Karen Wheeler, PhD, 2010
Title: Regulatory T Cells in Tolerance and Autoimmunity
The purpose of this research was to understand the mechanisms by which regulatory T cells (Treg) prevent the induction of autoimmune disease. First, we wished to elucidate whether regulatory T cells were present in day 3 thymectomized (d3tx) mice, which develop strain- and organ-specific autoimmune disease. It was thought that a paucity of Treg secondary to their late ontogeny relative to effector T cells (Teff) was central to disease induction. Contrary to this paradigm, we found that functional Treg were present in d3tx mice as well as normal 3 day old mice. Importantly, they dampened the severity of autoimmune ovarian disease (AOD) and dacryoadenitis (DA) in the endogenous host. Treg present in d3tx mice were distributed such that Treg that suppress AOD were preferentially found in the ovarian draining lymph node (LN). This disease-specific accumulation could belie a mechanism by which Treg suppress disease in a normal individual, but could also reflect the active autoimmune disease in the host.
We next determined whether LN-specific accumulation was present in normal mice. We took advantage of the organ specificity of d3tx disease and ability of Treg to suppress disease if given before 7 days of age. Using Treg isolated from the ovarian-draining LN (OLN), prostate-draining LN (PLN), or lacrimal gland draining LN (cervical LN; CLN), we tested the efficiency of LN-specific Treg to suppress disease by titrating down the dose from 0.5 x 10 6 to 0.003 x 106 . Indeed, Treg from the OLN, PLN, and CLN preferentially suppressed AOD, prostatitis, and DA, respectively. OLN Treg were enriched 15 times more than Treg from pooled LN, and PLN Treg 50 times more than CLN Treg. This disease-specific Treg accumulation was dependent on endogenous antigen. Effector T cells (Teff) isolated from the PLN, CLN, or mesenteric LN all elicited a similar patter of disease in Rag-/- recipients, indicating that Treg were unique in their LN-specific accumulation.
To rationalize the significance of regional LN enrichment of antigen-specific Treg, we hypothesized that, in the face of a "danger" signal (infectious or sterile inflammation), antigen-specific Treg in the organ draining LN were poised to recognize self-antigens presented by antigen presenting cells (APC) and prevent a subsequent autoimmune response. To test this, we exploited vasectomy, a widely used method of contraception that induces inflammatory granulomas, as a source of organ-specific sterile inflammation.
When Treg were depleted (CD25 Ab) from unilaterally vasectomized (UniVx) mice, frequent and bilateral EAO developed. A reduction of Treg from all LN was accompanied by T cell activation in the regional LN and a robust testis antibody response. CD4+ T cells that produced IFNγ predominated in the diseased testes. CD4+ T cells are sufficient for disease induction as in vivo depletion of CD4+ cells blocked the emergence of EAO and CD4+ cells from UniVx mice were able to adoptively transfer disease. We also identified sperm Zonadhesin (Zan) as the major orchitogenic autoantigen. By immunoblot, autoantibodies from UniVx mice prominently recognized Zan, and immunization with Zan in adjuvant induced EAO. As EAO induction in Vx men would be devastating, we depleted Treg two weeks following UniVx and found they were free of EAO. To test whether UniVx in fact induces tolerance to testicular antigens, we compared UniVx and Sham-vasectomized mice in their response to EAO induction by immunization with testis homogenate in CFA. Indeed, UniVx mice developed significantly lower EAO scores and autoantibody and T cell proliferative responses. Thus, Treg protect vasectomized mice from post-vasectomy EAO by blocking propagation of danger signals to pathogenic autoimmunity while granting heightened tolerance to testis antigens.
I believe the results of my research have uncovered new information that will contribute to the understanding of how Treg prevent autoimmune disease in the autoimmune and normal host. 1) Contrary to popular belief, functional Treg are present in d3tx mice with concurrent autoimmune disease, they temper ongoing disease, and accumulate in a regional LN- and organ-specific manner. 2) Regional LN specific accumulation of Treg can also be found in normal mice and is dependent on organ-antigens. 3) This distribution of Treg is unique as effector T cells do not have a similar distribution. 4) These organ-specific Treg function to prevent the propagation of autoimmune disease in the face of vasectomy, a model of organ-specific danger. And (5) vasectomy alone, which generates large epididymal granulomas, induces a state of "super-physiologic" tolerance to sperm and testicular antigens.