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Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1999)

Bottom Line: This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells.Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion.Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

View Article: PubMed Central - PubMed

Affiliation: Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova, Italy.

ABSTRACT
Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

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Northern blot analysis of NKp30 transcript expression and Zoo-Blot analysis. (A) Total RNA was isolated from cells of different origin. Lanes 1 and 2, polyclonal NK cell populations; lane 3, blank; lane 4, an NK cell line (NKL); lane 5, an NK cell line (NK3.3); lane 6, human monocytes; lane 7, a histiocytic lymphoma cell line (U937); lane 8, a T lymphoma cell line (Jurkat); lane 9, an acute promyelocytic leukemia cell line (HL60); and lane 10, an EBV-transformed B cell line (LCL721.221). 10 μg of each RNA preparation (2 μg of poly A+ RNA from polyclonal NK cell populations, lanes 1 and 2) were hybridized with the 421-bp NKp30 cDNA probe. The positions of 28S and 18S ribosomal RNA subunits are indicated on the left. (B) A Southern blot containing genomic DNA from humans, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and S. cerevisiae yeast was hybridized under low stringency condition with the 421-bp NKp30 cDNA probe.
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Figure 8: Northern blot analysis of NKp30 transcript expression and Zoo-Blot analysis. (A) Total RNA was isolated from cells of different origin. Lanes 1 and 2, polyclonal NK cell populations; lane 3, blank; lane 4, an NK cell line (NKL); lane 5, an NK cell line (NK3.3); lane 6, human monocytes; lane 7, a histiocytic lymphoma cell line (U937); lane 8, a T lymphoma cell line (Jurkat); lane 9, an acute promyelocytic leukemia cell line (HL60); and lane 10, an EBV-transformed B cell line (LCL721.221). 10 μg of each RNA preparation (2 μg of poly A+ RNA from polyclonal NK cell populations, lanes 1 and 2) were hybridized with the 421-bp NKp30 cDNA probe. The positions of 28S and 18S ribosomal RNA subunits are indicated on the left. (B) A Southern blot containing genomic DNA from humans, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and S. cerevisiae yeast was hybridized under low stringency condition with the 421-bp NKp30 cDNA probe.

Mentions: Searching EMBL/GenBank/DDBJ databases revealed that the clone 5C cDNA was identical to a previously identified alternatively spliced form of the 1C7 gene (available under accession no. AF031138). This gene has been mapped on human chromosome 6, in the TNF cluster of the MHC gene complex 38. To date, however, owing to the lack of specific mAb, neither the function nor the surface distribution of the putative product of 1C7 gene could be identified. Moreover, the 1C7 transcript could not be revealed by Northern blot on different tissues and cell lines. On the other hand, by reverse transcriptase (RT)-PCR, the 1C7 transcript could be amplified by RNA isolated from spleen (but not from other tissues) or certain lymphoid and myeloid cell lines. These data suggested that 1C7 transcripts could be poorly represented, or could be expressed at substantial levels only in a narrow range of cell types 39. Our present analysis of NKp30 expression by Northern blotting revealed a mRNA of ∼1 kb in polyclonal NK cell populations and NK cell lines, including NKL and NK3.3. On the contrary, consistent with the lack of reactivity with anti-NKp30 mAbs, no NKp30 mRNA could be detected in human monocytes or cell lines of different histotype, including U937, Jurkat, HL60, and LCL 721.221 cells (Fig. 8 A). In some of these cell lines that were negative for mRNA expression by Northern blot (and for anti-NKp30 mAb surface staining), it has been possible to detect transcripts when analyzed by the RT-PCR technique. This finding is likely to reflect a low level of NKp30 transcription resulting in lack of NKp30 surface expression. Moreover, Northern blot analysis of multiple human tissues showed selective expression of NKp30 transcript only in spleen cells (data not shown). Altogether, these data are consistent with the notion that NKp30 expression is largely NK specific.


Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Pende D, Parolini S, Pessino A, Sivori S, Augugliaro R, Morelli L, Marcenaro E, Accame L, Malaspina A, Biassoni R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1999)

Northern blot analysis of NKp30 transcript expression and Zoo-Blot analysis. (A) Total RNA was isolated from cells of different origin. Lanes 1 and 2, polyclonal NK cell populations; lane 3, blank; lane 4, an NK cell line (NKL); lane 5, an NK cell line (NK3.3); lane 6, human monocytes; lane 7, a histiocytic lymphoma cell line (U937); lane 8, a T lymphoma cell line (Jurkat); lane 9, an acute promyelocytic leukemia cell line (HL60); and lane 10, an EBV-transformed B cell line (LCL721.221). 10 μg of each RNA preparation (2 μg of poly A+ RNA from polyclonal NK cell populations, lanes 1 and 2) were hybridized with the 421-bp NKp30 cDNA probe. The positions of 28S and 18S ribosomal RNA subunits are indicated on the left. (B) A Southern blot containing genomic DNA from humans, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and S. cerevisiae yeast was hybridized under low stringency condition with the 421-bp NKp30 cDNA probe.
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Related In: Results  -  Collection

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Figure 8: Northern blot analysis of NKp30 transcript expression and Zoo-Blot analysis. (A) Total RNA was isolated from cells of different origin. Lanes 1 and 2, polyclonal NK cell populations; lane 3, blank; lane 4, an NK cell line (NKL); lane 5, an NK cell line (NK3.3); lane 6, human monocytes; lane 7, a histiocytic lymphoma cell line (U937); lane 8, a T lymphoma cell line (Jurkat); lane 9, an acute promyelocytic leukemia cell line (HL60); and lane 10, an EBV-transformed B cell line (LCL721.221). 10 μg of each RNA preparation (2 μg of poly A+ RNA from polyclonal NK cell populations, lanes 1 and 2) were hybridized with the 421-bp NKp30 cDNA probe. The positions of 28S and 18S ribosomal RNA subunits are indicated on the left. (B) A Southern blot containing genomic DNA from humans, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and S. cerevisiae yeast was hybridized under low stringency condition with the 421-bp NKp30 cDNA probe.
Mentions: Searching EMBL/GenBank/DDBJ databases revealed that the clone 5C cDNA was identical to a previously identified alternatively spliced form of the 1C7 gene (available under accession no. AF031138). This gene has been mapped on human chromosome 6, in the TNF cluster of the MHC gene complex 38. To date, however, owing to the lack of specific mAb, neither the function nor the surface distribution of the putative product of 1C7 gene could be identified. Moreover, the 1C7 transcript could not be revealed by Northern blot on different tissues and cell lines. On the other hand, by reverse transcriptase (RT)-PCR, the 1C7 transcript could be amplified by RNA isolated from spleen (but not from other tissues) or certain lymphoid and myeloid cell lines. These data suggested that 1C7 transcripts could be poorly represented, or could be expressed at substantial levels only in a narrow range of cell types 39. Our present analysis of NKp30 expression by Northern blotting revealed a mRNA of ∼1 kb in polyclonal NK cell populations and NK cell lines, including NKL and NK3.3. On the contrary, consistent with the lack of reactivity with anti-NKp30 mAbs, no NKp30 mRNA could be detected in human monocytes or cell lines of different histotype, including U937, Jurkat, HL60, and LCL 721.221 cells (Fig. 8 A). In some of these cell lines that were negative for mRNA expression by Northern blot (and for anti-NKp30 mAb surface staining), it has been possible to detect transcripts when analyzed by the RT-PCR technique. This finding is likely to reflect a low level of NKp30 transcription resulting in lack of NKp30 surface expression. Moreover, Northern blot analysis of multiple human tissues showed selective expression of NKp30 transcript only in spleen cells (data not shown). Altogether, these data are consistent with the notion that NKp30 expression is largely NK specific.

Bottom Line: This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells.Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion.Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

View Article: PubMed Central - PubMed

Affiliation: Istituto Nazionale per la Ricerca sul Cancro, 16132 Genova, Italy.

ABSTRACT
Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Show MeSH
Related in: MedlinePlus