Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.     

Characterization of a new minor lymphocyte stimulatory system. I. Cluster of self antigens recognized by "I-E-reactive" V beta s, V beta 5, V beta 11, and V beta 12 T cell receptors for antigen.

In the mouse, two sets of V beta gene products have been shown to be associated with T cell recognition of endogenous self Ag. One of these is the set of V beta associated with T cell reactivities to stimulatory Mls gene products, Mlsa (V beta 6, V beta 8.1, V beta 9) or Mlsc (V beta 3); another is the set of V beta, such as V beta 5, V beta 11, V beta 12, or V beta 17a, which were originally found to be related to I-E recognition. Although the Mls system has been well characterized, little is known about the nature of the ligands for the second set of V beta. In this work, we describe the evidence that the natural ligand or ligands of V beta 5, V beta 11, and V beta 12 may be novel Mls determinants that are recognized by naive T cells at a high precursor frequency and function as the ligand for clonal deletion of self-reactive T cells by negative selection. However, surprisingly, unlike the conventional Mls system, in which all V beta associated with Mlsa recognition or Mlsc recognition are uniformly deleted in those animals expressing the relevant Mls type, expression of these three V beta segregates independently among strains. Based on these observations, the nature of T cell recognition for this new Mls gene product(s) is discussed.     

Physiologic expression of two superantigens in the BDF1 mouse.

The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.     

A novel V beta 2-specific endogenous mouse mammary tumor virus which is capable of producing a milk-borne exogenous virus.

We have previously reported new Mtv loci, Mtv-48 and -51, in the Japanese laboratory mouse strains CS and NC. Here we show by backcross analysis that both Mtv-48 and -51 cosegregate with very slow deletion of T cells bearing V beta 2. The nucleotide sequences of the open reading frames in the 3' long terminal repeats of Mtv-48 and -51 were very similar to those of Mtv-DDO, mouse mammary tumor virus C4 [MMTV(C4)], and MMTV(BALB/cV), which encode V beta 2-specific superantigens. Furthermore, backcross female mice carrying Mtv-48 but not Mtv-51 were found to be able to produce milk-borne MMTV(CS), which can vigorously stimulate V beta 2-expressing T cells after local injection in vivo in an I-E-dependent manner. On the other hand, mice carrying Mtv-51 but not Mtv-48 could not produce such an MMTV in milk. The nucleotide sequences of MMTV(CS) open reading frame were completely matched with those of Mtv-48. These results indicate that the provirus Mtv-48 but not Mtv-51 is capable of producing a milk-borne virus of which the superantigen stimulates V beta 2-expressing T cells.     

Expression of Mouse Mammary Tumor Virus Superantigen mRNA in the Thymus Correlates with Kinetics of Self-Reactive T-Cell Loss

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is expressed at the surface of antigen-presenting cells in conjunction with major histocompatibility complex (MHC) type II molecules. The Sag-MHC complex is recognized by entire subsets of T cells, leading to cytokine release and amplification of infected B and T cells that carry milk-borne MMTV to the mammary gland. Expression of Sag proteins from endogenous MMTV proviruses carried in the mouse germ line usually results in the deletion of self-reactive T cells during negative selection in the thymus and the elimination of T cells required for infection by specific milk-borne MMTVs. However, other endogenous MMTVs are unable to eliminate Sag-reactive T cells in newborn mice and cause partial loss of reactive T cells in adults. To investigate the kinetics of Sag-reactive T-cell deletion, backcross mice that contain single or multiple MMTVs were screened by a novel PCR assay designed to distinguish among highly related MMTV strains. Mice that contained Mtv-17 alone showed slow kinetics of reactive T-cell loss that involved the CD4(+), but not the CD8(+), subset. Deletion of CD4(+) or CD8(+) T cells reactive with Mtv-17 Sag was not detected in thymocytes. Slow kinetics of peripheral T-cell deletion by Mtv-17 Sag also was accompanied by failure to detect Mtv-17 sag-specific mRNA in the thymus, despite detectable expression in other tissues, such as spleen. Together, these data suggest that Mtv-17 Sag causes peripheral, rather than intrathymic, deletion of T cells. Interestingly, the Mtv-8 provirus caused partial deletion of CD4(+)Vbeta12(+) cells in the thymus, but other T-cell subsets appeared to be deleted only in the periphery. Our data have important implications for the level of antigen expression required for elimination of self-reactive T cells. Moreover, these experiments suggest that mice expressing endogenous MMTVs that lead to slow kinetics of T-cell deletion will be susceptible to infection by milk-borne MMTVs with the same Sag specificity.     

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.     

Expression of the Mls-1a superantigen results in an increased frequency of V beta 14+ T cells.

Minor lymphocyte-stimulating (Mls) Ag are alloantigens that stimulate T cells expressing specific V beta regions. Recent studies have established that Mls Ag are examples of endogenous superantigens encoded by the products of endogenous mouse mammary tumor virus (MLV) genomes. In a mouse strain that expresses a given mammary tumor virus (Mls) Ag, reactive T cells expressing the corresponding V beta region are profoundly deficient, due at least in part to clonal deletion of the cells during their development. Expression of Mls and other endogenous superantigens, therefore, results in profound alterations in the ultimate repertoire of T cells in an animal. A role for endogenous superantigens in positive selection of T cells has not been previously established. Here we present evidence that expression of Mls-1a leads to a specific increase in the abundance of V beta 14+ T cells. Genetic studies indicate linkage of the effect to the Mls-1a gene. Neonatal tolerance studies argue against the possibility that the increase is due solely to the deletion of Mls-reactive V beta 14- T cells. The results are consistent with the Mls-1a product playing a role in the positive selection of V beta 14+ T cells.     

Characterization, chromosome assignment, and segregation analysis of endogenous proviral units of mouse mammary tumor virus.

In the course of analyzing sites of proviral integration in tumors induced by mouse mammary tumor virus (MMTV), we have isolated recombinant DNA clones corresponding to the 5' and 3' ends of four endogenous MMTV proviruses present in BALB/c and BR6 mice. This has permitted the structural characterization of each locus by detailed restriction mapping and the preparation of DNA probes specific for the cellular sequences flanking each provirus. These probes have been used to trace the segregation patterns of the proviruses, designated Mtv-8, Mtv-9, Mtv-17, and Mtv-21, in a panel of inbred strains of laboratory mice and to map Mtv-17 and Mtv-21 to mouse chromosomes 4 and 8, respectively. The unambiguous resolution of these four proviruses on Southern blots has greatly facilitated the analysis of other endogenous MMTV proviruses in these inbred mice.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

Mammary tumor induction loci in GR and DBAf mice contain one provirus of the mouse mammary tumor virus

The mammary tumor induction genes Mtv-1 in mouse strain DBAf and Mtv-2 in strain GR control the complete expression of the endogenous mouse mammary tumor virus (MMTV). We have used a combination of genetic, biochemical and molecular biological methods to identify and correlate specific copies of the endogenous MMTV proviral genes with the biological properties of the tumor induction genes Mtv-1 and Mtv-2. These Mtv induction genes contain specific MMTV proviral information, as was concluded from restriction enzyme analysis and molecular hybridization of DNAs of congenic mouse strains and of progenitors of backcross populations. The congenic strains differed from the parental strains GR and 020 only in the Mtv-2 gene, one lacking the Mtv-2 gene (GR/Mtv-2-) and one having obtained this gene (020/Mtv-2+). The gain or loss coincided with two Eco RI cellular DNA fragments containing MMTV DNA information. Since Eco RI cuts the exogenous proviral variant of MMTV DNA once, we assume that these two cellular DNA fragments contain one MMTV provirus. The same cellular DNA fragments containing MMTV DNA information segregated together with MMTV expression in the offspring population of the backcross. In a similar backcross analysis of the induction gene Mtv-1 it was also demonstrated that the Mtv-1 gene comprises one MMTV provirus. These data indicate that Mtv induction genes contain specific but different MMTV proviral genes and that nly a limited number of the MMTV proviruses present in the cellular DNA is associated with the control of proviral expression.     

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

MHC class II A alpha and E alpha molecules determine the clonal deletion of V beta 6+ T cells. Studies with recombinant and transgenic mice

Interactions between MHC class II genes and minor lymphocyte stimulating (Mls) associated products are responsible for clonally deleting self-reactive T cells in mice. Here we demonstrate the role of the intact I-A and I-E molecules as well as the individual A alpha and E alpha chains in the deletion of cells bearing the V beta 6 TCR. DBA/1 (H-2q, Mls-1a) mice were crossed with various inbred congenic, recombinant, and transgenic strains and the F1's were screened for V beta 6 expression. All I-E+ strains were fully permissive in deleting V beta 6+ T cells. I-E- strains expressing I-A b,f,s,k,p permitted only partial deletion, while I-Aq strains showed no deletion. Recombinant I-Aq and I-Af strains which expressed E kappa alpha chain in the absence of E beta chain showed a decrease in V beta 6+ T cells as compared to their H-2q and H-2f counterparts. Furthermore, transgenic mice expressing E kappa alpha Aq beta gene in an H-2q haplotype (E kappa alpha Aq beta?) gave similar results to that of the recombinants in deleting V beta 6 T-cells. The role of the 1-A molecule was also shown by the partial deletion of V beta 6+ T cells in H-2q mice expressing transgenic I-Ak molecules. These results demonstrate that the E alpha chain is important in the deletion of V beta 6 T-cells in Mls-1a mice. The role of A alpha chain is also implied by the permissiveness of E kappa alpha Aq beta but not Aq alpha Aq beta molecules in the deletion of V beta 6+ T cells.     

Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes

V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.     

Transgenic mouse mammary tumor virus superantigen expression prevents viral infection.

Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mother's milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.     

Bone marrow-derived cells are essential for intrathymic deletion of self-reactive T cells in both the host- and donor-derived thymocytes of fully allogeneic bone marrow chimeras

The fate of self-reactive T cells was examined in both the host- and donor-derived thymocytes of fully allogeneic bone marrow (BM) chimeras of two strain combinations of AKR/J (H-2k, IE+, Thy-1.1, Mls-1a2b) and C57BL/6 (H-2b, IE-, Thy-1.2, Mls-1b2b). Sequential appearance of host- and donor-derived T cells occurred in the thymus of both AKR----B6 and B6----AKR chimeras in which 5 x 10(6) of T cell-depleted BM cells were used to reconstitute recipients lethally irradiated with 950 rad. Thymocytes bearing V beta 6 high, which recognize MHC class II IE-binding Ag encoded by Mls-1a allele, were detected in neither host- nor donor-derived thymocytes of AKR-B6 chimeras in which Mls-1a and IE were expressed only by the BM-derived cells. Thymocytes bearing V beta 11high capable of recognizing IE were also deleted in the host- and donor-derived thymocytes of the AKR----B6 chimeras. One million of BM cells were inadequate to deletion of the B beta 6high and V beta 11high T cells in the host-derived thymocytes of these chimeras. On the other hand, significant number of V beta 6high and V beta 11high thymocytes were detected in both the host- and donor-derived thymocytes in B6----AKR chimeras where sufficient dose of IE- stem cells were used to reconstitute irradiated Mls-1aIE+ recipients. These results suggest that clonal deletion of the host- and donor-reactive T cells in both the host- and donor-derived thymocytes is an important mechanism for the induction of transplantation tolerance in allogeneic BM chimeras and that BM-derived APC may be essential for the intrathymic elimination of both the host- and donor-reactive T cells in the BM chimeras.