(A and B) Generation of memory CD4+ T cells. WT and IL-6RαT-KO mice, with or without Treg depletion, were immunized once with 2W peptide and LPS in IFA. The frequency of all antigen-specific 2W:I-Ab+ CD4+ memory T cells (A) or individual subsets of 2W:I-Ab+ CD4+ memory T cells (B) in the draining lymph nodes was measured 60 days later by flow cytometry. 2W:I-Ab+ CXCR5−PD-1− represent Th1 effector memory cells, CXCR5intPD-1− represent Th1 central memory cells, and CXCR5hi PD-1+ represent Tfh cells. Data are representative of at least three independent experiments. (C and D) Expansion and cytokine secretion of memory CD4+ T cells. WT and IL-6RαT-KO mice, with or without Treg depletion, were immunized with OVA and LPS in IFA. 60 days later, mice that were previously depleted of Tregs were depleted for a second time prior to re-immunization with OVA and LPS in IFA. 7 days after the second immunization, purified CD4+ T cells were restimulated and proliferation (C) and cytokine production (D) were measured by [3H]-thymidine incorporation and ELISA, respectively. Line graph and bar graph represent mean ± SEM.