Ari Waisman

Experimental autoimmune encephalomyelitis repressed by microglial paralysis

Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo.

Caspase-8 Serves Both Apoptotic and Nonapoptotic Roles

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Apoptosis of Oligodendrocytes via Fas and TNF-R1 Is a Key Event in the Induction of Experimental Autoimmune Encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, immunization with myelin Ags leads to demyelination and paralysis. To investigate which molecules are crucial for the pathogenesis of EAE, we specifically assessed the roles of the death receptors Fas and TNF-R1. Mice lacking Fas expression in oligodendrocytes (ODCs) were generated and crossed to TNF-R1-deficient mice. To achieve specific deletion of a loxP-flanked fas allele in ODCs, we generated a new insertion transgene, expressing the Cre recombinase specifically in ODCs. Fas inactivation alone as well as the complete absence of TNF-R1 protected mice partially from EAE induced by the immunization with myelin ODC glycoprotein. The double-deficient mice, however, showed almost no clinical signs of EAE after immunization. Histological analysis revealed that demyelination was suppressed in CNS tissue and that lymphocyte infiltration was notably reduced. We conclude that the death receptors Fas and TNF-R1 are major initiators of ODC apoptosis in EAE. Although only moderate reduction of lymphocyte infiltration into CNS tissue was observed, the absence of these receptors appears to confer protection from demyelination and development of clinical disease.

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen.

TGF-β Signalling Is Required for CD4+ T Cell Homeostasis But Dispensable for Regulatory T Cell Function

Signalling by the cytokine TGF-β regulates mature CD4+ T cell populations but is not involved in the survival and function of regulatory T cells.

Insulin-Like Growth Factor-1 Controls Type 2 T Cell-Independent B Cell Response

The IGF-1 receptor (IGF-1R) is expressed on T and B lymphocytes, and the expression of the insulin- and IGF-1-signaling machinery undergoes defined changes throughout lineage differentiation, offering a putative role for IGF-1 in the regulation of immune responses. To study the role of the IGF-1R in lymphocyte differentiation and function in vivo, we have reconstituted immunodeficient RAG2-deficient mice with IGF-1R−/− fetal liver cells. Despite the absence of IGF-1Rs, the development and ex vivo activation of B and T lymphocytes were unaltered in these chimeric mice. By contrast, the humoral immune response to the T cell-independent type 2 Ag 4-hydroxy-3-nitrophenyl acetyl-Ficoll was significantly reduced in mice reconstituted with IGF-1R-deficient fetal liver cells, whereas responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenyl acetyl-chicken globulin were normal. Moreover, in an in vitro model of T cell-independent type 2 responses, IGF-1 promoted Ig production potently upon polyvalent membrane-IgD cross-linking. These data indicate that functional IGF-1R signaling is required for T cell-independent B cell responses in vivo, defining a novel regulatory mechanism for the immune response against bacterial polysaccharides.

The B-cell antigen receptor integrates adaptive and innate immune signals.

Significance Clonal expansion of antigen-specific B cells during an immune response is necessary for effective antibody production. B cells must integrate signals from their clonally restricted B-cell antigen receptor (BCR) and from nonclonal coreceptors that provide contextual cues to the nature of the antigen. How this is accomplished is unclear. We found that B cells require expression of the BCR for mitogenic responses triggered by coreceptors that recognize innate or T-cell-derived signals. The signaling pathway used by the BCR to license coreceptor-induced proliferation is similar to the previously described BCR-dependent survival signal and, thus, couples B-cell survival and mitogenic responses in an unexpected manner that may have implications for B-cell lymphomagenesis. B cells respond to antigens by engagement of their B-cell antigen receptor (BCR) and of coreceptors through which signals from helper T cells or pathogen-associated molecular patterns are delivered. We show that the proliferative response of B cells to the latter stimuli is controlled by BCR-dependent activation of phosphoinositidyl 3-kinase (PI-3K) signaling. Glycogen synthase kinase 3β and Foxo1 are two PI-3K-regulated targets that play important roles, but to different extents, depending on the specific mitogen. These results suggest a model for integrating signals from the innate and the adaptive immune systems in the control of the B-cell immune response.

MHC‐restricted T cell receptor signaling is required for αβ TCR replacement of the pre T cell receptor

A developmental block is imposed on CD25+CD44– thymocytes at the β‐selection checkpoint in the absence of the pre T cell receptor (preTCR) α‐chain, pTα. Early surface expression of a transgenic αβu2004TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4+CD8+ double‐positive stage. We wanted to analyze whether a restricting MHC element is required for αβu2004TCR‐expressing double‐negative (DN) thymocytes to overcome the developmental block in pTα‐deficient animals. We used the HY‐I knock‐in model that endows thymocytes with αβu2004TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTα‐deficient background, this HY‐I TCR transgene ‘rescued’ CD25+CD44– thymocytes from apoptosis and enabled progression to later differentiation stages. On a non‐selecting MHC background, however, pTα‐deficient HY‐I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY‐TCR‐mediated rescue of pTα‐deficient thymocytes.

Immunity to T-Cell Receptor

A common theme found in studies with mouse models of autoimmune diseases is that pathogenic T cells are primarily responsible for the pathology. Such models include diabetes in non-obese diabetic (NOD) mice, experimental autoimmune encephalomyelitis (EAE)-a model of multiple sclerosis (MS), collagen II (CII)-induced arthritis (CIA)-a model of rheumatoid arthritis (RA). Pathogenic T cells in EAE utilize a restricted repertoire of genes encoding the T-cell receptor (TCR) (1). For example, upon immunization of H-2(u) mice with either myelin basic protein, or its immunodominant fragment, peptide Ac1-20, the Vβ8.2 TCR gene product is expressed in the majority of pathogenic T cells (2-4). The restricted usage of the Vβ8.2 TCR gene product has also been found in rats in which EAE was induced by a peptide of myelin basic protein (5). Similarly, a restriction in the TCR usage was found also in CII-induced arthritis in mice (6,7), and in the TCR alpha chain usage in non-obese diabetic (NOD) mice (8).

T-Helper Cells

T cells can be found in skin under steady-state conditions as well as in infl ammatory processes. T cells in skin play an important role in immune homeostasis as well as control of infectious, infl ammatory diseases or tumors. In addition, several important and frequent skin diseases such as psoriasis, atopic dermatitis, autoimmune disease, and contact allergy are initiated by T cells. In skin diseases, the majority of antigenspecifi c T cells can be found in the tissue, not the peripheral blood. Here, we present a protocol suitable for isolation of skin-resident (infl ammatory) T cells that can be used for an in-depth characterization of their frequency, function, and role for the respective infl ammatory condition.