Supplementary MaterialsS1 Fig: Generation of mutant mice. apoptosome[7, 8]. Absence of Apaf1[9, 10], Casp9[11, 12] or Apaf1-activating form of Cyt gene was disrupted with promoter-driven expression, to investigate the biological function of Apaf1 in T cells. Apaf1-deficient T cells showed resistance to mitochondria-dependent apoptosis but showed susceptibility to Fas-mediated apoptosis. We then performed the delayed-type hypersensitivity (DTH) assay, using ovalbumin (OVA)-specific T cell receptors (TCR)-expressing mice (OTII mice), and found that antigen-specific T cell activation leads Veralipride to enhanced proliferation and Th1-type immune responses in Lck-(carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone), did not reproduce the activation-related phenotypes observed in Apaf1-deficient T cells, indicating caspase-independent functions of Apaf1 during T cell activation. Our data suggested that Apaf1 in T cells is usually a negative regulator of immune responses. Materials and methods Generation of T cell-specific Apaf1-deficient mice The design of the conditional targeting vector for is usually shown in S1 Fig, in which exons 2 and 3 are flanked by two sites. The linearized targeting vector was electroporated into E14K ES cells and homologous recombinants were selected. The heterozygous mutant (transgenic (Tg) mice (RBRC01834, RIKEN BRC). Mice heterozygous for mutation (Tg mice and transgene-positive Tg mice and OTII mice were kindly provided by Dr. A. Yoshimura, Keio University, Japan. Successful disruption of gene was confirmed with genomic Southern blot analysis and absence of Apaf1 protein in Lck-(10 and 100 M, MBL) was added into the culture. DTH assay Seven days after immunization with OVA as above, mice were challenged s.c. at right footpad with 200 g of OVA in 20 l PBS. As a control, the same volume of PBS was injected into left footpad. Footpad thickness was measured with a dial vernier caliper (Teclock) on day 1 and 2. The magnitude of TRADD the DTH response was calculated as follows; footpad swelling (m) = thickness of OVA-injected footpad ? thickness of PBS-injected footpad. For histological analysis of the DTH lesions, paws were removed on day 2 and fixed with 10%-formaldehyde neutral buffer answer (Nacalai). After decalcification by a standard protocol, specimens were embedded in paraffin and were stained with hematoxylin-eosin (H&E). For analysis of Veralipride the tissue-infiltrating cells, paws were thoroughly minced with scissors and then were incubated at 37C for 1 hour in Hank’s answer made up of 1.0 mg/ml collagenase II (Worthington), 1.0 mg/ml dispase (Sigma-Aldrich) and 40 g/ml Dnase I (Roche). After removing debris with Veralipride 70 m cell-strainers, cells were re-suspended into 33.7% Percoll (GE Healthcare) and pelleted by centrifugation at 1,000 (10 and 100 M). Cell lysates were prepared, electrophoresed, and blotted. Tubulin, caspases 3, 7, and 9 were detected with Veralipride respective antibodies (anti-tubulin; Sigma Aldrich and anti-caspases; Cell Signaling Technology) and visualized using an enhanced chemiluminescence procedure (ImmunoStar LD, Wako). Statistical analysis Experiments were repeated at least three times. Values were expressed as means + SD. Differences between control (Apaf1-sufficient) and Apaf1-deficient samples were analyzed using unpaired re-stimulation and were higher in Lck-recall responses of Apaf1-deficient T cells.(A and B) LN cells from OVA-immunized with either OVA peptide or anti-CD3 antibody, Lck-(Fig 5A, middle panels). Additionally, percentages of CD69+ and CD44highCD62Llow cells in control Apaf1-sufficient OTII T cell populace were still lower over Apaf1-deficient OTII T cells in the presence of z-VAD-(Fig 5A, lower panels). Dexamethasone-induced apoptosis and caspase 3 activation in thymocytes was completely suppressed by z-VAD-at the same concentration (100 M, S4 Fig). Open in a separate windows Fig 5 Caspase-independent role of Apaf1 in T cell activation.LN cells from immunized (z-VAD) for 48 hours. (A) Cell proliferation, cell viability (Annexin V-negative and PI-negative), production of IFN- and IL-17, or expression of CD69, CD44, and CD62L were analyzed. Open columns; at 100 M (Fig 5B, cleaved Casp3). Cleaved form of caspase 7 was also detected in both Apaf1-sufficient T cells and Apaf1-deficient T cells almost similarly (cleaved Casp7) and z-VAD-at 100 M showed.