Mouse Mammary Tumor Virus Superantigen Research Articles

Bcl-2–Interacting Mediator of Cell Death Influences Autoantigen-Driven Deletion and TCR Revision

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Mouse mammary tumor virus (MMTV)-like DNA sequences in the breast tumors of father, mother, and daughter

BackgroundThe diagnosis of late onset breast cancer in a father, mother, and daughter living in the same house for decades suggested the possibility of an environmental agent as a common etiological factor. Both molecular and epidemiological data have indicated a possible role for the mouse mammary tumor virus (MMTV), the etiological agent of breast cancer in mice, in a certain percentage of human breast tumors. The aim of this study was to determine if MMTV might be involved in the breast cancer of this cluster of three family members.ResultsMMTV-like envelope (env) and long terminal repeat (LTR) sequences containing the MMTV superantigen gene (sag) were detected in the malignant tissues of all three family members. The amplified env gene sequences were 98.0%–99.6% homologous to the MMTV env sequences found in the GR, C3H, and BR6 mouse strains. The amplified LTR sequences containing sag sequences segregated to specific branches of the MMTV phylogenetic tree and did not form a distinct branch of their own.ConclusionThe presence of MMTV-like DNA sequences in the malignant tissues of all three family members suggests the possibility of MMTV as an etiological agent. Phylogenetic data suggest that the MMTV-like DNA sequences are mouse and not human derived and that the ultimate reservoir of MMTV is most likely the mouse. Although the route by which these family members came to be infected with MMTV is unknown, the possibility exists that such infection may have resulted from a shared exposure to mice.

A Defective Viral Superantigen-Presenting Phenotype in HLA-DR Transfectants Is Corrected by CIITA

Activation of T lymphocytes by mouse mammary tumor virus superantigen (vSAg) requires binding to MHC class II molecules. The subcellular location where functional interactions occur between MHC class II molecules and vSAgs is still a matter of debate. To gain further insight into this issue, we have used human epithelial HeLa cells expressing HLA-DR1. Surprisingly, the human cells were unable to present transfected vSAg7 or vSAg9 to a series of murine T cell hybridomas. The defect is not related to a lack of vSAg processing, because these cells can indirectly activate T cells after coculture in the presence of B lymphocytes. However, after IFN-gamma treatment, the HeLa DR1(+) cells became apt at directly presenting the vSAg. Furthermore, transfection of CIITA was sufficient to restore presentation. Reconstitution experiments demonstrated the necessity of coexpressing HLA-DM and invariant chain (Ii) for efficient vSAg presentation. Interestingly, inclusion of a dileucine motif in the DRbeta cytoplasmic tail bypassed the need for HLA-DM expression and allowed the efficient presentation of vSAg7 in the presence of Ii. A similar trafficking signal was included in vSAg7 by replacing its cytoplasmic tail with the one of Ii. However, sorting of this chimeric Ii/vSAg molecule to the endocytic pathway completely abolished both its indirect and direct presentation. Together, our results suggest that functional vSAgs-DR complexes form after the very late stages of class II maturation, most probably at the cell surface.

Mouse Mammary Tumor Virus and Human Breast Cancer

To the Editor: Wang et al. (1) described recently the cloning and sequencing of mouse mammary tumor virus (MMTV) superantigen (sag) from human breast cancers. They report that sag sequences exhibit high nucleotide homology with MMTV and that cloned sag proteins elicit in vitro activation of human T cells. This article gives the impression that an etiologic agent for sporadic cases of breast cancer has been identified by the scientific community. Sadly however, this is far from the truth and should be interpreted with caution, as a positive association between MMTV and human breast cancer is a highly controversial debate that has smoldered on and off for 60 years. Omitted in the Wang et al. article is the fact that only two independent groups have been able to repeat the original molecular observations of MMTV DNA in human breast cancers, whereas six have not (2). This may possibly have a rational explanation, such as differing geographic distribution of anetiologic agent, vector, or clustering of populations with differing infection or cancer susceptibilities. To advance the intellectual debate proponents of an infective hypothesis for sporadic cases of human breast cancer needs to seriously confront the following realities: (a) how does the virus cause infection of Homo sapiens given that human cells lack the necessary transferrin receptor for MMTV (3) and that MMTV is not an endogenous human retrovirus (2)? (b) for every established human oncogenic virus (high-risk human papillomaviruses, hepatitis B and C viruses, and human herpes virus type 8), immunosuppression increases cancer risk, which is not the case for human breast cancer; (c) unlike the MMTV murine model of breast hyperplasia, human breast-feeding does not predispose daughters to breast cancer (2, 4);and (d) pregnancy is protective against the risk of human breast cancer (2, 5), whereas the opposite is true for MMTV-caused murine hyperplasias. For the reasons outlined above, MMTV is not currently classified as an environmental carcinogen for humans and is unlikely ever to be categorized as such, although in fairness Wang et al. do fleetingly allude to this (1). Scientific articles, particularly those concerning such highly contentious issues, have a responsibility to be sufficiently nonpartisan as to make the limitations of the hypothesis being examined patently clear. This is essential to permit the impartial scientist to judge the true significance of the observations: caveat emptor!In Response: We feel compelled to respond to Mant and Cason's Letters to the Editor because it misrepresents our work on the relationship between mouse mammary tumor virus and human breast cancer. Their stated purpose was to clarify our findings so the readers of Cancer Research would not be misguided. Their criticism was not directed against the current article (1) but against the entire concept of mouse mammary tumor virus as a possible agent in human breast carcinogenesis. As discussed in several articles by our group, causation will only be claimed by us on proving that the virus is tumorigenic in human cells, fulfilling the requisite epidemiologic and virological proofs as well (2–5). Some of the requisites set forth have already been established by us and others (2–9).The question concerning receptors and infectivity was also discussed clearly (1). Furthermore, retroviruses can infect cells without cognate receptors using transactivation pathways, a mechanism that has not yet been well explored with mouse mammary tumor virus (10), and retroviruses are known to cross species (11).Mant and Cason (12) quoted in their review from articles in which env sequences were not detected. One of these is not applicable to our findings, because it concerns endogenous retrovirus expression (13). Although we agree that differences in population could explain some of the results (7, 14), it is likely that differences in technology were also involved. None of the articles cited (15–17) followed our exact methodology. Four laboratories have confirmed our results, one as yet unpublished.The question of endogenous versus exogenous sequences has been discussed in detail by us and others in previous publications (5–7).In humans, early pregnancy is a protective factor against breast cancer risk, but gestational breast cancer is one of the most virulent. High levels of hormones may be involved (discussed in ref. 18); therefore, the mouse and human cancers have similarities. To our knowledge, no extensive studies have been made to detect a mouse mammary tumor virus–like virus in milk.The participation of the immune system in mouse mammary tumor virus infections is unique and differentiates this virus from other tumor viruses.We believe that repeated confirmation of our work is significant and hope that in the future more laboratories will be able to successfully adapt our methodology.

Clonal isolation of different strains of mouse mammary tumor virus-like DNA sequences from both the breast tumors and non-Hodgkin's lymphomas of individual patients diagnosed with both malignancies.

In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin's lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin's lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail. DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin's lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced. MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin's lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree. The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin's lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.

Regions of mouse mammary tumor virus superantigen involved in interaction with the major histocompatibility complex class II I-A molecule.

To study the major histocompatibility complex class II I-E dependence of mouse mammary tumor virus (MMTV) superantigens, we constructed hybrids between the I-E-dependent MMTV(GR) and the I-E-independent mtv-7 superantigens and tested them in vivo. Our results suggest that, although the C-terminal third mediates I-A interaction, additional binding sites are located elsewhere in the superantigen.

Testing mouse mammary tumor virus superantigen as adjuvant in cytotoxic T-lymphocyte responses against a melanoma tumor antigen.

Cytotoxic T cells represent a powerful strategy for antitumor treatment. Depending on the route of injection, an important role for CD4 T cell-mediated help was observed in the induction of this response. For this reason, we investigated whether induction of a CTL response to the HLA-A2-restricted immunodominant peptide melanoma antigen Melan-A was improved by using rVVs expressing the CTL-defined epitope alone or in combination with an SAg. In the latter case, the few infected dendritic cells simultaneously presented an SAg and an antigen, i.e., peptide. Here, we show that the anti-Melan-A response was efficiently induced but not significantly improved by coexpression of the SAg.

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus.

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.

Human endogenous retrovirus IDDMK 1,222 and mouse mammary tumor virus superantigens differ in their ability to stimulate murine T cell hybridomas

Recently, a newly identified human HERV-K18 like endogenous retrovirus (IDDMK 1,222) has been associated to the etiology of type I diabetes (IDDM). Although the exact mechanism remains unclear, it was postulated that the 3′ end ORF product of the env gene of IDDMK 1,222 would trigger a Vβ7-specific human T cell expansion leading to their infiltration in the pancreas of afflicted patients and to the autoimmune destruction of the insulin-producing β cells. Since then, such superantigen (SAg)-like activity as well as the association between the IDDMK 1,222 virus and IDDM pathogenesis have been challenged. To further characterize functionally the putative IDDMK 1,222-encoded SAg, we have cloned from human DNA the identical 462bp ORF sequence originally described. The IDDMK 1,222 ORF fragment was transfected in the same human B cell line (Raji) originally used as APC to demonstrate the Vβ7 specificity. The immunostimulatory potential of IDDM ORF was tested on murine T cell hybridomas and compared to the well-characterized mouse mammary tumor virus Mtv7 SAg transfected in the same conditions. A panel of 16 T cell hybridomas encompassing 14 different Vβs was analyzed. We have failed to detect IDDMK 1,222-induced IL-2 production from any of these hybridomas, even those bearing the murine Vβ1 (mVβ1), Vβ4 or Vβ10 TcR β chains which are most closely related to the human Vβ7 (hVβ7). Our results suggest that IDDMK 1,222 ORF is devoid of superantigenic activity as defined by classical criteria.

Interleukin-4 up-regulates mouse mammary tumor virus expression yet is not required for in vivo virus spread.

The mouse mammary tumor virus (MMTV) superantigen induces T-cell production of cytokines, such as interleukin-4, which in turn increase MMTV transcription. However, interleukin-4 is not required for in vivo virus spread, because mice lacking interleukin-4 or the STAT6 transcription factor showed wild-type infection of lymphoid and mammary tissue. In spite of this, mammary tumor incidence was decreased in STAT6 null mice.

Association of mouse mammary tumor virus superantigen with MHC class II during biosynthesis.

Mouse mammary tumor viruses encode superantigens that interact with MHC class II proteins and stimulate T cells. We show here that presentation of mouse mammary tumor virus superantigen does not require DM. Furthermore, we have identified a strong class II peptide binding motif in the Mtv-7 superantigen, and we show that this motif is necessary for association with class II molecules in in vitro translation and in vivo functional assays. Our results suggest that endogenously synthesized viral superantigen can bind to MHC class II heterodimers during biosynthesis in the endoplasmic reticulum in a manner analogous to that used by the class II-associated invariant chain.

Detection of viral superantigen–class II MHC interactions at the cell surface

Mouse mammary tumor virus superantigens (vSAgs) must bind to class II MHC proteins to activate T cells. Although direct interaction of vSAgs with class II proteins has been demonstrated biochemically, the details of this interaction are largely unresolved. To facilitate the study of class II–vSAg interactions, a sensitive assay has been developed that can detect binding of vSAgs to class II proteins on the cell surface. The assay measures changes in vSAg surface expression upon enzymatic removal of a co-expressed glycan-phosphatidyl inositol-anchored form of the class II molecule IEk. Because the vSAgs are synthesized as integral membrane proteins that undergo proteolytic processing, an event that is likely required to eliminate membrane tethering, the data provide further evidence that a proteolytic fragment of vSAg is bound to class II proteins on the cell surface. The assay was utilized to identify mutant vSAgs that either did not associate with IEk molecules, or did not undergo furin-dependent proteolytic processing. Class II protein binding was detected using vSAg7 mutants that lacked furin endoprotease recognition sites, and after expression of vSAg in furin-deficient cells. The data demonstrate that furin-mediated processing is not necessary for association of vSAg7 with class II proteins, supporting previous studies that have indicated a role for alternative endoproteases in vSAg activation. However, because class II interactions were also noted in the apparent absence of proteases that are known to activate vSAgs, the data suggest that yet other proteases may process vSAgs in a fashion that does not necessarily lead to activation of T cells.

Expression of mouse mammary tumor virus superantigen accelerates tumorigenicity of myeloma cells.

To investigate whether superantigen (SAG) from endogenous mouse mammary tumor virus functions as an immunogenic or a tumorigenic factor in tumor development, the BALB/c myeloma cell line FO was transfected with the SAG gene from the 3' Mtv-50 long terminal repeat (LTR) open reading frame (ORF), the product of which was specific for Vbeta6. All five transfectants expressing Mtv-50 LTR ORF mRNA showed stimulatory activity for Vbeta6 T-cell hybridomas in vitro; this activity was inhibited by the addition of anti-Mtv-7 monoclonal antibody (MAb) or anti-major histocompatibility complex class II I-A(d) and I-E(d) MAb. All transfectants with the SAG gene grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation, whereas all clones, including mock transfectants, grew equally well in athymic nude mice. A significant fraction of Vbeta6 T cells selectively expressed activation markers, including CD44(high), CD62L(low), and CD69(high), and produced large amounts of interleukin 5 (IL-5) and IL-6 in BALB/c mice inoculated with transfectants. These results suggested that the expression of viral SAG enhances the tumorigenicity of a myeloma cell line through the stimulation of SAG-reactive T cells.

Mouse Mammary Tumor Virus Superantigen Expression Is Reduced by Glucocorticoid Treatment

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes are required for viral transmission and pathogenesis. Due to problems with detection and quantification of the superantigen protein, most reports about the mechanism of superantigen expression from the viral sag gene rely on the quantitative analysis of putative sag mRNAs. The description of multiple promoters as a source of putative sag mRNA has complicated the situation even further. All conclusions about the level of superantigen protein expression based on these data remain circumstantial. To test the effect of the glucocorticoid hormone dexamethasone on the total superantigen expression from an infectious MMTV provirus we used a quantitative assay that is based on a superantigen–luciferase fusion protein. MMTV gene expression from the major promoter in the 5′ long terminal repeat (LTR) is strongly induced in the presence of glucocorticoid hormones. We now demonstrate that, in the presence of dexamethasone, sag gene expression is reduced despite increased transcription from the MMTV 5′ LTR and increased amounts of putative sag mRNA initiated at the LTR promoter. These data show that the expression of the MMTV sag gene does not correlate with the activity of the major LTR promoter and thus differs from all other MMTV genes.

Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) haplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7 sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) but lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

Role of dendritic cells in the immune response induced by mouse mammary tumor virus superantigen.

After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.

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.

Chronic deletion, escape from deletion and activation of mouse mammary tumor virus superantigen-reactive T cells in C57BL/10 mice.

Though C57BL/10 mice express the mouse mammary tumor virus superantigens (sag) encoded by Mtv-8 and Mtv-9, it has been thought that these sag do not bind to the MHC class II molecule H2-Ab and consequently do not affect the T cell repertoire. However, we show that cells bearing TCR Vbeta chains specific for Mtv-8 and -9 sag are chronically deleted in C57BL/10 mice. Thymocytes and peripheral T cells escaping deletion by Mtv sag display a small reduction in the level of cell surface CD4. T cells escaping thymic deletion respond variably to endogenous Mtv sag with some, but not all, reactive populations appearing overrepresented in the activated/memory subset. The data suggest that in normal mice fine modulation of coreceptor expression levels may be a common way by which thymocytes escape elimination, that systems utilizing potentially Mtv sag-reactive TCR on a C57BL background may be inappropriate for the measurement of the affinity of TCR/MHC/peptide interactions required in thymic selection, and that detection of the activity of human sag may be aided by analysis of CD4 levels and activation markers on T cells in conjunction with studies of the frequency of cells bearing specific TCRVbeta chains.

Development of T-B cell collaboration in neonatal mice.

The neonatal immune response is impaired during the first weeks after birth. To obtain a better understanding of this immaturity, we investigated the development of T cell interactions with B cells in mice. For this purpose, we analyzed the immune response to three T-dependent antigens in vivo: (i) the polyclonal antibody response induced by vaccinia virus; (ii) the production of polyclonal and specific antibodies following immunization with hapten-carrier conjugates; (iii) the mouse mammary tumor virus superantigen (sAg) response involving an increase in sAg-reactive T cells and induction of polyclonal antibody production. After vaccinia virus injection into neonates, the polyclonal antibody response was similar to that observed in adult mice. The antibody response to hapten-carrier conjugates, however, was delayed and reduced. Injection with sAg-expressing B cells from neonatal or adult mice allowed us to determine whether B cells, T cells or both were implicated in the reduced immune response. In these sAg responses, neonatal T cells were stimulated by both neonatal and adult sAg-presenting B cells but only B cells from adult mice differentiated into IgM- and IgG-secreting plasma cells in the neonatal environment in vivo. Injecting neonatal B cells into adult mice did not induce antibody production. These results demonstrate that the environment of the neonatal lymph node is able to support a T and B cell response, and that immaturity of B cells plays a key role in the reduced immune response observed in the neonate.