Susceptibility to tumors induced by polyoma virus is conferred by an endogenous mouse mammary tumor virus superantigen.

A dominant gene carried in certain inbred mouse strains confers susceptibility to tumors induced by polyoma virus. This gene, designated Pyvs, was defined in crosses between the highly susceptible C3H/BiDa strain and the highly resistant but H-2k-identical C57BR/cdJ strain. The resistance of C57BR/cdJ mice is overcome by irradiation, indicating an immunological basis. In F1 x C57BR/cdJ backcross mice, tumor susceptibility cosegregates with Mtv-7, a mouse mammary tumor provirus carried by the C3H/BiDa strain. This suggests that Pyvs might encode the Mtv-7 superantigen (SAG) and abrogate polyoma tumor immunosurveillance through elimination of T cells bearing specific V beta domains. DNA typing of 110 backcross mice showed no evidence of recombination between Pyvs and Mtv-7. Strongly biased usage of V beta 6 by polyoma virus-specific CD8+ cytotoxic T lymphocytes in C57BR/cdJ mice implicates T cells bearing this Mtv-7 SAG-reactive V beta domain as critical anti-polyoma tumor effector cells in vivo. These results indicate identity between Pyvs and Mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's T cell repertoire.

Extrathymic clonal deletion of self-reactive cells in athymic mice.

The repertoire of T cells present in congenitally athymic mice was studied by flow cytometric analysis on populations of T cells expanded polyclonally in vitro. Athymic (BALB/c x C57BL/6)F1 mice have levels of potentially autoreactive V beta 3- and V beta 11-bearing T cells that are significantly higher than those of euthymic CB6F1 mice. Examination of potentially autoreactive cells in athymic AKR mice, however, yielded contrasting results. V beta 6+ cells, which are deleted intrathymically in normal AKR mice, are present in the repertoire of young (less than 6-wk-old) AKR nu/nu mice. Isolation of a cloned CD4+V beta 6+ cell line with Mls-1a reactivity from young AKR nu/nu mice indicates that the correlation between TCR usage and specificity is consistent with that described in euthymic mice and that this population contains autoreactive T cells that are not anergic. By 6 mo of age, however, cells expressing V beta 6 are no longer detectable. Inability to detect these cells is not simply caused by failure to expand these cells in culture, because freshly isolated populations from old nude mice exhibit the same selective absence of V beta 6-bearing cells. The data strongly suggest that extrathymic deletion, rather than clonal anergy, accounts for the apparent absence of autoreactive V beta 6-bearing cells in aged AKR nu/nu mice.

T-cell lymphomas emerging as epineoplasms in mice bearing transplanted polyoma virus-induced salivary gland tumors.

A subset of salivary epithelial tumors induced by mouse polyoma virus (PyV) has been designated lymphoepithelioma on the basis of a prominent lymphocytic component. Serial transplantation of this variant has previously been observed to result in lymphoma development. A recent repetition of this phenomenon allowed us to characterize the lymphoma cell populations with regard to phenotypic markers and PyV content. Lymphomas emerged in recipients of the third, fifth, sixth, and seventh transplant generations of the lymphoepithelioma. Most lymphomas were widely disseminated in hematopoietic and lymphoreticular tissues, and other sites as well. Flow cytometric analysis of lymphocyte populations from lymphomas in six recipients revealed that, while all lymphomas expressed phenotypic markers of immature cortical thymocytes, i.e., Thy-1, Pgp-1, Jlld, and CD5, they were not uniform with regard to other T-cell markers, notably CD4 and CD8. Varying levels of T-cell receptor markers CD3 and alpha/beta, as well as interleukin 2 receptor, were also noted. DNA blot analysis failed to detect PyV in lymphoma cells at a sensitivity level capable of detecting less than one intact copy per cell. It appears improbable the lymphoma was directly induced by PyV. Hypotheses invoking other mechanisms of lymphoma development are outlined.

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.

A role for L3T4+ T cells and their lymphokines in the generation of suppressor effector (TS3) cells.

The cellular requirements for the in vitro induction of antigen-specific suppressor T cells were examined. Previous reports indicated that Ia-bearing macrophages and anti-idiotypic B cells are required as accessory cells to facilitate the generation of suppressor effector (TS3) cells which regulate the response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten. The present study describes two distinct T cell populations which interact to generate antigen-specific TS3. Fractionation of the T cell populations with monoclonal antibody to the L3T4 determinant led to the identification of an NP-specific L3T4- TS3 progenitor population and an L3T4+ helper/inducer subset. In the presence of NP-coupled antigen, the L3T4+ subset could induce progenitor TS3 to differentiate into mature TS3 cells. The activity of the L3T4+ inducer population could be replaced with specifically activated cloned helper cells which were not NP-reactive since an I-Ab-restricted, insulin-reactive, L3T4+ clone was capable of supporting the generation of NP-specific TS3. Inducer activity appeared to be confined to the Th1 but not the Th2 subset. In addition, 18-hr supernatants from antigen-activated clones were capable of substituting for L3T4+ cells or T cell clones in TS3 induction cultures. The TS maturation/differentiation factor(s) active in these supernatants does not appear to be IL-1, IL-2, IL-3, or interferon-gamma alone since purified sources of these lymphokines failed to induce TS3 activity.

T cell recognition of Mlsc,x determinants.

Among a large number of cow insulin-specific T cell clones derived from both C57BL/10 and B10.A strains, several were found to react to non-MHC-linked gene products of a number of allogeneic strains. The stimulatory moiety for three of these clones correlates, in part, with expression of Mlsc, as defined by mouse strains C3H/HeJ and A/J. In addition, all three of these clones are stimulated by cells from strain PL/J, which has the poorly defined Mlsx allele. The data strongly suggest that Mlsx may, in fact, be Mlsc or is, at least, highly cross-reactive with Mlsc. Segregation analysis by using (B10.D2 X PL/J)F2 mice demonstrates that the Mlsx gene is genetically independent of the Mlsa linked Ly-9 marker on chromosome 1. Further studies with the use of these Mlsc,x-reactive clones reveal that they also recognize a gene product present in many mouse strains including DBA/2 which were previously phenotyped as Mlsa. However, testing of BxD recombinant inbred lines excludes Mlsa as being the stimulatory moiety. We therefore propose reclassification of the Mls phenotypes of several mouse strains based upon a two-locus model for Mls.

The relationship of Mlsx to Mlsc.

Among T cell clones with specificity for cow insulin and autologous class II MHC products, a significant number displayed interesting patterns of alloreactivity to non-MHC antigens. Four clones are described in this report. One is a typical Mlsa-reactive clone, while the other three proliferate to a variety of allogeneic spleen cells with reportedly different Mls phenotypes. These include PL/J stimulator cells, designated Mlsx, all strains reported to be Mlsc, and several strains previously typed as Mlsa. Little is known about Mlsx except that it does not appear to be cross-reactive with Mlsa. In this report, therefore, we attempt to investigate the reasons why these clones seem to be stimulated by a variety of different Mls phenotypes. Our conclusions are, first, that some of the strains previously typed as Mlsa may actually express a second Mls product, either c or x, in a manner analogous to the CBA/J strain (which expresses both Mlsa and Mlsc), and second, that Mlsc and Mlsx are cross-reactive. In preliminary experiments, we investigate the genetic relationship between Mlsc and Mlsx by analysis of backcrosses, and the extent of cross-reactive recognition of Mlsc and Mlsx by raising T cell clones which recognize one but not the other. Our preliminary conclusion is that Mlsc and Mlsx are cross-reactive, but represent distinct gene products.

Analysis of insulin-specific T cell clones. I. Strategy for production of clonotypic antibody.

A novel strategy for production of T cell clone-specific antiserum is described. Clones that are both antigen-specific and alloreactive can be injected in small numbers i.v. into a strain which expresses the appropriate alloantigens, inducing consistently high-titered antisera containing antibodies to the unique T cell receptor molecules on these cells. The antisera characterized in this report block activation of these cells by either antigen plus autologous class II products or alloantigen. Furthermore, in the absence of antigen, the antiserum strongly stimulates the specific clones to divide, and to synthesize and secrete various proteins. Consistent with the findings of other investigators, the antiserum immunoprecipitates a disulfide-linked dimer of 90,000 m.w. from the surfaces of cells with the appropriate specificity.

Specificity of T cell clones for antigen and autologous major histocompatibility complex products determines specificity for foreign major histocompatibility complex products.

We have analyzed a panel of T cell clones that corecognize defined epitopes of the insulin molecule in association with Ia for their patterns of recognition of alloantigens. A striking correlation is observed between recognition of the I-Ab gene product and cow insulin alpha loop and recognition of I-Eu of the PL/J haplotype. These results are consistent with the notion that reactions to foreign major histocompatibility complex (MHC) products reflect molecular mimicry by foreign class II antigens of 'physiologic' complexes formed by autologous class II MHC molecules and antigen.