TGF-β signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation - PubMed (original) (raw)

TGF-β signaling to T cells inhibits autoimmunity during lymphopenia-driven proliferation

Nu Zhang et al. Nat Immunol. 2012.

Abstract

T cell-specific deletion of the receptor for transforming growth factor-β (TGF-β) mediated by Cre recombinase expressed early in T cell development leads to early-onset lethal autoimmune disease that cannot be controlled by regulatory T cells. However, when we deleted that receptor through the use of Cre driven by a promoter that is active much later in T cell development, adult mice in which most peripheral CD4(+) or CD8(+) T cells lacked the receptor for TGF-β showed no signs of autoimmunity. Because of their enhanced responses to weak stimulation of the T cell antigen receptor, when transferred into lymphopenic recipients, naive TGF-β-unresponsive T cells underwent much more proliferation and differentiation into effector cells and induced lymphoproliferative disease. We propose that TGF-β signaling controls the self-reactivity of peripheral T cells but that in the absence of TGF-β signals, an added trigger such as lymphopenia is needed to drive overt autoimmune disease.

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Figures

Figure 1

Figure 1. Characterization of T cells in adult TGF-βRII KO mice

Mice with floxed alleles of TGF-βRII were bred to dLck-cre transgenic mice and examined at 7–9 weeks of age. (a) Surface staining for TGF-βRII and CD103 on gated splenic CD4+ and CD8+ T cells. (b) Splenic CD4+ and CD8+ T cells were stained for CD44 and CD62L. Numbers represent the percentage of cells falling in each quadrant. (c) Intracellular staining for T-bet in total and naive CD44loCD62L+ phenotype CD4+ and CD8+ T cells.

Figure 2

Figure 2. Dramatically increased lymphopenia induced proliferation of TGF-βRII KO T cells in _Rag1_−/− mice

(a) Schematic of the experiment. Naive T cells were purified from control (CD45.1) and KO (CD45.2) mice, mixed at a 1:1 ratio and CFSE labeled. 1×106 cells were transferred into sex-matched _Rag1_−/− recipient mice. 5.5 and 7 days later, recipient mice were sacrificed. (b) Purified naive CD4+ and CD8+ T cells were stained for CD44 and CD62L. (c) Representative CFSE profile of donor T cells in the spleen. (d) Relative KO T cell number was calculated for splenic CD4+ and CD8+ T cells separately. (e) The percentage of KLRG1+, Granzyme B+ and IFN-γ+ cells in the spleen was plotted for donor T cells. Each point represents an individual recipient mouse (* p<0.05 and ** p<0.01). Representative data from two independent experiments are shown.

Figure 3

Figure 3. TGF-βRII KO T cells induce lymphoproliferative disease in _Rag1_−/− recipient mice

1.5×106 naive T cells from control or KO mice were transferred separately into _Rag1_−/− recipient mice. (a) Recipient mice were weighed every week after T cell transfer (n=10 for both control and KO groups). (b) 5 weeks post transfer, spleen, lamina propria and liver lymphocytes of the recipient mice were analyzed by flow cytometry. CD4+ and CD8+ T cell number was calculated from the total cellularity and the percentage of T cells in each organ. Each point represents an individual recipient mouse (**p<0.01). Representative data from two independent experiments are shown.

Figure 4

Figure 4. Dramatically increased lymphopenia induced proliferation of TGF-βRII KO T cells in sublethally irradiated B6 mice

(a) Schematic of the experiment. Sex-matched recipient B6 mice were irradiated at 6.5G. On the following day, naive T cells were purified from control (CD45.1) and KO (CD45.1+CD45.2+) mice, mixed at a 1:1 ratio and CFSE labeled. 1×106 cells were transferred. 8 (b) and 14 (c and d) days later, recipient mice were sacrificed. (b) Representative CFSE profile of donor T cells in the spleen. (c) Relative KO T cell number was calculated for CD4+ and CD8+ T cells separately. (d) The percentage of IFN-γ+, KLRG1+, Granzyme B+, T-bet+ and Eomes+ cells in the spleen was plotted for donor T cells (** p<0.01). Each point represents an individual recipient mouse.

Figure 5

Figure 5. In vitro hyperproliferative response of TGF-βRII KO CD8+ T cells to weak stimulation

Equal numbers of naive OT-1 cells from control (CD45.1+CD45.2+) and KO (CD45.1) mice were mixed and CFSE labeled. T cell and NK cell depleted splenocytes (as APCs) from B6 (CD45.2) mice were incubated with 1 ng/ml of Ova peptide variants (N4, Y3, Q4, T4) plus 1 µg/ml LPS at 37°C for 1 hour. After extensive washing, an equal number of APCs and OT-1 T cells were mixed in the presence of 2.5 ng/ml hTGF-β1 with or without inflammatory cytokines (1000U/ml IFN-α, 20ng/ml IFN-γ or 20ng/ml IL-12). 50U/ml IL-2 was added one day later. (a) Starting population of mixed control and KO OT-1 T cells. (b) Relative KO OT-1 T cell number in the absence of inflammatory cytokines. (c) Flow cytometry profiles of T4 peptide-stimulated OT-1 T cells after a 4 day in vitro culture. 10 µg/ml αTGF-β neutralizing antibody was added during the culture as shown in the lower right panels. Representative data from three independent experiments are shown.

Figure 6

Figure 6. In vitro hyperproliferative response of WT CD8+ T cells to weak stimulation in the absence of TGF-β

Naive OT-1 cells from WT (CD45.1) mice were purified and CFSE labeled. T cell and NK cell depleted splenocytes (APCs) from B6 (CD45.2) mice were incubated with indicated concentration of Ova peptide variants plus 1 µg/ml LPS at 37°C for 1 hour. After extensive washing, 105 APCs and OT-1 T cells were mixed in the presence of 2.5 ng/ml hTGF-β1 or 10 µg/ml αTGF-β neutralizing antibody. 50U/ml IL-2 was added one day later. (a) Numbers of live OT-1 cells under each condition were determined everyday by flow cytometry with the addition of count standard beads. (b) CFSE profile of OT-1 cells after a 4 day in vitro culture. Representative data from two independent experiments are shown.

Figure 7

Figure 7. TGF-βRII KO CD4+ T cells induce autoimmune disease in _Rag1_−/− mice whereas KO CD8+ T cells only do so in the presence of CD4+ T cells

(a) Schematic of the experiment. 1×106 naive CD4+ or CD8+ T cells from control or KO mice were transferred into _Rag1_−/− recipient mice that were examined 5–7 weeks later. (b) Donor T cell number was calculated from the total cellularity and the percentage of each population in spleen and lamina propria. (c) and (d) Body weight was examined every week for the _Rag1_−/− mice receiving CD4+ T cells (c) and CD8+ T cells with or without CD4+ T cells (d). Each line represents an individual recipient mouse.

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