Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.
ABSTRACT Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.
Article: Cyclosporin A effects during primary and secondary activation of human umbilical cord blood T lymphocytes.[show abstract] [hide abstract]
ABSTRACT: Cyclosporin A (CsA), effective in prophylaxis and treatment of graft-vs-host disease (GVHD) after human allogeneic transplantation, blunts T-cell responses by inhibiting nuclear factor of activated T cells-1 (NFAT1) activation. This laboratory has shown that NFAT1 protein expression is severely reduced in human UCB (umbilical cord blood) T cells. Since UCB is increasingly used as a hematopoietic stem cell source in allogeneic transplantation, it is important to determine whether CsA sensitivity in UCB differs from that of adult T cells. Surface flow cytometric analysis, intracellular cytokine staining, flow cytometric analysis of cell death, and thymidine incorporation were used in this study to determine T-cell activation and effector functions during primary and secondary stimulation in the presence of CsA. Although we observed differential CsA sensitivity of T-cell activation marker (CD69, CD45RO, CD25) upregulation comparing UCB and adult, we did not observe any significant difference in CsA sensitivity of T-cell effector functions. Importantly, we observed reduced IFN-gamma and TNF-alpha expression in UCB T cells both in primary and secondary stimulation, as well as increased rates of activation-induced cell death (AICD). Thus, our studies do not support the previous hypothesis that reduced GVHD observed after UCB transplantation is attributable to increased CsA sensitivity of UCB T cells. Rather, reduced UCB T-cell cytokine production and increased AICD may be important cellular mechanisms underlying these favorable rates of GVHD in UCB transplant recipients.Experimental Hematology 08/2001; 29(7):903-9. · 2.90 Impact Factor
Article: Differential cyclosporin-A sensitivity on survival and activation of umbilical cord and adult peripheral blood CD4+ T cells.[show abstract] [hide abstract]
ABSTRACT: Enhanced cyclosporine-A (CsA) sensitivity of umbilical cord blood (CB) CD4(+) T cells may contribute to the lower incidence of graft-versus-host disease (GVHD) after mismatched CB transplantation. Interleukin (IL)-15, an IL-2 like gamma-chain signaling cytokine, may bypass CsA inhibition and play an important role in GVHD pathogenesis. The present study examines CsA sensitivity of IL-15-driven CB CD4(+) T cell survival, activation and proliferation, comparing adult peripheral blood (APB) CD4(+) T cell responses. We establish that (1) resting CB CD4(+) T cells were more susceptible to CsA-induced cell death than their adult counterpart; (2) IL-15 help support the survival of resting CB CD4(+) T cell under the influence of CsA, though to a lesser degree that observed in adults; (3) higher CsA sensitivity of CD25 up-regulation following TCR activation was observed in CB but more readily counteracted by IL-15 compared to adults; (4) IL-15-driven proliferation of TCR-activated CD4(+) T cells was more susceptible to CsA inhibition than corresponding adults; and (5) TCR-activated CB CD4(+) T cells surviving high dose CsA (5 microg/ml) in IL-15-supplemented cultures expressed significantly lower percentages of CD45RO(-)/CD25(+) subsets compared to corresponding APB. Taken together, the differential CsA sensitivity and IL-15 response between CB and APB CD4(+) T cells may contribute to the lessened GVHD severity in the setting of CB transplantation.Transplant Immunology 12/2007; 18(2):172-8. · 1.46 Impact Factor
Article: Ex vivo expansion, maturation, and activation of umbilical cord blood-derived T lymphocytes with IL-2, IL-12, anti-CD3, and IL-7. Potential for adoptive cellular immunotherapy post-umbilical cord blood transplantation.[show abstract] [hide abstract]
ABSTRACT: We investigated whether umbilical cord blood (UCB) T cells could be ex vivo expanded and activated in short-term culture for potential utilization as adoptive cellular immunotherapy post-umbilical cord blood transplantation (UCBT). Fresh UCB mononuclear cells (MNCs) were isolated by Ficoll density centrifugation. Cryopreserved UCB mononuclear cells were thawed and washed with 2.5% human serum albumin and 5% dextrose in isotonic saline. The nonadherent MNC fraction were then plated in a serum-free cocktail of IL-2, IL-12, and anti-CD3 with and without IL-7 for 48 hours. Proliferation, cytotoxicity, TH1 (IFN-gamma), CD25, and CD45RO assays were performed. Proliferation studies demonstrated a significant increase in the proliferative ability of the UCB MNCs incubated in anti-CD3, IL-2, IL-12, and IL-7 (fresh--p < 0.005, and thawed--p < 0.001). The combination of all four agonists significantly induced expression of CD45 RO (fresh--p < 0.05, and thawed--p < 0.001) in both the CD4(+) and CD8(+) T cells expressing CD25 (fresh UCB--p < 0.01 [CD4] and p < 0.005 [CD8], respectively; thawed UCB--p < 0.001 [CD4] and p < 0.001 [CD8]). Intracellular cytokine profiles also revealed a significant increase in the production of IFN-gamma (TH1 cells) (fresh UCB--p < 0.005, and thawed UCB--p < 0.001). The combination also significantly increased the killing of K562-labeled target cells (fresh--p < 0.0001, and thawed--0.731 +/- 0.03 vs 0.16 +/- 0.01) (p < 0.001). These data suggest that the ex vivo combination of IL-2, IL-12, anti-CD3, and IL-7 significantly enhances the proliferation, activation, maturation, and cytotoxic potential of UCB T cells of both fresh and thawed UCB MNC. Further studies, however, are required to determine whether these ex vivo--expanded MNC could also potentially exacerbate acute or chronic graft-vs-host disease and/or other toxicities if utilized post-UCBT.Experimental Hematology 03/2002; 30(3):245-51. · 2.90 Impact Factor