Physiologic self antigens rapidly capacitate autoimmune disease-specific polyclonal CD4+ CD25+ regulatory T cells

Abstract

Studies on CD4+ CD25+ regulatory T cells (Tregs) with transgenic T-cell receptors indicate that Tregs may receive continuous antigen (Ag) stimulation in the periphery. However, the consequence of this Ag encounter and its relevance to physiologic polyclonal Treg function are not established. In autoimmune prostatitis (EAP) of the day-3 thymectomized (d3tx) mice, male Tregs suppressed EAP 3 times better than Tregs from female mice or male mice without prostates. Importantly, the superior EAP-suppressing function was acquired after a 6-day exposure to prostate Ag in the periphery, unaffected by sex hormones. Thus, a brief exposure of physiologic prostate Ag capacitates peripheral polyclonal Tregs to suppress EAP. In striking contrast, autoimmune ovarian disease (AOD) was suppressed equally by male and female Tregs. We now provide evidence that the ovarian Ag develops at birth, 14 days earlier than prostate Ag, and that male Tregs respond to neonatal ovarian Ag in the Treg recipients to gain AOD-suppressing capacity. When d3tx female recipients were deprived of ovarian Ag in the neonatal period, AOD was suppressed by female but not by male Tregs, whereas dacryoadenitis was suppressed by both. We conclude that the physiologic autoAg quickly and continuously enhances disease-specific polyclonal Treg function to maintain self-tolerance.

Figure 1.

Identification of prostate autoAgs by autoAb from d3tx male B6AF1 mice with EAP. (A) Cytoplasmic Ag is detected by indirect immunofluorescence in anterior prostate lobe with serum IgG autoAb from d3tx mice with EAP (i; original magnification × 200). The reaction of serum from a d3tx mouse without EAP is shown in panel ii as a negative control (original magnification × 200). Histological studies were done using an Olympus BH2 microscope (Olympus, Melville, NY), and pictures were taken with an Olympus DP12 digital microscope camera. Images were taken with 20×/__ NA (panels __) and 40×/__ NA (panels __) objectives. (B) Correlation between EAP severity and level of serum autoAb to anterior prostate extract detected by enzyme-linked immunosorbent assay (ELISA) as arbitrary units. STx indicates sham thymectomized mice. (C) Two protein bands with molecular weight of 68 kDa and 110 kDa (EAPA1 and EAPA2, respectively) in B6AF1 anterior prostate extract were immunoprecipitated by serum autoAb from d3tx mice and the aire knockout mice with EAP, detected by SDS-PAGE. (D) Prostate-specific expression of EAPA1 and EAPA2 is determined by RT-PCR. Lane 6 contains prostate RNA, whereas the lanes with negative result contain RNA of kidney, stomach, lung, brain, testis, muscle, spleen, liver, heart, pancreas, and thymus. (E) The incidence of serum autoAb that immunoprecipitated EAPA1 (□) and EAPA2 (▪) correlated with EAP severity of d3tx mice, and EAPA2 Ab is detected more frequently.

Figure 2.

Histopathology of AOD and EAP in d3tx B6AF1 mice and comparison of suppression of the 2 autoimmune diseases by Tregs. (A) Prostatitis with lymphocytic infiltration in a d3tx mouse. (B) Normal anterior prostate of a d3tx mouse suppressed with 3 × 10⁵ male Tregs. (D) Severe oophoritis in a d3tx female mouse, with heavy inflammatory infiltrate and oocyte loss. (E) Normal ovary of a d3tx mouse suppressed by 5 × 10⁵ Tregs from normal female donors. Original magnification × 400 (A-B) and × 200 (D-E); hematoxylin and eosin. Histological studies were done using an Olympus BH2 microscope (Olympus, Melville, NY), and pictures were taken with an Olympus DP12 digital microscope camera. Images were taken with 20×/__ NA (panels __) and 40×/__ NA (panels __) objectives. (C) The cell dose response of EAP suppression by Tregs from male (□), female (○), and NOr male (▵) donors. (F) The cell dose response of AOD suppression by Tregs from male (□) and female (○) donors.

Figure 3.

Detection of EAPA1 and EAPA2 mRNA. Both the EAPA1 mRNA and EAPA2 mRNA are detected in the anterior prostate of NOr B6AF1 mice 4 days after implantation of DHT pellets. HPRT served as control.

Figure 4.

The ontogeny of prostate Ag and oocyte Ag expression in B6AF1 mice. The mRNA of the ovarian MATER Ag is detected from birth by RT-PCR (A), and the MATER Ag is detected in ovarian oocytes in 1-day-old ovary by immunofluorescence with serum autoAb from d3tx mice with AOD (original magnification × 400). (B) The RNAs of prostate EAPA1 and EAPA2 are detectable in prostates at and after 10 days of age by RT-PCR. (C) The EAPA1 or EAPA2 Ag is detected in the prostate at and after 14 days of age by indirect immunofluorescence with serum autoAb from d3tx male mice with EAP (D; original magnification × 200 for top panels and × 400 for bottom panels). Panels A-B are reproduced from Alard et al, with permission of the Journal of Immunology (Copyright 2001. The American Association of Immunologists, Inc).