Melanie D. Leech

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM+CD138hiTACI+CXCR4+CD1dintTim1int plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138+ plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

Cutting Edge: Th1 Cells Facilitate the Entry of Th17 Cells to the Central Nervous System during Experimental Autoimmune Encephalomyelitis

It has recently been proposed that experimental autoimmune encephalomyelitis, once considered the classical Th1 disease, is predominantly Th17 driven. In this study we show that myelin-reactive Th1 preparations devoid of contaminating IL-17+ cells are highly pathogenic. In contrast, Th17 preparations lacking IFN-γ+ cells do not cause disease. Our key observation is that only Th1 cells can access the noninflamed CNS. Once Th1 cells establish the experimental autoimmune encephalomyelitis lesion, Th17 cells appear in the CNS. These data shed important new light on the ability of Th1 vs Th17 cells to access inflamed vs normal tissue. Because the IL-17-triggered release of chemokines by stromal cells could attract many other immune cells, allowing Th17 cells to access the tissues only under conditions of inflammation may be a key process limiting (auto)immune pathology. This has major implications for the design of therapeutic interventions, many of which are now aiming at Th17 rather than Th1 cells.

Resolution of Der p1-Induced Allergic Airway Inflammation Is Dependent on CD4+CD25+Foxp3+ Regulatory Cells

Allergic airway inflammation (AAI) is characterized by airway hyperreactivity, eosinophilia, goblet cell hyperplasia, and elevated serum IgE, however, it is unclear what mediates natural resolution after cessation of allergen exposure. This is important because the outcome of subsequent allergen challenge may depend on the concurrent inflammatory milieu of the lung. Using a murine AAI model, we demonstrate that after exposure to a defined natural protein allergen, Der p1, the response in lungs and draining mediastinal lymph nodes (dMLN) peaks between 4 and 6 days then declines until resolution by 21 days. Der p1-specific serum IgE follows the same pattern while IgG1 continues to increase. Resolution of AAI is mediated by CD4+CD25+Foxp3+ regulatory T cells (Tregs), which appear in lungs and dMLN following airway challenge. Treg depletion exacerbated lung eosinophilia, increased dMLN IL-5 and IL-13 but not IL-10 secretion, and increased allergic Ab responses. Most convincingly, transfer of CD4+CD25+Foxp3+ T cells from Ag naive mice (natural Tregs) abolished AAI, decreased dMLN IL-5 and IL-13 secretion, increased dMLN IL-10 secretion, abolished IgE, and decreased IgG1 Abs. Blocking IL-10 receptor function in vivo did not block the anti-inflammatory function of transferred natural Tregs but did restore dMLN IL-5 and IL-13 secretion. Thus natural Tregs can control AAI in an IL-10 independent manner.

Contribution of Myelin Autoantigen Citrullination to T Cell Autoaggression in the Central Nervous System

Breakdown in immunological self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiological conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4+ T cell-driven model of multiple sclerosis, we used experimental autoimmune encephalomyelitis (EAE) induced with the immunodominant 35–55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathology. Immunization with an altered peptide ligand (APL) of pMOG with an Arg→citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, we found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathology if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology.

Myelin-reactive, TGF-β-induced regulatory T cells can be programmed to develop Th1-like effector function but remain less proinflammatory than myelin-reactive Th1 effectors and can suppress pathogenic T cell clonal expansion in vivo.

Interest in the use of regulatory T cells (Tregs) as cellular therapeutics has been tempered by reports of naturally occurring Tregs losing Foxp3 expression and producing IL-17, raising concerns over a switch to pathogenic function under inflammatory conditions in vivo. TGF-β–induced Tregs (inducible Tregs [iTregs]), generated in large numbers in response to disease-relevant Ags, represent the most amenable source of therapeutic Tregs. Using Foxp3-reporter T cells recognizing myelin basic protein (MBP), we investigated the capacity of iTregs to produce effector-associated cytokines under proinflammatory cytokine conditions in vitro and whether this translated into proinflammatory function in vivo. In contrast with naturally occurring Tregs, iTregs resisted conversion to an IL-17–producing phenotype but were able to express T-bet and to produce IFN-γ. iTregs initiated their T-bet expression during their in vitro induction, and this was dependent on exposure to IFN-γ. IL-12 reignited iTreg expression of T-bet and further promoted iTreg production of IFN-γ upon secondary stimulation. Despite losing Foxp3 expression and expressing both T-bet and IFN-γ, MBP-responsive IL-12–conditioned iTregs induced only mild CNS inflammation and only when given in high numbers. Furthermore, iTregs retained an ability to suppress naive T cell clonal expansion in vivo and protected against the development of experimental autoimmune encephalomyelitis. Therefore, despite bearing predictive hallmarks of pathogenic effector function, previously Foxp3+ iTregs have much lower proinflammatory potential than that of MBP-responsive Th1 cells. Our results demonstrate that autoprotective versus autoaggressive functions in iTregs are not simply a binary relationship to be determined by their relative expression of Foxp3 versus T-bet and IFN-γ.

Cutting Edge: IL-6–Dependent Autoimmune Disease: Dendritic Cells as a Sufficient, but Transient, Source

Mice lacking IL-6 are resistant to autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), which is driven by CNS-reactive CD4+ T cells. There are multiple cellular sources of IL-6, but the critical source in EAE has been uncertain. Using cell-specific IL-6 deficiency in models of EAE induced by active immunization, passive transfer, T cell transfer, and dendritic cell transfer, we show that neither the pathogenic T cells nor CNS-resident cells are required to produce IL-6. Instead, the requirement for IL-6 was restricted to the early stages of T cell activation and was entirely controlled by dendritic cell–derived IL-6. This reflected the loss of IL-6R expression by T cells over time. These data explain why blockade of IL-6R only achieves protection against EAE if used at the time of T cell priming. The implications for therapeutic manipulation of IL-6 signaling in human T cell–driven autoimmune conditions are considered.

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. DOI: http://dx.doi.org/10.7554/eLife.03416.001

Peptide-based immunotherapy of experimental autoimmune encephalomyelitis without anaphylaxis

Administration of peptide antigens in tolerogenic form holds promise as a specific treatment for autoimmune and allergic disorders. However, experiments in rodent autoimmune models have highlighted the risk of anaphylaxis in response to systemic peptide application once the aberrant immune response is underway. Thus, mice with clinical signs of experimental autoimmune encephalomyelitis (EAE) or diabetes have been reported to suffer fatal anaphylaxis upon administration of native autoantigenic peptides. Clearly, this might represent a significant barrier to the use of synthetic peptides in the treatment of ongoing human autoimmune conditions. Here we describe the development of an altered peptide ligand (APL) engineered to prevent anaphylaxis (no antibody binding) whilst retaining the ability to silence pathogenic myelin‐reactive T lymphocytes. Administration of the APL to mice with an ongoing anti‐myelin immune response did not cause anaphylaxis, but led to complete protection from the subsequent induction of EAE and, when given during ongoing EAE, led to a rapid remission of clinical signs. The approach of removing antibody recognition whilst maintaining the desired functional effect (in this case T cell tolerance) may be of value in other situations in which there is a risk of triggering anaphylaxis with peptide‐based drugs.

Combination peptide immunotherapy based on T‐cell epitope mapping reduces allergen‐specific IgE and eosinophilia in allergic airway inflammation

Peptide immunotherapy using soluble peptides containing allergen‐derived immunodominant T‐cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323–339) have been unable to demonstrate therapeutic effects in ovalbumin‐induced allergic airway inflammation. We have previously shown that intravenous application of p323–339 can effectively tolerise p323–339‐reactive T cells in a non‐allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323–339 immunotherapy in a C57BL/6 model of ovalbumin‐induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin‐responsive T‐cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin‐reactive T‐cell lines were generated from ovalbumin‐immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin‐induced allergic airway inflammation. Peptide immunotherapy using the 323–339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T‐cell epitope in ovalbumin was identified within the 263–278 sequence. Combination peptide immunotherapy, using the 323–339 and 263–278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin‐specific IgE, with apparent reductions in interleukin‐5 and interleukin‐13. Characterization of the T‐cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

PD-1 signalling in CD4 + T cells restrains their clonal expansion to an immunogenic stimulus, but is not critically required for peptide-induced tolerance

The ultimate outcome of T‐cell recognition of peptide–major histocompatibility complex (MHC) complexes is determined by the molecular context in which antigen presentation is provided. The paradigm is that, after exposure to peptides presented by steady‐state dendritic cells (DCs), inhibitory signals dominate, leading to the deletion and/or functional inactivation of antigen‐reactive T cells. This has been utilized in a variety of models providing peptide antigen in soluble form in the absence of adjuvant. A co‐inhibitory molecule of considerable current interest is PD‐1. Here we show that there is the opportunity for the PD‐1/PD‐L1 interaction to function in inhibiting the T‐cell response during tolerance induction. Using traceable CD4+ T‐cell receptor (TCR) transgenic cells, together with a blocking antibody to disrupt PD‐1 signalling, we explored the roles of PD‐1 in the induction of tolerance versus a productive immune response. Intact PD‐1 signalling played a role in limiting the extent of CD4+ T‐cell accumulation in response to an immunogenic stimulus. However, PD‐1 signalling was not required for either the induction, or the maintenance, of peptide‐induced tolerance; a conclusion underlined by successful tolerance induction in TCR transgenic cells genetically deficient for PD‐1. These observations contrast with the reported requirement for PD‐1 signals in CD8+ T‐cell tolerance.