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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: NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis.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.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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CD40 molecules are  able to activate NK-dependent  killing. P815 (open circles) and  CD40-P815 (closed circles) cells  were used as targets in a classical  chromium release assay. A and B  indicate % lysis obtained with  NK cell line NK3.3 and NK  clone JA2, respectively. C shows  a representative experiment in  which NK3.3 NK cell line was  tested in a classical chromium release assay in the presence of  CD40-transfected P815 target  preincubated with medium  (closed circles), with the IgM antiCD40 mAb 14G7 (open squares),  or with the control anti-CD57  mAb TIB-200 (open triangles).  Both mAb were used as 1:1,000  ascites dilution.
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Figure 1: CD40 molecules are able to activate NK-dependent killing. P815 (open circles) and CD40-P815 (closed circles) cells were used as targets in a classical chromium release assay. A and B indicate % lysis obtained with NK cell line NK3.3 and NK clone JA2, respectively. C shows a representative experiment in which NK3.3 NK cell line was tested in a classical chromium release assay in the presence of CD40-transfected P815 target preincubated with medium (closed circles), with the IgM antiCD40 mAb 14G7 (open squares), or with the control anti-CD57 mAb TIB-200 (open triangles). Both mAb were used as 1:1,000 ascites dilution.

Mentions: In an effort to assess the role of costimulatory molecules in NK activation, we studied the ability of the NK line NK3.3 and clone JA2 to recognize and kill P815 mastocytoma cells and their CD40 transfectants. Fig. 1 shows that the transfection of CD40 molecule is able, alone, to induce recognition and lysis of P815 targets by NK3.3 effectors (Fig. 1 A). Similar results were obtained with the NK clone JA2 (Fig. 1 B). Five NK clones were tested using the same target systems, giving comparable results (data not shown). Any interference due to possible clonal differences in the target susceptibility could be excluded by blocking experiments in which a chromium release assay was performed in the presence of targets pretreated with anti-CD40 IgM mAb 14G7 or with the isotypic control TIB-200 mAb (Fig. 1, C). These data indicated that a CD40-dependent pathway is functionally involved in the induction of cytotoxicity of human NK cell lines and clones. In contrast, fresh polyclonal NK cells that had not been exposed to IL-2 were not able to recognize the CD40-transfected targets (Fig. 2 A). However, PBMC depleted of plastic-adherent cells and activated with rIL-2 (1,000 IU/ml) were able to recognize and kill CD40 transfectants. P815 parental cells were not killed. The effect was detectable after 18 h of culture with IL-2, reached its maximum after 48 h and was still evident after 72 h of IL-2 treatment (Fig. 2 C). Depletion by magnetic beads of CD56+ but not CD3+ lymphocytes completely inhibited the killing of CD40-transfected P815 (Figs. 3, A and B). We conclude that a CD40-dependent cytotoxic pathway is functional, not only in human NK lines and clones, but also in polyclonal rIL-2–activated NK effectors, isolated from peripheral blood of normal individuals.


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)

CD40 molecules are  able to activate NK-dependent  killing. P815 (open circles) and  CD40-P815 (closed circles) cells  were used as targets in a classical  chromium release assay. A and B  indicate % lysis obtained with  NK cell line NK3.3 and NK  clone JA2, respectively. C shows  a representative experiment in  which NK3.3 NK cell line was  tested in a classical chromium release assay in the presence of  CD40-transfected P815 target  preincubated with medium  (closed circles), with the IgM antiCD40 mAb 14G7 (open squares),  or with the control anti-CD57  mAb TIB-200 (open triangles).  Both mAb were used as 1:1,000  ascites dilution.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: CD40 molecules are able to activate NK-dependent killing. P815 (open circles) and CD40-P815 (closed circles) cells were used as targets in a classical chromium release assay. A and B indicate % lysis obtained with NK cell line NK3.3 and NK clone JA2, respectively. C shows a representative experiment in which NK3.3 NK cell line was tested in a classical chromium release assay in the presence of CD40-transfected P815 target preincubated with medium (closed circles), with the IgM antiCD40 mAb 14G7 (open squares), or with the control anti-CD57 mAb TIB-200 (open triangles). Both mAb were used as 1:1,000 ascites dilution.
Mentions: In an effort to assess the role of costimulatory molecules in NK activation, we studied the ability of the NK line NK3.3 and clone JA2 to recognize and kill P815 mastocytoma cells and their CD40 transfectants. Fig. 1 shows that the transfection of CD40 molecule is able, alone, to induce recognition and lysis of P815 targets by NK3.3 effectors (Fig. 1 A). Similar results were obtained with the NK clone JA2 (Fig. 1 B). Five NK clones were tested using the same target systems, giving comparable results (data not shown). Any interference due to possible clonal differences in the target susceptibility could be excluded by blocking experiments in which a chromium release assay was performed in the presence of targets pretreated with anti-CD40 IgM mAb 14G7 or with the isotypic control TIB-200 mAb (Fig. 1, C). These data indicated that a CD40-dependent pathway is functionally involved in the induction of cytotoxicity of human NK cell lines and clones. In contrast, fresh polyclonal NK cells that had not been exposed to IL-2 were not able to recognize the CD40-transfected targets (Fig. 2 A). However, PBMC depleted of plastic-adherent cells and activated with rIL-2 (1,000 IU/ml) were able to recognize and kill CD40 transfectants. P815 parental cells were not killed. The effect was detectable after 18 h of culture with IL-2, reached its maximum after 48 h and was still evident after 72 h of IL-2 treatment (Fig. 2 C). Depletion by magnetic beads of CD56+ but not CD3+ lymphocytes completely inhibited the killing of CD40-transfected P815 (Figs. 3, A and B). We conclude that a CD40-dependent cytotoxic pathway is functional, not only in human NK lines and clones, but also in polyclonal rIL-2–activated NK effectors, isolated from peripheral blood of normal individuals.

Bottom Line: NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis.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.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