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Figure S4

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Purpose:Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response.Experimental Design:NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1.Results:In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells.Conclusions:We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.

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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.

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2–dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the twice-daily and thrice-daily regimens showed substantially reduced interindividual variability and lower median (interquartile range) values for sTXB2 (ng/mL) compared with the once-daily arm: 4 (2.1-6.7; n = 79), 2.5 (1.4-5.65, n = 79), and 19.3 (9.7-40; n = 85), respectively. Urinary PGIM was comparable in the 3 arms. Urinary TXM was reduced by 35% in both experimental arms. Patients in the thrice-daily arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75 to 100 once daily for cardiovascular prophylaxis appears to be largely inadequate in reducing platelet activation in the vast majority of patients with ET. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement with further reductions (EudraCT 2016-002885-30).

Background: Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is a valid treatment for hematological oncological or metabolic diseases. Despite its therapeutic efficacy, it is an aggressive treatment that impacts negatively on quality of life (QoL) and may result in Post-Traumatic Stress Disorder (PTSD) symptoms. The aim of this study is to explore rates and risk factors for PTSD symptoms, and fatigue in post-HSCT patients with hematological malignancies. Methods: A total of 123 patients after HSCT were evaluated for PTSD symptoms, QoL and fatigue. PTSD symptoms were assessed with the Impact of Event Scale- Revised (IES-R), QoL was measured with Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) and fatigue symptoms were assessed with Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F). Results: A total of 58.54% of the sample developed PTSD symptoms after transplant. Patients with PTSD symptoms reported significantly lower QoL total scores and significantly higher fatigue than those without PTSD symptoms (p < 0.001). The SEM analysis showed that worse QoL and fatigue affected PTSD symptomatology along different pathways. Fatigue was found as a major influencing factor of PTSD symptoms directly (β = 0.31 **), while QoL only through the mediation of fatigue at a lesser extent. (β = 0.33 *). Conclusions: Our findings indicate that QoL is a concurrent causative factor to the development of PTSD symptomatology through the mediating role of fatigue. Innovative interventions before transplantation to prevent PTSD symptoms should be investigated to improve survival and QoL in patients.

publishedVersion

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Purpose: Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response. Experimental Design: NOTCH activations was evaluated either in vitro and ex vivo in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1. Results: In vitro ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of in situ PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells. Conclusions: We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.

Donor lymphocyte infusion (DLI) is a treatment option to prevent or treat relapse after allogeneic hematopoietic cell transplantation (HCT). We here report data for 173 patients who received one or multiple DLIs after haploidentical-HCT with post-transplant cyclophosphamide (PTCY) at 47 EBMT centers from 2009 to 2018. Indication for DLI was: prophylactic for 59 (34.3%), preemptive for 20(11.6%), and therapeutic for 93(54.1%). For the prophylactic group, the median number of DLIs was 1 (IQR:1-2.5) with a median first dose of 0.1 × 10

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once-daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once- vs. twice- or three-times daily low-dose aspirin in inhibiting platelet thromboxane (TX) A2 production, as reflected by serum (s) TXB2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once-daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P

Key features of chronic lymphocytic leukemia (CLL) are defects in the immune system and the ability of leukemic cells to evade immune defenses and induce immunosuppression, resulting in increased susceptibility to infections and disease progression. Several immune effectors are impaired in CLL, including T and natural killer (NK) cells. The role of T cells in defense against CLL and in CLL progression and immunotherapy has been extensively studied. Less is known about the role of NK cells in this leukemia, and data on NK cell alterations in CLL are contrasting. Besides studies showing that NK cells have intrinsic defects in CLL, there is a large body of evidence indicating that NK cell dysfunctions in CLL mainly depend on the escape mechanisms employed by leukemic cells. In keeping, it has been shown that NK cell functions, including antibody-dependent cellular cytotoxicity (ADCC), can be retained and/or restored after adequate stimulation. Therefore, due to their preserved ADCC function and the reversibility of CLL-related dysfunctions, NK cells are an attractive source for novel immunotherapeutic strategies in this disease, including chimeric antigen receptor (CAR) therapy. Recently, satisfying clinical responses have been obtained in CLL patients using cord blood-derived CAR-NK cells, opening new possibilities for further exploring NK cells in the immunotherapy of CLL. However, notwithstanding the promising results of this clinical trial, more evidence is needed to fully understand whether and in which CLL cases NK cell-based immunotherapy may represent a valid, alternative/additional therapeutic option for this leukemia. In this review, we provide an overview of the current knowledge about phenotypic and functional alterations of NK cells in CLL and the mechanisms by which CLL cells circumvent NK cell-mediated immunosurveillance. Additionally, we discuss the potential relevance of using NK cells in CLL immunotherapy.

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P 95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

As previously reported, chronic lymphocytic leukemia (CLL) cells show constitutive Notch1/2 activation and express the Notchligand Jagged1. Despite increasing knowledge of the impact of Notch alterations on CLL biology and pathogenesis, the role of Jagged1 expressed in CLL cells remains undefined. In other cell types, it has been shown that after Notch engagement, Jagged1 not only activates Notch in signal-receiving cell, but also undergoes proteolytic activation in signal-sending cell, triggering a signaling with biological effects. We investigated whether Jagged1 expressed in CLL cells undergoes proteolytic processing and/or is able to induce Notch activation through autocrine/paracrine loops, focusing on the effect that CLL prosurvival factor IL-4 could exert on the Notch-Jagged1 system in these cells. We found that Jagged1 was constitutively processed in CLL cells and generated an intracellular fragment that translocated into the nucleus, and an extracellular fragment released into the culture supernatant. IL-4 enhanced expression of Jagged1 and its intracellular fragments, as well as Notch1/2 activation. The IL-4-induced increase in Notch1/2 activation was independent of the concomitant upregulated Jagged1 levels. Indeed, blocking Notch-Jagged1 interactions among CLL cells with Jagged1 neutralizing antibodies did not affect the expression of the Notch target Hes1. Notably, anti-Jagged1 antibodies partially prevented the IL-4-induced increase in Jagged1 processing and cell viability, suggesting that Jagged1 processing is one of the events contributing to IL-4-induced CLL cell survival. Consistent with this, Jagged1 silencing by small interfering RNA partially counteracted the capacity of IL-4 to promote CLL cell survival. Investigating the pathways whereby IL-4 promoted Notch1/2 activation in CLL cells independent of Jagged1, we found that PI3Kδ/AKT and PKCδ were involved in upregulating Notch1 and Notch2 proteins, respectively. Overall, this study provides new insights into the Notch-ligand system in CLL cells and suggests that targeting this system may be exploited as a novel/additional therapy approach for CLL.

After conventional (“unmanipulated”) allogeneic hematopoietic cell transplantation, eradication of residual disease, i.e., the so-called graft-versus-leukemia (GvL) effect, depends on donor T-lymphocytes which recognize host histocompatibility antigens on leukemic cells. However, this transplant is far from optimal because it is associated with high incidence of relapse and risk of graft-versus-host disease (GvHD). Recent clinical trials suggest that adoptive immunotherapy with regulatory and conventional T-lymphocytes prevents GvHD while allowing a GvL effect in acute leukemia patients undergoing T-cell-depleted-haploidentical transplantation. We discuss the clinical relevance of this new immunotherapeutic strategy and the mechanisms underlying the separation of GvL effect from GvHD.

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.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

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.

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p Disclosures No relevant conflicts of interest to declare.

The NOTCH and nuclear factor kappa B (NF-κB) pathways are both constitutively activated in Chronic Lymphocytic Leukemia (CLL). We first described the NOTCH1 PEST domain mutation in a CLL subgroup, but the activation of the NOTCH pathway in NOTCH1-unmutated cases remains unexplained. Here, we investigated whether genetic lesions in the NF-κB/NOTCH loop might support the NOTCH activation status by sequencing negative (TNFAIP3/A20) and positive (TRAF2, TRAF5, TNFRSF11A/RANK, MAP3K7/TAK1, and CARD11) regulators of NF-κB together with NF-κB targets on the NOTCH pathway, the NOTCH ligands Jagged1 and Jagged2, in CLL patients. The sequence analysis revealed four missense mutations for A20, TRAF2, TRAF5 and RANK1 genes, all causing a change in amino acid group from polar to non-polar, but functional domains were not involved. Specific predictive software analyses confirmed that the amino acid changes have a low-functional impact on the protein. Our results show that in CLL, NF-κB regulators and Jagged are both unmutated, suggesting that the Jagged-mediated interplay between NF-κB and NOTCH is independent of genetic lesions.

In high-risk acute leukemia patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), adoptive immunotherapy with T regulatory cells (Tregs) and T conventional cells (Tcons) prevented acute and chronic graft-versus-host disease (GvHD), favored post-transplant immunological reconstitution and was associated with a powerful graft-versus-leukemia (GvL) effect. With a particularly innovative approach, we developed a treatment with a Treg-protected donor lymphocyte infusion (DLI) for patients with early relapse after HSCT and we report here the results obtained in the first patient with APL (M3v) relapsed after a second matched allogeneic HSCT (15% blasts and 75% of donor cells in bone marrow). The patient received a first infusion of 2.5 × 106/kg Tregs derived from matched donor followed 7 days later by 5 × 106/kg Tcons. GvL effect was strongly evident as the percentage of leukemic cells decreased to 5%. A second infusion of Tregs (2.5 × 106/kg) and Tcons (2 × 106/kg) was performed. No GvHD was observed. Disease evaluation showed the absence of blastic cells at flow-cytometry, a normal caryotype and full donor chimerism. We also observed NOTCH1 down-regulation in peripheral blood. This new immunotherapy approach showed that Treg-protected DLI is effective in preventing GvHD and is associated with a strong GvL effect.

Purpose Richter syndrome (RS) is a rare event in chronic lymphocytic leukemia (CLL) that is influenced by biological factors and prior CLL treatments. Ibrutinib is a Bruton tyrosine kinase inhibitor that has shown remarkable efficacy in CLL; however, little is known about its relationship to RS. We report a case of ibrutinib efficacy against CLL in a patient with prolonged remission of RS. Methods The patient was diagnosed with CLL in 2003. Biological findings at onset included absent ZAP70 expression, mutated IGVH, and NOTCH1 mutation. He was treated with FCR with partial response. In 2013, he progressed to RS, not clonally related to the underlying CLL. The patient was treated with anthracycline- and platinum-based regimens, obtaining a complete remission. After 3 years, he presented a CLL progression with worsening lymphocytosis, anemia, thrombocytopenia, increased splenomegaly, and lymphadenopathies. Positron emission tomography-computed tomography scan excluded pathologic uptake. Thus, he was started on ibrutinib. Results At 12 months’ follow-up, we observed white blood cell normalization, increased hemoglobin and platelet levels, disappearance of lymphadenopathy, and spleen size reduction. Therapy was well-tolerated with no evidence of RS. Conclusion This case demonstrates sustained RS remission in a patient with CLL under ibrutinib therapy, thus improving our knowledge on the use of this new drug in CLL and beyond.

We developed a good manufacturing practices–compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons.

Notch1 signaling is involved in regulatory T (Treg)-cell differentiation. We previously demonstrated that, when cocultured with CD3+ cells, mesenchymal stem cells (MSCs) induced a T-cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4+ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI-I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC-induced CD4+CD25highFOXP3+ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF-β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg-cell induction mediated by MSCs.

The C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-? and interleukin-10 (IL-10), in addition to IL-1ß, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization., This work was supported by the Specific Targeted Research Projects MANASP (LSHE-CT-2006), contract 037899 (FP6), and SYBARIS (LSHE-CT-2006), contract 242220 (FP7), and the Italian Projects PRIN 2007KLCKP8_004 (L.R.) and 2007XYB9T9_001 (S.B.). C.C. and A. Carvalho were supported by Fundacao para a Ciencia e Tecnologia, Portugal (fellowship contracts SFRH/BD/65962/2009 and SFRH/BPD/46292/2008, respectively).

Introduction Multiple Myeloma (MM) is a hematologic malignancy characterized by proliferation of clonal plasmacells in the bone marrow and sometimes extramedullary sites which inevitably tend to relapse. Patients with relapsed/refractory MM (R/R MM) who are treated with conventional drugs as immunomodulating agents (IMiDs) and proteasoma inhibitors (PIs) have a poor survival. Based on the results of MM 003 phase III randomised trial, pomalidomide in association with dexamethasone has shown to improve R/R MM outcome. We conducted a single-center real life-based observational and pharmacological analysis to investigate the efficacy and tolerability of pomalidomide in a group of patients with R/R MM previously exposed to IMiDs and PIs. Method and Materials We included 34 patients (females 21) treated with pomalidomide (POMA) from January 2015 to May 2018 at the Hematology Department of "Santo Spirito" Hospital in Pescara, Italy. All patients were diagnosed by R/R MM according to IMWG guidelines and were classificated using ISS criteria. Prognostic cytogenetic value was identified by FISH analysis. POMA was administred at the oral daily dose of 3-4 mg (days 1-21) in combination with oral dexamethasone (days 1,8,15,22) at the dosage of 20 mg/day. Adverse events were evaluated according to the common terminology criteria for adverse reactions (CTCAE version 4.0). Overall survival (OS) and progression free survival (PFS) were designed according to Kaplan-Myer method. The adherence to therapy was determined using 'received daily dose/prescribed daily dose' method (RDD/PDD). Statistic analysis were conducted with GraphPad Prism 7.0; p values ≤ 0.05 were considered statistically significant. Results The clinical and biological characteristics of 34 patients are described in Tab.1. The median follow up after POMA therapy was 5 months (range, 1-32); at the end of this period 12 patients had completed at least 6 cycles of therapy (range, 1-21). Overall response rate (ORR) was 24%: 4 complete remission (CR), 3 very good partial response (VGPR), 1 partial response (PR); moreover 6 patients (18%) achieved a minimal response (MR). Clinical benefit rate (CBR) was 74%: ORR plus 6 MR and 11 stable disease (SD). At time of this report 12 patients (35%) are living. Among living patients, 4 are now receiving other treatments and 8 are still under POMA treatment: 4 are in CR, 1 is in PR, 1 in MR and 2 in SD. Median OS of all patients was 10 months, whereas median PFS was 6 months (Fig.1,A-B). Median PFS was notably lower in high cytogenetic risk patients (4 months) as compared to the PFS of standard cytogenetic risk patients (16 months) (p= 0.02) (Fig.1,D). Patients treated with at least 4 previous lines of therapy had a median PFS of 5 months, while those receiving until 3 previous lines of therapy had a median PFS of 24 months (p= 0.01) (Fig.1,C). Patients receiving a drug regimen lenalidomide-based as last treatment before starting POMA didn't show significant difference in PFS from those receiving other treatments (4 months vs 6 months) (p=NS). During treatment with POMA, 22 patients (65%) showed adverse drug reactions (ADRs) for a total of 77. Seventy-four ADRs were ≥ 2 grade. Forty nine ADRs (63%) were hematological and 43 ADRs (56%) occurred in the first 3 months of therapy. In particular Twenty-two of them occurred within the first month of therapy. Thirty two cases of ADRs (41%) were successfully managed with simple delay of treatment. As consequence, the adherence to therapy was not optimal (78%). However only 9 (12%) of the total ADRs were cause of end of treatment. Conclusions Despite the low sample size and short follow-up, our real life analysis contributes to show that pomalidomide therapy is a favorable option for patients with R/R MM, leading to good responses in terms of OS and PFS. The best results were obtained when POMA treatment was utilized at first relapses and in particular in patients with favorable genetic profile. About the safety, despite the frequent hematological toxicity, ADRs can be quite easily managed to allow few interruptions of the treatment and improve the adherence. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.