Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells

Abstract

Defects in the Bcl-2-regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25(low)Foxp3(+) cells in the thymus and peripheral lymph tissues. The increased CD25(low)Foxp3(+) population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen-specific Foxp3(+)Vβ5(+)CD4(+) T cells in BALB/c Bim(-/-) mice compared with control animals. The survival rate of the expanded CD25(low)Foxp3(+) cells was similar to that of CD25(high)Foxp3(+) CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25(low)Foxp3(+)CD4(+) T cells in vitro and in vivo. The expanded CD25(low)Foxp3(+)CD4(+) T cells from Bim(-/-) mice were anergic but also had weaker regulatory function than CD25(high)Foxp3(+) CD4(+) T cells from the same mice. Analysis of Bim(-/-) mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapes from thymic deletion in mice defective in the Bcl-2-regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-Ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4(+) T cells and thereby safeguard immunological tolerance.

Fig. 1. Inhibition of the ‘Bcl-2 regulated’ apoptotic pathway causes a marked increase in CD25^lowFoxP3⁺CD4⁺ SP thymocytes

Thymi were harvested from 6-10 week old mice. Thymocytes were first surface stained for CD4, CD8 and CD25 and then for intracellular FoxP3. (a) Comparison between Bim-/- and WT mice: (i) CD4 and CD8 expression on total thymocytes. (ii) Expression of CD25 and FoxP3 gated on CD4 SP thymocytes. (iii) Bar graphs show the mean numbers +/- SD of the indicated thymic sub-populations. Nine independent experiments were performed with similar results. (iv) Bar graphs show the mean numbers +/- SD of the indicated thymic sub-populations from the indicated genotypes of mice. Two independent experiments were performed with similar results. (b) Comparison between vavBcl-2 Tg and WT mice: (i) CD4 and CD8 expression on total thymocytes. (ii) Expression of CD25 and FoxP3 gated on CD4 SP thymocytes. (iii) Bar graphs show the mean numbers +/− SD of the indicated thymic sub-populations. Three independent experiments were performed. (c) Comparison between Bax−/−Bak−/− and control (WT) reconstituted mice: (i) CD4 and CD8 expression on total thymocytes from RAG-1−/− mice reconstituted with fetal liver cells from WT or Bax−/−Bak−/− mice. (ii) Expression of CD25 and FoxP3 gated on CD4 SP thymocytes. (iii) Bar graphs show the mean numbers +/−SD of the indicated thymic sub-populations. Three independent experiments were performed. Numbers in dot plots show the percentages of corresponding populations. Numbers in bar graphs indicate the fold-increase of a given population in the indicated mice compared to that in control WT animals. *P<0.05 compared to WT control.

Fig. 2. Inhibition of the ‘Bcl-2-regulated’ apoptotic pathway causes a marked increase in CD25^lowFoxP3^GFP+CD4⁺ thymocytes

(a) Thymocytes from 6–8 week old FoxP3GFP^KI or FoxP3GFP^KI/Bim−/− mice were surface stained for CD4, CD8, TCRβ and CD25. (i) TCRβ expression on total thymocytes; (ii) CD4 and CD8 expression gated on TCRβ⁺ thymocytes; (iii) CD25 and FoxP3^GFP expression gated on TCRβ⁺CD4⁺ SP thymocytes. Bar graphs show cell numbers +/−SD of the indicated thymic cell populations from 3 mice. NS, not significant; *P<0.05 compared to WT; **P<0.01 compared to WT. Four independent experiments were performed. (b) Thymocytes from 10 week-old FoxP3GFP^KI or FoxP3GFP^KI/vavBcl2 Tg mice were stained as in (a). Dot plots show expression of CD25 vs FoxP3 on gated CD4 SP thymocytes. Three independent experiments yielded similar results.

Fig. 3. Loss of Bim causes a selective increase in self-superantigen specific TCR Vβ5⁺ FoxP3⁺CD4⁺ thymocytes on the BALB/c background

Thymocytes from 3 individual 6–7 week old mice of the indicated genotypes were first surface stained for CD4, CD8, CD25 and individual TCR Vβ chains and then fixed, permeabilized and finally stained for intracellular FoxP3. (a) Percentages of subpopulations of CD4 SP thymocytes: [left panel] Mean +/− SEM of the percentages of CD4 SP thymocytes bearing the indicated TCR Vβ chain within total thymocytes. ***P<0.001 compared to WT mice. [right panel] Mean +/−SEM of the percentages of FoxP3⁺CD4⁺ cells within CD4⁺ SP thymocytes bearing the indicated TCR Vβ chain. *P<0.05, ***P<0.001 compared to WT mice. (b) Numbers of cells within the different sub-populations of CD4SP thymocytes: Mean +/− SD of the numbers of total thymocytes [left panel] and Mean +/− SEM of FoxP3⁺CD4 SP thymocytes bearing the indicated TCR Vβ chain [right panel]. *** P<0.001 compared to WT mice. (c) Usage of different vβ TCRs by FoxP3⁺ and FoxP3^- CD4 SP thymocytes from Bim−/− mice: Bar graph shows mean +/−SEM of the percentages of FoxP3⁺ and FoxP3⁻ CD4 SP thymocytes carrying different TCRvβ chains from 3 individual Bim−/−mice. Three experiments were performed with similar results.

Fig 4. Survival of CD25^lowFoxP3⁺CD4⁺ T cells is similar to that of CD25^highFoxP3⁺CD4⁺ T cells in vivo after adoptive transfer

2x10⁵ CellTrace-labeled CD4⁺ T cells were injected iv into C57BL/6-Ly5.1 recipient mice. 3 and 7 days after transfer, spleens were recovered. Transferred cells were identified as Ly5.2⁺CellTrace⁺ cells. (a) Dot plots show the expression of CD25 and FoxP3-GFP by CD4⁺ T cell sub-populations before and after transfer. (b) Bar graphs show the numbers of transferred cells (mean of 2–3 mice per group). (c) Histograms show Celltrace intensity of CD4⁺ T cell subpopulations. Two independent experiments were performed with similar results.

Fig. 5. CD25^lowFoxP3⁺ cells are hypo-responsive to TCR stimulation

(a) T cell proliferation: Distinct CD4⁺ T cell subpopulations from lymph nodes of FoxP3GFP^KI or FoxP3GFP^KI/vavBcl-2 Tg mice were sorted according to the levels of FoxP3-GFP and CD25 expression. These T cell subsets (2x10⁴ cells per well) were cultured in 96-well round-bottomed plates with 5 μg/mL anti-CD3 plus 2 μg/mL anti-CD28 Abs in the presence of 8x10⁴γ-irradiated and T cell depleted spleen cells (used as antigen presenting cells). The mean cpm +/− SD from 3–5 replicates per culture condition is shown. **P<0.01 compared to GFP^-CD25⁻CD4⁺ lymph node cells. Three experiments were performed with similar results. (b) Cytokine production: supernatants from the above cultures harvested after 3 days of incubation were examined for cytokine content. Data represent the mean +/− SEM from three replicate cultures per cell type and genotype. Three independent experiments were performed and produced similar results.

Fig. 6. CD25^lowFoxP3⁺CD4⁺ T cells are less potent suppressors of effector T cell activation compared to CD25^highFoxp3⁺CD4⁺ T cells

Suppression of effector T cell proliferation: CD25^lowFoxP3⁺CD4⁺ and CD25^highFoxP3⁺CD4⁺ cells (2x10⁴ cells) from lymph nodes of mice of the indicated genotypes were cultured with 2x10⁴ cells CFSE-labeled C57BL/6-Ly5.1 CD25^-CD4⁺ T cells as effectors in 200 μL medium and left untreated or stimulated with 5 μg/mL anti-CD3 Abs in the presence of 8x10⁴ spleen cells (used antigen presenting cells) for 3 days. After 3 days of culture, supernatants were harvested for measurement of cytokine content (Figure 8). Recovered cells were stained for Ly5.1, CD4 and CD25. Viable cells were enumerated by FACS analysis using PI exclusion and calibrating PE-beads. (a) FACS profile of CFSE dilution. Numbers in the plots indicate the percentages of proliferating cells. (b) Numbers of proliferating effector T cells. P values between samples that were compared are indicated. Three independent experiments were performed with similar results.

Fig 7. CD25^lowFoxP3⁺CD4⁺ T cells are less capable of suppressing IL-17 production compared to CD25^highFoxp3⁺CD4⁺ T cells

Cultures were set up as described for Fig. 6. Supernatants from 3-day cultures were harvested to measure the content of the indicated cytokines. Data show the mean concentrations of triplicate cultures for selected cytokines. *P<0.05, **P<0.01 compared to cultures with CD25^-FoxP3⁻CD4⁺ T cells. Two independent experiments produced similar results.

Fig. 8. IL-2R- but not TCR-stimulation can convert CD25^lowFoxP3⁺CD4⁺ T cells into CD25^highFoxP3⁺CD4⁺ T cells

(a) in vitro assay. T cells from FoxP3GFP^KI/vavBcl-2 Tg mice were sorted into three sub-populations according to the levels of CD25 and FoxP3 expression. Cells were cultured at a starting density of 2x10⁴ cells/well in 200 μL medium in round-bottom 96-well plates in the presence or absence of IL-2 (20 U/mL), or anti-CD3 plus anti-CD28 Abs. Cells were harvested after 36 h and stained for CD25. Dot plots show expression of FoxP3 and CD25 on freshly sorted viable cells before culture and on viable cells after 36 h culture. (b) in vivo assay: B6 mice were iv injected with 10⁵ CellTrace-labelled CD25^lowFoxP3⁺ CD4⁺ T cells from Foxp3GFP^KI/vavBcl-2 Tg mice. Half of mice were injected intra-peritoneally with 2 daily doses of IL-2 (1000U/dose). Mice were killed 60 h after the first dose of IL-2. Spleen cells from IL-2 treated and untreated mice were surface stained for CD4, CD8 and CD25. Contour plots show CD25 and Foxp3 expression on CellTrace-labelled CD4⁺ T cells. Histograms show for CellTrace violet intensity. Similar results were obtained from two independent experiments.