Figure 1.
IL-15 enhances the strength and duration of TCR/CD3-triggered proliferation by CD8+ and CD4+ T cells.
(A) Bulk splenocytes were stimulated with soluble anti-CD3 mAb and 3H-thymidine incorporation was measured (white circles: none; crosses: 1 ng/ml IL-15; black squares: 3 ng/ml IL-15). (B) CFSE-labeled splenocytes were stimulated using 0.1 µg/ml anti-CD3 mAb and supplemented or not with 3 ng/ml rhIL-15. Cells were stained for CD8 or CD4 after 2–3 days. Shown are representative density plots with inset values reflecting percentage and absolute numbers of CD4+ or CD8+ T cells in the viable cell gate and summarizing bar graphs. (C–F) CD8+ T cells (C,D) or CD4+ T cells (E,F) were stimulated with soluble anti-CD3 mAb and irradiated TdACs in the absence or presence of 3 ng/ml rhIL-15. 3H-thymidine incorporation (C,E; white circles: none, black squares: 3 ng/ml IL-15) or cell numbers (D,F; 0.1 µg/ml anti-CD3; white bars: none, black bars: 3 ng/ml IL-15) were measured. (G) CFSE-labeled CD4+ T cells were stimulated as in F. Shown are CFSE histogram overlays, gated on viable CD4+ T cells, of cultures without (grey) or with (black) exogenous rhIL-15. The heights of the profiles were normalized to the absolute viable CD4+ T cell numbers present in the culture, as determined by flow cytometry using fluorescent micro-beads as a reference. (H) Proliferation index of CD4+ T cells in cultures without (white squares) or with (black squares) supplemented IL-15 (3 ng/ml). For all panels, data are from triplicate wells in one representative of four repeat experiments. A, C: Symbols in line graphs reflect mean ± SD. D, F, H: Bar graphs represent the mean±SEM. In all relevant panels, statistical significance between IL-15-supplemented and non-supplemented cultures was calculated per anti-CD3 concentration using student t-test: ns = not significant, * P<0.05, ** P<0.01, *** P<0.001. All experiments were performed at least 3 times with similar results.
Figure 2.
IL-15 growth factor activity depends on TCR-signals, but is IL-2-dependent only in CD4+ T cells.
(A) CD8+ or (B) CD4+ T cells were stimulated with anti-CD3 mAb and irradiated TdACs in the absence or presence of 3 ng/ml rhIL-15, or 10 µg/ml anti-IL-2 mAb (JES6-5H4) or isotype control, as indicated. On day 4, 3H-thymidine incorporation was measured. Results are from one of five experiments. (C) IL-2+/+ or IL-2−/− CD4+ cells were stimulated with anti-CD3 mAb and irradiated IL-2+/+ and IL-2−/− splenocytes, respectively, in the absence (white circles) or presence of 1 ng/ml IL-2 (crosses) or 3 ng/ml IL-15 (black circles). (D) CD4+ cells were stimulated for 5 days with 0.1 µg/ml anti-CD3 and irradiated TdACs. On day 0, 1, 2, or 3, IL-15 alone or IL-15 plus anti-IL-2 mAb was added. On day 4, proliferation was measured by 3H-thymidine incorporation and represented as bar graphs (with left Y-axis) as ratio of cpmIL-15/cpmnone (black bars) or cpmIL-15 plus anti-IL-2/cpmnone (white bars). The line represents the percentage of reduction of the IL-15 effect by IL-2 depletion as calculated using the following formula: [(cpmIL-15– cpmIL-15 plus anti-IL-2)/cpmIL-15]×100. Results are from one representative out of three experiments.
Figure 3.
TCR-triggered IL-15-expanded CD4+ T cells remain dependent on IL-15 for expansion and survival.
CD4+ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdACs without or with 3 ng/ml IL-15. After 3 days, 50 ng/ml soluble IL-15Rα (sIL-15Rα, T1, 50 ng/ml) or control mutated sIL-15Rα (M4), lacking IL-15 binding capacity, were added. (A) Proliferation of CD4+ T cells, as determined by 3H-thymidine incorporation of triplicate cultures on day 5. (B) Viable CD4+ cell recovery on day 5, determined by flow cytometry. Bar graphs represent the mean±SEM. Statistical significance was calculated using student t-test: ns = not significant, ** P<0.01, *** P<0.001. Data are representative of three independent experiments.
Figure 4.
Exogenous IL-15 decreases the fraction but not the absolute number of CD25High CD4+ T cells.
(A) IL-15 alters TCR-induced gene expression of IL-2 and IL-15 receptor subunits. Purified CD4+ T cells were stimulated with 0.1 µg/ml anti-CD3 mAb and irradiated TdACs in the absence (white circles) or presence (black circles) of 3 ng/ml IL-15. At the indicated time points, CD4+ T cells were purified for PCR analysis of indicated gene transcripts and expressed as 2−ΔCt where ΔCt = Cttarget gene - mean Ctnormalization genes. Statistical significance was calculated using student t-test: ns = not significant, ** P<0.01, *** P<0.001. (B) Spleen CD4+ T cells were left unstimulated or stimulated with anti-CD3 in the presence or absence of IL-15 for two days, as indicated. Shown are histogram overlays of CD25 expression, gated on viable CD4+ T cells (d: day). (C) CFSE-labeled CD4+ T cells were stimulated with 0.1 µg/ml anti-CD3 and irradiated TdAC, without or with 3 ng/ml IL-15 for four or five days. Shown are flow cytometry dot plots of CFSE dye dilution versus CD25 expression in the viable CD4+ gate (left, shown percentages were calculated on viable CD4+ T cells) and summarizing bar graph of the CD25 high fraction (left). Statistical significance was calculated using student t-test: * P<0.05, ** P<0.01. (D) Absolute numbers of viable CD25High, CD25Int, CD25Negative and total CD4+ T cells, as determined by flow cytometry using micro-beads as reference. White and black bars represent stimulation with anti-CD3 in the absence or presence of 3 ng/ml rhIL-15, respectively. Statistical analysis was calculated using 2-way ANOVA and Bonferroni posttest: ns = not significant, *** P<0.001. Data shown are representative of three independent experiments. (E) CFSE-labeled CD4+ T cells were stimulated as in B. Shown are histogram overlays (top) of CD25 expression of the last generation of viable CD4+ T cells (by CFSE dilution) on day 5 after stimulation in the absence (dashed line) or presence (shaded) of 3 ng/ml rmIL-15. Bar graph (bottom) represents the mean fluorescenceIntensity of CD25 expression in the last generation, as shown in the overlays. Statistical significance was calculated using student t-test: * P<0.05, ** P<0.01.
Figure 5.
CD25High CD4+ T cells originate from natural CD25+ CD4+ T cells.
(A) Histogram showing CD25 expression pattern of unstimulated CD4+ T cells (day 0; top). Isolated CD25− and CD25+ CD4+ T cells were co-cultured in a cross-over setup with only one of the subsets carrying CFSE label. Three days after stimulation with 0.1 µg/ml anti-CD3 mAb and irradiated TdAC, CD25 expression and CFSE dye dilution were measured using flow cytometry and displayed as density plots (bottom). (B) CFSE-labeled total CD4+ T cells (top) or CD25+-depleted CD4+ T cells (bottom) were stimulated as in (A). Shown are flow cytometry density plots of CFSE dye dilution versus CD25 expression gated on viable CD4+ T cells. Percentages indicate the percentage of CD25High or CD25Int cells within all CD25+ positive CD4+ T cells. (C) Contour plots showing CD25 versus Foxp3 expression of unstimulated and day 4-stimulated CD4+ T cells. Numbers on the right reflect the percentages within the gates 1–5, gated on viable CD4+ T cells. (D) CFSE-labeled total CD4+ T cells (shaded) or CD25+-depleted CD4+ T cells (solid line) were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for 3 days. Shown are histogram overlays gated on viable CD4+ T cells and bar graph of proliferation index mean±SEM. (E) Total CD4+ T cells or CD25-depleted CD4+ T cells were stimulated with anti-CD3 (0.1 µg/ml) and irradiated TdACs for indicated time and proliferation was measured via 3H-thymidine incorporation. (B, D, E) Bar graphs represent the mean±SEM. Statistical significance was calculated by student t-test, ns: not significant, * P<0.05, ** P<0.01, *** P<0.001. Experiments performed at least 3 times with similar results.
Figure 6.
IL-15 blocks suppression by CD25High CD4+ T cells.
(A) CD4+ T cell numbers and (B) proliferation of total CD4+ T cells (white bars) or CD25-depleted CD4+ T cells (grey bars) stimulated with anti-CD3 mAb, irradiated TdACs and supplemented or not with IL-15. Data are expressed as absolute values (left panels) or fold increase of IL-15-supplemented versus non-supplemented cultures (right panels; 1 reflects no effect of IL-15 addition). Bar graphs represent the mean ± SEM. Statistical significance was calculated by student t-test, ns: not significant, * P<0.05, ** P<0.01, *** P<0.001. (C) CD25− CD4+ responder T cells (Tresp) were co-cultured with CD25High (left) or CD25Int (right) CD4+ T cells, FACS-purified from activated CD4+ T cell cultures as described in Materials and Methods, as putative suppressors at indicated ratios, prior to stimulation with 0.1 µg/ml anti-CD3, irradiated TdACs, and indicated cytokines for 3 days. Proliferation was measured via 3H-thymidine incorporation and the percent suppression was calculated as follows: % suppression = 100×(cpmResponder – cpmCoculture)/cpmResponder. Statistical significance between IL-15-supplemented and non-supplemented cultures was calculated using two-way ANOVA with Bonferroni posttest: ns: not significant, * P<0.05, ** P<0.01, *** P<0.001 (statistical analysis versus IL-2-supplemented cultures not shown). All experiments were performed at least 3 times with similar results.