The CMRF-35 mAb recognizes an antigen found on most leukocytes including monocytes, neutrophils, macrophages, dendritic cells, and subpopulations of lymphocytes and bone marrow cells. A cDNA expressing the CMRF-35 epitope was isolated by expression cloning and this predicts for a type I cell surface glycoprotein belonging to the Ig superfamily. Here we demonstrate that the CMRF-35 mAb recognizes an epitope more widely distributed on hemopoietic cells and cell lines than suggested by expression analysis of the CMRF-35 mRNA. Furthermore, we have isolated a novel cDNA (CMRF-35-H9) that encodes a protein product also recognized by the CMRF-35 mAb. This cDNA product is a type I cell surface glycoprotein with a single Ig V-like domain. Although the sequences of the extracellular V-like domains of the two molecules are very similar, there is little similarity between the remainder of their sequences. The two transcripts are expressed independently of each other, and their presence accounts for the discrepancy between CMRF-35 mAb binding and mRNA analysis. The cytoplasmic tail of CMRF-35-H9 contains motifs similar to the inhibitory motifs found in some leukocyte surface receptors. Their expression in hemopoietic cells suggests that these two molecules may play distinct but related roles in the regulation of leukocyte function.

The CMRF-35 monoclonal antibody recognizes an epitope found on at least two cell surface molecules, differentially expressed by many leukocytes. These molecules, the CMRF-35H (9) and CMRF-35A (CMRF-35) antigens are both members of the immunoglobulin (Ig) superfamily with a single V-like Ig domain. The function of these molecules is unknown, however the presence of putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain of the CMRF-35H molecule suggests that this molecule may play a regulatory role in leukocyte function. The CMRF-35H and CMRF-35A molecules show several similarities to the family of molecules containing ITIM or immunoreceptor tyrosine-based activatory motifs (ITAM) suggesting that CMRF-35H/CMRF-35A may be new members of this family. This would further indicate that, like other ITIM/ITAM containing molecules, CMRF-35H/CMRF-35A will also play an important role in the immune response. To further characterize these molecules, we have isolated genomic clones for the CMRF-35H gene and determined its intron-exon organization. The gene spans approximately 12 kb and consists of seven exons. Furthermore, this gene has been mapped to chromosome 17 and thus is not linked to the known human ITIM containing genes which map to human chromosome 19 or the recently characterized molecule, NKp44, localized to human chromosome 6.

Mast cell-mediated responses are likely to be regulated by the cross talk between activatory and inhibitory signals. We have screened human cord blood mast cells for recently characterized inhibitory receptors expressed on NK cells. We found that IRp60, an Ig superfamily member, is expressed on human mast cells. On NK cells, IRp60 cross-linking leads to the inhibition of cytotoxic activity vs target cells in vitro. IRp60 is constitutively expressed on mast cells but is down-regulated in vitro by the eosinophil proteins major basic protein and eosinophil-derived neurotoxin. An immune complex-mediated cross-linking of IRp60 led to inhibition of IgE-induced degranulation and stem cell factor-mediated survival via a mechanism involving tyrosine phosphorylation, phosphatase recruitment, and termination of cellular calcium influx. To evaluate the role of IRp60 in regulation of allergic responses in vivo, a murine model of allergic peritonitis was used in which the murine homolog of IRp60, LMIR1, was neutralized in BALB/c mice by mAbs. This neutralization led to a significantly augmented release of inflammatory mediators and eosinophilic infiltration. These data demonstrate a novel pathway for the regulation of human mast cell function and allergic responses, indicating IRp60 as a candidate target for future treatment of allergic and mast cell-associated diseases.