Correlation of human CD56+ cell cytotoxicity and IFN-gamma production.

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.

T cell lysis of murine renal cancer: multiple signaling pathways for cell death via Fas.

Activated T cells lyse the murine renal cancer Renca. We have examined the mechanism of tumor cell lysis with the use of T cells derived from C57BL/6, BALB/c, B6.gld, and B6.Pfp-/- mice. C57BL/6 and BALB/c T cells can lyse Renca cells through the use of both granule- and Fas ligand (FasL)-mediated pathways. However, B6.gld T cells predominantly use granule-mediated killing, whereas B6.Pfp-/- T cells use FasL. The lysis of Renca by Pfp-/- T cells is only partially inhibited by the caspase inhibitor ZVAD-FMK, suggesting that caspase-independent signaling is also important for Renca cell lysis. When the reactive oxygen scavenger butylated hydroxyanisole was used alone or in combination with ZVAD-FMK a substantial reduction of Renca lysis was observed. Therefore, the caspase-independent generation of reactive oxygen intermediates in Renca after Fas triggering contributes to the lysis of these cells.

Natural killer cell-endothelial cell interactions in xenotransplantation.

Interest in xenotransplantation derives from the documented need for more organs and tissues than can be expected from living or cadaveric donors. Although the barriers to xenotransplantation are formidable, the scientific rewards in addressing these problems have been significant. The first and most potent barrier to xenotransplantation is hyperacute rejection mediated by xenoreactive natural antibodies and serum complement. The majority of the xenoreactive antibodies appear to be directed at terminal galactose epitopes, especially gal alpha1-3 gal. Significant progress has been made in surmounting hyperacute rejection, and this has led to an examination of underlying mechanisms of delayed xenograft rejection. One of these delayed mechanisms concerns the potential role of graft recipient, natural killer (NK) cells. NK cells can cause variable, low-level cytotoxicity of xenogeneic endothelial cells in vitro that may be enhanced in the presence of xenoreactive IgG. The specificity of NK cell-mediated cytotoxicity appears to overlap with a major subset of xenoreactive natural antibodies. These cytotoxic interactions can be regulated by "humanizing" the endothelial cells through expression of the appropriate human MHC class I genes. More important, NK cells induce endothelial cell activation, which results in changing the nature of the endothelial cell surface from an anticoagulant surface to a procoagulant surface. These findings parallel those observed in allogeneic NK cell-endothelial cell interactions and suggest these important observations may be extended to NK cell-endothelial cell interactions in general.

The role of calnexin in NK-target cell interaction.

In this study, a relationship between target cell sensitivity to natural killing and target cell expression of the molecular chaperone++ calnexin was assessed. The NK-resistant cell line NKR was originally derived from the NK-sensitive, human T-cell line CEM and does not synthesize calnexin protein or mRNA. The cell lines CEM, NKR and 1B9 (NKR transfected with a calnexin cDNA) were compared in a number of assays. All the lines but CEM were resistant to NK in conventional 4 h cytotoxicity assay, but were highly sensitive to IL-2 activated NK. Incubation of NK cells with CEM but not with the other two lines led to increased expression of the NK cell activation marker CD69. Treatment of effector cells with PGE2 and TGF-beta resulted in an inhibition of NK activity and CD69 expression. The calnexin transfected clone 1B9 clone had intermediate ability to block cytotoxicity in cold target inhibition assay compared to CEM and NKR. Expression of the adhesion molecules CD44 and LFA-1alpha was significantly higher on both calnexin positive cell lines compared to NKR. These data suggest that calnexin controls the expression of some, but not all, target structures that are necessary for binding and activation of NK cells.

Conformation dependence of MHC class I in the modulation of target cell sensitivity to natural killing.

C1R.Aw68 delta 242 is a human B cell line expressing a mutant class I molecule that is defective in assembly and transport at 37 degrees C but is stably expressed at room temperature. This cell line has been utilized to study the conformation dependence of MHC class I in the modulation of target cell sensitivity to natural killing. Surface expression of MHC class I molecules was monitored by the antibodies W6/32 (detecting a pan-class I specificity that is beta 2-microglobulin and conformation dependent) and HC.10 (detecting free HLA-B heavy chain and a subset of HLA-A heavy chains). C1R.Aw68 delta 242 was cultured at reduced temperature to induce cell surface expression of class I molecules, and then the temperature was shifted to 37 degrees C. During the first 2 h at 37 degrees C, C1R.Aw68 delta 242 displayed a higher level of HC.10 reactivity than W6/32. Conjugation of C1R.Aw68 delta 242 to NK cells correlated inversely with W6/32 expression, but not with HC.10 reactivity as revealed by flow cytometry. The sensitivity of the C1R.Aw68 delta 242 cells to NK-mediated lysis was also examined as a function of temperature, and the level of C1R.Aw68 delta 242 cytolysis correlated inversely with W6/32 expression but not HC.10. The fact that both the conjugation rate and target cell cytolysis increased with decreased reactivity with the conformation-dependent antibody W6/32 and not with HC.10, is consistent with the hypothesis that NK cell inhibitory receptors (KIR) detect a conformation-dependent epitope(s).

Induction of procoagulant function in porcine endothelial cells by human natural killer cells.

NK cells may mediate effector functions other than target cell cytotoxicity. To explore such noncytotoxic effector mechanisms, we tested whether human PBL and purified NK (CD56+) cells might induce expression of tissue factor by cultured porcine aortic endothelial cells. Tissue factor is the major coagulation factor that binds to factor VIIa and initiates coagulation. The addition of freshly isolated NK cells but not T cells to endothelial cells resulted in the induction of tissue factor activity. NK-depleted (CD56-) effector cells did not induce tissue factor activity; however, the combination of CD56+ cells and NK-depleted cells induced tissue factor activity to the same extent as unseparated cells. PBL induced tissue factor mRNA in porcine endothelial cells and NK depletion resulted in a significant decrease of the induction. Induction of tissue factor activity in porcine endothelial cells by human NK cells required direct cell-to-cell contact, as transfer of supernatants from NK-endothelial cell cultures to secondary cultures did not induce tissue factor activity, and anti-LFA-1alpha Abs inhibited the induction of tissue factor activity. Induction of tissue factor activity in endothelial cells by NK cells may represent one of a variety of ways in which NK cells mediate noncytotoxic effects.

Differential expression of natural killer cell markers: human versus baboon.

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.

Human natural killer cells induce morphologic changes in porcine endothelial cell monolayers.

In this study, we have investigated the early in vitro effects of natural killer (NK) cells on porcine aortic endothelial cell (PAEC) monolayers. Incubation of effector cells containing about 70% CD56+ cells on PAEC monolayer led to time-dependent changes in PAEC monolayer morphology. As little as 20 min of incubation resulted in changes in PAEC shape and in the appearance of gaps between the cells. These effects have been observed for up to 6 hr, but not before 20 min or after 6 hr. When NK-depleted effector cells were used, no morphological changes were observed in comparison with the same effectors before depletion; if CD56+ cells were added back, the effects were comparable with those on nondepleted effector cells. There was no detectable NK cell-mediated cytolytic activity during the 1-6 hr of incubation of peripheral blood lymphocytes with PAEC monolayers. These data indicate that NK cells may participate in endothelial cell changes leading to xenograft rejection.