Control of T Lymphocyte Signaling by Ly108, a Signaling Lymphocytic Activation Molecule Family Receptor Implicated in Autoimmunity

Ming-Chao Zhong,André Veillette

doi:10.1074/jbc.m800209200

Abstract

The signaling lymphocytic activation molecule family of receptors has been implicated in the pathophysiology of autoimmunity in humans and mice. One member of the family, Ly108, was strongly linked to lupus susceptibility in mice. High expression of a Ly108 isoform, Ly108-1, was observed in lymphocytes of lupus-prone mice. Herein, we examined the molecular basis for the influence of Ly108 on lupus susceptibility by studying Ly108 signal transduction in T cells. We observed that Ly108 was able to mediate a tyrosine phosphorylation signal implicating Ly108, Vav-1, and c-Cbl in a manner strictly dependent on engagement of the extracellular domain of Ly108 and co-expression of the Src homology 2 (SH2) domain-containing adaptor signaling lymphocytic activation molecule (SLAM)-associated protein (SAP). Evaluation of T cells from mice carrying mutations in the SAP-FynT pathway indicated that Ly108-triggered protein tyrosine phosphorylation was due to the capacity of SAP to recruit FynT. Importantly, Ly108-1 was more apt at triggering tyrosine phosphorylation signals in T cells when compared with the predominant Ly108 isoform found in non-lupus-prone mice, Ly108-2. This difference was due in part to the presence in Ly108-1 of a unique intra-cytoplasmic tyrosine-based motif that promoted Ly108 signal transduction. Together these data provided a molecular explanation for the involvement of Ly108 in lupus susceptibility in mice.

Highlights

Susceptibility to autoimmune diseases such as systemic lupus erythematosus and diabetes is greatly influenced by genetic factors
Mouse Ly108 Is Expressed on T Cells and B Cells but Not on Most Mature natural killer (NK) Cells—Newly generated anti-Ly108 monoclonal antibody (mAb) 3E11 was used to study the expression of Ly108 on mouse immune cells (Fig. 1)
In the mouse, Ly108 is expressed on T cells, B cells, NK-T cells, and dendritic cells (DCs) but not on most NK cells

Summary

EXPERIMENTAL PROCEDURES

Cells—BI-141 is a mouse T-cell line [17]. Derivatives stably expressing various forms of Ly108 or Tac-Ly108 with or without SAP were generated by transfection [6]. Cells expressing Tac-SLAM or Tac-2B4 in combination with SAP were described [6, 18]. Fibroblasts expressing Ly108 were generated by transfection of DCEK cells (L929 fibroblasts stably expressing I-Ek) with a cDNA encoding a cytoplasmic domain-deleted version of. A mAb recognizing mouse Ly108 (mAb 3E11) was generated in rats using the Ly108-Fc fusion protein as immunogen This antibody recognizes Ly108 but not the other SRRs (supplemental Fig. 1). An isotype control was purchased from eBiosciences (San Diego, CA) For flow cytometry both antibodies were coupled to Alexa 647 according to the protocol outlined by the manufacturer (Invitrogen). For self-ligation of Ly108 on thymocytes, thymocytes (10 ϫ 106) from wild-type mice were incubated for 40 min at 37 °C with adherent DCEK fibroblasts expressing or not a cytoplasmic domain-truncated version of Ly108, previously plated in tissue culture dishes. Radioactive signals were quantitated using a Storm 860 PhosphorImager (GE Healthcare)

RESULTS