Adoptive Immunotherapy with Tregs Prevents GvHD and Favours Immune Reconstitution After HLA Haploidentical Transplants for Hematological Malignancies.

Abstract 4

Transplantation of large numbers of highly purified CD34+ cells from haploidentical relatives is a viable strategy for the cure of acute leukaemia at high risk of relapse (Aversa et al., NEJM 1998; JCO 2005). As extensive T cell depletion is required to prevent GvHD, the very narrow T cell repertoire in the inoculum delays recovery of immune response against pathogens, leading to a high incidence of infection-related deaths. Thus the key challenge is to improve immune recovery by administering allogeneic donor T cells without causing GvHD. Preclinical studies demonstrated that freshly isolated or ex vivo expanded T regulatory cells (Tregs) could be used to control GvHD following bone marrow transplantation. The present phase I/II clinical trial evaluated the impact of early infusion of freshly isolated donor CD4/CD25+ Tregs, followed by an inoculum consisting of donor mature T cells (Tcons) and positively immunoselected CD34+ cells, on GvHD prevention and immunological reconstitution. Twenty-two patients (10 male; 12 female; median age 40.5, range, 21 to 60) with AML (n=17; 8 in CR1 at high risk, 7 in ≥CR2 and 2 in relapse), ALL (n=4; 3 in CR1; 1 in relapse) and 1 with high grade NHL in relapse were enrolled from September 2008 onwards. The conditioning regimen consisted of 8Gy single fraction TBI, thiotepa (4 mg/kg×2), fludarabine (40mg/m²×4), and cyclophosphamide (35 mg/kg×2). All patients received CD4/CD25⁺ GMP immunoselected Tregs (CliniMACS, Miltenyi Biotec) (21/22 2×10⁶/kg bw; 1/22 1×10⁶/kg bw). Three days later they received positively immunoselected CD34+ cells (median 8.2, range 5.0-19.1) together with Tcons (16/22 1×10⁶/kg bw; 4/22 0.5 ×10⁶/kg bw; 2/22 did not receive Tcons). Immunoselected CD4/CD25⁺ Tregs (purity 91.5±4.5) consisted of CD25^high 25.6%±11.2; CD25^int 57.4%±5.9; CD25^low 8.5%±6; FoxP3 64%±1; CD127 14.9%±13.7 (mean±SD). As suggested by in vitro immunosuppressive assays and by immunophenotypic analysis, the contaminating cells in the Treg fraction were 50% of the CD25^int and 100% of the CD25^low, so that the infused Tregs:Tcons ratio was established at 1:1.5. No post-transplant prophylaxis against GvHD was used. 20/22 patients engrafted. Neutrophils reached 1×10⁹/L at a median of 15 days (range, 11 to 39 days). Platelets reached 25×10⁹/L and 50×10⁹/L at median of 13 and 15 days, respectively (range, 11 to 48 days, and 13 to 60 days). All engrafted patients showed persistent full donor-type chimerism in peripheral blood and bone marrow. Strikingly, no GvHD was observed in 17/20 valuable patients, 2/20 developed grade I cutaneous self-limited untreated GvHD and 1/20 developed grade III GvHD. This patient had received the fewest Tregs. Six patients died (1 bacterial sepsis, 2 VOD, 1 fungal pneumonia, 1 CNS aspergillosis and 1 GvHD/systemic toxoplasmosis). In contrast with our previous experience, the speed of immune recovery was enhanced. The CD4 and the CD8 counts reached, respectively, 50/μL medianly on days 34 (range, 19 to 63 days) and 24 (range, 15 to 87); 100/μL medianly on days 47 (range, 28 to 100 days) and 34 (range, 19 to 95); 200/μL on days 70 (range, 41 to 146 days) and 61 (range, 21 to 95). We also observed a rapid development of a wide T-cell repertoire and detection of high frequencies of specific CD4+ and CD8+ for opportunistic pathogens such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved (Ruggeri et al., Science 2002). In conclusion, in the setting of haploidentical transplantation infusion of freshly purified Tregs makes administration of high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution. Treg-based cellular therapy may represent an innovative strategy to improve the outcome of haploidentical transplants.