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. 2002 Apr 1;195(7):811-23.
doi: 10.1084/jem.20011508.

c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 have distinct roles in CD8(+) T cell activation

Dietrich Conze  1 , Troy Krahl, Norman Kennedy, Linda Weiss, Joanne Lumsden, Patricia Hess, Richard A Flavell, Graham Le Gros, Roger J Davis, Mercedes Rincón
Affiliations

VSports在线直播 - c-Jun NH(2)-terminal kinase (JNK)1 and JNK2 have distinct roles in CD8(+) T cell activation

"VSports" Dietrich Conze et al. J Exp Med. .

Abstract

The c-Jun NH(2)-terminal kinase (JNK) signaling pathway is induced by cytokines and stress stimuli and is implicated in cell death and differentiation, but the specific function of this pathway depends on the cell type. Here we examined the role of JNK1 and JNK2 in CD8(+) T cells. Unlike CD4(+) T cells, the absence of JNK2 causes increased interleukin (IL)-2 production and proliferation of CD8(+) T cells. In contrast, JNK1-deficient CD8(+) T cells are unable to undergo antigen-stimulated expansion in vitro, even in the presence of exogenous IL-2. The hypoproliferation of these cells is associated with impaired IL-2 receptor alpha chain (CD25) gene and cell surface expression VSports手机版. The reduced level of nuclear activating protein 1 (AP-1) complexes in activated JNK1-deficient CD8(+) T cells can account for the impaired IL-2 receptor alpha chain gene expression. Thus, JNK1 and JNK2 play different roles during CD8(+) T cell activation and these roles differ from those in CD4(+) T cells. .

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Figures (V体育ios版)

Figure 1.
Figure 1.
In vivo viral immune response in the JNK2-deficient mice. (A) Virus titers in the lung tissue of infected wild-type (WT) and Jnk2 − / − mice were determined at different time points during influenza infection. Open circles represent individual infected wild-type mice; filled circles represent individual infected Jnk2 − / − mice; (X) represents the geometric mean for wild-type mice; (*) represents the geometric mean for Jnk2 − / − mice. (B and C) Serum was collected from infected wild-type and Jnk2 − / − mice at different time points during influenza infection. Virus-specific IgG1 (B) and virus-specific IgG2A (C) Ab levels were determined by ELISA.
Figure 1.
Figure 1.
In vivo viral immune response in the JNK2-deficient mice. (A) Virus titers in the lung tissue of infected wild-type (WT) and Jnk2 − / − mice were determined at different time points during influenza infection. Open circles represent individual infected wild-type mice; filled circles represent individual infected Jnk2 − / − mice; (X) represents the geometric mean for wild-type mice; (*) represents the geometric mean for Jnk2 − / − mice. (B and C) Serum was collected from infected wild-type and Jnk2 − / − mice at different time points during influenza infection. Virus-specific IgG1 (B) and virus-specific IgG2A (C) Ab levels were determined by ELISA.
Figure 1.
Figure 1.
In vivo viral immune response in the JNK2-deficient mice. (A) Virus titers in the lung tissue of infected wild-type (WT) and Jnk2 − / − mice were determined at different time points during influenza infection. Open circles represent individual infected wild-type mice; filled circles represent individual infected Jnk2 − / − mice; (X) represents the geometric mean for wild-type mice; (*) represents the geometric mean for Jnk2 − / − mice. (B and C) Serum was collected from infected wild-type and Jnk2 − / − mice at different time points during influenza infection. Virus-specific IgG1 (B) and virus-specific IgG2A (C) Ab levels were determined by ELISA.
Figure 2.
Figure 2.
Activation of the JNK signaling pathway in CD8+ T cells. (A) Total CD8+ T cells (106 cells/ml) from wild-type mice were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Cells were harvested at the indicated time points, lysed, and cell extracts were assayed for JNK activity. Phosphorylated c-Jun was detected after SDS-PAGE by autoradiography. (B) Jnk expression was examined by Northern blot in CD8+ T cells stimulated as in A for the indicated time periods. Expression of 28S ribosomal RNA was used as a control. (C) JNK expression was examined by Western blot analysis in CD8+ T cells stimulated as in A for the indicated time periods. Actin expression was used as a control. (D) JNK activity was determined using cell extracts from freshly isolated (-) wild-type (WT) and Jnk2 − / − CD8+ T cells and wild-type and Jnk2 − / − CD8+ T cells stimulated as in A for 48 h.
Figure 2.
Figure 2.
Activation of the JNK signaling pathway in CD8+ T cells. (A) Total CD8+ T cells (106 cells/ml) from wild-type mice were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Cells were harvested at the indicated time points, lysed, and cell extracts were assayed for JNK activity. Phosphorylated c-Jun was detected after SDS-PAGE by autoradiography. (B) Jnk expression was examined by Northern blot in CD8+ T cells stimulated as in A for the indicated time periods. Expression of 28S ribosomal RNA was used as a control. (C) JNK expression was examined by Western blot analysis in CD8+ T cells stimulated as in A for the indicated time periods. Actin expression was used as a control. (D) JNK activity was determined using cell extracts from freshly isolated (-) wild-type (WT) and Jnk2 − / − CD8+ T cells and wild-type and Jnk2 − / − CD8+ T cells stimulated as in A for 48 h.
Figure 2.
Figure 2.
Activation of the JNK signaling pathway in CD8+ T cells. (A) Total CD8+ T cells (106 cells/ml) from wild-type mice were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Cells were harvested at the indicated time points, lysed, and cell extracts were assayed for JNK activity. Phosphorylated c-Jun was detected after SDS-PAGE by autoradiography. (B) Jnk expression was examined by Northern blot in CD8+ T cells stimulated as in A for the indicated time periods. Expression of 28S ribosomal RNA was used as a control. (C) JNK expression was examined by Western blot analysis in CD8+ T cells stimulated as in A for the indicated time periods. Actin expression was used as a control. (D) JNK activity was determined using cell extracts from freshly isolated (-) wild-type (WT) and Jnk2 − / − CD8+ T cells and wild-type and Jnk2 − / − CD8+ T cells stimulated as in A for 48 h.
Figure 2.
Figure 2.
Activation of the JNK signaling pathway in CD8+ T cells. (A) Total CD8+ T cells (106 cells/ml) from wild-type mice were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs. Cells were harvested at the indicated time points, lysed, and cell extracts were assayed for JNK activity. Phosphorylated c-Jun was detected after SDS-PAGE by autoradiography. (B) Jnk expression was examined by Northern blot in CD8+ T cells stimulated as in A for the indicated time periods. Expression of 28S ribosomal RNA was used as a control. (C) JNK expression was examined by Western blot analysis in CD8+ T cells stimulated as in A for the indicated time periods. Actin expression was used as a control. (D) JNK activity was determined using cell extracts from freshly isolated (-) wild-type (WT) and Jnk2 − / − CD8+ T cells and wild-type and Jnk2 − / − CD8+ T cells stimulated as in A for 48 h.
Figure 3.
Figure 3.
JNK2 is a negative regulator of CD8+ T cells activation. (A) Total CD8+ T cells (2.5 × 105 cells/ml) from wild-type (WT) and Jnk2 − / − mice were stimulated with immobilized anti-CD3 (5 μg/ml) alone or in combination with soluble anti-CD28 (1 μg/ml) mAbs. The proliferative response was examined by [3H]thymidine incorporation. (B) Cell death determined by the TUNEL assay in freshly isolated (unstimulated) wild-type and Jnk2 − / − CD8+ T cells and wild-type and Jnk2 − / − CD8+ T cells (106 cells/ml) stimulated with anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 24 h. Numbers represent the percentage of TUNEL-positive cells. TdT, terminal deoxynucleotidyltransferase. (C) Wild-type and Jnk2 − / − CD8+ T cells were stimulated as described in B for 48 h IL-2 production was determined by ELISA. (D) Cytokine gene expression was determined by RPA using RNA extracted from freshly isolated (−) Wild-type and Jnk2 − / − (J2) CD8+ T cells and wild-type and J2 CD8+ T cells stimulated (+) as described in B for 24 h (E) Wild-type and Jnk2 − / − CD8+ T cells were stimulated as in B for 48 h IFN-γ production was determined by ELISA. (F) CD8+ T cells from wild-type and Jnk2 − / − mice (2.5 × 105 cells/ml) were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAb in the presence of exogenous IL-2 (50 U/ml) or a neutralizing anti-IL-2 (10 μg/ml) mAb. The proliferative response was analyzed by [3H]thymidine incorporation.
Figure 4.
Figure 4.
JNK1 is required for CD8+ T cell activation. (A) JNK activity was determined using cell extracts from freshly isolated (−) wild-type (WT) and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h (B) CD8+ T cells (2.5 × 105 cells/ml) from wild-type and Jnk1 − / − mice were stimulated with anti-CD3 alone or in combination with soluble anti-CD28 mAbs. The proliferative response was examined by [3H]thymidine incorporation. (C) Cell death determined by the TUNEL assay in freshly isolated (unstimulated) wild-type and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated as in A for 24 h. Numbers represent the percentages of TUNEL-positive cells. TdT, terminal deoxynucleotidyltransferase. (D) Wild-type CD8+ T cells were stimulated as in B in the presence of different concentration of the JNK inhibitor (SP600125). The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 3.4 μM).
Figure 4.
Figure 4.
JNK1 is required for CD8+ T cell activation. (A) JNK activity was determined using cell extracts from freshly isolated (−) wild-type (WT) and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h (B) CD8+ T cells (2.5 × 105 cells/ml) from wild-type and Jnk1 − / − mice were stimulated with anti-CD3 alone or in combination with soluble anti-CD28 mAbs. The proliferative response was examined by [3H]thymidine incorporation. (C) Cell death determined by the TUNEL assay in freshly isolated (unstimulated) wild-type and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated as in A for 24 h. Numbers represent the percentages of TUNEL-positive cells. TdT, terminal deoxynucleotidyltransferase. (D) Wild-type CD8+ T cells were stimulated as in B in the presence of different concentration of the JNK inhibitor (SP600125). The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 3.4 μM).
Figure 4.
Figure 4.
JNK1 is required for CD8+ T cell activation. (A) JNK activity was determined using cell extracts from freshly isolated (−) wild-type (WT) and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h (B) CD8+ T cells (2.5 × 105 cells/ml) from wild-type and Jnk1 − / − mice were stimulated with anti-CD3 alone or in combination with soluble anti-CD28 mAbs. The proliferative response was examined by [3H]thymidine incorporation. (C) Cell death determined by the TUNEL assay in freshly isolated (unstimulated) wild-type and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated as in A for 24 h. Numbers represent the percentages of TUNEL-positive cells. TdT, terminal deoxynucleotidyltransferase. (D) Wild-type CD8+ T cells were stimulated as in B in the presence of different concentration of the JNK inhibitor (SP600125). The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 3.4 μM).
Figure 4.
Figure 4.
JNK1 is required for CD8+ T cell activation. (A) JNK activity was determined using cell extracts from freshly isolated (−) wild-type (WT) and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h (B) CD8+ T cells (2.5 × 105 cells/ml) from wild-type and Jnk1 − / − mice were stimulated with anti-CD3 alone or in combination with soluble anti-CD28 mAbs. The proliferative response was examined by [3H]thymidine incorporation. (C) Cell death determined by the TUNEL assay in freshly isolated (unstimulated) wild-type and Jnk1 − / − CD8+ T cells and wild-type and Jnk1 − / − CD8+ T cells (106 cells/ml) stimulated as in A for 24 h. Numbers represent the percentages of TUNEL-positive cells. TdT, terminal deoxynucleotidyltransferase. (D) Wild-type CD8+ T cells were stimulated as in B in the presence of different concentration of the JNK inhibitor (SP600125). The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 3.4 μM).
Figure 5.
Figure 5.
IL-2 does not restore impaired proliferation of Jnk1 − / − CD8+ T cells. (A) Wild-type (WT) and Jnk1 − / − CD8+ T cells (106 cells/ml) were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h IL-2 (left panel) and IFN-γ (right panel) production was determined by ELISA. (B) CD8+ T cells (106 cells/ml) from wild-type mice were stimulated as in A for 48 h in the presence of different concentration of the JNK inhibitor (SP600125). IL-2 production was determined by ELISA. (C) CD8+ T cells from wild-type and Jnk1 − / − mice (2.5 × 105 cells/ml) were stimulated as in A in the presence of exogenous IL-2 (50 U/ml). The proliferative response was examined by [3H]thymidine incorporation. (D) Total CD8+ T cells (2.5 × 105 cells/ml) from wild-type mice were stimulated as in A in the presence of different concentrations of the JNK inhibitor and exogenous IL-2. The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 7.4 μM).
Figure 5.
Figure 5.
IL-2 does not restore impaired proliferation of Jnk1 − / − CD8+ T cells. (A) Wild-type (WT) and Jnk1 − / − CD8+ T cells (106 cells/ml) were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h IL-2 (left panel) and IFN-γ (right panel) production was determined by ELISA. (B) CD8+ T cells (106 cells/ml) from wild-type mice were stimulated as in A for 48 h in the presence of different concentration of the JNK inhibitor (SP600125). IL-2 production was determined by ELISA. (C) CD8+ T cells from wild-type and Jnk1 − / − mice (2.5 × 105 cells/ml) were stimulated as in A in the presence of exogenous IL-2 (50 U/ml). The proliferative response was examined by [3H]thymidine incorporation. (D) Total CD8+ T cells (2.5 × 105 cells/ml) from wild-type mice were stimulated as in A in the presence of different concentrations of the JNK inhibitor and exogenous IL-2. The proliferative response was examined by [3H]thymidine incorporation. An IC50 was calculated by nonlinear regression (IC50 = 7.4 μM).
Figure 6.
Figure 6.
Impaired IL-2Rα gene expression in Jnk1 − / − CD8+ T cells. (A) CD8+ T cells from wild-type (WT) and Jnk1 − / − mice (106 cells/ml) were stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 48 h and stained with anti-IL-2Rα (CD25) mAbs. Cell surface expression of IL-2Rα was determined by flow cytometry. Numbers represent the percentage of cells in each quadrant. The mean fluorescence intensity for the upper right quadrant of each plot is indicated in bold italics. (B) Cell surface expression of IL-2Rα was determined on wild-type CD8+ T cells (106 cells/ml) stimulated as in A in the presence of the JNK inhibitor (SP600125; 5.6 μM). (C) Cytokine receptor gene expression was determined by RPA using RNA extracted from freshly isolated (−) wild-type and Jnk1 − / − (J1) CD8+ T cells and wild-type and J1 CD8+ T cells stimulated (+) as in A for 24 h. (D) Cytokine receptor gene expression was determined by RPA using RNA extracted from wild-type, J1, and Jnk2 − / − (J2) CD8+ T cells stimulated as in A for 36 h in the presence of a neutralizing anti-IL-2 (10 μg/ml) mAb. (E) Cytokine gene expression was determined by RPA using RNA extracted from wild-type CD8+ T cells stimulated as in A in the absence (−) or presence of exogenous IL-2 (50 U/ml) for 24 h. (F) Cytokine gene expression was determined by RPA using RNA extracted from wild-type and J1 CD8+ T cells stimulated as in A for 24 h in the presence of exogenous IL-2.
Figure 7.
Figure 7.
Reduced binding of AP-1 DNA binding in Jnk1 − / −CD8+ T cells. (A) DNA binding of transcription factors to the AP-1–like site in the IL-2Rα promoter (AP-1/IL-2Rα) was determined using nuclear extracts prepared from wild-type (WT), and Jnk1 − / − (J1) CD8+ T cells stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 36 h and a [32P]-end-labeled double stranded oligonucleotide spanning –365 to –347 of the IL-2Rα promoter. (B) Composition of the AP-1 complex detected in activated CD8+ T cells. Nuclear extracts were prepared from wild-type CD8+ T cells stimulated with immobilized anti-CD3 and soluble anti-CD28 mAbs as in A. DNA binding to the AP-1/IL-2Rα element was performed in the absence (−) or presence of cold AP-1, STAT, or CRE oligonucleotides. DNA binding to the AP-1/IL-2Rα element was also examined in the presence of anti-Jun family (αJun Family) antiserum. (C) AP-1, CREB, and NFAT DNA binding was determined using nuclear extracts from unstimulated wild-type, J1, and Jnk2 −/− (J2) CD8+ T cells and wild-type, J1, and J2 CD8+ T cells stimulated as in A using [32P]-end-labeled double stranded oligonucleotides containing either an AP-1, CREB, or NFAT consensus binding site. (D) AP-1 and CREB DNA binding was determined using nuclear extracts from wild-type and J1 CD4+ T cells stimulated with immobilized anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs for 36 h using [32P]-end-labeled double stranded oligonucleotides containing either an AP-1 or CRE consensus sequence. (E) Western blot of c-Jun and actin in wild-type, Jnk1 − / −, and Jnk2 − / −CD8+ T cells stimulated as in A for the indicated time periods. (F) CD8+ T cells were isolated from AP-1-luciferase and Jnk1 −/− × AP-1-luciferase reporter transgenic mice, stimulated with immobilized anti-CD3 and soluble anti-CD28 as in A for different time periods, harvested, and assayed from luciferase activity. UD, undetectable.
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