Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome.

The mouse Ly49 and human killer cell immunoglobulin-like receptors (KIR) gene clusters encode activating and inhibitory class I MHC receptors on natural killer (NK) cells. A direct correlation between the presence of multiple activating KIR and various human autoimmune diseases including diabetes has been shown. Previous studies have implicated NK cell receptors in the development of diabetes in the non-obese diabetic (NOD) inbred mouse strain. To assess the contribution of Ly49 to NOD disease acceleration the Ly49 gene cluster of these mice was sequenced. Remarkably, the NOD Ly49 haplotype encodes the largest haplotype and the most functional activating Ly49 of any known mouse strain. These activating Ly49 include three Ly49p-related and two Ly49h-related genes. The NOD cluster contains large regions highly homologous to both C57BL/6 and 129 haplotypes, suggesting unequal crossing over as a mechanism of Ly49 haplotype evolution. Interestingly, the 129-like region has duplicated in the NOD genome. Thus, the NOD Ly49 cluster is a unique mix of elements seen in previously characterized Ly49 haplotypes resulting in a disproportionately large number of functional activating Ly49 genes. Finally, the functionality of activating Ly49 in NOD mice was confirmed in cytotoxicity assays.

Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody.

Despite extensive study, the relative contribution of B7-1 and B7-2 molecules to the costimulation of cytotoxic T lymphocyte (CTL) activation remains controversial. We used blocking mAbs to B7-1 and B7-2 molecules to determine the role of these B7 family members in the in vitro induction of mouse nonspecific CTL in response to soluble anti-CD3 mAb. Optimal induction of anti-CD3-activated killer-T (AK-T) cells was found to require interactions with B7-2 on residual accessory cells in nylon wool-nonadherent spleen cell preparations during the first 12 h of culture in the presence of anti-CD3 mAb. Because B7-1 is not expressed at high enough levels on residual accessory cells in primary T cell cultures to be an effective ligand for CD28, we used LPS-stimulated B cells, which express substantial B7-1, in addition to B7-2, to determine the contribution of B7-1 to AK-T cell development. Compared with B7-2, the contribution of B7-1 to the costimulation of AK-T cells in this system was modest because anti-B7-1 mAb had only a minimal inhibitory effect on the generation of cytotoxicity, whereas anti-B7-2 mAb strongly inhibited AK-T cell development. Anti-CD3-induced cytotoxicity of T cells from CD4 knockout mice and CD4-depleted nylon wool-nonadherent spleen cells from wild-type mice was inhibited by anti-B7-2 mAb, implying that B7-2 is able to bind directly to CD28 on CD8+ T cells and costimulate their activation. B7-1 blockade, on the other hand, did not affect the costimulation of CD8+ T cells. Blockade of B7-2/ CD28 interactions with anti-B7-2 mAb strongly inhibited granzyme B, but not perforin or Fas ligand gene expression, suggesting an explanation for the inhibitory effect of anti-B7-2 mAb on AK-T cell development. These data indicate that B7-2 is superior to B7-1 as a costimulator of mouse AK-T cell induction.

Identification of the Ly49L protein: evidence for activating counterparts to inhibitory Ly49 proteins.

Previous studies have indicated that NK cells from different strains of inbred mice may express distinct Ly49 repertoires. Screening of NK cells from the CBA/J mouse for inhibitory and activating Ly49s revealed a novel DAP12-associated receptor that was immunoprecipitated with the Ly49G-specific mAb 4D11. Degenerate primers were designed to amplify and clone Ly49 cDNAs from CBA/J NK cells. A novel activating Ly49 cDNA was identified, which bears strong homology to the partially sequenced Ly49l gene found in C57BL/6 mice. Transfection of Ly49l into a DAP12+ cell line and subsequent immunoprecipitation experiments showed that Ly49L is likely the activating Ly49 detected by the 4DD11 antibody in CBA/J NK cells. Antibody-mediated cross-linking of Ly49L induced DAP12 phosphorylation, providing evidence that Ly49L is a functional activating receptor. Comparison of the extracellular domains of Ly49 family members indicates that all known activating members have an inhibitory counterpart with a highly related extracellular region.

Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody.

Cytotoxic T lymphocyte (CTL) development is regulated closely by an intricate series of signals provided by the T-cell receptor/CD3 complex, cytokines, and costimulatory ligand/receptor systems. In this study, we have explored the role of interleukin (IL)-12 and CD28 in mouse CTL development. Activation of T cells with anti-CD3 monoclonal antibody (mAb) in the presence of anti-CD86 mAb, which prevents CD28-CD86 interaction, led to decreased production of type 1 (IL-2, interferon-gamma) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as diminished expression of granzyme B (Gzm B) and reduced cytotoxic effector function. Cytolytic activity in T-cell cultures that were activated in the presence of anti-CD86-blocking mAb alone or in combination with anti-CD80 mAb could be restored by the addition of exogenous IL-12 at initiation of culture. The ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development was found to be dependent on the presence of IL-2 rather than interferon-gamma. IL-2 is required for IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb. Moreover, IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb is enhanced by IL-12. We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is a result of the interaction of IL-12 with IL-12Rbeta2 induced by low levels of IL-2 synthesized by T cells activated in a CD28-independent manner.

Anti-CD3-activated killer T cells: interferon-gamma and interleukin-10 cross-regulate granzyme B expression and the induction of major histocompatibility complex-unrestricted cytotoxicity.

We have investigated the effect of interferon-gamma (IFN-gamma) and interleukin (IL)-10 on granzyme B expression and the induction of major histocompatibility complex (MHC)-unrestricted cytotoxic activity in mouse T cell cultures following activation with anti-CD3 monoclonal antibody (mAb). First, metabolic inhibitors of granule-dependent and granule-independent cytolytic pathways were used to show that anti-CD3-activated killer T (AK-T) cells kill allogeneic P815 mastocytoma target cells primarily by the granule-dependent granzyme/perforin pathway. In comparison to control AK-T cells, lower levels of cytolytic activity were evident when AK-T cells were generated in the presence of anti-IFN-gamma neutralizing mAb or exogenous IL-10, whereas enhanced cytotoxicity was observed when AK-T cell cultures contained anti-IL-10 neutralizing mAb or exogenous IFN-gamma. In addition, granzyme B mRNA expression by AK-T cells was diminished when IFN-gamma bioactivity was neutralized or exogenous IL-10 was present in AK-T cell-cultures, whereas neutralization of IL-10 bioactivity or the addition of exogenous IFN-gamma resulted in increased expression of granzyme B mRNA. Similar results were obtained when granzyme B enzymatic activity in AK-T cell lysates was quantified using a colorimetric granzyme B assay. Altered cytotoxic potential, granzyme B mRNA expression, and granzyme B enzymatic activity following T cell activation in the presence of anti-IFN-gamma or anti-IL-10 neutralizing mAb or exogenous IFN-gamma or IL-10 could not be attributed to gross changes in T cell activation status or to altered percentages of CD4+ and CD8+ T cells in AK-T cell cultures. We conclude that IFN-gamma and IL-10 cross-regulate the induction of the granule-dependent cytolytic machinery of AK-T cells.

Anti-CD3-activated killer T cells: Interleukin-6 modulates the induction of major histocompatibility complex-unrestricted cytotoxicity and the expression of genes coding for cytotoxic effector molecules.

We have investigated the role of interleukin-6 (IL-6) in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxicity, as well as granzyme B, perforin, and Fas ligand gene expression, following mouse T lymphocyte activation with anti-CD3 monoclonal antibody (mAb). The generation of anti-CD3-activated killer-T (AK-T) cells was inhibited when anti-IL-6 neutralizing mAb was added at initiation of culture but not 24 h later, indicating that IL-6 is involved at an early stage of AK-T cell development. However, AK-T cell induction in the presence of exogenous IL-6 did not result in enhanced cytotoxicity, suggesting that saturating levels of IL-6 are normally synthesized in AK-T cell cultures. The inhibitory effect of IL-6 neutralization on AK-T cell generation could not be attributed to a defect in AK-T cell proliferation or to an inability of AK-T cells to recognize and adhere to P815 tumor target cells. However, IL-2 synthesis and CD25 expression were downregulated in AK-T cell cultures performed in the presence of anti-IL-6 mAb. In addition, IL-6 neutralization resulted in decreased expression of granzyme B and perforin, but not Fas ligand, mRNA. Exogenous IL-2 (50 U/ml) added at initiation of culture completely reversed the inhibitory effect of anti-IL-6 mAb on AK-T cell development, restoring CD25 expression and tumoricidal activity, as well as granzyme B and perforin mRNA expression, to control levels. We conclude that IL-6 modulates AK-T cell induction through an IL-2-dependent mechanism.

Ly49 gene expression in different inbred mouse strains.

Mouse natural killer cells express receptors for class I MHC in the form of the Ly49 family of proteins. The Ly49 family contains at least 13 expressed members (A, B, C, D, E, F, G, H, I, J, L, O, and P) and is further subdivided into activating and inhibitory subfamilies based on intracellular and transmembrane characteristics. The level of sequence identity between different members varies dramatically. However, comparison of the extracellular domain has revealed that several of the Ly49 molecules also form "pairs," where one member is activating and the other is inhibitory. Until recently, most Ly49 molecules described have come from the C57B1/6 strain of inbred mice. Using molecular cloning and immunochemical analysis we have found that different mouse strains express novel Ly49 molecules. Comparison of the allelic forms of some Ly49 molecules has shown that the dividing line between different genes and different alleles is blurred.

Cell biology and possible therapeutic applications of anti-CD3-activated killer-T cells (review).

Polyclonal T lymphocyte populations can be stimulated with anti-CD3 antibody to proliferate, secrete cytokines, and mediate MHC-unrestricted cytotoxic activity against a wide range of tumor target cells. Because anti-CD3-activated killer-T (AK-T) cells may be useful in the immunotherapy of human cancers, it is important to understand the signaling pathways and cell-surface structures involved in the induction and tumoricidal effector function of AK-T cells. Studies in the mouse model system have characterized the cytokines, signal transduction pathways, and costimulatory molecules involved in AK-T cell development. The recognition/adhesion and subsequent signaling events which lead to tumoricidal activity by mouse AK-T cells have also been defined. These findings, providing they translate accurately to the human system, may allow for the design of effective strategies to use AK-T cells for the treatment of human cancers. However, to date, the encouraging results obtained with anti-CD3 antibody/AK-T cell-based immunotherapies in mouse models of cancer have not been duplicated in clinical trials. The most likely explanation for this dis-appointing result is that tumor-reactive T lymphocytes in long term tumor-bearers fail to function correctly in the tumor microenvironment due to tumor-induced immune suppression and defects in key signal transduction molecules. It is clear that a detailed understanding of the inhibitory effect of established tumors on host T cells and the means to overcome tumor-induced immunosuppression are needed before anti-CD3 antibody/AK-T cell-based immunotherapies can be expected to succeed in the clinical setting.

The murine Ly49 family: form and function.

The activity of natural killer (NK) cells is regulated by surface receptors that recognize class I MHC. Murine NK cells express a large family of lectin-related receptors (Ly49s) to perform this function, while human NK cells utilize a separate group of proteins containing Ig-related domains (KIRs). Although these receptor families are not structurally related, the Ly49 family appears to be the functional equivalent of human KIRs, since it uses similar signal transduction pathways for either activation or inhibition of NK cell function. Therefore, lessons learned from the study of the murine MHC class I receptor system may be relevant to human NK function. This review summarizes the current state of knowledge of the Ly49 family.

Direct sequence comparison of two divergent class I MHC natural killer cell receptor haplotypes.

The murine Ly49 gene family encoding natural killer cell receptors for class I MHC is an example of a rapidly evolving cluster of immune response genes. Determining the genomic sequence of the 129S6/SvEvTac (129S6) Ly49 cluster and comparing it to the known sequence of the C57BL/6 (B6) region provided insight into the mechanisms of Ly49 gene evolution. 129S6 contains 20 Ly49, many of which are pseudogenes and 40% of the genes have no counterpart in the B6 genome. The difference in gene content between these two strains is primarily the result of distinct patterns of gene duplication. Phylogenetic analyses of individual exons showed that Ly49 genes form distinct sub-families and an ancestral haplotype can be surmised. Dotplot analysis supports limited allelism in the two haplotypes; however, large regions of variation punctuate these islands of co-linearity. These variable regions contain a high concentration of repetitive elements that are predicted to contribute to the dynamic evolution of this cluster. The extreme variation in Ly49 haplotype content between mouse strains provides a genetic explanation for the documented differences in natural killer cell phenotype, and also indicates that differences in natural killer cell function observed between B6 and 129-derived gene-targeted mice should be interpreted with caution.

Complete elucidation of a minimal class I MHC natural killer cell receptor haplotype.

The BALB/c inbred mouse is widely used in models of infectious disease, transplantation, and cancer. The differences in the immune responses of BALB/c compared to C57BL/6 mice are especially valuable for the identification of immune regulation genes. One striking immune variance between these mice is in the function of natural killer (NK) cells, and there is strong evidence implicating differential expression of Ly49 genes. In this study, the complete BALB/c Ly49 gene cluster has been sequenced and found to contain six functional genes and two pseudogenes. Compared to C57BL/6 mice, there is a 200 kb region absent in the BALB/c cluster including a complete lack of Ly49h-related genes, which explains the increased susceptibility of BALB/c to cytomegalovirus infection. In addition, there is no BALB/c Ly49d allele, explaining the inability of BALB/c NK cells to kill certain tumor cells. The Ly49 region has now been sequenced in three different inbred mouse strains, and comparisons indicate that the evolution of each haplotype is not straightforward and has involved large-scale deletions/insertions, gene recombination, and unequal crossing over between divergent haplotypes. This study confirms that relatively small murine class I MHC receptor haplotypes exist, analogous to observations made of human killer cell Ig-like receptor gene haplotypes.

Inhibition of CTL induction by rapamycin: IL-2 rescues granzyme B and perforin expression but only partially restores cytotoxic activity.

Rapamycin (RAP) is a potent inhibitor of CTL induction. Since RAP is known to block IL-2 signaling through the IL-2R, we hypothesized that RAP may interfere with CTL generation by inhibiting IL-2-induced expression of granzyme (Gzm) B, perforin, and/or Fas ligand (FasL). MHC-unrestricted mouse CTL induced in vitro with anti-CD3 mAb in the presence of RAP (1 ng/ml) exhibited dramatically reduced cellular proliferation and cytotoxicity against P815 tumor target cells. Gzm B mRNA expression and enzymatic activity in RAP-treated CTL were greatly reduced compared with those in control CTL. Perforin mRNA expression was also reduced by RAP. In contrast, RAP failed to inhibit FasL mRNA expression. RAP, therefore, inhibits induction of the perforin/Gzm B cytolytic pathway but spares Fas/FasL-mediated cytotoxicity. To determine whether RAP exerts a total blockade of the IL-2R signaling pathway, we induced CTL in the presence of both RAP and exogenous rIL-2 (100 U/ml). Under these conditions, Gzm B and perforin mRNA and protein expression as well as cellular proliferation were restored to at least control levels. Surprisingly, inhibition of cytotoxicity was only partially alleviated when CTL were induced in the presence of RAP plus rIL-2, even though CTL conjugated normally with target cells and had an intact granule secretory pathway. We conclude that 1) the inhibitory effect of RAP at the level of the IL-2R is incomplete; and 2) the suppressive effect of RAP on CTL induction is only partly due to inhibition of Gzm B and perforin gene expression.

Cyclosporin A inhibits 2-chloroadenosine-induced DNA cleavage in mouse thymocytes.

Incubation of mouse thymocytes with the adenosine analogue 2-chloroadenosine resulted in enhanced internucleosomal DNA fragmentation which could be inhibited by the immunosuppressive drug cyclosporin A. In order to be effective, cyclosporin A had to be added to thymocyte preparations at the same time as 2-chloroadenosine. Since cyclosporin A is a selective inhibitor of calcineurin, our data suggest a possible role for calcineurin as a signaling intermediate in the apoptotic pathway activated in thymocytes through adenosine receptors. However, at the present time we cannot exclude the possibility that the inhibitory effect of cyclosporin A on 2-chloroadenosine-induced apoptosis may be mediated through a calcineurin-independent process.

Pentoxifylline inhibits granzyme B and perforin expression following T-lymphocyte activation by anti-CD3 antibody.

Pentoxifylline (PTX), a methylxanthine derivative, is known to inhibit the production of the TH1 cytokines interleukin-2, tumour necrosis factor-alpha and interferon-gamma. Because these cytokines play an important role in promoting the development of cell-mediated immunity, we hypothesized that PTX would also interfere with the generation of cytotoxic effector cells in response to an immunological stimulus. In this study we used a mouse model system to investigate the effect of PTX on the induction of non-specific killer lymphocytes by anti-CD3 monoclonal antibody. Anti-CD3-induced T-cell proliferation, and the generation of anti-CD3-activated killer (AK) cells was inhibited in a dose-dependent fashion by PTX (25-100 micrograms/ml). The inhibitory effect of PTX could not be attributed to a defect in the recognition/adhesion phase of cytolysis because AK cells generated in the presence of PTX conjugated normally with P815 tumour target cells. However, AK cell expression of the cytoplasmic granule-associated cytolytic effector molecules granzyme B and perforin was markedly reduced when AK cells were induced in the presence of PTX. In eontrast, PTX had no effect on AK cell expression of Fas ligand, a cell-surface cytolytic effector molecule which is involved in granule-independent cytotoxicity. PTX thus has a profound inhibitory effect in vitro on the induction of granule-dependent cytolytic effector mechanisms in a mouse model system.

Characterization of the Ly49I promoter.

Fourteen potential Ly49 genes have been identified in the C57B1/6 mouse strain, and cDNAs containing a complete coding region have been isolated for 10 members of this gene family. Ly49 proteins are primarily expressed in natural killer (NK) cells. Although the sequence of the Ly49a promoter region has been published, no study of the cell-specific activity of the promoter has been reported. A 12-kb genomic fragment of the Ly49I gene was isolated and characterized by DNA sequencing. Approximately 5 kb of DNA sequence upstream of the first Ly49I exon was determined and this region was used to perform promoter analysis using luciferase reporter plasmid constructs. A core promoter was identified that was preferentially transcribed in a Ly49-expressing cell line, EL-4. Electrophoretic mobility shift assays using oligonucleotide probes from the core Ly49i promoter and comparable regions from the Ly49a promoter demonstrated the importance of TATA-related elements in generating EL-4 and NK cell-specific DNA/protein complexes.

Treatment of the P815 murine mastocytoma with cisplatin or etoposide up-regulates cell-surface Fas (CD95) expression and increases sensitivity to anti-Fas antibody-mediated cytotoxicity and to lysis by anti-CD3-activated killer-T cells.

We have investigated the effect of pre-treatment with the anti-cancer drugs cisplatin and etoposide on the susceptibility of P815 murine mastocytoma cells to lysis by murine spleen-derived anti-CD3-activated killer-T (AK-T) cells. A 20 hr pre-treatment with cisplatin (0.2-2 microg/ml) or etoposide (0.01-1 microg/ml) rendered P815 cells significantly more sensitive to AK-T cell-mediated lysis in a 4 hr 51Cr-release assay than untreated control tumor cells. At lower concentrations, pre-treatment with cisplatin or etoposide had no direct cytotoxic effects on P815 tumor cells, as measured by the MTT assay. AK-T cell-mediated killing of P815 tumor cells pre-treated with 2 microg/ml cisplatin or 1 microg/ml etoposide was only partially inhibitable by the Ca2+ chelator EGTA, suggesting that the Ca2+-independent Fas (CD95)/Fas ligand cytolytic pathway of AK-T cells contributes to cytotoxicity. In comparison to untreated control P815 cells, 2 microg/ml cisplatin- or 1 microg/ml etoposide-treated P815 cells exhibited increased expression of Fas mRNA and cell-surface Fas, which correlated with increased sensitivity to lysis by AK-T cells. In addition, pre-treatment with cisplatin or etoposide caused P815 tumor cells to become sensitive to the cytotoxic effects of anti-Fas antibody in a 4 hr 51Cr-release assay. Taken together, our results demonstrate that short-term exposure to concentrations of cisplatin and etoposide in the low cytotoxic range and below up-regulates Fas expression by P815 tumor cells, thereby facilitating cytotoxicity mediated through the Fas/Fas ligand cytolytic pathway.

Murine TRAIL (TNF-related apoptosis inducing ligand) expression induced by T cell activation is blocked by rapamycin, cyclosporin A, and inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and protein tyrosine kinases: evidence for TRAIL induction via the T cell receptor signaling pathway.

TRAIL (TNF-related apoptosis inducing ligand), like other members of the TNF family of proteins, is able to induce apoptosis in sensitive target cells. Recently, cell-surface TRAIL has been shown to be expressed by activated human and mouse T lymphocytes, raising the possibility that TRAIL might be involved in T cell-mediated cytotoxicity and/or immune regulation. In the present study we show by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis that activated, but not resting, mouse T cells express abundant TRAIL mRNA. TRAIL transcripts were detectable within 4 h of T cell activation. A panel of pharmacologic inhibitors was used to investigate the signal transduction pathways involved in TRAIL gene induction following T lymphocyte activation. TRAIL gene expression was sensitive to the src-like protein tyrosine kinase (PTK) inhibitor herbimycin A, as well as the more general PTK inhibitor genistein, suggesting the involvement of a src family PTK. The PKC inhibitors staurosporine and calphostin C, and the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002, also prevented TRAIL mRNA transcription by activated T cells, indicating a role for PKC and PI3-K. In addition, TRAIL induction was inhibited by cyclosporin A, implicating the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin. TRAIL expression was also blocked by rapamycin, which inhibits p70 S6 kinase involved in CD28 and interleukin (IL)-2 receptor signaling. However, TRAIL mRNA expression was not induced by IL-2, suggesting that TRAIL gene induction is not coupled to the IL-2 receptor. Data obtained by RT-PCR were confirmed at the protein level by immunoblotting with TRAIL-specific antibody. We conclude that TRAIL gene induction is initiated through a T cell receptor-associated signaling pathway similar to that responsible for the expression of cytokine genes such as IL-2.

Dose-dependent enhancing and inhibitory effects of A77 1726 (leflunomide) on cytotoxic T lymphocyte induction.

Leflunomide is an immunosuppressive prodrug which prevents allograft rejection in several animal model systems and may, therefore, have clinical application in organ transplant recipients. Although cytotoxic T lymphocytes (CTL) are an important component of the allograft rejection response, the effect of leflunomide on CTL development has not been thoroughly explored. In this study we have determined the effect of A77 1726, the active metabolite of leflunomide, on CTL induction in C57BL/6 mouse T cell cultures stimulated with anti-CD3 monoclonal antibody. Conjugate formation with P815 target cells, granzyme B enzymatic activity in CTL lysates, and P815 cytolysis in a 51Cr-release assay were used as determinants of in vitro CTL function. At high concentrations (10-20 microM), A77 1726 strongly inhibited CTL generation. In contrast, a low concentration (0.5 microM) of A77 1726 promoted CTL development. These dose-dependent opposing effects of A77 1726 on CTL induction could not be attributed to alterations in CD8+ lymphocyte percentages, interleukin-2 or CD25 expression, or the ability to conjugate with P815 target cells. However, both interferon-gamma and granzyme B expression were significantly decreased when CTL were induced in the presence of 10-20 microM A77 1726, and were slightly, but not always significantly, elevated in the presence of 0.5 microM A77 1726. We conclude that at high concentrations A77 1726 is a potent inhibitor of CTL induction, but a low concentration of A77 1726 enhances CTL development.

Class I MHC-binding characteristics of the 129/J Ly49 repertoire.

The Ly49 family of NK cell receptors and its MHC-binding characteristics have only been well characterized in C57BL/6 (B6) mice. Previous studies have shown that 129/J mice express unique Ly49 genes that are not found in the B6 strain. Screening of a 129/J cDNA library led to the discovery of 10 distinct full-length Ly49-related coding sequences (Ly49e, g, i, o, p, r, s, t, u, and v). Although 129/J mice share identical class I MHC (K(b) and D(b)) transcripts with B6 mice, only one Ly49 is identical in the two strains (Ly49E). In addition to the previously characterized Ly49P, two new activating Ly49 proteins were discovered, Ly49R and U. The MHC specificity of the total 129/J Ly49 repertoire was evaluated with soluble class I MHC tetramers and found to be distinct compared with the B6 Ly49 repertoire. Ly49V bound to many types of class I MHC, suggesting that Ly49V(+) NK cells may monitor host cells for a global down-regulation in MHC levels. An activating receptor, Ly49R, was shown to bind soluble class I molecules to a moderate degree, a result not previously observed for other activating Ly49 proteins. Furthermore, tetramer-binding results were confirmed functionally with cytotoxicity assays using sorted 129/J NK cells. This study shows that the Ly49 repertoire and its MHC-binding characteristics can be very different among inbred mouse strains. Ly49 divergence should be considered when using 129-derived embryonic stem cells for the production of gene-targeted mice, especially when an immune or NK-derived phenotype is under scrutiny.

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.