Stephen A. Newland

Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake.

Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.

Validated germline-competent embryonic stem cell lines from nonobese diabetic mice

Nonobese diabetic (NOD) mice provide an excellent model of type 1 diabetes. The genetic contribution to this disease is complex, with more than 20 loci implicated in diabetes onset. One of the challenges for researchers using the NOD mouse model (and, indeed, other models of spontaneous autoimmune disease) has been the high density of sequence variation within candidate chromosomal segments. Furthermore, the scope for analyzing many putative disease loci via gene targeting has been hampered by the lack of NOD embryonic stem (ES) cells. We describe here the derivation of NOD ES cell lines capable of generating chimeric mice after stable genetic modification. These NOD ES cell lines also show efficient germline transmission, with offspring developing diabetes. The availability of these cells will not only enable the dissection of closely linked loci and the role they have in the onset of type 1 diabetes but also facilitate the generation of new transgenics.

Epigenetic Changes at Il12rb2 and Tbx21 in Relation to Plasticity Behavior of Th17 Cells

Plasticity within Th cell populations may play a role in enabling site-specific immune responses to infections while limiting tissue destruction. Epigenetic processes are fundamental to such plasticity; however, to date, most investigations have focused on in vitro-generated T cells. In this study, we have examined the molecular mechanisms underpinning murine Th17 plasticity in vivo by assessing H3K4 and H3K27 trimethylation marks at Tbx21, Rorc, Il17a, Ifng, and Il12rb2 loci in purified ex vivo-isolated and in vitro-generated Th17 cells. Although both populations had largely comparable epigenetic signatures, including bivalent marks at Tbx21, freshly isolated ex vivo Th17 cells displayed restricted expression from Il12rb2 due to the presence of repressive chromatin modifications. This receptor, however, could be upregulated on isolated ex vivo Th17 cells after in vitro activation or by in vivo immunization and was augmented by the presence of IFN-γ. Such activated cells could then be deviated toward a Th1-like profile. We show that IL-12 stimulation removes H3K27 trimethylation modifications at Tbx21/T-bet leading to enhanced T-bet expression with in vitro Th17 cells. Our study reveals important potential phenotypic differences between ex vivo- and in vitro-generated Th17 cells and provides mechanistic insight into Th17 cell plasticity.

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig‐like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 μg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.

The Nonconventional MHC Class II Molecule DM Governs Diabetes Susceptibility in NOD Mice

The spontaneous destruction of insulin producing pancreatic beta cells in non-obese diabetic (NOD) mice provides a valuable model of type 1 diabetes. As in humans, disease susceptibility is controlled by the classical MHC class II genes that guide CD4+ T cell responses to self and foreign antigens. It has long been suspected that the dedicated class II chaperone designated HLA-DM in humans or H-2M in mice also makes an important contribution, but due to tight linkage within the MHC, a possible role played by DM peptide editing has not been previously tested by conventional genetic approaches. Here we exploited newly established germ-line competent NOD ES cells to engineer a loss of function allele. DM deficient NOD mice display defective class II peptide occupancy and surface expression, and are completely protected against type 1 diabetes. Interestingly the mutation results in increased proportional representation of CD4+Foxp3+ regulatory T cells and the absence of pathogenic CD4+ T effectors. Overall, this striking phenotype establishes that DM-mediated peptide selection plays an essential role in the development of autoimmune diabetes in NOD mice.

The Schistosoma mansoni T2 ribonuclease omega-1 modulates inflammasome-dependent IL-1β secretion in macrophages

The T2 ribonuclease omega-1 is a powerful Th2-inducing factor secreted by the eggs of the blood fluke Schistosoma mansoni. Omega-1 can modulate pattern recognition receptor-induced inflammatory signatures and alter antigen presentation by dendritic cells. Recent findings have suggested that component(s) contained in or secreted by S. mansoni eggs (soluble egg antigen) can also enhance IL-1β secretion by dendritic cells stimulated with pattern recognition receptor ligands. Here we show that omega-1 enhances IL-1β secretion in macrophages stimulated with Toll-like receptor 2 ligand, and propose omega-1 as the factor in soluble egg antigen capable of regulating inflammasome activity. This effect is dependent on the C-type lectin receptor Dectin-1, caspase-8 and the ASC inflammasome adaptor protein, highlighting the ability of omega-1 to regulate multiple pattern recognition receptor signalling pathways. These mechanistic insights into manipulation of host immunity by a parasite product have implications for the design of anti-inflammatory therapeutic drugs.

Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet

Splenic marginal zone B (MZB) cells, positioned at the interface between circulating blood and lymphoid tissue, detect and respond to blood-borne antigens. Here we show that MZB cells in mice activate a homeostatic program in response to a high-cholesterol diet (HCD) and regulate both the differentiation and accumulation of T follicular helper (TFH) cells. Feeding mice an HCD resulted in upregulated MZB cell surface expression of the immunoregulatory ligand PDL1 in an ATF3-dependent manner and increased the interaction between MZB cells and pre-TFH cells, leading to PDL1-mediated suppression of TFH cell motility, alteration of TFH cell differentiation, reduced TFH abundance and suppression of the proatherogenic TFH response. Our findings reveal a previously unsuspected role for MZB cells in controlling the TFH–germinal center response to a cholesterol-rich diet and uncover a PDL1-dependent mechanism through which MZB cells use their innate immune properties to limit an exaggerated adaptive immune response.

Type-2 innate lymphoid cells control the development of atherosclerosis in mice.

Type-2 innate lymphoid cells (ILC2) are a prominent source of type II cytokines and are found constitutively at mucosal surfaces and in visceral adipose tissue. Despite their role in limiting obesity, how ILC2s respond to high fat feeding is poorly understood, and their direct influence on the development of atherosclerosis has not been explored. Here, we show that ILC2 are present in para-aortic adipose tissue and lymph nodes and display an inflammatory-like phenotype atypical of adipose resident ILC2. High fat feeding alters both the number of ILC2 and their type II cytokine production. Selective genetic ablation of ILC2 in Ldlr−/− mice accelerates the development of atherosclerosis, which is prevented by reconstitution with wild type but not Il5−/− or Il13−/− ILC2. We conclude that ILC2 represent a major innate cell source of IL-5 and IL-13 required for mounting atheroprotective immunity, which can be altered by high fat diet.

PD-L1 blockade overrides Salmonella typhimurium-mediated diabetes prevention in NOD mice: No role for Tregs

Increasingly, evidence suggests that there is a strong environmental component to the development of the autoimmune disease type 1 diabetes. Our previous data showed that NOD mice are protected from developing diabetes after infection with Salmonella typhimurium and there is some evidence that changes within the DC compartment play a crucial role in this protective effect. This paper further characterises this Salmonella‐modulated protective phenotype. We find that, contrary to other infection‐mediated models of type 1 diabetes protection, there was no expansion of Foxp3+ Tregs. Furthermore, transcriptome analysis of DCs identified a distinct Salmonella‐induced signature in which the inhibitory receptor PD‐L1 was up‐regulated. This was confirmed by flow cytometry. In vivo blockade of the PD1/PD‐L1 interaction was found to ablate the protective function of Salmonella infection. These data provide evidence for a novel regulatory DC phenotype proficient at controlling autoreactive T cells for an extended duration in the NOD mouse model of diabetes.

Deletion of IRF8 (Interferon Regulatory Factor 8)-Dependent Dendritic Cells Abrogates Proatherogenic Adaptive Immunity.

Rationale: Despite an established role for adaptive immune responses in atherosclerosis, the contribution of dendritic cells (DCs) and their various subsets is still poorly understood. Objective: Here, we address the role of IRF8 (interferon regulatory factor 8)-dependent DCs (lymphoid CD8&agr;+ and their developmentally related nonlymphoid CD103+ DCs) in the induction of proatherogenic immune responses during high fat feeding. Methods and Results: Using a fate-mapping technique to track DCs originating from a DNGR1+ (dendritic cell natural killer lectin group receptor 1) precursor (Clec9a+/creRosa+/EYFP mice), we first show that YFPhiCD11chiMHCIIhi (major histocompatibility complex class II) DCs are present in the atherosclerotic aorta of low-density lipoprotein receptor–deficient (Ldlr−/−) mice and are CD11b–CD103+IRF8hi. Restricted deletion of IRF8 in DCs (Irf8flox/floxCd11cCre) reduces the accumulation of CD11chiMHCIIhi DCs in the aorta without affecting CD11b+CD103– DCs or macrophages but completely abolishes the accumulation of aortic CD11b–CD103+ DCs. Lymphoid CD8&agr;+ DCs are also deleted. This is associated with a significant reduction of aortic T-cell accumulation and a marked reduction of high-fat diet–induced systemic T-cell priming, activation, and differentiation toward T helper type 1 cells, T follicular helper cells, and regulatory T cells. As a consequence, B-cell activation and germinal center responses to high-fat diet are also markedly reduced. IRF8 deletion in DCs significantly reduces the development of atherosclerosis, predominantly in the aortic sinus, despite a modest increase in total plasma cholesterol levels. Conclusions: IRF8 expression in DCs plays a nonredundant role in the development of proatherogenic adaptive immunity.