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Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain Bcell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

In 1943, Luria and Delbrück used a phage resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing 2, 3, may be skewed by mutational “hot” or “cold” spots 3, 4. Both approaches are affected by numerous caveats 5, 6, 7 (see Supplemental Information). We devise a method, Maximum-Depth Sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in E. coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress; transcription-replication conflicts; and in the case of fluoroquinolones, direct damage to DNA.

Dendritic cells (DCs) orchestrate a repertoire of immune responses that bring about resistance to infection and silencing or tolerance to self. In the settings of infection and cancer, microbes and tumours can exploit DCs to evade immunity, but DCs also can generate resistance, a capacity that is readily enhanced with DC-targeted vaccines. During allergy, autoimmunity and transplant rejection, DCs instigate unwanted responses that cause disease, but, again, DCs can be harnessed to silence these conditions with novel therapies. Here we present some medical implications of DC biology that account for illness and provide opportunities for prevention and therapy.

αB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease. ©2007 Nature Publishing Group.

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are approved for cholesterol reduction, may also be beneficial in the treatment of inflammatory diseases. Atorvastatin (Lipitor) was tested in chronic and relapsing experimental autoimmune encephalomyelitis, a CD4(+) Th1-mediated central nervous system (CNS) demyelinating disease model of multiple sclerosis. Here we show that oral atorvastatin prevented or reversed chronic and relapsing paralysis. Atorvastatin induced STAT6 phosphorylation and secretion of Th2 cytokines (interleukin (IL)-4, IL-5 and IL-10) and transforming growth factor (TGF)-beta. Conversely, STAT4 phosphorylation was inhibited and secretion of Th1 cytokines (IL-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha) was suppressed. Atorvastatin promoted differentiation of Th0 cells into Th2 cells. In adoptive transfer, these Th2 cells protected recipient mice from EAE induction. Atorvastatin reduced CNS infiltration and major histocompatibility complex (MHC) class II expression. Treatment of microglia inhibited IFN-gamma-inducible transcription at multiple MHC class II transactivator (CIITA) promoters and suppressed class II upregulation. Atorvastatin suppressed IFN-gamma-inducible expression of CD40, CD80 and CD86 co-stimulatory molecules. l-Mevalonate, the product of HMG-CoA reductase, reversed atorvastatin's effects on antigen-presenting cells (APC) and T cells. Atorvastatin treatment of either APC or T cells suppressed antigen-specific T-cell activation. Thus, atorvastatin has pleiotropic immunomodulatory effects involving both APC and T-cell compartments. Statins may be beneficial for multiple sclerosis and other Th1-mediated autoimmune diseases.

Lipid mediators influence immunity in myriad ways. For example, circulating sphingosine-1-phosphate (S1P) is a key regulator of lymphocyte egress. Although the majority of plasma S1P is bound to apolipoprotein M (ApoM) in the high-density lipoprotein (HDL) particle, the immunological functions of the ApoM-S1P complex are unknown. Here we show that ApoM-S1P is dispensable for lymphocyte trafficking yet restrains lymphopoiesis by activating the S1P1 receptor on bone marrow lymphocyte progenitors. Mice that lacked ApoM (Apom(-/-)) had increased proliferation of Lin(-) Sca-1(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow. Pharmacological activation or genetic overexpression of S1P1 suppressed LSK and CLP cell proliferation in vivo. ApoM was stably associated with bone marrow CLPs, which showed active S1P1 signalling in vivo. Moreover, ApoM-bound S1P, but not albumin-bound S1P, inhibited lymphopoiesis in vitro. Upon immune stimulation, Apom(-/-) mice developed more severe experimental autoimmune encephalomyelitis, characterized by increased lymphocytes in the central nervous system and breakdown of the blood-brain barrier. Thus, the ApoM-S1P-S1P1 signalling axis restrains the lymphocyte compartment and, subsequently, adaptive immune responses. Unique biological functions imparted by specific S1P chaperones could be exploited for novel therapeutic opportunities.

B and T lymphocytes are the mediators of immunity, but their function is under the control of dendritic cells. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self-antigens), thereby minimizing autoimmune reactions. Once a neglected cell type, dendritic cells can now be readily obtained in sufficient quantities to allow molecular and cell biological analysis. With knowledge comes the realization that these cells are a powerful tool for manipulating the immune system.

Dendritic cells (DCs) have the unique capacity to initiate primary and secondary immune responses. They acquire antigens in peripheral tissues and migrate to lymphoid organs where they present processed peptides to T cells. DCs must therefore exist in distinct functional states, an idea that is supported by observations that they downregulate endocytosis and upregulate surface molecules of the class II major histocompatibility complex (MHC) upon maturation. Here we investigate the features of DC maturation by reconstituting the terminal differentiation of mouse DCs in vitro and in situ. We find that early DCs, corresponding to those found in peripheral tissues, exhibit a phenotype in which most class II molecules are intracellular and localized to lysosomes. Upon maturation, these cells give rise to a new intermediate phenotype in which intracellular class II molecules are found in peripheral non-lysosomal vesicles, similar to the specialized CIIV population seen in B cells. The intermediate cells then differentiate into late DCs which express almost all of their class II molecules on the plasma membrane. These variations in class II compartmentalization are accompanied by dramatic alterations in the intracellular transport of the new class II molecules and in antigen presentation. We found that although early DCs could not present antigen immediately after uptake, efficient presentation of the previously internalized antigen occurred after maturation, 24-48 hours later. By regulating class II transport and compartmentalization, DCs are able to delay antigen display, a property crucial to their role in immune surveillance.

A 1.3-million-year-long climate history from the Lake Malawi basin in eastern Africa displays a trend towards progressively wetter conditions superimposed on strong 100,000-year eccentricity cycles of temperature and rainfall since the Mid-Pleistocene Transition around 900,000 years ago. Climate variability during the Pleistocene epoch is fairly well known for the marine realm and Antarctica. But for much of the land surface — and Africa in particular — past climate variability remains unclear. Thomas Johnson et al. present a record of aridity for the past 1.3 million years from Lake Malawi, southeast Africa, and show a trend towards progressively wetter conditions superimposed on strong 100,000-year eccentricity cycles of temperature and rainfall since the Mid-Pleistocene Transition around 900,000 years ago. Carbon dioxide and dust, but not global ice volume, appear to have influenced climate variations at Lake Malawi over the past 500,000 years. African climate is generally considered to have evolved towards progressively drier conditions over the past few million years, with increased variability as glacial–interglacial change intensified worldwide1,2,3. Palaeoclimate records derived mainly from northern Africa exhibit a 100,000-year (eccentricity) cycle overprinted on a pronounced 20,000-year (precession) beat, driven by orbital forcing of summer insolation, global ice volume and long-lived atmospheric greenhouse gases4. Here we present a 1.3-million-year-long climate history from the Lake Malawi basin (10°–14° S in eastern Africa), which displays strong 100,000-year (eccentricity) cycles of temperature and rainfall following the Mid-Pleistocene Transition around 900,000 years ago. Interglacial periods were relatively warm and moist, while ice ages were cool and dry. The Malawi record shows limited evidence for precessional variability, which we attribute to the opposing effects of austral summer insolation and the temporal/spatial pattern of sea surface temperature in the Indian Ocean. The temperature history of the Malawi basin, at least for the past 500,000 years, strongly resembles past changes in atmospheric carbon dioxide and terrigenous dust flux in the tropical Pacific Ocean, but not in global ice volume. Climate in this sector of eastern Africa (unlike northern Africa) evolved from a predominantly arid environment with high-frequency variability to generally wetter conditions with more prolonged wet and dry intervals.

Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule alpha 4 beta 1, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-alpha 4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with alpha 4 beta 1 integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.

A better understanding of the molecules involved in immune responses has identified many potential targets for the treatment of autoimmune diseases. But although successful therapies have been found for immune disorders in animal studies, few have passed the much harder test of treating human diseases. So far, non-antigen-specific approaches, such as the blocking of tumour-necrosis factor, are achieving some success but the same is not true for antigen-specific approaches. Future therapies will probably include both non-antigen-specific strategies that target cytokines (cell-cell signalling molecules) or block the molecules that stimulate immune responses, and antigen-specific therapies that induce tolerance to self antigens.

THE role of infection in the pathogenesis of clinical relapses that occur in most autoimmune diseases, including multiple sclerosis, remains to be established1,2. Experimental autoimmune encephalomyelitis (EAE) serves as a model for multiple sclerosis, with episodes of relapsing paralysis3–9. In certain strains of mice, T-lym-phocytes expressing the V/β8 T-cell receptor (TCR)6–8 engage the amino-terminal epitope Ac 1–11 of myelin basic protein, leading to EAE. The bacterial superantigen staphylococcal enterotoxin B (SEB) activates V/β8-expressing T cells. Here we show that after immunization with Ac 1–11, or after transfer of encephalitogenic T-cell lines or clones reactive to Acl-11, SEB induces exacerbation or relapses of paralytic disease in mice that are in clinical remission following an initial episode of paralysis, and triggers paralysis in mice with subclinical disease. Tumour necrosis factor has a critical role in the mechanism underlying SEB-induced exacerbation of disease, because anti-tumour necrosis factor antibody given in vivo delays the onset of paralysis triggered by SEB. On reactivation of autoaggressive cells through their T-cell receptor, superantigens may induce clinical relapses of autoimmune disease.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

Dendritic cells and thymic epithelial cells perform important immunoregulatory functions by presenting antigens in the form of peptides bound to cell-surface major histocompatibility complex (MHC) molecules to T cells. Whereas B cells are known to present specific antigens efficiently through their surface immunoglobins, a comparable mechanism for the capture and efficient presentation of diverse antigens by dendritic cells and thymic epithelial cells has not previously been described. We show here that their antigen-presentation function is associated with the high-level expression of DEC-205, an integral membrane protein homologous to the macrophage mannose receptor and related receptors which are able to bind carbohydrates and mediate endocytosis. DEC-205 is rapidly taken up by means of coated pits and vesicles, and is delivered to a multivesicular endosomal compartment that resembles the MHC class II-containing vesicles implicated in antigen presentation. Rabbit antibodies that bind DEC-205 are presented to reactive T-cell hybridomas 100-fold more efficiently than rabbit antibodies that do not bind DEC-205. Thus DEC-205 is a novel endocytic receptor that can be used by dendritic cells and thymic epithelial cells to direct captured antigens from the extracellular space to a specialized antigen-processing compartment.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which a restricted cellular immune response has been observed. In order to establish whether such T cell responses are likely to be antigen-specific particularly with regard to myelin basic protein (MBP), we analysed T-cell receptor (TCR) gene rearrangements directly from MS brain plaques, using the polymerase chain reaction on reverse transcribed messenger RNA, and compared these with TCR of previously described MBP-specific T cell clones from MS and the rat model experimental allergic encephalomyelitis. Rearranged V beta 5.2 genes were detected in the brains of all patients who were HLA DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401. The V beta 5.2-D beta-J beta sequences in these MS brain plaques revealed five motifs. One of the common motifs was identical to that described for the VDJ region of a V beta 5.2 T-cell clone. This clone was from an MS patient who was HLA DRB1*1501, DQB1*0602, DPB1*0401, and it was cytotoxic towards targets containing the MBP peptide 89-106 (ref. 1). The deduced amino-acid sequence of this VDJ rearrangement, Leu-Arg-Gly, has also been described in rat T cells cloned from experimental allergic encephalomyelitis lesions, which are specific for MBP peptide 87-99 (ref. 2). VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TCR V beta 5.2 N(D)N rearrangements in MS lesions. The capacity of rat T cells with these VDJ sequences to cause experimental allergic encephalomyelitis and the prevalence of such sequences in demyelinated human lesions indicate that T cells with this rearranged TCR may be critical in MS.

The identification of activated T cells in the brain of individuals with multiple sclerosis (MS) indicates that these cells are critical in the pathogenesis of this disease. In an attempt to elucidate the nature of the lymphocytic infiltration, we used the polymerase chain reaction to amplify T-cell antigen receptor (TCR) V alpha sequences from transcripts derived from MS brain lesions. In each of three MS brains, only two to four rearranged TCR V alpha transcripts were detected. No V alpha transcripts could be found in control brains. Sequence analysis of transcripts encoded by the V alpha 12.1 region showed rearrangements to a limited number of J alpha region segments. These results imply that TCR V alpha gene expression in MS brain lesions is restricted.

AUTOIMMUNE diseases are characterised by an attack of an individual's lymphocytes against his own body's components, self-antigens. The immunogenicity of self-antigens can be defined operationally by their capacity to trigger lymphocytes bearing specific receptors. We have designed experiments to learn how a defined self-antigen can function as an immunogen in vitro and how the form of its presentation to self-recognising lymphocytes may abrogate this immunogenicity. We have found that myelin basic protein (BP) behaves as a self-immunogen when presented to lymphocytes by way of syngeneic macrophages. Moreover, recognition of this self-immunogen can be inhibited by the presence of the same self-antigen in soluble form during the primary lymphocyte–macrophage interaction.

Chronic relapsing paralysis and demyelination within the central nervous system (CNS), features associated with the human disease multiple sclerosis (MS), develop in mice after injection of murine T-cell clones specific for the autoantigen myelin basic protein (MBP). We examined the fine specificity of three independently derived encephalitogenic T-cell clones using synthetic polypeptides derived from portions of the N-terminal sequence of MBP. These clones appear functionally identical; they all respond to an epitope in the N-terminal nine amino acid residues in association with the same class II (I-A) molecules of the major histocompatibility complex (MHC). Both the N-terminal acetyl moiety and the first residue (Ala) are necessary for recognition. Only N-terminal MBP peptides recognized by these clones were found to cause encephalomyelitis (EAE) in vivo. These results show that the N-terminal MBP-specific T lymphocytes that mediate autoimmune encephalomyelitis are a small population with a limited repertoire; they all recognise the same combination of MHC and target.