Block of both TGF-β and IL-2 signaling impedes Neurophilin-1+ regulatory T cell and follicular regulatory T cell development

Abstract

Understanding the mechanisms that lead to autoimmunity is critical for defining potential therapeutic pathways. In this regard there have been considerable efforts in investigating the interacting roles of TGF-β and IL-2 on the function regulatory T cells. We have taken advantage of dnTGF-βRII Il2ra^-/- (abbreviated as Il2ra^-/-Tg) mouse model, which allows a direct mechanistic approach to define the relative roles of TGF-β and IL-2 on Treg development. Il2ra^-/-Tg mice spontaneously developed multi-organ autoimmune diseases with expansion of pathogenic T cells and enhanced germinal center response at 3-4 weeks of age. Importantly, peripheral Treg cells from Il2ra^-/-Tg mice demonstrated an activated Th1-like stable phenotype and normal in vitro suppressive function, while thymus Treg increased but manifested decreased suppressive function. Interestingly, neither thymus nor peripheral Treg cells of Il2ra^-/-Tg mice contained Neuropilin-1⁺ or PD-1^hi phenotype, resulting in defective follicular regulatory T (Tfr) cell development. Such defective Tfr development led to elevated follicular T helper cells, enhanced germinal center responses and increased plasma cell infiltration. These data demonstrate an important synergetic role of TGF-β and IL-2 in the generation, activation and stability of Treg cells, as well as their subsequent development into Tfr cells.

Figure 1

Multi-organ inflammation in Il2ra^−/−Tg mice. (a) Survival curve of Il2ra^−/−Tg (n=8), Il2ra^−/− (n=8), Il2ra^+/−Tg (n=8) and Il2ra^+/− (n=8) mice. Cell counts of (b) spleen, (c) peripheral (axillary, inguinal and brachial) lymph nodes of Il2ra^−/−Tg (n=12), Il2ra^−/− (n=12), Il2ra^+/−Tg (n=12) and Il2ra^+/− (n=12) mice. (d) Naive T cell (CD44⁻CD62L⁺) percentage and (e) IFN-γ secreting ability of CD4⁺ and CD8⁺ T cells in pLN from Il2ra^−/−Tg (n=5), Il2ra^−/− (n=5), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) mice. (f) Serum concentration of IFN-γ, TNF-α, IL-4 and IL-17A of Il2ra^−/−Tg (n=6), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) mice. Data are shown in mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001. (Student's t test)

Figure 2

Enhanced germinal center response in Il2ra^−/−Tg mice. Representative flow cytometry result of (a) follicular helper T cells (PD-1^hiCXCR5⁺ in CD4⁺CD19⁻) and (c) germinal center B cells (GL7⁺CD95⁺ in CD19⁺) and (e) plasma cells (CD19^loB220^-CD138^hi in total cells) in pLN of Il2ra^−/−Tg and littermate control mice. (b) Follicular helper T-cell percentage in CD4⁺ T cells and absolute number in pLN of Il2ra^−/−Tg (n=8), Il2ra^−/− (n=8), Il2ra^+/−Tg (n=8) and Il2ra^+/− (n=8) mice. (d) Germinal center B-cell percentage in B cells and number in pLN of Il2ra^−/−Tg (n=8), Il2ra^−/− (n=7), Il2ra^+/−Tg (n=7) and Il2ra^+/− (n=7) mice. (f) Plasma cell percentage and number in pLN of Il2ra^−/−Tg (n=6), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) mice. (g) Serum concentration of IgG1, IgG2a, IgG2b, IgG3 and IgM of Il2ra^−/−Tg (n=7), Il2ra^−/− (n=7), Il2ra^+/−Tg (n=7) and Il2ra^+/− (n=7) mice. Data are shown in mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001. (Student's t test)

Figure 3

Dysregulated Treg percentage and function in Il2ra^−/−Tg mice. (a) Representative flow cytometry result of Treg cell percentage in CD4 single-positive T cells in thymus and pLN of Il2ra^−/−Tg and control mice. (b) Statistical analysis of Treg percentage and absolute number in thymus and pLN of Il2ra^−/−Tg (n=9), Il2ra^−/− (n=9), Il2ra^+/−Tg (n=9) and Il2ra^+/− (n=9) mice. (c) Expression of GITR, CTLA-4 and Helios of Treg cells in thymus and pLN from Il2ra^−/−Tg and littermate control mice. (d) Foxp3 expression as indicated by GFP MFI of Treg cells in thymus and pLN of Il2ra^−/−Tg (n=9), Il2ra^−/− (n=9), Il2ra^+/−Tg (n=9) and Il2ra^+/− (n=9) mice. (e) Percentage of CD69⁺ Treg cells in thymus and pLN of Il2ra^−/−Tg (n=5), Il2ra^−/− (n=5), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) mice. (f) CFSE-labeled CD45.1⁺CD4⁺Foxp3-GFP^-CD62L⁺ T cells were co-cultured with CD45.2⁺CD4⁺ Foxp3-GFP⁺ Treg cells from thymus and spleen/pLN of Il2ra^−/−Tg and Il2ra^+/− mice in the presence of soluble anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) and mitomycin C treated supporter cells for 72 h. Percentage of proliferated cells among live target cells were analyzed. (g) Ki-67 staining of Treg cells in thymus and pLN of Il2ra^−/−Tg (n=5), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=4) mice. (h) Annexin V staining of Treg cells in thymus and pLN of Il2ra^−/−Tg (n=7), Il2ra^−/− (n=7), Il2ra^+/−Tg (n=7) and Il2ra^+/− (n=7) mice. Data are shown in mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001. (Student's t test)

Figure 4

Treg cells are skewed to Th1-like stable phenotype in Il2ra^−/−Tg mice. (a) Percentage of CXCR3⁺ Treg cells in thymus and pLN of Il2ra^−/−Tg (n=5), Il2ra^−/− (n=4), Il2ra^+/−Tg (n=4) and Il2ra^+/− (n=5) mice. (b) Percentage of Eomes⁺ Treg cells in thymus and pLN of Il2ra^−/−Tg (n=4), Il2ra^−/− (n=3), Il2ra^+/−Tg (n=3) and Il2ra^+/− (n=6) mice. (c) Representative flow cytometry result of Foxp3 and CXCR3, Eomes expression on CD4⁺CD8⁻ T cells from thymus and pLN of Il2ra^−/−Tg and control mice. (d) IFN-γ secreting ability of thymus and pLN Treg cells from Il2ra^−/−Tg (n=5), Il2ra^−/− (n=4), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) mice. (e) Ly6C expression of thymus and pLN Treg cells from Il2ra^−/−Tg (n=6), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) mice. (f) Percentage of Foxp3 TSDR CpG demethylation of thymus and spleen/pLN Treg cells from Il2ra^−/−Tg and Il2ra^+/− mice. Data are shown in mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001. (Student's t test)

Figure 5

Defective Nrp-1⁺ Treg and follicular regulatory T-cell development in Il2ra^−/−Tg mice. (a) Representative flow cytometry results of Nrp-1 expression on Treg and conventional CD4⁺ T cells from thymus and pLN of Il2ra^−/−Tg and littermate control mice. (b) Nrp-1⁺ percentage of Treg in thymus and pLN from Il2ra^−/−Tg (n=5), Il2ra^−/− (n=5), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) mice. (c) Representative flow cytometry results of PD-1 expression on Treg and conventional CD4⁺ T cells from thymus and pLN of Il2ra^−/−Tg and littermate control mice. (d) PD-1⁺ percentage of Treg in thymus and pLN from Il2ra^−/−Tg (n=5), Il2ra^−/− (n=5), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) mice. (e) Representative flow cytometry result of Tfr in Tfh cells in pLN of Il2ra^−/−Tg and littermate control mice. Percentage of Tfr cells (f) in Tfh cells and (h) in Treg cells in pLN of Il2ra^−/−Tg (n=6), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) mice. (g) Nrp-1 and Foxp3 expression of Tfh cells in pLN of Il2ra^−/−Tg and littermate control mice. Data are shown in mean±S.E.M. ***P<0.001. (Student's t test)

Figure 6

WT Treg cells suppress Il2ra^−/−Tg T cells after bone marrow chimera. (a) Strategy of bone marrow chimera: 5 × 10⁵ T-cell depleted bone marrow cells from CD45.1 Foxp3^GFP mice were mixed with 5 × 10⁵ T-cell depleted bone marrow cells from CD45.2 Il2ra^−/−Tg or littermate control mice and transferred intravenously to lethally irradiated CD45.1 CD45.2 recipient mice. Mice were killed 10 weeks after bone marrow transfer. (b) Naive T cell (CD44⁻CD62L⁺) percentage of pLN CD4⁺ and CD8⁺ T cells derived from Il2ra^−/−Tg (n=3), Il2ra^−/− (n=4), Il2ra^+/−Tg (n=4) and Il2ra^+/− (n=4) bone marrow. (c) Treg percentage in total CD4⁺ T cells after chimerism of Il2ra^−/−Tg (n=5), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) with WT bone marrow. (d) Tfh percentage of CD4⁺ T cells, (e) GC B percentage of B cells and (f) plasma cell percentage of total cells derived from Il2ra^−/−Tg (n=5), Il2ra^−/− (n=6), Il2ra^+/−Tg (n=6) and Il2ra^+/− (n=6) bone marrow. (g) Tfr cell percentage of Tfh cells after chimerism of Il2ra^−/−Tg (n=4), Il2ra^−/− (n=5), Il2ra^+/−Tg (n=5) and Il2ra^+/− (n=5) with WT bone marrow. Data are shown in mean±S.E.M. **P<0.01. (Student's t test)