Background/Purpose: CD11c-Flip-KO (HUPO) mice spontaneously develop inflammatory, erosive arthritis. We previously demonstrated that T regulatory cells (Tregs) were reduced in HUPO mice and that the adoptive transfer of Tregs isolated from spleens of control mice suppressed not only the arthritis but also the expression of autoreactive T cells. The purpose of these studies was to identify the mechanism(s) responsible for the reduction of Tregs in HUPO mice.

Methods: Immune cell phenotype and apoptosis/necrosis were assessed by flow cytometry employing multicolor fluorochrome-antibodies. Conventional dendritic cells (cDCs), Tregs or CD3⁺T cells are purified by positive or negative selection employing commercially available kits. In vitro cell culture or in vivo adoptive transfer were employed to examine T cell proliferation, Treg development and maintenance. Mice analyzed were HUPO vs. littermates, or Rag^-/- vs. HUPO-Rag^-/- as the lymphopenic recipients for in vivo homeostatic proliferation. Cytokines were determined by quantitative ELISA or rt-PCR.

Results: A marked reduction of IL-2 was identified in in vitro spleen culture supernatants of HUPO mice compared with littermate controls. Tregs were also reduced in the spleen cultures and the addition of IL-2 or cDCs increased Tregs but not other T cells. Further, cDCs from HUPO mice were deficient in TGFβ. In vivo, a significant increase of necrotic Tregs was identified in the spleens of young (4 weeks) and old (≥20 weeks) HUPO mice. Consistent with these observations central Tregs, which are highly IL-2 dependent, were greatly reduced in the spleens of young and old HUPO mice. Additional experiments were performed under lymphopenic conditions since the maintenance of Tregs is in part regulated by homeostatic proliferation. HUPO mice exhibit a reduction of cDCs, particularly the CD8a subset and of CD11c⁺ macrophages. Crossing HUPO and Rag^-/- resulted in HUPO-Rag^-/- line with mild non-progressive arthritis. Following the adoptive transfer of control CD3⁺ T cells no reduction of Treg homeostatic proliferation was observed in HUPO-Rag^-/- mice. However at 6 and 20 days following adoptive transfer Tregs became significantly reduced, in the absence of increased cell death, suggesting a reduction of maintenance or increased dedifferentiation of Tregs. Supporting this mechanism the MFI of Foxp3 expression was significantly reduced in the Tregs of HUPO mice and the control donor T cells in the HUPO-Rag^-/- recipients. Summary: Multiple mechanisms appear to contribute to the reduction of Tregs in HUPO mice. Increased Treg cell death and the reduction of central Tregs may be due to the reduction of IL-2. Increased dedifferentiation may also contribute to the reduction of Tregs in HUPO mice. Further studies with Foxp3-GFP Tregs will help determine the contributions of each mechanism.  

Conclusion: IL-2 and TGFβ are the essential cytokines for Treg differentiation and stability. Our observations suggest that IL-2 may be the effective treatment strategies for HUPO arthritis. The documented reduction of IL-2 in patients with RA suggests similar approach might be effective in patients with RA.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/mechanisms-regulating-the-loss-of-tregs-in-cd11c-flip-ko-mice-that-contribute-to-the-spontaneous-development-of-inflammatory-arthritis/