Fig 1.
Proliferation of T cells from humanized mice and human peripheral blood with or without cytokines.
Purified T cells (>95% CD3+) from humanized mice (A and B) or human peripheral blood (C) were labeled with CFSE and activated with plate-bound anti-CD3 and anti-CD28 for 3 days in the presence or absence of human IL-7 or IL-15. Cells were stained for CD4 and CD8 and analyzed by flow cytometry. Proliferation was assessed by CFSE dilution as compared to unstimulated purified T cells cultured without cytokines. (A) Representative CFSE histograms of stimulated (black line trace) and unstimulated (solid grey trace) CD4 or CD8 T cells from one humanized mouse. (B) Aggregated proliferation data from 10 mice that were constructed using 3 independent donor HSPCs. (C) Representative CFSE histograms of stimulated (black line trace) and unstimulated (solid grey trace) CD4 or CD8 T cells from one human peripheral blood sample. The numbers in A and C show percentages of cells that have diluted CFSE. * indicates P<0.05 by paired t test between no cytokine group and cytokine treated groups.
Fig 2.
Effects of cytokines on T cell proliferation during activation of cytokine-deprived human T cells.
Purified T cells from peripheral blood were cultured for 1 week in the absence of added cytokine. Both the cultured T cells and fresh purified T cells from the same donor were then labeled with CFSE, stimulated with plate-bound anti-CD3 and anti-CD28 for 3 days in the absence or presence of the indicated cytokines. Cells were stained for CD4 and CD8 and analyzed by flow cytometry. (A) Top and middle: Representative CFSE histograms of stimulated (black line trace) and unstimulated (solid grey trace) CD4 or CD8 T cells from one donor. Bottom: Viability of fresh and cultured cells following 3 days of stimulation. The numbers show percentages of cells that have diluted CFSE or in the gated region. (B) Aggregated data (mean ± SD) from 4 different donors. Cytokines are abbreviated to their corresponding number or alphabet and no cytokine is indicated by “C” for control. Cytokine concentrations used this experiment: IL2 (50U/ml), IL4 (100U/ml), IL-7 (10ng/ml), IL9 (50U/ml), IL-15 (10ng/ml), IL21 (10ng/ml), IFNγ (10ng/ml), and TNFα (10ng/ml). (C) Purified T cells from 3 donors were cultured for 1 week in the presence or absence of IL-7 (10ng/ml) or IL-15 (10ng/ml), live cells were then counted and cell counts normalized to cell number in the control cytokine-free condition, C indicates the control and IL-7 and IL-15 indicate culture with those cytokines. (D) CD4 and CD8 T cells from 3 donors were cultured for 24 hours without cytokine supplementation, labeled with CFSE and then activated with or without 10 ng/ml IL-7 for 3 days prior to analysis, C indicates control and IL-7 indicates treated cells. * indicates P<0.05 by paired T test between cytokine treatment to control without cytokines.
Fig 3.
Impaired CD25 upregulation by cytokine-deprived T cells.
Purified T cells from human peripheral blood were stimulated with plate-bound anti-CD3 and anti-CD28 for 2 days in the presence or absence of IL-7 or IL-15 or cultured for one day in the absence cytokine and then stimulated for 2 days. Cells were stained for CD3, CD4, CD8 and CD25 followed by flow cytometry. (A) Representative CD25 vs. CD8 staining profiles gating on CD3+ live cells. (B) Aggregate percentages of CD25+ CD4 and CD25+ CD8 T cells from 6 independent biological replicates. The numbers in A indicate the percentages of cells in the quadrants. * indicates P<0.05 by paired t test between no cytokine (C) cultured group and other groups.
Fig 4.
Impaired STAT5 phosphorylation in cytokine-deprived T cells.
(A) Western blotting showing phosphorylated and total ERK and AKT in human T cells following various treatments. Freshly purified human T cells from peripheral blood were either unstimulated or stimulated with anti-CD3 and anti-CD28 for 5 minutes in the absence (-) or presence of IL-7 or IL-15 (10ng/ml each). Alternatively, purified human T cells were cultured for 24 hours in the absence or presence of IL-7 or IL-15 and then treated as above. Cells were lysed and total proteins were fractionated on a SDS-PAGE and then blotted for total and phosphorylated ERK and AKT. GAPDH was used a loading control. Representative data from 3 independent donors is shown. (B) Western blotting of phosphorylated and total STAT5 in freshly isolated and cultured human T cells. The culture conditions and replicates are the same as in A. (C) T cell proliferation in the presence of different concentrations of IL-7 or IL-15. Purified human T cells were cultured in the indicated concentrations (ng/ml) of IL-7 or IL-15, labeled with CFSE and activated with plate-bound anti-CD3 and anti-CD28 for 3 days, control cells provided with no cytokine are indicated with a C. Data shown is representative of biological duplicates used for Western blotting. (D) Western blotting of phosphorylated and total STAT5 with reduced concentrations of IL-7 and IL-15 (ng/ml). The experiment was performed as in B except the concentrations of IL-7 and IL-15 were reduced. Data shown is representative of biological duplicates. (E) Inhibition of human T cell proliferation by STAT5 inhibitor. Freshly purified human T cells were stimulated with plate-bound anti-CD3 and anti-CD28 in the absence or presence of STAT5 inhibitor. Shown are CFSE histograms of CD4 and CD8 T cells with (black line trace) or without (solid grey trace) stimulation. (F) Aggregate data of 4 biological replicates of STAT5 inhibitor treatment. (G) TUNEL stained purified T cells (>90%) were stained to assess for apoptotic DNA fragmentation. Data shown are representative flow cytometry plots of 3 independent biological replicates. (H) Comparison of proliferation of fresh and IL-7 treated (24h) cells in the absence or presence of IL-2 blocking antibody. T cells were activated with anti-CD3 and anti-CD28 for 3 days. Biological triplicates are shown.
Table 1.
Cell counts from mice used in Fig 5.
Fig 5.
Rescue of T cell proliferation by IL-7 and IL-15 in humanized mice.
Humanized mice were hydrodynamically injected with control plasmid DNA or plasmid encoding IL-7 or IL-15. Seven days later, T cells purified from the spleens of treated mice were activated with plate-bound anti-CD3 and anti-CD28 in the absence or presence of IL-7 or IL-15. (A) CFSE histograms from one vector injected mouse, one IL-7 plasmid injected mouse and one IL-15 plasmid injected mouse. (B) Aggregated data from four vector plasmid-injected mice, four IL-7 plasmid-injected mice and five IL-15 plasmid-injected mice. Each group contains mice generated from 3 independent HSPCs. * = P<0.05 in a paired T test between control and treated condition.