Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

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 < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-ß, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Homocysteine and education but not lipoprotein (a) predict estimated 10-year risk of cardiovascular disease in blood donors: a community based cross-sectional study.

BACKGROUND: With aging of the population, screening and prevention health programs for blood donors will increasingly be a priority. We aimed at: assessing the 10 year-cardiovascular disease (CVD) risk in blood donors, according to Italian CUORE risk score (CRS); determining the association of homocysteine (Hcy), lipoprotein (Lp)(a) and socio-demographic or lifestyle variables with estimated 10-year CVD risk. METHODS: Between June 2015 and July 2017, 1,447 (61.2% men) unselected blood donors (aged 18-69 years) were enrolled at the Blood Transfusion Service of the Pescara General Hospital, Italy. The project entailed evaluation of unalterable (age and gender) and modifiable CV risk factors (total cholesterol, HDL, LDL, triglycerides, fasting glucose, smoking, hypertension). The educational attainment, socio-demographic and lifestyle behavior information were obtained through a structured self-report questionnaire, and Health-related quality of life (HRQoL) through the Short Form Survey (SF-12). Plasma Hcy and Lp(a) were determined in the fasting state. RESULTS: A CRS within the moderate-high risk range was reported in 21.7% donors. Multivariate logistic regression, after adjustment for clinical and demographic variables, showed that Hcy [OR (95% CI): 1.09 (1.04-1.13); p < 0.001) and low educational attainment [1.71 (1.09-2.73); p = 0.019] are independent risk factors for moderate-to-high CVD risk. Instead, Lp(a), evaluated in 774 donors, was > 30 mg/dL in 22.4% of the examined population, but without any significant correlation with CRS. CONCLUSIONS: Our study highlights a previously unappreciated need for CV risk assessment in blood donors, which may include evaluation of educational attainment as a non-traditional risk marker.

Daratumumab-based induction and autologous transplantation in concomitant multiple myeloma and chronic myeloid leukemia.

The coexistence of chronic myeloid leukemia (CML) and multiple myeloma (MM) is a rare clinical condition. By means of FISH and molecular analysis on both sorted CD138 plasma cells and cryopreserved CD34 stem cells, a distinct clonal origin of the hematological malignancies was demonstrated in our case. We report on the first patient diagnosed with CML and MM treated with daratumumab, bortezomib, thalidomide, and dexamethasone (Dara-VTd) induction, stem-cell collection, and autologous stem cell transplantation (ASCT). The co-administration of Dara-VTd and imatinib proved feasible and highly effective in the management of both CML and MM. Despite concerns with stem cell mobilization and collection in patients exposed to daratumumab, in our experience the use of higher cyclophosphamide dose 4 g/m2 together with plerixafor granted optimal stem cell mobilization and collection, irrespective of daratumumab, concomitant myeloid neoplasm, and imatinib. Moreover, ASCT was easily performed with a rapid hematological reconstitution.

Is the absence of JAK2 mutation a risk factor for bleeding in essential thrombocythemia? An analysis of 106 patients.

BACKGROUND: JAK2(V617F) mutation has been recognized as a possible thrombotic risk factor in essential thrombocythaemia (ET). It's role is probably due to an increased myeloid proliferation and white blood cells (WBC) activation. Only few data are available about the effect of JAK2(V617F) on hemorrhagic risk. The aim of our study was to evaluate the influence of the mutational status on hemorrhagic complication. METHODS: We retrospectively analysed laboratory and clinical findings of 106 consecutive patients with ET to evaluate possible relationships between thrombosis, abnormal bleeding, peripheral blood count, overexpression of PRV1 and JAK2(V617F) mutational status. RESULTS: ON UNIVARIATE ANALYSIS WE FOUND: an association between JAK2(V617F) mutation and thrombotic events before or at diagnosis (p<0.003, OR=4.44, 95% CI=1.74-12.4); no statistical correlation between the median value of JAK2(V617F) burden and an increased risk of thrombosis (p=0.4, 95% CI= -22.8-10.4); significant relationships between mutated status and higher haematocrit, high WBC count and low platelet count; and a strong correlation between JAK2(V617F) and PRV1 overexpression (p<0.0001). Moreover, the presence of the JAK2(V617F) mutation and a WBC count greater than 8.4 x 10(9)/L were found to be independent factors related to thrombotic complications in multivariable analysis (p<0.006, OR=3.85, 95% CI=1.3-11.9; and p<0.002, OR=2.8, 95% CI=1.08-7.03, respectively). The prognostic impact of JAK2 mutation status and WBC count on thrombosis was evaluated in the whole cohort. Only new cases occurring in patients without previous thrombotic events were recorded for the analysis. The multivariable analysis showed a statistical correlation between the presence of the mutation and a WBC count greater than 8.12 x 10(6)/L and an increased risk of thrombosis if no cytoreductive treatment was started at diagnosis (JAK2(V617F) p=0.02; WBC p=0.02; OR=4.97; 95% CI=1.04-23.8). Finally, wild-type JAK2 was associated with a higher haemorrhagic risk (p=0.02) in univariate analysis but only a platelet count greater than 1,022 x 10(9)/L was associated with an increased risk of bleeding in the multivariable analysis. CONCLUSION: Our data confirm the role of both JAK2(V617F) as factor associated with an increased risk of thrombosis at the diagnosis and during follow-up in no treated patients. Moreover a WBC count over 8.4 x 10(9)/L1 was also strictly associated to an increased risk of thrombosis. Regarding bleedings, our statistical analysis allows to exclude the mutation protective role on haemorrhage.

Inherited bleeding disorders: results from the Italian Regional Haemophilia Centre of Pescara.

BACKGROUND: Inherited bleeding disorders registries can be useful to improve health care of these rare disorders and document their natural history. MATERIAL AND METHODS: We analysed the epidemiological, diagnostic and therapeutic aspects of patients managed at an Italian Regional Haemophilia Centre, based in Pescara (in the Region of Abruzzo). RESULTS: This Regional Haemophilia Centre currently follows 376 patients: 248 with rare clotting factor defects, 33 with von Willebrand's disease, 75 with haemophilia A and 20 with haemophilia B. Three patients with severe haemophilia A have developed inhibitors. Among all the haemophiliacs, the prevalence of hepatitis C virus infection is 21% while the prevalence of human immunodeficiency virus infection is 5.3%. Among the whole haemophilic population referring to the Pescara Haemophilia Centre, 87.4% are treated with recombinant factors while 12 patients with severe haemophilia are receiving primary prophylaxis. CONCLUSION: In brief, an analysis of the epidemiological, clinical and therapeutic data collected at the Regional Haemophilia Centre of Pescara is a useful tool for monitoring and continuously improving the quality of care of patients with inherited bleeding disorders.