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Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.

B cells express MHC class II Ag in a constitutive fashion, whereas macrophages can do so only after induction by a variety of exogenous stimuli. In this study we describe interspecies somatic cell hybrids between the human B cell Raji and the murine macrophage cell P388 D1. This murine cell line does not express detectable levels of class II mRNA. Phenotypic, molecular, and karyotype analysis of a series of hybrids showed that murine macrophage class II genes can be expressed in a constitutive fashion under the control of the human B cell genome. This event is the consequence of de novo accumulation of class II specific mRNA and thus probably reflects activation of transcription. In certain cases the amount of murine class II Ag expressed on the surface of the hybrid cell was significantly higher than the one observed in the parental macrophage cells after induction with IFN-gamma and was not further modified by treatment with the murine lymphokine. Reversion from a murine class II-positive to class II-negative cell surface phenotype in the hybrids correlated with reduced expression of human markers and more important with segregation of human chromosomes. Interestingly, in this case certain hybrids still expressed detectable levels of murine class II mRNA and increased levels of murine invariant chain mRNA when compared with parental P388 D1 murine macrophage cells. These results indicate that constitutive class II gene expression behaves as a dominant trait in B cell x macrophage somatic cell hybrids. Possible mechanisms responsible of the different control of class II gene expression during cell type differentiation are discussed.

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.

In a previous series of studies, we had shown that the constitutive Ia expression in an immunoselected Ia-human B cell variant, RJ 2.2.5, could be restored by somatic cell hybridization with mouse B cells. These experiments allowed us to show the existence of a transacting activator factor(s) operating across species barriers and encoded by the aIr-1 locus located on mouse chromosome 16. The aim of the present study was to investigate whether the B cell constitutive Ia expression and the inducible Ia expression, as seen in macrophages treated with IFN-gamma, are controlled by similar intracellular factors. To this purpose, we constructed an interspecies somatic cell hybrid between the human Ia-RJ 2.2.5 B cells and the mouse Ia-P388 D1 macrophage cells. These murine cells transiently express Ia antigens when incubated with IFN-gamma. Our results show that RJ 2.2.5 X P388 D1 cell hybrids do not express either human or mouse class II gene products. Treatment with human recombinant IFN-gamma did not modify the MHC phenotype of either the hybrid cells or the human parental cells. On the other hand, treatment of the hybrid cells with murine recombinant IFN-gamma resulted in de novo expression of mouse Ia mRNA and corresponding cell surface antigens without, however, reinduction of the human class II-positive phenotype. Furthermore, treatment with the mouse lymphokine significantly increased the levels of human HLA class I mRNA and corresponding cell surface antigens in the hybrid cells, further reinforcing the notion of the existence of non-species-specific secondary mediators generated after receptor-ligand interaction in the IFN-gamma system. Together, these results indicate that in macrophages, the intracellular events taking place after binding of IFN-gamma with its own receptor and leading to the expression of a class II-positive phenotype do not operate via an activation of the aIr-1 locus and/or its products. Thus, at least in our experimental system, we can firmly establish a first, relevant distinction between constitutive and inducible class II gene expression. This difference, dictated by the specific differentiation program of each cell type, may be relevant for the understanding of the function of class II gene products.

Constitutive expression of major histocompatibility complex class II genes is acquired very early in B-cell ontogeny and is maintained up to the B-cell blast stage. Terminal differentiation in plasma cells is, however, accompanied by a loss of class II gene expression. In B cells this gene system is under the control of several loci encoding transacting factors with activator function, one of which, the aIr-1 gene product, operates across species barriers. In this report human class II gene expression is shown to be extinguished in somatic cell hybrids between the human class II-positive B-cell line Raji and the mouse class II-negative plasmacytoma cell line P3-U1. Since all murine chromosomes are retained in these hybrids and no preferential segregation of a specific human chromosome is observed, the results are compatible with the presence of suppressor factors of mouse origin, operating across species barriers and inhibiting class II gene expression. Suppression seems to act at the level of transcription or accumulation of class II-specific mRNA, since no human, and very few murine, class II transcripts are detectable in the hybrids.

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.