Journal: Immunology

Article Title: A role for cell‐autocrine interleukin‐2 in regulatory T‐cell homeostasis

doi: 10.1111/imm.13194

Figure Lengend Snippet: Efficiency of regulatory T‐cells (Treg) generation from wild‐type (WT) and interleukin (IL)2−/− genotypes in the thymus of mixed bone marrow chimeras. Thymocytes from mixed bone marrow chimeras were isolated and analysed by flow cytometry. Bone marrow chimeras were made with a wide range of WT:IL2−/− bone marrow cells. (a) WT:IL2−/− donor genotype ratios for Treg cells (CD4+ CD8− CD25+ FOXP3+) were calculated in each individual recipient mouse and plotted versus output chimerism in each recipient mouse (n = 57). For further analysis, the chimeras were divided into two groups, one with a chimerism ratio of < 1 (range: 0·1–0·9; n = 34), and the second with a ratio of > 1 (range: 1·1–36; n = 23). For each group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse, in Treg lineage cell frequencies in CD4SP thymocytes [(b), CD25+ FOXP3+ CD4+ CD8− Tregs; (c) CD25+ FOXP3− CD4+ CD8− Treg precursors; (d) CD25‐FOXP3+ CD4+ CD8− Treg precursors]. Student's t‐test was used to calculate P‐values. ns: P > 0·05. (e) IL2 mRNA levels, by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR), in sorted CD4SP, CD8SP, double‐negative (CD4− CD8−) and double‐positive (CD4+ CD8+) thymocytes from WT mice (n = 3).

Article Snippet: The following anti‐mouse antibodies were used for sorting and phenotyping: FOXP3 (FJK‐16s or 150D/E4) (AF488, PE, eFluor 450), Helios (22F6) (PE, eFluor 450), pStat5 (SRBCZX) APC, pJNK, CD8 α (53–6·7) (FITC, PE, APC, Pecy7), CD4 (RM4–5) (V500, PercP5.5, APC, eFlour 450), CD25 (PC61.5) (PE, APC‐eFluor 780, eFluor 450), CD62L (MEL‐14) (PE, APC, APC‐eFluor 780), CD44 (IM7) (APC, eFluorV450), CD45.2 (104) (FITC, PE, APC, V500, eFlour 450) and CD45.1 (A20) (PercP5.5, APC, eFlour 450, Pecy7) (from eBioscience, San Diego, CA, or from BD Biosciences, San Jose, CA).

Techniques: Isolation, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Immunology

Article Title: A role for cell‐autocrine interleukin‐2 in regulatory T‐cell homeostasis

doi: 10.1111/imm.13194

Figure Lengend Snippet: Relative levels of regulatory T‐cells (Treg) subsets of wild‐type (WT) and interleukin (IL)2−/− genotypes in peripheral lymphoid tissues of mixed bone marrow chimeras. Splenic cells, small or large intestinal lamina propria cells, or mesenteric lymph node cells (as indicated) from mixed bone marrow chimeras were isolated and analysed by flow cytometry. (a) WT:IL2−/− donor genotype ratios for Tregs (CD4+ CD25+ FOXP3+) were calculated in each individual recipient mouse and plotted versus output chimerism in each recipient mouse (n = 55). For further analysis, bone marrow chimeras were divided into two groups, one with a chimerism ratio of < 1 (range: 0·1–0·9; n = 32), and the second with a ratio of > 1 (range: 1·1–48; n = 23). For each chimera group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse. (b) Quantification of splenic Treg (CD4+ CD25+ FOXP3+) frequencies in total CD4 T‐cells of WT or IL2−/− genotypes in chimeric mice. (c,d) Quantification of naïve:memory ratios in CD4 (c) or CD8 (d) T‐cells in mixed bone marrow chimeras gated on either CD45.1 (WT) or CD45.2 (IL2−/−) donors. Naïve cells were gated as CD4+ CD44loCD25− or CD8+ CD44lo, while memory T‐cells were gated as CD4+ CD44hi CD25− or CD8+ CD44hi. For each chimera group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse in two groups with ratios of < 1 (range: 0·1–0·9; n = 10) or > 1 (range: 1·3–26; n = 7), respectively. Student's t‐test was used to quantify P‐values. ns: P > 0·05.

Article Snippet: The following anti‐mouse antibodies were used for sorting and phenotyping: FOXP3 (FJK‐16s or 150D/E4) (AF488, PE, eFluor 450), Helios (22F6) (PE, eFluor 450), pStat5 (SRBCZX) APC, pJNK, CD8 α (53–6·7) (FITC, PE, APC, Pecy7), CD4 (RM4–5) (V500, PercP5.5, APC, eFlour 450), CD25 (PC61.5) (PE, APC‐eFluor 780, eFluor 450), CD62L (MEL‐14) (PE, APC, APC‐eFluor 780), CD44 (IM7) (APC, eFluorV450), CD45.2 (104) (FITC, PE, APC, V500, eFlour 450) and CD45.1 (A20) (PercP5.5, APC, eFlour 450, Pecy7) (from eBioscience, San Diego, CA, or from BD Biosciences, San Jose, CA).

Techniques: Isolation, Flow Cytometry

Journal: Immunology

Article Title: A role for cell‐autocrine interleukin‐2 in regulatory T‐cell homeostasis

doi: 10.1111/imm.13194

Figure Lengend Snippet: Efficiency of regulatory T‐cells (Treg) generation from wild‐type (WT) and interleukin (IL)2−/− genotypes in the thymus of mixed bone marrow chimeras. Thymocytes from mixed bone marrow chimeras were isolated and analysed by flow cytometry. Bone marrow chimeras were made with a wide range of WT:IL2−/− bone marrow cells. (a) WT:IL2−/− donor genotype ratios for Treg cells (CD4+ CD8− CD25+ FOXP3+) were calculated in each individual recipient mouse and plotted versus output chimerism in each recipient mouse (n = 57). For further analysis, the chimeras were divided into two groups, one with a chimerism ratio of < 1 (range: 0·1–0·9; n = 34), and the second with a ratio of > 1 (range: 1·1–36; n = 23). For each group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse, in Treg lineage cell frequencies in CD4SP thymocytes [(b), CD25+ FOXP3+ CD4+ CD8− Tregs; (c) CD25+ FOXP3− CD4+ CD8− Treg precursors; (d) CD25‐FOXP3+ CD4+ CD8− Treg precursors]. Student's t‐test was used to calculate P‐values. ns: P > 0·05. (e) IL2 mRNA levels, by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR), in sorted CD4SP, CD8SP, double‐negative (CD4− CD8−) and double‐positive (CD4+ CD8+) thymocytes from WT mice (n = 3).

Article Snippet: Reagents The following anti‐mouse antibodies were used for sorting and phenotyping: FOXP3 (FJK‐16s or 150D/E4) (AF488, PE, eFluor 450), Helios (22F6) (PE, eFluor 450), pStat5 (SRBCZX) APC, pJNK, CD8 α (53–6·7) (FITC, PE, APC, Pecy7), CD4 (RM4–5) (V500, PercP5.5, APC, eFlour 450), CD25 (PC61.5) (PE, APC‐eFluor 780, eFluor 450), CD62L (MEL‐14) (PE, APC, APC‐eFluor 780), CD44 (IM7) (APC, eFluorV450), CD45.2 (104) (FITC, PE, APC, V500, eFlour 450) and CD45.1 (A20) (PercP5.5, APC, eFlour 450, Pecy7) (from eBioscience, San Diego, CA, or from BD Biosciences, San Jose, CA).

Techniques: Isolation, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Immunology

Article Title: A role for cell‐autocrine interleukin‐2 in regulatory T‐cell homeostasis

doi: 10.1111/imm.13194

Figure Lengend Snippet: Relative levels of regulatory T‐cells (Treg) subsets of wild‐type (WT) and interleukin (IL)2−/− genotypes in peripheral lymphoid tissues of mixed bone marrow chimeras. Splenic cells, small or large intestinal lamina propria cells, or mesenteric lymph node cells (as indicated) from mixed bone marrow chimeras were isolated and analysed by flow cytometry. (a) WT:IL2−/− donor genotype ratios for Tregs (CD4+ CD25+ FOXP3+) were calculated in each individual recipient mouse and plotted versus output chimerism in each recipient mouse (n = 55). For further analysis, bone marrow chimeras were divided into two groups, one with a chimerism ratio of < 1 (range: 0·1–0·9; n = 32), and the second with a ratio of > 1 (range: 1·1–48; n = 23). For each chimera group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse. (b) Quantification of splenic Treg (CD4+ CD25+ FOXP3+) frequencies in total CD4 T‐cells of WT or IL2−/− genotypes in chimeric mice. (c,d) Quantification of naïve:memory ratios in CD4 (c) or CD8 (d) T‐cells in mixed bone marrow chimeras gated on either CD45.1 (WT) or CD45.2 (IL2−/−) donors. Naïve cells were gated as CD4+ CD44loCD25− or CD8+ CD44lo, while memory T‐cells were gated as CD4+ CD44hi CD25− or CD8+ CD44hi. For each chimera group, paired data are shown for WT versus IL2−/− donor genotypes in each recipient mouse in two groups with ratios of < 1 (range: 0·1–0·9; n = 10) or > 1 (range: 1·3–26; n = 7), respectively. Student's t‐test was used to quantify P‐values. ns: P > 0·05.

Article Snippet: Reagents The following anti‐mouse antibodies were used for sorting and phenotyping: FOXP3 (FJK‐16s or 150D/E4) (AF488, PE, eFluor 450), Helios (22F6) (PE, eFluor 450), pStat5 (SRBCZX) APC, pJNK, CD8 α (53–6·7) (FITC, PE, APC, Pecy7), CD4 (RM4–5) (V500, PercP5.5, APC, eFlour 450), CD25 (PC61.5) (PE, APC‐eFluor 780, eFluor 450), CD62L (MEL‐14) (PE, APC, APC‐eFluor 780), CD44 (IM7) (APC, eFluorV450), CD45.2 (104) (FITC, PE, APC, V500, eFlour 450) and CD45.1 (A20) (PercP5.5, APC, eFlour 450, Pecy7) (from eBioscience, San Diego, CA, or from BD Biosciences, San Jose, CA).

Techniques: Isolation, Flow Cytometry