Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1)/mitogen-activated protein kinase kinase 4 (MKK4)-deficient T lymphocytes

Abstract

The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

Figure 2

Immunocytometric analysis of B cell populations in the bone marrow (left) and spleen (right) of 129/J, SEK1^+/+ chimeric, SEK1^−/− chimeric, and RAG2^−/− mice. Cells were isolated from 6-wk-old mice and double stained using anti-B220 (PE) and anti-CD43 (FITC), or anti-B220 (PE) and anti-sIgM (FITC). Percentages of positive cells within a quadrant are indicated. Note the partial block in the maturation from CD43⁺B220⁺ pro–B cells to CD43⁻B220⁺ pre–B cells in the bone marrow and the reduced number of sIgM⁺ B cells in the spleen of SEK1^−/− chimeric mice (see also Table 1). 10,000 viable cells were collected and analyzed on a FACScan^®. Total cell numbers were: 129/J bone marrow (total lymphoid cells isolated from one femur), 7.9 × 10⁶; 129/J spleen, 4.1 × 10⁷; SEK1^+/+ chimeric bone marrow, 8.3 × 10⁶; SEK1^+/+ chimeric spleen, 3.1 × 10⁷; SEK1^−/− bone marrow, 7.7 × 10⁶; SEK1^−/− chimeric spleen, 3.9 × 10⁷; RAG2^−/− bone marrow, 9.8 × 10⁶; RAG2^−/− spleen, 1.3 × 10⁷.

Figure 1

Proliferation (A and C) and IL-2 production (B and D) of SEK1^−/− chimeric (shaded bars) and SEK1^+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (10⁵ T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca²⁺ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1^−/− and SEK1^+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [³H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.

Figure 1

Proliferation (A and C) and IL-2 production (B and D) of SEK1^−/− chimeric (shaded bars) and SEK1^+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (10⁵ T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca²⁺ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1^−/− and SEK1^+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [³H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.

Figure 1

Proliferation (A and C) and IL-2 production (B and D) of SEK1^−/− chimeric (shaded bars) and SEK1^+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (10⁵ T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca²⁺ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1^−/− and SEK1^+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [³H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.

Figure 1

Proliferation (A and C) and IL-2 production (B and D) of SEK1^−/− chimeric (shaded bars) and SEK1^+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (10⁵ T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca²⁺ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1^−/− and SEK1^+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [³H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.

Figure 3

PCR analysis for SEK1 mutant and wild-type alleles in total bone marrow and sorted B220⁺CD43⁺ and B220⁺ CD43⁻ bone marrow cells from SEK1^−/− and SEK1^+/+ chimeric mice. Bone marrow cells were double stained with anti-B220 (PE) and anti-CD43 (FITC) and populations were sorted using a FACS^® power sorter (Coulter). Postsorting purity of CD43⁺ B220⁺ and B220⁺CD43⁻ cells was >98%. Purified B cell populations (5 × 10⁴ cells) were subjected to PCR analysis as described in Materials and Methods. Total bone marrow cells (10⁵) (Bone marrow) from 129/J, SEK1^−/− chimeric, SEK1^+/+ chimeric, and RAG2^−/− mice are shown as controls.

Figure 4

B cell activation and immunoglobulin production in SEK1^−/− mice. (A) Activation of splenic B cells. Purified splenic B cells (10⁵/well) from SEK1^−/− (shaded bars) and SEK1^+/+ (open bars) control mice were seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [³H]thymidine/well. The experiment shown is one of four experiments in which conditions for stimulation varied (time, cell concentration, concentration of activators). No significant differences (Student's t test; p > 0.05) were observed in the [³H]thymidine uptake between SEK1^−/− and SEK1^+/+ B cells in response to any of these conditions. [³H]thymidine uptake is shown in cpm ± SD. (B) SEK1^−/− mice produce normal levels of serum immunoglobulin subclasses. Sera were collected from two individual 6-wk-old SEK1^−/− (shaded bars) and two individual 6-wk-old SEK1^+/+ (open bars) chimeric mice. The concentrations of Ig subclasses are shown in μg/ml and were determined by ELISA. Standard deviations were <25 μg/ml.

Figure 4

B cell activation and immunoglobulin production in SEK1^−/− mice. (A) Activation of splenic B cells. Purified splenic B cells (10⁵/well) from SEK1^−/− (shaded bars) and SEK1^+/+ (open bars) control mice were seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [³H]thymidine/well. The experiment shown is one of four experiments in which conditions for stimulation varied (time, cell concentration, concentration of activators). No significant differences (Student's t test; p > 0.05) were observed in the [³H]thymidine uptake between SEK1^−/− and SEK1^+/+ B cells in response to any of these conditions. [³H]thymidine uptake is shown in cpm ± SD. (B) SEK1^−/− mice produce normal levels of serum immunoglobulin subclasses. Sera were collected from two individual 6-wk-old SEK1^−/− (shaded bars) and two individual 6-wk-old SEK1^+/+ (open bars) chimeric mice. The concentrations of Ig subclasses are shown in μg/ml and were determined by ELISA. Standard deviations were <25 μg/ml.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 5

Germinal center formation in SEK1^−/− and CD28^−/− mice. SEK1^−/−, SEK1^+/+, and CD28^−/− mice were immunized with VSV Indiana (2 × 10⁶ PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA⁺ cells localize to germinal centers and the marginal zone in (A) SEK1^+/+ and (B) SEK1^−/− mice. (C) Absence of germinal center formation and germinal center PNA⁺ B cells in VSV-infected CD28^−/− mice. Some PNA⁺ B cells are present in the marginal zone and the red pulp of CD28^−/− mice. (D–F) CD4⁺ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1^+/+ and (E) SEK1^−/− mice. (F) CD4⁺ T cells in the spleen of VSV-infected CD28^−/− mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1^+/+ and (H) SEK1^−/− mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28^−/− mice. (J) VSV-specific plasma cells in VSV-infected CD28^−/− mice.

Figure 6

Activation of SAPKs/JNKs in thymocytes (top) and lymph node T cells (bottom). (A) Thymocytes (top) and purified mesenteric lymph node T cells (bottom) were isolated from SEK1^+/+ and SEK1^−/− mice and cells (5 × 10⁶/lane) were activated with PMA (50 ng/ml) plus Ca²⁺ ionophore (1 μg/ml) for 0 and 10 min as described in Materials and Methods. SAPK/JNK were immunoprecipitated and assayed for in vitro kinase activity using glutathione-S-transferase–c-Jun as a substrate. Peripheral T cells were purified using affinity columns and purity of CD3⁺ T cells was >98% as determined by cytometry. One result representative of three independent experiments is shown. (B) Western blotting for p46 and p54 SAPK/JNK isoform expression in SEK1^+/+ (+/+) and SEK1^−/− (−/−) chimeric thymocytes and purified lymph node T cells. Thymocytes (10⁶/lane) and lymph node cells (5 × 10⁶/lane) were blotted for SAPK expression as described in Materials and Methods (23).

Figure 7

Proliferation (A) and IL-2 production (B) of SEK1^−/− (shaded bars) and SEK1^+/+ chimeric (open bars) thymocytes. Thymocytes (10⁵ T cells/well) were activated with plate-bound anti–CD3-ε (1 μg/ ml) and soluble anti-CD28 Abs (100 ng/ml; CD3-ε + CD28) or PMA (12.5 ng/ml) plus Ca²⁺ ionophore (100 ng/ml) (PMA + Ca). Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε/CD28 Abs are shown as controls. After 48 h of stimulation, proliferation was determined by [³H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. The low IL-2 production of thymocytes after PMA/Ca²⁺ ionophore stimulation is due the fact that PMA/Ca²⁺ ionophore also induces proliferation of CD4⁻CD8⁻TCR⁻ thymocytes, the majority of which does not produce IL-2. One result representative of three independent experiments is shown.