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 super-family. 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 ortho-logue. 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.     

Molecular characterization of the early activation antigen CD69: A type II membrane glycoprotein related to a family of natural killer cell activation antigens

CD69 is a disulfide-linked homo-dimer expressed on the surface of activated T cells, B cells, natural killer cells, neutrophils and platelets. Antibody cross-linking of CD69 in the presence of phorbol ester results in cellular activation events including proliferation and the induction of specific genes. Using an expression cloning strategy we have isolated cDNA encoding human CD69 from a CD4⁺ T cell clone. Transfection of the cDNA clone in CV-1/EBNA cells results in the expression of a covalently linked homodimer. The cDNA insert hybridizes to a 1.7-kb mRNA in phorbol 12-myristate 13-acetate- or phytohemoagglutinin-stimulated human T cells. Using the human clone we have isolated cDNA encoding mouse CD69, which, when expressed in human T cells allowed those cells to respond to anti-mouse CD69 antibodies by secreting interleukin-2 and interferon-γ. Sequence analysis showed that both mouse and human CD69 are type II membrane glycoproteins related to the NKR-P1 and Ly-49 families of natural killer cell activation molecules.     

CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells

Two families of major histocompatibility complex (MHC) class I-specific receptors are found on natural killer (NK) cells: immunoglobulin-like receptors and C-type lectin receptors. In mice, the latter category is represented by the Ly49 family of receptors, whereas in humans, NK cells express the distantly related CD94, which forms MHC class I-specific heterodimers with NKG2 family members. Humans also express the MHC class I-specific p50/p58/p70 family of immunoglobulin-like receptors, but these have not been identified in mice. Hence, there is no known instance of an MHC class I-specific receptor that is expressed by both human and murine NK cells. Here we report the cloning of CD94 from the CB.17 and C57BL/6 strains of mice. Mouse CD94 is 54 % identical and 66 % similar to human CD94, and is also a member of the C-type lectin superfamily. Mouse CD94 is expressed efficiently on the cell surface of cells transiently transfected with the corresponding cDNA, but surface CD94 was unable to mediate detectable binding to MHC class I-expressing ConA blasts. Notably, mouse CD94, like human CD94, has a very short cytoplasmic tail, suggesting the existence of partner chains that may play a role in ligand binding and signaling. Like many other C-type lectins expressed by NK cells, mouse CD94 maps to the NK complex on distal chromosome 6, synteneic to human CD94. We also demonstrate that mouse CD94 is highly expressed specifically by mouse NK cells, raising the possibility that mice, like humans, express multiple families of MHC class I-specific receptors on their NK cells. Murine homologs of human NKG2 family members have not yet been identified, but we report here the existence of a murine NKG2D-like sequence that also maps to the murine NK complex near CD94 and Ly49 family members.     

HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex

The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell-mediated lysis. The nonclassical HLA class I molecule HLA-G may mediate protection from NK cells. NK cells are known to express a number of HLA class I-specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I-negative 221 cell line either untransfected or transfected with HLA-G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A-negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA-G-specific inhibitory NKR, and that HLA-G-specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94⁺/NKG2A^? phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA-G specificity, thus suggesting the existence of a different, still unknown NKR.     

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 multigene 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.     

A High Dose of Intravenous Immunoglobulin Increases CD94 Expression on Natural Killer Cells in Women with Recurrent Spontaneous Abortion

Problem  A high dose of intravenous immunoglobulin (HIVIg) therapy is effective in various diseases such as autoimmune diseases, and also is expected to have efficacy in recurrent spontaneous abortion (RSA). The aim of this study was to understand immunological mechanisms of this therapy.
Method of study  By flowcytometric analyses, we examined phenotypic changes of a variety of immunological cells including natural killer (NK) cells, cytotoxic T cells, regulatory T cells and macrophages in peripheral blood of RSA women with HIVIg therapy ( n  = 8).
Results  Expression percentages of inhibitory CD94 on NK cells significantly ( P =  0.01) increased after the therapy (58.8 ± 21.4% versus 71.0 ± 17.6%).
Conclusion  Mechanisms of possible efficacy of HIVIg therapy for RSA may include enhancement of CD94 expression and subsequent suppression of NK cell cytotoxicity.     

A single-CRD C-type lectin is important for bacterial clearance in the silkworm

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca²⁺-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.     

C型凝集素受体CLEC-2的研究进展

C型凝集素样受体-2(CLEC-2)是一个非经典型的C型凝集素受体,主要表达在血小板、中性粒细胞等细胞表面,在马来西亚蝮蛇蛇毒蛋白rhodocytin和跨膜唾液酸糖蛋白poloplanin等相关配体的刺激下,能通过细胞质尾部的YXXL结构域形成二聚体,从而激活syk依赖的信号传导.最近有很多研究显示:CLEC-2在血小...     

Cloning of NKG2-F,a new member of the NKG2 family of human natural killer cell receptor genes

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.     

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR alphabeta lymphocytes

A subset of CD8(+) T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8(+) T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7(+) memory cells and CCR7(-) effector cells. Since NKG2A can only be induced on naive CD8(+) T cells while CD94(-) memory cells are refractory, it is likely that commitment to the CD94:NKG2A(+) subset occurs during the first encounter with antigen. CCR7(+)CD94:NKG2A(+) T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-gamma. These cells occur as a separate CD94:NKG2A(+) T cell lineage with a distinct TCR repertoire that differs from that of the other CD8(+)CD94(-) T cells activated in situ.     

Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis

Summary:  C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.     

The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules

CD94, a type II membrane protein containing a C-type lectin domain, has been shown to be involved in natural killer (NK) cell-mediated recognition of different HLA allotypes. The inhibitory form of the CD94 receptor has recently been identified by the specific monoclonal antibody (mAb) Z199. Herein, we demonstrate that the inhibitory receptor is in fact a complex formed by the covalent association of CD94 with the NKG2-A molecule (Mr ～ 43 kDa), another member of the C-type lectin superfamily, and that Z199 mAb specifically recognize NKG2-A molecules. Although the NKG2-A-encoding cDNA has been known for several years, the corresponding protein and its possible function remained undefined. Moreover, we show that the NKG2-B protein, an alternatively spliced product of the NKG2-A gene, can also assemble with CD94. Remarkably, both NKG2-A and NKG2-B proteins contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). This may provide the molecular basis of the inhibitory function mediated by the CD94/NKG2-A receptor complexes.     

Tyrosine kinase-dependent activation of human NK cell functions upon triggering through CD44 receptor

We recently showed evidence of CD44-mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca²⁺ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester-induced TNF-α secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44-stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44-mediated TNF-α production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44-mediated activation of NK cell functions.     

The role of TCR stimulation and TGF-beta in controlling the expression of CD94/NKG2A receptors on CD8 T cells

Following antigen recognition, murine CD8 T cells express CD94/NKG2A receptors. Our results show that this up-regulation occurs rapidly in vitro and is accompanied by an approximately 8-fold increase in CD94 and approximately 125-fold increase in NKG2A mRNA. In contrast, only a twofold increase in NKG2C mRNA is noted. The addition of TGF-beta, but not IL-10, IL-12 or IL-15, leads to a further increase in cell membrane expression of these receptors, as well as a approximately 6-fold increase in mRNA for both chains. TGF-beta also increases CD94/NKG2A expression on memory CD8 T cells that are re-exposed to antigen. The effect of TGF-beta on increasing CD94/NKG2A expression on both naive and memory CD8 T cells occurs only when there is a concurrent stimulation through the TCR. In contrast, TGF-beta does not increase expression of CD94/NKG2A on resting or activated NK cells. We also show by using purified CD8 T cells, that TGF-beta acts directly on these cells. These results implicate a role for both antigen and TGF-beta in increasing expression of inhibitory CD94/NKG2A receptors on CD8 T cells.     

HLA-Cw7 Zygosity Affects the Size of a Subset of CD158b+ Natural Killer Cells

Individuals with certain HLA class I genotypes are highly susceptible to disease after viral infection. Natural killer (NK) cells kill virus-infected cells through a mechanism involving HLA class I receptors. These facts may be connected if an individual's HLA genotype regulates the number and function of NK cells. We have observed that subjects homozygous for the HLA-B/C region of conserved major histocompatibility complex (MHC) extended haplotypes have lower NK cell activity and a significantly lower frequency of CD16⁺CD56⁺ NK cells than heterozygotes. The proportion of CD16^–CD56⁺ NK cells was unaffected by zygosity for the HLA-B/C region. We show here that the frequency of CD16⁺CD158b⁺, but not CD16^–CD158b⁺ NK cells, was significantly lower (p <0.026) in homozygotes for HLA-Cw7 (NK1 ligand) haplotypes than in heterozygotes. The frequencies of CD16⁺CD158a⁺ and CD16^–CD158a⁺ and CD16^–CD158a⁺ or CD16⁺NKB1⁺ and CD16^–NKB1⁺ NK cells were not different in these donor groups. These findings suggest that the proportion of NK cells coexpressing CD16 and CD158b, but not CD158a nor NKB1, is influenced by zygosity for the HLA-Cw7 (NK1 ligand) haplotype. Since NK cells are involved in protection from virus infection, a reduced size of a ligand-specific NK subset in individuals homozygous for some HLA-B/C haplotypes may help explain their increased susceptibility to virus-induced diseases.     

Jacalin: a lectin mitogenic for human CD4 T lymphocytes.

The major protein component of seeds from jackfruit is the lectin jacalin. Jackfruit crude extracts are known to stimulate human lymphocytes, but the mitogenic properties of purified jacalin have not been studied in detail so far. Study of the proliferative response of cell populations from normal human peripheral blood to purified jacalin showed it to be mitogenic through an interaction with lymphocytes by its lectin-binding site, as shown by inhibition by IgA. Jacalin failed to stimulate B cells to proliferate and to undergo plasma cell maturation. It induced a proliferation of CD4 (and not CD8) lymphocytes, as shown by phenotypic analysis of cells recovered after culture and by studies of the response of isolated T cell subpopulations. The proliferative response to jacalin was autologous monocyte-dependent. The kinetics of jacalin-induced DNA synthesis, expression of CD25 and interleukin-2 secretion was shifted by comparison with that induced by phytohaemagglutinin. The reason for the restricted responsiveness of CD4 T cells is presently unclear; jacalin bound to all blood cells and did not significantly co-cap with CD1, CD2, CD3, CD4, CD8 and CD38, and jacalin response was neither enhanced nor inhibited by antibodies to these surface antigens.