2) [19]

2) [19]. was decided using the paired Students t-test. NIHMS334832-product-01.tif (1.3M) GUID:?B1FC9216-0817-452E-85FA-C2369B67F9A4 02: FIGURE 2. Cellular source of IL-4 for IFN- induction in CD8+ T cells (A) Pan T cells were depleted or not of NKT cells by unfavorable selection using an NK1.1 mAb. Shown are two-color plots of DX5 mAb (CD49b) and CD1d-CGalCer tetramer staining from three mice of each genotype. (B) Pan-T cells or NKT cell-depleted pan-T cells from wild type C57BL/6 129 Sv mice or C57BL/6 mice (as in A) were stimulated with CD3 and CD28 mAb in the presence or absence of a neutralizing anti-IL-4 mAb. After 48 h, expression of IFN- upon CD44lo and CD44hi CD8+ T cell subsets was determined by circulation cytometry. Bar graphs show the imply percentage of IFN- positive cells + 1 SEM for CD44lo and CD44hi CD8+ T cells for each mouse genotype as decided in repeat experiments (n=5 mice each genotype). Statistical significance was decided using the two sample Students t-test. NIHMS334832-product-02.tif (13M) GUID:?0E9BA6AA-FD05-40D6-950C-5B46C72DBA46 03: FIGURE 3. Role of IL-4 in induction of CD8+ T cell cytotoxic function (A) Pan-T cells from IL-4-deficient and wild type control C57BL/6 mice were stimulated or not with anti-CD3 and CD28 mAb in the presence or absence of neutralizing anti-IL-2 and/or anti-IL-4 mAb. After 72 h, cells were restimulated in CD3 mAb coated (1 mg/well) wells (1 105 cells/well) in medium containing labeled CD107a mAb before staining for additional cell surface markers. Expression of cell surface CD107a upon CD8+ CD44lo and CD44hi T cell subsets was determined by circulation cytometry. At left are shown representative circulation cytometry plots of CD44 and CD107a staining. The bar graph at right shows the mean percentage of CD107a-positive cells + 1 SEM among CD8+ CD44hi cells under different conditions of activation in the two strains of mice as decided in repeat experiments (n=4 mice each genotype). (B) Purified CD8+ T cells from C57BL/6 mice were stimulated or not with CD3 and CD28 mAb and/or IL-4 as in (A) and expression of CD44 and CD107a was determined by circulation cytometry. At left are shown representative circulation cytometry plots of CD44 and CD107a staining. Bar graphs at right show the mean percentage of CD107a-positive cells + 1 SEM for CD44lo and CD44hi CD8+ T cells under different activation conditions as decided in repeat experiments (n=3 mice). (C) Pan-T cells from IL-4-deficient mice and wild type control C57BL/6 mice were stimulated as in (A). After 4 h restimulation, concentrations of Granzyme B in well supernatants were determined by ELISA. Shown is the mean concentration of Granzyme B +1 SEM for the different stimulation conditions and mouse genotypes as decided in repeat experiments (n=4 mice each genotype). NIHMS334832-product-03.tif (14M) GUID:?DF092284-D20C-42C8-A83A-2026AF01F572 04: FIGURE 4. Role Idarubicin HCl of Idarubicin HCl IL-4 in CD8+ T cell proliferation CFSE-labeled pan-T cells from C57BL/6 mice were stimulated or not with anti-CD3 and CD28 mAb in the presence or absence of neutralizing anti-IL-2 and/or anti-IL-4 mAb. After 96 h, CFSE fluoresence of CD8+ T cells was determined Idarubicin HCl by circulation cytometry. At left are shown representative circulation cytometry plots of CFSE fluoresence versus CD44 staining. The bar graph at right shows the mean percentage of live CD8+ T cells + 1 SEM that have undergone the indicated quantity of divisions under the different conditions of activation as decided in repeat experiments (n=4 mice). Statistical significance was decided using the paired Students t-test. NIHMS334832-product-04.tif (7.1M) GUID:?4BB0C053-A954-4589-87E9-BEDC86C55331 Abstract CD8+ T cell synthesis of IFN- 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- synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to TSPAN7 induce low levels of IFN- mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 activation was necessary for IFN- secretion. IL-4 induction of IFN- 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- 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- induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. Idarubicin HCl These novel findings point to a function for IL-4 as a direct regulator of IFN- expression in CD8+ T cells and reveal the molecular mechanisms involved. compared to mice singly-deficient for.