Continuous control of autoimmune disease by antigen-dependent polyclonal CD4+CD25+ regulatory T cells in the regional lymph node

Abstract

This study investigated the unresolved issue of antigen-dependency and antigen-specificity of autoimmune disease suppression by CD4+CD25+ T cells (T regs). Based on autoimmune ovarian disease (AOD) in day 3 thymectomized (d3tx) mice and polyclonal T regs expressing the Thy1.1 marker, we determined: (a) the location of recipient T cell suppression, (b) the distribution of AOD-suppressing T regs, and (c) the relative efficacy of male versus female T regs. Expansion of recipient CD4+ T cells, activation/memory marker expression, and IFN-gamma production were inhibited persistently in the ovary-draining LNs but not elsewhere. The cellular changes were reversed upon Thy1.1+ T reg depletion, with emergence of potent pathogenic T cells and severe AOD. Similar changes were detected in the regional LNs during autoimmune dacryoadenitis and autoimmune prostatitis suppression. Although the infused Thy1.1+ T regs proliferated and were disseminated in peripheral lymphoid organs, only those retrieved from ovary-draining LNs adoptively suppressed AOD at a suboptimal cell dose. By depriving d3tx recipients of ovarian antigens, we unmasked the supremacy of ovarian antigen-exposed female over male T regs in AOD suppression. Thus, disease suppression by polyclonal T regs depends on endogenous antigen stimulation; this occurs in a location where potent antigen-specific T regs accumulate and continuously negate pathogenic T cell response.

Figure 1.

Time course of AOD induction and dose response of suppression by T regs in d3tx B6AF1 mice. (A) AOD in d3tx mice of different ages. (B) AOD in 6-wk-old d3tx recipients of different numbers of Thy1.1⁺CD25⁺ T cells from normal female donors.

Figure 2.

The total cell number and recipient CD4⁺ T cell number in ovarian and inguinal LNs at 4 wk (top), and from 3 to 8 wk (bottom). Compared with d3tx/nOX control, the total cellularity and CD4⁺ T cell number in the d3tx mice were increased significantly in ovary-draining LNs, but not inguinal LNs. Compared with d3tx mice, profound reduction in the total cellularity and recipient CD4⁺ T cell number occurred in ovary-draining LNs but not inguinal LNs of the d3tx/suppressed mice. This was detectable at 4 wk and persisted to 8 wk.

Figure 3.

Ovarian immunohistology and ovarian LN histology of d3tx mice and the d3tx/suppressed mice. At 4 wk, the ovary of a d3tx mouse has heavy T cell infiltration (A), whereas the ovary of a d3tx/suppressed mouse is completely devoid of T cells (B) (Texas red–labeled anti-CD5 antibody plus DAPI). Findings are representative of 10 mice for each group. The ovary-draining LN of the d3tx mice is enlarged, and contains a germinal center and a broad T cell zone (C), whereas the ovary-draining LN of the d3tx/suppressed mice has an empty T cell zone and is devoid of germinal centers (D) (hematoxylin and eosin).

Figure 4.

Proliferation (A) and IFN-γ production (B) of Thy1.1-negative recipient CD4⁺ T cells in the ovary-draining LNs of d3tx versus d3tx/suppressed mice. BrdU uptake in 12 h by cells in d3tx mice greatly exceeds that of d3tx/suppressed mice; the difference is not found in the inguinal LN. IFN-γ–producing CD4⁺ T cell numbers of d3tx mice exceed those of d3tx/suppressed mice in ovarian LNs but not in inguinal LNs.

Figure 5.

Proliferation, distribution, and function of the polyclonal T reg cells in d3tx/suppressed mice. T regs proliferated and were distributed equally in ovarian LNs and nondraining LNs (A, B). However, the Thy1.1⁺ T regs (C) that were reisolated from the ovarian LNs (OLNs) but not the nondraining LNs (NDLNs) suppress AOD and oocyte antibody response in d3tx mice at 0.1 million cells per recipient (D). Conversely, dacryoadenitis was suppressed by T regs that were reisolated from NDLNs but not by T regs from OLNs (D). In D, closed circles denote mice with serum oocyte antibody. The NDLNs included the inguinal, axillary, brachial, and cervical LNs. T reg proliferation was determined by injecting 0.5 million CFSE-labeled Thy1.1⁺ T regs into 5-d-old d3tx B6AF1 mice, and studying them 23 d later. In A, donor cells (gated on CD4⁺Thy1.1⁺ cells) were analyzed for percentages that are undivided, have divided one to six times, or more than six times. The CFSE line graph represents data pooled from four to eight mice.

Figure 6.

Reversal of AOD suppression by in vivo and in vitro depletion of Thy.1 T reg cells. (A) Reversibility of ovarian LN suppression and AOD. (B) Thy1.1⁺ T regs in d3tx/suppressed mice were eliminated by a single injection of Thy1.1 mAb at 3 wk, and were studied at 7 wk. (C) To detect functional splenic effector T cells and T regs in the d3tx/suppressed mice, spleen cells were transferred to 5 d-old recipients with or without ex vivo depletion of the infused Thy1.1⁺ T regs. AOD in cell recipients was determined 12 d later. AOD in recipients of spleen cells from d3tx mice served as control.

Figure 7.

Ovarian antigen exposure in neonatal mice capacitates AOD-specific T reg function. In this experiment, each neonatal recipient received 0.1 million Thy1.1⁺ cells retrieved from the LNs of d3tx/suppressed mice. (A) AOD does not develop in the d3tx/nOX recipients of female T regs, whereas the d3tx/nOX recipients of male T regs developed AOD with the same incidence and severity as the AOD in control d3tx mice. (B) In contrast to AOD, dacryoadenitis in these d3tx/nOX recipients is inhibited completely by male or female T regs. (C) The ovarian graft of d3tx/nOX recipients of female T regs is normal histologically (arrow points to normal oocyte). (D) The ovarian graft of d3tx/nOX recipients of male T regs is infiltrated heavily by inflammatory cells, some replacing the oocyte (arrow; hematoxylin and eosin).