Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.     

IL-10 controls ultraviolet-induced carcinogenesis in mice

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.     

B7-mediated costimulation of CD4 T cells constrains cytomegalovirus persistence

Cytomegalovirus (CMV) utilizes multiple strategies to modulate immunity and promote lifelong, persistent/latent infection, including suppressing T cell activation pathways. Here we examined the role of B7 costimulatory ligands in establishing immune détente from both the host and virus perspectives. Mice lacking both B7.1 and B7.2 showed reduced early expansion of CMV-specific CD4 T cells, consequently allowing for enhanced levels of persistent virus replication. In turn, a CMV mutant lacking expression of the m138 and m147.5 gene products, which restrict B7.1 and B7.2 expression in infected antigen-presenting cells, induced a more robust CD4 T cell response and showed decreased persistence. Together, these data reveal a requirement for B7-mediated signaling in regulating the CMV-specific CD4 T cell response and establishing host-virus equilibrium.     

Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease

The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.     

B7 costimulation plays an important role in protection from herpes simplex virus type 2-mediated pathology

We have used mice lacking both B7-1 and B7-2 costimulation molecules (B7KO) to investigate the effects of B7 costimulation on herpes simplex virus type 2 (HSV-2) pathogenesis. B7KO mice infected intravaginally with virulent HSV-2 showed more severe genital and neurologic disease and higher mortality rates than their wild-type counterparts. These results suggest that B7 costimulation molecules play an important role in the development of primary immune responses protective against HSV-2.     

B7 costimulation molecules expressed from the herpes simplex virus 2 genome rescue immune induction in B7-deficient mice

The interaction between B7 costimulation molecules on antigen-presenting cells and CD28 on antigen-responsive T cells is essential for T-cell activation and maturation of immune responses to herpes simplex virus (HSV) infection. Vaccine-induced immune responses also depend upon adequate upregulation of B7 costimulation molecules, but this signal may be limiting for replication-defective virus vaccines. We investigated whether expression of B7 costimulation molecules by a prototypical replication-defective antiviral vaccine could enhance immune responses to the vaccine and whether B7-1 and B7-2 would be similarly effective. We altered an ICP8(-) replication-defective strain of HSV type 2 (HSV-2), 5BlacZ, to encode either murine B7-1 or B7-2. B7 molecule expression was detected on the surface of cells infected in vitro and at the RNA level in tissue of immunized mice. Immunization of B7-1/B7-2 knockout mice with B7-encoding virus modestly expanded the number of gamma interferon-producing T cells and significantly augmented class-switched HSV-specific antibody responses compared with the parental virus. Mice immunized with either B7-expressing virus showed less replication of challenge virus in the genital mucosa than mice immunized with 5BlacZ, markedly fewer signs of genital and neurological disease, and little weight loss. Virtually all mice immunized with B7-encoding virus survived challenge with a large dose of HSV-2, whereas most 5BlacZ-immunized mice succumbed to infection. These results indicate that protective immune responses can be enhanced by the inclusion of host B7 costimulation molecules in a prototypical replication-defective HSV vaccine against HSV-2 genital infection and that B7-1 and B7-2 induce immune responses with similar capacities to fight HSV-2 infection.     

Active antitumor immunotherapy, with or without B7-mediated costimulation, increases tumor progression in an immunogenic murine T cell lymphoma model

 BW-Sp3 is a BW-5147-derived T cell lymphoma with limited immunogenicity since, despite regression of the majority of subcutaneous tumors, an important fraction of the animals will die from metastases. In the present study, the BW-Sp3 cells were transfected with genes encoding B7-1 or B7-2, known to be involved in the induction of T cell responses. The resulting transfectants exhibited a reduced tumorigenicity and did not cause mortality in the syngeneic recipients. Furthermore, immunization with the B7-1 or B7-2 transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTL) that lysed both the transfectants and the wild-type BW-Sp3 cells. Since the B7 transfectants were completely rejected in syngeneic recipients and induced potent CTL recognizing the wild-type BW-Sp3 cells, these engineered cells were considered as candidates for immunotherapy. Vaccinations with the B7-1 or B7-2 transfectants could completely protect the animals from metastatic disease when subsequently challenged with wild-type BW-Sp3 cells. Furthermore, immunization with the B7 transfectants could prolong the survival time of mice that had been challenged intravenously with BW-Sp3 cells. Surprisingly, however, when these transfectants, as well as the wild-type BW-Sp3 cells, were used for vaccination of tumor-bearing animals, the presence of the subcutaneous BW-Sp3 tumors clearly interfered with the outcome of immunotherapy, resulting in increased malignancy, as reflected by a higher incidence of progressing tumors and a reduced survival rate. Possible implications for immunotherapy in humans are discussed. Received: 5 August 1997 / Accepted: 15 August 1997     

The role of B7 costimulation in T-cell immunity.

CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.     

Mechanisms of depressed reactivity to dinitrochlorobenzene and ultraviolet-induced tumors during ultraviolet carcinogenesis in BALB/c mice

Chronic treatment of BALB/c mice with ultraviolet (uv) radiation produces two distinct immunologic deficiencies. These deficiencies are apparent long before visible skin tumors are induced by the uv irradiation. One is reflected in a transient inability to develop delayed hypersensitivity to dinitrochlorobenzene and appears to be due to a defect in antigen processing. The other is expressed by the failure of mice to reject syngeneic uv-induced tumors, which are highly antigenic. This lack of tumor rejection can be passively transferred with lymphoid cells and seems to be due to the presence of specific suppressor lymphocytes.     

B7/CD28 costimulation of T cells induces a distinct proteome pattern

Effective immune strategies for the eradication of human tumors require a detailed understanding of the interaction of tumor cells with the immune system, which might lead to an optimization of T cell responses. To understand the impact of B7-mediated costimulation on T cell activation comprehensive proteome analysis of B7-primed T cell populations were performed. Using this approach we identified different classes of proteins in T cells whose expression is either elevated or reduced upon B7-1- or B7-2-mediated CD28 costimulation. The altered proteins include regulators of the cell cycle and cell proliferation, signal transducers, components of the antigen processing machinery, transporters, cytoskeletal proteins, and metabolic enzymes. A number of differentially expressed proteins are further modified by phosphorylation. Our results provide novel insights into the complexity of the CD28 costimulatory pathway of T cells and will help to identify potential targets of therapeutic interventions for modulating anti-tumor T cell activation.     

Reduced susceptibility to colitis-associated colon carcinogenesis in mice lacking plasma membrane-associated sialidase

Sialic acids are acidic monosaccharides that bind to the sugar chains of glycoconjugates and change their conformation, intermolecular interactions, and/or half-life. Thus, sialidases are believed to modulate the function of sialoglycoconjugates by desialylation. We previously reported that the membrane-associated mammalian sialidase NEU3, which preferentially acts on gangliosides, is involved in cell differentiation, motility, and tumorigenesis. The NEU3 gene expression is aberrantly elevated in several human cancers, including colon, renal, prostate, and ovarian cancers. The small interfering RNA-mediated knock-down of NEU3 in cancer cell lines, but not in normal cell-derived primary cultures, downregulates EGFR signaling and induces apoptosis. Here, to investigate the physiological role of NEU3 in tumorigenesis, we established Neu3-deficient mice and then subjected them to carcinogen-induced tumorigenesis, using a sporadic and a colitis-associated colon cancer models. The Neu3-deficient mice showed no conspicuous accumulation of gangliosides in the brain or colon mucosa, or overt abnormalities in their growth, development, behavior, or fertility. In dimethylhydrazine-induced colon carcinogenesis, there were no differences in the incidence or growth of tumors between the Neu3-deficient and wild-type mice. On the other hand, the Neu3-deficient mice were less susceptible than wild-type mice to the colitis-associated colon carcinogenesis induced by azoxymethane and dextran sodium sulfate. These results suggest that NEU3 plays an important role in inflammation-dependent tumor development.     

Reduced susceptibility of muscle-specific insulin receptor knockout mice to colon carcinogenesis

Insulin resistance is a risk factor for colon cancer, but it is not clear which of its metabolic sequelae are involved. The objective of this study was to determine whether increased adiposity and elevated circulating lipids commonly seen in insulin resistance promote colon carcinogenesis independent of changes in insulin. We made use of muscle-specific insulin receptor knockout (MIRKO) mice that exhibit elevated serum triglycerides (TG), free fatty acids (FFA), and fat mass but have similar body weights, circulating glucose, and insulin and insulin sensitivity to their wild-type littermates used as controls. Seven-week-old male MIRKO mice and controls received four weekly intraperitoneal injections of either 5 mg/kg azoxymethane (AOM) to induce aberrant crypt foci (ACF) or 10 mg/kg AOM to induce tumors and were killed at 24 or 40 wk of age, respectively. The MIRKO mice displayed hyperinsulinemia at 7 wk of age and reduced insulin sensitivity at 16 wk of age compared with controls. The previously reported MIRKO phenotype developed between 16 and 24 wk of age. By 40 wk of age, however, MIRKO mice were again insulin resistant. ACF development did not differ between MIRKO mice and controls, but MIRKO mice developed significantly fewer colon tumors. Our results suggest that circulating TG and FFA are not promoters of colon tumor development. Indeed, we show that the cumulative effects of the metabolic changes that occur with knockout of the insulin receptor in muscle are associated with reduced susceptibility to colon tumorigenesis.     

Viral interference with B7-1 costimulation: a new role for murine cytomegalovirus fc receptor-1

Murine CMV (MCMV), a beta-herpesvirus, infects dendritic cells (DC) and impairs their function. The underlying events are poorly described. In this study, we identify MCMV m138 as the viral gene responsible for promoting the rapid disappearance of the costimulatory molecule B7-1 (CD80) from the cell surface of DC. This was unexpected, as m138 was previously identified as fcr-1, a putative virus-encoded FcR. m138 impaired the ability of DC to activate CD8+ T cells. Biochemical analysis and immunocytochemistry showed that m138 targets B7-1 in the secretory pathway and reroutes it to lysosomal associated membrane glycoprotein-1+ compartments. These results show a novel function for m138 in MCMV infection and identify the first viral protein to target B7-1.     

Autoantibody responses and pathology regulated by B7-1 and B7-2 costimulation in MRL/lpr lupus

The activation of T lymphocytes requires both Ag-mediated signaling through the TCR as well as costimulatory signals transmitted through B7-1 and/or B7-2 with CD28. The interference of B7-mediated costimulatory signals has been proposed as one immunotherapeutic intervention for the prevention autoimmune disease. This study has examined autoantibody responses and autoimmune pathology in a murine model of human systemic lupus erythematosus (SLE), the MRL-lpr/lpr mouse, genetically deficient in B7-1 or B7-2, or in mice treated with B7-1/B7-2 blocking Abs. In contrast to other studies of murine models of SLE, MRL-lpr/lpr mice treated with B7 blocking Abs exhibit strong anti-small nuclear ribonucleoprotein (snRNP) and anti-DNA autoantibody responses with some changes in isotype switching as compared with untreated animals. All MRL-lpr/lpr mice deficient in B7-1 or B7-2 produce anti-snRNP and anti-DNA titers with isotypes virtually identical with wild-type animals. However, the absence of B7-2 costimulation did interfere with the spontaneous activation and the accumulation of memory CD4+ or CD8+ T lymphocytes characteristic of wild-type MRL-lpr/lpr mice. IgG and C3 complement deposition was less pronounced in the kidneys of B7-2 deficient MRL-lpr/lpr mice, reflecting their lessor degree of glomerulonephritis. By comparison, B7-1-deficient MRL-lpr/lpr mice had more severe IgG and C3 deposits in glomeruli.     

Impaired serum thyrotropin response to hypothyroidism in mice with disruption of neuromedin B receptor

Neuromedin B (NB), a neuropeptide highly concentrated in pituitary, has been proposed to be an inhibitor of thyrotropin (TSH) secretion. Previous study showed that mice with disruption of neuromedin B receptor (NBR-KO) have higher TSH release in response to thyrotropin-releasing hormone (TRH), although TSH seems to have decreased bioactivity. Here we examined in NBR-KO mice the response of TSH to thyroid hormone (TH) deprivation, obtained by methimazole treatment, or excess, obtained by acute and chronic TH administration. In response to hypothyroidism NBR-KO mice exhibited a lower magnitude increase in serum TSH compared to wild-type (WT) mice (1.7 vs. 3.3-times increase compared to euthyroid values, respectively, P<0.001). One hour after a single T4 injection (0.4 microg/100 g BW), WT and NBR-KO hypothyroid mice presented similar degree of serum TSH reduction (54%, P<0.05). However, 3 h after T4 administration, WT mice presented serum TSH similar to hypothyroid baseline, while NBR-KO mice still had decreased serum TSH (30% reduced in comparison to hypothyroid baseline P<0.05). T3 treatment of euthyroid mice for 21 days, with progressively increasing doses, significantly reduced serum TSH similarly in WT and NBR-KO mice. Also, serum T4 exhibited the same degree of suppression in WT and NBR-KO. In conclusion, disruption of neuromedin B receptor did not interfere with the sensitivity of thyroid hormone-mediated suppression of TSH release, but impaired the ability of thyrotroph to increase serum TSH in hypothyroidism, which highlights the importance of NB in modulating the set point of the hypothalamus-pituitary-thyroid axis at hypothyroidism.     

A critical role for B7/CD28 costimulation in experimental autoimmune encephalomyelitis: a comparative study using costimulatory molecule-deficient mice and monoclonal antibody blockade

The B7/CD28 pathway provides critical costimulatory signals required for complete T cell activation and has served as a potential target for immunotherapeutic strategies designed to regulate autoimmune diseases. This study was designed to examine the roles of CD28 and its individual ligands, B7-1 and B7-2, in experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the CNS. EAE induction in CD28- or B7-deficient nonobese diabetic (NOD) mice was compared with the effects of B7/CD28 blockade using Abs in wild-type NOD mice. Disease severity was significantly reduced in CD28-deficient as well as anti-B7-1/B7-2-treated NOD mice. B7-2 appeared to play the more dominant role as there was a moderate decrease in disease incidence and severity in B7-2-deficient animals. EAE resistance was not due to the lack of effective priming of the myelin peptide-specific T cells in vivo. T cells isolated from CD28-deficient animals produced equivalent amounts of IFN-gamma and TNF-alpha in response to the immunogen, proteolipid protein 56-70. In fact, IFN-gamma and TNF-alpha production by Ag-specific T cells was enhanced in both the B7-1 and B7-2-deficient NOD mice. In contrast, peptide-specific delayed-type hypersensitivity responses in these animals were significantly decreased, suggesting a critical role for CD28 costimulation in in vivo trafficking and systemic immunity. Collectively, these results support a critical role for CD28 costimulation in EAE induction.     

The role of B7 costimulation in CD4/CD8 T cell homeostasis

The effect of B7-mediated costimulation on T cell homeostasis was examined in studies of B7-1 (CD80) and B7-2 (CD86) transgenic as well as B7-deficient mice. B7 overexpression in transgenic mice resulted in marked polyclonal peripheral T cell hyperplasia accompanied by skewing toward an increased proportion of CD8 single-positive cells and a decreased proportion of CD4 single-positive cells in thymus and more markedly in peripheral T cells. B7-induced T cell expansion was dependent on both CD28 and TCR expression. Transgenic overexpression of B7-1 or B7-2 resulted in down-regulation of cell surface CD28 on thymocytes and peripheral T cells through a mechanism mediated by intercellular interaction. Mice deficient in B7-1 and B7-2 exhibited changes that were the reciprocal of those observed in B7-overexpressing transgenics: a marked increase in the CD4/CD8 ratio in peripheral T cells and an increase in cell surface CD28 in thymus and peripheral T cells. These reciprocal effects of genetically engineered increase or decrease in B7 expression indicate that B7 costimulation plays a physiological role in the regulation of CD4+ and CD8+ T cell homeostasis.     

Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.     

Different rates of ultraviolet-induced DNA damage in the epidermis and dermis of a platyfish model for carcinogenesis

The purpose of this study was to compare fluence-response relationships for the production of cyclobutane pyrimidine dimers in epidermal or dermal DNA of platyfishXiphophorus hybrids irradiated with UVB, and to determine photoreactivation from black light on dimers producedin situ. This was accomplished by quantitative gel electrophoresis of unlabeled DNA following extraction of the DNA and treatment with an enzyme specific for the detection of pyrimidine dimers. The dermis was the target tissue for UV-induced DNA damage inXiphophorus hybrid fish skin. Shapes of dimer-fluence response data following filtered sunlamp irradiation ( > 290 nm) or monochromatic wavelength 302 nm in the epidermis or dermis were different. In the epidermis there was an initial steep upward slope followed by a plateau, whereas in the dermis a linear relationship was observed. The final values of dimers at the high doses were, however, nearly equal in the epidermis and dermis exposed to either radiation. These differences in fluence-response relationships are probably attributable to the intertwining of the epidermis and to the shielding effect of the epidermal layer, with scales leading to a heterogenous population of cells which are exposed to different UV doses. Photoreversal of dimers was readily observed by black light irradiation in both epidermis and dermis irradiated with either > 290 nm or 302 nm.This research was supported by the Office of Health and Environmental Research of the U.S. Department of EnergyThe author is recipient of the National Academy of Sciences' Kobelt Fund Grant