IL-15 augments TCR-induced CD4⁺ T cell expansion in vitro by inhibiting the suppressive function of CD25High CD4⁺ T cells

Due to its critical role in NK cell differentiation and CD8(+) T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4(+) T cells. The increased levels of IL-15 found in several CD4(+) T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4(+) T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4(+) and CD8+ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4(+) T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15R alpha was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15R beta, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4(+) T cell suppression by a gradually expanding CD25(High)CD4(+) T cell subset that expresses Foxp3 and originates from CD4(+)CD25(+) Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology

Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Background: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 mu g and less) and has a sound reproducibility. Conclusions: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner

IL-15 augments TCR-Induced CD4+ T Cell Expansion In Vitro by Inhibiting the Suppressive Function of CD25High CD4+ T Cells

Due to its critical role in NK cell differentiation and CD8+ T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4+ T cells. The increased levels of IL-15 found in several CD4+ T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4+ T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4+ and CD8+ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4+ T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15Rα was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15Rβ, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4+ T cell suppression by a gradually expanding CD25HighCD4+ T cell subset that expresses Foxp3 and originates from CD4+CD25+ Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology

Exogenous IL-15 decreases the fraction but not the absolute number of CD25^High CD4⁺ T cells.

<p>(<b>A</b>) 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 = Ct_{target gene} - mean Ct_{normalization genes}. Statistical significance was calculated using student t-test: ns = not significant, ** <i>P</i><0.01, *** <i>P</i><0.001. (<b>B</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). (<b>C</b>) 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: * <i>P</i><0.05, ** <i>P</i><0.01. (<b>D</b>) Absolute numbers of viable CD25^High, CD25^Int, CD25^Negative 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, *** <i>P</i><0.001. Data shown are representative of three independent experiments. (<b>E</b>) CFSE-labeled CD4⁺ T cells were stimulated as in <i>B</i>. 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: * <i>P</i><0.05, ** <i>P</i><0.01.</p

IL-15 blocks suppression by CD25^High CD4⁺ T cells.

<p>(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, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. (C) CD25⁻ CD4⁺ responder T cells (Tresp) were co-cultured with CD25^High (left) or CD25^Int (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×(cpm_Responder – cpm_Coculture)/cpm_Responder. Statistical significance between IL-15-supplemented and non-supplemented cultures was calculated using two-way ANOVA with Bonferroni posttest: ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 (statistical analysis versus IL-2-supplemented cultures not shown). All experiments were performed at least 3 times with similar results.</p

CD25^High CD4⁺ T cells originate from natural CD25⁺ CD4⁺ T cells.

<p>(<b>A</b>) 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>B</b>) CFSE-labeled total CD4⁺ T cells (top) or CD25⁺-depleted CD4⁺ T cells (bottom) were stimulated as in (<b>A</b>). Shown are flow cytometry density plots of CFSE dye dilution versus CD25 expression gated on viable CD4⁺ T cells. Percentages indicate the percentage of CD25^High or CD25^Int cells within all CD25⁺ positive CD4⁺ T cells. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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>B, D, E</b>) Bar graphs represent the mean±SEM. Statistical significance was calculated by student t-test, ns: not significant, * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. Experiments performed at least 3 times with similar results.</p