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A new mechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction.

Carbone E, Ruggiero G, Terrazzano G, Palomba C, Manzo C, Fontana S, Spits H, Kärre K, Zappacosta S - J. Exp. Med. (1997)

Bottom Line: CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells.Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells.NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

View Article: PubMed Central - PubMed

Affiliation: Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Naples, Italy.

ABSTRACT
NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2-activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

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CD56+CD3− lymphocytes express CD40L after incubation  with IL-2. Staining profiles of o.n. cultured lymphocytes, obtained as  indicated in the Materials and Methods section, by using PE-labeled antiCD40L TRAP-1, FITC-labeled anti-CD3, or anti-CD56 mAbs in a classical double fluorescence assay. Results were evaluated as histograms  referring to TRAP-1 binding on CD3+ or CD56+ gated cell. The comparison with the binding obtained in the counterpart population (CD3−/ CD56−) was performed by histogram overlapping, as indicated. The analysis was performed on the same population of lymphocytes cultured o.n.  in the presence of medium (A and C) or with rIL-2 (B and D), respectively.
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Figure 5: CD56+CD3− lymphocytes express CD40L after incubation with IL-2. Staining profiles of o.n. cultured lymphocytes, obtained as indicated in the Materials and Methods section, by using PE-labeled antiCD40L TRAP-1, FITC-labeled anti-CD3, or anti-CD56 mAbs in a classical double fluorescence assay. Results were evaluated as histograms referring to TRAP-1 binding on CD3+ or CD56+ gated cell. The comparison with the binding obtained in the counterpart population (CD3−/ CD56−) was performed by histogram overlapping, as indicated. The analysis was performed on the same population of lymphocytes cultured o.n. in the presence of medium (A and C) or with rIL-2 (B and D), respectively.

Mentions: To better characterize the induction of the CD40-dependent cytotoxic pathway in the human NK lines and clones, we studied their expression of surface CD40L. Fig. 4 shows profiles obtained by staining the NK3.3 cell line (A) and JA2 NK clone (B) with the anti-CD40L mAb TRAP-1. Both NK populations expressed CD40L molecule. We also assessed the expression of CD40L on polyclonal NK effectors derived from peripheral blood of normal donors, by double staining with anti-CD40L mAb TRAP-1 and anti-CD3 or anti-CD56 mAb. A clear TRAP-1 binding on the CD3−CD56+ population was demonstrated after overnight (o.n.) incubation in the presence of rIL-2, but not with medium alone (Fig. 5). No binding was observed on fresh cells (data not shown). These data indicate that CD3−CD56+ NK effectors, activated with rIL-2, can express the CD40L molecule; this is likely to account for their ability to specifically recognize and kill CD40-expressing targets.


A new mechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction.

Carbone E, Ruggiero G, Terrazzano G, Palomba C, Manzo C, Fontana S, Spits H, Kärre K, Zappacosta S - J. Exp. Med. (1997)

CD56+CD3− lymphocytes express CD40L after incubation  with IL-2. Staining profiles of o.n. cultured lymphocytes, obtained as  indicated in the Materials and Methods section, by using PE-labeled antiCD40L TRAP-1, FITC-labeled anti-CD3, or anti-CD56 mAbs in a classical double fluorescence assay. Results were evaluated as histograms  referring to TRAP-1 binding on CD3+ or CD56+ gated cell. The comparison with the binding obtained in the counterpart population (CD3−/ CD56−) was performed by histogram overlapping, as indicated. The analysis was performed on the same population of lymphocytes cultured o.n.  in the presence of medium (A and C) or with rIL-2 (B and D), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196353&req=5

Figure 5: CD56+CD3− lymphocytes express CD40L after incubation with IL-2. Staining profiles of o.n. cultured lymphocytes, obtained as indicated in the Materials and Methods section, by using PE-labeled antiCD40L TRAP-1, FITC-labeled anti-CD3, or anti-CD56 mAbs in a classical double fluorescence assay. Results were evaluated as histograms referring to TRAP-1 binding on CD3+ or CD56+ gated cell. The comparison with the binding obtained in the counterpart population (CD3−/ CD56−) was performed by histogram overlapping, as indicated. The analysis was performed on the same population of lymphocytes cultured o.n. in the presence of medium (A and C) or with rIL-2 (B and D), respectively.
Mentions: To better characterize the induction of the CD40-dependent cytotoxic pathway in the human NK lines and clones, we studied their expression of surface CD40L. Fig. 4 shows profiles obtained by staining the NK3.3 cell line (A) and JA2 NK clone (B) with the anti-CD40L mAb TRAP-1. Both NK populations expressed CD40L molecule. We also assessed the expression of CD40L on polyclonal NK effectors derived from peripheral blood of normal donors, by double staining with anti-CD40L mAb TRAP-1 and anti-CD3 or anti-CD56 mAb. A clear TRAP-1 binding on the CD3−CD56+ population was demonstrated after overnight (o.n.) incubation in the presence of rIL-2, but not with medium alone (Fig. 5). No binding was observed on fresh cells (data not shown). These data indicate that CD3−CD56+ NK effectors, activated with rIL-2, can express the CD40L molecule; this is likely to account for their ability to specifically recognize and kill CD40-expressing targets.

Bottom Line: CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells.Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells.NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

View Article: PubMed Central - PubMed

Affiliation: Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Naples, Italy.

ABSTRACT
NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2-activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

Show MeSH
Related in: MedlinePlus