Negative Regulation of T Cell Receptor Signaling by Siglec-7 (p70/AIRM) and Siglec-9

Abstract

Siglec-7 (p70/AIRM) and Siglec-9 are "CD33"-related siglecs expressed on natural killer (NK) cells and subsets of peripheral T cells. Like other inhibitory NK cell receptors, they contain immunoglobulin receptor family tyrosine-based inhibitory motifs in their cytoplasmic domains, and Siglec-7 has been demonstrated to negatively regulate NK cell activation. Based on reports of the presence of these siglecs on T cells, we sought to determine if they are capable of modulating T cell receptor (TCR) signaling using Jurkat T cells stably and transiently transfected with Siglec-7 or Siglec-9. Following either pervanadate stimulation or TCR engagement, both Siglecs exhibited increased tyrosine phosphorylation and recruitment of SHP-1. Effects of Siglec-7 and -9 were also evident in downstream events in the signaling pathway. Both siglecs reduced phosphorylation of Tyr 319 on ZAP-70, known to play a pivotal role in up-regulation of gene transcription following TCR stimulation. There was also a corresponding decreased transcriptional activity of nuclear factor of activated T cells (NFAT) as determined using a luciferase reporter gene. Like all siglecs, Siglec-7 and -9 recognize sialic acid-containing glycans of glycoproteins and glycolipids as ligands. Mutation of the conserved Arg in the ligand binding site of Siglec-7 (Arg 124 ) or Siglec-9 (Arg 120 ) resulted in reduced inhibitory function in the NFAT/luciferase transcription assay, suggesting that ligand binding is required for optimal inhibition of TCR signaling. The combined results demonstrate that both Siglec-7 and Siglec-9 are capable of negative regulation of TCR signaling and that ligand binding is required for optimal activity.The human siglec family of cell receptors is composed of eleven members of the Ig superfamily, which are functionally related by their ability to bind sialic acid-containing carbohydrates of glycoproteins and glycolipids as ligands (1-3). The Siglecs are predominately and differentially expressed on a wide variety of white blood cells (2, 3), the notable exceptions being myelin-associated glycoprotein expressed in glial cells (4, 5) and Siglec-6 expressed in placenta (2, 6). The extracellular region has a variable number of C2-set Ig domains and a single homologous N-terminal "V-set" domain that binds to sialic acids (2). Crystal structure analysis of two Siglecs, sialoadhesin (Siglec-1) and Siglec-7, have revealed a shallow sialic acid binding pocket with a conserved sequence of six amino acids in the tip of the C-CЈ loop that influences the specificity for binding various sialoside sequences found in nature (7-10). Within this sequence, a conserved arginine residue coordinates with the C-1 hydroxyl group of the sialic acid and is required for binding as evidenced by Arg to Ala mutations that abrogate binding to sialic acid-containing ligands (11,12).Another characteristic feature of the siglecs is the presence of consensus immunoglobulin receptor family tyrosine-based inhibitory motifs (ITIM) 1 in the cytoplasmic domains of all but si...

Siglec-5 (CD170) Can Mediate Inhibitory Signaling in the Absence of Immunoreceptor Tyrosine-based Inhibitory Motif Phosphorylation

“…Thus, weak constitutive interactions between clustered Siglec-5 and SHP-1 and/or SHP-2 could be important in maintaining leukocytes in the quiescent state until activation is triggered via appropriate receptors. Siglec-5 clustering and inhibitory signaling could be mediated via cis interactions with appropriately sialylated activatory receptors, as shown recently for Siglecs-7 and -9 in transfected human Jurkat T cells (36) and CD33 expressed on human monocytes (37).…”

Siglec-7 Undergoes a Major Conformational Change When Complexed with the α(2,8)-Disialylganglioside GT1b

“…Siglec-7 is an inhibitory receptor expressed on NK cells and to a lesser extent, monocytes and a subset of CD8ϩ T cells (13,(17)(18)(19)(20)(21). In vitro binding studies have revealed that Siglec-7 shows a marked preference for glycoconjugates bearing ␣(2,8)-linked disialic acid and branched ␣(2,6)-linked sialic acid (22).…”

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

“…Although Siglec-5 is expressed predominantly on myeloid cells such as neutrophils and monocytes/macrophages, Siglec-10 is also expressed on B cells (6,10), and recent evidence suggests that Siglec-10 acts with properly glycosylated CD52 to allow binding and inhibition of T cell activation (51). Moreover, Siglec-9 is expressed on a subpopulation of CD8 T cells (54). Therefore, up-regulation of LGALS3BP may directly influence T cell activation against tumors and hamper the generation of an antitumoral Th1 immune response.…”