Figure 2.

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.