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αcd8β 53-5.8 antibody  (Bio X Cell)


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    Bio X Cell αcd8β 53-5.8 antibody
    αcd8β 53 5.8 Antibody, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/antibody+%CE%B1cd8%CE%B2+53%E2%80%935%2E8/%CE%B1cd8%CE%B2+53+5+8+antibody/pm36376335-434-0-15
    Average 90 stars, based on 1 article reviews
    αcd8β 53-5.8 antibody - by Bioz Stars, 2026-09
    90/100 stars

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    Related Articles

    Injection:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Generated:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Control:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Flow Cytometry:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Infection:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    BrdU Incorporation Assay:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Expressing:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Standard Deviation:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    RNA Sequencing:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Cell Culture:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Isolation:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    In Vitro:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Labeling:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    In Vivo:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Blocking Assay:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Ex Vivo:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Adoptive Transfer Assay:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Tumor Implantation:

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity
    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).



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    CXCR5 + <t>CD8</t> + T cells are generated in acute settings in the absence of follicular infection following protein immunisation and IAV challenge. Isolated, congenically marked (CD45.1/2) OT-I cells were transferred i.v into C57BL/6 mice that were then (A) immunised with OVA/alum intraperitoneally or (B) infected with x31-OVA intranasally. Flow cytometry of (A) spleen cells on day 7 post-immunisation with OVA/alum and (B) mLN cells on day 8 post-infection with x31-OVA, assessing CXCR5 expression on the indicated cell populations. Data are representative of at least three independent experiments with at least 4 mice. Data were analysed by a repeated measures one-way ANOVA. Mean ± SEM. **p < 0.01, ****p < 0.0001.
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    Image Search Results


    CXCR5 + CD8 + T cells are generated in acute settings in the absence of follicular infection following protein immunisation and IAV challenge. Isolated, congenically marked (CD45.1/2) OT-I cells were transferred i.v into C57BL/6 mice that were then (A) immunised with OVA/alum intraperitoneally or (B) infected with x31-OVA intranasally. Flow cytometry of (A) spleen cells on day 7 post-immunisation with OVA/alum and (B) mLN cells on day 8 post-infection with x31-OVA, assessing CXCR5 expression on the indicated cell populations. Data are representative of at least three independent experiments with at least 4 mice. Data were analysed by a repeated measures one-way ANOVA. Mean ± SEM. **p < 0.01, ****p < 0.0001.

    Journal: Frontiers in Immunology

    Article Title: CXCR5 + CD8 + T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection

    doi: 10.3389/fimmu.2021.626199

    Figure Lengend Snippet: CXCR5 + CD8 + T cells are generated in acute settings in the absence of follicular infection following protein immunisation and IAV challenge. Isolated, congenically marked (CD45.1/2) OT-I cells were transferred i.v into C57BL/6 mice that were then (A) immunised with OVA/alum intraperitoneally or (B) infected with x31-OVA intranasally. Flow cytometry of (A) spleen cells on day 7 post-immunisation with OVA/alum and (B) mLN cells on day 8 post-infection with x31-OVA, assessing CXCR5 expression on the indicated cell populations. Data are representative of at least three independent experiments with at least 4 mice. Data were analysed by a repeated measures one-way ANOVA. Mean ± SEM. **p < 0.01, ****p < 0.0001.

    Article Snippet: To deplete CD8 + T cells, mice were treated with 100 μg of αCD8β (clone 53-5.8, Bio X cell) in 200 μL PBS i.p 4 days prior to IAV infection and on day 4 (and again on day 12 where required) post-infection.

    Techniques: Generated, Infection, Isolation, Flow Cytometry, Expressing

    CXCR5 + CD8 + T cells generated in response to OVA/alum and IAV are distinct from their CXCR5 - counterparts. Isolated congenically marked (CD45.1/2) OT-I cells were transferred i.v into C57BL/6 mice that were immunised with OVA/alum i.p or infected with x31-OVA i.n the following day. On day 7 post-immunisation and day 8 post-infection, the spleens (OVA/alum) and mLNs (x31-OVA) were harvested and analysed by flow cytometry. (A, B) IFNγ and TNFα expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (A) OVA/alum and (B) x31-OVA. (C, D) Granzyme B (GzmB) expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (C) OVA/alum and (D) x31-OVA. (E) Ex vivo cytotoxicity assay assessing the specific killing of CXCR5 + and CXCR5 - OT-I cells FACS-sorted from the spleens of mice immunised i.p with OVA/alum 7 days prior. (F, G) CD127 and KLRG1 expression by CXCR5 + and CXCR5 - OT-I cells following (F) OVA/alum and (G) x31-OVA. (H, J) Tim-3 expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (H) OVA/alum and (J) x31-OVA. (I, K) TCF-1 expression by CXCR5 + and CXCR5 - OT-I cells, TFH, Pre-TFH, Naïve CD8 + T cells and B220 + B cells following (I) OVA/alum and (K) x31-OVA. (L, M) Expression of ICOS, BTLA, PD-1 and Ly108 by CXCR5 + and CXCR5 - OT-Is, TFH, Pre-TFH and Naïve CD8 + T cells following (L) OVA/alum and (M) x31-OVA. Refer to <xref ref-type= Figure 1 for gating of CXCR5 + and CXCR5 - OT-I cells and Naïve CD8 + T cells. Refer to Supplementary Figure 1A for gating of TFH and Pre-TFH populations. (A–D, F–M) Data are representative of at least two independent experiments with at least 4 mice. (E) Data are representative of two independent experiments with 3 mice. (A–D, F–M) Data were analysed by paired t-tests or (E) two-way ANOVA with Bonferroni’s multiple comparison test to compare CXCR5 + and CXCR5 - OT-I cells. (A–D, F–M) Mean ± SEM or (E) Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. " width="100%" height="100%">

    Journal: Frontiers in Immunology

    Article Title: CXCR5 + CD8 + T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection

    doi: 10.3389/fimmu.2021.626199

    Figure Lengend Snippet: CXCR5 + CD8 + T cells generated in response to OVA/alum and IAV are distinct from their CXCR5 - counterparts. Isolated congenically marked (CD45.1/2) OT-I cells were transferred i.v into C57BL/6 mice that were immunised with OVA/alum i.p or infected with x31-OVA i.n the following day. On day 7 post-immunisation and day 8 post-infection, the spleens (OVA/alum) and mLNs (x31-OVA) were harvested and analysed by flow cytometry. (A, B) IFNγ and TNFα expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (A) OVA/alum and (B) x31-OVA. (C, D) Granzyme B (GzmB) expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (C) OVA/alum and (D) x31-OVA. (E) Ex vivo cytotoxicity assay assessing the specific killing of CXCR5 + and CXCR5 - OT-I cells FACS-sorted from the spleens of mice immunised i.p with OVA/alum 7 days prior. (F, G) CD127 and KLRG1 expression by CXCR5 + and CXCR5 - OT-I cells following (F) OVA/alum and (G) x31-OVA. (H, J) Tim-3 expression by CXCR5 + and CXCR5 - OT-I cells and naïve CD8 + T cells following (H) OVA/alum and (J) x31-OVA. (I, K) TCF-1 expression by CXCR5 + and CXCR5 - OT-I cells, TFH, Pre-TFH, Naïve CD8 + T cells and B220 + B cells following (I) OVA/alum and (K) x31-OVA. (L, M) Expression of ICOS, BTLA, PD-1 and Ly108 by CXCR5 + and CXCR5 - OT-Is, TFH, Pre-TFH and Naïve CD8 + T cells following (L) OVA/alum and (M) x31-OVA. Refer to Figure 1 for gating of CXCR5 + and CXCR5 - OT-I cells and Naïve CD8 + T cells. Refer to Supplementary Figure 1A for gating of TFH and Pre-TFH populations. (A–D, F–M) Data are representative of at least two independent experiments with at least 4 mice. (E) Data are representative of two independent experiments with 3 mice. (A–D, F–M) Data were analysed by paired t-tests or (E) two-way ANOVA with Bonferroni’s multiple comparison test to compare CXCR5 + and CXCR5 - OT-I cells. (A–D, F–M) Mean ± SEM or (E) Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

    Article Snippet: To deplete CD8 + T cells, mice were treated with 100 μg of αCD8β (clone 53-5.8, Bio X cell) in 200 μL PBS i.p 4 days prior to IAV infection and on day 4 (and again on day 12 where required) post-infection.

    Techniques: Generated, Isolation, Infection, Flow Cytometry, Expressing, Ex Vivo, Cytotoxicity Assay, Comparison

    CD8 + T cells require CXCR5 expression to skew class switching from IgG1 to IgG2c in ASCs in response to OVA/alum. Congenically marked WT (CD45.1/2) or Cxcr5 -/- (CD45.2) OT-I cells (or PBS only for no transfer controls) were transferred i.v into separate B6.Ly5.1 (CD45.1) mice that were subsequently immunised with OVA/alum i.p. and harvested on day 7 post-immunisation for analysis. (A) Schematic outline of the experiment. (B) Gating strategy used for identification of CD138 + B220 int ASCs in spleens. Pre-gated on live, single cells. (C) Total number of ASCs. (D) Analysis of intracellular IgG1 and IgG2c in IgM - ASCs. (E, F) Frequency of (E) IgG1 + and (F) IgG2c + ASCs among total ASCs. (G) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells in spleens. Pre-gated on live, single, B220 + CD138 - cells. (H) Total number of GCB cells. (I) Analysis of intracellular IgG1 and IgG2c in total GCB cells. (J, K) Frequency of (J) IgG1 + and (K) IgG2c + GCB cells among total GCB cells. (C, E, F, H, J, K) Data are pooled from two independent experiments with a total of 6-10 mice per group. Data were analysed by ordinary one-way ANOVA. Mean ± SEM. *p < 0.05.

    Journal: Frontiers in Immunology

    Article Title: CXCR5 + CD8 + T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection

    doi: 10.3389/fimmu.2021.626199

    Figure Lengend Snippet: CD8 + T cells require CXCR5 expression to skew class switching from IgG1 to IgG2c in ASCs in response to OVA/alum. Congenically marked WT (CD45.1/2) or Cxcr5 -/- (CD45.2) OT-I cells (or PBS only for no transfer controls) were transferred i.v into separate B6.Ly5.1 (CD45.1) mice that were subsequently immunised with OVA/alum i.p. and harvested on day 7 post-immunisation for analysis. (A) Schematic outline of the experiment. (B) Gating strategy used for identification of CD138 + B220 int ASCs in spleens. Pre-gated on live, single cells. (C) Total number of ASCs. (D) Analysis of intracellular IgG1 and IgG2c in IgM - ASCs. (E, F) Frequency of (E) IgG1 + and (F) IgG2c + ASCs among total ASCs. (G) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells in spleens. Pre-gated on live, single, B220 + CD138 - cells. (H) Total number of GCB cells. (I) Analysis of intracellular IgG1 and IgG2c in total GCB cells. (J, K) Frequency of (J) IgG1 + and (K) IgG2c + GCB cells among total GCB cells. (C, E, F, H, J, K) Data are pooled from two independent experiments with a total of 6-10 mice per group. Data were analysed by ordinary one-way ANOVA. Mean ± SEM. *p < 0.05.

    Article Snippet: To deplete CD8 + T cells, mice were treated with 100 μg of αCD8β (clone 53-5.8, Bio X cell) in 200 μL PBS i.p 4 days prior to IAV infection and on day 4 (and again on day 12 where required) post-infection.

    Techniques: Expressing

    CD8 + T cells support induction of IgG2c responses against IAV. C57BL/6 mice were treated i.p. with either αCD8β or control antibody 4 days before and on day 4 after i.n infection with x31 and harvested on day 8 post-infection. Levels of serum x31-specific (A) IgM, (B) IgG, (C) IgG3, (D) IgG1 and (E) IgG2c assessed by ELISA. (F) Absorption summation (AbS) analysis of the x31-specific IgG2c ELISA data in (E) . AbS is determined by adding the absorbance values from all dilutions to obtain a single value for each biological replicate . (G) Gating strategy used for identification of CD138 + B220 int ASCs and IgG2c-switching in ASCs in the mLN. Cells in the top panel were pre-gated on live, single cells. (H, I) Quantification of the ASC response by (H) frequency and (I) number. (J–L) Analysis of the IgG2c + ASC response by (J) proportion of total ASCs, (K) frequency of total live cells and (L) number. (M) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells and IgG2c-switching in GCB cells in the mLN. Cells in the top panel were pre-gated on live, B220 + CD138 - cells. (N, O) Quantification of the GCB cell response by (N) frequency and (O) number. (P–R) Analysis of the IgG2c + GCB cell response by (P) proportion of total GCB cells, (Q) frequency of total live cells and (R) number. (A–F) Data are pooled from two independent experiments with a total of 12 mice per group (Ctrl and αCD8β) or 3 mice (Naïve) and the absorbance values for the ELISA data have been normalised to the average of the Ctrl group for each independent experiment prior to pooling the data. (H–L, N–R) Data are pooled from three independent experiments with 17-19 mice per group. (A–E) Data were analysed by two-way ANOVA with Bonferroni’s multiple comparison test or (F, H–L, N–R) unpaired t-tests. (F, H–L, N–R) Mean ± SEM or (A–E) Mean + SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

    Journal: Frontiers in Immunology

    Article Title: CXCR5 + CD8 + T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection

    doi: 10.3389/fimmu.2021.626199

    Figure Lengend Snippet: CD8 + T cells support induction of IgG2c responses against IAV. C57BL/6 mice were treated i.p. with either αCD8β or control antibody 4 days before and on day 4 after i.n infection with x31 and harvested on day 8 post-infection. Levels of serum x31-specific (A) IgM, (B) IgG, (C) IgG3, (D) IgG1 and (E) IgG2c assessed by ELISA. (F) Absorption summation (AbS) analysis of the x31-specific IgG2c ELISA data in (E) . AbS is determined by adding the absorbance values from all dilutions to obtain a single value for each biological replicate . (G) Gating strategy used for identification of CD138 + B220 int ASCs and IgG2c-switching in ASCs in the mLN. Cells in the top panel were pre-gated on live, single cells. (H, I) Quantification of the ASC response by (H) frequency and (I) number. (J–L) Analysis of the IgG2c + ASC response by (J) proportion of total ASCs, (K) frequency of total live cells and (L) number. (M) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells and IgG2c-switching in GCB cells in the mLN. Cells in the top panel were pre-gated on live, B220 + CD138 - cells. (N, O) Quantification of the GCB cell response by (N) frequency and (O) number. (P–R) Analysis of the IgG2c + GCB cell response by (P) proportion of total GCB cells, (Q) frequency of total live cells and (R) number. (A–F) Data are pooled from two independent experiments with a total of 12 mice per group (Ctrl and αCD8β) or 3 mice (Naïve) and the absorbance values for the ELISA data have been normalised to the average of the Ctrl group for each independent experiment prior to pooling the data. (H–L, N–R) Data are pooled from three independent experiments with 17-19 mice per group. (A–E) Data were analysed by two-way ANOVA with Bonferroni’s multiple comparison test or (F, H–L, N–R) unpaired t-tests. (F, H–L, N–R) Mean ± SEM or (A–E) Mean + SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

    Article Snippet: To deplete CD8 + T cells, mice were treated with 100 μg of αCD8β (clone 53-5.8, Bio X cell) in 200 μL PBS i.p 4 days prior to IAV infection and on day 4 (and again on day 12 where required) post-infection.

    Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Comparison

    CXCR5 + CD8 + T cells promote class switching to IgG2c in responding B cells following IAV infection. WT or Cxcr5 -/- OT-I cells (or PBS only for no transfer controls) were transferred i.v. into separate B6. Ifng -/- mice that were then infected with x31-OVA i.n. (A) Schematic outline of the experiment. (B) Gating strategy used for identification of CD138 + B220 int ASCs and IgG2c-switching in ASCs in the mLN. Cells in the top panel were pre-gated on live, single cells. (C) Quantification of ASC number. (D) Frequency of IgG2c + ASCs among total ASCs. (E) Fold change in IgG2c-induction relative to the no transfer control (dashed line). (F) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells and IgG2c-switching in GCB cells in the mLN. Cells in the top panel were pre-gated on live, single, B220 + CD138 - cells. (G) Quantification of GCB cell number. (H) Frequency of IgG2c + GCB cells among total GCB cells. (I) Fold change in IgG2c-induction relative to the no transfer control (dashed line). (J) Levels of serum x31-specific IgG2c on day 8 post-infection assessed by ELISA. (C–E, G–I) Data are pooled from two independent experiments with a total of 7-9 mice group or (J) representative of two independent experiments with 4-5 mice per group. (C–E, G–I) Data were analysed by ordinary one-way ANOVA or (J) two-way ANOVA with Bonferroni’s multiple comparison test. (C–E, G–I) Mean ± SEM or (J) Mean + SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

    Journal: Frontiers in Immunology

    Article Title: CXCR5 + CD8 + T Cells Shape Antibody Responses In Vivo Following Protein Immunisation and Peripheral Viral Infection

    doi: 10.3389/fimmu.2021.626199

    Figure Lengend Snippet: CXCR5 + CD8 + T cells promote class switching to IgG2c in responding B cells following IAV infection. WT or Cxcr5 -/- OT-I cells (or PBS only for no transfer controls) were transferred i.v. into separate B6. Ifng -/- mice that were then infected with x31-OVA i.n. (A) Schematic outline of the experiment. (B) Gating strategy used for identification of CD138 + B220 int ASCs and IgG2c-switching in ASCs in the mLN. Cells in the top panel were pre-gated on live, single cells. (C) Quantification of ASC number. (D) Frequency of IgG2c + ASCs among total ASCs. (E) Fold change in IgG2c-induction relative to the no transfer control (dashed line). (F) Gating strategy used for identification of B220 + IgD - GL7 + GCB cells and IgG2c-switching in GCB cells in the mLN. Cells in the top panel were pre-gated on live, single, B220 + CD138 - cells. (G) Quantification of GCB cell number. (H) Frequency of IgG2c + GCB cells among total GCB cells. (I) Fold change in IgG2c-induction relative to the no transfer control (dashed line). (J) Levels of serum x31-specific IgG2c on day 8 post-infection assessed by ELISA. (C–E, G–I) Data are pooled from two independent experiments with a total of 7-9 mice group or (J) representative of two independent experiments with 4-5 mice per group. (C–E, G–I) Data were analysed by ordinary one-way ANOVA or (J) two-way ANOVA with Bonferroni’s multiple comparison test. (C–E, G–I) Mean ± SEM or (J) Mean + SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

    Article Snippet: To deplete CD8 + T cells, mice were treated with 100 μg of αCD8β (clone 53-5.8, Bio X cell) in 200 μL PBS i.p 4 days prior to IAV infection and on day 4 (and again on day 12 where required) post-infection.

    Techniques: Infection, Control, Enzyme-linked Immunosorbent Assay, Comparison

    (a) Schematic of co-transfer experiment during LCMV Clone 13 infections. Chimeric mice were generated using the CHIME system. (b) TIDE assay on naïve CD8 + T cells for a control and Ptpn2 -targeting sgRNA. Representative of four independent experiments, n = 1 mouse. (c) Representative flow cytometry plot of co-transferred control or Ptpn2 -deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of eight independent experiments, n ≥ 4 mice. (d-e) Frequency of CD45.1 + transferred cells (d) and number (e) of control or Ptpn2 -deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (f) Quantification of BrdU incorporation for co-transferred control and Ptpn2- deleted P14 T cells 8, 15, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 3 mice. (g) Representative flow cytometry plots of Granzyme B expression from splenic control or Ptpn2 -deleted P14 CD8 + T cells co-transferred at day 8 post LCMV Clone 13 infection as in (c). Representative of two independent experiments, n ≥ 4 mice. (h) Quantification of (g) days 8, 15, 22, and 30 post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (d, e, f, h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a) Schematic of co-transfer experiment during LCMV Clone 13 infections. Chimeric mice were generated using the CHIME system. (b) TIDE assay on naïve CD8 + T cells for a control and Ptpn2 -targeting sgRNA. Representative of four independent experiments, n = 1 mouse. (c) Representative flow cytometry plot of co-transferred control or Ptpn2 -deleted P14 T cells in the spleen 8 days post LCMV Clone 13 infection. Representative of eight independent experiments, n ≥ 4 mice. (d-e) Frequency of CD45.1 + transferred cells (d) and number (e) of control or Ptpn2 -deleted P14 T cells in the spleen 8, 15, 22, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. (f) Quantification of BrdU incorporation for co-transferred control and Ptpn2- deleted P14 T cells 8, 15, and 30 days post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 3 mice. (g) Representative flow cytometry plots of Granzyme B expression from splenic control or Ptpn2 -deleted P14 CD8 + T cells co-transferred at day 8 post LCMV Clone 13 infection as in (c). Representative of two independent experiments, n ≥ 4 mice. (h) Quantification of (g) days 8, 15, 22, and 30 post LCMV Clone 13 infection. Representative of two independent experiments, n ≥ 4 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (d, e, f, h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: Generated, Control, Flow Cytometry, Infection, BrdU Incorporation Assay, Expressing, Standard Deviation

    (a) tSNE projection of single-cell RNA-seq profiles from 7,027 control or Ptpn2 -deleted P14 + CD8 + T cells responding to day 30 LCMV Clone 13 infection. Clusters are distinct colors. Representative of one experiment, n = 4 pooled mice. (b) Enrichment of gene signatures in the clusters. Representative of one experiment, n = 4 pooled mice. (c) Plots depicting the inter-cluster density for control or Ptpn2 -deleted cells. Representative of one experiment, n = 4 pooled mice. (d) Quantification of the proportion of control or Ptpn2 -deleted cells in each cluster. Error bars represent the 95% confidence interval and the dotted line represents the proportion of Ptpn2- deleted cells among all cells projected. Representative of one experiment, n = 4 pooled mice. (e-f) Signature enrichments of co-transferred control and Ptpn2 -deleted cells from the (e) progenitor or (f) terminally exhausted clusters. Representative of one experiment, n = 4 pooled mice. (g-h) GSEA curves for significantly enriched signatures in (g) Slamf6 + cells and (h) Tim-3 + cells for bulk RNA-seq of co-transferred control and Ptpn2 -deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (i-j) Row-normalized heat map of effector-associated genes in (i) Slamf6 + cells and (j) Tim-3 + cells for bulk RNA-seq of co-transferred control and Ptpn2 -deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (k) Quantification of frequency of tumor cells killed when co-cultured with control or Ptpn2 -deleted CD8 + T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (l) Quantification of frequency of tumor cells killed when co-cultured with Tim-3 + control or Tim-3 + Ptpn2 -deleted CD8 + T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. Statistical significance was assessed by the two-sided Wilcoxon rank sum test (b, e, f), two-sided binomial test (d), two-sided Kolmogorov-Smirnov test (g-h), and two-sided Student’s paired t-test (k-l) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a) tSNE projection of single-cell RNA-seq profiles from 7,027 control or Ptpn2 -deleted P14 + CD8 + T cells responding to day 30 LCMV Clone 13 infection. Clusters are distinct colors. Representative of one experiment, n = 4 pooled mice. (b) Enrichment of gene signatures in the clusters. Representative of one experiment, n = 4 pooled mice. (c) Plots depicting the inter-cluster density for control or Ptpn2 -deleted cells. Representative of one experiment, n = 4 pooled mice. (d) Quantification of the proportion of control or Ptpn2 -deleted cells in each cluster. Error bars represent the 95% confidence interval and the dotted line represents the proportion of Ptpn2- deleted cells among all cells projected. Representative of one experiment, n = 4 pooled mice. (e-f) Signature enrichments of co-transferred control and Ptpn2 -deleted cells from the (e) progenitor or (f) terminally exhausted clusters. Representative of one experiment, n = 4 pooled mice. (g-h) GSEA curves for significantly enriched signatures in (g) Slamf6 + cells and (h) Tim-3 + cells for bulk RNA-seq of co-transferred control and Ptpn2 -deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (i-j) Row-normalized heat map of effector-associated genes in (i) Slamf6 + cells and (j) Tim-3 + cells for bulk RNA-seq of co-transferred control and Ptpn2 -deleted cells day 8 post LCMV Clone 13 infection. Representative of one experiment, n = 2 mice with n = 2 technical replicates per mouse. (k) Quantification of frequency of tumor cells killed when co-cultured with control or Ptpn2 -deleted CD8 + T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (l) Quantification of frequency of tumor cells killed when co-cultured with Tim-3 + control or Tim-3 + Ptpn2 -deleted CD8 + T cells (2:1 effector: target ratio) isolated from day 8 LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. Statistical significance was assessed by the two-sided Wilcoxon rank sum test (b, e, f), two-sided binomial test (d), two-sided Kolmogorov-Smirnov test (g-h), and two-sided Student’s paired t-test (k-l) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: RNA Sequencing, Control, Infection, Cell Culture, Isolation

    (a) Quantification of frequencies of co-transferred control or Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection. Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 5 mice. (b) Representative flow cytometry plots of Slamf6 and Tim-3 expression on splenic CD8 + T cells day 4 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells. Representative of two independent experiments, n = 5 mice. (c) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets in (b). Representative of two independent experiments, n = 5 mice. (d) Quantification of Granzyme B expression of cells as in (a). Representative of two independent experiments, n = 5 mice. (e) Schematic of in vitro conversion and CTV proliferation assays using co-transferred control or Ptpn2- deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. (f) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28 with IL-2 and IFN-α) of control or Ptpn2 -deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (g-h) Quantification of frequency of divisions following in vitro stimulation (αCD3/CD28 with IL-2) of (g) CTV-labeled Slamf6 + or (h) CTV-labeled Tim-3 + , control or Ptpn2 -deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 3 mice (g), 5 mice (h). (i) Schematic of in vivo persistence assay of Tim-3 + cells. (j) Quantification of number of recovered Tim-3 + control or Ptpn2- deleted cells in the spleen day 6 post LCMV Clone 13 infection, following the transfer of these cells which were previously isolated at day 8 post LCMV Clone 13 infection. Representative of two pooled experiments, n = 8 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (a, c-d, f-h) or two-sided Student’s unpaired t-test (j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a) Quantification of frequencies of co-transferred control or Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection. Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 5 mice. (b) Representative flow cytometry plots of Slamf6 and Tim-3 expression on splenic CD8 + T cells day 4 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells. Representative of two independent experiments, n = 5 mice. (c) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets in (b). Representative of two independent experiments, n = 5 mice. (d) Quantification of Granzyme B expression of cells as in (a). Representative of two independent experiments, n = 5 mice. (e) Schematic of in vitro conversion and CTV proliferation assays using co-transferred control or Ptpn2- deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. (f) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28 with IL-2 and IFN-α) of control or Ptpn2 -deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 5 mice. (g-h) Quantification of frequency of divisions following in vitro stimulation (αCD3/CD28 with IL-2) of (g) CTV-labeled Slamf6 + or (h) CTV-labeled Tim-3 + , control or Ptpn2 -deleted CD8 + T cells isolated at day 8 post LCMV Clone 13 infection. Representative of two independent experiments, n = 3 mice (g), 5 mice (h). (i) Schematic of in vivo persistence assay of Tim-3 + cells. (j) Quantification of number of recovered Tim-3 + control or Ptpn2- deleted cells in the spleen day 6 post LCMV Clone 13 infection, following the transfer of these cells which were previously isolated at day 8 post LCMV Clone 13 infection. Representative of two pooled experiments, n = 8 mice. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-sided Student’s paired t-test (a, c-d, f-h) or two-sided Student’s unpaired t-test (j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: Control, Infection, Flow Cytometry, Expressing, In Vitro, Isolation, Labeling, In Vivo, Standard Deviation

    (a-c) Quantification of (a) Slamf6 + Tim-3 – , (b) Slamf6 + Tim-3 + , and (c) Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28) of control or Ptpn2 -deleted naive CD8 + T cells in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (d) Quantification of pSTAT1 expression of splenic CD8 + T cells day 6 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells following ex vivo restimulation with IFN-α. Representative of two independent experiments, n = 5 biological replicates. (e-f) Quantification of pSTAT1 in (e) Slamf6 + or (f) Tim-3 + cells following ex vivo IFN-α restimulation of co-transferred control and Ptpn2 -deleted cells as in (d). Representative of two independent experiments, n = 5 biological replicates. (g) Quantification of frequencies of co-transferred control and Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFNAR blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 3 biological replicates. (h) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFNAR blocking antibody (right). Representative of two independent experiments, n = 5 biological replicates. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFN-γ neutralizing antibody (right). Representative of two independent experiments, n = 5 biological replicates. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a-c) or two-sided Student’s paired t-test (d-i) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a-c) Quantification of (a) Slamf6 + Tim-3 – , (b) Slamf6 + Tim-3 + , and (c) Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28) of control or Ptpn2 -deleted naive CD8 + T cells in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (d) Quantification of pSTAT1 expression of splenic CD8 + T cells day 6 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells following ex vivo restimulation with IFN-α. Representative of two independent experiments, n = 5 biological replicates. (e-f) Quantification of pSTAT1 in (e) Slamf6 + or (f) Tim-3 + cells following ex vivo IFN-α restimulation of co-transferred control and Ptpn2 -deleted cells as in (d). Representative of two independent experiments, n = 5 biological replicates. (g) Quantification of frequencies of co-transferred control and Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFNAR blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 3 biological replicates. (h) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFNAR blocking antibody (right). Representative of two independent experiments, n = 5 biological replicates. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFN-γ neutralizing antibody (right). Representative of two independent experiments, n = 5 biological replicates. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a-c) or two-sided Student’s paired t-test (d-i) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: In Vitro, Control, Blocking Assay, Expressing, Infection, Ex Vivo, Standard Deviation

    (a) Representative flow cytometry plot of control or Ptpn2 -deleted OT-1 T cells in the tumor 7 days post MC38-OVA injection. Representative of two independent experiments, n = 4 biological replicates. (b) Quantification of frequencies of co-transferred control or Ptpn2 -deleted CD8 + T cells day 7 post MC38-OVA injection. Frequencies at day 7 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 4 biological replicates. (c-d) (c) GSEA TIL Slamf6 + vs. Tim-3 + Up top 50 and TIL Slamf6 + vs. Tim-3 + Down top 50 signature enrichment and (d) GSEA effector signatures for co-transferred control or Ptpn2 -deleted OT-1 T cells in MC38-OVA tumors 7 days post injection. Representative of one experiment, n = 3 pooled mice and 2 technical replicates. (e) Quantification of Granzyme B expression in co-transferred OT-1 CD8 + T cells day 7 post MC38-OVA implantation in the tumor, draining lymph node, and spleen for control and Ptpn2 -deleted co-transferred mix as in (a). Representative of two independent experiments, n ≥ 3 mice. (f) Schematic of adoptive transfer of either control or Ptpn2- deleted naive OT-1 CD8 + T cells separately to mice challenged with B16-OVA one day post transfer of T cells. (g) Tumor growth curves for B16-OVA tumors following transfer of naive OT-1 control or Ptpn2- deleted CD8 + T cells separately into WT recipients that were implanted with B16-OVA cells. Representative of two independent experiments, n = 8 mice. (h) Survival curves of mice in (g). Representative of two independent experiments, n = 8 mice. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 9 post B16-OVA implantation in the tumor for co-transferred control and Ptpn2 -deleted cells as in (a). Representative of two independent experiments, n = 7 mice. Bar graphs represent mean and error bars represent standard deviation (except for g where error bars represent standard error). Statistical significance was assessed by two-sided Student’s paired t-test (b, e, i), two-sided Kolmogorov-Smirnov test (c, d), two-way ANOVA (g), or two-sided log-rank Mantel-Cox test (h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a) Representative flow cytometry plot of control or Ptpn2 -deleted OT-1 T cells in the tumor 7 days post MC38-OVA injection. Representative of two independent experiments, n = 4 biological replicates. (b) Quantification of frequencies of co-transferred control or Ptpn2 -deleted CD8 + T cells day 7 post MC38-OVA injection. Frequencies at day 7 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 4 biological replicates. (c-d) (c) GSEA TIL Slamf6 + vs. Tim-3 + Up top 50 and TIL Slamf6 + vs. Tim-3 + Down top 50 signature enrichment and (d) GSEA effector signatures for co-transferred control or Ptpn2 -deleted OT-1 T cells in MC38-OVA tumors 7 days post injection. Representative of one experiment, n = 3 pooled mice and 2 technical replicates. (e) Quantification of Granzyme B expression in co-transferred OT-1 CD8 + T cells day 7 post MC38-OVA implantation in the tumor, draining lymph node, and spleen for control and Ptpn2 -deleted co-transferred mix as in (a). Representative of two independent experiments, n ≥ 3 mice. (f) Schematic of adoptive transfer of either control or Ptpn2- deleted naive OT-1 CD8 + T cells separately to mice challenged with B16-OVA one day post transfer of T cells. (g) Tumor growth curves for B16-OVA tumors following transfer of naive OT-1 control or Ptpn2- deleted CD8 + T cells separately into WT recipients that were implanted with B16-OVA cells. Representative of two independent experiments, n = 8 mice. (h) Survival curves of mice in (g). Representative of two independent experiments, n = 8 mice. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 9 post B16-OVA implantation in the tumor for co-transferred control and Ptpn2 -deleted cells as in (a). Representative of two independent experiments, n = 7 mice. Bar graphs represent mean and error bars represent standard deviation (except for g where error bars represent standard error). Statistical significance was assessed by two-sided Student’s paired t-test (b, e, i), two-sided Kolmogorov-Smirnov test (c, d), two-way ANOVA (g), or two-sided log-rank Mantel-Cox test (h) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: Flow Cytometry, Control, Injection, Expressing, Adoptive Transfer Assay, Standard Deviation

    (a) Schematic for MC38-WT tumor challenge in chimeric mice where approximately 50% of immune cells express a control sgRNA or a Ptpn2- targeting sgRNA. (b) Tumor growth curves for control or Ptpn2 -deleted chimeric mice following 1 × 10 6 cell MC38-WT challenge. Representative of two independent experiments, n ≥ 8 mice. (c-d) Quantification of (c) frequency and (d) number of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets in CD8 + T cells infiltrating day 9 MC38 tumors in control or Ptpn2 -deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (e-f) Quantification of (e) frequency and (f) number of Granzyme B-expressing CD8 + T cells infiltrating day 9 MC38 tumors implanted in control or Ptpn2 -deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (g) Quantification of frequency of Granzyme B + CD8 + T cells from the blood of control or Ptpn2 -deleted bone marrow chimeras day 14 post MC38 tumor implantation, pregated on CD8β + Vex + cells. Representative of two independent experiments, n = 5 mice. (h) Tumor growth curves for mice as in (a) challenged with 1 × 10 6 MC38-WT tumor cells following treatment with CD8-depleting antibody or isotype control. Representative of two independent experiments, n ≥ 9 mice. (i) Tumor growth curves for control or Ptpn2 -deleted bone marrow chimeras challenged with 1 × 10 6 B16 tumor cells treated with GVAX (green triangles) on days 1, 4 and αPD-1 (black triangles) on days 12, 14, 16, 18, 20, 22, 24, and 26. Representative of two independent experiments, n ≥ 9 mice. (j) Quantification of frequency of Granzyme B + CD8 + T cells from the blood of chimeras in (i) day 14 post B16 tumor implantation, pregated on CD8β + Vex + cells. . Representative of two independent experiments, n = 5 mice. Bar graphs represent mean and error bars represent standard deviation (except for b, h, i where error bars represent standard error). Statistical significance was assessed by two-way ANOVA (b-d, h-i), or two-sided Student’s unpaired t-test (e-g, j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Journal: Nature immunology

    Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

    doi: 10.1038/s41590-019-0480-4

    Figure Lengend Snippet: (a) Schematic for MC38-WT tumor challenge in chimeric mice where approximately 50% of immune cells express a control sgRNA or a Ptpn2- targeting sgRNA. (b) Tumor growth curves for control or Ptpn2 -deleted chimeric mice following 1 × 10 6 cell MC38-WT challenge. Representative of two independent experiments, n ≥ 8 mice. (c-d) Quantification of (c) frequency and (d) number of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets in CD8 + T cells infiltrating day 9 MC38 tumors in control or Ptpn2 -deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (e-f) Quantification of (e) frequency and (f) number of Granzyme B-expressing CD8 + T cells infiltrating day 9 MC38 tumors implanted in control or Ptpn2 -deleted bone marrow chimeras. Representative of two independent experiments, n ≥ 9 mice. (g) Quantification of frequency of Granzyme B + CD8 + T cells from the blood of control or Ptpn2 -deleted bone marrow chimeras day 14 post MC38 tumor implantation, pregated on CD8β + Vex + cells. Representative of two independent experiments, n = 5 mice. (h) Tumor growth curves for mice as in (a) challenged with 1 × 10 6 MC38-WT tumor cells following treatment with CD8-depleting antibody or isotype control. Representative of two independent experiments, n ≥ 9 mice. (i) Tumor growth curves for control or Ptpn2 -deleted bone marrow chimeras challenged with 1 × 10 6 B16 tumor cells treated with GVAX (green triangles) on days 1, 4 and αPD-1 (black triangles) on days 12, 14, 16, 18, 20, 22, 24, and 26. Representative of two independent experiments, n ≥ 9 mice. (j) Quantification of frequency of Granzyme B + CD8 + T cells from the blood of chimeras in (i) day 14 post B16 tumor implantation, pregated on CD8β + Vex + cells. . Representative of two independent experiments, n = 5 mice. Bar graphs represent mean and error bars represent standard deviation (except for b, h, i where error bars represent standard error). Statistical significance was assessed by two-way ANOVA (b-d, h-i), or two-sided Student’s unpaired t-test (e-g, j) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

    Article Snippet: To deplete CD8 + T cells, mice were injected i.p. with 100 μg αCD8β (53–5.8 BioXCell Cat# BE0223) or isotype (HRPN BioXCell Cat# BE0088) on days −3, 0, 3, 6, 9 and 200 μg αCD8α (2.43 BioXCell Cat# BE0061) or isotype (LTF-2 BioXCell Cat# BE0090) on days 12, 15, 18, 21, 24 (relative to MC38 tumor injection on day 0).

    Techniques: Control, Expressing, Tumor Implantation, Standard Deviation