was evaluated in this retrospective study. Forty consecutive children (median, 1.7 years; range, 0.9 to 3.2 years) with MPS I H received high-dose chemotherapy with or without radiation followed by BMT between January 27, 1989 and May 13, 1994. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients are alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute graft-versus-host disease (GVHD) was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD BMT because of graft rejection or failure. Of the 20 survivors, 13 children have complete donor engraftment, two children have mixed chimeric grafts, and five children have autologous marrow recovery. The BM cell dose was correlated with both donor engraftment and survival. Thirteen of 27 evaluable patients were engrafted at 1 year following URD BMT. Neither T-lymphocyte depletion (TLD) of the bone marrow nor irradiation appeared to influence the likelihood of engraftment. Ten of 16 patients alive at 1 year who received a BM cell dose greater than or equal to 3.5 x 10(8) cells/kg engrafted, and 62% are estimated to be alive at 3 years. In contrast, only 3 of 11 patients receiving less than 3.5 x 10(8) cells/kg engrafted, and 24% are estimated to be alive at 3 years (P = .05). The mental developmental index (MDI) was assessed before BMT. Both baseline and post-BMT neuropsychological data were available for 11 engrafted survivors. Eight children with a baseline MDI greater than 70 have undergone URD BMT (median age, 1.5 years; range, 1.0 to 2.4 years). Of these, two children have had BMT too recently for developmental follow-up. Of the remaining six, none has shown any decline in age equivalent scores. Four children are acquiring skills at a pace equal to or slightly below their same age peers; two children have shown a plateau in learning or extreme slowing in their learning process. For children with a baseline MDI less than 70 (median age, 2.5 years; range, 0.9 to 2.9 years), post-BMT follow-up indicated that two children have shown deterioration in their developmental skills. The remaining three children are maintaining their skills and are adding to them at a highly variable rate. We conclude that MPS I H patients with a baseline MDI greater than 70 who are engrafted survivors following URD BMT can achieve a favorable long-term outcome and improved cognitive function. Future protocols must address the high risk of graft rejection or failure and the impact of GVHD in this patient population.