Human memory, but not naive, CD4+ T cells expressing transcription factor T-bet might drive rapid cytokine production.

We found that after stimulation for a few hours, memory but not naive CD4(+) T cells produced a large amount of IFN-γ; however, the mechanism of rapid response of memory CD4(+) T cells remains undefined. We compared the expression of transcription factors in resting or activated naive and memory CD4(+) T cells and found that T-bet, but not pSTAT-1 or pSTAT-4, was highly expressed in resting memory CD4(+) T cells and that phenotypic characteristics of T-bet(+)CD4(+) T cells were CD45RA(low)CD62L(low) CCR7(low). After short-term stimulation, purified memory CD4(+) T cells rapidly produced effector cytokines that were closely associated with the pre-existence of T-bet. By contrast, resting naive CD4(+) T cells did not express T-bet, and they produced cytokines only after sustained stimulation. Our further studies indicated that T-bet was expressed in the nuclei of resting memory CD4(+) T cells, which might have important implications for rapid IFN-γ production. Our results indicate that the pre-existence and nuclear mobilization of T-bet in resting memory CD4(+) T cells might be a possible transcriptional mechanism for rapid production of cytokines by human memory CD4(+) T cells.

[IL-12-induced expression of TRAIL enhances the cytotoxicity of NK cells against Jurkat cells].

AIM: To study the mechanism underlying the IL-12-induced cytotoxic function of NK cells to Jurkat cells. METHODS: NK cells from peripheral blood mononuclear cells (PBMCs) were purified by magnetic sorting and stimulated with or without IL-12. The expression of genes on IL-12-treated and non-IL-12-treated NK cells was analyzed by gene chips and the expression of cytolytic molecules was evaluated by flow cytometry. RESULTS: Seventeen genes were up- (5/17) or down-regulated (12/17) on IL-12-treated NK cells compared with non-IL-12-treated NK cells (fold change≥10). IL-12-induced expression of TRAIL on NK cells mediated the cytotoxicity to Jurkat cells. The expression of TRAIL on subsets of CD56(+);CD16(+); and CD56(-);CD16(+); NK cells significantly increased after the stimulation with IL-12 and Jurkat cells expressed high level of TRAIL receptor 2 (TRAIL-R2). Importantly, the neutralizing mAbs against TRAIL (RIK-2) significantly inhibited the cytotoxicity of NK cells induced by IL-12. CONCLUSION: The expression of TRAIL on human NK cells induced by IL-12 was one of the major mechanisms of cytotoxicity to Jurkat cells.

[Differences between CD3⁺ TCRvα24⁺ NKT cell and CD3⁺ TCRvß11⁺ NKT cell in PBMC].

AIM: Clarified the differences between CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in their frequencies, subpopulations, phenotypes and biological functions, so as to fully understand the effects of NKT cells in immune responses. METHODS: PBMCs from blood donors were isolated and cell surface markers (CD3, TCRvα24, TCRvß11, CD4, CD8, CD45RA, CD62L, CCR7) and intracellular cytokines (IL-4, IFN-γ) were detected by flow cytometry directly or after stimulation with PMA plus Ionomycin. RESULTS: The mean frequencies of CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in PBMCs were 0.63% and 0.43% and they varied according to individuals. A small population of NKT cells coexpressed TCRvα24 and TCRvß11. The subpopulations of CD4(+); NKT 64.35%, CD8(+); NKT 19.04%, CD4(-);CD8(-); NKT 17.18% in human CD3(+);TCRvα24(+); NKT cells and CD4(+); NKT 53.69%, CD8(+); NKT 18.99%, CD4(-);CD8(-); NKT 29.74% in CD3(+);TCRvß11(+); NKT cells could be identified based upon the expressions of CD4 and CD8 molecules. There were no significant differences between relative subtypes. The frequency of CD45RA(+);CD3(+);TCRvß11(+); NKT cells(71.14%) was higher than the frequency of CD45RA(+);CD3(+);TCRvα24(+); NKT cells and the differences between them were significant. The differences between the frequencies of CD62L(+);CD3(+);TCRvα24(+); NKT cells(46.26%) and CD62L(+);CD3(+);TCRvß11(+); NKT cells(42.36%), the frequencies of CCR7(+);CD3(+);TCRvα24(+); NKT cells(9.24%) and CCR7(+);CD3(+);TCRvß11(+); NKT cells(8.22%) were not significant. There were no significant differences in the secretions of IL-4 by CD3(+);TCRvα24(+); NKT cells(13.01%) and CD3(+);TCRvß11(+); NKT cells(6.62%), and IFN-γ by CD3(+);TCRvα24(+); NKT cells(38.12%) and CD3(+);TCRvß11(+); NKT cells(26.95%). However, there were significant differences between the mean frequency of IFN-γ(+);IL-4(+);CD3(+);TCRvα24(+); NKT cells(12.65%) and that of IFN-γ(+);IL-4(+);CD3(+);TCRvß11(+); NKT cells(3.02%). CONCLUSION: There were some differences between CD3(+);TCRvα24(+); NKT cells and CD3(+);TCRvß11(+); NKT cells in their frequencies, phenotypes and productions of cytokines. In all, although their frequencies were low, the complicated phenotypes and high secretions of cytokines(IL-4 and IFN-γ) assigned NKT cells immunoregulatory effects.

A selective phosphodiesterase 4 (PDE4) inhibitor Zl-n-91 suppresses IL-17 production by human memory Th17 cells.

Th17 cells are highly proinflammatory and involved in the immunopathogenesis of severe autoimmune diseases. Selective phosphodiesterase 4 (PDE4) inhibitors, which elevate intracellular cAMP by inhibiting the hydrolysis of cAMP, have been demonstrated to be an effective anti-inflammatory agent in airway inflammatory diseases. In the present study, we assessed the effect of a selective PDE4 inhibitor Zl-n-91 on IL-17 production by PBMCs and by purified CD4(+) T cells following stimulation. The results for the first time demonstrated that the addition of Zl-n-91 into cell cultures of PBMCs and purified CD4(+) T cells could result in the suppression of IL-17 production at the protein and mRNA levels. Further analysis indicated that Zl-n-91 had a direct inhibitory effect on the IL-17 production by memory Th17 cells via the suppression of activation, proliferation and division of CD4(+) T cells. Our data suggested that Zl-n-91 might have beneficial effects in the treatment of IL-17-related autoimmune diseases.

[Correlation of BrdU incorporation with activation and cytokine expression of T cells].

AIM: To investigate the correlation of BrdU incorporation with the activation and cytokine expression of T cells. METHODS: PBMCs from healthy persons were isolated and stimulated by PMA plus Ionomycin at different periods of time, BrdU was then added to the cells one hour before the end of culture. The cells were harvested and stained with anti-BrdU, anti-cell surface and intracellular antibodies. Then the cells were washed and analyzed by flow cytometer. RESULTS: The peak of BrdU incorporation was observed in T cells after they were stimulated for 48 hours in vitro, but no further increase of the peak of BrdU incorporation was found after incubated for a longer period of time. The comparison made between BrdU incorporation and cell activation indicated CD69 expression reached the peak after stimulated for 8 hours whereas CD25 was at the peak after stimulated for 24 hours. Furthermore, no correlation between BrdU incorporation and cytokine production was observed. High frequency of IFN-gamma producing cells was detected after stimulated for 8 hours but no obvious increase was observed for a longer period of time. When PBMC were stimulated with OKT3 plus antiCD28, the percentage of BrdU(+) T cells was higher than that stimulated by PMA plus Ionomycin. Similarly, the percentage of BrdU(+) CD8(+) T cells was higher than that of BrdU(+) CD4(+) T cells. CONCLUSION: The percentage of BrdU(+) cells can be detected by flow cytometer to evaluate the proliferation of T cells. Only a few T cells proliferate after polyclonal stimulation and BrdU incorporation is dependent on stimulants and time of stimulation. Therefore, BrdU incorporation is not correlated with activation markers and cytokine production.

Phenotypic and functional heterogeneity of natural killer cells from umbilical cord blood mononuclear cells.

Natural killer cells (NK) from umbilical cord blood (CB) play an important role in allogeneic stem cell transplantation and defending infections of newborn. Based on the surface expression of CD56 and CD16 or inhibitory and activatory receptors, NK cells could be subdivided into various subsets with distinct functions. To investigate the biological characterization of NK subsets, the phenotypes and intracellular proteins in freshly isolated CB NK subsets were analyzed at the single cell level by flow cytometry in current study. The production of IFN-gamma and cytotoxicity against K562 target cells were also evaluated after stimulation with IL-12. The results showed that NK cells from CB could be divided into four subsets on the basis of CD56 and CD16 expression. Interestingly, CB NK cells expressed CD45RA but not CD45RO molecules that is similar to the naïve T cells. Moreover, CD27, a memory T cell marker, highly expressed on CD56(hi)CD16- NK cells. The killing-associated molecules, NKG2A, NKG2D, CD95 and the intracellular granzyme B and perforin were heterogeneously expressed among the 4 subsets. Addition of IL-12 into cultures resulted in the induction of IFN-gamma expression by CD56(hi)CD16- and CD56(lo)CD16- subsets and the enhancement of NK cytolytic activity. Taken together, this study elucidated the heterogeneity in phenotypes and biological functions of CB NK cells.

Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs.

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.

[Human peripheral blood CD56+ natural killer cell subsets and their phenotypic and biological properties].

OBJECTIVE: To characterize the phenotypic and biological properties of CD56(+) natural killer cells from human peripheral blood mononuclear cells (PBMCs). METHODS: Surface markers and intracellular cytotoxic molecules were stained with multi-color-labeled monoclonal antibodies and analyzed at the single cell level the relation between NK subsets and biological characteristics by flow cytometry. RESULTS: NK cells in PBMCs could be divided into two major populations, CD56(bright) and CD56(dim), based upon the expression of CD56 molecules. Both CD56(bright) and CD56(dim) expressed CD95 (Fas) with CD95(bright) and CD95(dim) subsets. CD56(dim) subsets had higher percentage of CD8, granzyme B and perforin expression compared to those of CD56(bright) subsets. In CD56(bright) and CD56(dim) subpopulations, CD95(bright) and CD8(+) subsets had higher percentage of granzyme B and perforin expression. CONCLUSION: CD56(+) NK cells in PBMCs are composed of distinct subpopulations, CD56(dim) and CD56(dim) CD8(+) NK subsets have higher percentage of granzyme B and perforin and may play an important role in the killing of target cells.