Expression of Major Histocompatibility Complex Class I-Related Chain (MIC) on Fresh and Cloned Myeloma Cells.

Introduction: Major histocompatibility complex class-I related chain (MIC) is a ligand for NKG2D, one of the activating NK receptor. It is expressed on various cancerous cells, and mediates NK cytolysis. Myeloma cells may be susceptible to immune therapy because interferon (IFN)-alpha or thalidomide are effective for a part of myeloma patients. It is also reported that one myeloma cell line is susceptible to NK cytolysis, but another lines are not, and this difference is not depend on its KIR incompatibility. We therefore examined the expression of MIC on myeloma cells, and its role on NK cell cytotoxicity.

Method: MIC expression was examined on two myeloma cell lines, U266 and RPMI8226, and fresh myeloma cells by flow cytometry (FCM) and reverse transcript polymerase chain reaction (RT-PCR). Modulation of MIC expression on these cell lines and fresh myeloma cells by the addition of various concentration of the drugs such as IFN-alpha, thalidomide, all-trans retinoic acid (ATRA), dexamethasone, and incadronate was examined by FCM and RT-PCR. The relationship between MIC expression and NK cytolysis was examine by 51Cr release assay.

Result: MIC was highly expressed on these two cell lines. MIC mRNA was also detected by RT-PCR in both cell lines. IFN-alpha, thalidomide, ATRA, incadronate and dexamethasone are known to be effective for MM in vivo or in vitro, we therefore examined the effect of these drugs on the expression of MIC on two myeloma cell lines. The cell lines were treated with or without these drugs at various concentrations, and then subjected to FCM. MIC expression was not changed after treatment of thalidomide, incadronate and dexamethasone. But unexpectedly, it was down-regulated by IFN-alpha and ATRA dose-dependently on both cell lines. To examine whether MIC expression on myeloma cell lines is involved in their susceptibility to NK cells, NK cell cytotoxicity against these cell lines was analyzed by 51Cr releasing assay. A substantial cytolysis was detected in U266 cells, while RPMI8226 did not show any relevant cytolysis, though the cell line express high level of MIC. Furthermore NK cytotoxicity was not inhibited by the addition of anti-MIC antibody. Then we examined the effect of IFN-alpha on the NK cytotoxicity, because IFN-alpha down-regulated the expression of MIC on myeloma cell lines. However, NK cytotoxicity was also induced by IFN-alpha in both cell lines. To examine whether MIC is expressed on fresh myeloma cells and is involved in the effect of the various drugs against myeloma cells, fresh samples from 12 MM patients were examined on the expression of MIC using two-color flow cytometric analysis. MIC was expressed on myeloma cells in one of 12 patients examined. The treatment with IFN-alpha, thalidomide and ATRA did not up-regulated the expression of MIC in all samples.

Conclusion: MIC is expressed on myeloma cell lines, but rarely expressed on fresh myelma cells. The reason of the discrepancy of MIC expression between fresh and cloned myeloma cells may be that myeloma cells are killed by host NK cells in vivo, provided the cells express MIC. In case myeloma cells express MIC in vivo, it may be that the molecule is not recognized by NKG2D on the NK cell.