Human class-I restricted T cell associated molecule is highly expressed in the cerebellum and is a marker for activated NKT and CD8+ T lymphocytes
Introduction
Various subpopulations of thymocytes representing a progression of developing T cells have been identified (Godfrey and Zlotnik, 1993). In previous studies, we undertook a systematic search for new genes expressed by thymocyte subsets in the mouse thymus. One subset that appeared particularly interesting was a very active population of CD4–CD8− thymocytes that could be differentiated from other thymocyte precursors through their expression of T cell receptor (Fowlkes et al., 1987). This subset was found to express a canonical T cell receptor repertoire (invariant T cell receptor α and β chains) (Lantz and Bendelac, 1994) and produce very large amounts of some cytokines, including IL4 and IFNγ, upon activation (Zlotnik et al., 1992). Together with a small CD4+ population, they are part of what are now known as NKT cells (Godfrey and Kronenberg, 2004). The unusual ability of NKT cells to produce large amounts of specific cytokines led us to study in more detail the molecules expressed by these cells upon activation. We confirmed that activated CD3+ CD4–CD8− thymocytes produce large amounts of IL4 and IFNγ mRNA, and also found that they express very large amounts of known chemokine transcripts, including CCL3/MIP1α, CCL4/MIP1β and CCL5/Rantes and XCL1/lymphotactin (Kelner et al., 1994, Kennedy et al., 1995). Among the most highly expressed transcripts was one encoding a membrane molecule, a novel member of the immunoglobulin supergene family, which we named Class-I restricted T cell activation molecule (CRTAM) because we found it specifically expressed only by activated CD8 or NKT cells. In this first description of CRTAM, we characterized the mouse molecule and confirmed that it was expressed, following activation, by CD8 and NKT cells (Kennedy et al., 2000).
In the present study, we characterize human CRTAM. We had previously observed that CRTAM mRNA was expressed in the CNS (Kennedy et al., 2000) and in a detailed survey of gene expression across > 70 normal human tissues we found the highest levels of CRTAM transcripts in the cerebellum. We then used antibodies specific for human CRTAM to determine which cell types express the protein and show that human CRTAM is strongly expressed in Purkinje neurons. Bioinformatics analyses indicate that CRTAM is a member of the nectin family of cell adhesion proteins that form homo- and heterodimers with each other. Nectin-like molecule 2 (Necl-2) was recently identified as the binding partner to CRTAM (Galibert et al., 2005) and, using our expression survey, we have found significant Necl-2 expression in the cerebellum raising the possibility that CRTAM/Necl-2 heterodimers participate in cell–cell interactions between Purkinje neurons and adjacent cells. We also used the CRTAM antibodies to further characterize CRTAM expression on human lymphocytes and show that this molecule, like its mouse counterpart, is expressed on activated CD8 and NKT cells. Thus, CRTAM holds promise as a marker for monitoring these activated cell types in inflammatory and pathogenic processes.
Section snippets
Isolation and activation of peripheral blood mononuclear cells
Heparinized peripheral blood samples were obtained from healthy donors at The Hospital Juarez de Mexico Blood Bank (Mexico City, Mexico). Total peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples using Ficoll-Paque Plus according to manufacturer's protocol (Amersham Biosciences, AB Uppsala, Sweden). Where indicated, CD4+ T cells, CD8+ T cells or CD14+ cells, were purified by cell-sorting (FACSVantage, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). To
CRTAM is a new member of the nectin-like gene family
We subjected the CRTAM protein sequence to PBLAST analysis and found significant alignment to members of the nectin and nectin-like families of adhesion proteins (Mizoguchi et al., 2002, Takai et al., 2003). The alignment shown in Fig. 1A indicates that CRTAM has approximately 30% identity and 55% similarity to the human nectins and is most closely related to the nectin-like gene group, see Fig. 1B. However, while all nectins and nectin-like proteins contain 3 Ig domains, CRTAM contains 2.
Discussion
Using sequence alignment, we have identified CRTAM as distant relative of the Nectin superfamily. Nectins are Ca2+-independent cell adhesion molecules, initially identified as viral receptors (Lopez et al., 1995, Takahashi et al., 1999). We report here that CRTAM shares other properties with this gene family. The human CRTAM gene is located in close proximity to two other Nectin superfamily members on the long arm of chromosome 11 (11q23.3); nectin 1 and Necl-2. The latter is now known to be
Acknowledgements
We thank Monica Tsang (R&D Systems) for making the anti-CRTAM monoclonals 210206 and 210213 available for these studies; Benjamin Ortiz-Lopez and Hector Romero-Ramires for excellent technical support; Victor Rosales-Garcia for cytometry assistance. This work was supported in part by a research grant 2003-CO1-139 from SSA/IMSS/ISSSTE-CONACYT. G.P.L. was recipient of a CONACYT scholarship (115040).
References (26)
- et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
Nectin-like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I restricted T-cell associated molecule
J. Biol. Chem.
(2005) - et al.
Control points in early T-cell development
Immunol. Today
(1993) - et al.
PDZ domains: structural modules for protein complex assembly
J. Biol. Chem.
(2002) - et al.
Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene
Gene
(1995) - et al.
Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily
Cell
(1989) - et al.
Biology and pathology of nectins and nectin-like molecules
Curr. Opin. Cell Biol.
(2004) - et al.
Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell–cell adhesion and transmembrane protein localization in epithelial cells
J. Biol. Chem.
(2003) - et al.
The roles of cadherins and nectins in interneuronal synapse formation
Curr. Opin. Neurobiol.
(2003) - et al.
Mouse NK1.1+ T cells: a new family of T cells
Immunol. Today
(1996)
The role of innate immunity in autoimmunity
J. Exp. Med.
A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family
Nature
Going both ways: immune regulation via CD1d-dependent NKT cells
J. Clin. Invest.
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2015, Molecular ImmunologyCitation Excerpt :CRTAM (Class-I-restricted T cell associated molecule), or CD355, is a member of the nectin-like protein superfamily. CRTAM is constitutively expressed in several non-immune tissues, including liver, lung, testis, kidney, intestine, and brain (Kennedy et al., 2000; Patino-Lopez et al., 2006). Several studies have shown that CRTAM expression in lymphocytes is restricted to cell activation, specifically for TCD8, NK, and TCD4 cells (Boles et al., 2005; Chan et al., 2012; Patino-Lopez et al., 2006; Valle-Rios et al., 2009).
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2013, StructureCitation Excerpt :The utility of the Brotherhood method was demonstrated using the nectin/nectin-like family as a specific example (see Experimental Procedures and Supplemental Experimental Procedures for additional examples of large families defined by the Brotherhood method). A recent report identified CRTAM as a distant relative to the nectin/nectin-like family (Patiño-Lopez et al., 2006); however, this similarity was inferred on the basis of low sequence identity and a weak BLAST similarity score between CRTAM and the nectin-like proteins. Our all-to-all BLAST comparison demonstrates that at this level of sequence similarity one cannot distinguish between true and false-positive connections.
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These authors contributed equally to this paper.