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

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Abstract

We have previously reported the characterization of a novel immunoglobulin supergene family member, designated class-I MHC-restricted T cell associated molecule (CRTAM). Here we further characterize human CRTAM and find that it is highly expressed in the cerebellum, notably in Purkinje neurons. We identify CRTAM as a new member of the nectin-like (Necls) family and find significant expression of Necl-2 (IGSF4), a protein known to bind CRTAM and another member of the nectin superfamily, in the cerebellum. These findings suggest that CRTAM/Necl-2 binding may contribute to neuronal interactions. We also show that, in the immune system, CRTAM expression is restricted to activated class-I MHC-restricted T cells, including NKT and CD8 T cells. CRTAM represents one of the most highly expressed surface markers of activated human CD8 T cells and NKT cells, suggesting it may have diagnostic uses in various human viral and autoimmune diseases.

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).

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    These authors contributed equally to this paper.

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