The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1

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Abstract

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.

Section snippets

Cloning of mCD96; cloning, expression, and purification of recombinant mCD96-hIgG1, mNectin-1/2/3-hIgG1, and mCD226-hIgG1

mCD96 was cloned using primers MUCD96.BAM and mucd96_full in PCR with cDNA from BALB/c spleen (MTC panel, CLONTECH): 2 min 94 °C, 25 cycles with 15 s 94 °C, 30 s 55 °C, 1 min 72 °C. The PCR fragment, cloned into pcDNA3, served as template in a subsequent PCR applying primers MUC96.BAM and mucd96_fus_bam to generate an ectodomain fragment for in frame cloning into vector phIgG1 (containing the cDNA for domains 3 + 4 of hIgG1).

PCR products representing the ectodomains of mNectin-1, mNectin-2, mNectin-3,

Cloning of mCD96 and binding to mCD155

Using splenic cDNA from BALB/c mice, a full-length clone spanning the putative mCD96 open-reading frame was generated by PCR. The obtained product was sequenced and found to be identical to the published sequence (Accession No. Y029156). An alignment of the human and murine protein sequence reveals 56% conservation at the amino acid level (Supplementary Fig. S1).

Apart from CD226, CD155 binds to CD96 expressed on human NK cells [4]. To investigate whether the CD155/CD96 interaction is conserved

Acknowledgments

This work was funded by DFG-Grant BE1886/2-1 from Deutsche Forschungsgemeinschaft to G. Bernhardt. S. Seth is a recipient of a Georg Christoph Lichtenberg Stipend provided by the Ph.D. program Infection Biology sponsored by the Ministry of Science and Culture of Lower Saxony.

We thank O. Pabst for critically reading the manuscript.

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These authors contributed equally to this work.

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