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Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day −4 followed by 1 × 106/kg donor conventional T cells on day −1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor–type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #{"type":"clinical-trial","attrs":{"text":"NCT03977103","term_id":"NCT03977103"}}NCT03977103.

Introduction Adoptive immunotherapy with CD4+CD25+FOXP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) has been employed after allogeneic hematopoietic stem cell transplantation (HSCT) to prevent Graft-versus-Host Disease (GvHD) while mantaining Graft-versus-Leukemia effect in the absence of any further immune suppressive GvHD prophylaxis (Di Ianni, Blood 2011; Martelli, Blood 2014). A recent study of age-adapted haploidentical HSCT with Treg/Tcon adoptive immunotherapy (Treg/Tcon haplo-HSCT) resulted in an unprecedented 75% chronic GvHD (cGvHD)/leukemia-free survival (CRFS) in acute myeloid leukemia patients. Acute GvHD (aGvHD) occured in 33% of patients and was the most frequent complication of the procedure (Pierini, Blood Advances 2021). Here, we analyzed clinical and histological characteristics of aGvHD and evaluated its impact on outcomes in patients who received Treg/Tcon haplo-HSCT. Methods We retrieved data of patients who underwent Treg/Tcon haplo-HSCT at Perugia University Hospital and who were evaluable for aGvHD from January 2015 until June 2021 (clinicaltrials.gov: NCT03977103). Histological features and lymphoid infiltration by immunohistochemistry were analyzed in gut samples of patients who received diagnostic colonscopy and biopsy for gastrointestinal symptoms. Results A total of 105 patients engrafted after Treg/Tcon haplo-HSCT. Mean age at transplant was 48 years (range 18-70). Acute leukemia was the most frequent diagnosis (78/105 myeloid, 23/105 lymphoblastic), 4 patients had myelodisplastic syndrome and 2 multiple myeloma. Twenty-seven (26%) patients had active disease at the time of HSCT. Fourteen (13%) patients received Treg/Tcon haplo-HSCT as a second HSCT procedure. Thirty-one (29%) patients developed grade II-IV aGvHD. Four patients had grade II aGvHD, 23 grade III, and 4 grade IV. Almost all patients (90%) presented gut involvement, while skin was involved in 64% of them and liver in 35%. To explain why gut symptoms such as diarrhea and abdominal pain were more frequently present than symptoms of skin or liver involvement, we evaluated 40 histological samples from 34 patients who received colonoscopy for suspected aGvHD. Twenty-two of them (65%) received a clinical diagnosis of aGvHD and pharmacological immune suppression was promptly started. Clinical diagnosis did not always match histological findings: biopsies were suggestive of aGvHD in 20/22 patients who required treatment, but also in 8/12 patients who did not. We found no difference in median CD3+ cell infiltration among patients who did not need immune suppressive treatments and patients with aGvHD (460 cells/mmq vs 446 cells/mmq). However, FOXP3+ Tregs preferentially infiltrated gut mucosa of patients who resolved clinical symptoms with no need of immune suppressive treatments (120 cells/mmq vs 59 cells/mmq, p = 0.008; FOXP3/CD3 ratio: 0.27 vs 0.18, p = 0.004). This observation suggested Treg defective homing to gut mucosa of aGvHD patients. Indeed, we found infused Tregs expressed lower levels of gut homing receptor α4β7 in comparison with Tcons (p< .001). Despite severe presentation at diagnosis, aGvHD resolved in the majority of patients (71%), and immune suppressive therapy could be completely withdrawn after a relatively short-course (median: 98 days, range 48-404). Sixteen/31 patients (52%) were steroid refractory and required further treatments after first-line (e.g. Ruxolitinib). Only two of them developed moderate/severe cGvHD and one is alive and off-therapy. Indeed, aGvHD diagnosis did not result in higher incidence of non-relapse mortality (cumulative incidence of 29% in aGvHD patients vs 27% in controls; p=non significant, ns, Fig.1A), relapse (13% vs 20%; p=ns, Fig.1B), or cGvHD (6.5% vs 3%; p=ns, Fig.1C). In fact, CRFS of aGvHD patients was similar to CRFS of patients that did not develop aGvHD (Fig. 1D, p=ns, median follow up of live patients: 1246 days). Conclusions Treg/Tcon haplo-HSCT is followed by low rates of relapse and cGvHD and results in good CRFS in high risk leukemia patients. aGvHD after Treg/Tcon haplo-HSCT preferentially involves the gut where Treg homing is defective. Strategies that enhance Treg homing in the gut (e.g. low-dose IL-2) may help reduce aGvHD after Treg/Tcon haplo-HSCT. In conclusion, despite aGvHD occurred in 29% of patients, it was responsive to treatments and it did not result in disease relapse or cGvHD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Since their discovery CD4+FOXP3+ regulatory T cells (Tregs) represented a promising tool to induce tolerance in allogeneic hematopoietic cell transplantation. Preclinical models proved that adoptive transfer of Tregs or the use of compounds that can favor their function in vivo are effective for prevention and treatment of graft-vs.-host disease (GvHD). Following these findings, Treg-based therapies have been employed in clinical trials. Adoptive immunotherapy with Tregs effectively prevents GvHD induced by alloreactive T cells in the setting of one HLA haplotype mismatched hematopoietic transplantation. The absence of post transplant pharmacologic immunosuppression unleashes T-cell mediated graft-vs.-tumor (GvT) effect, which results in an unprecedented, almost complete control of leukemia relapse in this setting. In the present review, we will report preclinical studies and clinical trials that demonstrate Treg ability to promote donor engraftment, protect from GvHD and improve GvT effect. We will also discuss new strategies to further enhance in vivo efficacy of Treg-based therapies.

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4+Foxp3+ regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most powerful therapy for patients with high risk of relapse. In spite of that, no matter the donor source or conditioning regimen used, leukemia relapse is still the leading cause of HSCT failure. In HLA-haploidentical HSCT, we recently applied a clinical protocol consisting of total body irradiation (TBI)-based conditioning regimen and a peripheral blood CD34+ cell graft combined with the adoptive transfer of naturally occurring regulatory T cells (Tregs) and conventional T cells (Tcons). No post-transplant pharmacologic GvHD prophylaxis was given. Such protocol was associated with low GvHD and relapse rate (Martelli et al., Blood 2014). To further reduce leukemia relapse in Treg/Tcon-based haploidentical HSCT (Treg/Tcon haplo-HSCT) we used high dose hyper-fractionated TBI (HF-TBI) in the conditioning regimen. We also extended Treg/Tcon haplo-HSCT to patients that are unfit (because of previous comorbidities) and/or too old to withstand high intensity regimens. In these patients the extra-hematologic toxicity of irradiation was reduced with the use of targeted total marrow and lymph node irradiation (TMLI). 40 patients with high risk acute leukemia (36 AML, 4 ALL) received Treg/Tcon haplo-HSCT. All but 3 patients were transplanted in complete remission. 12 younger patients (median age: 28, range: 20-43) received HF-TBI, while 28 older or unfit patients (59, 40-70) received TMLI in the conditioning regimen. HF-TBI (14.4 Gy) was administered in 12 fractions, 3 times a day for 4 days. TMLI was administered by means of Helical Tomotherapy HI-ART (9 fractions, 2 times a day for 4.5 days). Irradiation was followed by chemotherapy with Thiotepa, Fludarabine, and Cyclophosphamide. 2 × 106/kg freshly isolated CD4+CD25+FOXP3+ Tregs were transferred 4 days before the infusion of 1 × 106/kg Tcons and a mega-dose of CD34+ hematopoietic stem cells. No post-transplant pharmacologic GvHD prophylaxis was given. 38/40 patients engrafted. 12 (31%) developed aGvHD grade ³2 (10 are alive and off-therapy). 6 (16%) died because of transplant related complications (2 because of aGvHD, 2 infections, 1 veno-occlusive disease, 1 intracranial hemorrhage). Strikingly, despite the high risk diseases, no patient relapsed after a median follow up of 13 months (range 1-36, Fig. A). Further, only 1 patient developed cGvHD. Thus, cGvHD/Leukemia-free survival was 82% (Fig. B). Treg adoptive transfer allows for the safe infusion of an otherwise lethal dose of donor alloreactive Tcons in the absence of any other form of immune suppression. Our results demonstrate that the potent graft versus leukemia effect of Treg/Tcon adoptive transfer was boosted by high dose marrow irradiation. Thus, this study proves that the right combination of haploidentical Treg/Tcon immunotherapy plus a powerful conditioning regimen can fully eradicate leukemia.

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.

INTRODUCTION Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models. METHODS Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay. Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies. RESULTS We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p CONCLUSION The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD. Disclosures No relevant conflicts of interest to declare.

Background: Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Tregs) effectively prevents conventional T cell (Tcons) mediated Graft versus Host Disease (GvHD) while it does not impair graft-versus-leukemia effect in haploidentical hematopoietic cell transplantation (haplo-HCT). Moreover Treg immunotherapy promotes fast donor T cell recovery after transplant. Recent studies showed that mouse bone marrow (BM) Tregs localize in the hematopoietic stem cell (HSC) niche, where they contribute to HSCs maintenance and promote donor engraftment and B cell lymphopoiesis. We are investigating if human Tregs promote B cell reconstitution and immunity in preclinical models and in haplo-HCT patients. Methods: Human sample analysis: B cell reconstitution was analysed monthly by FACS in BM and peripheral blood (PB) samples from 66 patients who underwent either Treg/Tcon haplo-HCT (45 patients), or T-cell depleted haplo-HCT (8 patients) or haplo-HCT with post-transplant cyclophosphamide (PTCy, 13 patients). Diagnosis was acute leukemia in 52 patients, lymphoma in 11 and multiple myeloma in 3. PB total immunoglobulin (Ig), anti-Cytomegalovirus (CMV) IgM and CMV viremia were monitored. Humanized mouse model: donor derived human Tregs and purified CD34+ HSCs were co-infused in sublethally irradiated (2 Gy) immune-deficient NSG mice. Donor engraftment and B cell reconstitution were analysed by FACS and histology. Results: B cell reconstitution was faster after Treg/Tcon haplo-HCT when compared to other haplo-HCT protocols. B cell counts were higher in PB of patients that received Treg/Tcon haplo-HCT (p To further assess whether HSC-derived B cells were functional in patients that received Treg/Tcon haplo-HCT, we analysed total Ig production and specific responses to CMV reactivation. Post-transplant hypogammaglobulinemia was rapidly corrected in Treg/Tcon haplo-HCT patients. Total IgM were higher compared to other haplo-HCT protocols and reached normal levels by 3 months after transplant (96±155 mg/dL, Fig.1B). CMV reactivation rate was lower (47% vs 79%) and it occurred later after Treg/Tcon immunotherapy (51±19 vs 40±48 days). New production of anti-CMV specific IgM was documented in 43% of CMV seropositive patients 98±48 days after Treg/Tcon haplo-HCT, while anti-CMV specific IgM were almost undetectable after other haplo-HCT protocols within the first 6 months after transplant (Fig.1C). Such potent B cell responses coupled with T cell reconstitution resulted in a reduction of CMV second reactivations (Fig.1D p Conclusions: Adoptive transfer of human Tregs boosts donor HSC engraftment and facilitates the reconstitution of functional HSC-derived donor B cells. Such results suggest that Treg/Tcon haplo-HCT patients could be early vaccinated after transplant. Treg/Tcon immunotherapy promotes control of infections and B cell immunity in patients undergoing haplo-HCT. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemias (AML) with complex karyotype (≥ 3 abnormalities) and/or monosomy 7/del(7q) are a subset of AML with extremely poor prognosis. Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment, but relapse rate is high (most often above 50%) and negatively impacts on patients' survival. (Ciurea et al. Cancer 2018) Such poor results are even questioning the role of HSCT and rise the need of new transplant procedures for these patients. To this end, we are performing a clinical trial of haploidentical HSCT with adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) in the absence of post-transplant pharmacologic immune suppression (Treg-Tcon haplo-HSCT). Such approach allows for an extremely low relapse rate in patients with high-risk acute leukemias transplanted in complete remission. (Martelli et al. Blood 2014) In this study we analyzed the outcome of all the patients that were referred to our center from January 1st 2007 to May 31st 2018 with a diagnosis of AML with complex karyotype and/or monosomy 7/del(7q) and that were fit for receiving an induction treatment. 49 patients were included. Median age at diagnosis was 52; 22 were female, 27 were male. 20/49 had chromosome 7 abnormalities (isolated in 12 patients; in a complex karyotype in 8 patients). 11 patients received a hypomethylating agent as first line treatment, while the other 38 patients received high-dose chemotherapy. We performed 30 HSCT procedures in 25 patients; 5 patients received a second transplant (4 after disease relapse and 1 after rejection). 6 underwent a HLA-matched HSCT, 6 a T-depleted haploidentical HSCT, and 18 a Treg-Tcon haplo-HSCT. Median age at transplant was 42 with a median follow up of 40 months. Regarding disease status at transplant, 26 HSCT were performed in patients that were in 1st or 2nd hematological remission and 4 in patients with refractory disease. Cytogenetic analysis (karyotype and/or FISH analysis) revealed the presence of abnormal clones in 11 patients and multiparametric flow cytometry analysis detected minimal residual disease in 16 patients. Thus, most of the patients (17/30, 57%) were transplanted with detectable disease. 15/49 patients were disease-free and alive at the time of the analysis, all of them after receiving HSCT (HSCT vs Non-HSCT, p In conclusion, our results not only confirm that HSCT is the only potentially curative treatment for AML with complex karyotype and/or chromosome 7 involvement, but also demonstrate that Treg-Tcon haplo-HSCT allows for a strong antileukemic effect, the only needed for a long term control of such unfavorable AMLs with high disease burden at transplant. While it is necessary to confirm these results in a larger cohort of patients, Treg-Tcon haplo-HSCT could represent the best transplant option for this subset of very high-risk AML patients. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

In patients with acute leukemia (AL) at high risk of relapse, because of unfavorable cytogenetics, molecular markers and disease status, the most powerful therapy is hematopoietic stem cell transplantation. In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented graft-versus-host-disease (GvHD) (Di Ianni et al., Blood 2011) and was associated with low transplant-related mortality (TRM) and relapse rate (Martelli et al., Blood 2014) in patients with AL conditioned with single-dose TBI. However, patients that are heavily pretreated (e.g., because of multiple induction chemotherapy cycles, previous autologous or allogeneic transplant, previous irradiation, infections, etc.) and elderly patients are not expected to withstand a high-intensity TBI-based conditioning regimen. For these patients with an indication to transplant we, therefore, designed a chemotherapy only-based conditioning regimen. We enrolled 24 patients with high risk AL (19 AML and 5 ALL, median age: 46, range 21-64). Twenty-two patients were transplanted in CR and 2 in relapse. For 5 patients this was the second transplant. Patients received a conditioning regimen that included thiotepa (10 mg/Kg on days -10 and -9), fludarabine (200 mg/sqm from day -10 to -7) and melphalan (80-120 mg/sqm on day -5). Fiftheen patients also received ATG (6 mg/kg on days -24 and -23). Seven patients also received cyclophosphamide (20 mg/Kg on days -8 and -7) (and no ATG). Two patients received only Treosulfan (36 gr/sqm from day -8 to -6), fludarabine and thiotepa. On day -4 patients were received an infusion of 2x10e6/kg nTregs (93% ± 8 SD FoxP3+ cells). On day 0 they received CD34+ cells (mean 9.5x10e6/kg ±3.1 SD) and Tcons (1x10e6/kg). No post-transplant pharmacologic immune suppressive GvHD prophylaxis was given. Twenty-two of the 24 patients engrafted. CD4+ and CD8+ cell counts reached 200/µL on days 56 (range 30-100) and 35 (range 20-120) respectively with a wide T-cell repertoire as analyzed by Spectratyping. Eight of the 22 engrafted patients (36%) developed ≥ grade II acute GvHD. Three of the 8 patients died, 3 are alive with cGvHD and 2 are currently under immunosuppressive therapy. The incidence of TRM was 32% (7/22). Only one patient (in relapse at the time of transplant) relapsed. At a median follow-up of 24 months (range 5-44), 62% of the patients are alive and leukemia-free. The mechanisms underlying Treg suppression of GVHD with no loss of GVL activity were clarified by our murine transplant models. NSG mice received human myeloid or lymphoblastic leukemia and HLA mismatched Tregs/Tcons. Mice that received leukemia and Tcons (without Tregs) cleared leukemia but died of GvHD. Human T cells harvested from their bone marrow, liver and gut displayed a CD8+ phenotype and mediated alloreactivity against human leukemia. Mice treated with Tregs alone died of leukemia. No human T cells were found in their bone morrow. Human CD4+ T cells were instead recovered in the liver and gut. They had retained their regulatory function as they inhibited mixed lymphocyte reaction. Mice that received human leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. Human T cells harvested from liver and gut were composed of CD4+/FOXP3- T cells (60%) and CD8+ T cells (40%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against human leukemia. In contrast, human T cells harvested from bone marrow were still predominantly CD8+ and still displayed potent alloreactivity against human leukemia. In conclusion, peripheral blood Tregs used for adoptive immunotherapy were predominantly CD45RO+ (and negative for the bone marrow homing receptor CXCR4) and, when infused in mice, migrated to GvHD target organs (i.e., liver and gut) where they blocked Tcons and prevented GvHD. However, they were unable to migrate to the bone marrow, thereby allowing Tcons to exert unopposed alloantigen recognition and leukemia killing. These results demonstrate haploidentical transplantation with a chemotherapy-based conditioning regimen and Treg/Tcon immunotherapy is not only feasible in fragile patients but also exerts a powerful anti-leukemic effect. Apparently, the Treg/Tcon immunotherapy plays a key role in the GvL effect. Disclosures Pierini: Stanford University: Patents & Royalties.