A major role for Bim in regulatory T cell homeostasis

Abstract

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4(+) T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bim(lo) Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

Figure 1. T_reg accumulate in aged mice and exhibit increased survival in vitro

(A) The percentages of FoxP3⁺CD4⁺ T cells were determined in the spleens and peripheral LNs of 7 young (1.5-month old) and 6 old (22-month old) C57Bl/6 mice. Lines represent means. Statistical differences were evaluated using unpaired t-tests. (B) Thymocytes from 7 young (1.5-month old) and 6 old (22-month old) mice were analyzed by flow cytometry. Cellularity was assessed for each thymus. The percentage of FoxP3⁺ cells was analyzed in CD4⁺ SP thymocytes. Lines represent medians. Statistical differences were evaluated using Mann-Whitney tests. (C). Naïve (CD62L⁺CD44⁻GFP⁻CD4⁺), effector memory (EM, CD62L⁻CD44⁺GFP⁻CD4⁺) and central memory (CM, CD62L⁺CD44⁺GFP⁻CD4⁺) non-T_reg were sorted from a pool of spleen and pLN from young (2-month old, N=3, full line) and middle-aged (12-month old, N=4, dotted line) FoxP3-IRES-GFP knock-in Balb/c mice. 5×10⁵ naïve, EM or CM cells were stimulated with immobilized anti-CD3 Ab and soluble anti-CD28, in presence of IL-2 and TGF-β1. GFP expression was analyzed by flow cytometry after 5 days of culture in all cell populations. (D) Young (1.5-month old) and old (18-month old) mice were injected with BrdU every day for 3 days. The percentage of BrdU⁺ cells was analyzed in gated splenic T_reg (CD4⁺FoxP3+). Values shown are mean % (± SE) of BrdU⁺ cells in each group. (E) LN cells were harvested from 12 young (1.5-month old) and 11 old (20-month old) mice and cultured in S-MEM media containing 10% FBS, in the absence of exogenous cytokines. After 24 hrs in culture, cells were stained for CD4, CD25 and Propidium Iodide (PI). The percentage of dead cells (PI⁺) was analyzed in gated non-T_reg (CD4⁺CD25⁻) and T_reg (CD4⁺CD25⁺). Values shown are mean % (± SE) of PI⁺ cells in each group. p values represent the difference between old and young mice (unpaired t-tests).

Figure 2. Bim expression is decreased in T_reg from old and middle-age mice

(A) Spleen cells from either BimKO or C57BL/6 (WT) mice were stained for CD4, FoxP3 and intracellularly for Bim, and analyzed by flow cytometry. Histogram shows the fluorescence signal for Bim in BimKO (light line) and WT (dark line) mice. (B, C) Spleen cells from 12 young (1.5-month old) and 11 old (20-month old) mice were stained for CD4, FoxP3 and Bim, and analyzed by flow cytometry. Bim MFI was analyzed in gated T_reg (CD4⁺FoxP3⁺) versus gated non-T_reg (CD4⁺FoxP3⁻CD44^hi). Representative Bim staining in (A) T_reg and (B) non-T_reg in young (thin lines) and old (thick lines) mice. (C) Mean (± SE) Bim MFI in T_reg and non-T_reg. p values represent differences in Bim expression in young versus old mice, in each cell subset (unpaired t-tests). (D) Spleen cells from 3 young (6–8 wk old) and 3 middle-aged (10-month old) mice were stained for CD4, FoxP3, and Bim. Bim expression in gated T_reg (CD4⁺FoxP3⁺) is shown in representative animals. The p value represents the difference between young and middle-aged mice (unpaired t-test). Results are representative of two independent experiments.±

Figure 3. Increased frequency of FoxP3⁺ T cells in middle-aged Bim KO mice

(A, B, C) Groups of 2 month and 6 month old C57BL/6 (N=9 total; white symbols) and Bim KO mice (N==11 total; black symbols), were injected i.p. with BrdU for 6 days. Results show the mean (±SE) (A) frequency and total numbers of (B) CD4⁺ FoxP3⁺ vs (C) CD4+FoxP3⁻ T cells in the spleen. p-values represent the differences between WT and Bim KO mice (unpaired t-test). (D,E) Groups of Rag-2p-GFP transgenic mice (N=3) and Bim KO-Rag-2-GFP (N=3) mice were bled every 2 weeks. Results show the mean (±SE) percent of CD4⁺ FoxP3⁺ cells that were GFP+ from Rag-2GFP or Bim KO-Rag-2-GFP in the peripheral blood. (E) Mice were sacrificed at 17 weeks of age. Results show the percent (±SE) of GFP+ versus GFP− CD4+ LN T cells that were FoxP3⁺. (F) Results show the percent (±SE) of FoxP3⁺ cells that are BrdU⁺ in the spleens of Bim KO versus C57BL/6 mice after in vivo treatment with BrdU.

Figure 4. CD127 expression is increased and functional on T_reg from aged mice but is not required for their survival

Spleen cells from 7 young (1.5-month old) and 6 old (22-month old) mice were stained with antibodies against CD4, FoxP3, CD127, and CD25 and analyzed by flow cytometry. (A) Mean % (± SE) of CD127⁺ cells in gated T_reg (CD4⁺FoxP3⁺) are shown. p-value denotes significant difference between young and old mice (student’s t-tests). (B, C) Old (18-month old, n=5) mice were treated with IL-7 IC every other day for one week. Results show the mean (± SE) absolute numbers of CD4⁺FoxP3⁻ and CD4⁺FoxP3⁺ cells. Spleen cells were harvested and stained with antibodies against CD4, FoxP3 and Bim and analyzed by flow cytometry. (C) Results show the mean fluorescence intensity of the Bim stain in CD4+ FoxP3-vs CD4+FoxP3+ cells from PBS (open bars) vs IL-7-treated (filled bars) mice. p-values represent the difference between untreated and IL-7 IC-treated mice (student’s t-test). Results are representative of 2 independent experiments. (D, E) Groups of 2 month (N=4) or 12 month old (N=4) C57BL/6 mice were injected with either anti-IL7R (black bars) or control antibody (ctl Ab; white bars). Results show the mean (±SE) total numbers of splenic (D) CD4⁺FoxP3⁺ and (E) CD4⁺FoxP3⁻. p-values represent the difference between control and anti-IL-7R-treated mice (student’s t-test).

Figure 5. Proliferation and accrual of Bim^lo T_reg

(A). Young BL/6 mice were injected with BrdU for 3 days, sacrificed and spleen cells analyzed for Bim expression and BrdU incoporation by intracellular flow cytometric staining. Results show the mean fluorescence intensity of the Bim signal in either CD4+FoxP3+ or CD4+FoxP3− cells that were either BrdU− (open bars) or BrdU+ (closed bars) (± SE) (B–D) Young BL/6 mice (N = 3/group) were injected i.p., with either 3×10⁴ U hIL-2 or PBS twice daily for 4 days. Results show the mean (±SE) absolute number of (B) CD4⁺ FoxP3⁺ T cells; (C) CD4⁺FoxP3⁻ T cells; or (D) CD4⁺FoxP3⁺ T cells that are either Bim^lo or Bim^hi. (E, F) Groups of 8 week old C57BL/6 mice were injected twice daily with either PBS or IL-2 for 3 days and on the day before sacrifice were injected twice with 0.6mg BrdU. (E) Results show percent (±SE) of FoxP3+ versus FoxP3− cells that are BrdU+ (F). Results show the percent (±SE) of BrdU⁻ or BrdU⁺ cells that were Bim^lo in either FoxP3⁻ (open bars) versus FoxP3⁺ (closed bars) T cells in IL-2 treated mice. (G). Groups of BL/6 and BimKO mice (n=4 mice/group/timepoint) were injected with IL-2 twice daily for three days and with BrdU the last 36 hours of IL-2 treatment. Groups of BL/6 and BimKO mice were sacrificed either one or seven days after the last IL-2/BrdU injection. Results show the percentage of FoxP3+BrdU+ cells (± SE) remaining on day 7 relative to the amount observed on day 1 after cessation of IL-2/BrdU treatment.