IFN-γ+ CD4 memory T cells maintain CXCR5 expression, but little T-bet.

BAC-In mice were infected and splenocytes were analyzed by flow cytometry on day 60 post-infection. (A) CD4+Ifng/Thy1.1+ memory (CD44hiCD127+, green box, gate set on CD4+) T cells were gated and expression of CXCR3, T-bet, CXCR5, and Bcl6 was measured. Numbers represent mean percentages. (B, C) Surface staining of CD4+ T cells with CD4, CD44, CD127, and Thy1.1 (yellow) was followed by intracellular staining with Bcl-6 (green), T-bet (red), and nuclei with DAPI (purple). Cells were analyzed by imaging flow cytometry. (B) Histogram showing expression of Ifng/Thy1.1 on single focused cells. Bar graphs show MFI of Bcl6 and T-bet within Thy1.1 gates (C) Representative images of individual Ifng/Thy1.1 high and low cells showing Bcl6 and T-bet expression and localization in relation to the DAPI stained nucleus. Bright field (BF; left) and DAPI (nucleus)/Bcl6/T-bet merged images also shown (right). (D) Real time PCR analysis of bcl6, tbx21, prdm1, and eomes of CD4+Thy1.1+ sorted T cells. Results were normalized to control gene rp18s. RNA from FACS Sorted naïve (CD44loCD25-) cells from uninfected, aged-matched BAC-In mice was used as control. Data are representative of three (A) and one (B, C, D) independent experiments with 3–4 animals per group. Statistical significance shown using Students t-test. Error bars represent the SEM; ★p ★★p