Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production

Abstract

During the immune response to pathogens and autoantigens, CD8T cells are exposed to numerous inflammatory agents including the cytokine IL-12. Previous studies have focused on how IL-12 regulates T cell functions when present during or after the activation of the T cell receptor (TCR). However, recent studies suggest that prior exposure to IL-12 also alters the TCR responsiveness of murine T cells. Whether similar phenomena occur in human activated CD8T cells and the mechanisms mediating these effects remain unexplored. In this study, we observed that pretreatment of human activated CD8T cells with IL-12 results in increased cytokine mRNA and protein production following subsequent TCR challenge. The potentiation of TCR-mediated cytokine release was transient and required low doses of IL-12 for at least 24h. Mechanistically, prior exposure to IL-12 increased the TCR induced activation of select MAPKs and AKT without altering the activation of more proximal TCR signaling molecules, suggesting that the IL-12 mediated changes in TCR signaling are responsible for the increased production of cytokines. Our data suggest that prior treatment with IL-12 potentiates human CD8T cell responses at sites of infection and inflammation, expanding our understanding of the function of this clinically important cytokine.

Keywords: CD8T cells; Human T cells; IL-12; TCR signaling.

Figure 1. Human activated CD8 T cells pretreated with IL-12 have increased IFN-γ and TNF-α production following TCR stimulation

(A-D) Human activated CD8 T cells were exposed to media alone (no cytokine) or to different recombinant cytokines (50 ng/mL) for 24 h, washed, and then stimulated with 1 μg/mL of plate bound anti-CD3 antibodies for 24 h. IFN-γ and TNF-α production were determined in the cell culture supernatants by ELISA. Graphs are shown as the mean ±SEM of values from three to seven different donors. Data were statistically compared to no cytokine cells using a two-tail Student's t test. *p<0.05; **p<0.01; ***p<0.001; no symbol=not significant.

Figure 2. The potentiation of TCR-mediated cytokine production by human activated CD8 T cells is transient and requires low physiological doses of IL-12 for at least 24 hours

Human activated CD8 T cells were left untreated (no cytokine) or (A) treated for various times with IL-12 (50 ng/mL), (B) exposed for 24 h to various doses of IL-12, or (C) treated with IL-12 for 24 h (50 ng/mL), washed and rested for various times. After treatment, the cells were washed and stimulated with 1 μg/mL of plate bound anti-CD3 antibodies for 24 hours. Then IFN-γ production was determined by ELISA. (D) Human activated CD8 T cells were left untreated or treated with IL-12 for 24 h (50 ng/mL), washed and immediately stimulated with different doses of plate bound anti-CD3 antibodies for 24 hours. IFN-γ production was then determined by ELISA. Graphs are shown as the mean ±SEM of values from three to five different donors. Data were statistically compared to no cytokine cells using a two-tail Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s. or no symbol=not significant.

Figure 3. Human activated CD8 T cells have variable surface expression of IL-12R β1 and β2

Human activated CD8 T cells were stained with anti-IL-12 R β1 or anti-IL-12R β2 and isotype controls and the expression of the IL-12R was then analyzed using flow cytometry. (A) Representative histograms show the expression of IL-12 receptor β1 or β2 following gating on live lymphocytes (based on forward and side scatter). Black line represents staining with IL-12R antibody and gray line represents staining with isotype controls. (B) The median fluoresce intensities (Median FI) of the expression of the IL-12 receptor subunits were determined and plotted as Mean±SD of three separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001

Figure 4. Prior exposure to IL-12 increases the frequency of human activated CD8 T cells making IFN-γ and TNF-α upon TCR stimulation

Human activated CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/mL). Cells were then washed and stimulated with 1 μg/mL of plate bound anti-CD3 antibodies for 18 h with BFA added for the last 5 h. Intracellular protein levels of IFN-γ and TNF-α were determined by flow cytometry. 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 fluorescence intensities of cytokine expression for IFN-γ and TNF-α single producing cells and IFN-γ/TNF-α double producing cells were then determined. Data are shown as (A) representative plots, (B) percentage of T cells producing IFN-γ or TNF-α alone or IFN-γ and TNF-α together, or (C) median fluorescent intensity of IFN-γ or TNF-α in single (SP) and double (DP) producing cells. Each data set is shown as the mean ± SEM of four separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant.

Figure 5. The IL-12 pretreatment increases IFN-γ and TNF-α mRNA expression after TCR stimulation in human activated CD8 T cells

Human activated CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/mL), washed, and stimulated with or without 1 μg/mL of plate bound anti-CD3 antibodies for 6 and 18 h. The expression of IFN-γ and TNF-α mRNA were then determined by qPCR at these times. 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 three to four separate donors.

Figure 6. The IL-12 mediated enhancement of cytokine production in human activated CD8 T cells is not due to residual STAT4 synergizing with TCR signals

Human activated CD8 T cells were left untreated or pretreated with 50 ng/mL of IL-12 for different times. At the indicated times, cells were lysed and immunoblotting for total and phosphorylated STAT4 was performed in whole cell lysates as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as representative blots (A) and (B-C) mean ± SEM of normalized results of three separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant.

Figure 7. Prior exposure to IL-12 does not alter the activation of proximal TCR signaling molecules in human activated CD8 T cells

Human activated CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/mL). Cells were then washed and stimulated for various times using 3 μg/mL of crosslinked anti-CD3. Then the phosphorylation of signaling molecules was assessed in whole cell lysates by immunoblotting as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A and C) representative blots and (B and D) mean ± SEM of normalized results of three separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant.

Figure 8. IL-12 pretreatment enhances the TCR-induced activation of select MAP kinases and AKT in human activated CD8 T cells

Human activated CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/mL). Cells were then washed and stimulated for various times using 3 μg/mL of crosslinked anti-CD3. Then, the phosphorylation of signaling molecules was assessed in whole cell lysates by immunoblotting as described in the materials and methods section. Results were normalized to GAPDH and the maximal level of activation of no cytokine cells. Data are shown as (A) representative blots and (B) mean ± SEM of normalized results of three to seven separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant.

Figure 9. IL-12 pretreatment increases the expression of SOS1 and the phosphorylation of MKK3/MKK6 downstream of the TCR

Human activated CD8 T cells were left untreated or pretreated with IL-12 for 24 h (50 ng/mL). Cells were then washed and stimulated for various times using 3 μg/mL of crosslinked anti-CD3. The phosphorylation or total expression of signaling molecules was assessed in whole cell lysates by immunoblotting and the results were normalized to GAPDH. Upon normalization, the average of the total expression of SOS1 at each time point was calculated. Data are shown as (A) representative blots and (B-C) mean ± SEM of normalized results of three to seven separate donors. Data were analyzed with two-tail, unpaired Student's t test. *p<0.05; **p<0.01; ***p<0.001; n.s.=not significant.