SummaryInterferon (IFN) ",/, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-0t or -[3 suppresses MHC class II expression. The mechanism of IFN-13-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-~/-induced MHC class II transcription. We studied the status of IFN-~/-induced CIITA messenger P, NA (mI(NA) accumulation and CIITA-driven transactivation in IFN-13-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-~ treatment did not suppress IFN-~-induced accumulation of CIITA mR~A. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-[3. These results suggest that IFN-[3 acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) was essential for IFN-[3 to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-~ or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-[3-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.

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Recently, a non-DNA binding protein, class II transactivator (CIITA), has been shown to be required for constitutive and IFN-gamma-inducible class II MHC transcription. The cytokine TGF-beta inhibits IFN-gamma-induced class II MHC expression at the transcriptional level. In this study, we provide evidence that TGF-beta blocks IFN-gamma-induced CIITA mRNA accumulation. TGF-beta down-regulates class II MHC and CIITA mRNA accumulation in human astroglioma and fibrosarcoma cell lines, but TGF-beta does not destabilize the CIITA message, suggesting an effect at the transcriptional level. In cells that stably overexpressed CIITA, leading to a constitutive class II MHC-positive phenotype, the inhibitory effect of TGF-beta on class II MHC was abrogated, but the cells remained responsive for expression of TGF-beta-inducible genes. Cell lines that possessed defects in TGF-beta signaling also became refractory to inhibition of IFN-gamma-induced CIITA and class II MHC expression. Our data indicate that TGF-beta suppresses IFN-gamma-induced class II MHC expression by inhibiting accumulation of CIITA mRNA.

Cell surface proteins play an important role in multidrug resistance (MDR). However, the identification involving chemoresistant features for cell surface proteins is a challenge. To identify potential cell membrane markers in hematologic cancer MDR, we used a cell-and antibody-based strategy of subtractive immunization coupled with cell surface comparative screening of leukemia cell lines from sensitive HL60 and resistant HL60/DOX cells. Fifty one antibodies that recognized the cell surface proteins expressed differently between the two cell lines were generated. One of them, the McAb-5D12 not only recognizes its antigen but also block its function. Comparative analysis of immunofluorescence, flow cytometry, and mass spectrum analysis validated that the membrane antigen of McAb-5D12 is a nucleoprotein-polypyrimidine tract binding protein associated splicing factor, PSF. Our results identified that PSF overexpressed on the membrane of sensitive cells compared with resistant cells and its relocation from the nuclear to the cell surface was common in hematological malignancy cell lines and marrow of leukemia patients. Furthermore, we found that cell surface PSF contributed to cell sensitivity by inhibiting cell proliferation. The results represent a novel and potentially useful biomarker for MDR prediction. The strategy enables the correlation of expression levels and functions of cell surface protein with some cell-drug response traits by using antibodies.