αcd8β antibody (clone 53-5.8)  (Bio X Cell)

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

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

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

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

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