The Regulation And Mechanism Of Bacillus Calmette-Guérin On Human Nature Killer Cells

ObjectiveBacillus Calmette-Guérin (BCG) is a nonvirulent form of the live Mycobacterium bovis. Usually, BCG was used to prevent tuberculosis infection and to treat clinical tumors. Althought the immunotherapy with BCG for cancer is successful, the precise mechanism is still unclear. Up to now, the studies of BCG have focused on its effect on some of the immune cells such as T lymphocyte and antigen presenting cells. However, the effect of BCG on natural killer cells (NK cells) is still not well known. NK cell is one of the main components of innate immune system and plays an important role in mediating both early protection against viruses or tumor cells and regulating immune functions. Our study has focused on the effect and mechanism of BCG on human nature killer cells.Subjects and Methods1. Subjects: 43 healthy volunteers consisting of 23 men and 20 women were studied. Mean age was 30.3 years (range, 21- 43 years ) with negative anti-Mycobacterium tuberculosis antibody. Our study was conducted according to the principles expressed in the Helsinki Declaration with informed consent obtained from each subject. 2. Methods: 10-20 ml venous blood sample was collected into a tube containing sodium heparin (15 ~ 25 U/ml) by sterile operation . Peripheral blood mononuclear cells (PBMC) were prepared using Ficoll density gradient centrifugation.2.1 PBMC were cultured in the presence or absence of BCG. The production of IFN-γand TNF-αfrom cell-free culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). The frequency of IFN-γand granzyme B producing cells were analyzed at the single-cell level by enzyme-linked immunospot assay ( ELISpot ). The cytolytic activity of PBMC against K562 target cells sensitive to natural killer cells was detected by MTT reduction assay.2.2 Purified NK cells were obtained from PBMC by negative selection using immunomagnetic beads. PBMC and NK cells were cultured without any stimuli or with BCG, IL-12 or PMA plus Ionomycin. The level of IFN-γand TNF-αin supernatants was measured by ELISA. The cytolytic activity of NK cells was detected by MTT reduction assay.2.3 PBMC were cultured with or without different doses of BCG, and the level of IL-12p40 in the supernatants was measured by ELISA. The surface expression of IL-12Rβ1 on NK cells was detected by flow cytometry. The effect of anti-IL-12Rβ1 monoclonal antibody (2B10) on the production of IFN-γand granzyme B of BCG exposed PBMC were detected by ELISA and ELISpot assays.3. Statistical analysisStatistics were determined using independent-samples t-test by SPSS13.0 software (Statistical Package for Social Sciences) , with p<0.05 considered significant.Results1. BCG significantly induced IFN-γand TNF-αproduction by PBMC in a dose-dependent manner.2. When PBMC was stimulated with BCG , the frequency of granzyme B producing cells was higher than that from the unstimulated PBMC(incubated in RPMI1640) (P<0.05). BCG could enhance the cytotoxic activity of PBMC against K562 target cells, increased with effector:target ratio.3. No IFN-γproduction was detected when purified NK cells were stimulated with BCG alone. However, BCG could significantly enhance IFN-γproduction when purified NK cells were stimulated with IL-12. Moreover, purified NK cells did not produce TNF-αwhen cultured with BCG, IL-12 or BCG plus IL-12, respectively.4. The cytotoxic activity of BCG-stimulated purified NK cells was slightly higher than that of unstimulated NK cells but no significant difference was observed (P>0.05).5. BCG could induce IL-12 production by PBMC in a dose-dependent manner and enhance IL-12Rβ1 expression on different subsets of NK cells.6. IFN-γproduction by PBMC which were exposed to BCG plus 2B10 was significantly lower than those exposed to BCG alone (P <0.001) . Similar results were shown in the analysis of the frequency of granzyme B producing cells at the same conditions (P <0.05).Conclusion1. BCG can induce the production of IFN-γand TNF-αby PBMC in a dose-dependent manner and the costimulation with BCG and IL-12 has a synergistic effect significantly. Moreover, BCG can increase the frequency of granzyme B producing cells in PBMC and enhance the cytotoxic activity against K562 target cells.2. BCG alone can't induce IFN-γand TNF-αproduction by purified NK cells, but it can augment IL-12-induced IFN-γproduction by purified NK cells. BCG alone doesn't enhance the cytotoxic activity of purified NK cells.3. BCG can induce IL-12 production by PBMC and augment IL-12Rβ1 expression on the surface of NK cells. Anti-IL-12Rβ1 mAb (2B10) partially inhibits BCG-induced IFN-γproduction and completely inhibits BCG-induced granzyme B releasing by PBMC.Taken together, our studies indicate that BCG can indirectly promote cytotoxic effect of NK cells and cytokine production by NK cells, and the production of endogenous IL-12 and up-regulation IL-12Rβ1 expression on the surface of NK cells are the possible regulating mechanisms in the immune response.