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T-cell STAT3 is required for the maintenance of humoral immunity to LCMV.

McIlwain DR, Grusdat M, Pozdeev VI, Xu HC, Shinde P, Reardon C, Hao Z, Beyer M, Bergthaler A, Häussinger D, Nolan GP, Lang KS, Lang PA - Eur. J. Immunol. (2014)

Bottom Line: STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types.As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum.These effects were associated with reduced control of viral replication and prolonged infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.

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Generation of LCMV-specific Tfh cells requires STAT3. (A) Quantity of B220+, CD3+, CD4+, CD8+, and NK1-1+ cells were compared in naive spleens from Lck-Cre−Stat3fl/fl (WT) vs. Lck-Cre+Stat3fl/fl (Stat3−/−) mice. (B–C) Quantification of CFSE dilution indicative of proliferation for Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl (B) CD8+ T cells or (C) CD4+ T cells (both pregated on CD3+) in vitro. Percentage of cells in each division cycle for negatively selected, CFSE labeled Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl T cells stimulated with 5 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 72 h as measured by flow cytometry. (D–H) Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl mice were infected with 2 × 103 PFU LCMV-Docile. (D) Quantity of gp33 tetramer-specific CD8+ T cells in the spleen at days 12, 20, 40, and 65 after infection was measured (% of total CD8+ T cells). (E) Quantity of IFN-γ-producing CD8+ T cells present at days 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp33. (F) Quantity of IFN-γ-producing CD4+ T cells present at days 8, 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp61. (G) Quantity of virus-specific CXCR5+ cells in spleen at days 8, 12, and 20 postinfection was measured (% of gp66-tet+CD4+ T cells). (H) Quantification of CXCR5hiBCL-6hi virus-specific cells in spleen at days 12 and 20 postinfection (% of gp66-tet+CD44+CD4+ T cells). (A–H) Data are shown as mean ± SEM from (A) n = 5–6 mice/group, (B and C) n = 7 mice/group, (D–F) n = 4–7 mice/group, (G) n = 6 mice/group, (H) n = 3–4 mice/group. Data are pooled from two independent experiments (A–C), or pooled from one or two independent experiments per time point (D–H). Statistical significance between groups was determined by Student's t-test.
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fig01: Generation of LCMV-specific Tfh cells requires STAT3. (A) Quantity of B220+, CD3+, CD4+, CD8+, and NK1-1+ cells were compared in naive spleens from Lck-Cre−Stat3fl/fl (WT) vs. Lck-Cre+Stat3fl/fl (Stat3−/−) mice. (B–C) Quantification of CFSE dilution indicative of proliferation for Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl (B) CD8+ T cells or (C) CD4+ T cells (both pregated on CD3+) in vitro. Percentage of cells in each division cycle for negatively selected, CFSE labeled Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl T cells stimulated with 5 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 72 h as measured by flow cytometry. (D–H) Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl mice were infected with 2 × 103 PFU LCMV-Docile. (D) Quantity of gp33 tetramer-specific CD8+ T cells in the spleen at days 12, 20, 40, and 65 after infection was measured (% of total CD8+ T cells). (E) Quantity of IFN-γ-producing CD8+ T cells present at days 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp33. (F) Quantity of IFN-γ-producing CD4+ T cells present at days 8, 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp61. (G) Quantity of virus-specific CXCR5+ cells in spleen at days 8, 12, and 20 postinfection was measured (% of gp66-tet+CD4+ T cells). (H) Quantification of CXCR5hiBCL-6hi virus-specific cells in spleen at days 12 and 20 postinfection (% of gp66-tet+CD44+CD4+ T cells). (A–H) Data are shown as mean ± SEM from (A) n = 5–6 mice/group, (B and C) n = 7 mice/group, (D–F) n = 4–7 mice/group, (G) n = 6 mice/group, (H) n = 3–4 mice/group. Data are pooled from two independent experiments (A–C), or pooled from one or two independent experiments per time point (D–H). Statistical significance between groups was determined by Student's t-test.

Mentions: STAT3 is critically required for development as homozygous Stat3 deficient mice arrest early during embryogenesis [19]. In an attempt to understand the role of STAT3 in T cells we investigated a model employing transgenic Lck-Cre to conditionally inactivate Stat3 across all T-cell lineages [20, 21]. Lck-Cre− Stat3fl/fl (referred to as WT in figures for simplicity) mice were indistinguishable from Lck-Cre+ Stat3fl/fl (also referred to as Stat3−/−) littermate controls in terms of appearance and long-term survival (Supporting Information Fig. 1A). We investigated basal levels of B cells, T cells and NK cells in the spleen of these mice, finding no major defects in cell population numbers associated with presence or absence of STAT3 (Fig.1A). Similarly, thymic T-cell populations were largely equivalent for Lck-Cre+ Stat3fl/fl and Lck-Cre− Stat3fl/fl mice indicating grossly normal T-cell development (Supporting Information Fig. 1B, C; also see general flow cytometry gating strategy in Supporting Information Fig. 2).


T-cell STAT3 is required for the maintenance of humoral immunity to LCMV.

McIlwain DR, Grusdat M, Pozdeev VI, Xu HC, Shinde P, Reardon C, Hao Z, Beyer M, Bergthaler A, Häussinger D, Nolan GP, Lang KS, Lang PA - Eur. J. Immunol. (2014)

Generation of LCMV-specific Tfh cells requires STAT3. (A) Quantity of B220+, CD3+, CD4+, CD8+, and NK1-1+ cells were compared in naive spleens from Lck-Cre−Stat3fl/fl (WT) vs. Lck-Cre+Stat3fl/fl (Stat3−/−) mice. (B–C) Quantification of CFSE dilution indicative of proliferation for Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl (B) CD8+ T cells or (C) CD4+ T cells (both pregated on CD3+) in vitro. Percentage of cells in each division cycle for negatively selected, CFSE labeled Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl T cells stimulated with 5 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 72 h as measured by flow cytometry. (D–H) Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl mice were infected with 2 × 103 PFU LCMV-Docile. (D) Quantity of gp33 tetramer-specific CD8+ T cells in the spleen at days 12, 20, 40, and 65 after infection was measured (% of total CD8+ T cells). (E) Quantity of IFN-γ-producing CD8+ T cells present at days 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp33. (F) Quantity of IFN-γ-producing CD4+ T cells present at days 8, 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp61. (G) Quantity of virus-specific CXCR5+ cells in spleen at days 8, 12, and 20 postinfection was measured (% of gp66-tet+CD4+ T cells). (H) Quantification of CXCR5hiBCL-6hi virus-specific cells in spleen at days 12 and 20 postinfection (% of gp66-tet+CD44+CD4+ T cells). (A–H) Data are shown as mean ± SEM from (A) n = 5–6 mice/group, (B and C) n = 7 mice/group, (D–F) n = 4–7 mice/group, (G) n = 6 mice/group, (H) n = 3–4 mice/group. Data are pooled from two independent experiments (A–C), or pooled from one or two independent experiments per time point (D–H). Statistical significance between groups was determined by Student's t-test.
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fig01: Generation of LCMV-specific Tfh cells requires STAT3. (A) Quantity of B220+, CD3+, CD4+, CD8+, and NK1-1+ cells were compared in naive spleens from Lck-Cre−Stat3fl/fl (WT) vs. Lck-Cre+Stat3fl/fl (Stat3−/−) mice. (B–C) Quantification of CFSE dilution indicative of proliferation for Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl (B) CD8+ T cells or (C) CD4+ T cells (both pregated on CD3+) in vitro. Percentage of cells in each division cycle for negatively selected, CFSE labeled Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl T cells stimulated with 5 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 72 h as measured by flow cytometry. (D–H) Lck-Cre−Stat3fl/fl vs. Lck-Cre+Stat3fl/fl mice were infected with 2 × 103 PFU LCMV-Docile. (D) Quantity of gp33 tetramer-specific CD8+ T cells in the spleen at days 12, 20, 40, and 65 after infection was measured (% of total CD8+ T cells). (E) Quantity of IFN-γ-producing CD8+ T cells present at days 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp33. (F) Quantity of IFN-γ-producing CD4+ T cells present at days 8, 12, 20, 40, and 65 after infection following in vitro restimulation with virus specific peptide gp61. (G) Quantity of virus-specific CXCR5+ cells in spleen at days 8, 12, and 20 postinfection was measured (% of gp66-tet+CD4+ T cells). (H) Quantification of CXCR5hiBCL-6hi virus-specific cells in spleen at days 12 and 20 postinfection (% of gp66-tet+CD44+CD4+ T cells). (A–H) Data are shown as mean ± SEM from (A) n = 5–6 mice/group, (B and C) n = 7 mice/group, (D–F) n = 4–7 mice/group, (G) n = 6 mice/group, (H) n = 3–4 mice/group. Data are pooled from two independent experiments (A–C), or pooled from one or two independent experiments per time point (D–H). Statistical significance between groups was determined by Student's t-test.
Mentions: STAT3 is critically required for development as homozygous Stat3 deficient mice arrest early during embryogenesis [19]. In an attempt to understand the role of STAT3 in T cells we investigated a model employing transgenic Lck-Cre to conditionally inactivate Stat3 across all T-cell lineages [20, 21]. Lck-Cre− Stat3fl/fl (referred to as WT in figures for simplicity) mice were indistinguishable from Lck-Cre+ Stat3fl/fl (also referred to as Stat3−/−) littermate controls in terms of appearance and long-term survival (Supporting Information Fig. 1A). We investigated basal levels of B cells, T cells and NK cells in the spleen of these mice, finding no major defects in cell population numbers associated with presence or absence of STAT3 (Fig.1A). Similarly, thymic T-cell populations were largely equivalent for Lck-Cre+ Stat3fl/fl and Lck-Cre− Stat3fl/fl mice indicating grossly normal T-cell development (Supporting Information Fig. 1B, C; also see general flow cytometry gating strategy in Supporting Information Fig. 2).

Bottom Line: STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types.As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum.These effects were associated with reduced control of viral replication and prolonged infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.

Show MeSH
Related in: MedlinePlus