Murine CTLL-2 cells respond to mIL12: prospects for developing an alternative bioassay for measurement of murine cytokines IL12 and IL18.
Cell line-based bioassays are becoming increasingly popular for assessment of biological activities of cytokines primarily because these are easy to perform and are not subject to donor variation. A well characterised cell line with world wide availability would further minimise the inter-assay variations. C57BL/6 mice derived T cell line; CTLL-2 fits this criterion. We explored the potential of CTLL-2 cells to develop a bioassay to detection of murine (m) IL12 and mIL18. Both cytokines have shown significant activity against a number of cancers and importantly, act synergistically via mutual upregulation of each other's receptors. The preliminary flow cytometric analyses of immunostained CTLL-2 cells showed that approximately 65% expressed mIL12 and approximately 5% expressed mIL18 receptors suggesting that these may respond to mIL12. As predicted, cells incubated with different doses of mIL12 or mIL18 for 72 h were responsive to mIL12 and not to mIL18. However, when pre-treated with mIL12 for 24 h prior to incubation with mIL18, there was a significant enhancement in response. The sensitivity of the response was comparable to that obtained using the conventional splenocyte-based IFNgamma release assay. The cytokine specificity of the response was proven unequivocally when significant reduction in CTLL-2 response was observed in the presence of the relevant neutralising antibodies. Finally, we could successfully detect lowest doses of approximately 0.1 pg/microL mIL12 or 40 pg/mL of mIL18 in cell supernatants in a cytokine specific manner, which is lower than the resting levels of these cytokines in mouse sera. Again the sensitivity was comparable to that observed in the conventional IFNgamma release assay. Hence, we have demonstrated the potential of CTLL-2-based bioassay to detect biologically active mIL12 and mIL18 in biological samples accurately and reproducibly.
Khatri
Aparajita
A
Prince of Wales Hospital and University of New South Wales, Oncology Research Centre, Level 2, Clinical Sciences Building, Prince of Wales Hospital, Barker Street, Randwick, NSW, 2031, Australia. a.khatri@unsw.edu.au
Husaini
Yasmin
Y
Russell
Pamela J
PJ
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D003124:Colorimetry; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D018664:Interleukin-12 / Q000032:analysis* / Q000378:metabolism / Q000502:physiology*; D020382:Interleukin-18 / Q000032:analysis* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D053707:Receptors, Interleukin-12 / Q000096:biosynthesis / Q000235:genetics; D053724:Receptors, Interleukin-18 / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000737:chemistry
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D020382:Interleukin-18; D053707:Receptors, Interleukin-12; D053724:Receptors, Interleukin-18; D013778:Tetrazolium Salts; D018664:Interleukin-12