Background/Purpose: Abatacept is a recombinant fusion protein comprising cytotoxic T lymphocyte antigen 4 (CTLA4) and Fc region of immunoglobulin (Ig), which is efficacious against rheumatoid arthritis (RA). Its mechanism of action is believed to be competitive inhibition of T-cell co-stimulation mediated through binding of CD28 to CD80/CD86 on antigen-presenting cells. On the other hand, it has been shown that binding of CTLA4-Ig to CD80/CD86 on monocytes, macrophages, and osteoclast precursors is capable of transmitting intracellular signaling pathways. This signaling is potentially involved in the therapeutic action of abatacept, but detailed mechanisms still remain unclear. This study is aimed to investigate direct effects of CTLA4-Ig on circulating monocytes, which might contribute to its efficacy for suppressing pathogenic process of RA.

Methods: This study enrolled 34 RA patients and 13 controls, including patients with non-inflammatory rheumatic disorders or healthy individuals. Circulating monocytes were isolated from peripheral blood mononuclear cells by negative selection with magnetic-activated cell sorting system, and were cultured in the presence or absence of CTLA4-Ig, CD28-Ig, or Ig alone for 24 hours. In some experiments, anti-CD80, CD86, and/or isotype-matched control monoclonal antibody (mAb) were added in the culture. The recovered cells were subjected to flow cytometry to evaluate expression levels of CD14, CD16, CD32, CD40, CD54, CD62L, CD64, CD80, CD86, CCR2, CXCR2, CD273, CD274, and CD275, and IL-1β, IL-6, IL-8, IL-10, IL-12p70, IFNγ, MCP-1, TNFα were measured in culture supernatants by multiplex particle-based flow cytometric assay. Expression of candidate molecules was further examined by immunoblots using total cellular extracts of monocytes cultured with CTLA4-Ig or CD28-Ig. Statistical analysis was maded using non-parametric Mann-Whitney U test.

Results: In a pilot study using 5 RA patients and 5 controls, we selected CD64, CD80, CD86, CCR2, and CXCR2 as candidate molecules whose expression levels were modulated by CTLA4-Ig stimulation in a fashion different from CD28-Ig stimulation. The validation study involving 20 RA patients and 8 controls demonstrated that treatment with CTLA4-Ig significantly down-regulated expression of CD64 or Fcγ receptor I in circulating monocytes, but stimulation with CD28-Ig had no effect. The CD64 down-regulation induced by CTLA4-Ig was not found in cultures with control Ig alone, and was confirmed by immunoblots. This effect was observed in both RA patients and controls, while CD64 expression levels on circulating monocytes tended to be higher in RA patients compared with controls. Finally, CTLA4-mediated CD64 down-regulation was completely abolished by anti-CD86 mAb, but not by anti-CD80 mAb.

Conclusion: Efficacy of abatacept is associated with a high titer of anti-citrullinated protein antibodies, which form immune complexes and induce activation of monocytes/macrophages through binding to Fcγ receptors. These findings suggest that therapeutic effects of abatacept on RA are mediated, in part, through down-regulation of Fcγ receptor I on circulating monocytes via direct binding to CD86.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ctla4-signaling-down-regulates-fc%ce%b3-receptor-i-expression-on-circulating-monocytes-a-potential-mechanism-of-action-of-abatacept-for-ra/