Fig. 7

CD40L/CD40 and TCR/MHC I interactions required for CD8⁺ Tfh function. a CD40L expression in CXCR5⁺PD-1⁺CD8⁺ T cells. Splenocytes from LCMV-infected wild-type mice were stained with anti-CD4, anti-CD8, anti-CXCR5, anti-PD-1 and anti-CD40L antibodies. Numbers indicate percentages of CD40L⁺ cells (left) and bar graphs show the average MFI of CD40L expression in the indicated CD8⁺ populations (right). b CD40L expression in CXCR5⁺PD-1⁺CD8⁺ T cells following activation. Splenocytes from LCMV-infected wild-type mice were stimulated with PMA/ionomycin and stained as in a. Numbers indicate percentages of CD40L⁺ cells (left) and bar graphs show the average MFI of CD40L expression in the indicated CD8⁺ populations (right). c, d Requirement of CD40/CD40L interaction for CXCR5⁺PD-1⁺CD8⁺ T cells to help B cells. B cells sorted from LCMV-infected wild-type or CD40-deficient mice were cultured without T cells or with CXCR5⁺PD-1⁺CD8⁺ or CD4⁺ T cells sorted from LCMV-infected wild-type mice (c). Wild-type B cells were cultured with CXCR5⁺PD-1⁺CD8⁺ or CD4⁺ T cells sorted from LCMV-infected wild-type mice with or without anti-CD40L neutralizing antibodies (d). Bar graphs show the IgG1 levels in the supernatant of the indicated co-cultures. e, f Requirement of TCR/MHC I interaction for CXCR5⁺PD-1⁺CD8⁺ T cells to help B cells. B cells sorted from LCMV-infected C57BL/6J or bm1 mice were cultured without T cells or with CXCR5⁺PD-1⁺CD8⁺ or CD4⁺ T cells sorted from LCMV-infected C57BL/6J mice and the inset shows the zoomed area (e). Bar graphs show the IgG1 levels in the supernatant of the indicated co-cultures. CD8⁺ T cells sorted from LCMV-infected wild-type C57BL/6J or bm1 mice were adoptively transferred into Rag1-deficient mice along with B cells from LCMV-infected bm1 mice (f). The recipients were infected with LCMV and IgG production was measured 30 days later. Dot plots show the IgG1 levels in the sera of recipients adoptively transferred with the indicated cells. Bars represent the mean values and each dot represents an individual data value (a–e) or recipient (f) and horizontal bars indicate mean values. Mean ± SD is shown. P-values were calculated with the unpaired two-tailed Student’s t-test. Data shown are representative of two (a–f) independent experiments. Source data are available as a source data file