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We examined the roles of B7-1 (CD80) and B7-2 (CD86) in a model of allergic pulmonary inflammation and airway hyperresponsiveness (AHR) by using mice with germline deletions of the B7-1 and/or B7-2 molecules. Multiple parameters of the allergic response were affected to varying degrees by the absence of B7-1 and/or B7-2. Mice lacking both B7-1 and B7-2 had no elevation of serum immunoglobulin E, lack of airway eosinophilia, and no AHR. These same disease parameters were also reduced in mice lacking either B7-1 or B7-2. Lack of B7-1 and/or B7-2 resulted in an increase in T-helper 1 cytokine production. Our observations suggest that whereas B7-2 is quantitatively more significant in the induction of this response, B7-1 and B7-2 may have complementary roles in mediating the development of allergic pulmonary inflammation.

The past year has seen significant advances in our understanding of the role of the B7–CD28/CTLA-4 pathway in regulating the responses of self-reactive T cells, giving impetus to manipulation of this pathway for treating human autoimmune diseases. Recent studies have demonstrated that B7–CD28 costimulation has critical roles in stimulating both the initiation and effector phases of autoimmunity and that CD28 regulates the threshold for activation of self-reactive T cells. Recent work has also revealed critical roles for CTLA-4 in limiting the extent of Th1/Th2 cell differentiation and in downregulating the responses of self-reactive T cells during both the initiation and progression of autoimmune disease.

The wild mouse ecotropic retrovirus, Cas-Br-E, induces progressive, noninflammatory spongiform neurodegenerative disease in susceptible mice. Functional genetic analysis of the Cas-Br-E genome indicates that neurovirulence maps to the env gene, which encodes the surface glycoprotein responsible for binding and fusion of virus to host cells. To understand how the envelope protein might be involved in the induction of disease, we examined the regional and temporal expression of Cas-Br-E Env protein in the central nervous systems (CNS) of mice infected with the highly neurovirulent chimeric virus FrCas E . We observed that multiple isoforms of Cas-Br-E Env were expressed in the CNS, with different brain regions exhibiting unique patterns of processed Env glycoprotein. Specifically, the expression of gp70 correlated with regions showing microglial infection and spongiform neurodegeneration. In contrast, regions high in neuronal infection and without neurodegenerative changes (the cerebellum and olfactory bulb) were characterized by a gp65 Env protein isoform. Sedimentation analysis of brain region extracts indicated that gp65 rather than gp70 was incorporated into virions. Biochemical analysis of the Cas-Br-E Env isoforms indicated that they result from differential processing of N-linked sugars. Taken together, these results indicate that differential posttranslational modification of the Cas-Br-E Env is associated with a failure to incorporate certain Env isoforms into virions in vivo, suggesting that defective viral assembly may be associated with the induction of spongiform neurodegeneration.

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-γ and TNF-α in response to the immunogen, proteolipid protein 56–70. In fact, IFN-γ and TNF-α 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.

All mammals produce heparin, a negatively charged glycosaminoglycan that is a major constituent of the secretory granules of mast cells which are found in the peritoneal cavity and most connective tissues. Although heparin is one of the most studied molecules in the body, its physiological function has yet to be determined. Here we describe transgenic mice, generated by disrupting the N-deacetylase/N-sulphotransferase-2 gene, that cannot express fully sulphated heparin. The mast cells in the skeletal muscle that normally contain heparin lacked metachromatic granules and failed to store appreciable amounts of mouse mast-cell protease (mMCP)-4, mMCP-5 and carboxypeptidase A (mMC-CPA), even though they contained substantial amounts of mMCP-7. We developed mast cells from the bone marrow of the transgenic mice. Although these cultured cells contained high levels of various protease transcripts and had substantial amounts of mMCP-6 protein in their granules, they also failed to express mMCP-5 and mMC-CPA. Our data show that heparin controls, through a post-translational mechanism, the levels of specific cassettes of positively charged proteases inside mast cells.

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896–8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.

The functional significance of B7 co-stimulation in T-cell activation was described first in the context of preventing the induction of anergy. The functions of this pathway are far more complex than initially appreciated in view of the existence of two B7 molecules which have specificities for both CD28 and CTLA-4, which serve to amplify and terminate T-cell responses respectively Mice lacking B7 co-stimulators and CD28 and CTLA-4 co-stimulatory receptors are helping to clarify the functions of this key immunoregulatory pathway In this review we will focus on the role of B7 co-stimulation in the activation and differentiation of CD4 + helper cells and CD8 + cytotoxic cells. The contribution of B7 co-stimulation to CD4 + responses depends upon the activation history of the T-cell and the strength of the T-cell antigen receptor signal. B7 co-stimulation contributes to interleukin (IL)-2 production by both naive and previously activated CD4- T cells. B7 co-stimulation is most critical for the differentiation of naive CD4 + T cells to IL-4 producers, but predominately influences IL-2 production by previously activated CD4 + cells. B7 co-stimulation is important in development of cytotoxic T cells through both effects on T-helper cells and by direct co-stimulation of CD8 + cells.

There have been significant recent advances in our understanding of the role of the B7-CD28/CTLA-4 costimulatory pathway in T-cell activation and self-tolerance. Recent studies have begun to clarify how signaling through this pathway can influence cytokine production. The critical role for CTLA-4 in regulating T-cell activation and autoreactivity has been demonstrated, revealing a previously unsuspected means by which costimulation is involved in the maintenance and breakdown of self-tolerance. In vivo studies indicate the therapeutic potential of manipulating this important, but complex, immunoregulatory pathway.

Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.

Resting dendritic cells can induce T cell tolerance. This process requires engagement of the costimulatory molecules PD-1 and CTLA-4 on T cells.

The CBFbeta subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFbeta associates with DNA-binding CBFalpha subunits and increases their affinity for DNA. Genes encoding the CBFbeta subunit (CBFB) and one of the CBFalpha subunits (CBFA2, otherwise known as AML1) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 (AML1) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFbeta subunit is required for CBFalpha2 function in vivo.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

Signaling via the B7:CD28/CTLA4 pathway can provide a potent co-stimulatory signal to T cells. Recent developments indicate that signaling through this pathway is more complex than previously thought. Analysis of the B7- 1-deficient mouse strain revealed the existence of additional functional CTLA4 counterreceptor(s) and indicated that despite the powerful co- stimulatory effects of B7-1 in vitro, in vivo the absence of B7-1 resulted in only a partial effect. This finding stimulated the cloning of the CTLA4 counterreceptor B7-2, which has been found to be the major, early activating co-stimulator in this pathway. B7-1 and B7-2 interact with two receptors, CD28 and CTLA4, on T cells. While both B7-1 and B7-2 display a very restricted pattern of expression on antigen-presenting cells (APCs), they are differentially expressed on distinct APC populations. The constitutive or early expression of B7-2 has led to the hypothesis that B7-2 may participate in the initiation of the immune response, thereby playing a pivotal role in the decision between T-cell activation and anergy, whereas mB7-1, being expressed later, may serve to amplify or regulate the immune response.

A general principle of lymphocyte activation is that functional responses of mature T and B lymphocytes require at least two signals. The first signal is provided by antigen recognition, and is responsible for ensuring that the resultant immune response is specific for the eliciting antigen. For T lymphocytes, the second signal is provided by proteins, called co-stimulators, that are expressed on the surfaces of antigen-presenting cells (APCs). Only professional APCs, such as dendritic cells, macrophages, and B lymphocytes, express co-stimulators, and this expression is enhanced by inflammatory reactions that often accompany infections or the administration of antigens with adjuvants. Therefore, the requirement for co-stimulators ensures that T lymphocytes are activated at the correct place—where foreign antigens are presented by professional APCs—and the correct time—when the introduction of antigen elicits the warning sign of local inflammation. T lymphocytes that encounter the first signal, antigen, in the absence of co- stimulators either fail to respond or are rendered anergic, i.e., unresponsive to subsequent interactions with the antigen. It is postulated that because of this, antigens that are presented on nonprofessional APCs or antigens, including self antigens, that do not normally elicit local inflammation fail to stimulate any specific T cells that may be present. Thus, the absence of co- stimulators on normal, “resting” tissue APCs may induce and maintain T-cell tolerance to self antigens that may be presented by these APCs. Conversely, the aberrant expression of co-stimulators on tissue APCs may contribute to the development of autoimmune reactions.

The generation of an antigen-specific T-cell response requires that the T lymphocyte receive two signals from the antigen presenting cell. The specificity of this response is provided by antigen presented to the T lymphocyte and involves stimulation of the T lymphocyte via the T-cell receptor (TCR)/CD3 complex. The second, or costimulatory signal, can be provided by ligation of the B-lymphocyte activation antigens B7-1 (CD80) and B7.2 (CD86) to TCR antigen CD28. The cDNAs for both CD80 and CD86 have been isolated and are predicted to encode type 1 membrane proteins of the immunoglobulin (Ig) superfamily. The predicted protein is composed of a signal peptide followed by two Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail. Here we report that the genomic organization of CD86 reflects its functional structure, and is similar to that found for CD80. The gene is composed of eight exons which span more than 22 kilobases. The predicted protein functional domains of signal peptide, extracellular IgV- and IgC-like regions, and transmembrane domain coincide with the genomic structure. Two independent sequences had been reported for CD86 cDNA which differed in their 5'untranslated (UT) regions. We find CD86 exons 1 and 2 correspond to these alternate 5'UT sequences. Splicing of exon 1 or 2 with the signal peptide encoding exon 3 would produce mRNA transcripts complementary to the reported cDNA clones. Exons 4 and 5 correspond to IgV- and IgC-like extracellular domains, respectively. Exon 6 encodes the transmembrane region and beginning of the cytoplasmic tail. Exons 7 and 8 encode the remainder of the cytoplasmic tail and 3'UT sequences.

PURPOSE. Given that dry eye disease (DED) is associated with T cell-mediated inflammation of the ocular surface and that PD-L1 is an important negative or inhibitory regulator of immune responses constitutively expressed at high levels by corneal epithelial cells, the authors studied the expression and function of PD-L1 in DED. METHODS. Dry eye was induced in untreated wild-type mice, PD-L1(-/-) mice, and wild-type mice treated with anti-PD-L1 antibody by exposing these mice to a desiccating environment in the controlled environment chamber modified with subcutaneous administration of scopolamine. Real-time PCR was used to quantify the expression of chemokine gene transcript levels of multiple CC and CXC chemokine ligands and receptors. Epifluorescence microscopy was used to evaluate corneal infiltration of CD3(+) T cells after immunohistochemical staining. RESULTS. The increased expression of specific chemokine ligands and receptors in PD-L1(-/-) corneas of normal mice is associated with significant increases in T-cell homing into these corneas. Similar, and more enhanced, increases in T-cell infiltration were observed in PD-L1(-/-) DED mice or DED mice treated with anti-PD-L1 antibody compared with controls. In addition, the authors found significantly decreased expression of PD-L1 by corneal epithelial cells in DED and significantly increased corneal fluorescein staining score with PD-L1 functional blockade using anti-PD-L1 antibody. CONCLUSIONS. Downregulation of corneal epithelial PD-L1 amplifies dry eye-associated corneal inflammation and epitheliopathy by increasing the expression of chemokine ligands and receptors that promote T-cell homing to the ocular surface.

We have generated mice carrying a mutation of the gene encoding the low affinity NGF receptor p75NGFR by targeted mutation in embryonic stem cells. Mice homozygous for the mutation were viable and fertile. Immunohistochemical analyses of the footpad skin of mutant mice revealed markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers. The defective innervation was correlated with loss of heat sensitivity and associated with the development of ulcers in the distal extremities. Complicated by secondary bacterial infection, the ulcers progressed to toenail and hair loss. Crossing a human transgene encoding p75NGFR into the mutant animals rescued the absent heat sensitivity and the occurrence of skin ulcers and increased the density of neuropeptide-immunoreactive sensory innervation of footpad skin. The mutation in the gene encoding p75NGFR did not decrease the size of sympathetic ganglia or the density of sympathetic innervation of the iris or salivary gland. Our results suggest that p75NGFR has an important role in the development and function of sensory neurons.

Publisher Summary This chapter discusses mutations in the mouse through breeding to homozygosity transgenic mice, resulting from infection of embryos with retroviruses. To date, four recessive lethal mutations have been identified; these manifest their effect at quite different stages of development. Of 70 strains bred to homozygosity, four were found to have recessive lethal mutations. The identity of only one of the four mutated genes is known. In the Movl3 strain, a Moloney murine leukemia virus (M-MuLV) provirus has integrated in the first intron of the αl(I) collagen gene. The effect of this provirus on transcription of this gene and the resulting death of homozygotes at the 12th day of gestation from the rupture of major vessels is well documented. A second mutation (Mov34) was found to arise from the integration of an M-MuLV-derived provirus into an as yet unidentified gene. Embryos homozygous for the Mov34 integration were found to develop normally to blastocysts, and then die after implanting into the uterine wall.

Purpose Mammalian programmed cell death (PD)-1 is a membrane-associated receptor regulating the balance between T-cell activation, tolerance, and immunopathology; however, its role in neurons has not yet been defined. The hypothesis that PD-1 signaling actively promotes retinal ganglion cell (RGC) death within the developing mouse retina was investigated. Methods Mature retinal cell types expressing PD-1 were identified by immunofluorescence staining of vertical retina sections; developmental expression was localized by immunostaining and quantified by Western blot analysis. PD-1 involvement in developmental RGC survival was assessed in vitro using retinal explants and in vivo using PD-1 knockout mice. PD-1 ligand gene expression was detected by RT-PCR. Results PD-1 is expressed in most adult RGCs and undergoes dynamic upregulation during the early postnatal window of retinal cell maturation and physiological programmed cell death (PCD). In vitro blockade of PD-1 signaling during this time selectively increases the survival of RGCs. Furthermore, PD-1-deficient mice show a selective increase in RGC number in the neonatal retina at the peak of developmental RGC death. Lastly, gene expression of the immune PD-1 ligand genes Pdcd1lg1 and Pdcd1lg2 was found throughout postnatal retina maturation. Conclusions These findings collectively support a novel role for a PD-1-mediated signaling pathway in developmental PCD during postnatal RGC maturation.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57B1/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50 % of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the IV mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak IV alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.

Many different T-cell co-stimulatory molecules have been recently discovered. Determining exactly how many there are, why there are so many and what they all do is the next task ( 1365–1369 ).

RETROVIRUSES are involved in several human neurological diseases with varying pathological features1–3. Whether these diseases are due to a direct effect of the virus on nervous system cells is unknown. To gain insight into the pathogenesis of one retroviral neurological disease, we are studying the murine neurotropic retro-virus, Cas-Br-E, which causes lower motor neuron disease associated with spongiform degenerative changes in brain and spinal cord4. Central nervous system (CNS) injury seems to be due to direct viral action5, but the precise target cells of the virus are uncertain. After blood-borne virus enters the CNS it is found in capillary endothelial cells5–7. No microscopic evidence for virus within glia or neurons has been found in some studies6, whereas virus or incomplete particles have been observed in CNS cells in other studies5,8. Here we identify the neuron as a major target for Cas-Br-E in the CNS, suggesting that this disease may be a direct result of viral infection of neurons. We also show that envelope protein (Env, encoded by the env gene), a major determinant of neurovirulence9,10, cannot be detected in neurons but is present in non-neuronal cells, although spliced env messenger RNA is synthesized in CNS tissue. This suggests that a post-transcriptional step in Cas-Br-E Env protein synthesis is impaired and that the neurological disease may be a consequence of abortive replication of virus in neurons. This may explain the failure to find neuronal infection in other neurological diseases by conventional methods of virus detection.

Asthma is a major public health problem that has increased markedly in prevalence in the past two decades. In several mouse models, natural killer T cells (NKT) have recently been found to be required for the development of airway hyperreactivity (AHR), a cardinal feature of asthma. Moreover, in patients with persistent severe asthma, a significant number of NKT cells are present in the lungs. While T cell receptor signaling is required to activate NKT cells, the costimulatory requirements for NKT cell activation and function are not clear. In this study we found that ICOS was highly expressed on both naïve and activated NKT cells and that expression of ICOS was much higher on the CD4+ NKT than CD4− NKT cells. The function of NKT cells from ICOS−/− mice was impaired, as adoptive transfer of these cells into NKT cell deficient Ja18−/− mice failed to restore AHR, whereas transfer of wildtype NKT cells fully restored AHR in the Ja18−/− recipients. The failure of ICOS−/− NKT to induce AHR may be due to a great extent to the absence of cytokine production by ICOS−/− NKT cells. Additionally, ICOS−/− mice challenged with α-GalCer did not develop AHR, but adoptive transfer of WT NKT cells into ICOS−/− mice restored AHR. Finally, ICOS may be required for CD4+ NKT cell survival in the periphery, as the number of CD4+ NKT cells was significantly lower in spleen and liver of ICOS−/− mice. These results indicate that costimulation by ICOS is required for the function and homeostasis of CD4+ NKT cells, which play a major role in development of AHR.

Cytotoxic T lymphocyte Antigen 4 (CTLA-4) is an important cell surface molecule that inhibits T cell activation. Four splice variants of CTLA-4 have been identified, indicating the regulatory complexity of CTLA-4. In this study, we characterized the biology of the shortest splice variant 1/4WT CTLA-4 and its functional correlation to full length (FL CTLA-4). 1/4WT CTLA-4 mRNA was found in quiescent CD4+ T cells and upregulated after immunization with MOG35-55 peptide. The kinetic expression of 1/4WT was opposite to FL CTLA-4. To test functions of 1/4WT CTLA-4, we transduced CD4 T cells with a retroviral vector coexpressing 1/4WT CTLA-4 and the green fluorescent protein (GFP). FL CTLA-4 was downregulated on the cell surface by 1/4WT overexpression, which consequently enhanced antigen specific T cell responses by enhancing T cell proliferation and cytokine production in vitro. This functional correlation between 1/4WT and FL CTLA-4 had profound influence on effector CD4 T cell function. Mice adoptively transferred with MOG35-55 specific CD4 T cells containing ectopically expressed 1/4WT CTLA-4 showed dramatically exacerbated EAE disease, increased CD4 T cell proliferation, and increased IFN gamma and IL-17 production. These data suggest that 1/4WT CTLA-4 plays an important role in regulating T cell activation by affecting expression and/or function of FL CTLA-4.

Pathways in the B7:CD28 family regulate T cell activation and tolerance. The demonstration of B7 dependent responses in CD28/CTLA-4−/− T cells together with studies pointing to stimulatory and inhibitory functions for PD-L1 and PD-L2, have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 can bind to one another with an affinity greater than the affinity of B7-1 for CD28 but less than CTLA-4. The B7-1:PD-L1 interaction can be shown in both biophysical and cell-based assays, and is functionally significant. We demonstrate that this interaction is restricted to B7-1 and PD-L1. B7-2 does not interact with PD-L1, nor does PD-L2 interact with B7-1. We have identified anti-PD-L1 antibodies with either dual-specificity, which can block two interactions (PD-L1:B7-1 and PD-L1:PD-1), or mono-specificity, which can block only one (PD-L1:B7-1). The effects of this pathway on T cell proliferation and cytokine production will be discussed. Our findings give increased significance to B7-1 and PD-L1 on T cells, and add an additional dimension to immunoregulatory functions of the B7:CD28 family.

Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

Although B7 on APCs has a well-recognized role in T cell costimulation, little is known about the functional significance of constitutive and activation-induced B7 expression that also occurs on T cells. To analyze the role of B7 on T cells, B7-1/B7-2-deficient mice (B7 double knockout) and mice overexpressing B7-2 exclusively on T cells (B7-2 transgenic) were used as T cell donors for allogeneic transplant recipients, and graft-vs-host disease (GVHD) was assessed. B7 double-knockout T cells resulted in significant GVHD acceleration compared with wild-type T cells. Conversely, B7-2 transgenic donor T cells mediated reduced GVHD mortality compared with wild-type T cells. Data indicated that B7 expression on T cells down-regulated alloresponses through CTLA-4 ligation. This study is the first to provide definitive in vivo data illustrating the importance of T cell-associated B7 as a negative regulator of immune responses in a clinically relevant murine model of GVHD. The up-regulation of B7 on T cells may be an important component of normal immune homeostasis.

Rationale T cell co-stimulatory pathways are critical regulators of T cell activation and tolerance. The B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized of these. The key immunoregulatory role of this pathway has prompted searches for additional B7 and CD28 family members. Several new co-stimulatory pathways in this family have been identified and these have important roles in regulating T cell activation and tolerance. However, the extent of these families and the identities of close relatives in the human genome have not been fully described. Methods We searched the NIH human genome protein database using a new multiple sequence analysis tool of our design. Putative family members were analyzed using structurally-biased multiple sequence alignment and three-dimensional structural models were constructed. Results Novel candidate members and distantly related relatives of the CD28/ICOS/CTLA-4 receptor superfamily and B7/ICOSL ligand superfamily were found. Sequence alignments recapitulated functionally important regions of these families as validated in the co-crystal structures. Conclusions We have identified potentially new members of the B7:CD28 family that may be important for immune activation and tolerance. Multiple sequence alignments and structure modeling demonstrate regions important for protein-protein interactions, and facilitate understanding of the disease-causing role of human polymorphisms.

The contribution of the costimulatory molecules B7-1 and B7-2 to the in vivo differentiation of Th cells remains controversial. The infection of resistant and susceptible strains of mice with the parasite Leishmania majorprovides a well-established model for studying in vivo differentiation of CD4 + T cells. We have infected B7-1/B7-2-deficient mice on the BALB/c and 129 background with L. major and subsequently examined different parameters of infection and cytokine responses upon restimulation of lymph node cells in vitro. BALB/c B7-2-deficient and B7-1/B7-2-double deficient mice are resistant to L. major, whereas BALB/c B7-1-deficient mice remain as susceptible as wild-type BALB/c mice. Differential expression of B7-1 and B7-2 can explain the distinct roles observed for these B7 costimulators in L. major infection.

Transgenic Mov-14 mice, which carry the provirus of Moloney murine leukemia virus (Mo-MuLV) in the germ line and begin to produce infectious virus on embryonic day 14, were used to evaluate the ability of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to cross the placenta and protect embryos from viremia. We have used the Mov-14 model previously to demonstrate the antiviral efficacy and lack of teratogenicity of transplacental therapy with 3'-azido-3'-deoxythymidine (zidovudine, ZDV). PMEA was administered to pregnant females by daily intraperitoneal injection or by osmotic pump. In contrast to ZDV, PMEA was either noneffective in preventing viremia in the offspring or embryotoxic, depending on the dose. The specific toxic effects seen were resorption of pregnancy, low birth weight, and neonatal death. Histopathological analysis of neonatal mice exposed to PMEA showed severe lymphoid depletion of the thymus. We conclude that PMEA therapy is contraindicated for use during pregnancy.

The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments M1 and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except M1 and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type 3. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycine- and the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.

Infection of mouse embryos at 8 days of gestation with a replication-defective retrovirus carrying the human c-Ha-ras-1 oncogene led to efficient and rapid induction of hyperplastic lesions. Twenty-four percent of viable off-spring developed abnormal growths after infection with purified virus. The lesions contained a single integrated provirus and produced viral RNA and the Ha-ras oncogene product (p21). The latency period between the time of infection and appearance of the lesions suggested that secondary alterations in addition to activated ras were necessary for neoplasms to develop. The earliest and most abundant growths were cutaneous and appeared from 4 to 36 weeks of age, with a median of 4 weeks of age. A number of subcutaneous lesions also developed over the same time span but at a median of 18 weeks of age. The rapid development of cutaneous lesions in response to transduction of the ras oncogene contrasts with other studies in which adult skin required secondary treatment with promoters prior to ras induction of epithelial hyperplasia. These results demonstrate that infection of midgestation mouse embryos allows rapid analysis of oncogene potency in skin.

We have examined the effect of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and cycloheximide on the CV-1 cell cytoskeleton. Within a few hours after producing an inhibition of cellular protein synthesis, all these agents specifically disrupted the organization of the vimentin filament system with no discernable effect on microtubules or microfilaments during the period of observation. Furthermore, just as the inhibition of protein synthesis by cycloheximide is reversible, so was the disruption of vimentin filaments by cycloheximide.