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. 2006 Dec;34(12):1730-40.
doi: 10.1016/j.exphem.2006.08.001.

"VSports app下载" Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system

Hongfang Wang  1 , L Jeanne Pierce, Gerald J Spangrude
Affiliations

Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system

Hongfang Wang et al. Exp Hematol. 2006 Dec.

V体育官网入口 - Abstract

Objective: The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system VSports手机版. .

Methods: Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. V体育安卓版.

Results: IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation V体育ios版. Flt3 ligand promoted proliferation without affecting differentiation. .

Conclusion: These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation VSports最新版本. .

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Figures

Figure 1
Figure 1
Differentiation of Thy1.1neg cells in OP9 and OP9-DL1 co-cultures without added cytokines. Panel (A) T cell differentiation on OP9 and OP9-DL1 cell lines. Purified Thy1.1neg cells were cultured on OP9 or OP9-DL1 stromal cell lines in the absence of exogenous cytokines. T cell markers (CD4 and CD8, respectively) were analyzed by flow cytometry on day 12 and day 20 after co-culture. (B) NK cell and B cell development on OP9 and OP9-DL1 cell lines. The same samples as shown in (A) were stained with NK1.1 and CD19. Each culture was initiated with 1,200 cells, which expanded to about 23,000 cells by day 12; no further increase in cell number was noted at day 20. Numbers in the plots represent the percentage of cells within the indicated quadrants. The data is a representative example of three independent experiments.
Figure 2
Figure 2
Effects of exogenous cytokines on T cell development. (A) Growth curve of Thy1.1neg cells in OP9-DL1 co-cultures with different combinations of cytokines. Thy1.1neg cells were cultured on OP9-DL1 stromal cells with different combinations of Flt3L, IL-7 and SCF. Cell numbers were counted at the indicated time points, and fold cell expansion was calculated. Error bars indicate standard error of the mean values of fold cell expansion obtained in separate experiments. Differences between Flt3L alone (n = 3) and Flt3L + IL-7 (n = 6) are significant at all time points (p < 0.03). Differences between Flt3L + IL-7 (n = 6) and Flt3L + IL-7 + SCF (n = 5) are significant at days 12, 16, 20, and 23 (p < 0.03). (B) Effect of IL-7 on T cell development. Thy1.1neg cell progeny were analyzed at different time points after co-culture with OP9-DL1. Expression of CD4 and CD8 was analyzed by flow cytometry. (C) Effect of IL-7 on αβ and γδ T cell development. Expression of αβ TCR and γδ TCR was analyzed at indicated time points. Numbers in the plots represent the percentage of cells within the indicated quadrants. (D) IL-7 promotes differentiation from the DP stage to the CD8 SP stage. Cultures of Thy.1.neg cells maintained on OP9-DL1 in the presence of Flt3L were split on day 23 and replated on OP9-DL1 in the presence of Flt3L or with Flt3L plus IL-7. Cultures were evaluated for CD4 and CD8 expression 3 days later.
Figure 3
Figure 3
Antibody blocking of endogenous IL-7 inhibits T cell development. (A) Effect of neutralizing antibody against IL-7 on DN cell development. Multicolor analysis of differentiating cells cultured on OP9-DL1 cells with Flt3L was performed at the indicated times. CD4 and CD8 DN cells were gated and analyzed for the expression of CD44 and CD25. On day 23, the cells cultured on OP9-DL1 cells in the presence of anti-IL-7 were washed and returned to culture either under the original conditions or in the presence of exogenous Flt3L and IL-7. (B) Growth curves of Thy1.1neg cells plated on OP9-DL1 cells in the presence of Flt3L (5 ng/ml), either alone or in the presence of exogenous IL-7 (5 ng/ml) or of the indicated concentrations of rabbit anti-IL-7. (C) Dosage effect of neutralizing antibody against IL-7 on T cell differentiation. Cells were co-cultured with OP9-DL1 cells and Flt3L with exogenous IL-7, without exogenous IL-7, or in the presence of control immunoglobulin or the indicated concentrations of anti-IL-7. At day 12, 16, 20, and 23, multicolor analysis was performed as indicated in (A). (D) Effect of anti-IL-7 on DP T cell development. Cells were co-cultured with OP9-DL1 cells and Flt3L in the presence of exogenous IL-7, in the absence of exogenous IL-7, or in the presence of control immunoglobulin or anti-IL-7. CD4 and CD8 expression was analyzed at the indicated time points. The data is representative of two independent experiments.
Figure 4
Figure 4
Exogenous soluble SCF inhibits T lineage differentiation of Thy1.1neg cells. (A) Effect of SCF on αβ T cell differentiation. Upper panel: T cell differentiation of Thy1.1neg cells on OP9-DL1 with the addition of Flt3L and IL-7. Lower panel: T cell differentiation of Thy1.1neg cells on OP9-DL1 with addition of Flt3L, IL-7 and SCF. CD4 and CD8 expression was analyzed at different time points. (B) Effect of SCF on αβ and γδ T cell differentiation. αβ TCR and γδ TCR expression was analyzed as described for Panel A. (C) Effect of SCF on DN cell development. Multicolor analysis of differentiating cells cultured on OP9-DL1 cells was performed at the indicated times. CD4 and CD8 DN cells were gated and analyzed for the expression of CD44 and CD25. (D) Effect of SCF withdrawal on DN cell development. SCF was withdrawn from the culture medium on day 23, and expression of CD44 and CD25 was analyzed on gated DN cells. (E) Dose effect of SCF on T cell differentiation. Cells were co-cultured with OP9-DL1 cells without SCF, or in the presence of SCF at the indicated concentrations. At day 23, expression of CD44 and CD25 was analyzed on gated CD4 and CD8 DN cells. The data are representative examples of three to six independent experiments except panel E, which is from one experiment.
Figure 5
Figure 5
Blocking endogenous SCF function promotes T lineage development. (A) Growth curve of Thy1.1neg cells on OP9-DL1 with different combinations of cytokines and inhibitors. Cell numbers were counted at each time point, and the average fold expansion was calculated. Error bars indicate standard error of the mean values of fold cell expansion obtained in separate experiments. Significant differences (p < 0.04) were observed between Flt3L + IL-7 + ACK4 (n = 4) and Flt3L + IL-7 + ACK2 (n = 3) and between Flt3L + IL-7 (n = 4) and Flt3L + IL-7 + imatinib (n = 3) at all times shown, while differences between Flt3L + IL-7 and Flt3L + IL-7 + ACK4 or between Flt3L + IL-7 + ACK2 and Flt3L + IL-7 + imatinib were not statistically significant at any time (p > 0.1). (B) Blocking of SCF function does not prevent αβ and γδ T cell development. Thy1.1neg cells were cultured on OP9-DL1 with and without SCF inhibitors. Expression of αβ TCR and γδ TCR was analyzed as indicated. (C) Blocking of SCF function does not inhibit DP T cell development. Kinetic expression of T cell surface markers CD4 and CD8 was analyzed at the indicated times after co-culture.
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References

    1. Radtke F, Wilson A, Mancini SJ, MacDonald HR. Notch regulation of lymphocyte development and function. Nat Immunol. 2004;5:247–253. - PubMed
    1. Jaleco AC, Neves H, Hooijberg E, et al. Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation. J Exp Med. 2001;194:991–1002. - PMC - PubMed
    1. Schmitt TM, Zuniga-Pflucker JC. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity. 2002;17:749–756. - PubMed
    1. Schmitt TM, de Pooter RF, Gronski MA, Cho SK, Ohashi PS, Zuniga-Pflucker JC. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol. 2004;5:410–417. - PubMed
    1. Moore TA, Zlotnik A. Differential effects of Flk-2/Flt-3 ligand and stem cell factor on murine thymic progenitor cells. J Immunol. 1997;158:4187–4192. - PubMed

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