Mechanism(s) of Action of the Novel Immunoregulatory Molecule, CD200

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

Keywords: CD200
immunotolerance
Advisor: Gorczynski, Reginald M.