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p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1997)

Bottom Line: Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones.The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A.Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Istologia ed Embriologia Generale, Università di Genova, Genova, Italy.

ABSTRACT
Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

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Related in: MedlinePlus

Analysis of the expression of p46 molecules in peripheral blood NK cells. In these  experiments freshly isolated PBMCs either unfractionated (a–d)  or depleted of both adherent cells  and CD3+ lymphocytes (e–g)  were analyzed by two-color immunofluorescence and FACS®  analysis for the expression of p46  surface molecules in combination with the indicated surface  markers. Cells were stained with  mAbs to the indicated molecules  followed by FITC or PE-conjugated isotype-specific goat anti– mouse second reagent. The contour plots were divided into quadrants  representing unstained cells (lower left), cells with only red fluorescence  (upper left), cells with red and green fluorescence (upper right), and cells  with only green fluorescence (lower right).
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Figure 3: Analysis of the expression of p46 molecules in peripheral blood NK cells. In these experiments freshly isolated PBMCs either unfractionated (a–d) or depleted of both adherent cells and CD3+ lymphocytes (e–g) were analyzed by two-color immunofluorescence and FACS® analysis for the expression of p46 surface molecules in combination with the indicated surface markers. Cells were stained with mAbs to the indicated molecules followed by FITC or PE-conjugated isotype-specific goat anti– mouse second reagent. The contour plots were divided into quadrants representing unstained cells (lower left), cells with only red fluorescence (upper left), cells with red and green fluorescence (upper right), and cells with only green fluorescence (lower right).

Mentions: To obtain an NK cell–enriched lymphocyte population, PBMCs were depleted of plastic-adherent cells and subsequently incubated with anti-CD3 (JT3A) mAb for 30 min at 4°C followed by treatment with goat anti–mouse–coated Dynabeads (Dynal, Oslo, Norway) for 30 min at 4°C. The resulting CD3− lymphocyte populations contained ∼1% CD3+ cells, 20% HLA-DR+ cells, and 80% CD56+ cells. These cells were used for FACS® analysis of CD3-depleted populations (see Fig. 3) and for cytolytic activity against K562 and P815 target cells (see Fig. 6 a). In another set of experiments, PBMCs were stained for both anti-CD3 and anti-p46 mAb and subsequently cells lacking the expression of p46 or CD3 or both markers (CD3−, p46−) were sorted and analyzed for cytolytic activity against K562 target cells (see Fig. 6 b).


p46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A - J. Exp. Med. (1997)

Analysis of the expression of p46 molecules in peripheral blood NK cells. In these  experiments freshly isolated PBMCs either unfractionated (a–d)  or depleted of both adherent cells  and CD3+ lymphocytes (e–g)  were analyzed by two-color immunofluorescence and FACS®  analysis for the expression of p46  surface molecules in combination with the indicated surface  markers. Cells were stained with  mAbs to the indicated molecules  followed by FITC or PE-conjugated isotype-specific goat anti– mouse second reagent. The contour plots were divided into quadrants  representing unstained cells (lower left), cells with only red fluorescence  (upper left), cells with red and green fluorescence (upper right), and cells  with only green fluorescence (lower right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211712&req=5

Figure 3: Analysis of the expression of p46 molecules in peripheral blood NK cells. In these experiments freshly isolated PBMCs either unfractionated (a–d) or depleted of both adherent cells and CD3+ lymphocytes (e–g) were analyzed by two-color immunofluorescence and FACS® analysis for the expression of p46 surface molecules in combination with the indicated surface markers. Cells were stained with mAbs to the indicated molecules followed by FITC or PE-conjugated isotype-specific goat anti– mouse second reagent. The contour plots were divided into quadrants representing unstained cells (lower left), cells with only red fluorescence (upper left), cells with red and green fluorescence (upper right), and cells with only green fluorescence (lower right).
Mentions: To obtain an NK cell–enriched lymphocyte population, PBMCs were depleted of plastic-adherent cells and subsequently incubated with anti-CD3 (JT3A) mAb for 30 min at 4°C followed by treatment with goat anti–mouse–coated Dynabeads (Dynal, Oslo, Norway) for 30 min at 4°C. The resulting CD3− lymphocyte populations contained ∼1% CD3+ cells, 20% HLA-DR+ cells, and 80% CD56+ cells. These cells were used for FACS® analysis of CD3-depleted populations (see Fig. 3) and for cytolytic activity against K562 and P815 target cells (see Fig. 6 a). In another set of experiments, PBMCs were stained for both anti-CD3 and anti-p46 mAb and subsequently cells lacking the expression of p46 or CD3 or both markers (CD3−, p46−) were sorted and analyzed for cytolytic activity against K562 target cells (see Fig. 6 b).

Bottom Line: Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones.The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A.Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Istologia ed Embriologia Generale, Università di Genova, Genova, Italy.

ABSTRACT
Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.

Show MeSH
Related in: MedlinePlus