Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type I IFN-producing CD4 Valpha14i NKT cells facilitate priming of IL-10-producing CD8 T cells by hepatocytes.

doi: 10.4049/jimmunol.178.4.2083

Figure Lengend Snippet: FIGURE 6. The cytokine response of OT-I CD8 T cells primed by HC with or without NKT cell help. A, OT-I CD8 T cells (105/well) were cocul- tured with GalCer- and SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). Cells were harvested after a 72-h culture, incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml), washed, and tested for IL-10 secretion in a 3-h capture assay. Cells were labeled with a PE-conjugated anti-IL-10 mAb and an anti-CD8 mAb. Data from a representative of five independent experiments are shown. B, OT-I CD8 T cells (105/well) were cocultured for 72 h with GalCer-/ SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). CD8 T blasts were harvested, purified, and restimu- lated in vitro for 48 h with peptide-pulsed DC. IFN-, IL-2, and IL-10 in 48-h supernatants of these secondary cultures were determined by ELISA. Mean values of triplicates (SEM) of a representative of four independent experiments are shown.

Article Snippet: IL-10-producing CD8 T cells were identified by a cytokine capture assay (catalog no. 130-090-489; Miltenyi Biotec) combined with surface staining with the allophycocyanin-conjugated anti-CD8 mAb 53-6.7 (catalog no. 553035; BD Biosciences).

Techniques: Incubation, Labeling, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor Bhlhe40 is a switch of inflammatory versus antiinflammatory Th1 cell fate determination

doi: 10.1084/jem.20170155

Figure Lengend Snippet: Bhlhe40 suppresses IL-10 production by Th1 cells. (A) Gene expression values (RPKM) of Il10, Maf , and Ikzf3 from RNA-Seq analysis of C57BL/6 WT and Bhlhe40 cKO Th1 cells ( n = 2). (B) Sorted naive CD4 T cells cultured under Th1-polarizing conditions with plate-bound anti-CD3/CD28 for 4 d and restimulated with PMA-ionomycin for 4 h. Flow cytometric analysis of IFN-γ/IL-10 production (top) and IL-10 expression (bottom left) by CD4 + CD44 hi WT and cKO Th1 cells. Gray solid, WT; red line, cKO. Bottom right: IL-10 production from three independent experiments (≥2 mice per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. (C) Sorted naive CD4 T cells cultured under indicated conditions with plate-bound anti-CD3/CD28 for 4 d and restimulated with PMA-ionomycin for 4 h. Flow cytometric analysis of IFN-γ/IL-10 production by CD4 + CD44 hi WT and Bhlhe40 cKO cells. Data are representative of two independent experiments (≥2 mice per group).

Article Snippet: Total PEC samples were cultured with T. gondii –soluble tachyzoite antigen (STAg, 5 µg/ml) in complete RPMI for 3 d. The samples were centrifuged and the supernatant was used to measure the levels of IFN-γ and IL-10 using mouse Quantikine ELISA kits for IFN-γ and IL-10 (R&D systems).

Techniques: Expressing, RNA Sequencing Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor Bhlhe40 is a switch of inflammatory versus antiinflammatory Th1 cell fate determination

doi: 10.1084/jem.20170155

Figure Lengend Snippet: Bhlhe40 expression in T cells is required for colitis induction. (A) Rag1 −/− mice received sorted naive CD4 + CD25 − CD45RB hi T cells from either WT (red dots) or Bhlhe40 cKO (black squares) or injected i.v. with PBS (blue diamonds) as the control group. Mice were weighed weekly. Statistical significance of body weight of Bhlhe40 cKO versus WT (mean ± SEM, n = 5) at different time points was determined by a two-tailed unpaired Student’s t test. Data are representative of three independent experiments. (B) Graphical representation of the absolute number of CD4 T cells harvested from spleen and mesenteric lymph node (MLN) of Rag1 −/− mice, in which sorted naive CD4 + CD25 − CD45RB hi T cells from either WT (dots) or Bhlhe40 cKO mice (squares) were i.v. transferred for 4 wk ( n = 3 per group) are shown. Data are representative of two independent experiments. (C) Percentages of IFN-γ + and IL-17A + cells among the CD4 + CD44 hi T cells in spleen and MLN from Rag1 −/− mice received WT (dots) or Bhlhe40 cKO transfer (squares) cells for 8 wk (mean ± SEM, n = 5, right). Data are representative of three independent experiments. (D) Sorted naive CD4 T cells from WT or Bhlhe40 cKO were transferred into Rag1 −/− mice. 2 wk after transfer, CD4 T cells were sorted from the spleens and RNAs were prepared from sorted cells. Real time PCR analysis of Ifng and Il10 mRNA was performed (mean ± SEM, n = 5). Data are representative of two independent experiments. (E) Sorted naive CD4 T cells from C57BL/6 or Bhlhe40 cKO were transferred into Rag1 −/− mice. 2 wk after transfer, cells from MLN were restimulated with PMA plus ionomycin, and then intracellular staining for IFN-γ and IL-10 was performed. Flow plots were gated on CD4 + CD44 hi cells. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Data are representative of two independent experiments.

Article Snippet: Total PEC samples were cultured with T. gondii –soluble tachyzoite antigen (STAg, 5 µg/ml) in complete RPMI for 3 d. The samples were centrifuged and the supernatant was used to measure the levels of IFN-γ and IL-10 using mouse Quantikine ELISA kits for IFN-γ and IL-10 (R&D systems).

Techniques: Expressing, Injection, Two Tailed Test, Real-time Polymerase Chain Reaction, Staining

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor Bhlhe40 is a switch of inflammatory versus antiinflammatory Th1 cell fate determination

doi: 10.1084/jem.20170155

Figure Lengend Snippet: Bhlhe40 cKO mice are susceptible to T. gondii infection. (A) Bhlhe40 WT and cKO mice were infected i.p. with a mean of 15–20 ME49 cysts of T. gondii . Survival of infected mice was monitored. The survival curves shown are one representative of two independent experiments preformed ( n = 11 per group). (B) T. gondii –infected mice were treated i.p. with anti–IL-10R or control IgG1 at 2 d before and 2 d after infection. Survival of infected mice (WT with control IgG1, dots; cKO with control IgG1, squares; cKO with anti–IL-10R, diamonds) was monitored daily ( n = 7–8). The data shown are the pooled results from two independent experiments. (C) T. gondii –infected mice were treated i.p. with anti–IL-10R or control IgG1 at 2 d before and 2 d after infection. Parasite burden was determined by counting infected cells in cytospin smears obtained from PECs on day 8 (mean ± SEM, n = 3–6). *, P < 0.05; Student’s t test.Data are representative of two independent experiments, and similar results were obtained in a third experiment analyzed on day 13. (D) PECs from 13-d infected mice were stained with tetramer to assess the frequency of T. gondii AS15 peptide-specific CD4 T cells. These PECs were also cultured with STAg for 3 d. IFN-γ and IL-10 production by T. gondii antigen-specific cells were measured by ELISA (mean ± SEM, n = 3–6). ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test. Data are representative of two independent experiments.

Article Snippet: Total PEC samples were cultured with T. gondii –soluble tachyzoite antigen (STAg, 5 µg/ml) in complete RPMI for 3 d. The samples were centrifuged and the supernatant was used to measure the levels of IFN-γ and IL-10 using mouse Quantikine ELISA kits for IFN-γ and IL-10 (R&D systems).

Techniques: Infection, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay