Search

Introduction: Cytomegalovirus (CMV) reactivation and disease are important risk factors after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and strongly affect morbidity and mortality after transplant. CMV-specific T cell reconstitution controls CMV reactivation and protects against serious adverse events but a protective level of CMV-specific T cell response or standardized method for its monitoring have not been yet determined. Methods: We designed a prospective, single-center observational study to assess if the kinetic and quality of CMV specific T-cell reconstitution impact the incidence and severity of CMV reactivations. We have enrolled 84 consecutive patients affected by hematological malignancies receiving allo-HSCT followed by Cyclophosphamide and Rapamycin between December 2017 and February 2019. Here we report preliminary data on the first 61 patients. Patients received allo-HSCT from family (siblings=10; HLA haploidentical=24), unrelated HLA-matched (n= 24) donors or cord blood (CB, n=3). The CMV serostatus of host (H) and donor (D) pairs was: H+/D+(n=40, 65%), H+/D-(n=20, 33%) and H-/D+ (n=1, 2%); H-/D-(7% of the overall transplanted population at our center) were excluded. CMV DNAemia was assessed weekly in whole blood (WB). Absolute numbers of polyclonal and CMV-specific T cells were quantified by flow cytometry using Troucount™Tubes (BD) and Dextramer®CMV-Kit (Immudex), respectively, in the graft and fresh WB at days -7, +30, +45, +60, +90, +120, +150, +180 and +360. Dextramer CMV kit includes reagents for the identification of CMV-specific lymphocytes restricted for several HLA class I molecules: A*01:01/*02:01/*03:01/*24:02 and B*07:02/*08:01/*35:01. These alleles allowed the longitudinal evaluation of 54 out of 61 (89%) patients. Results: At a median follow-up of 226 days post-HSCT, 31 (57%) patients experienced a CMV-related clinically relevant event (CRE, median +63 days), including 8 patients (15%) with CMV disease (median +59 days). Univariate analyses showed that the incidence of CMV clinically-relevant reactivation (CRE) was influenced by H/D CMV serostatus (0.90 in H+/D- versus 0.44 in H+/D+pairs, p=0.015) and by previous acute Graft-versus-Host Disease (aGvHD) requiring systemic immunosuppression (0.82 in aGvHD grade II-IV versus 0.52 in aGvHD grade 0-I, p=0.051). The disease status at transplant, the donor type (HLA-matched versus HLA-haploidentical/CB donors), donor's or host's age did not significantly affect the probability to develop CRE. For each time-point, we compared the absolute number of CMV-specific lymphocytes in patients experiencing or not a subsequent CRE. Our data demonstrate that higher levels of CMV-specific CD8+T cells in the donor apheresis and at +45 days after allo-HSCT are associated with reduced risk of subsequent CRE (median CMV-specific CD8+cells/kg in the apheresis=5x103in CRE-positive patients (CRE+) and 5x105in CRE-negative patients (CRE-), p=0.012; median CMV-specific CD8+at +45 days=0.14 cells/μL in CRE+and 1.21 cells/μL in CRE-, p=0.034). Furthermore, patients with any Dextramer positivity at +45 days displayed a lower incidence of CRE compared with subjects who were negative (CRE probability: 0.5 vs 1.0, p=0.003). Conversely, the absolute number of neither polyclonal CD3+CD8+T lymphocytes nor total CD3+T cells correlate with subsequent CRE. Taking advantage of the HLA mismatched-HSCT setting, we then dissected CMV-specific T-cell response according to HLA restriction elements (H/D=shared n=45, D-restricted n=14, H-restricted n=11). In H+/D+pairs, we observed a fast and similar kinetic of reconstitution of CMV-specific lymphocytes restricted by H/D and D HLAs. Conversely, in H+/D-pairs, we detected only CMV-specific CD8+lymphocytes restricted for H/D haplotypes. Host-restricted cells remained undetectable for the first 180 days after HSCT. Conclusion: Early after allo-HSCT and in the donor apheresis, the level of CMV-specific CD8+T cells measured by Dextramer staining differs in patients experiencing or not subsequent CRE. Furthermore, our findings indicate that CMV reactivations can prime H/D-restricted T cells presumably educated in the donor thymus; conversely, D- and H-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education respectively. Disclosures Brix: Immudex: Employment. Bonini:Kite/Gilead: Consultancy; Intellia Therapeutics: Consultancy; Intellia Therapeutics: Research Funding; Novartis: Consultancy; GSK: Consultancy; Allogene: Consultancy; Molmed: Consultancy; TxCell: Consultancy; -: Patents & Royalties: Adoptive T cell therapy field.

Background. Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML); nonetheless, relapse remains the major cause of death after such therapeutic option. Patients and Methods . We investigated the expression of Inhibitory Receptors (IR; i.e. PD-1, CTLA-4, TIM-3, LAG-3 and KLRG1) on different T-cell subsets infiltrating the bone marrow (BM) of 8 healthy donors (HD) and 32 allogeneic HSCT recipients diagnosed with Acute Myeloid Leukemia, collected at relapse (median 251 days) or at complete remission (CR) 1 year after HSCT. Inclusion criteria were: a diagnosis of acute myeloid leukemia or myelodysplastic syndrome, a relapse-free survival of at least 4 months after allogenein HSCT, absence of active GvHD, CMV infections or other complications at the time of sampling. Samples were analysed by multi-parametric flow cytometry for the expression of inhibitory receptors on T-cell subsets and the results were validated with BH-SNE, an unbiased dimensionality reduction algorithm. We exploited HLA-mimicking fluorescent molecules loaded with a specific epitope to screen anti-tumour and anti-viral T cells whereas the T-cell receptor repertoire was assessed by TRAC and TRBC RNA sequencing and the relative frequency of each T-cell receptor calculated. To evaluate T-cell function and specificity, CD107a expression, cytokine profiles and killing of autologous blasts were quantified. Results. After Haploidentical-HSCT PD-1, CTLA-4, 2B4 and Tim-3 were expressed at higher percentage when compared to HD, independently from the clinical outcome. In contrast, after HLA-matched HSCT, patients who relapsed displayed a higher frequency of BM-infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To gain insights on the inhibited T-cell subpopulation identified in the BM of relapsing patients, we separately profiled the different T-cell memory subsets: in both HD and CR patients the IR expression was confined to effector memory and effectors whereas at relapse PD-1, 2B4, KLRG1 and Tim-3 were also expressed in BM-infiltrating central memory (TCM) and memory stem T cells (TSCM, p Interestingly, the TCR repertoire of BM-infiltrating T cells at relapse displayed a restricted clonality, suggesting that immune inhibitory signals are active on discrete and specific T-cell clones. To gain further insights on such clones, we assessed the IR expression profile on CD8 T cells specific for viral (CMV) and tumor-associated antigens (including peptides from WT1, EZH2 and PRAME). We observed a higher IR expression and co-expression on tumor-specific T cells when compared to viral-specific CD8 cells, particularly in case of patients who experienced post-transplant relapse. In accordance, IRpos sorted T cells harvested from relapsing patients showed a restricted TCR repertoire and, when challenged with autologous leukemic blasts, proved enriched in leukemic specificities as shown by higher expression of the activation marker HLA-DR (p Conclusion. These results highlight a wide, yet reversible, immunological dysfunction likely mediated by AML blasts in the BM of patients relapsing after allogeneic HSCT, that is particularly evident on memory T cells specific for tumor antigens. This suggest and open new therapeutic opportunities for AML. Figure. Figure. Disclosures Bondanza: Novartis: Employment. Vago:GENDX: Research Funding; Moderna TX: Research Funding. Bonini:Intellia Therapeutics: Research Funding.

Background Despite the constant improvement in the outcome of allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Acute Myeloid Leukemia (AML), relapses remain frequent, warranting investigation on their biological bases. After haploidentical HSCT up to one third of AML relapses feature selective genomic loss of the HLA haplotype targeted by alloreactive donor T cells (Vago, N Engl J Med, 2009; Crucitti, Leukemia, 2015), evading their control and gaining the ability to outgrow. Yet, Natural Killer (NK) cells mediate alloreactivity in response to loss of specific HLA allotypes from target cells: thus, in theory, HLA loss relapses should represent excellent targets for donor NK cell recognition. Here we investigated the dynamics of NK cells in this unique immunogenetic context, to understand the biological bases of their failure in preventing the emergence of HLA loss variants. Methods We took into consideration 23 patients who after T cell replete haploidentical HSCT experienced HLA loss relapses. NK cell alloreactivity was predicted according to the Perugia algorithm (Ruggeri, Science, 1999). Killer Cell Immunoglobulin-like Receptor (KIR) typing was performed using a commercially-available kit, and KIR B-content estimated using the EMBL-EBI calculator. The phenotypic features of peripheral blood NK cells were assessed by multiparametric flow cytometry, and high dimensional single-cell analysis was performed using the viSNE bioinformatic tool. Results Based on donor-recipient HLA typing, at the time of HSCT NK cell alloreactivity in the graft-versus-leukemia direction was predicted in 10/23 patients who experienced HLA loss relapses (43.5%). In 7/23 additional patients (30.4%), conditions for predicted NK cell alloreactivity were fulfilled at time of relapse, upon genomic loss of the mismatched HLA haplotype from AML blasts. In all cases KIR genotyping confirmed the presence in the donor repertoire of the necessary KIR genes. Only 3/17 HSC donors were homozygous for KIR A haplotypes, encoding preferentially inhibitory KIR genes, and most carried equal or higher numbers of activating KIR genes than expected (Cooley, Blood, 2010). Thus, the absence of NK cell-mediated control of HLA loss variants can not be explained by an unfavorable immunogenetic asset of HSC donors. Therefore we characterized the phenotypic features of NK cells circulating in the peripheral blood of 7 patients at the time of HLA loss relapse (median time after HSCT 307 days, range 147-703), and compared them to their counterparts in healthy individuals (n=6), and matched-paired transplanted patients in remission (n=6), or at the time of non HLA loss ("classical") relapse (n=7). We analyzed a total of 27 markers involved in NK cell target recognition (KIRs, NKG2A, NKG2C, SIGLEC7, SIGLEC9), activation (NKp30, NKp44, NKp46, NKG2D, 2B4, DNAM1), maturation (CD57, CD16, CD62L), and exhaustion (PD1, TIM3, KLRG1). At the time of HLA loss relapse, NK cells had recovered a mature phenotype, although with a slightly higher frequency of CD56bright cells. In all cases in which NK cell alloreactivity had been predicted we detected the single-KIR+ NK cells of interest, without significant differences between patients and controls. However NK cells from transplanted patients expressed lower levels of the SIGLEC9 (p Conclusions Even at late timepoints after partially-incompatible allo-HSCT, when HLA loss relapses typically occur, the reconstituted NK cell repertoire displays profound differences from its counterpart in healthy subjects, hinting for defective immunosurveillance. Therapeutic protocols employing freshly isolated mature donor NK cells should thus be further investigated for the prevention and treatment of HLA loss relapses. Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Figure 1. The same viSNE map, obtained by analysis of the entire dataset, is differentially colored to evidence the spatial distribution, and thus phenotypic similarity, of NK cells from each cohort (upper row) or the intensity of expression of the indicated markers (lower row). Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Human herpesvirus type 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic hematopoietic stem cell transplantation (AlloSCT). HHV-6 is a member of the beta herpesvirus subfamily (genus Roseolovirus). HHV-6 infection is recognized as the cause of a febrile disease and exanthem subitum in early childhood. Approximately 60% of solid organ transplant and 40% of patients after alloSCT experienced HHV-6 reactivation. Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, some clinical reports suggest that HHV-6 can facilitate the occurrence of severe clinical complications of alloSCT, increasing transplant-related mortality. METHODS: From January 2009 to February 2013, we retrospectively evaluated 54 consecutive adult patients (median age 50 years) who developed positivity to HHV-6 after alloSCT for high-risk hematological malignancies. Stem cell donors were family haploidentical (37), HLA identical sibling (8), unrelated volunteer (6), cord blood (3). The viral load was determined by quantitative PCR (Nanogen Advanced Diagnostic S.r.L) in cell-free body fluids such as plasma, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), bone marrow (BM) aspirates or in gastrointestinal biopsies. RESULTS: Median time from alloSCT to HHV-6 reactivation was 34 days (range: 0-705). Thirty-one patients presented HHV-6 positive in plasma, 9/54 in BM, 33/54 in gut biopsies or BAL, 7/54 in CSF. At the time of viral positivity all pts were receiving acyclovir as viral prophylaxis except five. Twenty-nine patients had acute graft versus host disease (GvHD). Twenty-two out of these twenty-nine patients experienced a grade III-IV acute GvHD, requiring high dose steroids in twenty-six cases. A concomitant CMV positivity was detected in 15/54 patients. The median absolute count of CD3+ lymphocytes was 207 cells/mcl. In 52/54 cases we reported HHV-6 clinical manifestations: fever (43), skin rash (22), hepatitis (19), diarrhoea (24), encephalitis (10), BM suppression (18), delayed engraftment (11). HHV-6 positivity led to antiviral pharmacological treatment in 37/54 cases, using as first choice therapy foscarnet. Amongst the total fifty-four patients with documented HHV-6 positivity thirty-one solved the clinical event. However the mortality rate was relatively high in this population (overall survival (OS) ±SE at 1 year after HHV-6 reactivation was 38% ± 7%), mainly related to severe infections or GvHD. A better OS is significantly associated with CD3+ cells ≥200/mcl at the time of HHV-6 reactivation (fig 1) (OS at 1 year 63% compared to 11% for patients with CD3 CONCLUSIONS: This retrospective study confirms a correlation of HHV-6 with high morbidity and mortality rates after alloSCT, thus suggesting a regular HHV-6 monitoring in alloSCT recipients. The regular monitoring of HHV-6 DNA, using a real-time PCR assay, may be useful for identifying active HHV-6 infection and for the introduction of a pre-emptive treatment, possibly reducing the incidence of the most severe clinical complications. Despite HHV-6 detection typically occurred early after alloSCT, a better immune reconstitution has the potential to improve clinical outcome. Figure 1: Overall survival after alloSCT in HHV-6 positive patients: green line showed patients with more than 200/mcl CD3+ cells, blue line the ones with less than 200/mcl CD3+ cells at HHV-6 reactivation. P value is provided by Log Rank test. Figure 1:. Overall survival after alloSCT in HHV-6 positive patients: green line showed patients with more than 200/mcl CD3+ cells, blue line the ones with less than 200/mcl CD3+ cells at HHV-6 reactivation. P value is provided by Log Rank test. Disclosures Bonini: MolMed S.p.A.: Consultancy.

Background: Post transplant immune reconstitution plays a major role in determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is currently monitored with different techniques in different Centers, with the aim of identifying clinically relevant immunological biomarkers. However, it is unclear which and how many of these immunological tests are currently performed on a routine basis, and which ones have the potential to predict patient outcome, and possibly guide patient care after allo-HSCT. Methods: The EBMT Cellular Therapy & Immunobiology Working Party (CTIWP) conducted a survey to identify current policies to monitor immune reconstitution in patients undergoing allo-HSCT and possibly reach a general consensus. This study followed the EBMT study guidelines. All EBMT Centers were invited to participate. Each participating Center received a questionnaire on the availability of specific immunomonitoring assays, specifying the use in clinical practice and/or within investigational trials. Assays were based on relatively simple and readily available parameters such as absolute lymphocyte counts (ALC) to more complex cellular and molecular tests. Moreover, the Centers were asked to define the transplant platform (HLA-identical sibling, matched unrelated donor, haploidentical and/or cord blood) on which each test is generally performed. Results: Policies for post-transplant immunomonitoring have been reported by 35 participating EBMT Centers active in 14 Countries and performing allo-HSCT from HLA identical related (35 centers), matched unrelated (33), haploidentical (34), unrelated cord blood (27). Complete blood counts and immunoglobulins are routinely tested for patients' care by all centers. Relative proportions of T cell subsets are currently tested by flow-cytometry as "standard of care" or "investigational" by 82% and 17% of centers respectively. B cell and NK cell counts are quantified routinely by 46% and 23% of Centers, and investigationally by 40% of Centers. The availability of molecular tests (STR, qPCR, Fish) to measure post-transplant engraftment are reported by all Centers, except two, as a standard of care measure. T cell receptor-expressing circles (TRECs) and/or K-deleting recombination excision circles (KRECs) are quantified within selected clinical trials by 37% of Centers. Interestingly, 60% of Centers evaluate, mostly as an investigational measure, antigen specific T cell responses by: proliferation assays (49%), interferon-gamma enzyme-linked immunospot-Elispot (49%), intracellular cytokine staining (46%) and tetramer/dextramer staining (37%). Most of these Centers test responses to Cytomegalovirus and Epstein Barr Virus, and 5 Centers use at least one of these assays on a routine basis. About half of the participating Centers (43%) commonly test antigen-specific antibodies, mainly as responses to vaccines, and not routinely. T-cell receptors (TCR) and B-cell receptors (BCR) repertoires are measured by spectratyping in 14 out of 35 Centers (4 as clinical practice and 10 in selected trials), or, in selected trials, by next generation sequencing (in 11 out of 35 the participating Centers). Conclusions: Results of this survey indicate that country- and center expertise are associated with heterogeneous and distinct protocols, and underline the clinical need to harmonize methods and to provide practical recommendations for monitoring post-transplant immune reconstitution, both for routine purposes and investigational studies. Adequate reporting and connection between individual Centers exploiting these data will foster collaborative and comparative research studies, with the ultimate goals of improving patient care and refining our understanding of the immunological correlates to clinical outcome. Acknowledgments: R. Ram, M. A. Diaz, G. McQuaker, D. Russo, E. Faber, P. Chiusolo, C. Rössig, S. M. Martin, A. Anagnostopoulos, M. Stelljes, K. Orchard, P. Jindra, A. Sampol, K. Patrick, M. A. Bekadja, J. Gayoso, A. Olivieri, J. Passweg, E. Jost, H Labussiere-Wallet, Y Koc, A. Lange, I. Garcia Cadenas, N. Kröger, A. Biondi, N. Milpied, D. Olive, E. Lanino, G. Stuhler, J.H. Dalle, J.R. Cabrera Marín, F. Ciceri, D. Uckan-Cetinkaya, R. Parody Porras, G. Kriván. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

BACKGROUND: Although Human herpesvirus 6 (HHV6) reactivation in healthy individuals usually occurs without significant morbidity, in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is associated with severe clinical manifestations and increased transplant-related mortality (TRM). The role of HHV6 in transplant-related complications remains in question, considering that both latent and active viral infection can occur. Moreover, only limited experiences are reported on HHV6-specific immune responses after HSCT, and their correlation with clinical outcome is largely unexplored. METHODS: From February 2013 to October 2015, we conducted a prospective observational study to investigate HHV6 reactivation in 213 consecutive adult patients (median age 52 years) who received allo-HSCT for high-risk hematological malignancies (57% acute leukemia) in our institute. Stem cell donors were family haploidentical (104), HLA identical sibling (39), unrelated (63), cord blood (7). Stem cell source was mainly T-cell replete PBSCs (87%). Viral load was weekly monitored by quantitative PCR in plasma within the first month after HSCT. Numbers of IFNγ-producing HHV6-T-cells were determined by enzyme-linked immunospot assay (ELISPOT). We challenged patients PBMC against a library of overlapping peptides covering the entire sequence of the immunodominant virus protein U54, expressed during the lytic cycle of virus replication. Patients were evaluated at a median of 34 days after HSCT (HHV6-; 57 patients) for controls or by the 4th day after the first HHV6 DNAemia (median 32 days) for reactivating patients (HHV-6+; 54 patients). RESULTS: HHV6-reactivation occurred in 56% of patients at 100 days, with a median time of 28 days after HSCT. HHV6 was detected in plasma for 86% of patients, while 33% resulted positive in other materials: 9 BM aspirates, 39 gut biopsies, 3 BAL, 5 CSF. All patients received acyclovir as prophylaxis. Only 41% of reactivating patients presented a clinically relevant HHV6 infection (HHV6 positivity in presence of HHV6-related clinical symptoms and/or HHV6-disease). Clinical manifestations were: fever (25), skin rash (37), hepatitis (19), diarrhoea (28), encephalitis (5), BM suppression (30). According to center guidelines, antiviral treatment was given in 23% of reactivating patients, for uncontrolled clinically relevant HHV6 infection. Overall survival (OS) was not different in HHV6 reactivating patients compared to controls (p=0,2). Relapse incidence and TRM were not affected by HHV6. All HSCT recipients showed a better OS with CD3+ cells≥200/mcl at 30 days (p The number of IFNγ-producing HHV6-specific T-cells was significantly higher in HHV6 reactivating patients (p= 0.0149; mean number of specific T-cells 43.48 per 10^5 PBMC) than in non-reactivating patients (specific T-cells 12.57 per 10^5 PBMC), especially in the presence of active and clinically relevant HHV6 infection (p CONCLUSIONS: In this study, we observed that active disease status before HSCT, haploidentical donors, especially using PT-Cy, CMV reactivation, GvHD and lower CD3+ counts at 30 days, are strong predictors of HHV6 reactivation. HHV6-specific T-cells, detectable by ELISPOT assay despite extremely low T-cell numbers and immunosuppressive therapy, are significantly associated with active and clinically relevant HHV6 infections, representing a new and promising tool to unravel the role of HHV6 positivity in allo-HSCT recipients. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Background:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only cure for high-risk acute myeloid leukemia (AML). Unfortunately, relapse still remains the major cause of death after HSCT. We investigated if T-cell dysfunction is associated to post-transplant relapse. Patients and Methods: To this,we longitudinally analyzed the T-cell dynamics in bone marrow (BM) and peripheral blood (PB) of 32 AML patients receiving HSCT from HLA identical (HLAid, 20 pts) or HLA haploidentical (haplo, 12 pts) donors. Samples were analysed by multi-parametric flow cytometry to investigate the expression of inhibitory receptors (IRs) on CD4 and CD8 T-cell subsets defined by CD45RA, CD62L and CD95 expression, and to assess the proportion of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Results were also analyzed with the BH-SNE algorithm, an unbiased computational method for the analysis of FACS data. To evaluate T-cell effector functions, the CD107a degranulation assay was performed and the production of cytokines (IL-2, IFNg and TNFa) was measured by intracellular staining. BM and PB were collected 60 days after HSCT and at relapse (median 237 days; 16 pts) or, when complete remission was maintained (CR; 16 pts), at 1 year. Samples from 8 healthy donors (HD) were used as controls. Results:After transplant, BM and PB T cells showed a lower CD4/CD8 ratio (p We next investigated the expression of several IRs as T-cell exhaustion markers. After haplo-HSCT, PD-1, CTLA-4, 2B4 and Tim-3 were significantly upregulated in BM and PB T cells at all time-points, compared to HD and independently from the clinical outcome. Conversely, after HLAid-HSCT, at the late time-point, patients who relapsed, displayed a higher frequency of BM infiltrating T cells expressing PD-1, CTLA-4 and Tim-3 than CR pts (p To verify whether the T-cell exhaustion phenotypic profile at relapse associates with functional impairment, we evaluated T-cell effector functions upon polyclonal stimulation. Strikingly, we observed a lower degranulation ability of CD8 cells at relapse when compared to CR (p Conclusions: After HSCT, the molecular signature of exhausted CD8 cells in relapsing pts includes PD-1, CTLA-4, 2B4 and Tim-3. The expression of IRs on early differentiated central memory and memory stem T cells at relapse suggests a wide, though reversible, immunological dysfunction mediated by AML relapsing blasts. Disclosures Bondanza: TxCell: Research Funding; MolMed SpA: Research Funding; Formula Pharmaceuticals: Honoraria. Ciceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Introduction: The T cell memory compartment is multi-faceted and encompasses multiple subsets with divergent properties. In addition to central memory (TCM) and effector memory (TEM) cells, the spectrum of immunological memory has been recently extended with the identification of memory stem T cells (TSCM). Gene expression profiling, corroborated by in vitro and in vivo experimental results, posits TSCM upstream TCM and TEM in T-cell ontogeny. However, TSCM role in immune reconstitution (IR) following allogeneic HSCT remains largely unknown. Here, we tracked TSCM dynamics in patients undergoing haploidentical HSCT. By this process, we unconventionally exploited HSCT as model system to provide novel insights into the contribution of TSCM to a mounting immune response. Patients and Methods: We characterized T cell dynamics during the first month post HSCT in 20 patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft, and GvHD prophylaxis consisting of cyclophosphamide (PT-Cy day 3,4), MMF and sirolimus from day 5. Results: Upon infusion, naïve T cells (TN) cells gradually disappeared from circulation, and by day 8 post HSCT the peripheral compartment was composed only by memory lymphocytes. At day 8, TSCM cells were the most represented circulating subset, and their frequency was significantly higher than that of the infused graft (LK). To investigate whether such high TSCM frequency was due to expansion of TSCM cells infused or to their direct in vivo generation, T cell subset proliferation was analyzed. At day 3 upon infusion (in the absence of immunosuppression), all memory subsets robustly proliferated, with TSCM cells displaying significantly higher frequencies of Ki-67+ cells compared to all other subsets. In sharp contrast, TN cells were scantly Ki-67+, in line with the longer timeframe required for their priming. PT-Cy abrogated proliferation in all subsets. T cell counts however did not drop between day 5 and 8 post HSCT, and TSCM cells increased in percentages and absolute numbers. We thus explored whether TSCM cells were resistant to PT-Cy, but failed to record ALDH activity, the major mechanism of Cy inactivation, in CD3+ cells. neither within LK nor upon infusion. Accordingly, we detected high percentages of apoptotic cells within all memory subsets at day 5 post HSCT. Nonetheless, at day 8 significantly lower percentages of TSCM cells were Annexin V+ compared to TCM/TEM, while by day 15 post-HSCT all memory subsets displayed very low levels of apoptosis. Thus, we reasoned that direct conversion of TN into TSCM could be the preferential mechanism underlying TSCM expansion. To prove this, we exploited the TCR sequences harbored by each individual T cell as surrogate clonal markers. Purified T cell subsets from LK and harvested 30 days post HSCT in 3 consecutive patients were subjected to TCR sequencing. Sequences harbored by LK-TN were retrieved in all memory subsets at day 30 after HSCT, showing that TN were able to generate the complete spectrum of immunological memory, including TSCM. Quantitative analysis revealed that only clonotypes originally harbored by LK-TN were significantly increased in counts at day 30 post-HSCT. In contrast, sequences unique to LK-TSCM, LK-TCM or LK-TEM were similar or reduced in counts at day 30 post-HSCT compared to LK, indicating that either PT-Cy efficiently dampened their expansion and/or that such memory lymphocytes persisted unchallenged upon in vivo infusion. Overall, in vivo fate mapping through TCR sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells upon HSCT, highlighting TSCM as privileged players in the diversification of immunological memory. We next validated these results at the antigen-specific level. In suitable 5 patients, we recorded the presence of WT1 or PRAME-specific TN cells within donor LK. Upon HSCT, tumor-specific T cells increased in frequency and displayed a memory phenotype, comprising TSCM. Finally, by quantifying patient serum cytokines, we found that the degree of IL-7 availability at day 1 post HSCT correlated with the extent of TSCM expansion at day 8. Conclusions: These data provide novel insights into TSCM biology and ongoing analyses will define correlations with clinical events. Longer immunological follow-up will advance our understanding of the contribution of early TSCM generation to the overall success of post HSCT IR. Disclosures Bordignon: MolMed: Employment. Bonini:MolMed S.p.A.: Consultancy.

BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output. TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation. The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.

Background Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. Results We studied 14 adult patients who underwent allo-HSCT (haploidentical HSCT: n=11; HLA-identical HSCT n=3) and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-2.8x108) for high-risk hematologic malignancies between 1995 and 2010. At a median follow-up of 8,7 years (range 3-17), all patients are in complete remission. Five out of 14 patients experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. We observed a complete recovery of NK cells, comprising of mature (CD56+CD16+) and immature (CD56+CD16-) NK cells. Interestingly the proportion of B cells circulating long-term in patients was significantly higher than that observed in age-related healthy controls (p TK-cells were detected in the majority of analyzed patients (90%), at low levels (median=0,43%±6,9%). Ex vivo selection of pure TK-cells after polyclonal stimulation and NGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. The proportion of TK-cells detectable at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first 3 months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Of notice TK-cells could be retrieved also in patients successfully treated with ganciclovir for GvHD, thus confirming the selective action of ganciclovir only on proliferating TK-cells. Accordingly, ganciclovir sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. While infused TK-cells displayed a predominant effector memory phenotype, gene modified T cells persisting long-term were enriched for central memory (CD45RA-CD62L+) and stem memory (CD45RA+CD62L+CD95+) phenotypes, suggesting the higher ability of these T cell subsets to persist and shape the immunological profile long-term in treated patients. Conclusion These data show that a complete donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 14 years in treated patients. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures: Valtolina: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Employment. Bonini:MolMed S.p.A: Consultancy.

Background Human herpesvirus type 6 (HHV-6) is a member of the beta herpesvirus subfamily (genus Roseolovirus) and two distinct variants have been described: types A and B. HHV-6 infection is recognized as the cause of a febrile disease and exanthem subitum in early childhood. The infection rarely causes serious events in healthy individuals, but viral reactivation in immunocompromised patients is frequently associated with severe clinical manifestations. Above all HHV-6 is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic hematopoietic stem cell transplantation (AlloSCT). Approximately 60% of solid organ transplant and 40% of patients undergoing alloSCT experience HHV-6 reactivation, mainly of variant B. Reported clinical manifestations of HHV-6 infection in transplanted patients are skin rash, interstitial pneumonia, bone marrow suppression and encephalitis. Moreover, some clinical reports suggest that HHV-6 can facilitate the occurrence of severe clinical complications of alloSCT, increasing transplant-related mortality. Methods From January 2009 to February 2013, we retrospectively evaluated 54 consecutive adult patients (median age 50 years) who developed positivity to HHV-6 after alloSCT for high-risk hematological malignancies. Stem cell donors were family haploidentical (37), HLA identical sibling (8), unrelated volunteer (6), cord blood (3). The viral load was determined by quantitative PCR (Nanogen Advanced Diagnostic S.r.L) in cell-free body fluids such as plasma, bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), bone marrow (BM) aspirates or in gastrointestinal biopsies. Results Median time from alloSCT to HHV-6 reactivation was 34 days (range: 0-705). Thirty-one patients presented HHV-6 positive in plasma, 9/54 in BM, 33/54 in gut biopsies or BAL, 7/54 in CSF. At the time of viral positivity all pts were receiving acyclovir as viral prophylaxis except five. Twenty-nine patients had acute graft versus host disease (GvHD). Twenty-two out of these twenty-nine patients experienced a grade III-IV acute GvHD, requiring high dose steroids in twenty-six cases. A concomitant CMV positivity was detected in 15/54 patients. The median absolute count of CD3+ lymphocytes was 262 cells/mcl. In 52/54 cases we reported HHV-6 clinical manifestations: fever (43), skin rash (22), hepatitis (19), diarrhoea (24), encephalitis (10), BM suppression (18), delayed engraftment (11). HHV-6 positivity led to antiviral pharmacological treatment in 37/54 cases, using as first choice therapy foscarnet. Amongst the total fifty-four patients with documented HHV-6 positivity thirty-one solved the clinical event. However the mortality rate was relatively high in this population (only 30% of patients were alive), mainly related to severe infections or GvHD. A better overall survival is significantly associated with CD3+ cells higher than 200/mcl (p-value 0.011) and time after alloSCT more than 2 months (p-value 0.035). In this analysis the overall survival was not significantly influenced by steroids administration, presence of acute GvHD, plasma viral load and organ involvement. Conclusions This retrospective study further demonstrates the correlation between HHV-6 reactivation and high morbidity and mortality rates in patients after alloSCT. Despite HHV-6 detection typically occurred in the first month after AlloSCT, a better immune reconstitution has the potential to improve the outcome. The regular monitoring of HHV-6 DNA, using a real-time PCR assay, may be useful for identifying active HHV-6 infection and for the introduction of a pre-emptive treatment, possibly reducing the incidence of the most severe clinical complications. Disclosures: Bonini: MolMed SpA: Consultancy.

Abstract 4191 Introduction: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a readily available therapeutic option for patients suffering from high-risk hematological malignancies who lack an HLA-compatible donor or for whom there is no time to find one. To avoid GVHD, haplo-HSCT has been classically performed under conditions of strict T-cell depletion, in the form of CD34-selected (“naked”) grafts. Unfortunately, naked haplo-HSCT is followed by a profound state of immune incompetence, which associates with high non-relapse mortality (NRM) rates, mainly due to opportunistic infections. Over the last decade, different strategies have been developed in order to speed-up immune reconstitution after haplo-HSCT, including the delayed infusion of donor T cells (DLI), the selective depletion of T-cell subsets from the graft or the use of an unmanipulated graft followed by novel strategies of immune suppression. Aim: To analyze in depth the early phenomena of immune reconstitution following haplo-HSCT in order to find early surrogate biomarkers of NRM. Results: From 2004 to 2010, we prospectively studied multiple parameters of T-cell immune reconstitution in 89 pts treated at our Center with haplo-HSCT. Time-points of analysis were pre-transplant, at day 30, day 90 and day 180 post-transplant. Underlying diseases included: high-risk AML 53 pts (60%), ALL 11 pts (12%), CLL 2 pts (2%), tyrosine kinase inhibitors-resistant CML 4 pts (5%), Hodgkin lymphoma 6 pts (7%), NHL 2 pts (2%), MDS with high International Prognostic Scoring System 7 pts (7%), other 4 pts (4%). Eighteen patients (20%) were given prophylactic suicide gene-modified DLI starting at day 30 post-transplant, while 64 patients (80%) received an unmanipulated graft followed by rapamycin until day 100. Overall, the incidence of grade III-IV acute GvHD was as low as 12%. Chronic extensive GvHD developed in 28% of pts. Compared with historical naked haplo-HSCT, the recovery of T-cell counts was accelerated: at day 90, median CD3+ cells were 378 per μL (0–2817), CD4+ 127 (0–804), CD8+ 173 (0–1922). Higher T-cell counts, however, did not clearly associate with lower NRM rates. After the initial expansion of effector memory cells (CD45RA−/CD62L−) in patients given DLI (P95%) pts that did not reactivate the virus at later time points. Strikingly, while in pts with 1000 spots/mL, this was 0% (P Conclusions: These results clearly indicate that the early reconstitution of T-cell immunity to CMV after haplo-HSCT does not only protect from subsequent viral reactivation, but also surrogates for lower NRM rates. Moreover, they warrant the investigation of a CMV-specific IFN-γ ELISPOT cut-off value of 1000 spots/mL as a predictive biomarker in larger, multicenter series. Disclosures: No relevant conflicts of interest to declare.

Abstract 1968 BACKGROUND: The genetic modification of T cells with a suicide gene grants a mechanism of control of Graft-versus-Host Disease (GvHD), allowing safe infusion of donor lymphocytes after partially HLA-incompatible Hematopoietic Stem Cell Transplantation (HSCT). In the TK007 phase I-II clinical trial, which enrolled a total of 54 adults with hematologic malignancies, 22 of the 28 treated patients experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the Herpes Simplex Virus Tymidine Kinase suicide gene (TK cells; Ciceri and Bonini et al., Lancet Oncology, 2009). In these patients, after a first wave of circulating TK cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naïve lymphocytes, leading us to hypothesize a thymus-dependent development of T cells, occurring only upon TK cell engraftment. METHODS: Thymic function was investigated in a total of 31 patients enrolled in the TK007 trial (median age 55 years), which were compared to a cohort of adult patients receiving non T cell-depleted haploidentical transplantation (n=31), and to healthy pediatric and adult subjects. T cell subsets and the proportion of CD31+ recent thymic emigrants amongst CD4 naïve T cells were measured by immunophenotypic analysis. Single joint T cell Receptor Excision Circles (sjTREC) were quantified by qPCR. The volume of the biologically active thymus was assessed by chest CT scans. Serum concentration of cytokines was assessed by a multiplex luminex-based assay. Pathogen-specific immunity was quantified by interferon-γ ELISpot. RESULTS: After the infusion of TK cells we documented a significant increase in peripheral blood sjTRECs as compared to the pre-HSCT determination (p = 0.02), suggesting an improved thymic output. Importantly, in line with that, only in TK007 patients almost the totality of CD4 naïve T cells circulating after transplantation were CD31+, thus bona fide recent thymic emigrants (89.54±9.55% at immune reconstitution, 81.84±15.9% at 6 months after HSCT, and 79.55±16.66% at 12 months after HSCT). Accordingly, a substantial expansion of the active thymic tissue was observed at chest tomography scans as compared to the pre-HSCT counterparts (p < 0.0001). A peculiar observation, possibly linked to the renewal of thymic activity and unique to the TK007 patients who achieved immune reconstitution, was the documentation of a peak in the serum level of interleukin-7, reproducibly occurring after each infusion of suicide gene-modified cells and anticipating the appearance of the newly generated T cells. Ultimately, the development of a wide repertoire of T cells in the patient thymus from donor precursors ensured a long-term protective immunity against pathogens, as exemplified by the preservation of a physiological and protective response against viruses both ex vivo and in vivo, even after the elimination of the infused TK cells in case of GvHD. CONCLUSIONS: Our data from TK007 patients show that the infusion of genetically modified donor T cells after transplantation can drive the recovery of thymic activity in adults, leading to long-term immune reconstitution. On the lead of the encouraging biological and clinical results of the phase I-II clinical trial, demonstrating a dramatic decrease in late infectious mortality, a multicenter, phase III clinical trial (TK008 study) to assess the efficacy of TK cells in the context of haploidentical HSCT for leukemia started in 2010 at the San Raffaele Institute, and is currently expanding to multiple centers throughout Europe and US. Main endpoints of this randomized phase III trial are disease free survival and overall survival. The first TK008 patients randomized to receive suicide gene-modified cells showed recovery of thimyc activity and concomitantly achieved a rapid and robust T cell immune reconstitution. Disclosures: Bonini: MolMed SpA: Consultancy.

Abstract 833 Introduction: Hematopoietic Stem Cell Transplantation (HSCT) from partially HLA-matched (haploidentical) family donors represents a promising therapy for high-risk leukemia, but requires appropriate strategies to control the adverse reactions mediated by the partially incompatible, transplanted immune system. In a recent phase II study (TK007 study), we demonstrated that the infusion of donor lymphocytes transduced with the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows to control Graft-versus-Host Disease (GvHD) and to rapidly provide an effective and polyclonal anti-infective T cell repertoire (Ciceri and Bonini et al., Lancet Oncology, 2009). Even though their engraftment is necessary to achieve these effects, HSV-Tkpos cells represent the minority of lymphocytes circulating in treated patients. Therefore, in the present study, we investigated the putative role of HSV-Tkpos cells in promoting thymic activity and T cell development from graft progenitors. Methods: Twenty-eight adult patients underwent haploidentical HSCT and infusion of purified suicide gene-modified donor T cells for high-risk hematologic malignancies in the TK007 study. Thymic function was investigated in a selected cohort of this study (n=14) and in a control group who underwent unselected T cell-replete haploidentical transplantation with an ATG-rapamycin-mycophenolate-based GvHD prophylaxis (n=31), after validation in healthy pediatric and adult controls. T cell subsets and the proportion of CD31+ recent thymic emigrants (RTEs) amongst CD4+ naïve T cells were measured by immunophenotypic analysis. Single joint T cell Receptor Excision Circles (sjTREC) were quantified by qPCR. Thymic output was correlated with thymic volume, as assessed by CT scans. Post-transplantation pathogen-specific immune response was quantified by ELISpot. Alloreactivity against leukemic blasts was studied by mixed lymphocyte cultures. Results: Post-transplantation recovery of naïve CD45RA+CD62L+ T cells occurred in patients treated with gene modified T cells, reaching values of healthy controls in approximately one year. At the time of immune reconstitution (median 76 days after HSCT, defined as CD3+ cells > 100/ml peripheral blood), 76.5% of circulating T cells did not carry the HSV-Tk suicide gene, and the CD4+ naïve subset was largely comprised of cells recently originated from the thymus (90.5±3.2%). This observed frequency of CD31+ RTEs in these patients was significantly higher than that measured in the same patients before HSCT (60.7±6%, p=0,0087) or in patients analyzed 90 days after T cell-replete haploidentical HSCT (31.0±6.3%, p Conclusions: These data show that the infusion of suicide gene-modified T cells prompts the renewal of thymic activity, which contributes to the recovery of a polyclonal T cell repertoire protective against pathogens. Contextually, the infused transduced cells mediate also a direct antitumor effect through their recognition of allogeneic determinants on leukemic cells. A phase III clinical trial (TK008 study) to assess the efficacy of HSV-Tkpos cells in the context of haploidentical HSCT for leukemia started in 2010 in Italy, and is currently expanding to multiple centers throughout Europe. Disclosures: Bonini: MolMed S.p.A.: Consultancy.

Abstract 46 The broader application of haploidentical stem cell transplantation (haplo-HCT), is limited by the delayed immune reconstitution (IR) secondary to the procedures for GvHD prophylaxis. This ultimately results in a high-rate of infectious complications and non-relapse mortality. We dynamically analyzed immunoreconstitution (IR) in patients undergoing haplo-HCT for acute leukemias enrolled in two different phase I-II clinical trials aimed at improving IR. In the first trial (TK007), 28 patients (out of 50 enrolled) received suicide-gene transduced donor T cells at day +42 after a T-cell depleted graft, in the absence of post-transplant immunosuppression. In the second trial (TrRaMM), 40 patients received an unmanipulated graft and a rapamycin-based GvHD prophylaxis. T-cell immune reconstitution was more rapid in TrRaMM than in TK007 patients, with a threshold of CD3 cells>100/μl reached at days +30 and +90, respectively. In both trials IR was mainly composed of Th1/Tc1 lymphocytes with an inverted CD4/CD8 ratio. While in TrRaMM patients we observed an early expansion of naïve and central memory T cells, producing high amounts of IL-2, in TK patients IR was mainly composed of activated effectors. Furthermore, in TrRaMM patients we detected high levels of CD4+CD25+CD127- T regulatory cells (up to 15% of circulating T lymphocytes) that persisted after rapamycin withdrawal, and was significantly superior to that observed in TK patients and in healthy controls. Interestingly, in contrast to the different kinetics of T-cell reconstitution, no differences were observed in time required to gain protective levels of CMV-specific T cells, as shown by ψIFN ELISPOT analysis. Protective frequencies of CMV-specific lymphocytes were observed 3 months after HCT in both groups, a time-point that in TrRaMM patients corresponds to the average time of rapamycin withdrawal. In both trials the number of circulating CMV-specific T cells was inversely correlated to the number and severity of subsequent CMV reactivations and days of antiviral therapy. GvHD was diagnosed in 16 TrRaMM patients (40%) and in 10 TK patients (35% of patients who received TK cells). Severity of GvHD was different in the two cohort of patients with 5 TrRaMM patients (12,5%) and only 2 TK patients (7%) with grade III-IV GvHD. Of interest, in the TrRaMM group CMV-specific immunity was significantly hampered by the immunosuppressive treatment required to treat GvHD. On the contrary, in the TK group, the administration of ganciclovir was able to activate the suicide machinery and control GvHD without impairing viral-specific T-cell immunocompetence. These results matched with the kinetics of CMV reactivations. We observed that while in TrRaMM patients 80% of viral reactivations occurred after the immunosuppressive therapy, in TK patients no significant differences could be assessed before and after therapy. IFN-ψ ELISPOT might thus be a relevant and predictive test to guide patient-specific clinical monitoring and antiviral treatment. Overall, these results show that early immune reconstitution can be promoted in haplo-HCT by different strategies associated with a wide range of alloreactive potential. The risks and benefits associated with alloreactivity should guide the therapeutic choice tuned on patient disease status and co-morbidities. Disclosures: Bordignon: Molmed Spa: Employment.