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The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

Monogenic diseases can often manifest diverse clinical phenotypes and cause diagnostic dilemmas. While monoallelic loss-of-function variants in TNFAIP3 (Haploinsufficiency of A20; HA20) cause a highly penetrant autoinflammatory disease, the variable expressivity suggest a role for additional genetic and environmental disease modifiers. Here, we identify critically ill children who inherited a family-specific TNFAIP3 deletion from one of their otherwise healthy parents. Each of the probands also inherited in trans a subtle loss-of-function I207L TNFAIP3 variant that is common in Oceania, originally introgressed from Denisovans. Modelling this compound heterozgous state in mice under specific pathogen free conditions demonstrated a reduced threshold to break immune tolerance. Exaggerated immune responses were precipitated by inheriting the two genetic hits on the TNFAIP3 checkpoint coupled with increasing the microbial challenge to immune tolerance, either by co-housing with pet store mice carrying a wild microbial burden or by transient dietary exposure to a chemical that diminishes the intestinal mucin barrier separating gut microbes from immune sensing systems. These data illuminate second-hit genetic and environmental modifiers contributing to complex inflammatory and autoimmune disease. Increased mechanistic understanding of the presence and contribution of disease modifiers will aid diagnostic and prognostic patient stratification and potentially reveal novel therapeutic opportunities.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Injection of Interleukin-2 (IL-2) complexed with a particular anti–IL-2 monoclonal antibody (mab) JES6-1 has been shown to selectively expand CD4(+)Foxp3(+) T regulatory T cells (Tregs) in vivo. Although the potency of this approach with regard to transplantation has already been proven in an islet transplantation model, skin graft survival could not be prolonged. Since the latter is relevant to human allograft survival, we sought to improve the efficiency of IL-2 complex (cplx) treatment for skin allograft survival in a stringent murine skin graft model. Here, we show that combining low doses of IL-2 cplxs with rapamycin and blockade of the inflammatory cytokine IL-6 leads to long-term (>75 d) survival of major histocompatibility complex-different skin allografts without the need for immunosuppression. Allograft survival was critically dependent on CD25(+)FoxP3(+) Tregs and was not accompanied by impaired responsiveness toward donor alloantigens in vitro after IL-2 cplx treatment was stopped. Furthermore, second donor-type skin grafts were rejected and provoked rejection of the primary graft, suggesting that operational tolerance is not systemic but restricted to the graft. These findings plus the lack of donor-specific antibody formation imply that prolonged graft survival was largely a reflection of immunological ignorance. The results may represent a potentially clinically translatable strategy for the development of protocols for tolerance induction.

Chlamydia trachomatis (CT) is a common sexually transmitted bacterial infection, that in women can cause pelvic inflammatory disease and infertility. No preventative vaccine has been developed against CT. Immunity to CT is primarily mediated by Th1 CD4+ T cells. We are defining immunoprevalent CT proteins in a well-defined cohort of CT seropositive women with the goal of defining novel vaccine immunogens. We screened 30 women one month after a CT-positive test by cultured IFN-γ ELISpot. Ten-day short-term cell lines (STCL) were generated against overlapping peptides spanning 21 CT antigens. The threshold for a positive CT-specific T cell response (≥ 300 spot-forming cells, SFU, per 106 cells) was defined following a screening of 12 CT seronegative donors. CT− specific T cell responses were detected in 27/30 CT-seropositive women. On average, women harbored T cell responses to two CT proteins (range 0–6). Strikingly, CT858 (CPAF) elicited a T cell response in 16/30 women with an average of 966 IFN-g SFU/106 cells (range 300–3,633 SFU/106 cells). Data to date suggest CPAF-specific T cell responses are predominantly CD4-restricted. In preliminary studies, we have also detected CT-specific T cell responses in men with documented CT infection, including a T cell response to CPAF. We are currently mapping CPAF T cell epitopes and expanding our screen to other CT secreted proteins. In summary, CT858 (CPAF) is an immunoprevalent antigen in women and a promising vaccine immunogen. Supported by UNC Chlamydia Vaccine Initiative (U19AI144181)

CD8+ T cell immunity is essential to the control of HIV viremia. We examined the safety and immunogenicity of MVA-vectored vaccines expressing highly conserved HIV regions in a first-in-man Phase I study in people living with HIV on ART. Participants received a single intramuscular dose of MVA.tHIVconsv3 (M3), MVA.tHIVconsv4 (M4), combined M3+M4 or saline in a 7:7:7:3 ratio. M3 and M4 span the same 6 HIV regions but differ by approximately 10% of amino acids; a design to increase vaccine coverage of circulating HIV variants. We employed ex vivo IFN-g ELISpot assays to measure changes in HIV-specific T cell magnitude and breadth to M3 and/or M4 immunogens following vaccination. We also examined whether M3, M4 or M3+M4 vaccination increased the ability of CD8+ T cells inhibit HIV in vitro replication. The M&M study is fully enrolled but presently is blinded. Analysis of blinded data show that vaccination was safe and well tolerated. Vaccination induced strong increases in the T cell response to M3, M4 vaccine immunogens producing a 2- to 18-fold increase in magnitude in 16/20 participants tested to date. M3/M4-specific T cell breadth also increased across participants. Vaccine-associated T cell responses mostly remained elevated (>2-fold increase) for at least 70 days post-vaccination visit. Vaccination was also associated with clear and sustained increases in in vitro virus inhibition. The percentage of the total HIV T cell response targeting conserved HIV regions in participants increased on average from 40 to 60% post-vaccination, suggesting M3/M4/M3+4 vaccination successfully produced a sustained shift in T cell immunodominance. Unblinded data will be presented at this meeting. Supported by U01AI131310

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.

NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role NF-κB plays in normal metabolism. Here, to define this role in the context of beta cell function and metabolic control, we deleted the canonical NF-κB transcription factor RelA/p65 specifically in pancreatic beta cells to generate βP65KO mice. RelA deficiency resulted in complete loss of stimulus dependent inflammatory gene upregulation, consistent with its known role to govern inflammation. However, RelA deletion also rendered mice glucose intolerant due to a functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as βP65KO islets failed to secrete insulin ex vivo in response to a glucose challenge, and were unable to restore metabolic control when transplanted into secondary diabetic recipients. Maintenance of glucose tolerance required RelA, but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knock-out of the essential activator of NF-κB, NEMO, or beta cell overexpression of the negative NF-κB regulator, A20, did not cause severe glucose intolerance. Thus, basal RelA activity performs an essential and islet-intrinsic role to maintain normal glucose homeostasis. Genome wide bioinformatic mapping revealed the presence of RelA binding sites in the promoter regions of specific metabolic genes, and in the majority (~70%) of islet enhancer hubs that are responsible for shaping beta cell type-specific gene expression programmes. Indeed, islet specific metabolic genes Slc2a2 and Capn9, identified within the large network of islet enhancer hub genes showed dysregulated expression in βP65KO islets. These data demonstrate an unappreciated role for RelA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism.

IL-17–producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine. We found γδT-17 cells to be enriched, not depleted, in IL-2–deficient mice. The absence of IL-2 also resulted in higher IL-17 production and the emergence of IL-17+IFN-γ+ double producers. Furthermore, the addition of IL-2 to in vitro cultures of sorted γδT-17 cells was able to moderate IL-17 and affect differentiation into polyfunctional cytokine-producing cells. Interestingly, the Vγ6+ subset was more susceptible to the effects of IL-2 than Vγ4+ γδT-17 cells. We also found that unlike other γδ T cells, γδT-17 cells do not produce IL-2, but express Blimp-1, a known transcriptional repressor of IL-2. Although IL-2 was able to induce robust proliferation of γδT-17 cells, it did not sustain viability, negatively impacting their survival via downregulation of the IL-7R. Taken together, these data indicate that IL-2 can augment the γδT-17 response in favor of short-lived effectors with limited plasticity, particularly in the presence of IL-1β and IL-23. In this way, IL-2 may act to curtail the innate-like response of γδT-17 cells upon arrival of IL-2–producing adaptive immune cells at the site of inflammation.

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded byTNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish IκB-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.One Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

CD4+ T helper (Th) cells that express the gut homing chemokine receptor CCR9 are increased in the peripheral blood of patients with inflammatory bowel disease and Sjögren's syndrome and in the inflamed lesions of autoimmune diseases that affect the accessory organs of the digestive system. However, despite the important role of the GIT in both immunity and autoimmunity, the nature of CCR9-expressing cells in GIT lymphoid organs and their role in chronic inflammatory diseases remains unknown. In this study, we analyzed the characteristics of CCR9+ Th and T follicular helper (Tfh) cells in GIT associated lymphoid tissues in health, chronic inflammation and autoimmunity. Our findings reveal an association between the transcriptome and phenotype of CCR9+ Th in the pancreas and CCR9+ Tfh cells from GIT-associated lymphoid tissues. GIT CCR9+ Tfh cells exhibited characteristics, including a Th17-like transcriptome and production of effector cytokines, which indicated a microenvironment-specific signature. Both CCR9+ Tfh cells and CCR9+ Th cells from GIT-associated lymphoid tissues migrated to the pancreas. The expression of CCR9 was important for migration of both subsets to the pancreas, but Tfh cells that accumulated in the pancreas had downmodulated expression of CXCR5. Taken together, the findings provide evidence that CCR9+ Tfh cells and Th cells from the GIT exhibit plasticity and can accumulate in distal accessory organs of the digestive system where they may participate in autoimmunity.

This study investigated the mechanistic basis behind the reported superior efficacy of high-avidity CD8 T cells. CMV and HIV are both chronic viral infections, but while CMV-specific CD8 T cells can mediate lifelong viral control, in untreated HIV infection HIV-specific CD8 T cells progressively lose function. Using in vitro studies of human cells, we show that avidity-dependent downregulation of the CD8 co-receptor directly programs metabolism, due to a novel association between CD8 and the glucose transporter GLUT1. We used flow cytometry to profile ex vivo and virus-specific CD8 T cells from HIV-infected individuals on antiretroviral therapy. Ex vivo, cells expressing low levels of CD8 (CD8dim) expressed more CD69 but less cell surface GLUT1, and took up less glucose (2-NBDG) than CD8bright T cells. Following antigen stimulation, CD3, CD8, and GLUT1 were downregulated from the cell surface in an avidity-dependent manner. CMV-specific CD8 T cells, which were of higher avidity, downregulated these proteins to a greater extent than lower-avidity HIV-specific CD8 T cells. GLUT1 downregulation strongly correlated with CD8 but not CD3 downregulation. Antibody-mediated downregulation of CD8 from the cell surface resulted in reduced glucose uptake and increased fatty acid (Bodipy) uptake, independent of CD3. Finally, CD3, CD8, and GLUT1 downregulation by HIV-specific CD8 T cells was impaired following viral escape mutations that reduced CD8 T cell avidity. We confirmed this finding in a transduction setting with a single clonal TCR. Our data reveal a novel function of the CD8 co-receptor, linking the avidity and metabolism of CD8 T cells.

This study investigated the function of memory CD8 T cells in HIV-infected people durably suppressed on antiretroviral therapy (HIV+ cART). We assessed bulk and virus-specific memory CD8 T cells in HIV+ cART and HIV-seronegative individuals (HIV−) by flow cytometry. We observed a population of CD3+ CD8dim CD14− CD16− (CD8dim) T cells that was expanded as a percentage of total CD8 T cells in both HIV− and CMV-seropositive individuals. Bulk memory CD8dim T cells expressed significantly higher CD69 and less MHC Class I and CD127 ex vivo than CD8bright T cells, suggesting recent activation. CD8dim T cells expressed less GLUT1 and PGC-1α and took up less glucose (2-NBDG) and lipid (Bodipy) than CD8bright T cells, indicating relatively lower metabolic activity. Multimer reactivity was used to examine CMV-, EBV- and HIV-specific CD8 T cells ex vivo. Virus-specific populations were consistently CD8high. However, after peptide stimulation, antigen-specific CD8 T cells down-regulated CD3 and CD8. CMV-specific CD8 T cells down-regulated CD3 and CD8 more than HIV-specific cells. CD3 and CD8 downregulation were strongly correlated with the functional avidity of the response. A strong correlation between GLUT1 down-regulation and CD8 down-regulation was also observed, suggesting an association between CD8 expression and metabolic activity. These results suggest that the expanded CD8dim population in HIV+ cART individuals, who are >90% CMV-seropositive, may be driven by ongoing activation of high-avidity CMV-specific CD8 T cells. They also suggest that different virus-specific CD8 T cell populations differentially downregulate components of the TCR complex and metabolism after antigen stimulation, possibly to avoid excessive activation.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Summary: The archetypal T cell-dependent antigen is sheep red blood cells (SRBCs), which have defined much of what we know about humoral immunity. Early studies using solubilized or sonicated SRBCs argued that the intact structure of SRBCs was important for optimal antibody responses. However, the reason for the requirement of intact SRBCs for the response to polyvalent protein antigen remained unknown. Here, we report that the immune response to SRBCs is driven by cytosolic recognition of SRBC RNA through the RIG-I-like receptor (RLR)-mitochondrial anti-viral signaling adaptor (MAVS) pathway. Following the uptake of SRBCs by antigen-presenting cells, the MAVS signaling complex governs the differentiation of both T follicular cells and antibody-producing B cells. Importantly, the involvement of the RLR-MAVS pathway precedes that of endosomal Toll-like receptor pathways, yet both are required for optimal effect. : Immunization with sheep red blood cells (SRBCs) has been used for almost a century to study antibody generation. Loetsch et al. now show that, after engulfment, SRBC RNA accesses antigen-presenting cell RNA-sensing pathways to stimulate the immune system.

T follicular regulatory (Tfr) cells control the magnitude and specificity of the germinal centre reaction, but how regulation is contained to ensure generation of high-affinity antibody is unknown. Here we show that this balance is maintained by the reciprocal influence of interleukin (IL)-2 and IL-21. The number of IL-2-dependent FoxP3+ regulatory T cells is increased in the peripheral blood of human patients with loss-of-function mutations in the IL-21 receptor (IL-21R). In mice, IL-21:IL-21R interactions influence the phenotype of T follicular cells, reducing the expression of CXCR4 and inhibiting the expansion of Tfr cells after T-cell-dependent immunization. The negative effect of IL-21 on Tfr cells in mice is cell intrinsic and associated with decreased expression of the high affinity IL-2 receptor (CD25). Bcl-6, expressed in abundance in Tfr cells, inhibits CD25 expression and IL-21-mediated inhibition of CD25 is Bcl-6 dependent. These findings identify a mechanism by which IL-21 reinforces humoral immunity by restricting Tfr cell proliferation., IL-21 is central to follicular helper T cell function and germinal centre responses. Here the authors show that IL-21 signalling directly inhibits T follicular regulatory cells by limiting Bcl-6-dependent IL-2 receptor expression.

SUMMARY The development of autoimmune disease type 1 diabetes (T1D) is determined by both genetic background and environmental factors. Environmental triggers include RNA viruses, particularly coxsackie-virus (CV), but how they induceT1D is not understood. Here, we demonstrate that deletion of the transcription factor hypoxia-inducible factor-1α (HIF-1α) from β cells increases the susceptibility of non-obese diabetic (NOD) mice to environmentally triggered T1D from coxsackieviruses and the β cell toxin streptozotocin. Similarly, knockdown of HIF-1α in human islets leads to a poorer response to coxsackievirus infection. Studies in coxsackievirus-infected islets demonstrate that lack of HIF-1α leads to impaired viral clearance, increased viral load, inflammation, pancreatitis, and loss of β cell mass. These findings show an important role for β cells and, specifically, lack of β cell HIF-1α in the development of T1D. These data suggest new strategies for the prevention of T1D., Graphical Abstract, In Brief Lalwani et al. describe a role for β cell hypoxia-inducible factor-1α (HIF1a) in determining whether β cell injury is followed by resolution and normal function or ongoing injury, autoimmunity, and type 1 diabetes.

Micro-RNAs (miRNAs) are frequently dysregulated in a range of human malignancies, many have been shown to act either as tumour supressors or oncogenes and several have been implicated in breast cancer. However, breast cancer is a diverse disease and little is known about the relationships between miRNA expression, clinical outcome and tumour subtype. We used locked nucleic acid probe in situ hybridization (LNA-ISH) to visualize, in tissue micro-arrays (TMAs) of 2919 formalin-fixed paraffin-embedded (FFPE) archival breast tumours, the expression of two key miRNAs that are frequently lost in a range of solid malignancies, let-7b and miR-205. These miRNAs were also quantified by quantitative reverse transcription PCR in cores of FFPE tissue from 40 of these cases, demonstrating that LNA-ISH is semi-quantitative. The tumours in the TMAs were assigned to subtypes based on their immunohistochemical (IHC) staining with ER, PR, HER2, CK5/6 and EGFR. let-7b expression was shown to be associated with luminal tumours and to have an independent significant positive prognostic value in this group. miR-205 is associated with tumours of ductal morphology and is of significant positive prognostic value within these tumours. We propose that the expression of miR-205 may contribute to ductal tumour morphology. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The selection of affinity-matured Ab-producing B cells is supported by interactions with T follicular helper (Tfh) cells. In addition to cell surface–expressed molecules, cytokines produced by Tfh cells, such as IL-21 and IL-4, provide B cell helper signals. In this study, we analyze how the fitness of Th cells can influence Ab responses. To do this, we used a model in which IL-21R–sufficient (wild-type [WT]) and –deficient (Il21r−/−) Ag-specific Tfh cells were used to help immunodeficient Il21r−/− B cells following T-dependent immunization. Il21r−/− B cells that had received help from WT Tfh cells, but not from Il21r−/− Tfh cells, generated affinity-matured Ab upon recall immunization. This effect was dependent on IL-4 produced in the primary response and associated with an increased fraction of memory B cells. Il21r−/− Tfh cells were distinguished from WT Tfh cells by a decreased frequency, reduced conjugate formation with B cells, increased expression of programmed cell death 1, and reduced production of IL-4. IL-21 also influenced responsiveness to IL-4 because expression of both membrane IL-4R and the IL-4–neutralizing soluble (s)IL-4R were reduced in Il21r−/− mice. Furthermore, the concentration of sIL-4R was found to correlate inversely with the amount of IgE in sera, such that the highest IgE levels were observed in Il21r−/− mice with the least sIL-4R. Taken together, these findings underscore the important collaboration between IL-4 and IL-21 in shaping T-dependent Ab responses.

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.

The alternative pathway of complement is activated continuously in vivo through the C3 'tick-over' pathway. This pathway is triggered by the hydrolysis of C3, resulting in the formation of C3 convertase. This, in turn, generates C3b, which mediates many of the biological functions of complement. Factor H, the main regulator of this activation, prevents formation and promotes dissociation of the C3 convertase enzyme, and, together with factor I, mediates the proteolytic inactivation of C3b. Factor H deficiency, described in 29 individuals from 12 families and in pigs, allows unhindered activation of fluid-phase C3 and severe depletion of plasma C3 (ref. 11). Membranoproliferative glomerulonephritis (MPGN) occurs in factor H-deficient humans and pigs. Although MPGN has been reported in other conditions in which uncontrolled activation of C3 occurs, the role of C3 dysregulation in the pathogenesis of MPGN is not understood. Here we show that mice deficient in factor H (Cfh(-/-) mice) develop MPGN spontaneously and are hypersensitive to developing renal injury caused by immune complexes. Introducing a second mutation in the gene encoding complement factor B, which prevents C3 turnover in vivo, obviates the phenotype of Cfh(-/-) mice. Thus, uncontrolled C3 activation in vivo is essential for the development of MPGN associated with deficiency of factor H.

Gene-targeted C1q-deficient mice have been shown to develop a syndrome reminiscent of human systemic lupus erythematosus with antinuclear Abs and proliferative glomerulonephritis. Initial phenotypic analysis conducted in (129 × C57BL/6) hybrid mice showed that background genes were a significant factor for the full expression of the autoimmune disease. To assess the contribution of background genes in the expression of the autoimmune phenotype, the disrupted C1qa gene was backcrossed for seven generations onto C57BL/6 and MRL/Mp+/+ strains. These were intercrossed with C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains to generate C1q-deficient substrains. In C1q-deficient C57BL/6 mice, no evidence of an autoimmune phenotype was found, and C1q deficiency in both the C57BL/6.lpr/lpr and MRL/Mp-lpr/lpr strains did not modify the autoimmune phenotype observed in wild-type controls. However, in C1q-deficient MRL/Mp+/+ animals an acceleration of both the onset and the severity of antinuclear Abs and glomerulonephritis was seen. Disease was particularly pronounced in females, which developed severe crescentic glomerulonephritis accompanied by heavy proteinuria. In addition, the C1q-deficient MRL/Mp+/+ mice had an impairment in the phagocytic clearance of apoptotic cells in vivo. These data demonstrate that the expression of autoimmunity in C1q-deficient mice is strongly influenced by other background genes. The work also highlights the potential value of the C1q-deficient MRL/Mp+/+ strain as a tool with which to dissect further the underlying mechanisms of the autoimmune syndrome associated with C1q deficiency.

The role of the complement system in host defense againstSalmonellainfection is poorly defined. Bacterial cell wall O-antigen polysaccharide can activate the alternative pathway in vitro. No studies, however, have elucidated the role of the classical pathway in immunity toSalmonellaspp. in vivo. C1q-deficient mice (C1qa−/−) on a 129/Sv genetic background and strain-matched controls were infected intraperitoneally and intravenously withSalmonella entericaserovar Typhimurium and monitored over a 14-day period. After inoculation by either route, theC1qa−/−mice were found to be significantly more susceptible toSalmonellainfection. Hepatic and splenic bacterial counts, performed at various time points, showed increased numbers of colonies in complement-deficient mice compared to controls. Analysis of blood clearance showed no difference between the two experimental groups during the first 15 min. However, after 20 min and until 6 h postinfection, numbers of circulating bacteria were significantly higher in complement-deficient mice. In vitro experiments using either resident or thioglycolate-elicited peritoneal macrophages showed a significant increase in the number of bacteria inside C1q-deficient macrophages compared to controls irrespective of the serum used for opsonizing the bacteria. These findings could not be explained either by an increased bacterial uptake, analyzed in vitro and in vivo using green fluorescent protein-tagged salmonellae, or by a defect in the respiratory burst or in NO production. The data presented here suggest the possibility of novel pathways by which C1q may modulate the pathogenesis of infectious diseases caused by intracellular pathogens.

Untreated HIV infection is characterized by generalized immune activation, T cell dysfunction, and ultimately the exhaustion of HIV-specific CD8 T cell responses. Durable virus suppression resulting from anti-retroviral therapy (ART) is associated with greatly improved immune function, but some phenotypic and functional abnormalities persist even after years of suppressive ART. We have observed that, relative to HIV-seronegative individuals, CD8 T cells from HIV+ participants on ART are skewed toward a more effector-like (T-bethigh Eomeslow) phenotype and have significantly reduced proliferative capacity in response to antigenic stimulation. Metabolic pathways play a key role in shaping T cell maturation, phenotype, and function, and the ability to produce ATP via both glycolytic and oxidative phosphorylation (mitochondrial) pathways is critical to T cell function and longevity. However, whether underlying metabolic abnormalities contribute to residual CD8 T cell dysfunction in HIV+ individuals on ART has not been fully explored. Here we compare the metabolic phenotype of T cells from HIV+ participants on ART with those from HIV-seronegative participants. We are using MitoTracker® dyes to assess mitochondrial mass and polarization in T cell memory subsets, ex vivo and in response to antigen, and measuring expression of the glucose transporter GLUT1 and the mitochondrial biogenesis master regulator PGC-1α in different CD8 T cell effector subsets. Our findings will inform whether phenotypic and functional abnormalities in CD8 T cells from HIV+ individuals on ART reflect underlying metabolic dysfunction and if metabolic interventions could improve T cell function.

Micro-RNAs (miRNAs) are frequently dysregulated in a range of human malignancies, many have been shown to act either as tumour supressors or oncogenes and several have been implicated in breast cancer. However, breast cancer is a diverse disease and little is known about the relationships between miRNA expression, clinical outcome and tumour subtype. We used locked nucleic acid probe in situ hybridization (LNA-ISH) to visualize, in tissue micro-arrays (TMAs) of 2919 formalin-fixed paraffin-embedded (FFPE) archival breast tumours, the expression of two key miRNAs that are frequently lost in a range of solid malignancies, let-7b and miR-205. These miRNAs were also quantified by quantitative reverse transcription PCR in cores of FFPE tissue from 40 of these cases, demonstrating that LNA-ISH is semi-quantitative. The tumours in the TMAs were assigned to subtypes based on their immunohistochemical (IHC) staining with ER, PR, HER2, CK5/6 and EGFR. let-7b expression was shown to be associated with luminal tumours and to have an independent significant positive prognostic value in this group. miR-205 is associated with tumours of ductal morphology and is of significant positive prognostic value within these tumours. We propose that the expression of miR-205 may contribute to ductal tumour morphology.

Membranoproliferative glomerulonephritis (MPGN) type II (dense deposit disease) is an inflammatory renal disease characterized by electron-dense deposits and complement C3 on the glomerular basement membrane. There is no effective therapy. We investigated the role of C5 activation in a model of MPGN that develops spontaneously in complement factor H-deficient mice (Cfh−/−). At 12 months there was a significant reduction in mortality, glomerular cellularity, neutrophil numbers, and serum creatinine levels inCfh−/−mice deficient in C5. Excessive glomerular neutrophil numbers, frequently seen in patients with MPGN during disease flares, were also observed inCfh−/−mice after the administration of an antiglomerular basement membrane antibody. This exaggerated injurious phenotype was absent inCfh−/−mice deficient in C5 but not inCfh−/−mice deficient in C6, indicating a key role for C5 activation in the induction of renal lesions. Importantly, the renal injury was completely reversed inCfh−/−mice pretreated with an anti-murine C5 antibody. These results demonstrate an important role for C5 in both spontaneous MPGN and experimentally induced nephritis in factor H-deficient mice and provide preliminary evidence that C5 inhibition therapy might be useful in human MPGN type II.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.

CD59 is a complement regulatory protein that inhibits the terminal part of the complement system, the membrane attack complex (MAC), a mediator of renal injury. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in experimentally induced accelerated nephrotoxic nephritis, a model of immune complex-mediated glomerulonephritis. After accelerated nephrotoxic nephritis was induced by administration of sheep nephrotoxic globulin, mCd59a-/- mice and strain-matched controls on two genetic backgrounds, 129/Sv x C57BL/6 and 129/Sv, were examined. For both, mCd59a-/- mice developed significantly greater glomerular cellularity than wild-type (WT) mice at day 5 after administration. At day 10 post-administration, mCd59a-/- mice exhibited more glomerular thrombosis than WT mice (thrombosis score, 1.8 [range, 1.4 to 4.0] versus 0.8 [range, 0.2 to 1.5] quadrants thrombosed per glomerulus, respectively; P = 0.0006). In the majority of experiments, mCd59a-/- mice also had significantly more proteinuria than controls; however, there was no difference in serum creatinine or albumin. Quantitative immunofluorescence of kidney sections revealed significantly more C9 (as a marker of MAC) deposition within glomeruli of mCd59a-/- mice than WT controls (P < 0.001). There was no difference in deposition of C3 and sheep IgG between the two experimental groups. The lack of CD59a, by allowing unregulated MAC deposition, exacerbates the renal injury in this model of immune complex-mediated glomerulonephritis.