Po-Jen Cheng

Evaluation of cytotoxic function and apoptosis in interleukin (IL)-12/IL-15-treated umbilical cord or adult peripheral blood natural killer cells by a propidium-iodide based flow cytometry

Both deficient natural killer (NK) cell effector function and increased propensity to apoptosis of neonatal NK cells contribute to the increased susceptibility to infection in the neonates. Interleukin (IL)‐12 and IL‐15 are two immunoregulatory cytokines known to enhance cytolytic function of neonatal NK cells. The present study aims to simultaneously investigate the effect of IL‐12/IL‐15 on K562 cytotoxicity as well as NK cells apoptosis of enriched umbilical cord blood (CB) and adult peripheral blood (APB) NK cells, using flow cytometric cytotoxicity assays. The results indicated that (i) prior to cytotoxicity assays, CB NK cells underwent greater degree of spontaneous apoptosis than did APB NK cells; (ii) both IL‐12 and IL‐15 inhibited the spontaneous apoptosis of CB NK cells, while IL‐15 promoted the apoptosis in APB NK cells; (iii) the deficient K562 cytotoxicity of CB NK cells could be enhanced to levels comparable with that of APB NK cells by IL‐15; (iv) IL‐15 increased the percentages of apoptosis in NK–K562 conjugates in a dose‐dependant manner in both CB and APB with a greater effect seen with APB NK cells; (v) target‐induced apoptosis was observed with APB NK cells which were further enhanced with IL‐15. However, CB NK cells, unstimulated or IL‐15‐activated, were resistant to K562‐induced apoptosis. Thus, the multi‐parameter flow cytometry analysis not only demonstrates better for the deficient CB NK function but also provides greater details for cytotoxic mechanisms of NK cells.

The regulatory function of umbilical cord blood CD4(+) CD25(+) T cells stimulated with anti-CD3/anti-CD28 and exogenous interleukin (IL)-2 or IL-15.

The abundance of CD4+ CD25+ regulatory T cells in umbilical cord blood (UCB) might contribute to the decreased severity of graft‐vs.‐host disease (GVHD) for UCB transplantation. This study aims to characterize the phenotypes and suppressive function of UCB CD4+ CD25+ T cells under the influence of anti‐CD3/anti‐CD28 (CD3/CD28) and exogenous interleukin (IL)‐2 or IL‐15. Higher percentages of CD4+ CD25high and FoxP3+ cells were detected in UCB compared to their adult counterparts. IL‐15 was as effective as IL‐2 in enhancing the proliferation of CD3/CD28 stimulated UCB CD4+ CD25+ T cells. Phenotypically, IL‐2/IL‐15‐stimulated UCB CD4+ CD25+ T cells expressed higher level of CTLA‐4, GITR, membrane bound transforming growth factor‐β (mTGF‐β), and especially Foxp‐3 than controls. IL‐2/IL‐15‐stimulated UCB CD4+ CD25+ T cells also produced much higher IL‐10 and TGF‐β than controls; while IL‐2/IL‐15‐stimulated UCB CD4+ CD25− T cells showed increased TGF‐β, but not IL‐10 production. IL‐2/IL‐15‐cultured UCB CD4+ CD25+ T cells showed comparable suppressor activity on allogeneic adult CD4+ T‐cell proliferation compared to controls, partly through a contact‐dependent fashion. Taken together, IL‐2/IL‐15‐stimulated UCB CD4+ CD25+ T cells show distinct regulatory T‐cell phenotypic and functional features, and may be applied for the alleviation of GVHD severity following UCB transplantation.

Azithromycin Inhibits IL-5 Production of T Helper Type 2 Cells from Asthmatic Children

Background: Childhood asthma is a type 2 helper T (Th2) cell-driven inflammatory airway disease characterized by recurrent episodes of airway obstruction. Azithromycin (AZM), a macrolide antibiotic exhibiting anti-inflammatory activity aside from its antibacterial effect, may prove beneficial for asthmatic children. This study aimed to determine the effect of AZM on Th2 cells from atopic asthmatic children and non-atopic controls. Methods: CD4+ cells were isolated from peripheral blood mononuclear cells of 9 patients with asthma and 9 non-atopic individuals. Cells were activated as Th0 and differentiated into Th2 cells. The effect of AZM on activated CD4+ cells was evaluated with respective cell proliferation and cytokine production. Results: Th0 and Th2 CD4+ T cells from atopic asthmatic children produced greater interleukin (IL)-5 (Th2 cytokine) but lower interferon (IFN)-γ (Th1 cytokine) compared to the non-atopic controls, respectively. AZM inhibited IL-5 production of Th0 and Th2 cells from atopic asthmatics in a dose-dependent fashion, without significantly affecting their IL-13 and IFN-γ production. A similar effect was observed in non-atopic controls except that AZM did inhibit IFN-γ production of their Th0 cells. AZM at a higher dose decreased cell viability by inhibiting CD4+ T cell proliferation and enhanced their apoptosis, an effect similarly observed in Th0 and Th2 cells, and did not differ between asthmatic children and controls. Conclusion: Our finding that AZM preferentially downregulates IL-5 production suggests its therapeutic potentials in controlling childhood asthma.

Effect of interleukin-15 on anti-CD3/anti-CD28 induced apoptosis of umbilical cord blood CD4+ T cells.

Abstract: Objectives: Interleukin‐15 (IL‐15) has potential therapeutic advantage for patients receiving umbilical cord blood (CB) transplantation. The present study aims to examine the ability of IL‐15 to modulate the survival, maturation, and function of anti‐CD3/anti‐CD28 stimulated CB CD4+ T cells, in comparison with responses from adult peripheral blood (APB) CD4+ T cells.

Effect of Influenza A Infection on Umbilical Cord Blood Natural Killer Function Regulation With Interleukin-15

BACKGROUND Influenza A is a major pathogen of humans and has the potential to cause worldwide pandemics. Natural killer (NK) cells are important effector cells in the innate immune response against viruses, including influenza A. Infants are more susceptible to severe influenza A viral infection, possibly attributed in part to their defective NK function. METHODS We compared the NK responses to influenza using umbilical cord blood (UCB) and adult peripheral blood (APB) mononuclear cells and purified NK cells. RESULTS Influenza A induced dose-dependent apoptosis of NK cells with down-regulation of NKp46 expression, which was more pronounced in UCB. Both UCB and APB NK cells responded to influenza infection by up-regulating CD69 and CD107a expression, a process further enhanced by interleukin (IL) 15. Influenza exposure also down-regulated perforin expression and K562 cytotoxicity in UCB NK cells, which was partially restored by IL-15. The production of interferon (IFN) γ and tumor necrosis factor (TNF) α by NK cells in responding to influenza was further enhanced by IL-15. CONCLUSIONS Our findings show differential NK responses between newborns and adults. IL-15 may be beneficial in combating influenza by enhancing cytotoxic function and IFN-γ production.

Regulation of CD28 expression on umbilical cord blood and adult peripheral blood CD8+ T cells by interleukin(IL)-15/IL-21.

Interleukin (IL)-15 and IL-21, both belonging to common γ-chain-signaling cytokine family, have an important role to maintain homeostatic proliferation of CD8(+) T cells. CD28, an essential co-stimulatory molecule on T cells, may be a marker of replicative senescence. We investigated the effect of IL-15 and IL-21, alone or in combination, on activation, apoptosis, cytokine production and cytotoxic function of magnetic bead purified umbilical cord blood (UCB) and adult peripheral blood (APB) CD8(+) T cells with regards to their CD28 expression. We established that (1) IL-15-induced CD8(+) T cell proliferation was associated with a preferential expansion of CD28(-) population in UCB, which could be partially counteracted by IL-21; (2) UCB CD8(+) T cells were more readily responsive to IL-15 compared to their adult counterparts in terms of CD69 expression, with the majority of CD69-bearing CD8(+) T cells were CD28(-); (3) IL-21 further promoted interferon-gamma, but not tumor necrosis factor-alpha production from IL-15 treated CD8(+) T cells; (4) IL-21 also synergized with IL-15 to enhance perforin and granzyme B expression of CD8(+) T cells, especially in APB CD8(+)CD28(-) subsets; (5) IL-21 resulted in CD8(+) T cells apoptosis both in APB and UCB cells, mainly in CD8(+)CD28(-) subsets. Taken together, we demonstrate differential IL-15/IL-21 response in UCB CD8(+) T cells with regards to CD28 expression. Our results suggest that combining IL-21 and IL-15 immunotherapy may be better than IL-15 alone to ameliorate graft-versus-host disease while preserving antitumor effect in the post-UCB transplantation period.

Susceptibility to Fas and tumor necrosis factor-α receptor mediated apoptosis of anti-CD3/anti-CD28-activated umbilical cord blood T cells

Decreased severity of graft‐versus‐host disease after mismatched umbilical cord blood (UCB) transplantation may be attributed in part to the increased propensity to apoptosis of UCB T cells following activation. Interleukin (IL)‐15, a pleiotropic cytokine that is essential for T‐cell proliferation and survival, may serve as promising immunomodulative therapy post‐CB transplantation for its anti‐apoptotic effect. This study aimed to determine the kinetics of Fas or tumor necrosis factor‐α receptor (TNFR) mediated caspase‐3 expression and apoptosis of anti‐CD3/anti‐CD28 activated UCB T cells in the influence of IL‐15. Activated caspase‐3 expression was analyzed by Western blotting and the percentage of apoptotic cells was determined by annexin‐V/propidium iodide (PI) flow cytometric staining. Significant expression of Fas and TNFR2 was detected on anti‐CD3/anti‐CD28 pre‐activated UCB T cells. These cells were susceptible to anti‐Fas but not TNF‐α‐induced apoptosis. Kinetic study shows that caspase‐3 expression became evident at 6th–8th h following anti‐Fas stimulation, while early apoptotic cells with annexin‐V+/PI− expression appeared at 12th–16th h. IL‐15, though successful in decreasing apoptosis in pre‐activated UCB T cells, failed to completely prevent Fas‐mediated caspase‐3 expression and apoptosis of CB T cells. The pre‐activated UCB and adult peripheral blood T cells behaved similarly with regard to death receptor expression, caspase‐3 expression and apoptosis upon Fas‐engagement. Although IL‐15 promotes overall activated UCB T‐cell survival, it did not particularly prevent Fas‐mediated activation‐induced cell death.

Interleukin-15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood.

Invariant natural killer T cells (iNKT cells) are innate-like non-conventional T cells restricted by the CD1d molecule that are unique in their ability to play a pivotal role in immune regulation. Deficient iNKT function has been reported in patients receiving umbilical cord blood (UCB) transplantation. We sought to determine the effect of interleukin (IL)-15 on α-galactosylceramide (α-GalCer)-expanded iNKT cell function from UCB and adult peripheral blood (APB) mononuclear cells (MNCs). Fresh APB and UCB MNCs were cultured with IL-15 (50 ng/ml) in the presence or absence of α-GalCer (100 ng/ml) for 10 days. Cells were harvested for examination of cell yield, apoptosis, cytokine production and cytotoxic function of Vα24(+)/Vβ11(+) iNKT cells. We observed that α-GalCer-expanded APB and UCB iNKT cells and such expansion was further enhanced with IL-15. The percentage of CD3(+)CD56(+) NKT-like cells in both APB and UCB MNCs was increased with IL-15 but not with α-GalCer. Apoptosis of UCB iNKT cells was ameliorated by IL-15. Although APB and UCB iNKT cells secreted lower IFN-γ, it could be enhanced with IL-15. The expression of perforin in APB iNKT cells can also be enhanced with IL-15. UCB Vα24(+)Vβ11(+) iNKT cells further augmented K562 cytotoxicity mediated by IL-15. Taken together, these results demonstrated the relative functional deficiencies of α-GalCer induced UCB iNKT cells, which can be ameliorated by IL-15. Our findings suggest a therapeutic benefit of IL-15 immunotherapy during the post-UCB transplant period when iNKT function remains poor.

Effect of FK506 on the interleukin 15-driven proliferation and apoptosis of anti-CD3-activated umbilical cord blood T cells

BACKGROUND Increased susceptibility of umbilical cord blood (CB) T cells to FK506 immunosuppression may contribute to the lessened severity of graft-vs-host disease in CB transplantation. OBJECTIVE To investigate the FK506 sensitivity of interleukin 15 (IL-15)- and IL-2-driven proliferation and apoptosis of anti-CD3-stimulated CB T cells compared with adult peripheral blood (APB) T cells. METHODS Surface flow cytometric analysis (CD25 and CD95), carboxyfluorescein diacetae succinimidyl ester staining to track CD3+ T-cell division, and flow cytometric analysis of apoptotic cell death using Annexin V were performed to determine the effect of FK506 on CD3+ T-cell activation and apoptosis after anti-CD3 stimulation in the presence of IL-15 or IL-2. RESULTS IL-15 is superior to IL-2 in promoting CD25 expression and proliferation of anti-CD3-stimulated CB and APB T cells. Although IL-15-driven proliferation evaluated by carboxyfluorescein diacetae succinimidyl ester staining revealed comparable sensitivity to FK506 in anti-CD3-stimulated CB and APB T cells, IL-15-driven CD25 up-regulation in CB T cells was more sensitive to FK506 inhibition than APB T cells. FK506 down-regulated anti-CD3-induced apoptosis in CB and APB T cells (P < .01). However, the FK506 sensitivity of anti-CD3-induced T-cell apoptosis was lost in IL-15-supplemented CB cultures (P = .51) but not in corresponding APB cultures (P = .002). The IL-15-enhanced Fas expression on CB T cells (CD95) was decreased by FK506, similar to that observed with adults. CONCLUSIONS We observed differential FK506 sensitivity of IL-15-driven CD25 up-regulation and apoptotic response comparing CB and APB T cells. This finding suggests the potential therapeutic benefit of FK506 in ameliorating graft-vs-host disease by decreasing IL-15-driven donor T-cell proliferation without inhibiting associated activation-induced apoptosis during CB transplantation.