Mast Cells Man the Barrier
Autoreactive T cells activated in peripheral lymphoid organs must cross the blood-brain barrier (BBB) to initiate experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis. Mast cells have been implicated in EAE development, but their mechanism of action is not known. Because these cells densely populate regions of the meninges, Sayed et al. (p. 6891) hypothesized that mast cells might regulate BBB permeability. To address this possibility, they induced EAE in mast cell-deficient mice and found that, although myelin-specific Th1 and Th17 cells were effectively activated in the periphery, there was a defect in their entry into the CNS. In addition, the few T cells that entered the CNS were not strongly reactivated in the parenchyma. Reconstitution of meningeal mast cells restored disease to wild-type levels, indicating the importance of these local mast cells in EAE pathogenesis. Surprisingly, histamine produced by mast cells was not involved in EAE development. Instead, mast cell-derived TNF was required for the recruitment of neutrophils into the CNS during EAE induction. The authors proposed that meningeal mast cells indirectly promote the breach of the BBB through their control of an early influx of neutrophils into the CNS, and inhibition of meningeal mast cell activity could be a useful avenue to explore in multiple sclerosis treatment.
Inhibiting Factor B
The complement system effectively attacks a wide variety of pathogens but can also damage host tissue if activated inappropriately. Although all three major pathways of complement activation can lead to tissue damage, the alternative pathway is a particularly important target for therapeutic modulation. Kadam and Sahu (p. 7116) therefore screened for small peptide inhibitors of factor B (fB), the key protease of the alternative pathway of complement activation. Peptides able to bind to fB were identified from phage display libraries. Common motifs from these peptides were used to design cyclic peptides, and, of these, a peptide dubbed Complin effectively inhibited the activation of fB. The structural and sequence requirements for Complin's inhibitory activity were identified, and this peptide was found to bind to both the Ba and Bb fragments of fB. However, Complin did not block the interaction between fB and C3b, suggesting that it acted instead by inhibiting factor D-mediated cleavage of fB. Interestingly, Complin could also block the activation of the classical complement pathway component C2 and could inhibit the classical and lectin pathways of complement activation. This novel small molecule inhibitor thus acts on all three pathways of complement activation and could be useful for further therapeutic development, as well as for basic studies of fB and C2 activation.
The development of a diverse immune receptor repertoire requires tight regulation of V(D)J recombination. Chromatin remodeling and consequent modulation of access to recombination signal sequences (RSSs) by RAG1 and 2 proteins are key to this regulation, but very little is known about how nucleosomes are organized on Ag receptor loci. Kondilis-Mangum et al. (p. 6970) therefore extensively characterized nucleosome organization on the TCR loci of primary mouse thymocytes. To do this, the authors analyzed DNA associated with mononucleosomes prepared from thymocyte chromatin and compared nucleosome positioning in accessible versus inaccessible RSSs in Tcra and Tcrb alleles. Wild-type alleles were compared with alleles lacking specific promoters or enhancers known to be necessary for RSS accessibility, and all alleles analyzed demonstrated similarly highly organized arrays of nucleosomes. The positioning of these nucleosomes was partially established by the local DNA sequence, but there was no consistent relation between nucleosome localization and RSS location. However, accessible alleles showed a loss of some nucleosomes and a repositioning of others, compared with inaccessible alleles. Some of these effects were demonstrated to be a direct result of germline transcription. Taken together, these data indicate that promoter and enhancer gene segments work together to regulate nucleosomal occupancy and positioning and thereby control RSS accessibility and ultimately the TCR repertoire.
Bacterial Spores Boost B Cells
Commensal bacteria in the gut are important for the proper development of many components of the immune system. In rabbits, some bacterial species have been shown to promote GALT development, but the mechanism by which this occurs is not known. Severson et al. (p. 6782) have found that some proteins on the surface of Bacillus spores can resemble superantigens in their ability to polyclonally stimulate B cells and thereby drive GALT development. scFv-Ig proteins composed of single-chain Ab fragments containing rabbit Ig VH and VL domains demonstrated binding to multiple proteins from Bacillus anthracis spores via a superantigen-like binding site. These spores could also bind to surface Ig on human B cells, and binding was found to be dependent on the Vκ region of both rabbit and human Abs. As suspected, on the basis of their superantigen-like binding, B. anthracis spores were able to polyclonally activate human B cells in vitro. In vivo, molecules on the surface of B. anthracis spores were able to promote the development of B cell follicles in GALT in rabbits. These data demonstrate a mechanism by which bacterial spores can modulate the development of the adaptive immune system.
Sustaining Gut Regulation
Both unregulated T cell activation and activity of the pathogen recognition receptor NOD2 are involved in the chronic intestinal inflammation associated with Crohn's disease (CD). In this issue, Rahman et al. (p. 7247) link these two mechanisms by demonstrating the involvement of NOD2 signaling in human regulatory T cell (Treg) activity. CD patients with disease-associated polymorphisms in the NOD2 gene had significantly fewer Tregs in the lamina propria than other CD patients. Human Tregs expressed NOD2, and the NOD2 ligand muramyl dipeptide (MDP) induced NF-κB activation, indicating that NOD2 was functionally expressed. Ligation of NOD2 with MDP protected Tregs from Fas-mediated apoptosis, and interference with NOD2 activity removed this protection. Demonstrating in vivo relevance of this observation, Tregs from a patient bearing a disease-related polymorphism of NOD2 were not protected from Fas-mediated apoptosis. MDP stimulation of Tregs induced the upregulation of X-IAP, indicating a possible mechanism of MDP-mediated anti-apoptotic activity. Thus, NOD2 regulates human Treg homeostasis, which may explain its involvement in CD.
The Importance of Being Specific
Difficulties inherent in the development of therapies using polyclonal natural regulatory T cells (nTregs) have led to interest in finding ways to manipulate induced Ag-specific Tregs (iTregs) to suppress inappropriate immune responses. To define the role of Ag specificity in iTreg-mediated suppression of autoimmunity, Zhang et al. (p. 6629) generated iTregs specific for the encephalitogenic Ag proteolipid peptide (PLP) 139–151 and analyzed their activity. Compared with nTregs, these cells, dubbed 139-iTregs, had decreased CD62L and increased CD103 expression, indicative of an effector/memory Treg phenotype. Following adoptive transfer, these 139-iTregs migrated to draining lymph nodes and proliferated in response to immunization with PLP139–151, but not with the linked Ag PLP178–191. 139-iTregs were able to suppress PLP139–151-induced experimental autoimmune encephalomyelitis more efficiently than were polyclonal iTregs but could not suppress immune responses to other encephalitogenic peptides. These cells also inhibited the proliferation of PLP139–151-specific T cells but did not affect that of T cells with other specificities. These data indicate that, at least for 139-iTregs, Ag specificity is required for both Treg activation and subsequent suppression of target cell immune responses. It may therefore be possible to generate Ag-specific iTregs that do not induce bystander suppression for therapeutic use.
Altering epigenetic modifications of DNA, such as methylation, can potently affect gene expression. In fact, inhibition of DNA methylation in T cells can be sufficient to induce the development of systemic lupus erythematosus. MicroRNAs (miRNAs) modulate posttranscriptional gene repression, and miRNA dysregulation has been described in diseases including lupus. Pan et al. (p. 6773) therefore sought to determine whether miRNAs affected DNA hypomethylation in lupus and found that two miRNAs, miR-21 and miR-148a, were upregulated in CD4+ T cells from both mice and humans with lupus. Both of these miRNAs downregulated the expression of DNA methyltransferase (DNMT) 1, suggesting that they affected methylation. Interestingly, miR-21 indirectly targeted DNMT1 by inhibiting its upstream regulator RASGRP1, whereas miR-148a directly acted on the DNMT1 coding region and not its 3′UTR. These miRNAs induced upregulation of methylation-sensitive, autoimmune-associated genes, including those encoding CD70 and LFA-1. Finally, specific inhibitors of miR-21 and miR-148a attenuated DNA hypomethylation in CD4+ T cells from lupus patients, indicating the potential therapeutic relevance of this study. These observations of miRNA-mediated regulation of DNA methylation are important to the understanding of lupus pathogenesis and to future approaches to treatment.
MHC and the T Cell Community
The development of the naive T cell repertoire is shaped by selection processes in the thymus, but how the expression of specific MHC alleles may impact human repertoire development is unclear. To address this issue, Legoux et al. (p. 6731) used a peptide:MHC multimer-based sorting approach to analyze the frequencies of naive CD8+ T cells specific for several defined tumor and viral Ags in the context of the MHC class I molecule HLA-A*0201 (A2) in individuals unexposed to the Ags tested. The frequencies of Ag-specific A2-restricted CD8+ T cells were remarkably consistent among A2-expressing individuals for each Ag. Interestingly, A2-restricted CD8+ T cells specific for the Ags tested were also observed in A2-negative individuals, and the impact of A2 expression on Ag-specific T cell frequency varied between Ags. Further complicating the picture, CD4+ T cells specific for A2-restricted Ags were identified in all donors, even in those lacking TAP, indicating that these cells were not selected by MHC class I molecules in the thymus. The frequencies of these CD4+ T cells correlated with those of naive CD8+ T cells specific for the same Ag. These data emphasize the involvement of MHC cross-reactivity in human T cell repertoire development and have important implications for understanding the relationship between CD4+ T cell help and the development of CD8+ T cell responses.
Summaries written by Jennifer Hartt Meyers, Ph.D.