Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients’ bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II–IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus–mycophenolate–ATG-F–rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.
Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option for many patients with hematologic malignancies.1 Unfortunately, a considerable proportion of these patients do not have, or do not find in time, a suitable human leukocyte antigen (HLA)-matched related (MRD) or unrelated (MUD) donor, especially in the contexts of highly aggressive, rapidly proliferating diseases.2
Conversely, almost all patients who are candidate to transplantation have a partially HLA-compatible (haploidentical) family donor promptly available.3 The major limitation of this transplant modality has traditionally been the high risk of severe graft-versus-host disease (GvHD), due to alloreactions mediated by donor T cells recognizing the mismatched HLA haplotype.4 Extensive graft T-cell depletion proved effective in preventing GvHD in this context, but came at the price of an unacceptably high incidence of infectious complications.5, 6 Cell-based strategies aimed at improving post-transplantation immune recovery showed a beneficial impact in abating infectious mortality, but the required expertise in cell manipulation limited the clinical implementation of haploidentical HSCT to highly specialized centers.7, 8 Moreover, T-cell-depleted approaches to haploidentical HSCT yielded very poor results in patients affected by refractory diseases, in which relapses almost invariably occurred.9
In more recent years, the implementation of novel regimens of pharmacologic immune suppression, such as the use of high-dose post-transplantation cyclophosphamide, enabled the infusion of T-cell replete bone marrow from haploidentical donors with a strikingly low incidence of acute and chronic GvHD (aGvHD and cGvHD).10, 11, 12 These novel transplantation protocols have sparkled a renewed interest toward the clinical implementation of haploidentical transplantation in routine donor selection algorithms: still, the use of bone marrow as graft source is perceived as a limitation by several centers, for its significant logistic and economic hurdles.13
Here, we report the results of a clinical study investigating the feasibility of unmanipulated haploidentical peripheral blood stem cell (PBSC) transplantation in patients affected by high-risk hematological malignancies, exploring as GvHD prophylaxis the association of sirolimus, mycophenolate mofetil (MMF), rituximab and antithymocyte globulin Fresenius (ATG-F). Sirolimus, also known as rapamycin, is a macrolide characterized by peculiar immunosuppressive features, as it preferentially inhibits effector T cells, resulting in the relative expansion of natural T regulatory lymphocytes (Tregs).14 Natural Tregs, being endowed with suppressive activity not requiring previous antigen exposure, are attractive candidates for the clinical modulation of undesired immune responses, including autoimmunity and transplantation reactions.15 In mouse models of HSCT, the adoptive transfer of purified natural Tregs prevents GvHD while sparing a significant graft-versus-leukemia effect.16, 17 Resistance of Tregs from the cytotoxic activity of cyclophosphamide has been recently reported after T-cell replete bone marrow haploidentical HSCT,18 and the infusion of purified Tregs after umbilical cord blood (UCB) and haploidentical HSCT to prevent GvHD has produced promising results.19, 20 Besides their effects on Tregs, sirolimus and analogues exert a direct antitumor activity against many malignancies.21 In the context of unmanipulated haploidentical HSCT, sirolimus has been previously tested only in association with calcineurin inhibitors (tacrolimus), a combination which might hamper Treg expansion.22
In this study, we show that a calcineurin inhibitor-free sirolimus-based GvHD prophylaxis promotes the in vivo expansion of Tregs, and permits PBSC transplantation from haploidentical donors.
Materials and methods
Patients and donors
Starting from March 2007, patients of age 1–70 years with high-risk hematological malignancies in advanced phase were enrolled to a prospective, non-randomized, non-controlled, open-label phase II clinical trial approved by the San Raffaele Ethical Committee (TrRaMM protocol, Eudract 2007-5477-54). Following ad interim analysis, since June 2009 inclusion was extended to patients with acute leukemia in early phase. Patients were stratified by disease and status at the time of transplantation according to the disease risk index (DRI) validated by Armand et al.:23 as the index was developed for patients at first HSCT, patients with a previous allogeneic transplant were considered as a distinct category. Comorbidities at the time of transplantation were evaluated according to Hematopoietic Cell Transplantation-Comorbidity Index.24 HLA compatibility among donor–recipient pairs was assessed by 10 loci molecular typing (HLA-A, -B, -C, -DRB1, -DQB1), and predicted natural killer (NK) cell alloreactivity according to the model developed by the Perugia group.25
Conditioning regimen and GvHD prophylaxis
All patients received myeloablative conditioning based on treosulfan (14 g/m2) on days −6 through −4 and fludarabine (30 mg/m2) on days −6 through −2.
GvHD prophylaxis consisted of in vivo T-cell depletion by ATG-F (10 mg/kg) on days −4 through −2; sirolimus (orally, monitored two times a week to maintain a target therapeutic plasma level of 8–15 ng/ml) from day −1 and MMF (15 mg/kg t.i.d. orally or i.v.) from day 0. In the absence of GvHD or disease relapse, sirolimus and MMF were tapered to discontinuation at 3 months and at 1 month after HSCT, respectively.
Rituximab was administered in a single 500 mg dose on day −1, as secondary prophylaxis of Epstein–Barr virus (EBV) reactivation and as an additional agent to deplete B cells in vivo and potentially reduce the incidence of cGvHD.
PBSCs were mobilized with subcutaneous granulocyte-colony-stimulating factor 10 μg/kg daily for 4–6 days, collected by leukoapheresis and infused without any ex vivo manipulation, to achieve a target stem cell dose of 4–10 × 106 CD34+ cells per kg of patient body weight.
Prophylaxis against viral infections consisted of ganciclovir from day −6 to day −2 and aciclovir from day 0; prophylaxis against fungal infections consisted of voriconazole. Cytomegalovirus (CMV) and EBV reactivations were monitored weekly in peripheral blood plasma samples by quantitative PCR. Patients with a CMV DNA titer superior to 1000 copies per ml of peripheral blood plasma were subjected to anti-viral treatment.
Engraftment and chimerism
Neutrophil engraftment was defined as neutrophil counts ⩾0.5 × 109/l for more than three consecutive days, and platelet engraftment was defined as platelet counts ⩾20 × 109/l for more than seven consecutive days in the absence of transfusions. Post-transplantation disease follow-up comprised monthly bone marrow evaluations for the first 6 months after HSCT, and thereafter two times a year, until 5 years after transplant. In most of the patients with acute leukemia, an additional bone marrow evaluation was performed 9 months after HSCT. Hematopoietic chimerism was assessed on bone marrow aspirate samples by performing in parallel short-tandem repeats analysis (AmpFISTR Profiler Plus PCR Kit; Applied Biosystem, Foster City, CA, USA) and genomic HLA typing.26
Immune reconstitution was evaluated by fluorescence-activated cell sorting (FACS) (Beckman Coulter, Brea, CA, USA; FC500) and analyzed with the FCS Express software (De Novo Software, Los Angeles, CA, USA). Absolute cell counts were determined on the CD45bright/SSClow population with fluorochrome-conjugated monoclonal antibodies to CD3, CD4, CD8, CD16 and CD56, and TrueCount beads (Beckman Coulter). In a group of patients, selected based on sample availability, we performed more detailed phenotypic and functional analyses. The relative distribution of naive (CD45RA+CD62L+), central memory (CD45RA−CD62L+), effector memory (CD45RA−CD62L−) and terminal effector (CD45RA+CD62L−) T cells was determined among the CD4 and CD8 subsets. Frequencies of circulating Tregs, defined as CD4+CD25+Foxp3+CD127− cells, were assessed by intracellular immunophenotype, and expressed as % of CD4+ T cells. Based on their frequencies, Treg absolute counts were calculated from CD4+ T-cell absolute counts. Samples were analyzed on a FACSCanto I (Becton Dickinson, Franklin Lakes, NJ, USA) with the FlowJo software (Tree Star Inc., Ashland, OR, USA). Frequencies of CMV-specific T cells were assessed upon specific stimulation of T cells with CMV antigen by interferon-γ ELISpot (Zeiss KS Reader, Zeiss Inc., Oberkochen, Germany).27 The rate of CMV reactivations for each month of post-transplantation follow-up was calculated as follows: number of CMV reactivations/patients at risk at that time-point.
Molecular and functional analysis of Treg cells
The percentage of Foxp3 Treg-specific demethylated region (TSDR) was evaluated on peripheral blood mononucleated cell (PBMC)-derived genomic DNA by quantitative PCR and normalized for CD3 content. Given the negligible contribution of CD8 T cells to the overall levels of Foxp3 TSDR demethylation, normalization for CD3 provide an accurate estimate of the overall frequency of Tregs among T cells.28 To test their suppressive activity, CD4+/CD25bright T cells (Tregs) were FACS purified. CD8+ cells were FACS sorted from the negative fraction and labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. CFSE-stained T cells (responder cells) were stimulated with anti-Biotin MACSiBead particles preloaded with biotinylated CD2-, CD3- and CD28-directed antibodies (Tregs Suppression Inspector beads; Miltenyi Biotech, Bergisch Gladbach, Germany), in the absence or presence of autologous purified Tregs, at a responder/Treg ratio of 3:1. After 6 days of culture at 37 °C, cell division was quantified by FACSCanto I (Becton Dickinson) with the FlowJo software (Tree Star Inc.), and the percentage of proliferating cells was calculated according to the formula: T cells (>1 cycle)/total T cells × 100.14
Overall survival (OS), transplant-related mortality (TRM), progression-free survival (PFS), aGvHD and cGvHD, and relapse incidence (RI) were defined according to the EBMT (European Group for Blood and Marrow Transplant) criteria.29 Kaplan–Meier method was used to calculate OS and PFS, and estimates were provided together with 95% confidence interval. Incidence of CMV reactivations, TRM, aGvHD and cGvHD, and relapse were calculated with competing risk analysis, considering overall mortality as competing event for CMV reactivations, both relapse and TRM as competing events for aGvHD and cGvHD, and as reciprocal competing events.30 All patients were considered evaluable for the analysis of aGvHD, and those who had a documented engraftment were evaluable for cGvHD. aGvHD was graded according to the Glucksberg criteria,31 cGvHD was classified according to the Seattle and National Institutes of Health (NIH, Bethesda, MD, USA) criteria.32, 33 The multivariate analyses were performed using Cox proportional-hazards regression model; variables used were as follows: patient age, DRI, Hematopoietic Cell Transplantation-Comorbidity Index, donor/patient CMV serostatus, occurrence of post-transplantation CMV reactivations (tested as a time-dependent variable), number of donor–recipient HLA mismatches and predicted NK alloreactivity. P-value was considered significant if <0.05. Statistics were performed with SPSS version 13.0 and R version 2.12.2, package ‘cmprsk’.
Between March 2007 and June 2012, 121 patients were transplanted according to the TrRaMM protocol (median age 48, range 10–72 years). Data were collected until 30 June 2013, when analysis was started. Median follow-up of survivors was 38 months (range: 3–70 months days). Patient and donor’s characteristics are summarized in Table 1.
Transplantation procedure and hematopoietic engraftment
PBSCs were collected in a median of 2 (range 1–3) procedures of leukapheresis, and infused without ex vivo manipulation. Median CD34+ and CD3+ cell doses were 7.3 × 106/kg (range 2.1–11.6) and 3.0 × 108/kg (range 1.0–8.0), respectively. The conditioning regimen induced absolute grade 4 neutropenia in all patients. Median time of engraftment was 17 days for neutrophils (range 11–61) and 19 for platelets (range 7–154). At 30 days after HSCT, neutrophil engraftment occurred in 93±3% of the patients (Figure 1a), and platelet engraftment in 76±8% (Figure 1b). At first bone marrow evaluation, performed 30 days after HSCT, 98/104 patients (94%) presented donor chimerism equal to or above 95%, whereas the remaining six displayed disease persistence. No case of immune rejection or persistent mixed chimerism was documented. Median time of discharge after transplantation was 28 days (range 16–94).
Kinetics of immune reconstitution
All evaluable patients achieved rapid T and NK cell reconstitution, showing at day 30 after HSCT median cell counts of 294 CD3+ cells per μl (range 4–2146 cells per μl) and 114 CD3−CD56+CD16+ cells per μl (range 2–1404 cells per μl). B-cell reconstitution was much slower, possibly because of the inclusion of rituximab in the conditioning regimen (Figure 2a). T-cell repertoire was skewed toward CD8+ cells, as commonly observed after allogeneic HSCT (Figure 2b). All T-cell differentiation stages were represented as early as 30 days after HSCT, with a predominant central memory phenotype in CD4+ cells during the first 3 months (Figure 2c).
Recovery of anti-viral T-cell immunity
Post-transplantation recovery of interferon-γ-producing CMV-specific lymphocytes was heavily affected by host/donor CMV serostatus: whereas most pos/pos pairs had a detectable response as early as 30 days after HSCT and reached values not significantly different from those of healthy subjects by day 180, kinetics were significantly slower in the neg/pos and pos/neg subgroups (Figure 3a). Accordingly, also the rate over time and cumulative incidence of CMV reactivations differed greatly between the three subgroups of patients, with the host pos/donor neg group experiencing most high-titer reactivations requiring therapy (Figures 3b and c). No cases of post-transplant lymphoproliferative disorder or high-titer EBV viremia were documented, possibly because of the combined effect of rapid T-cell recovery and inclusion of rituximab in the conditioning regimen.
GvHD and prophylaxis withdrawal
Oral administration of MMF and sirolimus could be permanently discontinued in 101 and 91 patients, respectively. Median time of withdrawal was 37 days after HSCT (range 10–200 days) for MMF and 111 days for sirolimus (range 10–553 days). Sirolimus discontinuation was due to tapering in 51 out of 91 patients (56%), impending relapse in 29 (32%), incomplete control of GvHD in 6 (7%) and neurological toxicity in 5 (5%). We did not document cases of transplant-associated microangiopathy requiring sirolimus discontinuation, or of veno-occlusive disease. At 1 year after HSCT, 48 out of 53 live patients (90%) had discontinued any form of pharmacologic immunosuppression.
Cumulative incidence of grade II–IV and grade III–IV aGvHD at day 150 after HSCT were 35±9% and 22±8%, respectively (Figure 4a). Cumulative incidence of cGvHD at 2 years after HSCT was 47±11% (Figure 4b). According to the revised Seattle criteria, 43 patients were diagnosed with cGvHD (13 limited and 30 extensive). Among the 13 patients with limited cGvHD, 8 required a short course of pharmacologic treatment with steroids (n=3) or sirolimus (n=5), upon which 6 completely resolved all sign of disease (75%). Among the 30 patients with extensive cGvHD, 21 required pharmacologic treatment with steroids (n=4), sirolimus (n=7) or both (n=10), upon which 14 completely resolved all sign of disease (33%). According to the NIH Consensus Criteria, 22 patients were reclassified as having classic cGvHD, 20 patients as having overlap syndrome and 1 patient as having late-onset aGvHD.
Treg cell dynamics
To verify the hypothesis that an immunosuppressive regimen based on the combination of ATG-F and sirolimus could promote in vivo the expansion of natural Tregs, we measured their frequencies and absolute counts in the peripheral blood of patients at day 30, when all patients were receiving sirolimus, and at day 180, after immunosuppression was discontinued. At day 30, natural Tregs, identified as CD4+CD25+Foxp3+CD127− cells, represented on average 9.9% (range: 0.2–37.2) of circulating CD4+ T cells, a percentage significantly higher than that observed in healthy subjects (P=0.0026). Conversely, at day 180 only 3.2% (range 0–14.5) of circulating CD4+ lymphocytes displayed a Treg phenotype (Figure 5a). Absolute counts of Tregs showed a similar trend (Figure 5b). The increased frequency of Tregs observed at 30 days after HSCT was confirmed by molecular quantification of Foxp3 TSDR in patients and healthy controls (Figure 5c). To demonstrate their functional activity, Tregs were purified from selected patients at 30 days after HSCT and challenged in a suppression assay against autologous activated T cells: patient Tregs proved as efficient as those harvested from healthy subjects in inhibiting T-cell polyclonal proliferation (Figure 5d). Interestingly, patients who experienced grade 0–I aGvHD had higher frequencies of circulating Tregs at day 30 after HSCT, as compared with those experiencing grade II aGvHD or severe aGvHD (grade III–IV; Figure 5e).
Tumor response and relapse
Relapse incidence was 36±9% at 1 year after HSCT and 48±9% at 3 years (Figure 6a). In the subgroup of patients affected by acute myeloid leukemia, we documented 33 cases of relapse after HSCT: interestingly, 9 (27% of relapses) occurred in extramedullary sites and 11 (33% of relapses) were due to immune escape leukemic variants characterized by selective genomic loss of the HLA haplotype mismatched between patient and donor (Figure 6b).34 Leukemia relapse with loss of mismatched HLA occurred at a median of 375 days after HSCT, extramedullary relapses at a median of 205 days and ‘classical’ bone marrow relapses at a median of 65 days after HSCT. No difference in relapse incidence was observed according to the presence or absence of predicted NK alloreactivity in the graft-versus-host direction (P=0.66), not even in the subgroup of patients with acute myeloid leukemia in complete remission (P=0.2), possibly because of the competing presence of donor T cells and GvHD prophylaxis.35
TRM and survival
TRM at 1 and 3 years after HSCT were 30±8% and 31±8%, respectively (Figure 7a). Causes of the 36 TRM events were infections in 26 cases (bacterial in 23, fungal in 2 and toxoplasmosis in 1), encephalopathy of unknown origin in 3 cases, sudden cardiac arrest in 1 and aGvHD in 8 cases.
OS was 47±8% at 1 year after HSCT and 25±8% at 3 years after HSCT; PFS was 34±8% at 1 year after HSCT and 20±7% at 3 years after HSCT (Figure 7b). PFS at 3 years in patients with low or intermediate DRI was 29±15%, with high DRI 20±12% and with very high DRI 6±12% (P=0.02 by log-rank test; Figure 7c). Interestingly, we observed some long-term survivors also in patients who had relapsed after a previous allogeneic HSCT (PFS at 3 years 15±15%).
Multivariate analysis for PFS, OS, TRM and relapse incidence showed that DRI (very high vs low/intermediate), Hematopoietic Cell Transplantation-Comorbidity Index (⩾3 vs 0–2) and the number of donor/recipient HLA mismatches (1–3 vs ⩾4) had a significant and independent effect on PFS, OS and relapse incidence (P<0.05). As observed in several recent reports,36, 37 CMV reactivations requiring treatment were found to be significantly associated with protection from relapse, and the presence of four or more HLA mismatches between patient and donor was associated with increased incidence of relapse and with a reduced OS and PFS. No significant association was found for TRM.
Allogeneic HSCT represents the only curative option for patients with chemorefractory leukemia, granting up to 30% long-term clinical remissions in the HLA-matched donor setting.38, 39, 40 Still, a considerable proportion of patients with active disease have no access to compatible donors: over the recent few years, the development of novel promising strategies of immunomodulation has sparkled great interest in the clinical implementation of HSCT from haploidentical family members as an alternative curative option for all these patients.10, 11, 12 In our study, we present a novel protocol for haploidentical HSCT based on a myeloablative conditioning regimen with full-dose treosulfan, the infusion of unmanipulated PBSC grafts and pharmacologic immune suppression with ATG-F, rituximab and oral administration of sirolimus and mycophenolate.
The conditioning regimen was well tolerated, and engraftment was rapid and robust, with achievement of stable full donor chimerism in the majority of patients. Also, reconstitution of adaptive T-cell immunity was quick, with high numbers of CD4 and CD8 lymphocytes documented as early as 30 days after HSCT. Recovery of a specific T-cell immunity and the profile of CMV reactivations were heavily influenced by host/donor serostatus, matching the results reported in other transplantation settings upon sirolimus prophylaxis41 and comparing favorably with the historical experience in the T-cell-depleted haploidentical context.42, 43 Nevertheless, overall mortality rate due to infections in our experience was high, an observation that might at least in part be explained by the heavily pretreated patient cohort and by the considerable incidence of cGvHD documented in our protocol.
The immune repertoire reconstituting after allogeneic HSCT was highly enriched in functional donor-derived Tregs, possibly as an effect of the combined activity of ATG-F and sirolimus in the absence of calcineurin inhibitors. Currently, different strategies to take advantage of Treg-suppressive effect in human allogeneic transplantation are investigated, mostly relying on ex vivo cell manipulation:19, 20 in our study, this goal was achieved without additional cellular therapies, reducing the economic impact of the procedure. Also, the toxicity profile of the GvHD prophylaxis we adopted resulted extremely favorable, and with a tight monitoring of sirolimus plasma level, we registered negligible incidence of drug-related adverse events. Moreover, the induction of Treg cells upon sirolimus administration was reversible upon drug withdrawal, suggesting that a time-wise in vivo modulation of alloreactivity might be pursued by this approach.
Still, despite the presence of high numbers of circulating Tregs, the documented incidence of aGvHD and cGvHD in our protocol was higher as compared with current protocols of haploidentical transplantation based on bone marrow as graft source,10, 11, 12 but still comparable to the historical results obtained in matched unrelated donor HSCT with PBSCs.44, 45 This difference may reflect the higher dose of T cells infused (almost one logarithm more) in PBSC transplantation and the more pronounced intrinsic alloreactivity of peripheral blood circulating T cells.46
On the other hand, T-cell-mediated alloreactivity in our protocol might have also been associated with a graft-versus-leukemia effect, as suggested by two observations. The first is that several patients affected by chemoresistant disease, high or very high DRI score, or that had relapsed after a previous allogeneic HSCT achieved and maintained long-term complete remission. The second indication of an antileukemic effect mediated by circulating alloreactive T cells in our study is the exceptionally high proportion of leukemic relapses localized to immunoprivileged extramedullary sites or characterized by genomic loss of mismatched HLA, both expression of mechanisms adopted by leukemic cells to evade immune recognition.34, 47
Still, post-transplantation relapse remains a formidable challenge, especially for patients with advanced disease. The favorable toxicity profile associated with most recent approaches to haploidentical HSCT, comprising ours, might support intensification of the conditioning regimens, the implementation of pre-emptive pharmacologic therapy with demethylating agents,48 or adoptive cell therapy approaches, such as those based on donor T cells genetically redirected against cancer cells.49, 50
In conclusion, our study provides a novel and manageable platform to partially HLA-mismatched transplantation, allowing the use of PBSC grafts, and exploiting pharmacologic immune suppression for a transient and controlled expansion of functional Treg cells.
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This study was supported by the Italian Ministry of Health, by the Italian Ministry of University and Research, by the Cariplo Foundation (to KF), by the Associazione Italiana per la Ricerca sul Cancro (My First AIRC Grant to AB, Start-Up Grant to LV, Investigator Grant to ChB and to KF), and by the European Community (ERANET-TRANSCAN grant to AB). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
JP and FC designed the study and analyzed the data. JP, AFo, DC, LV, MTLS, CM, AC, AA, SMar, SMas, FG, AL, EG, FLu, MC, MT, MGR, SR, MBe, CC, MM, FP, MZ, FLo and FC contributed to patient clinical care and data collection. AFo, LV, MN, SO, MBa, AFe, MRC, GO, KF, AB and ChB contributed to the immune monitoring data collection and interpretation. RC and LC performed the statistical analyses. JP, AFo, LV, AB, ChB and FC wrote the paper.
Dr Chiara Bonini is a scientific consultant of MolMed SpA, Milan, Italy. Professor Claudio Bordignon is an employee of MolMed SpA, Milan, Italy. The remaining authors declare no conflict of interest.
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Peccatori, J., Forcina, A., Clerici, D. et al. Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors. Leukemia 29, 396–405 (2015). https://doi.org/10.1038/leu.2014.180
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