Immunogenetics (1996) 43:352-359 © Springer-Veflag 1996 Ana-Maria Lennon • Catherine Ottone Ad Peijnenburg • Chantal Hamon-Benais Fr6d~ric Colland • Sam Gobin - Peter van den Elsen Marc Fellous • Rosa Bono • Catherine Alca'ide-Loridan The RAG cell line defines a new complementation group of MHC class II deficiency Received: 24 October 1995 / Revised: 13 December 1995 Abstraet We previously described RAG, a mouse adeno- carcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN- 7, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induc- tion of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN-7 of DMA, DMB, and the invariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designated F16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e. g., BLS cell lines), and which belong to five different complemen- tation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when trans- fected with the cDNA encoding human CIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA. A.-M. Lennon - C. Ottone • C. Hamon-Benais • E Colland M. Fellous • C. Alca'/de-Loridan(r~) Unit6 d'Immunog6n6tiqueHumaine, INSERM U276, Institut Pasteur, 25, rue du Dr. Roux, 75724 PARIS cedex t5, France A. Peijenburg • S. Gobin • E van den Elsen Department of Immunohematologyand Bloodbank, University Hospital Leiden, POB9600, 2300 RC. Leiden, The Netherlands R. Bono Departamento de Biologia,Facultad de Ciencias, Universidadde Chile, Santiago, Chile Introduction Class II major histocompatibility (MHC) antigens are involved in the presentation of antigens to CD4 + T tymphocytes (Germain 1994). These antigens are ex- pressed constitutively in B lymphoblastoid cell lines (B-LCLs), and are inducible by IFN- 7 in macrophages, dendritic and epithelial cells (Glimcher and Kara 1992). This expression pattern is mainly regulated at the transcrip- tional level. Nucleotide sequencing of the promoters of MHC class II genes has revealed that they contain several conserved sequences, referred to as the W, X, and Y boxes (Dorn et al. 1987). The functional role of these sequences in the expression of MHC class II antigens has been demon- strated by deletion analysis (Boss and Strominger 1986; Koch et al. 1989). Three additional proteins, the invariant chain (Ii), DMA, and DMB are required for a proper peptide loading of the MHC class II antigens and complex translocation to the plasma membrane (Germain 1994; Fling et al. 1994; Sloan et al. 1995). The promoters of the corresponding genes also contain the X and Y boxes, leading to a coordinate expres- sion of these proteins and the MHC class II antigens (O'Sullivan et al. 1986; Kelly et al. 1991). A valuable approach for studying the regulatory me- chanisms of MHC class II gene expression has involved the use of cell lines established from patients affected by a severe combined immunodeficiency (SCID) also named Bare Lymphocyte Syndrome (BLS), or MHC class II defi- ciency. The BLS syndrome is characterized by the absence of MHC class II antigen expression in all the cell types studied, even though the MHC class II genes are intact (dePrtval et al. 1985; B~nichou and Strominger 1991; Hume and Lee 1989; Lambert et al. 1993). Fusion of these various cell lines has defined four BLS complementa- tion groups (groups A, B, C, and E; Btnichou and Stro- minger 1991; Hume and Lee 1989; Peijnenburg et al. 1995). In addition, a fifth complementation group (group D) has been identified using an experimentally derived B-LCL mutant (Gladstone and Pious 1978).