1. MP cells consist of rapidly proliferating CD5hi and relatively quiescent CD5lo cells, both of which require CD28 signaling for Ki67 expression.
MP cells consist of rapidly proliferating CD5hi and relatively quiescent CD5lo cells, both of which require CD28 signaling for Ki67 expression. (A and B) MP cells are composed of proliferating CD5hi and relatively quiescent CD5lo cells in both SPF and GF mice. (A) Representative dot plots of CD5 and Ki67 expression on splenic MP cells. (B) Histograms of CD5 expression by MP cells used to define 10 bins. The graph depicts the frequency (mean ± SD) of Ki67+ fraction among MP cells of each CD5 bin (n = 3 to 4). Data are representative of two independent experiments. (C and D) CD5hi MP cells die and proliferate more rapidly than do CD5lo MP cells. (C) Splenic CD5hi and CD5lo MP and naïve cells were analyzed for BrdU incorporation and 7-AAD staining 1 hour after in vivo BrdU pulse of wild-type mice. The numbers in the dot plots show the frequency of BrdU+ (S phase) and BrdU− 7-AAD+ (G2-M phase) cells among each CD4+ T cell subpopulation, whereas the bar graph indicates the frequency (mean ± SD) of cells in S and G2-M phase (n = 3). (D) Splenic MP and naïve cells were stained with amine-reactive dye and annexin V. Dot plots indicate the frequency of amine-reactive dye+ annexin V+ (dead) and amine-reactive dye− annexin V+ (early apoptotic) cells among each CD4+ T cell subpopulation, whereas the bar graph shows the frequency (mean ± SD) of dead and early apoptotic cells (n = 3). (E) CD5 expression is maintained in MP conversion. CD5lo and CD5hi naïve CD4+ T cells from CD45.1 (blue line) and CD45.2 (red line) mice, respectively, were mixed at a 1:1 ratio and transferred into 1-day-old neonatal and 8-week-old adult CD45.1/2 mice, and 3 weeks later, their CD5 expression was reanalyzed. Representative histograms show CD5 expression by naïve and MP donor cells and were overlaid with respective host populations shown in gray. Data are representative of four to eight animals. (F) Short-term MP cell proliferation requires CD28 but not TCR signaling. Wild-type mice were injected intraperitoneally with anti–I-Aβ, CTLA4-Ig, or control IgG on days 0 and 2, and splenic MP cells were analyzed on day 4. The graph indicates the frequency (mean ± SD) of Ki67+ cells among MP cells in each CD5 bin (n = 3 to 4). Data are representative of two independent experiments. (G) CD28 is required for optimal Ki67 expression on MP cells. The graph shows the frequency (mean ± SD) of Ki67+ population among MP cells from wild-type and CD28 KO mice (n = 3). *P < 0.05, **P < 0.01, ***P <
Takeshi Kawabe et al. Sci. Immunol. 2017;2:eaam9304
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