Lineage stability and effector function of CD4+FOXP3+ regulatory T cells

Abstract

This work focuses on regulatory T (Treg) cell biology in the context of defining the role of forkhead box protein 3 (FOXP3) isoform expression and Treg mediated suppression via CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). Treg cells are crucial in maintaining immunological tolerance and they in part act by suppressing dendritic cells (DCs). Previous studies have demonstrated that Treg cells express the co-inhibitory receptor CTLA-4, which is essential for Treg cells ability to control the expression of the costimulatory molecules CD80/CD86 on DCs. In study I we generated a novel mouse strain that exclusively expresses the naturally occurring ligand-independent CTLA-4 isoform (liCTLA-4) in Treg cells only. This isoform cannot control CD80/CD86 expression by direct binding to these molecules. One of the key findings in this study is that these Ctla4ex2fl/flFoxp3-Cre mice did not develop a lymphoproliferative phenotype early in life. When we extended our study, we saw that older Ctla4ex2fl/flFoxp3-Cre mice (6 months) developed an inflammatory phenotype in the lung. Interestingly, Ctla4ex2fl/flFoxp3-Cre mice had an increased number of Treg cells, in particular Treg cells lacking CD25 expression. The dramatic increase of Treg cells could potentially compensate for any functional impairment due to altered CTLA-4 expression. Thus, we isolated Treg cells for in vitro assays to further investigate Treg cell function using an DC suppression assay that we established. We found that Treg cells isolated from Ctla4ex2fl/flFoxp3-Cre were able to control CD80 and CD86 expression. However, Treg cells isolated from Ctla4ex2fl/flFoxp3-Cre failed to support upregulation of PD-L2 on suppressed DCs.

The transcription factor FOXP3 is a key regulator for Treg cell differentiation and is required to maintain their suppressive phenotype. FOXP3 occurs as four distinct isoforms, full-length FOXP3 and isoforms lacking exon 2 and or exon 7. In study II we determined the FOXP3 isoform expression in plaques and blood from patients suffering from atherosclerotic disease. We found a positive correlation between FOXP3D2 splice variant expression and plaque stability in plaque tissue that was not apparent in blood. Another key finding from this study is that during Treg cell activation the overall upregulation of FOXP3 expression is mainly due to an increased expression of the isoform FOXP3D2. In study III and IV we reported on two patients with a novel frameshift mutation in the FOXP3 gene (NM_014009.3:c.305delT) located in exon 2 which results in a premature stop codon and consequently the loss of any isoforms expressing exon 2. Both patients had a very mild IPEX phenotype, which strongly suggests FOXP3D2 alone is able to at least partially maintain Treg cell development and function. Investigating the female carrier, we saw approximately 20% of the Treg pool had the c.305delT mutation not matching the expected 50% from random X-chromosome inactivation. RNA sequencing allowed us to identify a set of genes, several which previously have been demonstrated to regulate FOPX3 expression, that are specifically regulated by full-length FOXP3. This gene set may explain the loss of c.305delT Treg cells and we currently favor the view that they regulate the lineage stability of Treg cells.