Rhoanne C. McPherson

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.

Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Summary Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8+ T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8+ T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK’s immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8+ T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

BackgroundThe DNA methylation profiles of mammalian cell lines differ from those of the primary tissues from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblasts to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs, suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.

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

T-bet Expression by Foxp3(+) T Regulatory Cells is Not Essential for Their Suppressive Function in CNS Autoimmune Disease or Colitis

Accumulation of T regulatory (Treg) cells within the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) is essential for the resolution of disease. CNS Treg cells have been shown to uniformly express the Th1-associated molecules, T-bet and CXCR3. Here, we report that the expression of T-bet is not required for the function of these Treg within the CNS. Using mice that lacked T-bet expression specifically within the Treg compartment, we demonstrate that there was no deficit in Treg recruitment into the CNS during EAE and no difference in the resolution of disease compared to control mice. T-bet deficiency did not impact on the in vitro suppressive capacity of Treg. Transfer of T-bet-deficient Treg was able to suppress clinical signs of either EAE or colitis. These observations demonstrate that, although Treg can acquire characteristics associated with pathogenic T effector cells, this process is not necessarily required for their suppressive capacity and the resolution of autoimmune inflammation.

Adaptive immune responses in CNS autoimmune disease: mechanisms and therapeutic opportunities

The processes underlying autoimmune CNS inflammation are complex, but key roles for autoimmune lymphocytes seem inevitable, based on clinical investigations in multiple sclerosis (MS) and related diseases such as neuromyelitis optica, together with the known pathogenic activity of T cells in experimental autoimmune encephalomyelitis (EAE) models. Despite intense investigation, the details of etiopathology in these diseases have been elusive. Here we describe recent advances in the rodent models that begin to allow a map of pathogenic and protective immunity to be drawn. This map might illuminate previous successful and unsuccessful therapeutic strategies targeting particular pathways, whilst also providing better opportunities for the future, leading to tailored intervention based on understanding the quality of each individual’s autoimmune response.

Eliminating roles for T-bet and IL-2 but revealing superior activation and proliferation as mechanisms underpinning dominance of regulatory T cells in tumors

Foxp3+ regulatory T cells (Tregs) are often highly enriched within the tumor-infiltrating T cell pool. Using a well-characterised model of carcinogen-induced fibrosarcomas we show that the enriched tumor-infiltrating Treg population comprises largely of CXCR3+ T-bet+ ‘TH1-like’ Tregs which are thymus-derived Helios+ cells. Whilst IL-2 maintains homeostatic ratios of Tregs in lymphoid organs, we found that the perturbation in Treg frequencies in tumors is IL-2 independent. Moreover, we show that the TH1 phenotype of tumor-infiltrating Tregs is dispensable for their ability to influence tumor progression. We did however find that unlike Tconvs, the majority of intra-tumoral Tregs express the activation markers CD69, CD25, ICOS, CD103 and CTLA4 and are significantly more proliferative than Tconvs. Moreover, we have found that CD69+ Tregs are more suppressive than their CD69− counterparts. Collectively, these data indicate superior activation of Tregs in the tumor microenvironment, promoting their suppressive ability and selective proliferation at this site.

Induction of Passive EAE Using Myelin-Reactive CD4+ T Cells

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS) often used as a model for the early inflammatory stages of multiple sclerosis and also as a model of organ-specific autoimmune disease.This protocol describes the induction of passive EAE in mice, either using T cells isolated from mice primed with myelin antigens, or through the use of naïve TCR transgenic T cells activated in vitro in the presence of myelin-derived antigens.

A Context-Dependent Role for αv Integrins in Regulatory T Cell Accumulation at Sites of Inflammation

Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3+ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.