Assembly and annotation of human chromosome 2q33 sequence containing the CD28, CTLA4, and ICOS gene cluster: analysis by computational, comparative, and microarray approaches.

Human chromosome 2q33 is an immunologically important region based on the linkage of numerous autoimmune diseases to the CTLA4 locus. Here, we sequenced and assembled 2q33 bacterial artificial chromosome (BAC) clones, resulting in 381,403 bp of contiguous sequence containing genes encoding a NADH: ubiquinone oxidoreductase, the costimulatory receptors CD28, CTLA4, and ICOS, and a HERV-H type endogenous retrovirus located 366 bp downstream of ICOS in the reverse orientation. Genomic microarray expression analysis using differentially activated T-cell RNA against a subcloned CTLA4/ICOS BAC library revealed upregulation of CTLA4 and ICOS sequences, plus antisense ICOS transcripts generated by the HERV-H, suggesting a potential mechanism for ICOS regulation. We identified four nonlinked, polymorphic, simple repetitive sequence elements in this region, which may be used to delineate genetic effects of ICOS and CTLA4 in disease populations. Comparative genomic analysis of mouse genomic Icos sequences revealed 60% sequence identity in the 5' UTR and regions between exon 2 and the 3' UTR, suggesting the importance of ICOS gene function.

Differential expression of inducible costimulator-ligand splice variants: lymphoid regulation of mouse GL50-B and human GL50 molecules.

The process of immunological costimulation between APC and T cells is mediated by protein ligand:receptor interactions. To date, costimulatory receptors known to be expressed by T cells include the structurally related proteins CD28 and the inducible costimulator (ICOS). The ligands to human and mouse ICOS, human GL50 (hGL50), and mouse GL50 (mGL50) were recently cloned and demonstrated to have sequence similarity to the CD28 ligands B7-1 and B7-2. Examination of mGL50 cDNA transcripts by 3'RACE revealed an alternatively spliced form, mGL50-B, that encoded a protein product with a divergent 27-aa intracellular domain. Both mGL50- and mGL50-B-transfected cells exhibited binding to human and mouse ICOS-Ig fusion protein, indicating that the alternate cytoplasmic domain of mGL50-B does not interfere with extracellular interactions with ICOS receptor. Flow cytometric and RT-PCR analysis of BALB/c and RAG1(-/-) mice splenocytes demonstrate that freshly isolated B cells, T cells, macrophages, and dendritic cells express both splice variant forms of ICOS ligand. Comparative analyses with the human ICOS ligand splice variants hGL50 and B7-H2 indicate that differential splicing at the junction of cytoplasmic exon 6 and exon 7 may be a common method by which GL50-ICOS immunological costimulatory processes are regulated in vivo.

Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor.

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.

Complete sequence determination of the mouse and human CTLA4 gene loci: cross-species DNA sequence similarity beyond exon borders.

CTLA4 (CD152), a receptor for the B7 costimulatory molecules (CD80 and CD86), is considered a fundamental regulator of T-cell activation. In this paper, we present the complete primary structure of the mouse and human CTLA4 gene loci. Sequence comparison between the mouse and the human CTLA4 gene loci revealed a high degree of sequence conservation both for homologous noncoding regions (65-78% identity) and for coding regions (72-98% identity), with an overall score of 71% over the entire length of the two genes. Of the CTLA4 genomic regions aligned, five simple repetitive elements were found in the mouse locus, whereas two simple repetitive sequences were localized on the human locus. RNA blot analysis of mouse and human primary tissues indicated that both CTLA4 and T-cell receptor transcripts were found in most organs with generally higher levels in lymphoid tissues. The conservation of CTLA4 gene patterning raises the possibility that constrained gene evolution of CTLA4 may be linked to conserved transcriptional control of this locus.