Post-Transcriptional Regulation of Interleukin-7 Receptor Expression

Division Of Basic Sciences - Nci

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Abstract

IL-7 is critical for the generation and maintenance of T cells. However, IL-7 is not produced by T cells themselves, such that T cells depend on exogenous IL-7 that is produced by stromal cells and dendritic cells. Importantly, the in vivo abundance of IL-7 is scarce and thought to be developmentally fixed. Thus, T cells must constantly compete for IL-7. Based on these observations, IL-7 signaling has been considered to be primarily controlled by the regulation of IL-7 receptor expression and not by the abundance of its ligand. The functional IL-7 receptor is a heterodimer composed of the IL-7-specific IL-7R alpha-chain (IL-7Ra) and the shared gc. Notably, T cell activation and cytokine signaling dramatically alter IL-7Ra expression, and we maintain an active research program studying the post-transcriptional regulatory mechanisms of its expression. Recently, our studies have made advances in understanding the role and expression of the shared gc receptor, which is crucial for signaling of the IL-7 receptor complex. Specifically, we conducted an in-depth analysis of an inborn error of human gc, where this particular genetic variation (c.961_962insC; p.Leu321fsX327) resulted in a frameshift mutation and introduction of a premature stop codon that generates a truncated form of gc proteins. In fact, the Leu321fsX327 mutation shortens the intracellular domain from 85 amino acids into 38 amino acids and then adds a neoepitope of 6 amino acids, i.e., PAARLQ. More importantly, this genetic variation destroys a critical region in the gc cytosolic tail, known as box 2. The gc box 2 region is defined as a stretch of 14 amino acids downstream of the PROX domain (cytoplasmic residue 5-37), which corresponds to amino acid 38-52, and that is critical for the binding of the tyrosine kinase JAK3. While this human genetic variation causes primary immunodeficiency because it is unable to transduce signaling by gc family cytokines (lack of JAK3 binding), it is unknown whether this variant can bind IL-7 and can internalize IL-7 upon ligand binding, or how it interferes with IL-7Ra and other proprietary receptors of the gc family. To experimentally address these questions, we generated mice with the same single nucleotide variation as found in humans using CRISPR/Cas9 technology. Leu321fsX327 mice phenocopied the human immunodeficiency as they were lymphopenic, lacked mature B cells, iNKT cells, gdT cells, and NK cells, and contained only small numbers of T cells. Thymopoiesis was also severely impaired, and the overall thymocyte profile was similar to that of germline gc-deficient mice. Unlike gc-KO mice, however, Leu321fsX327 thymocytes expressed large amounts of gc, and their surface gc levels exceeded those of WT cells. These results affirmed that the cytosolic tail of gc is critical for gc cytokine signaling, but they also indicated that the gc intracellular region is involved in controlling the transport and possibly recycling of surface gc protein. We aim to further address the mechanism how the surface abundance of gc proteins is controlled in a post-translational manner. Additionally, these results validate the use of Leu321fsX327 mice as an animal model to study the syntenic human gc point mutation, and we will make use of this new experimental system to address these issues. IL-7 and other gc family cytokines not only control the survival of T cells but their phenotypic and functional differentiation as well. In this regard, we found that gc cytokines control the expression of integrins, including the expression of integrin alphaE, also commonly referred to as CD103. Integrin CD103 binds to E-cadherin, a cell adhesion molecule that is predominantly expressed on epithelial cells in barrier sites such as the gut, lung or skin. Thus, CD103 had been considered critical to sequester tissue-resident memory CD8 T cells in barrier tissues. Importantly, we noted that naive CD8 T cells circulating through lymphoid organs also express copious amounts of CD103. However, the role of CD103 for CD8 T cells that reside and circulate outside of barrier sites has been unclear. By analyzing CD103-deficient or CD103-overexpressing mice, and generating mice with conditional deletion of E-cadherin, we identified a previously unappreciated role of CD103 in naïve CD8 T cells for their acquisition of memory-phenotype and effector function in lymph nodes. Importantly, we discovered that the gc family cytokine IL-15 is a negative regulator of CD103 expression, and that STAT5 signaling is downstream of IL-15 to suppress CD103 mRNA and protein expression. As IL-15 also drives and maintains CD8 memory cell differentiation, there is a direct correlation of CD103 downregulation and CD8 T cell differentiation. In this regard, we identified new subsets of dendritic cells expressing E-cadherin and producing type I IFN cytokines being responsible for driving the differentiation of memory CD8 T cells. As a corollary, conditional deletion of E-cadherin on DCs resulted in diminished effector CD8 T cell differentiation and increased tumor susceptibility while the forced expression of CD103 markedly bolstered the effector functions and anti-tumor activity of CD8 T cells. Collectively, these results demonstrate a previously unappreciated mechanism of cytokine-controlled CD103 expression in T cell immunity.