Extended Data Figure 6. Characterization of Cd40lgSrtA/Y T cells.
a, Upregulation of CD86 on B cells by CD40L-SrtA. B1-8hi λ+ B cells were pulsed with the indicated concentrations of NP-OVA and co-cultured with Cd40lgSrtA/Y CD4-Cre OT-II or WT OT-II CD4+ T cells for 18 hours. Cells were analyzed by flow cytometry. Graph shows percentage of CD86+ B cells when co-cultured with Cd40lgSrtA/Y CD4-Cre OT-II or Wt OT-II T cells in the presence of varying concentrations of NP-OVA. b, Germinal center formation in Cd40lgSrtA/Y and Cd40lgSrtA/Y CD4-Cre mice. C57BL/6J, Cd40lg+/Y and Cd40lgSrtA/Y CD4-Cre mice were immunized subcutaneously with 20 μg of NP-OVA in alum at the base of the tail. Inguinal LNs were analyzed by flow cytometry 12 days post-immunization. Dot plots show the absence of germinal center formation in both Cd40lgSrtA/Y and Cd40lgSrtA/Y CD4-Cre mice, suggesting impaired ability of Cd40lgSrtA/Y T cells to activate B cells. A similar phenotype is observed regardless of the presence of Cre recombination, likely due to the addition of a translated LoxP site to the C-terminus of the CD40L protein. c, In vivo expansion of Cd40lgSrtA/Y CD4-Cre OT-II CD4+ T cells. 5 × 105 Cd40G5/G5 DCs pulsed ex vivo with OVA323-339 were injected subcutaneously into the hind footpad of C57BL/6J recipients. Eighteen hours later, 3 × 105 CFSE labeled CD40lgSrtA/Y CD4-Cre OT-II (or Cd40lg+/Y OT-II as control) CD4+ T cells were transferred intravenously. Popliteal LNs were analyzed by flow cytometry 72 hours after T cell transfer. Histogram plots show comparable expansion of both transferred T cell populations, as indicated by CFSE dilution. Data representative of two independent experiments.