CD4+ T cells deficient in signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) exhibit a selective impairment in adhesion to antigen presenting B cells but not dendritic cells (DC), resulting in defective germinal center formation. However, the nature of this selective adhesion defect remained unclear. We found that whereas T:DC interactions were primarily integrin-dependent, T:B cell interactions had both an early integrin-dependent phase and a sustained phase that also required SAP. We further found that the SLAM family member, CD84, was required for prolonged T:B cell contact, optimal T follicular helper function, and germinal center formation in vivo. Moreover, both CD84 and another SLAM member, Ly108, mediated T cell adhesion and participated in stable T:B cell interactions in vitro. Our results reveal insight into the dynamic regulation of T:B cell interactions and identify SLAM family members as critical components of sustained T:B cell adhesion required for productive humoral immunity.

SUMMARY X-linked lymphoproliferative syndrome, characterized by fatal responses to Epstein-Barr virus infection, is caused by mutations affecting the adaptor SAP, which links SLAM family receptors to downstream signaling. Although cytotoxic defects in SAP-deficient T cells are documented, the mechanism remains unclear. We show that SAP-deficient murine CD8+ T cells exhibited normal cytotoxicity against fibrosarcoma targets, yet had impaired adhesion to and killing of B cell and low avidity T cell targets. SAP-deficient cytotoxic lymphocytes showed specific defects in immunological synapse organization with these targets, resulting in inefficient actin clearance. In the absence of SAP, signaling through the SLAM family members Ly108 and 2B4 resulted in increased recruitment of the SHP-1 phosphatase, associated with altered SHP-1 localization and decreased activation of Src kinases at the synapse. Hence, SAP and SLAM receptors regulate positive and negative signals required for organizing the T:B cell synapse and setting thresholds for cytotoxicity against distinct cellular targets.

The promyelocytic zinc finger transcription factor (PLZF) is required for the development of activated phenotypes in NKT and other innate T lymphocytes. Although strong TCR stimulation has been implicated in the induction of PLZF, the factors regulating PLZF expression are incompletely understood. We show here that costimulation of pre-selection double positive thymocytes through the Signaling lymphocyte activation molecule (SLAM) family receptor Ly108 markedly enhanced PLZF expression compared to that induced by T cell receptor (TCR) stimulation alone. Costimulation with Ly108 increased expression of Egr-2 and binding of Egr-2 to the promoter of Zbtb16, which encodes PLZF, and resulted in PLZF levels similar to those seen in NKT cells. In contrast, costimulation with αCD28 failed to enhance Egr-2 binding and Zbtb16 expression. Moreover, mice lacking Ly108 showed decreased numbers of PLZF expressing CD4+ T cells. Together, these results support a potential role for Ly108 in the induction of PLZF.

Ly108 (CD352) is a member of the Signaling Lymphocyte Activation Molecule (SLAM) family of receptors that signals through SLAM-associated protein (SAP), an SH2 domain protein that can function by the recruitment of Src family kinases or by competition with phosphatases. Ly108 is expressed on a variety of hematopoietic cells and with especially high levels on developing thymocytes. We find that Ly108 is constitutively tyrosine phosphorylated in murine thymi in a SAP- and Fyn kinase-dependent manner. Phosphorylation of Ly108 is rapidly lost after thymocyte disaggregation, suggesting dynamic contact-mediated regulation of Ly108. Similar to recent reports, we find at least 3 isoforms of Ly108 mRNA and protein in the thymus, which are differentially expressed in the thymi of C57Bl/6 and 129S6 mice that express the lupus-resistant and lupus-prone haplotypes of Ly108, respectively. Notably, the recently described novel isoform, Ly108-H1, is not expressed in mice having the lupus-prone haplotype of Ly108, but is expressed in C57Bl/6 mice. We further provide evidence for differential phosphorylation of these isoforms; the novel Ly108-H1 does not undergo tyrosine phosphorylation, suggesting it functions as a decoy isoform that contributes to the reduced overall phosphorylation of Ly108 seen in C57Bl/6 mice. Our studies suggest that Ly108 is dynamically regulated in the thymus and shed light on Ly108 isoform expression and phosphorylation.

Promyelocytic zinc finger transcription factor (PLZF) is an important transcription factor expressed by natural killer T (NKT) cells and a variety of other innate T cells. Development of these PLZF positive cells require the adaptor molecule SLAM- associated protein (SAP) and the interaction of the signaling lymphocyte activation molecule (SLAM) family member receptors in addition to TCR stimulation. However, it remains unclear how these interactions influence the development of innate thymocytes. We have found that costimulation of pre-selection double positive (PS-DP) thymocytes with an agonist to the SLAM family receptor Ly108 greatly enhances PLZF expression compared to TCR stimulation alone. Furthermore, PLZF expressing cells were reduced in Ly108 deficient mice and increased in transgenic mice expressing a hyperactive Ly108 isoform. Costimulaiton with Ly108 also increased the expression of Egr-2 and the binding of Egr-2 to the PLZF (Zbtb16) promoter. Surprisingly costimulation with CD28 was unable to enhance Egr-2 nor PLZF expression in vitro suggesting that Ly108 has a unique role in PLZF expression. This work provides a novel mechanism by which innate thymocyte development is regulated via Ly108 and possibly other SLAM family receptor interactions.