Differential requirement for IL-2 and IL-15 during bifurcated development of thymic regulatory T cells

Abstract

The developmental pathways of regulatory T cells (T(reg)) generation in the thymus are not fully understood. In this study, we reconstituted thymic development of Zap70-deficient thymocytes with a tetracycline-inducible Zap70 transgene to allow temporal dissection of T(reg) development. We find that T(reg) develop with distinctive kinetics, first appearing by day 4 among CD4 single-positive (SP) thymocytes. Accepted models of CD25(+)Foxp3(+) T(reg) selection suggest development via CD25(+)Foxp3(-) CD4 SP precursors. In contrast, our kinetic analysis revealed the presence of abundant CD25(-)Foxp3(+) cells that are highly efficient at maturing to CD25(+)Foxp3(+) cells in response to IL-2. CD25(-)Foxp3(+) cells more closely resembled mature T(reg) both with respect to kinetics of development and avidity for self-peptide MHC. These population also exhibited distinct requirements for cytokines during their development. CD25(-)Foxp3(+) cells were IL-15 dependent, whereas generation of CD25(+)Foxp3(+) specifically required IL-2. Finally, we found that IL-2 and IL-15 arose from distinct sources in vivo. IL-15 was of stromal origin, whereas IL-2 was of exclusively from hemopoetic cells that depended on intact CD4 lineage development but not either Ag-experienced or NKT cells.

Figure 1. Reconstitution of T_reg development in FoxP3^GFP TetZap70 chimeras following induction of Zap70 expression

FoxP3^GFP TetZap70 chimeras were generated by reconstituting Rag1^−/− hosts with bone marrow of FoxP3^GFP TetZap70 mice (see Materials and Methods). Following reconstitution, after 6 weeks, mice were placed on dox food to induce Zap70 expression and thymi and lymph nodes from groups of mice (for days 2-8, n=7, 8, 15, 13, 17,12 and 9 respectively) analysed at different times. (A) Density plots are of CD25 vs FoxP3^GFP amongst CD4 SP thymocytes (top row) or CD4⁺ TCR^hi lymph node cells (bottom row) at the days indicated after dox food administration started. Numbers indicate percentage of cells that were CD25⁺FoxP3^GFP−, CD25⁺FoxP3^GFP+ or CD25⁻FoxP3^GFP+. (B) Line graphs show frequency of CD4 SP (black lines, left Y axis scale) and FoxP3^GFP+ CD4 SP (red lines, right Y axis scale) amongst total thymocytes at different days after inducing Zap70 expression. Data are pooled from six independent experiments.

Figure 2. T_reg and their precursors develop with distinct kinetics

Following their reconstitution, FoxP3^GFP TetZap70 chimeras were placed on dox food to induce Zap70 expression and thymi analysed at between days 3 and 6. (A) Density plots are of CD4 vs CD8 by total live cells (top row), FoxP3^GFP vs GITR by CD4 SP TCR^hi thymocytes, CD25 vs FoxP3^GFP by CD4SP TCR^hi GITR^hi thymocytes. (B) Line graph shows frequency of CD25⁺ FoxP3^GFP− (open circles), CD25⁻ FoxP3^GFP+ (black squares) and CD25⁺ FoxP3^GFP+ cells (open squares) amongst TCR^hi CD5⁺ CD4 SP thymocytes. Data are pooled from six independent experiments.

Figure 3. Regulation of T_reg development by co-stimulatory and TCR signaling

FoxP3^GFP TetZap70 chimeras were placed on dox food to induce Zap70 expression. Groups of mice were treated with either anti-CD154 (CD40L) blocking mAb or anti-CD80 and anti-CD86 blocking mAb in combination on d-1, d1, d3 and d5 after dox feeding. Controls received PBS injections. Thymi were analysed on day 6. (A) Bar chart shows the frequency of CD25⁺ FoxP3^GFP−, CD25⁻ FoxP3^GFP+ and CD25⁺ FoxP3^GFP+ cells amongst CD4 SP thymocytes of chimeras treated with anti-CD154 (open bars), six days after Zap70 induction as compared with controls treated with PBS (filled bars). (B) Bar chart shows the frequency of CD25⁺FoxP3^GFP−, CD25⁻ FoxP3^GFP+ and CD25⁺ FoxP3^GFP+ amongst CD4 SP thymocytes of chimeras treated with anti-CD80 and anti-CD86 (open bars), six days after Zap70 induction compared with controls given PBS injection (filled bars). (C) Zap70 induction was transiently induced in FoxP3^GFP TetZap70 chimeras following a single i.p. injection of Met. Control chimeras were fed dox food. At days four and six after Zap70 induction, thymi were analysed for the presence of FoxP3^GFP+ T_reg. Left bar chart shows frequency of CD4 SP thymocytes in dox or met treated mice at days four and six after treatment. Right bar chart shows frequency of FoxP3^GFP+ cells amongst CD4 SP TCR^hi thymocytes at days four and six after Zap70 induction. Density plots are of CD25 vs FoxP3^GFP amongst CD4 SP TCR^hi thymocytes from dox and Met treated chimeras, six days after induction of Zap70. Data are pooled from three (A-B) or two (C) independent experiments.

Figure 4. Differential roles for IL-2 and IL-15 for the development of T_reg and precursor populations

FoxP3^GFP TetZap70 chimeras were generated using either Rag1^−/−, Il15ra^−/−Rag1^−/− or Il2^−/−Rag1^−/− hosts. (A-C) FoxP3^GFP TetZap70 chimeras of Rag1^−/− and Il15ra^−/−Rag1^−/− hosts were placed on dox food. Groups of mice were additionally injected with anti-IL-2 blocking mAb or PBS as control at days −1, 1, 3 and 5 after dox feeding. Thymi were analysed at day 6. (A) Density plots are of CD25 vs FoxP3^GFP by CD4 SP TCR^hi thymocytes from FoxP3^GFP TetZap70 chimeras of Rag1^−/− hosts. Bar chart shows frequencies of CD25⁺ FoxP3^GFP− (black fill bars), CD25⁻ FoxP3^GFP+ (hatched bars) and CD25⁺ FoxP3^GFP+ (empty bars) amongst CD4 SP thymocytes of chimeras treated with anti-IL-2, six days after Zap70 induction. (B) Density plots are of CD25 vs FoxP3^GFP by CD4 SP TCR^hi thymocytes from chimeras of Rag1^−/− or Il15ra^−/−Rag1^−/− hosts six days after dox feeding. Bar chart shows frequencies of CD25⁺FoxP3^GFP− (black fill bars), CD25⁻ FoxP3^GFP+ (hatched bars) and CD25⁺ FoxP3^GFP+ (empty bars) amongst CD4 SP thymocytes from Rag1^−/− or Il15ra^−/−Rag1^−/− FoxP3^GFP TetZap70 chimeras. (C) Groups of Rag1^−/− or Il15ra^−/−Rag1^−/− FoxP3^GFP TetZap70 chimeras were additionally treated with anti-IL-2. Bar chart shows frequencies of CD25⁻ FoxP3^GFP+ (hatched bars) and CD25⁺ FoxP3^GFP+ (empty bars) amongst CD4 SP thymocytes from Rag1^−/− or Il15ra^−/−Rag1^−/− FoxP3^GFP TetZap70 chimeras treated or not with anti-IL-2 blocking mAb. (D) Il2^−/−Rag1^−/− and Rag1^−/− FoxP3^GFP TetZap70 chimeras were fed dox and T_reg development analysed at day 6. Bar chart shows frequencies of CD25⁺FoxP3^GFP− (black fill bars), CD25⁻ FoxP3^GFP+ (hatched bars) and CD25⁺ FoxP3^GFP+ (empty bars) cells amongst CD4 SP thymocytes from the indicated chimeras. Data are representative of two (D) or three (A-C) independent experiments.

Figure 5. Mature T_reg arise from both CD25⁺ FoxP3^GFP− and CD25⁻ FoxP3^GFP+ precursors

(A) Nur77^GFP reporter expression was analysed amongst subsets of CD4 SP thymocytes from either Nur77^GFP mice, Nur77^GFP TetZap70 chimeras fed dox food for 6 days, or WT control mice as control for GFP detection. Thymocytes were fixed and stained for intracellular FoxP3 protein. Density plots are of FoxP3 vs CD25 amongst CD4SP of the indicated strain. Histograms are of Nur77^GFP expression by CD25⁻FoxP3⁻ (grey fill), CD25⁺FoxP3⁻ (blue line), CD25⁻FoxP3⁺ (black line) and CD25⁺FoxP3⁺ cells (red line) from Nur77^GFP− WT control mice (top), Nur77^GFP mice (middle row) and Nur77^GFP TetZap70 chimeras (bottom row). Colour coded numbers indicate MFI of GFP for the corresponding histogram on each plot. Data are representative of four independent experiments. (B) CD25⁺ FoxP3^GFP− and CD25⁻ FoxP3^GFP+ populations were purified by cell sorting from CD4 SP of Zap70^WT FoxP3^GFP mice and cultured with or without exogenous IL-2 for 24h and phenotype assessed. Density plots are of CD25 vs FoxP3^GFP immediately following sorting (post sort), and after 24 h culture with (+) and without (−) IL-2. Data are representative of three independent experiments. (C) Purified CD25⁺ FoxP3^GFP− and CD25⁻ FoxP3^GFP+ CD4 SP thymocytes were sorted from Zap70^WT FoxP3^GFP mice and injected directly into the thymus of CD45.1 congenic control hosts. 48h later, thymi were recovered and phenotype of donor populations determined. Density plots are of CD25 vs FoxP3^GFP immediately following cell sorting (Post-sort), and 48h after transfer (Post-transfer). Data are representative of two independent experiments.

Figure 6. CD25⁺FoxP3⁺ T_reg development is most efficient in the presence established CD4 lineage development

Chimeras were generated by reconstituting irradiated Rag1^−/− hosts with either bone marrow from FoxP3^GFP TetZap70 donors alone (single) or a 1:1 mixture of bone marrow from CD45.1 WT and FoxP3^GFP TetZap70 donors (mixed). Following reconstitution, chimeras were fed dox food and groups of mice analysed at different times after dox feeding. (A) Density plots are of CD25 vs FoxP3^GFP amongst CD45.2 CD4 SP TCR^hi TetZap70 donor populations in single or mixed chimeras. (B) The line graph is frequency of FoxP3^GFP cells amongst CD45.2 TetZap70 donor CD4 SP thymocytes in either single (open circles) or mixed (filled square) chimeras. (C) Bar chart shows the fraction of TetZap70 CD4 SP FoxP3^GFP+ cells that are CD25^hi in either single or mixed bone marrow chimeras at the days indicated. Data are pool of four independent experiments. (D) Mixed chimeras were additionally treated with anti-IL-2 blocking mAb at days −1, 1, 3 and 5 after dox feeding, and thymus analysed at day 6. Density plot is of CD25 vs FoxP3^GFP amongst CD45.2 TetZap70 donor CD4 SP thymocytes from control or anti-IL-2 treated mice. Bar chart shows mean frequency of CD25⁻FoxP3^GFP+ and CD25⁺FoxP3^GFP+ cells amongst CD45.2 TetZap70 donor CD4 SP thymocytes from control or anti-IL-2 treated mice. Data are representative of three independent experiments. (E) Mixed bone marrow chimeras were generated using bone marrow from FoxP3^GFP TetZap70 donors together with bone marrow from either OTII Rag1^−/−, OTI Rag1^−/−, CD45.1 WT, Ikk2^fx/fx R26R^EYFP CD4^Cre or control Ikk2^fx/WT R26R^EYFP CD4^Cre donors. Following reconstitution, mice were fed dox food and thymus analysed at day 6. Bar chart shows the fraction of TetZap70 donor FoxP3^GFP+ CD4 SP thymocytes that were CD25⁺. Data are pooled from six independent experiments.