The signalling signature downstream of the notch receptor in CD4⁺CD25^highregulatory T cells in RA defines anergic cells: insight into resistance to anti-tumour necrosis factor therapies

Background

We have previously reported on the abnormal expression of the different members of the Jagged/Notch signalling pathway in early rheumatoid arthritis (RA) patients. HES1 and Deltex are two major signalling molecules resulting from the transduction of notch signals. We have shown that signalling through the Jagged/Notch pathway is involved in the development of an anergic phenotype in a T-cell clone model in vitro. We have also used this model to establish a Notch signalling signature characterising a T-cell suppression reaction. We analysed the Notch signature of effecter CD4⁺ T cells and CD4⁺CD25^high regulatory T cells in RA patients and compared it with healthy controls.

Methods

The HA1.7 CD4⁺ T-cell clone develops an anergic phenotype associated with high CD25 expression when stimulated with its cognate peptide in the absence of co-stimulation. These cells were used in a suppression assay of fresh HA1.7 (CD25^low) cells, activated by anti-CD3/CD28 antibodies in proliferation assays. Real-time PCR for the quantification of gene expression was undertaken. CD4⁺CD25^- T cells and CD4⁺CD25^high regulatory T cells were sorted using flow cytometry from 50 ml blood from five RA patients with early, disease-modifying anti-rheumatic drug naive disease and seven healthy controls. Gene expression was quantified by real-time PCR.

Results

During a suppression reaction of fresh HA1.7 cells (CD25^low) by anergic HA1.7 expressing high levels of CD25, the expression of HES1 was induced; however, Deltex was inhibited. This signature is unique and differs from either an activation (downregulation of both) or an anergy signalling signature (upregulation of both). Results from a similar quantification in RA patients, suggest that CD4⁺CD25^high T cells in RA have the signature of anergic cells with high levels of both HES1 and Deltex, whereas control regulatory cells display a signature comparable with previously published data (low HES1, high Deltex) [1]. Preliminary results in RA patients, pre and post TNF-blockade, suggest that the response to therapy is associated with a normalisation of the Notch signature post treatment to resemble the signature of control activated regulatory T cells (upregulated HES1 and downregulated Deltex).

Conclusions

CD4⁺CD25^high regulatory T cells in RA appear anergic and may therefore not be able to suppress an undesirable auto-immune reaction very early in the RA disease process. Altogether, our results on the abnormal baseline expression of Jagged/Notch molecules and these atypical signalling signatures suggest that the Jagged/Notch pathway may be involved in the pathogenesis of RA.