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Abstract 4195 Directed family cord blood (DCB) storage provides hemopoietic stem cell source for transplantation (HSCT) for families with an existing or a potentially future recipient with HSCT curable disease (D). The National CB Banks Network in Italy (ITCBN) has a leading role in providing public DCB service for high-risk families, in compliance to GITMO directives for eligibility criteria (HSCT curable D: malignant MD, non MD, inherited ID). To provide best cost-effective practices recommendations it is important to report on DCB procedures and HSCT rate (HSCT-R) among public Banks. By 12.12.2008 almost 1800 DCB units were stored in 18 Italian Banks and 104 (9%) issued for HSCT. The present survey aims at summarizing the over 15 yrs DCB experience among 5 ITCBN Banks active since 1997 (range 1990-1997), and including 670 DCB units. Results Preliminary analysis reports a 94% overall compliance to eligibility criteria directives, and overall HSCT- R for an alive sibling of 12% (63/522); the 63 HSCT were 97% matched, for curing ID in 84% and with 72 % overall survival outcome. Different policies among Banks were compared (Bank vs others: 1) eligibility criteria distribution : Bank PV06 DCB for MD Conclusion. Advice for public long-term DCB storage should depend on HLA compatibility, potential recipient, disease progression, and likelihood/ timing of using DCB; Italian CB Banks are actively cohoperating to find best public banking practices to ensure a CDB storage system that is ethical, cost effective and responsive to patient needs. Disclosures: No relevant conflicts of interest to declare.

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.

CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- 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 immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.