FOXP3 and CTLA-4 : how isoforms regulate immunological tolerance

The maintenance of immunological tolerance is vital for preventing the immune system to damage normal tissues and physiological function of the body. CD4+FOXP3+ regulatory T (Treg) cells can suppress immune responses in a dominant manner and are essential for immunological tolerance. Although many pathways and molecules have been attributed to the suppressive function of Treg cells, the exact nature of the Treg cell-mediated suppression program is still elusive.

In this thesis, I aimed to study the role of different protein isoforms, in particular FOXP3 and CTLA-4 isoforms, in the function of Treg cells and uncover the consequences of Treg cellmediated suppression on dendritic cells (DCs).

In paper I the aim was to understand the regulation and functional consequences of FOXP3 isoform expression in chronic inflammatory diseases in human. We found that FOXP3 isoforms were differentially expressed in biopsies from IBD patients. Moreover, the proinflammatory cytokine IL-1β promoted the exclusion of FOXP3 exon 7, which favoured the differentiation of naïve CD4+ T cells into Th17 cells.

In paper II we generated a mouse model (Foxp3δ2δ7 mouse) where Treg cells exclusively express an FOXP3 isoform lacking both exon 2 and exon 7 (FOXP3δ2δ7). We found that FOXP3δ2δ7 was unable to confer suppressive function of Treg cells in vivo. Homozygote Foxp3δ2δ7 mice phenocopied Foxp3 deficient scrufy mice and died from severe autoimmune disorder starting from 3 weeks of age. The Foxp3δ2δ7 mouse may provide a useful alternative for studying FOXP3 isoform function in Treg cells in vivo.

In paper III we investigated the role of CTLA-4 in Treg cell-mediated suppression on DCs. We generated a mouse model (Ctla4ex2fl/flFoxp3-Cre mouse) where Treg cells exclusively express an isoform of CTLA-4 that lacks the extracellular binding domain encoded by Ctla4 exon 2. Ctla4ex2fl/flFoxp3-Cre mice were born healthy and only started to display inflammatory lesions in environmental surfaces such as the lung and intestine after 4 months of age. In vitro co-culture experiments demonstrated that Treg cells from Ctla4ex2fl/flFoxp3- Cre mice were fully capable of inhibiting the up-regulation of CD80/CD86 on DCs. DCs cocultured with Ctla4ex2fl/flFoxp3-Cre Treg cells had decreased ability to support effector T cell proliferation. Unexpectedly, we also found that DCs up-regulated PD-L2 when co-cultured with wild type Treg cells in a CTLA-4 dependent manner. Collectively these data suggest that CTLA-4 mediated trans-endocytosis is a dispensable mechanism for Treg cell-mediated suppression and that Treg cell-mediated suppression on DCs is a multi-faceted process involving both CTLA-4 dependent and independent mechanisms.

In summary, these studies showed that it is necessary to investigate FOXP3 and Treg cell function on an isoform basis, and that Treg cell-mediated suppression on DCs is a multi-faceted program that involves both CTLA-4 dependent and independent mechanisms.

List of scientific papers

I. Mailer RKW, Joly A-L, Liu S, Elias S, Tegnér J, Andersson J. IL-1β promotes Th17 differentiation by inducing alternative splicing of FOXP3. Scientific Reports 5:14674, 2015.
https://doi.org/10.1038/srep14674

II. Joly A-L, Liu S, Dahlberg CIM, Mailer RKM, Westerberg LS, Andersson J. Foxp3 lacking exon 2 and 7 is unable to confer suppressive ability to regulatory T cells in vivo. Journal of Autoimmunity, 63:23-30, 2015.
https://doi.org/10.1016/j.jaut.2015.06.009

III. Liu S, Seitz C, Joly A-L, Klocke K, Wing K, Andersson J. Multi-faceted inhibition of dendritic cell function by CD4+Foxp3+ regulatory T cells. [Manuscript]