Clonal deletion of T cell repertoires with specific T cell receptor Vß chains by two endogenous superantigens in NC/Nga mice.

Superantigens (SAgs) are powerful T-cell stimulatory proteins. Because an atopic dermatitis (AD) model NC/Nga mice had two endogenous SAgs, namely minor lymphocyte-stimulating locus-1a (Mls-1a) and mouse mammary tumor virus (MMTV)(SHN), SAg-responsive T-cells bearing Vß5.1, Vß6, Vß8.1, Vß8.2, Vß8.3, Vß9, and Vß11 should be endogenously deleted. Here, we discuss that the endogenous SAgs-expression may be involved in AD-sensitivity in NC/Nga mice.

Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.

Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.IMPORTANCE Bacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction by retrotranslocation, which is part of a protein quality control system known as ER-associated degradation (ERAD) that is essential to cellular health. Through poorly understood mechanisms, certain pathogen-derived proteins are retrotranslocated but not degraded. We demonstrate here that MMTV SP retrotranslocation from the ER membrane avoids degradation through a unique process involving interaction with cellular p97 ATPase and failure to acquire cellular proteasome-targeting sequences.

Inherently variable responses to glucocorticoid stress among endogenous retroviruses isolated from 23 mouse strains.

Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Expression of a dominant T-cell receptor can reduce toxicity and enhance tumor protection of allogeneic T-cell therapy.

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of 'dominant' T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a 'dominant' T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in-vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in-vitro selection of allogeneic T cells expressing high levels of a 'dominant' T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in-vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.

MMTV superantigens coerce an unconventional topology between the TCR and MHC class II.

Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecule, MHCII-associated peptide, and TCR. We used covalently linked peptide/MHCII complexes to demonstrate that vSAG presentation is tolerant to variation in the protruding side chains of the peptide, but can be sensitive to the nature of the protruding N-terminal extension. An original approach in which vSAG was covalently linked to either MHCII chain confirmed that vSAG binds outside the peptide binding groove. Also, whereas the C-terminal vSAG segment binds to the MHCII α-chain in a conformation-sensitive manner, the membrane-proximal N-terminal domain binds the ß-chain. Because both moieties of the mature vSAG remain noncovalently associated after processing, our results suggest that vSAG crosslinks MHCII molecules. Comparing different T cell hybridomas, we identified key residues on the MHCII α-chain that are differentially recognized by the CDR3ß when engaged by vSAG. Finally, we show that the highly conserved tyrosine residue found in the vSAg TGXY motif is required for T cell activation. Our results reveal a novel SAG/MHCII/TCR architecture in which vSAGs coerce a near-canonical docking between MHCII and TCR that allows eschewing of traditional CDR3 binding with the associated peptide in favor of MHCII α-chain binding. Our findings highlight the plasticity of the TCR CDRs.

Immunotherapy of radioresistant mammary tumors with early metastasis using molecular chaperone vaccines combined with ionizing radiation.

In the current study, exposure of mammary tumor cells derived from mice transgenic for the polyomavirus middle T oncogene to ionizing radiation resulted in the generation of a tumor cell population that preferentially expressed cancer stem cell markers. In addition, these cells were more resistant to subsequent radiation treatments and appeared to acquire an enhanced capacity for dissemination to the lungs of mice. Therefore, we tested an immunotherapy approach to the treatment of local and disseminated mammary tumor cells in a murine model using a recently developed molecular chaperone-based vaccine that specifically targets the radioresistant subpopulation of tumor cells. Heat shock protein 70-peptide complexes (Hsp70.PC-F) were extracted from fusions of dendritic cells and radiation-enriched tumor cells, and the resulting chaperone vaccines were used to treat mice with pre-existing lung metastases. Immunization of mice with the Hsp70.PC-F vaccine resulted in a T cell-mediated immune response, including a significant increase in CD4 and CD8 T cell proliferation and the induction of effector T cells capable of targeting radioresistant tumor cells. Importantly, the growth of primary tumors was inhibited, and the number of tumor cells metastasizing to lung was reduced significantly by combining chaperone vaccine with radiotherapy. These results indicate that Hsp70.PC-F vaccine can induce specific immunity to radioresistant populations of mammary tumor cells and, thus, can complement radiotherapy, leading to synergistic killing.

Bone marrow stromal cell antigen 2 (BST-2) restricts mouse mammary tumor virus (MMTV) replication in vivo.

BACKGROUND: Bone marrow stromal cell antigen 2 (BST-2) is a cellular factor that restricts the egress of viruses such as human immunodeficiency virus (HIV-1) from the surface of infected cells, preventing infection of new cells. BST-2 is variably expressed in most cell types, and its expression is enhanced by cytokines such as type I interferon alpha (IFN-α). In this present study, we used the beta-retrovirus, mouse mammary tumor virus (MMTV) as a model to examine the role of mouse BST-2 in host infection in vivo. RESULTS: By using RNA interference, we show that loss of BST-2 enhances MMTV replication in cultured mammary tumor cells and in vivo. In cultured cells, BST-2 inhibits virus accumulation in the culture medium, and co-localizes at the cell surface with virus structural proteins. Furthermore, both scanning electron micrograph (SEM) and transmission electron micrograph (TEM) show that MMTV accumulates on the surface of IFNα-stimulated cells. CONCLUSIONS: Our data provide evidence that BST-2 restricts MMTV release from naturally infected cells and that BST-2 is an antiviral factor in vivo.

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine.

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.

Successful transmission of a retrovirus depends on the commensal microbiota.

To establish chronic infections, viruses must develop strategies to evade the host's immune responses. Many retroviruses, including mouse mammary tumor virus (MMTV), are transmitted most efficiently through mucosal surfaces rich in microbiota. We found that MMTV, when ingested by newborn mice, stimulates a state of unresponsiveness toward viral antigens. This process required the intestinal microbiota, as antibiotic-treated mice or germ-free mice did not transmit infectious virus to their offspring. MMTV-bound bacterial lipopolysaccharide triggered Toll-like receptor 4 and subsequent interleukin-6 (IL-6)-dependent induction of the inhibitory cytokine IL-10. Thus, MMTV has evolved to rely on the interaction with the microbiota to induce an immune evasion pathway. Together, these findings reveal the fundamental importance of commensal microbiota in viral infections.

Mouse mammary tumor virus in anti-mitochondrial antibody producing mouse models.

BACKGROUND & AIMS: A human betaretrovirus resembling the mouse mammary tumor virus (MMTV) has been characterized in primary biliary cirrhosis (PBC) and associated with aberrant pyruvate dehydrogenase complex (PDC)-E2-like expression. As MMTV is prevalent in mice as either an exogenous or endogenous infection, we tested the hypothesis that MMTV is linked with anti-mitochondrial antibody (AMA) production in models with severe immune dysfunction. METHODS: Evidence for MMTV was assessed in the liver and spleen of mice by PCR and immunochemistry and PDC-E2-like protein by immunochemistry. ELISA and Western blot were used to investigate AMA and anti-MMTV antibody production. RESULTS: Increased MMTV gag or env expression was detected in the livers of AMA producing mice including NOD.c3c4, CD4 directed dominant negative TGF-ß receptor II and IL-2 receptor α knockout mice as well as the NOD parental strain when compared to healthy strains and biliary disease control mice. The NOD.c3c4 mice expressed MMTV surface and capsid proteins and aberrant PDC-E2-like protein in the bile ducts, whereas IL-2 receptor α knockout mice, NOD.c3c4 and the NOD mice expressed MMTV proteins and aberrant PDC-E2-like protein in the spleen. A significant correlation between anti-MMTV antibody production and AMA production was observed in the sera of NOD and NOD.c3c4 mice (p<0.0001). CONCLUSIONS: The association of betaretroviral protein production and aberrant PDC-E2-like protein expression in the NOD.c3c4, NOD, and the IL-2 receptor α knockout mice is comparable to observations in patients with PBC. The correlation of AMA and anti-MMTV suggests the hypothesis that MMTV infection may trigger the production of AMA.

Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.

Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.

Vital role for CD8+ cells in controlling retroviral infections.

Antiviral adaptive immune defenses consist of humoral and cell-mediated responses, which together eliminate extracellular and intracellular virus. As most retrovirus-infected individuals do not raise efficient protective antivirus immune responses, the relative importance of humoral and cell-mediated responses in restraining retroviral infection is not well understood. We utilized retrovirus-resistant I/LnJ mice, which control infection with mouse mammary tumor virus (MMTV) and murine leukemia virus (MuLV) via an adaptive immune mechanism, to assess the contribution of cellular responses and virus-neutralizing antibodies (Abs) to the control of retroviral infection. We found that in retrovirus-infected CD8-deficient I/LnJ mice, viral titers exceed the neutralizing capability of antiviral Abs, resulting in augmented virus spread and disease induction. Thus, even in the presence of robust neutralizing Ab responses, CD8-mediated responses are essential for full protection against retroviral infection.

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

Peripheral CD4(+)Vß5(+) 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 TCRß rearrangement and results in a population of postrevision CD4(+)Vß5(-) T cells expressing revised TCRß 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 Vß5 transgenic (Tg) mice both impair peripheral deletion. Vß5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vß5 chain and a revised TCRß 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 Vß5 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.

Increases in IgA(+) B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vß region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

Junctional diversity prevents negative selection of an antigen-specific T cell repertoire.

Endogenous mouse mammary tumor proviruses (MMTV; Mtv loci) deletes Vbeta6 expressing T cells in the thymus of Mtv-7(+) DBA/2 (H2(d)) mice through negative selection. We found that in Mtv-7(-) BALB/c (H2(d)) mice, Vbeta6 is a dominant V gene used in T cell responses to an 18 amino acid long peptide antigen: EYKEYAEYAEYAEYAEYA [abbreviated as K5 or EYK(EYA)(5)]. It was therefore surprising to find that despite the deletion of Vbeta6+ T cells, vigorous K5 specific T cell responses that use Vbeta6 can be raised in DBA/2 mice. Sequence analysis of Vbeta6 junctional diversity in K5 specific T cell lines revealed that the DBA/2 K5 repertoire compensates for the loss of most Vbeta6 T cells by overusing and amplifying Vbeta6+ T cells escaping central deletion and peripheral tolerization. In order to address the inability of some Vbeta6 T cells to recognize Mtv-7(+) we analyzed a panel of BALB/c Vbeta6 expressing T cell hybridomas. This data supported the argument that certain Vbeta6 junctional sequences preclude Mtv recognition and allows their escape from central deletion in DBA/2 mice. These cells are not anergic and can be activated with cognate peptide antigen in periphery. We suggest that junctional diversity at the V region of some of the T cell receptors does not allow these cells to recognize self-superantigens with high enough affinity and thus they escape negative selection in the thymus. These results for the first time provide a molecular explanation of how the immune system compensates for "hole in the repertoire" caused by deletion of the majority of T cells carrying certain V region segments.

Detection of human mammary tumor virus proteins in human breast cancer cells.

Mouse mammary tumor virus (MMTV) has been proven to induce mammary cancer in mice. MMTV-like env gene sequences have been detected in one-third of the human breast tumors studied. The whole proviral structure with 95% homology to MMTV was found in two human breast tumors and was designated as human mammary tumor virus (HMTV). HMTV viral particles with betaretroviral features have been isolated. In addition, a retrovirus called human betaretrovirus (HBRV), homologous to the mentioned retroviruses, has been isolated from tissues of patients with primary biliary cirrhosis. In this report, the expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. By contrast, HMTV proteins did not react with antibody against the MMTV Env Gp52 protein. All the antibodies detected MMTV proteins with exception of two out of four monoclonal antibodies against HMTV Env. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis. This report shows the expression of HMTV proteins for the first time in human breast cancer cells using a panel of antibodies against HMTV, HBRV and MMTV proteins. This should be taken into consideration when MMTV antibodies are used to detect HMTV proteins in human tissues.