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RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Four monoclonal antibodies reacting with distinct human Ia antigenic determinants have been used to demonstrate the coexpression of four distinct subsets, NG1, NG2, H40+-3/4+, and DC1 H40--3/4+, in the Ia pool of DR heterozygous or homozygous B cell lines. By two-dimensional peptide mapping the four subsets within the same Ia pool displayed structurally different beta as well as alpha subunits. The beta chain of the NG1 subset was shown to display considerable structural polymorphism when analyzed in two cell lines with distinct DR but similar DC phenotype, LG2 (DR1,1-DC1) and Raji (DR3, W6-DC1). In contrast, the beta chains of NG2, DC1 H40+-3/4+, and DC1 H40--3/4+ subsets of LG2 cells were shown to be very similar to their homologous Raji cell counterparts, thus indicating a relatively low structural polymorphism. Furthermore, the alpha chains of either one of the four subsets expressed in LG2 cells displayed very high structural similarities to the homologous counterparts in the Raji Ia pool, thus suggesting a relatively low polymorphism for the large Ia subunits described in this study. A striking feature deduced from this study was the selective subunit association of the distinct alpha-beta heterodimers.

Structural analysis by two-dimensional peptide maps (2D-PM) of the human Ia molecular pool expressed on the cell surface of two distinct lymphoblastoid cell line, LG-2 and Raji, revealed the existence of a novel MHC class II molecular heterodimer that differs at the level of both alpha and beta subunits from the previously described DP, DQ, and DR antigens. These differences were also seen at the level of two-dimensional electrophoresis (2D-PAGE) of biosynthetically labeled intact molecules, although to a lesser extent, due to the intrinsic limitations of this technique in resolving fine structural differences. We have designated this new class II antigen as the fourth Ia subset. The fourth Ia subset seems to represent a small proportion of the human Ia pool. Comparative analysis by 2D-PM of the two cell lines showed the presence of structural variations in the alpha chains of the fourth Ia subset, suggesting the existence of polymorphism for these subunits. Cell surface iodination did not show appreciable labeling of the fourth subset beta chain in LG-2 cells, and this prevented analysis of the structural polymorphism of this subunit. Furthermore, for the first time, we have shown that DP alpha chains display distinct peptide maps in LG-2 and Raji cells, thus suggesting the presence of structural polymorphism for these Ia subunits also. The DQ1 alpha and beta allelic products present in LG-2 cells (DQ homozygous) did not show appreciable structural variation when compared with the homologous allelic products present in Raji cells (DQ heterozygous). Finally, we have confirmed the absence of polymorphism for the DR alpha subunits. By 2D-PM, relatively low structural variation was instead found for the highly polymorphic DR beta subunits expressed in the two cell lines, suggesting that cell surface iodination preferentially labels constant domains of DR beta chains.

Raji, a human B lymphoma line, expresses high levels of major histocompatibility complex (MHC) class II antigens. Conversely, none of the detectable human Ia antigens is present in RJ 2.2.5, an immunoselected Raji variant. Clonal analysis, biochemical characterization, and nucleic acid hybridization studies of hybrids between mouse spleen cells and RJ 2.2.5 show that MHC class II gene expression is regulated in trans by a factor which, as judged by dominance studies, has the characteristics of an activator. Such a positive trans acting factor is expressed in mouse spleen cells, and is able to implement MHC class II gene expression across species boundaries. Expression of this factor in spleen cells strongly suggests that it plays a role in in vivo regulation of Ia expression. Additional data suggest that different subsets of class II genes such as DR and DQ may, in part, be regulated by different mechanisms. It has also been possible to show that the amount of In chain-specific mRNA, present at reduced levels in RJ 2.2.5 cells compared to the parental Raji cells, drastically increased in human X mouse cells hybrids reexpressing human Ia antigens, suggesting that the In chain gene and the class II genes, although located on different chromosomes, are regulated in a concerted fashion, either directly through the same implementing factor, or indirectly through a cascade mechanism.

An E beta DNA protein complex termed complex A, whose binding activity has recently been shown to correlate with both constitutive and regulated class II expression in murine cell lines, is also present in a human B cell, Raji. The DNA involved in complex A, which includes three previously defined transcriptional motifs, W, X, and Y, is a cis-acting transcription element in Raji cells. Both complex A binding activity and transcriptional activity of its target sequence are absent in an Ia- mutant subclone of Raji, RJ 2.2.5. This cell line, whose defect is complemented by a locus on mouse chromosome 16, reexpresses both class II and complex A upon transfection with mouse genomic DNA. We suggest that factors that form complex A or that regulate complex A formation account for the molecular lesion in this cell line.

We have studied the effect on the interleukin (IL-) 2-dependent human T cell growth of two distinct monoclonal antibodies (Mab), D1-12 and 4F2, with specificity for common determinant of human Ia antigens and for a differentiation antigen expressed on all activated T cells, respectively. Strong inhibition of cell growth was found in cultures supplemented with the anti-Ia D1-12 Mab but not in cultures supplemented with 4F2 Mab. These results were obtained when either total mixed leukocyte culture (MLC) T cells or an MLC-derived T cell clone were used as indicator cell systems for IL-2 activity. The inhibition of cell growth appears to be mediated by a direct interaction of D1-12 Mab with the cells and not by a direct inactivation of the growth factor, as addition of the antibody to murine MLC T cells, which do not express the determinant defined by D1-12 Mab, resulted in no inhibition of their proliferation induced by the same source of human IL-2.

Two monoclonal antibodies, D1-12 and BT 2.2, recognizing two distinct subsets of human Ia molecules, NG1 and NG2, respectively, present in all individuals irrespective of their HLA-DR phenotype, have been used to immunoselect cell variants from the lymphoblastoid cell line Raji. Results showed that, irrespective of the monoclonal antibody used for immunoselection, the cell variants analyzed in this study had lost the expression of both D1-12-and BT 2.2-specific antigenic determinants. Moreover, the expression of antigenic determinants specific for a third family of Ia molecules, the DC-1 subset, were also lost in the cell variants. In contrast, expression of HLA A, B, and C common structures, as recognized by the W6.32 monoclonal antibody, as well as expression of surface immunoglobulins, were not affected. Possible mechanisms inducing such a coordinate loss of expression of several families of human Ia molecules are discussed.