Mouse endogenous superantigens: Ms and Mls-like determinants encoded by mouse retroviruses.

Commonly used inbred mouse strains express different combinations of integrated mouse mammary tumor proviruses (MMTV). This appendix summarizes the proviruses that have been detected. The reported functional properties of those MMTV proviral products which have been identified as superantigens are also summarized, including the ability to elicit primary or secondary T cell responses and to induce Vb-specific clonal deletion during T cell differentiation. In addition, the amino acid sequences of putative ORF gene products of different MMTV are compared.

Direct binding of the Mtv7 superantigen (Mls-1) to soluble MHC class II molecules.

The superantigen encoded by the mouse mammary tumor virus (MMTV) is a potent stimulator of T cells when bound to MHC class II molecules. Recent data from this laboratory have shown that the Mtv7 superantigen, Mls-1, elicits a strong T cell response when presented by HLA-DR. To expand these observations further, we have produced the 28 kDa extracellular domain and the 18 kDa carboxy-terminal subfragment of the Mls-1 protein in E. coli and studied their interaction with human MHC class II molecules in vitro. In this report, we demonstrate direct binding of these recombinant forms of the Mls-1 protein to soluble HLA-DR1 and HLA-DR4, but not to HLA-A2. Our data imply a unique class II interaction site of retroviral superantigens that is not shared with bacterial superantigens.

Superantigens of mouse mammary tumor virus.

Superantigens (SAgs) are proteins of microbial origin that bind to major histocompatibility complex (MHC) class II molecules and stimulate T cells via interaction with the V beta domain of the T cell receptor (TCR). Mouse mammary tumor virus (MMTV) is a milk-transmitted type B retrovirus that encodes a SAg in its 3' long terminal repeat. Upon MMTV infection, B cells present SAg to the appropriate T cell subset, which leads to a strong "cognate" T-B interaction. This immune reaction results in preferential clonal expansion of infected B cells and differentiation of some of these cells into long-lived memory cells. In this way a stable MMTV infection is achieved that ultimately results in infection of the mammary gland and virus transmission via milk. Thus, in contrast to many microorganisms that attempt to evade the host immune system (reviewed in 1), MMTV depends upon a strong SAg-induced immune response for its survival. Because of their ability to stimulate very strong T cell responses in MHC-identical mice, minor lymphocyte stimulatory (Mls) antigens, discovered more than 20 years ago, are now known to be SAgs encoded by endogenous MMTV proviruses that have randomly integrated into germ cells. The aim of this review is to combine the extensive biology of Mls SAgs with our current understanding of the life cycle of MMTV.

Identification of HLA-DRB1 and HLA-DQB1 identical individuals by a cytokine-based mixed lymphocyte culture.

Cytokine determination in MLC is under discussion as providing more sensitive and specific information regarding host-graft compatibility, and is therefore suggested to represent a new method for transplantation medicine. Little is known, however, about the stimulatory influence of HLA class II antigens and minor lymphocyte-stimulating antigens (Mls). Our results demonstrate that cytokine determination in MLC is suitable to detect identical alleles of HLA-DRB1 and HLA-DQB1. Among more than 100 random MLC experiments, we observed one cytokine pattern similar to the cytokine release detected in a control MLC of HLA-identical siblings, which showed marginal or no secretion of IL-2, sIL-2R, IFN-gamma, TNF-alpha, and IL-6. HLA-typing of these two nonreactive individuals elevated identical HLA-DRB1 and HLA-DQB1 regions, while they differed in the HLA-DP locus. This suggests that HLA-DP has no stimulatory influence on cytokine release. Further investigation of the stimulatory capacity of HLA-DR and DQ showed that HLA-DR is more effective in inducing IFN-gamma release than HLA-DQ. To evaluate the stimulatory influence of human Mls, i.e., human endogenous retroviruses (HERV), we analyzed HERV sequences of nonreactive individuals. Both individuals showed identical HERV patterns. A third individual, who had shown distinct cytokine release in MLC with both nonreactive individuals, differed in the HERV fragments. In conclusion, cytokine determination in MLC is a new method of evaluating the biological relevance of stimulatory antigens after allogeneic stimulation detecting all individual diversities in one experiment.

Expression of Mtv-7 sag gene in vivo using a retroviral vector results in selective inactivation of superantigen reactive T cells.

T cells expressing specific TCR V beta chains are intrathymically eliminated in mice expressing the murine Mls (minor lymphocyte stimulating) superantigens. Recently, in vitro studies have shown that the endogenous mouse mammary tumor virus (MMTV)-7 sag gene encodes Mls-1 Ag. The demonstrated ability of MMTV superantigen proteins to react with TCRs has led to the postulate that other infectious retroviruses may use superantigen-like molecules to modify the host's immune system. In this report, successful retrovirus-mediated Mtv-7 sag gene transfer into pluripotent hematopoietic stem cells is described. In two different strains of Mls-1- host mice (CBA/Ca and BALB/c) reconstituted with Mtv-7 sag gene expressing bone marrow cells, low levels of ectopic Mtv-7 sag gene expression on syngeneic donor hematopoietic stem cell-derived population alone can induce partial clonal deletion of Mls-1 reactive V beta 6+ and V beta 8.1+ T cells, and complete clonal inactivation of V beta 8.1+ T cells.

Delay in expression of a mammary tumor provirus is responsible for defective clonal deletion during postnatal period.

A gene-encoding ligand for deletion of T cells bearing TcRV beta 6 and V beta 8.1 cosegregates a new mammary tumor provirus locus, Mtv-50 in NC mice. The sequence of the open reading frame (ORF) in the 3' long terminal repeat (LTR) of Mtv-50 was strikingly similar to those of Mtv-7, Mtv-43 and exogenous mouse mammary tumor virus (SW) with properties of minor lymphocyte stimulating antigen 1a. Consistent with previous reports, clonal deletion of mature thymocytes bearing TcRV beta 6 was defective during the early postnatal period of mice carrying Mtv-50. Appreciable levels of mRNA corresponding to common Mtv ORF and Mtv-6 ORF were expressed in the neonatal thymus, while little, if any, mRNA corresponding to Mtv-50 ORF was detected in the thymus at the early postnatal stage. Delay in expression of Mtv-50 ORF during the postnatal period may be responsible for the failure of clonal deletion of V beta 6-T cells in the early postnatal life of mice carrying Mtv-50.

Thymic repertoire selection by superantigens: presentation by human and mouse MHC molecules.

The initial report of T cell receptor (TCR) V beta-specific thymic selection in mice showed association with expression of H-2E molecules and affected V beta 17a T cells which were present in CD4+8+ double positive thymocytes but deleted from the CD4+ and CD8+ single positive populations. Similar deletions were subsequently reported for V beta 8.1+ and V beta 6+ T cells in Mls-1a mouse strains and for V beta 3+ T cells in Mls-2a/3a strains. The 'Mls antigens' are most effectively presented by H-2E molecules but certain alleles of H-2A molecules can also present these endogenous superantigens. Expression of Mls antigens can cause both V beta-specific thymic deletion and stimulation of peripheral T cells from Mls-negative strains. Another category of 'Mls-like' antigens cause only V beta-specific thymic deletion in H-2E+ strains, affecting V beta 5+ and V beta 11+ T cells. The non-MHC ligands responsible for each of these effects are superantigens analogous to the exogenous bacterial superantigens, which also show TCR V beta-specific stimulatory effects when presented by MHC class II positive antigen-presenting cells. The genes encoding endogenous superantigens in mice were shown to co-segregate with mouse mammary tumour virus integrations (Mtv) and to be the Mtv-LTR orf genes. In vitro translation of Mtv-LTR orf genes identified their products as type II integral membrane glycoproteins with the polymorphic C terminus outside the cell. These polymorphisms correlate with specificity for the different TCR V beta chains. Virtually all TCR V beta-specific negative selection in the mouse thymus can be accounted for by the expression of Mtv or MMTV (the infectious counterparts of Mtv proviral integrants) LTR-orf proteins, presented with H-2E or certain H-2A alleles. It is unlikely that TCR V beta-specific positive selection is due to endogenous superantigens since it does not segregate with Mtv genomes. In humans, HLA-DR molecules appear to be homologous with H-2E in mice whereas HLA-DQ are the homologues of H-2A. H-2E negative mice transgenic for HLA-DR alpha chain express a mouse/human heterodimeric molecule which presents Mtv superantigens causing TCR V beta-specific deletion. Such trans-species class II molecules are also effective in TCR V beta-specific positive selection of V beta 2+, V beta 6+ and V beta 10+ T cells. Taken together, these results show that human MHC class II molecules can interact with the murine T cell repertoire.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of minor lymphocyte stimulatory-1a antigen from activated CD4+ or CD8+ T cells.

Minor lymphocyte stimulatory (Mls) Ag are super Ag that stimulate a high proportion of T cells of a specific TCR V beta family. One of the super Ag, Mls-1a, which is recognized mainly by TCR V beta 6+ and V beta 8.1+ T cells, has recently been linked to the response to product of the open reading frame in 3'-long terminal repeat of endogenous mammary tumor virus, MTV-7. It is quite certain that B cells are able to produce and also to present the Mls-1a Ag. However, it remains to be determined whether other cell types, especially T cells, produce Mls-1a Ag. In this study using highly purified T cell subpopulations, capacity to produce Mls-1a Ag was analyzed by calculating the proportion of Mls-1a reactive V beta 6+ or V beta 8.1+ T cells in responding cell populations. We found that nonstimulated CD8+ T cells produced a low amount of Mls-1a Ag, and the capacity to do so was considerably increased by stimulation with immobilized anti-TCR mAb. By contrast, nonstimulated CD4+ T cells did not produce Mls-1a Ag at all. Even when CD4+ T cells were activated via TCR signaling with immobilized anti-TCR mAb, CD4+ T cells did not produce Mls-1a Ag. However, CD4+ T cells primed with conventional Ag in vivo produced Mls-1a Ag on restimulation with that specific Ag in vitro. These findings indicate that not only CD8+ T cells but also CD4+ T cells can produce Mls-1a Ag on appropriate stimulation, although different mechanisms for Mls-1a production may operate between the CD4+ and CD8+ T cells.

Mouse intestinal epithelial cells express the self superantigen Mls1a.

In previous studies, we demonstrated that intestinal epithelial cells of the mouse small intestine could present exogenous antigen to specific CD4+ T cell hybridomas. We now report on the ability of normal enterocytes to present the self superantigen Mls1a. Enterocytes from Mls1a but not from Mls1b strains stimulated interleukin-2 production through a V beta 6+ T cell hybridoma specific for Mls1a determinants. Antibody inhibition experiments showed that enterocytes presented Mls determinants via a major histocompatibility complex class II-dependent mechanism. Furthermore, the ability of enterocytes to activate V beta 6+ Mls1a-specific T cells was inhibited by monoclonal antibodies against the Orf protein encoded by an Mtv-7 provirus which is associated with Mls1a expression. These findings provide evidence for the first time that Mls determinants are expressed on normal enterocytes and support the theory of a possible role of these cells in extrathymic selection of T cell receptor V beta repertoire of intraepithelial T lymphocytes.

The host response in graft versus host disease. II. The emergence of host protective cells is in part determined by background genomic compatibility.

Murine graft versus host (GVH) disease takes two forms depending on the parental/F1 strain combination employed. In an accompanying paper (Singh et al., Clin. Immunol. 151, 1993) many of the clinical features of these two forms of GVH disease are described. In addition to these clinical characteristics, acute lethal GVH (ALGVH) disease is characterized by diminished natural killer cell activity, whereas chronic GVH disease is characterized by normal or increased natural killer cell activity. Previously we have reported that this marked disparity in disease expression can be attributed to radiosensitive host cells which protect the F1 mouse from parental anti-F1 cytotoxicity (CTX) in mice undergoing chronic GVH (CGVH) disease. These cells fail to functionally emerge in mice undergoing ALGVH disease. We now report that the background genome, presumably the minor lymphocyte stimulatory loci, of the donor cells determines whether these host cells emerge and thereby dictates the form of GVH disease which is induced. C57BL/6 (B6) cells (H-2b, minor lymphocyte stimulatory locus (Mls)b) and B10.D2 cells (H-2d, Mlsb) were found to induce ALGVH disease when adoptively transferred to [C57BL/6xDBA/2]F1 (B6D2) (H-2b/d, Mls-1a/b, Mls-2a/b) recipient mice. DBA/2 cells (H-2d, Mls-1a, Mls-2a) and Balb/c cells (H-2d, Mls-1a, Mls-2b) induced CGVH disease in B6D2 mice. Using Mls congenic strains we have demonstrated that donor cell reactivity against Mls-2a was necessary and sufficient to induce ALGVH disease as determined by anemia, lymphopenia, anti-F1 cytotoxicity, and loss of cytotoxicity against allogeneic targets. Such Mls-2a reactivity correlated with the impaired induction of a host protective cell capable of vetoing self-directed CTX. Failure of this host protective cell to emerge in turn correlated with donor anti-host CTX and the emergence of ALGVH disease.

Involvement of multiple factors in the clonal deletion of self-reactive T cells.

Self-tolerance is primarily induced by the elimination of potentially self-reactive T cells during early development of the T cell repertoire. In the mouse, endogenous mouse mammary tumor viruses (MMTV), including minor lymphocyte-stimulating antigens and milk-transmitted exogenous MMTV, have been known to function as self-antigens inducing the clonal deletion of self-reactive T cells in a V beta-specific manner. We investigated the factors involved in the deletion of V beta 17a-bearing T cells. The results indicated that in addition to the previously reported V beta 17a deletion ligand, Mtv-3, there is a nonmilkborne gene product which progressively deletes V beta 17a (and V beta 3)-bearing T cells during aging. This suggests that clonal deletion is mediated by multiple factors and that a clonal deletion element associated with aging may play a significant role in shaping the T cell repertoire.

Anti-Mlsa-like effect of CBA/J anti DBA2 antibody in in vitro MLRs between Mls incompatible combinations.

Antisera from CBA/J (H-2k,Mlsa) mice hyperimmunised with DBA2 (H-2d,Mlsa) and/or AKR (H-2k,Mlsa) splenocytes, can block in vitro MLRs against Mlsa determinants. These two antisera are furthermore specifically cytotoxic against targets expressing H-2 components but not against Mlsa-bearing targets. In these experimental models the anti-Mlsa-like effects of CBA/J anti-DBA2 and CBA/J anti AKR antibodies seem to be associated with an autoreactive immune response in CBA/J mice elicited by DBA2 and AKR splenocytes, respectively. In this report we propose that anti-Mlsa antibodies can also be generated in a situation in which Mlsa is presented as 'altered self' in conjunction with allo-determinants.

Frequent deletion of the transgene in T cell receptor beta chain transgenic mice.

TCR V beta 8.1 transgenic mice were generated using a genomic TCR V beta gene construct under the control of its promoter and enhancer. Among three lines of transgenic mice, one line expressed the transgenic TCR on only approximately 70% of peripheral T cells, while the other two lines expressed it on almost all mature T cells. T cells which lacked expression of the transgenic TCR beta chain expressed endogenous TCR beta chains. The molecular basis underlying the lack of transgene expression in T cells of this line of transgenic mice was investigated. The transgenic TCR- cells were isolated by two methods. First, Thy-1+ V beta 8.1/8.2- cells were purified from peripheral T cells using cell sorting. Second, transgenic TCR- T cell clones were established. In both cases, Southern blotting indicated that V beta 8.1- T cells had deleted the transgenic TCR gene. Thus, deletion of the transgenic TCR can occur in a high proportion of T cells, which allows rearrangement and expression of endogenous TCR beta chains.

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process.

An extensive comparison of TCR alpha beta V-region usage by CD8 beta-CD4+CD8+ intraepithelial lymphocytes (IEL), CD4-CD8+ IEL, and lymph node (LN) T cell subsets in three minor lymphocyte stimulating (Mls)-disparate, MHC-identical mouse strains revealed novel TCR selection patterns. In cases where forbidden V regions were expressed by CD8 beta- CD4-CD8+ IEL, the same TCRs were deleted from CD8 beta- CD4+CD8+ IEL, indicating that lack of CD8 beta expression was not solely responsible for forbidden V-region expression. These results also suggested that CD4 may be involved in negative selection of CD4+CD8+ IEL TCRs. In C57BR/cdJ (Mls-1b2b) mice, a major increase in V beta 3+CD4+CD8+ IEL but not in other IEL or LN subsets was noted suggesting a subset-specific expansion of V beta 3+ cells. Negative selection of V beta 14+ cells in only the CD4+CD8+ IEL subset further supported the existence of intestine-specific TCR selection processes. Analysis of V-region expression of CD8 beta + and CD8 beta-CD4-CD8+ IEL subsets revealed that forbidden V-region expression was not strictly confined to the CD8 beta- subset in all cases. Overall, the data point to a dynamic, gut-specific TCR selection process that may be antigen driven.

Tissue distribution of Mtv-7-like exogenous retroviral transcripts and clonal deletion of V beta 6+ T cells in Mls-1b BALB/c mice.

We have previously described an Mls-1a-like clonal deletion of mature CD4+ T cells which express V beta 6 and V beta 8.1 chains of the TCR in half of the mice of a BALB/c, Mls-1b colony (BALB/c IC). This occurs in the absence of the Mtv-7 provirus which is responsible for the clonal deletion in Mls-1a mice. We developed a polymerase chain reaction assay in order to study the presence of retroviral transcripts homologous to the viral superantigen gene (vSAG) of Mtv-7 and Mtv-6 in various tissues. Mtv-7 homologous transcripts were present in the mammary glands of lactating BALB/c IC mice and in the thymuses and/or spleens of BALB/c IC virgin mice with deletion of V beta 6+ lymph node T cells, and not in BALB/c IC with normal V beta 6 expression. These results indicate that this BALB/c colony is infected with an exogenous mouse mammary tumour virus (MMTV) whose vSAG is similar to Mtv-7, as recently reported. Thymectomies performed at 4-5 weeks of age (at least 4 weeks before detection of clonal deletion), did not affect the occurrence of clonal deletion in peripheral lymph nodes when tested 20 weeks later. This suggests that clonal deletion can be achieved without further intrathymic contact with the antigen. Since MMTV is transmitted through milk and is likely to be present in the gut, we evaluated the percentage of V beta 6+CD4+ T cells within the gut intraepithelial lymphocyte (IEL) population. Mice with normal V beta 6 expression in lymph nodes may show partial deletion of V beta 6+CD4+ IEL.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

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.