Programmed cell Death-1 (PD-1) plays an important role in peripheral T cell tolerance, therefore PD-1 deficient (PD-1 KO) mice develop strain-specific autoimmune phenotypes. C57BL/6 (B6) background PD-1 KO mice develop glomerulonephritis after 24-48 weeks of age, which is similar to human endocapillary proliferative glomerulonephiritis observed in lupus nephritis. The differentiation of Th cells in PD-1 KO mice was not fully elucidated. The purpose of this study is to clarify the effect of T cell specific T-bet overexpression on autoimmune disease in PD-1 KO mice.

 

Methods:  

1) T-bet overexpressing PD-1 KO mice were generated by crossing T-bet transgenic (T-bet Tg) mice under the promoter of CD2 gene with PD-1 KO mice (PD-1 KO x T-bet Tg mice ; P/T mice). 2) In wild-type (WT) mice, PD-1 KO mice, T-bet Tg mice and P/T mice, the pathological evaluation of the kidneys was performed with H·E, PAS and PAM staining at 6-8 weeks of age. Deposition of IgG and C3 in kidneys was analyzed with immunofluorescence staining. 3) The histological analyses were evaluated on the heart, spleen, mesenteric LN, lung, liver, pancreas, salivary gland and lacrimal gland with H·E staining and immunofluorescence staining in WT mice, PD-1 KO mice, T-bet Tg mice and P/T mice. 4) Proportion of lymphocytes subset and cytokine production by CD4⁺ T cells in spleen was analyzed by FACS. 5) FACS analysis was performed to evaluate transcription factor expression on CD4⁺ T cells in spleen.

 

1) Most of P/T mice died within 10 weeks. 2) Glomerulonephritis was not observed in WT mice, PD-1 KO mice, T-bet Tg mice and P/T mice. Deposition of IgG and C3 was observed in glomeruli in PD-1 KO mice, but not in WT mice, T-bet Tg mice and P/T mice. 3) Splenomegaly and infiltration of mononuclear cells in liver were observed only in P/T mice. Immunofluorescence staining revealed that infiltrating cells were CD3⁺ T cells. 4) FACS analysis showed that the total cell number was increased in P/T mice (15.3 ± 3.11 x10⁷cells), compared with WT mice (6.41 ± 0.81 x10⁷cells, P<0.05 by Mann-Whitney U-test), PD-1 KO mice (8.46 ± 1.77 x10⁷cells, P=0.086) and T-bet Tg mice (7.12 ± 2.32 x10⁷cells, P=0.086), and IFN-γ production on CD4⁺ T cells observed in P/T mice (20.5 ± 3.42%) was higher than that in WT mice (3.56 ± 0.63%, P<0.05) and PD-1 KO mice(6.85 ±1.17%, P<0.05). 5) Percentage of Foxp3⁺ cells in CD4⁺ Treg cells was significantly decreased in P/T mice (2.47 ± 1.31%), compared with WT mice (13.4 ± 1.39%), PD-1 KO mice (16.9 ± 2.15%) and T-bet Tg mice (19.1 ± 2.55%) (P<0.05).

 

In P/T mice, the reduced percentage of Foxp3⁺CD4⁺ Treg cells induced systemic inflammation, resulting in short-life span.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/foxp3-treg-cells-decreased-in-overexpression-of-t-bet-in-pd-1-deficient-mice/