Background/Purpose:  We have previously identified CD4⁺CD25^–Foxp3^– regulatory T cells (Treg) that characteristically express lymphocyte-activation gene 3 (LAG3) produce IL-10. Recently we reported that these Treg regulate humoral immunity and ameliorate lupus pathologies in MRL/lprmice with producing large amounts of TGF-β3. However, the immunological significance of the co-existence of IL-10 and TGF-β3 has not been elucidated. Here we examined the synergistic role of IL-10 and TGF-β3 in the control of lupus pathogenesis.

Methods: In vivo, 4-hydroxy-3-nitrophenylacetyl (NP)- keyhole limpet hemocyanin (KLH) immunized C57BL/6 mice were intravenously injected with plasmid pCAGGS-Mock, pCAGGS-Il10, pCAGGS-Tgfb1, or pCAGGS-Tgfb3 vectors. NP-specific antibody titers were quantified by ELISA. Lupus-prone MRL/lpr mice were also injected with each pCAGGS-Mock, pCAGGS-Tgfb1, or pCAGGS-Tgfb3 vectors and comparative analyses of splenomegaly, autoantibody production, and renal pathology were conducted. In vitro, B cells stimulated with lipopolysaccharides (LPS) were cultured in the presence or absence of IL-10 and TGF-β3, and the proliferation and antibody production were assessed. Further, the comprehensive gene expression analyses by next generation sequencing (NGS) analysis and immunoblotting in each condition were conducted.

Results:  NP-specific IgG antibody titer was significantly suppressed in NP-KLH immunized mice only in cases with simultaneous administration of pCAGGS-Il10 and pCAGGS-Tgfb3. In MRL/lpr mice whose serum IL-10 levels were quite higher than MRL/+ mice, not pCAGGS-Tgfb1, but pCAGGS-Tgfb3 ameliorated lupus-like phenotypes, such as splenomegaly, glomerulonephritis, and anti-dsDNA antibody production. In in vitroexperiments, although LPS stimulated B cells either with IL-10 or TGF-β3 enhanced the proliferation and antibody production, conversely, the simultaneous addition of IL-10 and TGF-β3 suppressed them. Hierarchical clustering of NGS data revealed that the genes of “stimulated B cells with IL-10 and TGF-β3” and “no stimulated B cells”, but not “stimulated B cells either with IL-10 or TGF-β3”, located within the same cluster. Immunoblot analyses confirmed the specific down-regulation of mammalian target of rapamycin (mTOR) target molecules in “stimulated B cells with IL-10 and TGF-β3”.

Conclusion: We revealed that IL-10 and TGF-β3 synergistically suppressed humoral immune responses through suppressing mTOR signaling. Also, in lupus pathology, TGF-β3 exhibited the superior therapeutic effects compared to TGF-β1. The combination of IL-10 and TGF-β, especially TGF-β3, could be a novel therapeutic approach to systemic autoimmune diseases including lupus.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/synergistic-immunoregulatory-effects-of-il-10-and-tgf-%ce%b2-on-humoral-immunity/