ALBERT

Description: Post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (AL) patients. Attempts to manipulate donor T cell alloreactivity to spare normal tissues while killing leukemic cells have been largely unsuccessful. In the search for strategies to separate the GvL effect from GVHD, we investigated the role of a thymic-derived CD4+CD25+ FoxP3+ regulatory T-cell subpopulation (Tregs) that physiologically helps maintain immunological self-tolerance and immune homeostasis. Evidence from murine bone marrow (BM) transplantation across major histocompatibility barriers showed co-infusion of conventional T lymphocytes (Tcons) with Tregs suppressed lethal GVHD without impairing Tcon activity against malignant diseases. In 69 high-risk AL patients who had received an HLA haploidentical T cell-depleted hematopoietic transplant and no post-transplant immunosuppressive GvHD prophylaxis, adoptive immunotherapy with donor Tregs (2 × 106/kg) and Tcons (1 × 106/kg) protected patients from GvHD (Di Ianni et al., Blood 2011) and largely prevented post-transplant leukemia relapse. In fact, only 5% of patients relapsed (Martelli et al., Blood 2014). However, also because such patients had advanced stage disease, TRM was still in the range of that of T cell-depleted haplo transplants, (i.e. 40%, Aversa et al., JCO 2005). A current cohort of AL patients was conditioned with total body irradiation (TBI) (8Gy as single dose TBI or 13.5 Gy as fractionated TBI), cyclophosphamide (30mg/kg), thiotepa (8mg/kg) and fludarabine (200mg/m2). To date, 24 high-risk AL patients (7 ALL, 17 AML) have been enrolled. Twenty-two of the 24 patients engrafted, one AML patient relapsed, 4/22 developed aGvHD (4/4 are alive) and one developed cGvHD, TRM was exceptionally low (Fig. 1). At a median follow up of 2 years, 19 patients are alive. Consequently, probabilities of leukemia-free and leukemia/cGVHD-free survivals are good (Fig. 1). Hypotheses have been put forward to explain the ability of Tregs to prevent of GvHD while preserving the GvL effect mediated by Tcons. However, the mechanism is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 BM homing receptor and preferentially localize to the BM while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010). Since Tregs that are recovered from peripheral blood and are used for adoptive immunotherapy are CD45RO+ and largely CxCR4 negative, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the BM combined with regulated T cell alloreactivity at the periphery. In order to verify such hypothesis, we infused NSG mice with human leukemia and human Tregs and Tcons. Mice that received leukemia and haploidentical Tcons (without Tregs) cleared leukemia but died of GvHD. T cells harvested from their BM, spleen and liver were predominantly CD8+ and displayed alloreactivity against leukemia. Mice that received leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+T cells (60%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against leukemia. In contrast, T cells harvested from BM were still predominantly CD8+ and still displayed alloreactivity against leukemia suggesting Tcons had retained their alloantigen recognition. Finally, in mice treated with Tregs alone, T cells were recovered only in the spleen and liver, they displayed a CD4+ phenotype and inhibited mixed lymphocyte reaction. No T cells were found in the BM. In contrast, when we infused human purified CD45RA+ naïve Tregs (that display the CXCR4 BM homing receptor), mice died of leukemia progression despite the co-infusion of large doses of Tcons. In the BM we isolated CD8+ T cells that displayed no ability to kill leukemia and CD4+ T cells that displayed regulatory function. Thus, naïve Tregs homed to the BM and blocked the GvL effect of T cons. When CXCR4 was blocked with an anti-CXCR4 antibody, naïve Tregs could not home to the BM and T cons killed leukemia. In conclusion, Treg-Tcon adoptive immunotherapy confines the graft-versus-host alloreaction to the hematopoietic system and, consequently, allows a GvL effect without GvHD. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect transplantation outcomes when the mother acts as donor for the child. Indeed, survival after T cell-depleted HLA haploidentical haematopoietic transplantation was improved using the mother as donor (vs all other family members) (Stern et al., Blood 112:2990, 2008; Kruchen et al., BMT 50:1367 2015). However, maternal donors were associated with increased incidence of GvHD and decreased survival after un-manipulated HLA haploidentical blood and marrow grafts (Wang Y et al., Blood 124:843, 2014). Patients and Methods A retrospective EBMT registry-based study was performed in a combined series of adult (n=333) and pediatric (n=105) patients with acute leukemia (AML=268, ALL=160, Mixed phenotype=10) who received transplant from one MHC mismatched family donor. Forty-four percent (233) of patients were in I or II complete remission (CR) at the time of transplant, 193 were in chemo-resistant relapse or in CR 〉 2. Median age was 32.7 (range: 0.67-70). Seventy-one percent of patients received ex-vivo T cell depleted transplants. Twenty-two patients were given bone marrow and peripheral blood stem cells, the others were given peripheral blood cells only. The mother was used as donor in 98 patients. Results Eighty patients developed acute GvHD ≥ grade II. At a median follow up of 58.7 months, 130 patients are alive without disease, 146 relapsed and 152 died of non-relapse mortality (NRM). Univariate analyses of factors influencing relapse-free survival (RFS) showed age, disease status at transplant, Karnofsky score and ex-vivo T cell depletion impacted significantly. As transplantation outcomes from family members other than mothers did not differ from one another, such transplants were combined for analyses. When compared with transplantation from all other family members (n=330), transplantation from mother donors (n=98) was associated with lower relapse incidence (RI) (28% vs 39%, P=0.06) and non-relapse mortality (NRM) (30% vs 40%, P=0.09) and, consequently, better relapse-free survival (RFS) (43% vs 21%, P 〈 0.001). Multivariate analyses showed transplantation from mother donors was an independent factor predicting better RFS (other donors vs mothers: HR: 1.42; CI: 1.01-2.00; P=0.04) and lower RI (other donors vs mothers: HR: 1.93; CI: 1.16-3.22; P = 0.01). In addition, transplantation in relapse (vs remission) predicted worse RFS (HR: 2.33, CI: 1.80-3.00, P 〈 0.001) such as ≥18 (vs 〈 18) adversely impacted on RFS (HR: 1.40, CI: 1.00-1.96, P=0.04). Discussion Our retrospective analyses in 438 HLA haploidentical hematopoietic transplants for acute leukemia patients (pediatric and adult) show that transplantation from mother donors, when compared with transplantation from any other family member, is an independent factor predicting better outcomes, i.e., better RFS and lower RI. Mothers should therefore be preferred when selecting an HLA haploidentical family donor. Further clinical and preclinical studies are needed to unveil the mysteries underlying mother-to-child immune interaction during pregnancy and its bearing on the reproductive success of the human species. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Description: Introduction and Aim: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is increasingly offered to patients with high-risk acute myeloid (AML) or lymphoid leukemia (ALL). Unfortunately, graft manipulation employed to overcome the HLA barrier significantly delays immune reconstitution, posing the patients at risk of infections. Accordingly, non-relapse mortality after haplo-HSCT clearly extends beyond day 100 post-transplant. Over the years, different approaches have been investigated to speed-up immune reconstitution. In the absence of validated immune biomarkers, it is however difficult to evaluate the clinical impact of accelerated immune reconstitution. The aim of this EBMT retrospective study is to explore immune-cell counts early after haplo-HSCT as predictive of its overall outcome. Methods and Patients: Among AML and ALL patients in the EBMT database who underwent haplo-HSCT in the period 2001-2012, criteria for study entry were survival beyond day 100 and availability of differential immune-cell counts (CD3+, CD4+, CD8+ T cells, CD19+ B cells, CD16+/CD56+ NK cells) within this period. Accordingly, statistical analysis was landmarked at day 100. Of 259 patients meeting these criteria (age 2-70, median 33), 67 (26%) were children. The underlying disease was AML in 162 cases (63%), while ALL in the remaining (including 5 cases of bi-phenotypic leukemia). Fifty-two percent of patients were transplanted in CR1. The stem-cell source was G-CSF mobilized peripheral blood in all but one patient (〉99%) and 171 received TBI (66%). The graft was manipulated in 199 patients (78%), including CD34-selection (50%), ex vivo T-cell depletion (15%) or both (13%). Female-to-male transplants were 68 (26%), while 204 (79%) recipients were CMV seropositive. Sustained hematopoietic engraftment was reached in 246 patients (95%) Results: The estimated overall survival at 2yrs was43%. The estimated cumulative incidence of death due to relapse was 33%, while that of death due to other causes was 35% (51% of those were infections) The occurrence of grade III-IV GVHD and of chronic GVHD was 9% and 18% (7% extensive), respectively. As expected, overall survival was better in children (62% vs 36%, P=0.002 by Log-rank), who clearly had a lower incidence of death due to causes other than relapse compared with adults (10% vs 37%, P=0.0001). Negative prognostic factors for overall survival were any disease state other than CR1 at time of transplantation (P=0.002) and CMV seropositivity (P=0.009). Type of leukemia, TBI or graft manipulation had no effect on the outcome. By day 100 post-transplant, patients reached the following median immune-cell counts: 100 CD3+ T cells (range 0-2576), 30 CD4+ T cells (0-1714), 48 CD8+ T cells (0-1880), 276 CD16+/CD56+ NK cells (18-3581), 21 CD19+ B cells (0-790). Importantly, CD3+ counts above the first quartile (1Q) of the entire data set (29 cells per microL) were significantly associated with a better overall survival (P=0.0005 by Log rank) and a lower incidence of death due to causes other than relapse (P=0.002 by Gray test). The same held true for CD8+ counts (1Q: 15 cells per microL; P=0.003 on overall survival; P=0.0004 on death due to other causes). CD4+ counts also showed similar correlations, but at higher values (above the median). None of the other immune-cell counts analyzed correlated with clinical outcome. Strikingly, when challenged in multivariate analysis taking into account age category, CMV seropositivity, graft manipulation and CR1 status at transplant, CD3+ and CD8+ counts above the 1Q adjusted to fit optimal cut-off points were still significantly associated with a better overall survival (P=0.006 and P=0.015, respectively), but only CD8+ values associated with a lesser risk of death due to causes other than relapse (P=0.026). Conversely, similarly adjusted median CD4+ counts failed to show any association. Conclusions: Contrary to what is generally accepted, these results indicate that an accelerated CD8+, but not CD4+, T cell reconstitution associates with a more favorable clinical outcome after haplo-HSCT, likely due to its protective role against opportunistic viral infections. Moreover, they suggest that yet to be validated CD8+ cut-off points, rather than the commonly used arbitrary value of 200 CD4+ T cells per microL, should be considered as surrogate biomarkers in clinical trials. Disclosures Bonini: MolMed S.p.A: Consultancy.