NILR-1, a novel immunoglobulin-like receptor expressed by neutrophilic granulocytes, is encoded by a leukocyte receptor gene complex on rat chromosome 1.

Several receptors expressed by subsets of leukocytes and with sequence homology to the killer cell inhibitory receptors have recently been identified both in man and mouse. Here we describe a rat cDNA that encodes a novel receptor of this group, designated neutrophil immunoglobulin-like receptor-1 (NILR-1). The predicted 58.7-kDa mature NILR-1 protein is a type I integral membrane protein, with three C2-type immunoglobulin superfamily domains, a transmembrane region devoid of charged amino acids, and a cytoplasmic tail containing four immunoreceptor tyrosine-based inhibition motif-like regions. NILR-1 shows greatest sequence homology to the mouse paired immunoglobulin-like receptor-B and members of the human leukocyte immunoglobulin-like receptor/immunoglobulin-like transcript group of receptors. As shown by Northern blot analysis, NILR-1 was transcribed by neutrophilic granulocytes. Although weaker transcription was found with a macrophage cell line, no signal was detected with peritoneal macrophage or spleen RNA. Linkage analysis localized Nilr1 to chromosome 1, closely linked to a locus encoding a rat NKp46 orthologue. The two loci define a rat leukocyte receptor gene complex, in a region syntenic to human chromosome 19q13.4 and the proximal part of mouse chromosome 7, that harbors the human and mouse leukocyte receptor gene complexes.

Identification of a human member of the Ly-49 multigene family.

Three classes of multigene family-encoded receptors enable NK cells to discriminate between polymorphic MHC class I molecules: Ly-49 homodimers, CD94/NKG2 heterodimers and the killer cell inhibitory receptors (KIR). Of these, CD94/NKG2 has been characterized in both rodents and humans. In contrast, Ly-49 family members have hitherto been found only in rodents, and KIR molecules only in the human. In this report, we describe a human cDNA, termed Ly-49L, that constitutes the first human member of the Ly-49 multi-gene family. Compared with rodent Ly-49 molecules, the Ly-49L sequence contains a premature stop codon and predicts a truncated protein that lacks the distal part of a C-terminal lectin domain. Evidence is presented that the premature stop codon results from incomplete excision of the intron between the first two lectin domain exons. Splice variants predicting a full-size Ly-49L protein were not detected. As demonstrated by Northern blot analysis, Ly-49L was transcribed by IL-2-activated NK cells, but not by freshly isolated B or T cells. PCR screening of a 22-clone yeast artificial chromosome contig localized the LY49L locus to the human NK gene complex on chromosome 12p12-p13. Southern blot analysis of genomic DNA showed a simple pattern with a full-length Ly-49L probe at low stringency hybridization conditions, suggesting that Ly-49L may be the only human member of the Ly-49 multigene family.

Molecular characterization of rat NKR-P2, a lectin-like receptor expressed by NK cells and resting T cells.

The gene for a rat NK lectin-like receptor (NKLLR), named NKR-P2, has been cloned and characterized. Sequence analysis shows that it represents the orthologue of human NKG2D and that the two molecules form a distinct NKLLR family, no more related to NKG2A/B, -C or -E than to other NKLLR families. Nkrp2 is a single-copy gene containing seven introns, mapping to the rat NK gene complex. Rat NKR-P2 differs from the human orthologue in that its cytoplasmic tail contains 13 additional amino acids, encoded by a separate exon. Splice variants lacking this exon were not detected in T cells or NK cells. NKR-P2 is strongly expressed by NK cells. In contrast to other NKLLR, it is also strongly expressed by resting thoracic duct CD4+ and CD8+ T cells, but not by thymocytes or other hemopoietic cells.

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

Two genes in the rat homologous to human NKG2.

Two different lectin-like receptors for MHC class I molecules have so far been identified on natural killer (NK) cells, the Ly-49 homodimeric receptors in mice and the NKG2/CD94 heterodimeric receptors in humans. The recent identification of a rat CD94 orthologue implied that NK cell receptors equivalent to NKG2/CD94 also exist in rodents. Here we describe the cDNA cloning of two rat genes homologous to members of the human NKG2 multigene family. The deduced rat NKG2A protein contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM), whereas the cytoplasmic tail of rat NKG2C lacks ITIM. The genes map to the rat NK gene complex and are selectively expressed by NK cells. The expression is strain dependent, with high expression in DA and low in PVG NK cells, correlating with the expression of rat CD94. Ly-49 genes have previously been identified in the rat, and the existence of rat NKG2 genes in addition to a CD94 orthologue suggests that NK cell populations utilize different C-type lectin receptors for MHC class I molecules in parallel.

Molecular characterization of a gene in the rat homologous to human CD94.

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly-49 homodimers in rodents. CD94, NKG2 and Ly-49 belong to the C-type lectin superfamily. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly-49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2% identical to human CD94. The single-copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human orthologue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC-encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C-type lectin receptors for MHC class I molecules in parallel.

Comparing macrophages and dendritic leukocytes as antigen-presenting cells for humoral responses in vivo by antigen targeting.

Immunotargeting is a novel technique whereby antigen is directed against antigen-presenting cells (APC) by conjugation to specific monoclonal antibodies (mAb). In this study we have employed the technique to investigate the efficiency of macrophages as APC compared with constitutively major histocompatibility complex (MHC) class II-positive cells, i.e. dendritic leukocytes and B cells, in vivo. We first studied the organ retention of the radiolabeled conjugates by gamma counting, and their distribution within the draining lymph nodes by autoradiography. We could confirm that the conjugates reached the cells at which they were aimed. We then measured primary and secondary humoral responses. The results confirmed previous findings that targeting with mAb against MHC class II, i.e. to dendritic leukocytes, strongly enhanced the primary humoral response. In contrast, anti-IgD conjugates, directed against B cells gave only weak primary responses. Although conjugates directed against macrophages were retained for a longer time than the other conjugates, the primary humoral response was virtually abolished. The secondary responses, however, were at least as strong as those obtained in animals primed with control conjugates, whereas animals primed with anti-MHC class II conjugates showed little if any amplification of the secondary response. The discrepancies between the various conjugates could not be ascribed to TH1 versus TH2 responses as IgG1, IgG2a, IgG2b and IgE titers all co-varied in single animals. A possible explanation for the observed results is that macrophages fail to induce cytokine production for terminal differentiation of B cells to plasma cells, whereas conversely, upon presentation by dendritic leukocytes most stimulated B cells mature to plasma cells, leaving less progeny for immunological memory.

Targeting antigens to antigen presenting cells.

Antigen may be targeted to antigen presenting cells (APC) by conjugating the antigen to monoclonal antibodies directed against surface molecules on APC. By now several laboratories have shown that immunotargeting enhances humoral responses, depending upon the targeted ligand or cell type, with low doses of antigen and without the use of adjuvants. There is also preliminary evidence that the method may be used to bias immune responses in desired directions, possibly also to induce tolerance. In addition to its use as an experimental tool for exploring immune reactions the method could in the future also be clinically important, e.g. in vaccination. In this article we give a brief account on work so far published with this novel method and discuss possible mechanisms behind its immunopotentiating effects.