Andrea Velardi

Treatment of High-Risk Acute Leukemia with T-Cell–Depleted Stem Cells from Related Donors with One Fully Mismatched HLA Haplotype

BACKGROUNDnIn this study we tried to achieve successful transplantation in patients with acute leukemia with the use of hematopoietic stem cells from donors who shared only one HLA haplotype with the recipient (a full-haplotype mismatch). To prevent graft failure, large doses of T-cell-depleted hematopoietic stem cells were transplanted after a conditioning regimen of enhanced myeloablation and immunosuppression was administered to the recipient.nnnMETHODSnForty-three patients with high-risk acute leukemia who were scheduled for transplantation received total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. The graft consisted of peripheral-blood progenitor cells that had been mobilized in the donor with recombinant granulocyte colony-stimulating factor and also, in 28 cases, bone marrow. Bone marrow from the donor was depleted of T lymphocytes by processing with soybean agglutinin and E-rosetting. T-cell depletion of peripheral-blood mononuclear cells was achieved by E-rosetting followed by positive selection of CD34+ cells. No post-transplantation prophylaxis against graft-versus-host disease (GVHD) was administered.nnnRESULTSnIn all the patients, full donor-type engraftment was achieved. In none of the patients who could be evaluated did acute or chronic GVHD develop. Regimen-related toxicity was minimal. Eleven of the 23 patients with acute lymphoblastic leukemia had a relapse, as did 2 of the 20 patients with acute myeloid leukemia. Transplantation-related mortality was 40 percent. After a median follow-up of 18 months (range, 8 to 30), 12 of the 43 patients were alive and free of disease. All surviving patients had a good quality of life.nnnCONCLUSIONSnThe main limitations of transplantation of bone marrow from donors who are matched with the recipient for only one HLA haplotype GVHD and graft failure - can be overcome. Since most patients have a relative with one haplotype mismatch, advances in this method will increase the availability of hematopoietic-cell transplantation as curative therapy for acute leukemia.

The NOTCH1/CD39 axis: a Treg trip-switch for GvHD.

Regulatory T cells (Tregs) suppress alloimmune reaction such as graft versus host disease (GvHD)1 and promote tolerance induction to allogeneic organ transplants.2 In high-risk acute leukaemia patients undergoing full-haplotype mismatched transplantation we demonstrated that adoptive immunotherapy with Tregs conventional T cells (Tcons) almost completely prevented acute and chronic GvHD, favoured post-transplant immunological reconstitution and was associated with a powerful graft-versus-leukaemia (GvL) effect.3, 4, 5 Interestingly, GvHD severity and mortality was markedly reduced by inactivation of NOTCH signalling in donor T cells by means of humanised antibodies and conditional genetic models.6, 7, 8 The present study attempted to unravel the connection between Tregs and NOTCH signalling in Tcons for GvHD prevention. We discovered that NOTCH1 downregulation on Tcons is a new Treg mechanism of action and showed that Tregs use the CD39 pathway to modulate NOTCH1 expression on Tcons.

Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes.

The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components. No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).

Next generation HLA-haploidentical HSCT.

Relapse is still the major cause of failure of allogeneic stem cell transplantation in high-risk acute leukemia patients. Indeed, whoever the donor and whatever the transplantation strategy, post-transplant relapse rates are ~30%, which is hardly satisfactory. The present phase 2 study analyzed the impact of adoptive immunotherapy with naturally occurring FoxP3+ T-regulatory cells (2 × 106 per kg) and conventional T lymphocytes (1 × 106 per kg) on prevention of GvHD and leukemia relapse in 43 high-risk adults undergoing full-haplotype mismatched transplantation without any post-transplant immunosuppression. Ninety-five percent of patients achieved full-donor type engraftment. Only 6/41 patients (15%) developed ⩾grade II acute GvHD. Specific CD4+ and CD8+ for opportunistic pathogens emerged significantly earlier than after standard T-cell-depleted haplo-transplantation. The probability of disease-free survival was 0.56. At a median follow-up of 46 months (range 18–65 months), only 2/41 evaluable patients have relapsed. The cumulative incidence of relapse was significantly lower than in historical controls (0.05 vs 0.21; P=0.03). These results demonstrate that the immunosuppressive potential of Tregs can be used to suppress GvHD without loss of the benefits of GvL activity. Humanized murine models provided insights into the mechanisms underlying separation of GvL from GvHD.

58 O - Successful engraftment of t-cell-depleted HLA-incompatible transplants

Since March 1993, 48 patients (mean age 22 years, range 2–51) with high-risk or advanced stage leukemia (14 AML, 31 ALL, 3 CML) have been transplanted. 26 were in hematological remission (5 CR I, 20 CRxa0≥xa0II. 2nd CP of CML) and 22 (11 AML, 9 ALL. 2 BT) in chento-resistant relapse at the time of transplant. All donors were HLA-haploidentical “three loci” incompatible family members. The first 36 patients received a conditioning regimen that included single TBI (8xa0Gy), 25xa0mg/kg rabbit ATG. 10xa0mg/kg thiotepa and 100xa0mg/kg cyclophosphamide (Cy). In the last 12 Cy was replaced by fludarabine (40xa0mg/m2/day for 6 days) and thiotepa increased to 13xa0mg/kg. T-cell-depletion of the bone marrow and the PBPCs by the soybean agglutinin and E-rosetting technique was the sole prophylaxis for GvffD in the first 36 patients, while a CD34-selection of E-rosetted PBPCs bas been used for the last 12 cases. 46 patients engrafted. GvHD occurred in 6; 9 re1apsed. 18 survive. 16 event-free at a median follow. Up of 12 months (range 1–33) The details of the clinical data will be presented.