M. F. Martelli

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.

Haploidentical 'megadose' CD34+ cell transplants for patients with acute leukemia.

The two workshops on Haploidentical Stem Cell Transplantation were held in Italy and in the USA to communicate the achievements to date in this field. In the past decade, the histoincompatibility barrier, which was responsible for a high incidence of rejection in extensively T cell-depleted transplants, has successfully been overcome. There is no doubt that the turning point in the history of the T cell-depleted mismatched transplant was the infusion of a megadose of stem cells, a principle which had been amply demonstrated in animal models. To date, the results obtained show that remarkable progress has been made in several areas. Full donor-type engraftment is successful and sustained in over 90% of recipients, when megadoses of highly purified CD34+ cells are given after highly immunoand myelo-ablative conditioning regimens containing total body irradiation plus thiotepa, fludarabine and anti-thymocyte globulin (ATG) or based on chemotherapy alone (mainly in children). Graft-versus-host disease is almost fully prevented when using CD34+ cell selection devices. Following such procedures, the graft is extensively depleted of T cells, so that the mean T cell content does not exceed 1–2 × 104 cells/kg body weight. It is worth noting that the inclusion of ATG or OKT3 in conditioning regimens also contributes in reducing the incidence of graft-versus-host disease, because it exerts an in vivo T cell depletion of the inoculum. Furthermore, efficient purification of the graft has the corollary of reducing B lymphocyte contamination, thus almost completely preventing B lymphoproliferative disorders. Growing evidence suggests that cells within the CD34+ cell population are endowed with marked veto activity. The term ’veto’ relates to the ability of cells to neutralize cytotoxic T lymphocyte precursors (CTLp) directed against their antigens. When purified CD34+ cells are added to bulk mixed lymphocyte cultures, they suppress the development of CTLp into mature CTL against matched stimulators but not against stimulators from a third party. This effect can be exhibited only if the cells are added within the first 48 h of mixed leukocyte reaction (MLR). Therefore, the veto effect of these cells seems to be directed against CTLp, but does not affect differentiated anti-donor CTL. Preliminary data suggest that the veto activity of human CD34+ cells, similarly to other veto cells described in the literature, is mediated by apoptosis (Gur et al,

Haploidentical hematopoietic stem cell transplantation: state of art

For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2- or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present overview of the 7th symposium on haplidentical transplantation that took place at the Weizmann Institute on February 2014, shows how these obstacles to successful transplantation can now be overcome. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for rebuilding immunity, reducing leukemia relapse and improving survival after transplantation. In addition, new modalities for immune tolerance induction following nonmyeloablative conditioning are discussed, showing new options for treatment of elderly patients who cannot tolerate myeloablative conditioning protocols, as well as novel strategies for immune tolerance and chimerism induction as a platform for cell therapy and organ transplantation.

Optimizing a photoallodepletion protocol for adoptive immunotherapy after haploidentical SCT

In adults, one-haplotype-mismatched haematopoietic SCT (haploidentical HSCT) is associated with slow immune recovery due to decaying thymic function and extensive T-cell depletion of the graft. Although essential for preventing GVHD, T-cell depletion underlies the major reasons for transplant failure: leukemia relapse and infections, with infection-related mortality accounting for about 40% of non-leukemic deaths. Adoptive T-cell therapy would be helpful for these patients but to administer it without causing GVHD, alloreactive T cells need to be eliminated from donor T lymphocytes before infusion. In a preclinical study, to address this problem, we determined the efficacy of photodynamic purging of alloreactive T cells, by investigating combinations of parameters in order to achieve maximum allodepletion, preservation of T-regulatory cells and of pathogen and leukemia-specific T-cell responses in donor-vs-recipient MLR. We also needed to identify an optimal method to quantify the Ag-specific T-cell repertoires. Optimal procedures were identified. In particular, we compared limiting-dilution analyses (LDA) of proliferating T cells with H3-thymidine incorporation by bulk T cells and with flow cytometry CD25 expression, which is accepted as a T-cell activation marker. This study demonstrated that LDA is a reliable, predictable and sensitive method for measuring alloreactive, pathogen- and leukemia-specific T-cell frequencies.

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.