Genetic disruption of p38α Tyr323 phosphorylation prevents T-cell receptor–mediated p38α activation and impairs interferon-γ production by Ludmila Jirmanova, Dandapantula N. Sarma, Dragana Jankovic, Paul R. Mittelstadt, and Jonathan D. Ashwell Blood Volume 113(10):2229-2237 March 5, 2009 ©2009 by American Society of Hematology

Generation of p38αY323F mice. Generation of p38αY323F mice. (A) A 9.3-kb genomic fragment of p38α locus (top) was cloned with the use of AvaI- and EcoRI-specific restriction sites and used to create the targeting vector (middle), into which a Y323F mutation and LoxP-flanked neomycin resistance gene were inserted. Insertion of the Y323F mutation created a new BclI restriction site in the knockin allele. Positions of the forward (Fwd) and reverse (Rev) primers used in long template PCR are shown (bottom). (B) Genotyping of p38αY323F mice by long template PCR, followed by digestion with BclI restriction enzyme generated a nondigested 4172-bp wild-type (WT/WT) band and 3546-bp knockin (Y323F/Y323F) band together with a small 626-bp band (not shown). Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

T-cell and thymocyte subsets. T-cell and thymocyte subsets. (A,B) Lymph nodes, spleen, and (C) thymus from wild-type and p38αY323F mice were homogenized. Cells were counted, then stained for CD4, CD8, and B220 (n = 10; bars indicate mean ± SEM). (C) Double-negative thymocytes were stained for CD44 and CD25 and the number of cells in different developmental stages are shown (n = 4; bars indicate mean ± SEM; P value was calculated with the Student t test). DN1 = CD44+CD25−, DN2 = CD44+CD25+, DN3 = CD44−CD25+, DN4 = CD44−CD25−. Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

TCR stimulation with or without costimulation activates p38α through the alternative pathway. TCR stimulation with or without costimulation activates p38α through the alternative pathway. (A) T cells purified from spleen and lymph nodes of wild-type (WT) or p38αY323F mice were stimulated with plate-bound anti (α)-CD3 and/or α-CD28 antibodies (Abs) for 30 minutes. Whole lysates from 4 × 106 cells were immunoblotted with antibodies recognizing p38 phosphorylated on Tyr323 (P-Y323-p38), Thr180/Tyr182 (P-p38), or total p38. (B) WT and p38αY323F T cells were stimulated with α-CD3/CD28 Abs, PMA, or sorbitol, and phospho-p38 was detected in whole cell lysates. (C) p38α or (D) p38β were immunoprecipitated from WT and p38αY323F T cells stimulated with α-CD3/CD28 Abs or PMA, and in vitro kinase (IVK) assays of immune complexes with ATF-2 as a substrate were performed. (E) B cells purified from spleen of WT or p38αY323F mice were stimulated with anti-IgM antibody (10 μg/mL) for 10 minutes, p38α specifically immunoprecipitated from whole cell lysates, and kinase assays performed as in panel C. Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

CD28 signaling does not stimulate p38 through the classic MAPK pathway. CD28 signaling does not stimulate p38 through the classic MAPK pathway. T cells purified from lymph nodes and spleen of wild-type or p38αY323F mice were stimulated with plate-bound α-CD3/CD28 for 5 minutes (MEK1/2 detection) or 30 minutes (MKK3/6). Phosphorylation of MEK1/2 and MKK3/6 was detected in whole cell lysates with the use of phosphospecific antibodies. Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

Onset of proliferation is delayed in p38αY323F T cells stimulated through TCR/CD28. Onset of proliferation is delayed in p38αY323F T cells stimulated through TCR/CD28. (A) Wild-type (■; WT) and p38 αY323F (□; Y323F) T cells were stimulated with plate-bound anti-CD3/CD28 antibodies or with PMA plus ionomycin (PMA/Iono) for the indicated times, fixed, and RNA were stained with pyronin Y. Error bars represent SEM (3 replicates per condition). (B) WT and p38αY323F T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for the indicated times, fixed, and DNA (Hoechst 33342) and RNA (Pyronin Y) were stained. The percentages of positive cells are indicated for each upper quadrant (results are representative of 2 independent experiments). (C) WT and p38αY323F (Y323F) T cells were purified from spleen and lymph nodes, labeled with CFSE, and stimulated with plate-bound α-CD3/CD28 antibodies or PMA/Iono for indicated times. One of 4 independent experiments is shown. (D) Summary of percentage of nondivided cells and mitotic indexes of WT and p38αY323F (Y323F) T cells from panel C, n = 4; bars indicate mean (± SEM); P value was calculated using the Student t test. Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

TCR/CD28-induced IFN-γ expression is decreased in p38αY323F T cells. TCR/CD28-induced IFN-γ expression is decreased in p38αY323F T cells. (A) Wild-type (solid line) and p38αY323F (dashed line) T cells were stimulated as indicated for 48 hours and IFN-γ and IL-2 expression were detected by intracellular staining (A) or ELISA (B). (C) TNF-α expression was detected by intracellular staining in T cells stimulated with α-CD3/CD28 for 48 hours. (D) T cells were treated with IL-12 plus IL-18 or PMA/Iono for 48 hours, and then IFN-γ production was analyzed by intracellular staining. Shaded histograms present stimulated wild-type cells stained with matched isotype control antibody only. One of 3 independent experiments is shown. Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology

p38αY323F T cells polarize into Th1 cells in vivo but produce less IFN-γ upon TCR stimulation. p38αY323F T cells polarize into Th1 cells in vivo but produce less IFN-γ upon TCR stimulation. (A) T-bet and GATA-3 expressions were analyzed in whole cell lysates of WT and p38αY323F T cells stimulated with plate-bound anti-CD3/CD28 Abs and harvested on the indicated days. (B) WT and p38αY323F mice were immunized with 20 μg of STAg and, 12 days later, splenocytes were restimulated with either anti-CD3 or PMA/Ion. IL-4 and IFN-γ in CD4+ T cells were analyzed by intracellular staining. The numbers indicate the frequency of positive cells in each quadrant. The data are representative for 1 of 2 independent experiments. (C) The expression of IFN-γ per responding cell (MFI) and (D) the percentage of CD4+ IFN-γ–positive T cells from individual immunized animals from 2 independent experiments are summarized (bars indicate the mean ± SEM; P value was calculated using the Student t test). Ludmila Jirmanova et al. Blood 2009;113:2229-2237 ©2009 by American Society of Hematology