IL-12 mediated enhancement of cytokine production following TCR stimulation is mediated by a function of both transcriptional and post-transcriptional effects.

<p>(A) IL-12R β1 and IL-12R β2 expression was measured on human activated CD4 T cells by flow cytometry. Black line represents staining with IL-12R antibody and gray line represents staining with isotype-matched control antibody. Blots are representative of five different donors. (B-D) Human activated CD4 T cells were left untreated or pretreated with 50 ng/mL IL-12 for 6 h and then subsequently stimulated with 6 μg/mL plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 18 h. Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-TCR stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as (B) representative plots or (C and D) mean ± SEM of five to nine separate donors. (E) Gene expression of IFN-γ, TNF-α, IL-4, IL-13 and IL-10 were determined by qPCR in IL-12 pretreated and untreated activated CD4 T cells stimulated with or without 6 μg/mL of plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 18 h. Data were normalized to that of mRNA encoding β-actin and presented relative to that of no cytokine cells. Results are shown as mean ± SEM of five separate donors. Data were analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p

Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism

<div><p>Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism.</p></div

IL-12 pretreated human CD4 T cells have enhanced production of a range of cytokines following TCR stimulation.

<p>Human activated CD4 T cells were left untreated or pretreated with 50 ng/mL of IL-12 for 6 h and then subsequently stimulated with various doses of plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 24 h. Protein levels of (A) IFN-γ, (B) TNF-α, (C) IL-4, (D) IL-13, and (E) IL-10 were assessed by ELISA. Data for each treatment in an individual experiment were normalized to the maximum amount produced by no cytokine cells. The mean normalized value ± SEM from four to five different donors is shown. Data was analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p

IL-12 pretreated human activated CD4 T cells undergo metabolic reprogramming towards oxidative metabolism.

<p>Human activated CD4 T cells were treated with or without 50 ng/mL IL-12 for 6 h. Cells were plated onto poly-l-lysine-coated plates and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed at basal state and following addition of indicated compounds. (A) and (B) graphs are representative of 4 different donors and used to calculate the (C) basal respiration, (D) basal ECAR, (E) maximal respiratory capacity, and (F) maximal ECAR. Data were analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p

IL-12 mediated enhancement of cytokine production following TCR stimulation is mediated by a function of both transcriptional and post-transcriptional effects.

<p>(A) IL-12R β1 and IL-12R β2 expression was measured on human activated CD4 T cells by flow cytometry. Black line represents staining with IL-12R antibody and gray line represents staining with isotype-matched control antibody. Blots are representative of five different donors. (B-D) Human activated CD4 T cells were left untreated or pretreated with 50 ng/mL IL-12 for 6 h and then subsequently stimulated with 6 μg/mL plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 18 h. Protein levels of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 were determined by analysis of intracellular-cytokine staining. Live lymphocytes were gated based on forward and side scatter. Quadrants were set so the baseline cytokine production of non-TCR stimulated cells was less than 1%. The frequencies and median fluoresce intensities of cytokine expression were then determined. Data are shown as (B) representative plots or (C and D) mean ± SEM of five to nine separate donors. (E) Gene expression of IFN-γ, TNF-α, IL-4, IL-13 and IL-10 were determined by qPCR in IL-12 pretreated and untreated activated CD4 T cells stimulated with or without 6 μg/mL of plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 18 h. Data were normalized to that of mRNA encoding β-actin and presented relative to that of no cytokine cells. Results are shown as mean ± SEM of five separate donors. Data were analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p

IL-12 pretreatment increases the activation of select signaling molecules downstream of the TCR.

<p>Human activated CD4 T cells were incubated with or without IL-12 (50 ng/mL for 6 h) and then stimulated with anti-TCR (anti-CD3 clone OKT3) and anti-CD4 (clone RPA-T4) for various times. The phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) and (C) representative blots from different donors and (B) and (D) mean ± SEM of normalized results of six to nine different donors. The loading controls shown for each figure correspond to at least one of the blots shown but for the quantification each blot was quantified with its respective control Data were analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p

Exposure to IL-12 selectively alters how human activated CD4 T cells respond to TCR stimulation.

<p>Human activated CD4 T cells were left untreated (no cytokine) or treated (A) for 6 h with various cytokines (50 ng/mL), (B) various doses of IL-12, or (C) various times with 50 ng/mL of IL-12. (A-C) After pretreatment, cells were washed and stimulated with 2 μg/mL of plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 24 h. (D) Human activated CD4 T cells were pretreated for 6 h with 50 ng/mL of IL-12, rested for various times and then stimulated with plate bound anti-TCR antibodies (2 μg/mL) (anti-CD3 clone OKT3) for 24 h. IFN-γ production was determined by ELISA and results were normalized to the amounts produced by no cytokine. Graphs are shown as the mean ±SEM of normalized values from four to seven different donors. (E and F) Human activated CD4 T cells were incubated with or without 50 ng/ml IL-12 for 6 h and then stimulated with titrating doses of plate bound anti-TCR antibodies (anti-CD3 clone OKT3) for 24 h. The IFN-γ production was determined by ELISA. Data from five separate donors were normalized, plotted using GraphPad Prism and the EC₅₀ values calculated. Data in (A), (B), and (D) was statistically compared to no cytokine cells using an unpaired one-way ANOVA. Data in (C) and (F) were statistically compared to no cytokine cells with a two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. or no symbol = not significant.</p

Prior exposure to IL-12 alters select TCR-induced signaling pathways in human activated CD4 T cells.

<p>Human activated CD4 T cells were (A and B) treated with or without IL-12 (50 ng/mL) for different times or (C and D) incubated with or without 50 ng/mL of IL-12 for 6 h and then stimulated with anti-TCR (anti-CD3 clone OKT3) and anti-CD4 (clone RPA-T4) antibodies for various times. The phosphorylation of signaling molecules was determined in whole cell lysates by immunoblotting. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A and C) representative blots from different donors and (B and D) mean ± SEM of normalized results of three to six separate donors. The loading controls shown for each figure correspond to at least one of the blots shown but for the quantification each blot was quantified with its respective control. Data were analyzed with two-tail, unpaired Student’s t test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; n.s. = not significant.</p