Overexpression of Fibrinogen-Like Protein 2 Promotes Tolerance in a Fully Mismatched Murine Model of Heart Transplantation

Abstract

Both authors contributed equally to this paper.Fibrinogen-like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2 Tg ) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2 À/À mice. Immune characterization showed fgl2Tg T cells were hypoproliferative to stimulation with alloantigens or anti-CD3 and anti-CD28 stimulation, and fgl2Tg Tregs had increased immunosuppressive activity compared with fgl2 +/+ Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2Tg recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2Tg recipients without any immunosuppression. Tolerant fgl2Tg grafts had increased numbers and proportions of Tregs and tolerant fgl2Tg mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2Tg recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg-to-T cell ratio and acceptance of fully mismatched allografts.Abbreviations: BM, bone marrow; BMDC, bone marrow-derived dendritic cells; CAG, cytomegalovirus early enhancer/chicken b actin; CHO, Chinese hamster ovary; ConA, Concanavalin A; DC, dendritic cell; EGFP, enhanced green fluorescent protein; FGL2, fibrinogenlike protein 2; Foxp3, forkhead box P3; FReD, fibrinogen-related domain; IL, interleukin; MLR, mixed lymphocyte reaction; rFGL2, recombinant FGL2; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMNC, splenic mononuclear cell; TIGIT, T cell immunoglobulin and ITIM domain; Treg, regulatory T cell

Soluble fibrinogen like protein 2 (sFGL2), the novel effector molecule for immunoregulation

“…In tolerant cardiac and liver allografts, expression of sFGL2 as well as other tolerogenic set of genes such as forkhead box P3 (Foxp3), cytotoxic T lymphocyte-associated protein 4 (CTLA4), and killer cell lectin-like receptor G1 (Klrg1) was significantly elevated [72]. Numbers and immunosuppressive activity of CD4+ Tregs were significantly elevated in cardiac allografts of fgl2 transgenic mice, whereas proliferative ability of effector T cells to alloantigen stimulation was remarkably decreased [73]. Rapamycin-induced tolerance in C3H/HeJ mice transplanted with MHC-mismatched hearts from BALB/cJ mice was associated with elevated plasma levels of sFGL2, and FoxP3/FGL2 dual positive CD4+CD25+ Tregs were remarkably increased in tolerant allografts.…”

Overexpression of fibrinogen-like protein 2 protects against T cell-induced colitis

“…The generation of fgl2 Tg mice has been previously reported[20]. Briefly, a prothrombinase inactive fgl2 gene was inserted into the iZ/EG targeting vector, which was electroporated into R1 ES cells[21].…”

“…Cells were incubated in the presence of Concanavalin A at a final concentration of 1 μg/mL for 3 d at 37 °C in RPMI-10. To measure proliferation, 1 μCu of 3 H-thymidine was added to culture supernatants and incubated for 18 h. Percent suppression was calculated as previously described[20]. …”

“…The C-terminal of FGL2 contains a FRED domain, which accounts for the immunomodulatory activity and is important in regulating dendritic cell (DC) maturation, T cell proliferation and B cell apoptosis[12,17]. We and others have reported that Treg that express high levels of FGL2 lead to tolerance of fully mismatched heart allografts[18-20]. Studies showed that tolerant grafts contained large numbers of Treg that co-expressed Foxp3 and FGL2, whereas rejecting grafts contained Foxp3 + Treg that were FGL2 negative.…”

“…To further determine the role of FGL2 in immune function, we generated ubiquitous FGL2 over-expressing mice ( fgl2 Tg )[20]. Here we used these mice to determine the importance of FGL2 to the pathogenesis of IBD.…”