Studies on SLAM Family member Ly108, A Receptor involved in Thymocyte Development and Autoimmunity

Ly108 is a member of the SLAM family of receptors, which are self-ligands and are expressed on a variety of hematopoietic cells with especially high levels on developing thymocytes. SLAM receptors signal through SAP, an SH2-domain containing adaptor protein that functions by recruiting Src family kinases or by competing with phosphatases. The importance of SLAM receptors is highlighted in the primary human immunodeficiency XLP1, which is caused by mutations affecting SAP. Elements within the genetic locus encoding the SLAM family have been correlated with development of autoimmunity and polymorphisms that affect Ly108 splicing have a strong correlation with lupus development in mice. Ly108 is highly expressed on thymocytes and, along with SAP, is also implicated in the development of innate-like T cells such as NKT cells. Despite its high expression in thymus and implication in autoimmunity, Ly108 and its downstream signaling have not been well characterized. The aim of this study was to biochemically characterize Ly108 in the thymus and investigate differences in Ly108 from lupus-prone and -resistant mouse strains. The second part of my study focused on the physiological effect of signaling from Ly108 during thymocyte development. Using immunoprecipitations of intact thymi, I found that Ly108 is constitutively tyrosine phosphorylated in a SAP and Fyn kinase-dependent manner. Surprisingly, phosphorylation of Ly108 was rapidly lost within minutes of thymic disaggregation, suggesting dynamic contact-mediated regulation of Ly108 signaling. In contrast, re-induction of Ly108 phosphorylation occurred with slow kinetics. Cloning Ly108 from C57Bl/6 mice revealed at least 3 isoforms, including a novel isoform. These isoforms were differentially expressed in the thymi of C57Bl/6 and 129S6 mice which express the lupus-resistant and -prone haplotypes respectively. Notably, the novel isoform, which was also recently cloned by another group and shown to have lupus ameliorating properties, was absent in mice having the lupus-prone haplotype, but was expressed in lupus-resistant C57Bl/6 mice. By generating an antibody that was specific for distinct isoforms of Ly108, I further provided evidence for differential phosphorylation of these isoforms. Importantly, the novel lupus-ameliorating isoform was refractory to tyrosine phosphorylation in thymi or cell lines, suggesting it could function as a decoy isoform, thereby leading to mitigated Ly108 phosphorylation seen in C57Bl/6 mice. Based on this study, we predict that Ly108 signaling is tightly regulated in the thymus with quick OFF and slow ON kinetics that offers stringency to thymocyte interactions--only interactions of significant duration trigger productive Ly108 signaling. For the second part of my dissertation research, I studied the physiological relevance of Ly108 signaling in thymocytes. Using in vitro stimulation assays, I found that Ly108 signaling potentiates and prolongs TCR signaling that developing thymocytes encounter in the thymus. Apart from amplifying TCR signaling, Ly108 signaling transmits a unique signal that results in induction of a potent transcription factor of NKT cells called Promyelocytic Leukemia Zinc Finger, or PLZF. Using in vitro stimulation of pre-selection CD4 CD8 double positive (PS-DP) thymocytes, followed by Chromatin IPs, we show that Ly108 stimulation induces PLZF and is associated with sequential expression and maintenance of the NFAT-dependent Early Growth Response protein called Egr-2. This effect is specific to Ly108 costimulation and is not recapitulated by other costimulatory molecules such as CD28. Given the dramatic effects of Ly108 stimulation on PLZF induction, the rapid dephosphorylation (with relatively slow induction of phosphorylation) of Ly108 suggests that Ly108 activity might be tightly controlled in the thymus. A stringent control of Ly108 phosphorylation might prevent the full induction of PLZF unless contact is maintained between developing double positive cells, as may occur during the selection of NKT and other hematopoietically selected innate T cells. Thus, the rapid dephosphorylation Ly108 could provide a safety mechanism that would allow only certain cells to develop and maintain high PLZF expression. This would limit the number of CD4+T cells that develop innate-like characteristics, the uncontrolled activity of which might contribute to autoimmunity. Molecular dissection of signaling pathways that are downstream of Ly108 and other SLAM family members is an important step to help elucidate pathways responsible for innate-like T cell development and for systemic autoimmune pathogenesis