IL-4 acts as a potent stimulator of IFN-γ expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet

Abstract

CD8+ T cell synthesis of IFN-γ is an important component of the CD8+ T cell immune response. In short-term cultures of murine pan-T cells, we found that IL-4 was the principal cytokine responsible for driving IFN-γ synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to induce low levels of IFN-γ mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 stimulation was necessary for IFN-γ secretion. IL-4 induction of IFN-γ was explained by its ability to induce Eomesodermin and T-bet transcription factors whose expression was further increased by CD3/CD28. Expression of Eomesodermin, T-bet and IFN-γ induced by IL-4 was partially dependent upon activation of MAPK and PI3K but independent of the canonical IL-4-activated transcription factor, STAT6. In contrast, expression of IFN-γ induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. These novel findings point to a function for IL-4 as a direct regulator of IFN-γ expression in CD8+ T cells and reveal the molecular mechanisms involved.

Fig. 1

Role of IL-2 and IL-4 in induction of IFN-γ expression in CD8+ T cells. Experiments were performed with T cells from littermate C57BL/6 × 129 Sv (A–C) or C57BL/6 (D) mice. (A) Pan-T cells or purified CD8+ T cells were stimulated or not with CD3 and CD28 mAb. After 48 h, expression of IFN-γ upon CD44^lo and CD44^hi CD8+ T cells was determined by flow cytometry. At left are shown representative two color flow cytometry plots of CD44 versus IFN-γ staining. Numbers indicate percentage of CD8+ T cells in each quadrant. Bar charts at right show the mean percentage of IFN-γ positive cells + 1 SEM for CD44^lo and CD44^hi CD8+ T cells determined in repeat experiments (n=5 mice). (B) Pan-T cells were stimulated as in (A) in the presence or absence of neutralizing anti-IL-2 and/or anti-IL-4 antibodies. Representative flow cytometry plots of CD44 versus IFN-γ staining are shown at left. Bar charts show mean percentage of IFN-γ positive cells + 1 SEM for CD44^lo and CD44^hi CD8+ T cells under each stimulation condition determined in repeat experiments (n=5 mice). (C) Purified CD8+ T cells were stimulated as in (A) in the presence or absence of recombinant IL-2 or IL-4. Representative flow cytometry plots of CD44 versus IFN-γ staining are shown at left. Bar graphs at right show the mean percentage of IFN-γ positive cells + 1 SEM for CD44^lo and CD44^hi CD8+ T cells under the indicated stimulation conditions determined in repeat experiments (n=4 mice). (D) Pan-T cells from C57BL/6 IL-4-deficient (IL-4 KO) and wild type control C57BL/6 mice were stimulated as in (A) in the presence or absence of neutralizing anti-IL-2 and/or anti-IL-4 mAb. Expression of IFN-γ upon CD44^lo and CD44^hi CD8+ T cells was determined by flow cytometry after 48 h of culture. At top are shown representative flow cytometry plots of CD44 versus IFN-γ staining. Bar graphs show the mean percentage of IFN-γ positive cells + 1 SEM for CD44^lo and CD44^hi CD8+ T cells for each mouse genotype and condition of stimulation as determined in repeat experiments (n=5 mice each genotype). Statistical significance was determined using the paired Student’s t-test.

Fig. 2

IL-4 induced synthesis of IFN-γ in CD8+ T cells with and without concurrent CD3/CD28 stimulation. All experiments were performed with T cells from C57BL/6 × 129 Sv mice. (A) Purified CD8+ T cells were stimulated or not with IL-4 and/or CD3 and CD28 mAb for 48 h. Expression of IFN-γ in CD44^lo and CD44^hi CD8+ T cells was determined by flow cytometry. At left are shown representative flow cytometric plots of CD44 vs IFN-γ staining. Bar graph at right shows the mean percentage of IFN-γ positive cells + 1 SEM for CD44^lo and CD44^hi CD8+ T cells under different stimulation conditions as determined in repeat experiments (n=7 mice). (B) Purified CD8+ T cells were stimulated as in (A). Concentrations of IFN-γ in well supernatants were determined by ELISA. Shown is the mean concentration of IFN-γ + 1 SEM for each stimulation condition as determined repeat experiments (n=4 mice). N.D., not detectable. (C) Purified CD8+ CD44^lo T cells were stimulated or not with IL-4 and/or CD3 and CD28 mAb for 6 h. Relative expression levels of IFN-γ mRNA were determined by real time RT-PCR. Results are expressed as fold change in IFN-γ expression compared to non-stimulated T cells. Similar results were obtained in a repeat experiment. In (A) and (B) statistical significance was determined using the paired Student’s t-test.

Fig. 3

IL-4 induction of Eomes and T-bet transcription factors in CD8+ T cells. Pan-T cells or purified CD8+ T cells from C57BL/6 × 129 Sv mice were stimulated or not with CD3 and CD28 mAb in the presence or absence of a neutralizing IL-4 mAb or with or without IL-4 or with IL-4 alone as indicated. After 48 h, expression of CD44 and Eomes (A) or T-bet (B) upon CD8+ T cells was determined by flow cytometry. At left are shown representative flow cytometry plots of CD44 versus transcription factor staining. Bar graphs at right show the mean percentage of transcription factor positive cells + 1 SEM within CD44^lo and CD44^hi CD8+ T cell populations under the different stimulation conditions as determined in repeat experiments (A, n= at least 5 mice; B, n= at least 4 mice). (C) Shown are two-color flow cytometry plots of Eomes versus T-bet expression on CD44^lo and CD44^hi populations from A and B.

Fig. 4

Role of STAT6 in IL-4-mediated induction of IFN-γ expression in CD8+ T cells. Purified CD8+ T cells from STAT6-deficient (STAT6 KO) or wild type C57BL/6 control mice were stimulated or not with CD3 and CD28 mAb and/or IL-4. After 48 h, expression of IFN-γ (A), Eomes and T-bet (B) within CD44^lo and CD44^hi CD8+ T cell populations was determined by flow cytometry. Bar graphs show the mean percentage of IFN-γ or transcription factor-positive cells + 1 SEM for each condition of stimulation and mouse strain as determined in repeat experiments (n= at least 3 mice each genotype). Statistical significance was determined using the paired Student’s t-test.

Fig. 5

Roles of ERK and PI3K activation in IL-4-mediated induction of IFN-γ expression in CD8+ T cells. Purified CD8+ T cells from C57BL/6 mice were stimulated or not with CD3 and CD28 mAb and/or IL-4 in the presence or absence of the ERK inhibitor PD98059 (A, C) or the PI3K inhibitor wortmannin (B, D). After 48 h, expression of IFN-γ (A), Eomes (B) and T-bet (C) within CD44^lo and CD44^hi CD8+ T cell populations was determined by flow cytometry. Bar graphs show the mean percentage of IFN-γ or transcription factor-positive cells + 1 SEM for each condition of stimulation as determined in repeat experiments (n= at least 3 mice each genotype). Statistical significance was determined using the paired Student’s t-test. (E) Purified CD8+ T cells from STAT6-deficient or wild type C57BL/6 control mice were stimulated or not as in (A–D). After 48 h, the concentration of IFN-γ in well supernatants was determined by ELISA. Shown is the mean concentration of IFN-γ + 1 SEM for each condition of stimulation as determined in repeat experiments (n=3). Statistical significance was determined using the two sample Student’s t-test.