Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: IRF4 expression in WT CD8+ T cells is positively correlated with the production of effector molecules. Splenocytes from WT B6 mice were stimulated with 2 µg/ml soluble anti‐CD3e mAb for 24 h, followed by flow cytometric analysis. All contour plots were gated on living CD8+ T cells. (A) Schematic of the experimental design. (B,C) Representative contour plots and bar graphs display the IRF4 expression of CD127+KLRG1−, CD127−KLRG1− and CD127−KLRG1+ CD8+ T‐cell subsets. (D,E) Representative contour plots and bar graphs show the expression of IFN‐γ and granzyme B in CD8+ T‐cell subpopulations that express high, medium or low levels of IRF4. Data are mean ± SD (n = 5). **p < 0.01, ****p < 0.0001 (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Expressing

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: Irf4‐deficient CD8+ T cells are capable of recognizing BALB/c alloantigens. Irf4‐deficient or control CD8+ T cells were stimulated by B6 or BALB/c APCs for 72 h, supplemented with or without murine IL‐2. All contour plots were gated on the living CD8+ T cells. (A,B) % CD62L−CD44+ cells among the living CD8+ T cells in response to B6 or BALB/c stimulators. (C,D) % CD69+ cells among the living CD8+ T cells in response to B6 or BALB/c stimulators. (E,F) % Nur77+Ki67+ cells among the living CD8+ T cells in response to B6 or BALB/c stimulators. Data are mean ± SD (n = 5). ns, no significance; **p < 0.01, ****p < 0.0001 (unpaired Student's t‐test). APC, antigen‐presenting cell

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques:

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: IRF4 deletion in CD8+ T cells abrogates their function in rejecting skin allografts. Lymphopenic B6.Rag1−/− recipients were adoptively transferred with either 5 × 105 Irf4‐deficient CD8+ T cells isolated from Irf4 fl/fl Cd4‐Cre mice, or 5 × 105 control CD8+ T cells obtained from Irf4 fl/fl mice. One day later, B6.Rag1−/− recipients were transplanted with fully MHC‐mismatched skin allografts from BALB/c donors. (A) Schematic of the experimental design. Tx, transplantation. (B) The percentage of graft survival post‐allogeneic tail skin transplantation (n = 5). **p < 0.01; Mann–Whitney test. (C) Representative images of a rejected skin graft on a recipient that received control CD8+ T cells (left image), and accepted skin grafts on recipients that received Irf4‐deficient CD8+ T cells (middle and right images) at indicated days post‐skin grafting

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Isolation, Transplantation Assay, MANN-WHITNEY

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: IRF4 deletion in CD8+ T cells completely abolishes their terminal effector differentiation in transplant recipients. B6.Rag1−/− recipients were adoptively transferred with either Irf4‐deficient (Irf4 fl/fl Cd4‐Cre) or control (Irf4 fl/fl) CD8+ T cells, followed by BALB/c skin transplantation. Adoptively transferred CD8+ T cells in spleens and DLNs were analysed at day 14 post‐skin grafting. All contour plots were gated on the living transferred CD8+ T cells. (A,B) Representative contour plots and bar graph display the % CD127−KLRG1+ terminal effector cells among the transferred CD8+ T cells. (C,D), Representative contour plots and bar graph display the % CD62L−CX3CR1+ effector cells among the transferred CD8+ T cells. Data are mean ± SD (n = 3). ****p < 0.0001 (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Transplantation Assay

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: Irf4‐deficient CD8+ T cells barely produce proinflammatory cytokines and cytotoxic molecules in transplant recipients. At day 14 post‐BALB/c skin grafting, the adoptively transferred Irf4‐deficient and control CD8+ T cells in B6.Rag1−/− recipients were analysed. All contour plots were gated on the living transferred CD8+ T cells. (A,B) % IFN‐γ+ TNF‐α+ cells among the transferred CD8+ T cells in spleens and DLNs. (C,D) Percentage of granzyme B+ cells among the transferred CD8+ T cells in spleens and DLNs. (E,F) % IL‐2+ and % granzyme A+ cells among the transferred CD8+ T cells. Data are mean ± SD (n = 3). *p < 0.05; **p < 0.01, ***p < 0.001; ****p < 0.0001 (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques:

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: Irf4‐deficient CD8+ T cells exhibit low frequency and low Ki67 expression in transplant recipients. At day 14 post‐BALB/c skin grafting, the adoptively transferred Irf4‐deficient and control CD8+ T cells in B6.Rag1−/− recipients were analysed. All contour plots and overlay histograms were gated on the living transferred CD8+ T cells in spleens. (A,B) Representative plots and bar graph display the percentage of the transferred CD8+ T cells among CD45+ immune cells. (C‐D) Representative plots and bar graphs display MFI Ki67 among the transferred CD8+ T cells. Data are mean ± SD (n = 3). **p < 0.01 (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Expressing

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: IRF4 deficiency in CD8+ T cells affects the expression of key transcription factors that control terminal effector differentiation. At day 14 post‐BALB/c skin grafting, the adoptively transferred Irf4‐deficient and control CD8+ T cells in B6.Rag1−/− recipients were analysed. All overlay histograms and contour plots were gated on the living transferred CD8+ T cells in spleens. Representative overlay histograms and bar graphs show T‐bet (A,B) and ID2 (C,D) expression levels of the transferred CD8+ T cells. Representative contour plots and bar graphs show % TCF1+ (E,F) and % EOMES+ (G,H) cells among the transferred CD8+ T cells. Data are mean ± SD (n = 3). *p < 0.05; **p < 0.01, ***p < 0.001; (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Expressing

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: Naïve Irf4‐deficient CD8+ T cells barely differentiate into terminal effector cells and fail to reject skin allografts. B6.Rag1−/− recipients were adoptively transferred with naïve Irf4‐deficient or control CD8+ T cells, and transplanted with BALB/c tail skins or left without transplantation. (A) Schematic of the experimental design. (B) Representative contour plots display the purified CD44low naïve CD8 T cells prior to adoptive transfer. (C) The percentage of graft survival post‐transplantation (n = 6). ***p < 0.001; Mann–Whitney test. (D) Representative H&E staining images (×200) of the skin grafts on recipients that received naïve control (left image) or naïve Irf4‐deficient (right image) CD8+ T cells at day 14 post‐grafting. (E,F) Representative contour plots and graphs show % CD8+ cells among CD45.2+ immune cells, and the absolute number of CD8+ cells in spleens on day 14 post‐grafting. (G,H) Representative contour plots and the graph display % CD127−KLRG1+ cells among the transferred CD8+ T cells. Data are mean ± SD (n = 4). ns, no significance; *p < 0.05; ***p < 0.001; ****p< 0.0001 (unpaired Student's t‐test). Tx, transplantation

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Transplantation Assay, Purification, Adoptive Transfer Assay, MANN-WHITNEY, Staining

Journal: Immunology

Article Title: Interferon regulatory factor 4 deficiency in CD8 + T cells abrogates terminal effector differentiation and promotes transplant acceptance

doi: 10.1111/imm.13258

Figure Lengend Snippet: Irf4‐deficient CD8+ T cells fail to acquire terminal effector phenotypes in recipients with long‐term accepted allografts. B6.Rag1−/− recipients were adoptively transferred with either Irf4‐deficient or control CD8+ T cells, followed by BALB/c skin transplantation. Adoptively transferred CD8+ T cells in spleens were analysed by flow cytometry at day 100 post‐skin grafting. (A,B) Representative contour plots and bar graphs show % CD8+ T cells among CD45.2+ immune cells (A,B left bar graph), and absolute CD8+ T cells in spleens (B right bar graph). (C‐J) Contour plots were gated on the living transferred CD8+ T cells in spleens. Representative contour plots and bar graphs show % CD127−KLRG1+ (C,D), CD62L−CX3CR1+ (E,F), IFN‐γ+ (G,H), TCF1+ (I,J left bar graph) and EOMES+ (i,j right bar graph) cells among the adoptively transferred CD8+ T cells. Data are mean ± SD of n ≥ 3 animals per group. ns, no significance; *p < 0.05; **p < 0.01; ****p < 0.0001 (unpaired Student's t‐test)

Article Snippet: In brief, CD8 + T cells from cell cultures or transplant recipients were stained with the above antibodies and analysed on an LSR II or Fortessa flow cytometer (BD Biosciences) by using a previously described method.

Techniques: Transplantation Assay, Flow Cytometry