Diego G. Silva

Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA

Immune responses are normally targeted against microbial pathogens and not self-antigens by mechanisms that are only partly understood. Here we define a newly discovered pathway that prevents autoimmunity by limiting the levels on T lymphocytes of a co-stimulatory receptor, the inducible T-cell co-stimulator (ICOS). In sanroque mice homozygous for an M199R mutation in the ROQ domain of Roquin (also known as Rc3h1), increased Icos expression on T cells causes the accumulation of lymphocytes that is associated with a lupus-like autoimmune syndrome. Roquin normally limits Icos expression by promoting the degradation of Icos messenger RNA. A conserved segment in the unusually long ICOS 3′ untranslated mRNA is essential for regulation by Roquin. This segment comprises a 47-base-pair minimal region complementary to T-cell-expressed microRNAs including miR-101, the repressive activity of which is disrupted by base-pair inversions predicted to abrogate miR-101 binding. These findings illuminate a critical post-transcriptional pathway within T cells that regulates lymphocyte accumulation and autoimmunity, and highlights the therapeutic potential of partially antagonising the ICOS pathway.

Interferon-γ Excess Leads to Pathogenic Accumulation of Follicular Helper T Cells and Germinal Centers

Overactivity of the germinal center (GC) pathway resulting from accumulation of follicular helper T (Tfh) cells causes autoimmunity, underscoring the need to understand the factors that control Tfh cell homeostasis. Here we have identifed posttranscriptional repression of interferon-γ (Ifng) mRNA as a mechanism to limit Tfh cell formation. By using the sanroque lupus model, we have shown that decreased Ifng mRNA decay caused excessive IFN-γ signaling in T cells and led to accumulation of Tfh cells, spontaneous GC, autoantibody formation, and nephritis. Unlike ICOS and T-bet deficiency that failed to rescue several autoimmune manifestations, interferon-γ receptor (IFN-γR) deficiency prevented lupus development. IFN-γ blockade reduced Tfh cells and autoantibodies, demonstrating that IFN-γ overproduction was required to sustain lupus-associated pathology. Increased IFN-γR signaling caused Bcl-6 overexpression in Tfh cells and their precursors. This link between IFN-γ and aberrant Tfh cell formation provides a rationale for IFN-γ blockade in lupus patients with an overactive Tfh cell-associated pathway.

Roquin differentiates the specialized functions of duplicated T cell costimulatory receptor genes CD28 and ICOS.

During evolutionary adaptation in the immune system, host defense is traded off against autoreactivity. Signals through the costimulatory receptor CD28 enable T cells to respond specifically to pathogens, whereas those through the related costimulatory receptor, ICOS, which arose by gene duplication, are critical for affinity maturation and memory antibody responses. ICOS ligand, unlike the pathogen-inducible CD28 ligands, is widely and constitutively expressed in the immune system. Here, we show that crosstalk between these two pathways provides a mechanism for obviating the normal T cell dependence on CD28. Several CD28-mediated responses-generation of follicular helper T cells, germinal center formation, T helper 1 cell-dependent extrafollicular antibody responses to Salmonella and bacterial clearance, and regulatory T cell homeostasis-became independent of CD28 and dependent on ICOS when the E3 ubiquitin ligase Roquin was mutated. Mechanisms to functionally compartmentalize ICOS and CD28 signals are thus critical for two-signal control of normal immune reactions.

Identification of T Cell-Restricted Genes, and Signatures for Different T Cell Responses, Using a Comprehensive Collection of Microarray Datasets

We used a comprehensive collection of Affymetrix microarray datasets to ascertain which genes or molecules distinguish the known major subsets of human T cells. Our strategy allowed us to identify the genes expressed in most T cell subsets: TCR αβ+ and γδ+, three effector subsets (Th1, Th2, and T follicular helper cells), T central memory, T effector memory, activated T cells, and others. Our genechip dataset also allowed for identification of genes preferentially or exclusively expressed by T cells, compared with numerous non-T cell leukocyte subsets profiled. Cross-comparisons between microarray datasets revealed important features of certain subsets. For instance, blood γδ T cells expressed no unique gene transcripts, but did differ from αβ T cells in numerous genes that were down-regulated. Hierarchical clustering of all the genes differentially expressed between T cell subsets enabled the identification of precise signatures. Moreover, the different T cell subsets could be distinguished at the level of gene expression by a smaller subset of predictor genes, most of which have not previously been associated directly with any of the individual subsets. T cell activation had the greatest influence on gene regulation, whereas central and effector memory T cells displayed surprisingly similar gene expression profiles. Knowledge of the patterns of gene expression that underlie fundamental T cell activities, such as activation, various effector functions, and immunological memory, provide the basis for a better understanding of T cells and their role in immune defense.

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell‐mediated (Th1) immunity (e.g. Freunds complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non‐toxic adjuvant that is able to stimulate both cell‐mediated and humoral immunity. Inulin‐derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf‐life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin‐based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin‐derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.

Macrophage migration inhibitory factor exhibits a pronounced circadian rhythm relevant to its role as a glucocorticoid counter-regulator

In humans, maximal expression of T helper 1 cytokines coincide with the nocturnal nadir of plasma cortisol, whereas T helper 2 cytokine responses are dominant during day‐time. The pro‐inflammatory cytokine, macrophage migration inhibitory factor counter‐regulates glucocorticoid‐mediated immune suppression. To determine the relationship between cortisol and macrophage migration inhibitory factor, healthy volunteers had blood drawn hourly for 24 h for measurement of plasma cortisol and basal‐ and stimulated‐macrophage migration inhibitory factor. Similar to cortisol, macrophage migration inhibitory factor peaked during the late morning whereas interferon‐γ, tumour necrosis factor‐α, interleukin‐1 and interleukin‐12 demonstrated a nocturnal peak. After oral cortisone, plasma macrophage migration inhibitory factor rose 2–4‐fold, whereas the other cytokines decreased. There was no correlation between cortisol during the insulin tolerance test and plasma macrophage migration inhibitory factor. The late morning peak of macrophage migration inhibitory factor, by antagonizing cortisol‐mediated pro‐inflammatory cytokine suppression may prolong the duration of early morning inflammation. These observations explain the beneficial role of macrophage migration inhibitory factor neutralization in models of inflammatory arthritis.

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.

Anti-islet autoantibodies trigger autoimmune diabetes in the presence of an increased frequency of islet-reactive CD4 T cells

OBJECTIVE To define cellular mechanisms by which B cells promote type 1 diabetes. RESEARCH DESIGN AND METHODS The study measured islet-specific CD4 T cell regulation in T-cell receptor transgenic mice with elevated frequencies of CD4 T cells recognizing hen egg lysozyme (HEL) autoantigen expressed in islet β-cells and thymic epithelium under control of the insulin-gene promoter. The effects of a mutation in Roquin that dysregulates T follicular helper (Tfh) cells to promote B-cell activation and anti-islet autoantibodies were studied, as were the effects of HEL antigen–presenting B cells and passively transferred or maternally transmitted anti-islet HEL antibodies. RESULTS Mouse anti-islet IgG antibodies—either formed as a consequence of excessive Tfh activity, maternally transmitted, or passively transferred—caused a breakdown of tolerance in islet-reactive CD4+ cells and fast progression to diabetes. Progression to diabetes was ameliorated in the absence of B cells or when the B cells could not secrete islet-specific IgG. Anti-islet antibodies increased the survival of proliferating islet-reactive CD4+ T cells. FcγR blockade delayed and reduced the incidence of autoimmune diabetes. CONCLUSIONS B cells can promote type 1 diabetes by secreting anti-islet autoantibodies that act in an FcγR-mediated manner to enhance the expansion of islet-reactive CD4 T cells and cooperate with inherited defects in thymic and peripheral CD4 T–cell tolerance. Cooperation between inherited variants affecting CD4 T–cell tolerance and anti-islet autoantibodies should be examined in epidemiological studies and in studies examining the efficacy of B-cell depletion.

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell‐dependent (TD) antibody responses yields memory cells and long‐lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self‐antigens. GC also form in T‐independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T‐dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4α. This is consistent with formation of long neurite (axon and dendrite)‐like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD‐GC B cells including Basp1, Shh, Sult4α, Sc4mol are also preferentially expressed in post‐GC B cell neoplasms.

The Role of Endoplasmic Reticulum Stress in Nonimmune Diabetes: NOD.k iHEL, a Novel Model of β Cell Death

Abstract: The final common pathway in diabetes development is β cell apoptosis. We herein describe a novel diabetes model based on transgenic NOD.k iHEL mice, wherein male mice develop diabetes due to nonimmune‐mediated β cell death. Histology and electron microscopy confirm endoplasmic reticulum (ER) abnormalities that are consistent with endoplasmic stress caused by the HEL transgene. The NOD.k iHEL model may be particularly useful for studying mechanisms of β cell death secondary to ER stress and also for testing potential therapies designed to protect β cells from stress‐induced apoptosis. The observation that only male NOD.k iHEL mice develop diabetes and exhibit ER abnormalities is intriguing and suggests these mice may be useful in deciphering the link between hyperandrogenism, insulin resistance, and diabetes.