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The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore‐forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7‐fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.

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.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

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.

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.

RJ 2.2.5, a variant of the human B-lymphoma cell line Raji, does not express the HLA-DR, -DQ, and -DP class II (or Ia) histocompatibility antigens, as a result of a defect in the transcription of the corresponding genes. This defect is corrected after fusion of RJ 2.2.5 cells with mouse Ia-positive cells. Previous work showed that the trans-acting transcriptional activator supplied by the mouse cells is encoded by a locus on mouse chromosome 16. We show here that reexpression of human major histocompatibility complex class II genes by RJ 2.2.5 cells can also be achieved by stable integration of mouse genomic sequences into the RJ 2.2.5 genome after DNA-mediated gene transfer.

Mouse mammary tumor virus (MMTV) infects B lymphocytes and expresses a superantigen on the cell surface after integration of its reverse-transcribed genome. Superantigen-dependent B- and T-cell activation becomes detectable 2 to 3 days after infection. We show here that before this event, B cells undergo a polyclonal activation which does not involve massive proliferation. This first phase of B-cell activation is T cell independent. Moreover, during the first phase of activation, when only a small fraction of B cells is infected by MMTV(SW), viral DNA is detected only in activated B cells. Such a B-cell activation is also seen after injection of murine leukemia virus but not after injection of vaccinia virus, despite the very similar kinetics and intensity of the immune response. Since retroviruses require activated target cells to induce efficient infection, these data suggest that the early polyclonal retrovirus-induced target cell activation might play an important role in the establishment of retroviral infections.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore‐forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7‐fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.