Figure 4

Effect of DTA-mediated deletion of memory and regulatory CD4⁺ T cells on CD4⁺ T cell homeostasis. (a) Naïve, (b) memory and (c) regulatory CD4⁺ T cell BrdU incorporation during a 6-day administration period, and expression of Ki67 nuclear antigen. Numbers within the plots denote the percentage of positive CD4⁺ T cells and are representative of 4-6 mice per group. P = 0.011 for Ki67 staining in memory CD4⁺ T cells; P = 0.006 for BrdU incorporation and P = 0.0004 for Ki67 staining in regulatory CD4⁺ T cells. (d) Loss of BrdU⁺ naïve, memory or regulatory CD4⁺ T cells 3 days after cessation of a 6-day BrdU administration period. Values are the percentage (± SEM) of BrdU⁺ cells in each subset on day 3 after cessation of BrdU administration minus the percentage of BrdU⁺ cells at the peak (day 6 of the administration period), and are representative of three mice per group. (e) CD45.2⁺Tnfrsf4^Cre/+ R26^Dta/+ (DTA) and CD45.1⁺C57BL/6 (B6) bone marrow (BM) cells were injected separately (single) or mixed together at 1:1 ratio (mixed) into non-irradiated Rag1^-/- recipients and lymphoid organs were analyzed 12 weeks later. CD25 and CD44 expression in gated DTA BM-origin or B6 BM-origin CD4⁺ T cells in these recipients is shown. Numbers within the plots denote the percentage of positive CD4⁺ T cells and are representative of 4-8 mice per group analyzed in two independent experiments. (f-h) 1 × 10⁶ purified CD4⁺ T cells from Tnfrsf4^Cre/+R26^Yfp/+ (YFP/+) or Tnfrsf4^Cre/+R26^Yfp/Dta(YFP/DTA) mice were adoptively transferred into Rag1^-/- recipients and followed over time. (f) Percentage of YFP⁺ cells in CD4⁺ T cells in the blood. (g) Percentage of CD4⁺ T cells in blood mononuclear cells. (h) Total numbers of naïve, memory and regulatory (reg) CD4⁺ T cells in lymphoid organs at the end of the 7-week observation period. P < 0.0001 for memory CD4⁺ T cells; P = 0.006 for regulatory CD4⁺ T cells. Values in (f-h) are the means (± SEM) of five mice per group. Numbers within bars in (h) denote the absolute number, ×10^-6, of each cell type. (i) 5 × 10⁶ purified naïve (CD44^loCD25^-) CD45.1⁺ CFSE-labeled wild-type CD4⁺ T cells were adoptively transferred into Tnfrsf4^Cre/+R26^Dta/+ (DTA host) and control Tnfrsf4^Cre/+R26^+/+ (WT host) recipient mice. CFSE dilution and CD44 expression on gated CD45.1⁺ donor CD4⁺ T cells isolated from the spleens and lymph nodes of recipients 6 days after transfer are shown. Numbers within the plots denote the percentage of CFSE^-CD44^hiCD4⁺ T cells and are representative of three mice per group.