Interleukin-2 (IL-2) in a complex with a specific anti-IL-2 antibody (JES6-1) induces potent expansion of regulatory T cells (Tregs). Using this IL-2 complex (IL-2cx) before experimental mouse lung transplant (Tx), acute rejection was virtually absent, and a viable engrafted lung was maintained up to 60 days in a fully MHC-mismatched mouse model (ISHLT meeting, San Diego, 2017). Flow cytometry data showed Foxp3+CD4+CD25+ Tregs in IL-2cx-treated mice to be significantly higher within allografts on day 5 up to 60, while in contralateral naïve lungs and spleens only on day 5 and 15, compared to non-treated controls. The next question concerned a causal association between IL-2cx, Tregs and tolerance of lung allograft. Here, we evaluated this question in a conditional Treg-deficient mouse model.

Transplant (Tx) tolerance is a state of anergy by the recipient to Tx-related alloantigen. Lung allo-Tx has the worst allograft survival outcome when compared to all other transplantable solid organs. Interleukin (IL-) 2 promotes rejection through enhanced T cell-cytotoxicity. However, when binding IL-2 in complex (cplx) with a neutralizing anti-IL-2 antibody, it can induce expansion of regulatory T (Treg) cells. We therefore evaluated here the impact on allograft outcome of IL-2 cplx on experimental mouse lung Tx outcome.

Background Fos-related antigen 2 (Fra2) is a transcription factor belonging to the Fos family proteins which is part of the AP-1 transcription complex. We recently described a Fra2 transgenic (tg) mouse model which develops a multi-organ inflammatory phenotype affecting skin, lungs, thymus, liver and salivary glands. We have observed abnormalities in the T cell compartment, particularly in regulatory T (Treg) cells, which led us to hypothesize that Fra2 tg mice develop a T cell driven autoimmune phenotype. Objectives To demonstrate the autoimmune phenotype of Fra2 tg mice and to characterize the mechanisms leading to Treg cell abnormality. Methods We used previously generated Fra2 tg overexpressing mice. T lymphocyte populations were analyzed by flow cytometry for expression of activation markers and secretion of cytokines. We transferred purified CD4+ T cells into Rag2-/- mice lacking T and B cells, and we generated Rag2-/-Fra2 tg mice. Bone marrow cells were transferred into lethally irradiated recipients to create Fra2-WT bone marrow chimeric mice. Results Fra2 tg mice backcrossed onto a Rag2-/- background did not develop inflammatory manifestations (n=6), demonstrating the dependence on T and/or B cells of the autoimmune phenotype. In line with this, the transfer of purified CD4+ cells from 16 week-old Fra2 tg mice into Rag2-/- recipients was sufficient to transfer the disease phenotype (n=3). Analysis of T cell populations from Fra2 tg mice showed the presence of activated CD4+ and CD8+ cells in the spleen and lungs. After in vitro stimulation, we found that CD4+ T cells from Fra2 tg mice produced the Th2 cytokines IL-4, IL-5 and IL-13. Thus, these data strongly suggest a T cell-driven autoimmune disease in these mice. We previously reported a striking decrease of Treg cells in Fra2 tg mice, which might explain the autoimmune phenotype observed. Supporting this idea, we found that 3 week-old mice were devoid of organ manifestations and of T cell activation, but presented the same defect in the Treg cell population (n=6, p<0.001). Analysis of thymuses from these young tg mice showed an abnormal development of thymic Treg (tTreg) cells. In particular, we could observe a normal population of tTreg precursors (CD4+CD8-CD25+FoxP3-), but a strong decrease in mature tTreg cells (CD4+CD8-CD25+FoxP3+, n=4), suggesting a perturbation in the transition from tTreg precursors to mature tTreg cells in Fra2 tg mice. We also found that in vivo stimulation with IL-2 failed to induce the proliferation of Treg cells in Fra2 tg mice compared to WT mice, suggesting that Fra2 overexpression affects IL-2 sensitivity of T cells. Finally, Fra2-WT bone marrow chimera mice also displayed a decreased percentage of Tregs confirming a cell-intrinsic and hematopoietic role of Fra2 in Treg cell development. Conclusions Our data suggest that Fra2 controls tTreg cell development, possibly by modulating IL-2 signaling in T cells, which leads to autoimmunity in this mouse model. This new pathway could be targeted in a translational approach to modulate the capacity of T cells to differentiate in Tregs during autoimmune disease. Disclosure of Interest F. Renoux Grant/research support from: Swisslife, M. Stellato: None declared, D. Impellizzieri: None declared, A. Vogetseder: None declared, R. Huang Employee of: Sanofi-Genzyme, A. Subramaniam Employee of: Sanofi-Genzyme, C. Dees: None declared, J. Distler Shareholder of: 4D Science, Grant/research support from: Anamar, Active Biotech, Array Biopharma, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, GSK, Novartis, Sanofi-Aventis, UCB, Consultant for: Actelion, Active Biotech, Anamar, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, G. Kania: None declared, O. Boyman: None declared, O. Distler Grant/research support from: Actelion, Bayer, Boehringer Ingelheim, Pfizer, Sanofi, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, ChemomAb, EpiPharm, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, Mepha, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Pfizer, Sanofi, Serodapharm, Sinoxa, Speakers bureau: AbbVie, iQone Healthcare, Mepha

Treatment protocols of tolerance induction have been proposed in the past, however, with incomplete or short-term success only. We have previously shown that the immunomodulatory cytokine interleukin-2 (IL-2) in complex (cx) with a particular neutralizing anti-IL-2 antibody induces potent expansion of regulatory T (Treg) cells. Here, we evaluated if Treg cell-selective IL-2cx could induce lung allotransplant tolerance in a fully MHC-mismatched mouse model of lung transplantation (Tx).

Background Decreased number or altered function of regulatory T cells (Tregs) has been reported in many inflammatory rheumatic diseases, and Tregs are considered promising therapeutic targets for autoimmune diseases. It is thus of high importance to delineate the pathways controlling Tregs biology and the onset of autoimmunity. Fra2 is a transcription factor belonging to the Fos family proteins which takes part in the AP-1 transcription complex. The role of Fra2 in Tregs is so far unknown. Objectives To characterize the potential role of Fra2 in controlling Tregs and autoimmunity in Fra2 transgenic mice. Methods Fra2 transgenic mice were generated, in which the Fra2 transcription factor is ubiquitously overexpressed under the control of an MHCI promotor. T lymphocyte populations were analyzed by flow cytometry, and pathological manifestations in multiple organs by histology. Bone marrow cells were transferred into lethally irradiated recipients to create Fra2-wt chimeric mice. Results At 3 weeks of age, Fra2 mice showed a striking decrease of the Treg population (CD4+CD25+FoxP3+ in wt and Fra2 littermates spleens: 11.35 and 5.75% of CD4+, respectively, p=0.0005, Mann-Whitney test, n=6). The strong decrease in Tregs was stable over time and also observed in CD4+ single positive thymocytes, indicating that Fra2 mice have a defect in natural Treg development. Interestingly, from 7 weeks on, we could also detect the appearance of activated T cells (CD4+ and CD8+, with CD62LhighCD44highCD127low profile) which represented up to 60% of total T cells by 16 weeks of age. While the phenotype of young mice was limited to decreased Tregs, macroscopic and histological observations in 16 week-old mice showed the presence of an extensive multi-organ autoimmune phenotype (see figure). Perivascular inflammatory infiltrates, containing T cells, B cells and numerous granulocytes were observed in the lung and liver. Extensive inflammation and fibrosis were observed in the thymus. Spleens were also enlarged with pronounced extramedullary haematopoiesis. Fra2 mice also developed dermatitis on eyelids and back-skin, with an increase in dermal and epidermal thickness. Finally, duodenum was enlarged due to acute inflammation and reactive hyperplasia. To understand whether the effect of Fra2 is T cell intrinsic or extrinsic, we also performed bone marrow transfer experiments in which irradiated wt recipient mice received Fra2 transgenic bone marrow. Interestingly, chimera mice displayed a decreased percentage of Tregs in blood, spleen, but also thymus, confirming an intrinsic role of Fra2 in natural Treg development. Conclusions These data suggest that Fra2 overexpression inhibits natural Treg development, resulting in 1: diminished Tregs, 2: activation of effector T cells and 3: development of inflammation in multiple organs. This is the first evidence for a role of Fra2 in controlling Treg development and autoimmunity. This murine model also provides a unique opportunity to delineate the function of the Fra2 pathway in T cells and Tregs and its impact on autoimmunity. Disclosure of Interest F. Renoux: None declared, M. Stellato: None declared, E. Pachera: None declared, D. Impellizzieri: None declared, C. Dees: None declared, J. Distler: None declared, G. Kania: None declared, O. Boyman: None declared, O. Distler Grant/research support from: Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa