Background/Purpose:

Depletion of B cells using a monoclonal antibody against CD20 expressed on mature B cells has proven efficacious in patients with rheumatoid arthritis (RA). We have previously shown in our mouse model of RA, proteoglycan induced arthritis (PGIA), that depletion of B cells leads to enhanced T regulatory cell (Tregs) numbers and activity essential in the suppression of arthritis. This increase in Treg numbers and function correlated with a decrease in CD4⁺ T cell IFN-γ production. In this study, we investigate the effects of B cells on the generation of Tregs and how IFN-γ contributes to this process.

Methods:

An anti-CD20 mAb was used to deplete B cells in FOXP3^egfp reporter mice 5 days after immunization intraperitoneally (i.p.) with a recombinant protein for the G1 domain of the human proteoglycan in adjuvant. Mice were sacrificed 4 days after B cell depletion. Naïve and memory CD4⁺CD25^– T cells based on CD62L expression were isolated from wild type (WT) and IFN-γ knockout (IFN-γ^-/-) mice and cultured under Treg differentiating conditions in the presence of  proinflammatory cytokines IL-12 and IFN-γ. Flow cytometry was performed and Tregs were phenotyped as CD4⁺ Foxp3⁺ cells. ELISA was used to measure cytokine production. In the in vivo transfer model CD4⁺CD62L⁺Foxp3^– T cell were sorted from transgenic animals and transferred into congenic animals that were later immunized and B cell depleted. Mixed bone marrow chimeras were created by transferring bone marrow cells from B cell deficient and IFN-γ^-/- mice in a 1:1 ratio into irradiated recipients. These mice were later immunized i.p. with G1 in adjuvant 3 times at 3 week intervals. Paws were examined every other day for arthritis.

Results:

We show that in vitro IFN-γ suppresses the differentiation of CD4⁺CD25^–CD62L⁺ T cells into Foxp3⁺ Tregs. CD4⁺CD25^–CD62L^– T cells were resistant to Treg differentiation and this resistance correlates with copious IFN-γ production. Through an in vivo transfer model of transgenic T cells into congenic mice, we find that CD4⁺CD62L⁺Foxp3^– T cells are converted into Tregs more effectively in B cell depleted mice as compared to the control group. To determine if IFN-γ is responsible for the suppression of Treg generation, we setup an in vitro Treg differentiation assay using WT and IFN-γ^-/- CD4⁺CD25^–CD62L⁺ T cells in the presence of TGF-β and WT or IFN-γ^-/- B cells. We find that IFN-γ^-/-B cells resulted in an increase in Treg differentiation in comparison to WT B cells. Chimeras with a specific deficiency in IFN-γ in B cells also show an increase in the percentage of Tregs.

Conclusion:

Our findings demonstrate that B cell derived IFN-γ can negatively regulate the generation of Tregs in arthritis.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cell-derived-ifn-%ce%b3-contributes-to-the-negative-regulation-of-t-regulatory-cell-differentiation-in-arthritis/