Abstract
During a search for immunomodulatory receptors in the chicken genome, we identified a previously cloned chicken sequence as CRTAM homologue by its overall identity and several conserved sequence features. For further characterization, we generated a CRTAM specific mab. No staining was detectable in freshly isolated cell preparations from thymus, bursa, caecal tonsils, spleen, blood and intestine. Activation of splenocytes with recombinant IL-2 increased rapid CRTAM expression within a 2 h period on about 30% of the cells. These CRTAM+ cells were identified as CD8+ γδ T lymphocytes. In contrast, CRTAM expression could not be stimulated on PBL with IL-2, even within a 48 h stimulation period. As a second means of activation, T cell receptor (TCR) crosslinking using an anti-αβ-TCR induced CRTAM on both PBL and splenocytes. While CRTAM expression was again rapidly upregulated on splenocytes within 2 h, it took 48 h to reach maximum levels of CRTAM expression in PBL. Strikingly, albeit the stimulation of splenocytes was performed with anti-αβ-TCR, CRTAM expression after 2 h was mainly restricted to CD8+ γδ T lymphocytes, however, the longer anti-TCR stimulation of peripheral blood lymphocytes (PBL) resulted in CRTAM expression on αβ T lymphocytes. In order to characterize the potential ligand we cloned and expressed chicken Necl-2, a member of the nectin and nectin-like family which is highly homologous to its mammalian counterpart. Three independent assays including a reporter assay, staining with a CRTAM-Ig fusion protein and a cell conjugate assay confirmed the interaction of CRTAM with Necl-2 which could also be blocked by a soluble CRTAM-Ig fusion protein or a CRTAM specific mab. These results suggest that chicken CRTAM represents an early activation antigen on CD8+ T cells which binds to Necl-2 and is upregulated with distinct kinetics on αβ versus γδ T lymphocytes.
Highlights
CD8+ T cells and natural killer (NK) cells display a number of receptors that are important in the recognition of target cells
Identification of chicken CRTAM In an attempt to identify novel Ig-like receptors with immunomodulatory potential we found a previously cloned gene designated chicken thymic activation and developmental sequence (cTADS)
CRTAM is transiently expressed on activated cells Since CRTAM is a receptor known to be upregulated upon activation, we evaluated if activation of chicken splenocytes would lead to CRTAM expression
Summary
CD8+ T cells and natural killer (NK) cells display a number of receptors that are important in the recognition of target cells. One group of receptors expressed by both cell types belongs to the immunoglobulin superfamily (IgSF) and includes CD226 (DNAM1), TIGIT (WUCAM), CD96, and CRTAM (CD355) [1,2] These receptors bind to members of the nectin and nectin-like family, which are IgSF members, too and mediate Ca2+ independent homophilic and heterophilic adhesion. CRTAM was initially identified in a study devised to characterize upregulated genes in NK-T cells and received its name as ‘‘class I-restricted T cell-associated molecule’’ to denote its restricted expression pattern on CD8+ T cells. It has been further characterized as an important activation marker for both CD8+ T cells and NK-T cells [3]. Its intracytoplasmic domain contains a carboxyl–terminal class I PSD-95/Disc-large/ZO-1 (PDZ)-binding motif, capable of interacting with PDZ domain
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