Functional Analysis and Gene Expression Profile of Umbilical Cord Blood Regulatory T Cells.
Authors: Torelli Giovanni Fernando , Maggio Roberta , Peragine Nadia , Chiaretti Sabina , De Propris Maria Stefania , Screnci Maria , Lucarelli Barbarella , Mascolo Maria Grazia , Iori Anna Paola , Girelli Gabriella , Guarini Anna , Foà Robert 
Published: October 2019

Abstract
Abstract 4085
Poster Board III-1020

Introduction
Umbilical cord blood (CB) stem cells are now broadly used in the unrelated stem cell transplant setting and comparative studies with different stem cell sources have shown that CB transplant is characterized by a lower risk of graft-versus-host disease (GVHD). The immaturity of CB T cells has been generally regarded as the main contributing factor accounting for this phenomenon; the possible role played by CB regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is now under investigation, but very scare data are so far available. Aim of this study was to analyze and compare the functional properties and the gene expression profile of Tregs expanded from CB units with those expanded from the peripheral blood (PB) of adult normal donors.

Methods
Tregs were purified from mononuclear cells obtained from 23 CB units and from the PB of 13 adult normal donors using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with the anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml) MoAbs in the presence of IL-2 (100 U/ml). Immunophenotypic analyses were performed before and after expansion. To assess their suppressive functions, expanded Tregs were seeded with autologous effector T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The IL-10 production capacity of expanded Tregs was tested using an ELISA assay. The two-sided student t test was used to evaluate the significance of differences between groups. Gene expression profile experiments were performed using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analyses were carried out using the dChip software; a t test was used to evaluate the presence of specifically expressed classes of genes. Functional annotation analysis was performed using the DAVID software.

Results
CB and PB Tregs presented similar immunophenotypic appearances before and after expansion. Im particular, after expansion they presented a comparable expression of surface CD4, CD25, CD62L, CCR5 and CD45RO, and of cytoplasmic CTLA-4 and Foxp3, while they both were negative for the CD45RA antigen, thus indicating the loss of their naïve features. On the contrary, Tregs obtained from CB (n=23) presented a much higher expansion capacity compared to those obtained from PB (n=13): mean fold increase (range), CB 10.3 (1.6-24), PB 3.9 (1.5-10), p 0.003. CB expanded Tregs (n=6) exerted a potent suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC, that resulted inferior even though not significantly compared to that exerted by PB expanded Tregs (n=5): mean fold reduction (range), CB 7.8 (2.5-15.1), PB 14.3 (1.5-23.7), p 0.14. Tregs expanded from CB (n=4) and PB (n=1) presented a high and comparable in vitro IL-10 production capacity: mean pg/ml (range), CB 326.5 (226-426), PB 382. Gene expression profile analysis showed a higher number of upregulated genes in Tregs expanded from CB (n=2) compared to Tregs expanded from PB (n=3); among them, a significant enrichment of genes involved in cell proliferation, cell cycle checkpoints, signal transduction, cell differentiation, apoptosis, TGF-β receptor pathway and the GrNH pathway was observed. This suggests that CB Tregs retain a more undifferentiated program and are characterized by the high expression of genes which might provide an advantage in cell expansion. Finally, when looking at the Foxp3 gene expression levels, no difference was observed between the two populations.

Conclusions
These results demonstrate that Tregs contained in CB retain an expansion potential superior to that of Tregs isolated from the PB of normal donors, as confirmed by functional analyses and gene profile. Tregs expanded from CB and PB seem to exert a potent and comparable suppressive function of the proliferative effect in mixed lymphocyte reaction assays. The maintaining of the modulatory properties after expansion is confirmed by the expression of the Foxp3 gene and protein, and by the production of IL-10. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T-cell response.

Disclosures:
No relevant conflicts of interest to declare.