Control of CD8αα intestinal intraepithelial lymphocyte development by TGF-β

Intestinal intraepithelial lymphocytes (IEL) reside within the single cell epithelial layer of the intestine1, and are instrumental in safeguarding the integrity of mucosal immune responses. IEL contain two phenotypically distinct populations: conventional IEL, expressing the T cell receptor (TCR)αβ and either the co-receptor CD4 or heterodimers of CD8αβ, and unconventional IEL, which are either TCRαβ+ or TCRγδ+ and bear unique CD8αα homodimers. TCRαβ+CD8αα+ IEL remain a somewhat elusive cell type, with their function and development still poorly understood. TCRαβ+CD8αα+ IEL exhibit an “activated but resting” phenotype and have been suggested to have a regulatory role within the gut1. They express high levels of CTLA-4, PD-1, Lag3 and transforming growth factor-β (TGF-β) and inhibitory NK receptors2-4, molecules associated with immunoregulation. TCRαβ+CD8αα+ IEL prevent colitis when co-transferred with CD4+CD45RBhigh T cells into immuno-compromised mice5. Lymphocytic choriomeningitis virus (LCMV) infection of transgenic mice which express an LCMV-reactive TCR plus a cognate antigen trans-gene reinforced the regulatory potential of TCRαβ+CD8αα+ IEL; upon infection, TCRαβ+CD8αα+ IEL showed signs of activation, however, unlike conventional IEL they did not become cytotoxic nor secrete pro-inflammatory cytokines3. The developmental pathway followed by TCRαβ+CD8αα+ IEL has been debated6; once thought to be of extra-thymic origin, TCRαβ+CD8αα+ IEL are now generally considered to arise in the thymus7, 8, where the TCRαβ+CD8αα+ IEL precursors develop under the condition of a high affinity self-antigen-agonist4, 9. “Forbidden” TCRs have been identified in the TCRαβ+CD8αα+ IEL population10. However, the molecular pathways and regulatory factors controlling the development of TCRαβ+CD8αα+ IEL remain largely undetermined. Interleukin 15 (IL-15) was suggested to be required for their development, but IL-15 is not needed in the thymus, instead it maintains TCRαβ+CD8αα+ IEL in the gut11. The thymic “agonist selection” process utilized for the differentiation of TCRαβ+CD8αα+ IEL is shared with Foxp3+ regulatory T cells (Tregs)12 and CD1-d restricted NK T cells (iNKT)13. Since both Foxp3+ Tregs14 and iNKT15 require TGF-β and TCR ligation to develop and differentiate, we questioned whether TGF-β was also required for the development of TCRαβ+CD8αα+ IEL. We show here that TGF-β controls TCRαβ+CD8αα+ IEL development. Mice with either a null mutation in TGF-β1 (Tgfb1-/-) or a T cell-specific deletion of TGF-β receptor I (TβRI) lacked TCRαβ+CD8αα+ IEL. In contrast, transgenic mice that over-expressed TGF-β1 had an increased population of TCRαβ+CD8αα+ IEL. Mechanistically, we show that TGF-β prevents apoptosis by regulating the expression of Bcl-2 and Bim and induces CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors. TGF-β regulation of CD8α expression was not limited to the IEL, but was rather a more general phenomenon, as TGF-β induced the re-expression of repressed CD8α in lineage committed peripheral CD4+ T cells. CD8α expression was associated with suppression of Th-POK up-regulation and prevention of Runx3 down-regulation in CD4+ T cells. Thus we demonstrate a previously unrecognized role for TGF-β-signaling in the development of TCRαβ+CD8αα+ IEL and in the expression of CD8α in TCRαβ+ cells.