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.

Cellular Strategies for Separating GvHD from GvL in Haploidentical Transplantation.

GvHD still remains, despite the continuous improvement of transplantation platforms, a fearful complication of transplantation from allogeneic donors. Being able to separate GvHD from GvL represents the greatest challenge in the allogeneic transplant setting. This may be possible through continuous improvement of cell therapy techniques. In this review, current cell therapies are taken into consideration, which are based on the use of TCR alpha/beta depletion, CD45RA depletion, T regulatory cell enrichment, NK-cell-based immunotherapies, and suicide gene therapies in order to prevent GvHD and maximally amplify the GvL effect in the setting of haploidentical transplantation.

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.

A multiple regression analysis on factors influencing haematopoietic progenitor cell collection for autologous transplantation.

Autologous hematopoietic stem cell (HSC) transplantation today is the standard treatment for a wide variety of haematological and oncological diseases. HSC are collected from peripheral blood by leukapheresis (HPC-A) following chemotherapy and/or growth factor-mediated mobilization. The ideal HPC-A collection allows to reach the CD34(+) target dose through a single, tailored leukapheresis. The aim of this paper was to find out which collection parameter might play a key role in obtaining a CD34 dose >4×10(6)/kg with a reduced number of leukapheresis. To address this issue, a multivariate logistic regression was carried out on several operational and laboratory parameters from 943 HPC-A collections performed in 600 hematological and oncological patients. We observed a CD34(+) cells collection efficiency (CE) >50% when patient's pre-apheresis total WBC count was lower than 12.5×10(6)/mL. At the same time, the likelihood of reaching the CD34(+) cells target dose/kg increased from 6 to 3 times when the pre-apheresis WBC count ×10(6)/mL t was below 4.3 (OR=6.1; 2.6-14.1) and between 4.3 and 7 (OR=2.8; 1.4-5.7) respectively when compared to a pre-apheresis WBC count >36×10(6)/mL.

Homocysteine modulates the CD40/CD40L system.

OBJECTIVES: This study evaluated the impact of hyperhomocysteinemia (HHcy) on the CD40/CD40 ligand (CD40L) dyad in vivo and in vitro. BACKGROUND: Hyperhomocysteinemia is associated with an increased incidence of atherothrombosis, although the molecular mechanisms of this association are incompletely defined. The CD40L pair triggers inflammatory signals in cells of the vascular wall, representing a major pathogenetic pathway of atherosclerosis. METHODS: We used a commercially available enzyme-linked immunosorbent assay kit to evaluate circulating levels of soluble (s) CD40L in 24 patients with HHcy and 24 healthy subjects. We also used real-time polymerase chain reaction and flow cytometry to determine expression levels of CD40 and vascular cell adhesion molecule (VCAM)-1 in human umbilical vein endothelial cells (HUVECs) and of CD40L in human platelets. RESULTS: The sCD40L levels were significantly increased in HHcy patients (median [interquartile range] 8.0 [0.7 to 10.5] ng/ml vs. 2.1 [1.9 to 2.3] ng/ml, p = 0.0001). Positive correlations were noted between log sCD40L and log homocysteine (Hcy) (R = 0.68, p < 0.0001) or log sVCAM-1 (R = 0.41, p < 0.005). Homocysteine significantly stimulated CD40 mRNA expression in HUVECs (p = 0.033). Consistently, 24-h exposure to Hcy increased the percentage of CD40-expressing cells (p = 0.00025). Homocysteine also significantly enhanced CD40L expression in platelets (p = 0.025) to a comparable extent as that of thrombin. Notably, Hcy increased VCAM-1 protein expression induced by CD40L in HUVECs (p = 0.0046). CONCLUSIONS: The present results uncover a potential molecular target of Hcy, namely the CD40/CD40L dyad. Collectively, they indicate that upregulation of CD40/CD40L signaling may represent a link between HHcy and an increased risk of cardiovascular disease.