Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 activates the STAT1 and STAT3 signaling pathway, leading to the enhanced survival of CD8 T cells in vitro. (A) Purified polyclonal CD8 cells were cultured for 1 h in medium alone (Media) or medium supplemented with recombinant murine IL-21 (+ IL21). Cells were stained with anti-CD8 and anti-pSTAT1, anti-pSTAT3, or anti-pSTAT5 intracellularly. (B–C) Purified polyclonal CD8 cells from WT or STAT1−/− mice were retrovirally infected with empty vector (Control) or STAT3-D vector (STAT3-D) and stimulated with soluble anti-CD3 and anti-CD28 in medium alone (Media) or medium supplemented with IL-21 (+ IL21). (B) 4 days later, cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ CD8 cells is indicated. Plots are gated on GFP+ cells. (C) GFP+ cells were sorted and mRNA was extracted and quantitative PCR was used to measure the expression of Bcl-2 and Bcl-xL. mRNA abundance was normalized to β-actin. Data are representative of two independent experiments. * = p ≤.001.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vitro, Purification, Cell Culture, Recombinant, Staining, Infection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: Intrinsic IL-21 signaling is required for the priming of VV-specific CD8 T cells in vivo. 1 × 107 purified polyclonal naïve CD8 T cells (Thy1.2+) from either WT or IL-21R−/− mice were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 106 pfu, i.p.). 7 days later, splenocytes were harvested for analysis. (A–B) Splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-IFN-γ intracellularly. (A) Percentages of IFN-γ-producing CD8+ T cells among each respective group are indicated. Plots are gated on CD8+ T cells. (B) The mean absolute numbers ± SD of IFN-γ-producing cells per spleen are indicated (n = 4 per group). (C-D) Splenocytes were stained with anti-CD8, anti-Thy1.2, anti-CD62L, and Annexin V. (C) Percentage of Annexin V+ cells among transferred CD62Llow cells are indicated. Naive plot is gated on CD62Lhigh cells. WT and IL21R−/− plots are gated on Thy1.2+CD8+CD62Llow cells. (D) The mean absolute numbers ± SD of Annexin V+CD62Llow cells among those transferred are indicated (n = 4 per group). Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vivo, Purification, Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD8 T cell clonal expansion in response to VV infection is compromised in the absence of IL-21 signaling. 1 × 104 purified naïve clone 4 CD8 T cells (Thy1.2+) from either WT or IL-21R−/− mice were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, spleen and other lymphoid and nonlymphoid organs were harvested for analysis of expansion and function of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8 and anti-Thy1.2, and anti-IFNγ intracellularly. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated (left panels); the percentage of IFN-γ-producing clonotypic cells among total CD8 T cells is indicated with the numbers in parentheses showing the MFI (×102) of IFN-γ-producing clonotypic CD8 T cells (right panels). (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Infection, Purification, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 signaling is required for the survival, but not the activation or proliferation, of CD8 T cells. (A–B) 1 × 106 purified naïve clone 4 CD8 T cells from WT or IL-21R−/− mice were transferred to congenic recipients that were subsequently infected with rVV-HA (5 × 106 pfu, i.p.). Some mice were left uninfected (Naïve). (A) 24 h post-infection, splenocytes were harvested and stained with antibodies to CD8, Thy1.2, and the activation markers CD44 or CD69. Percentages of CD44high and CD69high are indicated. (B) 3 days post-infection, in vivo division of CFSE-labeled clonotypic cells in the spleen was analyzed. (C) 1 × 104 purified naïve clone 4 CD8 T cells from either WT or IL-21R−/− mice was transferred into recipients that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, splenocytes were stained with anti-CD8 and anti-Thy1.2 and analyzed for the expression of surface markers and the production of the effector molecules. The percentages of TNF-α-producing, CD62Llow, CD122high and Annexin V+ clonotypic CD8 T cells are indicated. All plots are gated on CD8+Thy1.2+ cells. Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Activation Assay, Purification, Infection, Staining, In Vivo, Labeling, Expressing

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD4 T help for CD8 T cell clonal expansion and survival in vivo is critically dependent on IL-21 signaling. Thy1.1+ B10.D2 mice were treated with the CD4-depleting antibody, GK1.5 on day −4. On day 0, 1 × 104 naïve clone 4 CD8 T cells (Th1.2+) from either WT or IL-21R−/− mice were transferred into these mice (CD8). In some mice, 1 × 105 naïve 6.5 CD4 T cells were also transferred (CD8 + CD4). After cell transfer, mice were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, spleen and other lymphoid and nonlymphoid organs were harvested for analysis of expansion of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8 and anti-Thy1.2. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated. (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 3 per group). (C) Splenocytes were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ clonotypic CD8 T cells is indicated.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vivo, Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: Defective CD8 memory formation in the absence of IL-21 signaling. 1 × 104 naïve clone 4 CD8 T cells from either WT or IL-21R−/− mice was transferred into recipients that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). (A–B) 42 days later, spleen and other lymphoid and non-lymphoid organs were harvested for analysis of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-IFN-γ intracellularly. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated (left panels); the percentage of IFN-γ-producing clonotypic cells among total CD8 T cells is indicated (right panels). (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). (C–D) 42 days post-infection with rVV-HA, mice were boosted with Ad-HA, and 5 days post-boost infection, lymphocytes were analyzed for clonotypic CD8 T cells. (C) The percentage of total clonotypic CD8 T cells among total splenic lymphocytes is indicated (left panels); the percentage of IFN-γproducing clonotypic cells among total splenic CD8 T cells is indicated (right panels). (D) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 signaling promotes activation of the STAT1 and STAT3 pathways and upregulation of Bcl-xL in vivo. 1 × 104 purified naïve clone 4 CD8 T cells (Thy1.2+) from either WT or IL-21R−/− were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.), of left uninfected (Naive). (A) 5 days post-infection, splenocytes were stimulated with PMA and Ionomycin and then stained with anti-CD8, anti-Thy1.2, as well as anti-pSTAT1, anti-pSTAT3, anti-STAT1, anti-STAT3, or an isotype control intracellularly. The percentage of pSTAT1+ and pSTAT3+ among clonotypic CD8 T cells is indicated. (B) 7 days post-infection, splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-Bcl-xL intracellularly. MFI of clonotypic CD8 T cells is indicated. Plots are gated on CD8+Thy1.2+ cells. Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Activation Assay, In Vivo, Purification, Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD4 T cell help for CD8 T cell survival is mediated by a soluble factor in vitro. (A–B) WT or CD4−/− mice were infected with rVV-HA (1 × 107pfu) intravenously, or left uninfected (Naive). 24 h later, CD8+CD11c+B220− DCs were purified by FACS, and cultured with purified naïve clone 4 HA-specific CD8 T cells for 4 days. (A) Division of CFSE-labelled clonotypic CD8 T cells. (B) Annexin V staining. Percentage of Annexin V+ clonotypic CD8 T cells is indicated. (C-D) Polyclonal CD8 T cells were stimulated with soluble anti-CD3 and anti-CD28 or left unstimulated for 4 days. In some wells, polyclonal CD4 T cells were added by either mixing with the CD8 T cells or placing in a transwell support. (C) Divsion of CFSE-labeled CD8 T cells. (D) Annexin V staining. Percentage of Annexin V+ CD8 cells is indicated. All plots are gated on CD8+ T cells. Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vitro, Infection, Purification, Cell Culture, Staining, Labeling

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD4 T cell help for the survival of activated CD8 T cells in vitro is mediated by IL-21. (A) Polyclonal CD4 T cells were stimulated with soluble anti-CD3 and anti-CD28 or left unstimulated. 36 h later, RNA was extracted and real-time quantitative PCR was used to measure the expression of IL-2, IL-7, IL-15, and IL-21. mRNA abundance was normalized to β-actin. Data are presented as the fold increase of the corresponding cytokine mRNA relative to that of unstimulated cells. (B) Polyclonal WT or IL-21R−/−CD8 T cells were stimulated with soluble anti-CD3 and anti-CD28 or left unstimulated as a control for 4 days. Recombinant murine IL-21 was added where indicated. Cells were stained with anti-CD8 and Annexin V. Percentages of Annexin V+CD8+ T cells are indicated. (C) CD8+CD11c+B220− DCs were purified from uninfected (Naive) and rVV-HA infected (rVV-HA) spleens and cultured for 4 days with purified naïve clone 4 HA-specific CD8 T cells from WT or IL-21R−/− clone 4 HA-TCR mice. Purified naïve 6.5 HA-specific CD4 T cells were added where indicated. Cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ clonotypic CD8 T cells is indicated. Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Recombinant, Staining, Purification, Infection, Cell Culture

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD4 T cell help for the survival of activated CD8 T cells in vitro is mediated by IL-21. (A) Polyclonal CD4 T cells were stimulated with soluble anti-CD3 and anti-CD28 or left unstimulated. 36 h later, RNA was extracted and real-time quantitative PCR was used to measure the expression of IL-2, IL-7, IL-15, and IL-21. mRNA abundance was normalized to β-actin. Data are presented as the fold increase of the corresponding cytokine mRNA relative to that of unstimulated cells. (B) Polyclonal WT or IL-21R−/−CD8 T cells were stimulated with soluble anti-CD3 and anti-CD28 or left unstimulated as a control for 4 days. Recombinant murine IL-21 was added where indicated. Cells were stained with anti-CD8 and Annexin V. Percentages of Annexin V+CD8+ T cells are indicated. (C) CD8+CD11c+B220− DCs were purified from uninfected (Naive) and rVV-HA infected (rVV-HA) spleens and cultured for 4 days with purified naïve clone 4 HA-specific CD8 T cells from WT or IL-21R−/− clone 4 HA-TCR mice. Purified naïve 6.5 HA-specific CD4 T cells were added where indicated. Cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ clonotypic CD8 T cells is indicated. Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Recombinant, Staining, Purification, Infection, Cell Culture

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: Intrinsic IL-21 signaling is required for the priming of VV-specific CD8 T cells in vivo. 1 × 107 purified polyclonal naïve CD8 T cells (Thy1.2+) from either WT or IL-21R−/− mice were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 106 pfu, i.p.). 7 days later, splenocytes were harvested for analysis. (A–B) Splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-IFN-γ intracellularly. (A) Percentages of IFN-γ-producing CD8+ T cells among each respective group are indicated. Plots are gated on CD8+ T cells. (B) The mean absolute numbers ± SD of IFN-γ-producing cells per spleen are indicated (n = 4 per group). (C-D) Splenocytes were stained with anti-CD8, anti-Thy1.2, anti-CD62L, and Annexin V. (C) Percentage of Annexin V+ cells among transferred CD62Llow cells are indicated. Naive plot is gated on CD62Lhigh cells. WT and IL21R−/− plots are gated on Thy1.2+CD8+CD62Llow cells. (D) The mean absolute numbers ± SD of Annexin V+CD62Llow cells among those transferred are indicated (n = 4 per group). Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vivo, Purification, Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD8 T cell clonal expansion in response to VV infection is compromised in the absence of IL-21 signaling. 1 × 104 purified naïve clone 4 CD8 T cells (Thy1.2+) from either WT or IL-21R−/− mice were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, spleen and other lymphoid and nonlymphoid organs were harvested for analysis of expansion and function of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8 and anti-Thy1.2, and anti-IFNγ intracellularly. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated (left panels); the percentage of IFN-γ-producing clonotypic cells among total CD8 T cells is indicated with the numbers in parentheses showing the MFI (×102) of IFN-γ-producing clonotypic CD8 T cells (right panels). (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Infection, Purification, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 signaling is required for the survival, but not the activation or proliferation, of CD8 T cells. (A–B) 1 × 106 purified naïve clone 4 CD8 T cells from WT or IL-21R−/− mice were transferred to congenic recipients that were subsequently infected with rVV-HA (5 × 106 pfu, i.p.). Some mice were left uninfected (Naïve). (A) 24 h post-infection, splenocytes were harvested and stained with antibodies to CD8, Thy1.2, and the activation markers CD44 or CD69. Percentages of CD44high and CD69high are indicated. (B) 3 days post-infection, in vivo division of CFSE-labeled clonotypic cells in the spleen was analyzed. (C) 1 × 104 purified naïve clone 4 CD8 T cells from either WT or IL-21R−/− mice was transferred into recipients that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, splenocytes were stained with anti-CD8 and anti-Thy1.2 and analyzed for the expression of surface markers and the production of the effector molecules. The percentages of TNF-α-producing, CD62Llow, CD122high and Annexin V+ clonotypic CD8 T cells are indicated. All plots are gated on CD8+Thy1.2+ cells. Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Activation Assay, Purification, Infection, Staining, In Vivo, Labeling, Expressing

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: CD4 T help for CD8 T cell clonal expansion and survival in vivo is critically dependent on IL-21 signaling. Thy1.1+ B10.D2 mice were treated with the CD4-depleting antibody, GK1.5 on day −4. On day 0, 1 × 104 naïve clone 4 CD8 T cells (Th1.2+) from either WT or IL-21R−/− mice were transferred into these mice (CD8). In some mice, 1 × 105 naïve 6.5 CD4 T cells were also transferred (CD8 + CD4). After cell transfer, mice were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). 7 days later, spleen and other lymphoid and nonlymphoid organs were harvested for analysis of expansion of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8 and anti-Thy1.2. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated. (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 3 per group). (C) Splenocytes were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ clonotypic CD8 T cells is indicated.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vivo, Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: Defective CD8 memory formation in the absence of IL-21 signaling. 1 × 104 naïve clone 4 CD8 T cells from either WT or IL-21R−/− mice was transferred into recipients that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.). (A–B) 42 days later, spleen and other lymphoid and non-lymphoid organs were harvested for analysis of clonotypic CD8 T cells. (A) Splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-IFN-γ intracellularly. The percentage of total clonotypic CD8 T cells among total lymphocytes is indicated (left panels); the percentage of IFN-γ-producing clonotypic cells among total CD8 T cells is indicated (right panels). (B) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). (C–D) 42 days post-infection with rVV-HA, mice were boosted with Ad-HA, and 5 days post-boost infection, lymphocytes were analyzed for clonotypic CD8 T cells. (C) The percentage of total clonotypic CD8 T cells among total splenic lymphocytes is indicated (left panels); the percentage of IFN-γproducing clonotypic cells among total splenic CD8 T cells is indicated (right panels). (D) The mean absolute numbers ± SD of clonotypic T cells per spleen, combined six peripheral lymph nodes (PLN), whole liver, or whole lung are indicated (n = 4 per group). Data shown are representative of four independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Infection, Staining

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 activates the STAT1 and STAT3 signaling pathway, leading to the enhanced survival of CD8 T cells in vitro. (A) Purified polyclonal CD8 cells were cultured for 1 h in medium alone (Media) or medium supplemented with recombinant murine IL-21 (+ IL21). Cells were stained with anti-CD8 and anti-pSTAT1, anti-pSTAT3, or anti-pSTAT5 intracellularly. (B–C) Purified polyclonal CD8 cells from WT or STAT1−/− mice were retrovirally infected with empty vector (Control) or STAT3-D vector (STAT3-D) and stimulated with soluble anti-CD3 and anti-CD28 in medium alone (Media) or medium supplemented with IL-21 (+ IL21). (B) 4 days later, cells were stained with anti-CD8 and Annexin V. Percentage of Annexin V+ CD8 cells is indicated. Plots are gated on GFP+ cells. (C) GFP+ cells were sorted and mRNA was extracted and quantitative PCR was used to measure the expression of Bcl-2 and Bcl-xL. mRNA abundance was normalized to β-actin. Data are representative of two independent experiments. * = p ≤.001.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: In Vitro, Purification, Cell Culture, Recombinant, Staining, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

Journal:

Article Title: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection

doi: 10.4049/jimmunol.1003009

Figure Lengend Snippet: IL-21 signaling promotes activation of the STAT1 and STAT3 pathways and upregulation of Bcl-xL in vivo. 1 × 104 purified naïve clone 4 CD8 T cells (Thy1.2+) from either WT or IL-21R−/− were adoptively transferred into congenic B10.D2 mice (Thy1.1+) that were subsequently infected with rVV-HA (5 × 105 pfu, i.p.), of left uninfected (Naive). (A) 5 days post-infection, splenocytes were stimulated with PMA and Ionomycin and then stained with anti-CD8, anti-Thy1.2, as well as anti-pSTAT1, anti-pSTAT3, anti-STAT1, anti-STAT3, or an isotype control intracellularly. The percentage of pSTAT1+ and pSTAT3+ among clonotypic CD8 T cells is indicated. (B) 7 days post-infection, splenocytes were stained with anti-CD8, anti-Thy1.2, and anti-Bcl-xL intracellularly. MFI of clonotypic CD8 T cells is indicated. Plots are gated on CD8+Thy1.2+ cells. Data are representative of three independent experiments.

Article Snippet: mAbs (all from BD Biosciences unless indicated) used for staining were PE-Cy5-conjugated anti-CD8; FITC-conjugated anti-B220, -CD8, -CD44, -CD69, -CD62L,-CD122, IFN-γ, -TNFα, Thy1.2, -pSTAT1, -pSTAT3, and -pSTAT5; PE-conjugated anti-Thy1.2, -CD11c, -Bcl-xL (Santa Cruz Biotechnology), -mouse IgG (Southern Biotech), and annexin V; biotin-conjugated anti-Thy1.2; APC-conjugated streptavidin; and purified anti-STAT1 and -STAT3 (Cell Signaling).

Techniques: Activation Assay, In Vivo, Purification, Infection, Staining