Cutting edge: CD28 and c-Rel-dependent pathways initiate regulatory T cell development

Abstract

Regulatory T cell (Treg) development proceeds via a two-step process in which naive CD4(+) thymocytes are first converted into CD4(+)CD25(+)CD122(+)GITR(+)Foxp3(-) Treg progenitors, followed by a second step in which IL-2 converts these Treg progenitors into CD4(+)Foxp3(+) Tregs. The costimulatory molecule CD28 is required for efficient Treg development. However, the stage at which CD28 affects Treg development remains undefined. In this article, we demonstrate that Cd28(-/-) mice lack Treg progenitors. Furthermore, the P(187)YAP motif in the cytoplasmic tail of CD28, which links CD28 to Lck activation, is required for this process. In contrast, the Y(170)MNM motif, which links CD28 to PI3K activation, is not required for Treg progenitor development. Finally, the CD28/Lck pathway was shown to activate the NF-kappaB family of transcription factors. We demonstrate that c-Rel, but not NF-kappaB1, promotes the development of Treg progenitors. Thus, a CD28/c-Rel-dependent pathway is involved in initiating Treg development.

Fig. 1

The CD28 Lck binding motif P¹⁸⁷YAP is required for Treg progenitor development. (A) The gating strategy to identify Tregs and Treg progenitors is shown. The CD122⁺GITR⁺ gate used to identify Treg progenitors was based on staining observed for these markers on CD4⁺Foxp3⁺ Tregs. Shown are CD4⁺Foxp3⁺CD122⁺GITR⁺ Tregs (top right) and CD4⁺CD25⁺CD122⁺GITR⁺Foxp3⁻ Treg progenitors (bottom right) from the thymus of a C57Bl/6 mouse. (B) The frequency of CD4⁺CD25⁺CD122⁺GITR⁺Foxp3⁻ Treg progenitors (left) and CD4⁺Foxp3⁺ Tregs (right) within the CD3⁺CD4⁺CD8⁻ thymocyte subset is shown. Error bars represent standard error of the mean. Data is representative of three independent experiments with 6 mice per group. p ≤ 0.05; determined by Anova using Bonferroni correction.

Fig. 2

The CD28 Lck binding motif is required for Treg progenitor development in a cell-intrinsic manner. (A) Thymii were isolated and stained as described in the methods. Contour plots depict mutant (CD45.1−) and WT (CD45.1+) cells gated on CD4⁺CD25⁻ Foxp3⁻ thymocytes (top row), CD4⁺CD25⁺CD122⁺GITR⁺Foxp3⁻ Treg progenitors (middle row) and CD4⁺Foxp3⁺ Tregs (bottom row). (B) Comparison of the ratio of WT to mutant cells between mixed chimeras in CD4⁺CD25⁻Foxp3⁻ thymocytes (left), Treg progenitors (middle), and CD4⁺Foxp3⁺ Tregs (right). Ratios were normalized by comparison to the ratio of WT to mutant cells in double positive thymocytes to control for differences in the initial input ratio of WT to mutant cells. Error bars represent standard error of the mean. Data is representative of three independent experiments with 11-15 recipient mice per group. p ≤ 0.002 determined by unpaired t-test.

Fig. 3

c-Rel but not NFκB1 is required for Treg progenitor development. (A) Shown are contour plots gated on CD4⁺CD25⁺CD122⁺GITR⁺Foxp3⁻ Treg progenitors from WT control (far left), nfκb1^-/- x c-rel^-/- (middle left), nfκb1^-/- (middle right) and c-rel^-/- (far right). (B) The frequency of Treg progenitors (left) and CD4⁺Foxp3⁺ Tregs (right) in CD3⁺CD4⁺CD8⁻ thymocytes is shown. Data is representative of two independent experiments with three mice per group. p ≤ 0.05; determined by Anova using Bonferroni correction.

Fig. 4

c-Rel but not NFκB1 is required in a cell intrinsic manner for Treg progenitor development. (A) Contour plots depict the ratio of mutant (CD45.1−) versus wild-type cells (CD45.1+) gated on CD4⁺CD25⁻Foxp3⁻ thymocytes (left), CD4⁺CD25⁺CD122⁺GITR⁺Foxp3⁻ Treg progenitors (middle) and CD4⁺Foxp3⁺ Tregs (bottom). (B) Comparison of the ratio of wild-type to mutant cells between mixed chimeras in CD4⁺CD25⁻Foxp3⁻ thymocytes (left), Treg progenitors (middle), and CD4⁺Foxp3⁺ Tregs (right). Ratios were normalized by comparison to the ratio of WT to mutant cells in double positive thymocytes to control for differences in the initial input ratio of WT to mutant cells. Error bars represent standard error of the mean. Data is representative of two independent experiments with 8 nfκb1^-/- vs. WT, 9 nfkb1^-/- x c-rel^-/- vs. WT, and 4 c-rel^-/- vs. WT mixed bone marrow chimeric mice. p < 0.02 determined by unpaired t-test.