Arthritogenic self-reactive CD4+ T cells acquire an FR4hiCD73hi anergic state in the presence of Foxp3+ regulatory T cells - PubMed (original) (raw)

Arthritogenic self-reactive CD4+ T cells acquire an FR4hiCD73hi anergic state in the presence of Foxp3+ regulatory T cells

Ryan J Martinez et al. J Immunol. 2012.

Abstract

Rheumatoid arthritis develops in association with a defect in peripheral CD4(+) T cell homeostasis. T cell lymphopenia has also been shown to be a barrier to CD4(+) T cell clonal anergy induction. We therefore explored the relationship between clonal anergy induction and the avoidance of autoimmune arthritis by tracking the fate of glucose-6-phosphate isomerase (GPI)-reactive CD4(+) T cells in the setting of selective T cell lymphopenia. CD4(+) T cell recognition of self-GPI peptide/MHC class II complexes in normal murine hosts did not lead to arthritis and instead caused those T cells to develop a Folate receptor 4(hi)CD73(hi) anergic phenotype. In contrast, hosts selectively depleted of polyclonal Foxp3(+)CD4(+) regulatory T cells could not make GPI-specific CD4(+) T cells anergic and failed to control arthritis. This suggests that autoimmune arthritis develops in the setting of lymphopenia when Foxp3(+)CD4(+) regulatory T cells are insufficient to functionally inactivate all autoreactive CD4(+) T cells that encounter self-Ag.

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Figures

Figure 1

Figure 1

Naïve KRN CD4+ T cells cause autoimmune arthritis in T cell lymphopenic hosts. Host mice were given 104 TCR-transgenic KRN CD4+ T cells on day 0 and were then monitored for signs of arthritis and weight loss. A, Representative rear paws of B6×B6.g7 (top) and TCRα−/− B6×B6.g7 (bottom) at day 10 after adoptive transfer. B, Arthritis, as assessed by change in measured ankle thickness (mm) of individual B6 (●), WT B6×B6.g7 (■), and TCRα−/− B6×B6.g7 (□) adoptive transfer recipient mice. C, Clinical disease scores of individual B6, B6×B6.g7, and TCRα−/− B6×B6.g7 hosts 10 days after adoptive transfer of either non-transgenic B6, KRN, or Rag1−/− KRN CD4+ T cells, with median score indicated by the bars. D, Daily weight measurements in individual mice, expressed as change in body weight (gm). Statistically significant differences (P < 0.05; Student’s t test) between the WT B6×B6.g7 and TCRα−/− B6×B6.g7 groups are indicated by an asterisk.

Figure 2

Figure 2

KRN CD4+ T cells cause an increase in the amount of anti-GPI antibody and numbers of GPI specific B plasmablasts in hosts expressing the I-A_g7_ allele. A, Mean anti-GPI IgG1 titers ±SEM were determined by ELISA for B6 (●), WT B6×B6.g7 (■), and TCRα−/− B6×B6.g7 (□) animals. B, Mean disease activity scores in the same experiment. C, Polyclonal spleen and lymph node B cells either from normal naïve mice (left), or from day 10 KRN T cell adoptive transfer recipient WT B6×B6.g7 (middle) and TCRα−/− B6×B6.g7 (right) mice, stained for intracellular immunoglobulin H+L chain accumulation and GPI-binding capacity. Plots and tracings are representative of multiple mice (> 6 animals per group).

Figure 3

Figure 3

KRN CD4+ T cell number, phenotype, and function following adoptive transfer into WT or TCRα−/− B6×B6.g7 hosts. A, Mean ±SEM KRN CD4+ T cell number in combined spleen and lymph nodes from WT B6×B6.g7 (■) and TCRα−/− B6×B6.g7 (□) recipients. The open square at T = 0 represents historical post-adoptive transfer cell count; asterisks indicate significant differences between groups (P < 0.05). B, KRN CD4+ T cell FSc, CD44, CCR7, CD27, FR4 and CD73 expression at day 10. Plots are representative of greater than 6 animals per group. C,D, Purified KRN T cell mRNA expression levels on days 5 (C) and 10 (D) after adoptive transfer into either WT (red) or TCRα−/− (green) B6×B6.g7 hosts. Freshly isolated naïve KRN T cells are also shown as a control (blue). E, Purified KRN CD4+ T cells were exposed for 7 days to GPI/I-A_g7_ in WT (shaded tracing) or TCRα−/− (open tracing) B6×B6.g7 hosts, recovered and labeled with CFSE, and then examined for CFSE dye–dilution 3 d after adoptive transfer into B6×B6.g7 secondary hosts. Digits above the tracing indicate the number of times a cell has divided. F, Average cell division rate ±SEM (n = 7) for groups as indicated. P values based on Student’s t test.

Figure 4

Figure 4

Partial reconstitution of TCRα−/− B6×B6.g7 mice with polyclonal Foxp3+ CD4+ T cells prevents autoimmune arthritis by inducing clonal anergy in the GPI-reactive CD4+ T cells. A, Polyclonal CD4+ T cell counts in TCRα−/− B6×B6.g7 mice on day 20 after partial reconstitution, as indicated. Counts from tolerant WT B6.B6.g7 mice are also shown for comparison. B, Mean clinical score in each experimental group ±SEM. P values are based on the Mann-Whitney U test. C, Absolute number of KRN CD4+ T cells recovered from host animals following partial polyclonal CD4+ T cell reconstitution, as in panel A. D, In a separate experiment, FR4− and CD73− levels (top) were measured on KRN CD4+ T cells recovered from partially reconstituted TCRα−/− B6×B6.g7 mice (as indicated) 10 d after the KRN adoptive transfer. Flow cytometric measurement of IFNγ and IL-2 intracellular accumulation was also performed (bottom) on ex vivo PMA plus ionomycin stimulated KRN CD4+ T cells. E, Percentage of KRN CD4+ T cells recovered from various hosts (as in panel D) capable of synthesizing IL-2, IFNγ, and/or TNFα in various combinations. In panels A and C, filled symbols represent animals developing at least one arthritic joint; open symbols are animals that remained free of disease. Bars indicate the mean cell count, and P values are as indicated, using Student’s t test.

Figure 5

Figure 5

Polyclonal CD25+ Foxp3+ CD4+ T regulatory cells inhibit the accumulation of FR4− CD73− GPI-specific CD4+ T effector cells. A, Identification of endogenous (top) and KRN (bottom) CD25+ Foxp3+ CD4+ T regulatory cells following partial polyclonal CD4+ T cell reconstitution of TCRα−/− B6×B6.g7 mice. B, Absolute number of endogenous Foxp3+ CD4+ T regulatory cells identified in partially reconstituted hosts. C,D, Correlation between endogenous Foxp3+ T regulatory cells and either total KRN CD4+ T cell number (C) or FR4− CD73− KRN CD4+ T cell number (D). In panel B, filled symbols represent animals developing at least one arthritic joint; open symbols are animals that remained free of disease. Bars indicate the mean cell count, and P values are as indicated, using Student’s t test.

Figure 6

Figure 6

Lymphopenic hosts reconstituted with CD25+ FoxP3+ CD4+ T cells are protected from autoimmune arthritis. On day 0, lymphopenic TCRα−/− B6×B6.g7 animals were given 106 purified CD25+ Foxp3+ or CD25− Foxp3− CD4+ polyclonal T cells from syngeneic _Foxp3_DTR (Foxp3-GFP-DTR) B6×B6.g7 donors, or were given no cells, as indicated in the panels. On day 10, all mice received 104 KRN CD4+ T cells. Arthritis severity measurements and T cell analysis were performed on day 20. A, Average arthritis clinical score ±SEM. P values based on the Mann-Whitney U test. B, KRN CD4+ T cell expression of FR4 and CD73 in representative animals. C, KRN FR4− CD73− CD4+ T cell absolute number. Bars represent the mean cell number, with filled symbols indicating arthritic animals, and open symbols indicating animals free of disease. P values using Student’s t test.

Figure 7

Figure 7

Ablation of polyclonal CD4+ CD25+ Foxp3+ regulatory T cells prevents clonal anergy development and leads to autoimmune arthritis. Hosts were either WT B6×B6.g7 or lymphopenic TCRα−/− B6×B6.g7 as indicated in the panels. On day 0, some of the lymphopenic animals received 106 polyclonal CD4+ T cells from syngeneic _Foxp3_DTR B6×B6.g7 donors. Diphtheria toxin (DT) was administered to some mice on days 8, 10, and 12. On day 10, 104 KRN CD45.1+ CD4+ T cells were adoptively transferred into most mice. Arthritis severity measurements and T cell analysis were performed on day 20. A, Average arthritis clinical score ±SEM in experimental groups as indicated. Significance determined by Mann-Whitney U test. B, Representative KRN CD4+ T cell FR4 and CD73 expression. C, KRN FR4− CD73−CD4+ T cell absolute numbers. Bars represent the mean cell number, with filled symbols indicating arthritic animals, and open symbols indicating animals free of disease. P values using Student’s t test. D, Foxp3+ GFP+ CD4+ T cells in _Foxp3_DTR-reconstituted TCRα−/−B6×B6.g7 mice, either with or without DT treatment.

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