Constitutional mutations of the WT1 gene, encoding a zinc-finger transcription factor involved in renal and gonadal development, are found in most patients with Denys-Drash syndrome (DDS), or diffuse mesangial sclerosis (DMS) associated with pseudohermaphroditism and/or Wilms tumor (WT). Most mutations in DDS patients lie in exon 8 or exon 9, encoding zinc finger 2 or zinc finger 3, respectively, with a hot spot (R394W) in exon 9. We analyzed a series of 24 patients, 10 with isolated DMS (IDMS), 10 with DDS, and 4 with urogenital abnormalities and/or WT. We report WT1 heterozygous mutations in 16 patients, 4 of whom presented with IDMS. One male and two female IDMS patients with WT1 mutations underwent normal puberty. Two mutations associated with IDMS are different from those described in DDS patients. No WT1 mutations were detected in the six other IDMS patients, suggesting genetic heterogeneity of this disease. We analyzed genotype/phenotype correlations, on the basis of the constitution of a WT1 mutation database of 84 germ-line mutations, to compare the distribution and type of mutations, according to the different symptoms. This demonstrated (1) the association between mutations in exons 8 and 9 and DMS; (2) among patients with DMS, a higher frequency of exon 8 mutations among 46, XY patients with female phenotype than among 46,XY patients with sexual ambiguity or male phenotype; and (3) statistically significant evidence that mutations in exons 8 and 9 preferentially affect amino acids with different functions.

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OBJECTIVEBoth the early steps and the high recurrence of autoimmunity once the disease is established are unexplained in human type 1 diabetes. Because CD8+ T cells are central and insulin is a key autoantigen in the disease process, our objective was to characterize HLA class I–restricted autoreactive CD8+ T cells specific for preproinsulin (PPI) in recent-onset and long-standing type 1 diabetic patients and healthy control subjects.RESEARCH DESIGN AND METHODSWe used HLA-A*02:01 tetramers complexed to PPI peptides to enumerate circulating PPI-specific CD8+ T cells in patients and characterize them using membrane markers and single-cell PCR.RESULTSMost autoreactive CD8+ T cells detected in recent-onset type 1 diabetic patients are specific for leader sequence peptides, notably PPI6–14, whereas CD8+ T cells in long-standing patients recognize the B-chain peptide PPI33–42 (B9–18). Both CD8+ T-cell specificities are predominantly naïve, central, and effector memory cells, and their gene expression profile differs from cytomegalovirus-specific CD8+ T cells. PPI6–14–specific CD8+ T cells detected in one healthy control displayed Il-10 mRNA expression, which was not observed in diabetic patients.CONCLUSIONSPPI-specific CD8+ T cells in type 1 diabetic patients include central memory and target different epitopes in new-onset versus long-standing disease. Our data support the hypothesis that insulin therapy may contribute to the expansion of autoreactive CD8+ T cells in the long term.

OBJECTIVE-A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8 ϩ T-cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence.RESEARCH DESIGN AND METHODS-Seven 8-to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24, and -B8 were selected from databases. HLA-A2-restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/␤2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-␥ (IFN-␥) ELISpot assay. RESULTS-FiveHLA-A2-restricted peptides were immunogenic in transgenic mice. Murine T-cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-␥ in response to six preproinsulin peptides covering residues 2-25 within the preproinsulin region. In most patients, the response was against several class Irestricted peptides. T-cells recognizing preproinsulin peptide were characterized as CD8 ϩ T-cells by staining with peptide/ HLA-A2 tetramers.CONCLUSIONS-We defined class I-restricted epitopes located within the leader sequence of human preproinsulin through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8

The first signs of autoimmune activation leading to b-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPI B15-23 T-cell receptor-transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4 + regulatory T cells expressing transforming growth factor-b and in increased effector CD8 + T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions.Islet destruction by autoreactive T cells is the hallmark of type 1 diabetes (T1D). Intense research efforts are therefore ongoing to develop immunotherapies aimed at blunting islet autoimmunity. Antigen (Ag)-specific immunotherapies are particularly attractive due to their selectivity and safety (1) but have met with limited success. Several attempts have focused on tolerogenic vaccination with b-cell Ags derived from preproinsulin (PPI) (2), which is the target initiating the autoimmune cascade in nonobese diabetic (NOD) mice (3) and likely also in humans (2). A recent clinical trial employing intranasal insulin in slow-onset T1D patients did not result in C-peptide preservation, despite successful induction of insulin-specific immune tolerance (4). These results suggest that the timing of intervention may be too late and that the Ag spreading that follows early b-cell destruction leads to a diversification of autoimmune reactions beyond insulin, thus making tolerance restoration to this sole Ag insufficient. The same problem is encountered in prevention trials. Despite absence of clinical disease, selection of at-risk patients based on positivity for multiple autoantibodies (auto-Abs) underscores the presence of an autoimmune reaction that has already spread to several Ags (5). Prospective cohorts of genetically at-risk children further highlighted that b-cell autoimmunity initiates very early, possibly already during

Streptococcus pneumoniae is an important cause of acute otitis media (AOM). The aim of this study was to evaluate trends in antibiotic resistance and circulating serotypes of pneumococci isolated from middle ear fluid of French children with AOM during the period 2001-2011, before and after the introduction of the PCV-7 (2003) and PCV-13 (2010) vaccines. Between 2001 and 2011 the French pneumococcal surveillance network analysed the antibiotic susceptibility of 6683 S. pneumoniae isolated from children with AOM, of which 1569 were serotyped. We observed a significant overall increase in antibiotic susceptibility. Respective resistance (I+R) rates in 2001 and 2011 were 76.9% and 57.3% for penicillin, 43.0% and 29.8% for amoxicillin, and 28.6% and 13.0% for cefotaxime. We also found a marked reduction in vaccine serotypes after PCV-7 implementation, from 63.0% in 2001 to 13.2% in 2011, while the incidence of the additional six serotypes included in PCV-13 increased during the same period, with a particularly high proportion of 19A isolates. The proportion of some non-PCV-13 serotypes also increased between 2001 and 2011, especially 15A and 23A. Before PCV-7 implementation, most (70.8%) penicillin non-susceptible pneumococci belonged to PCV-7 serotypes, whereas in 2011, 56.8% of penicillin non-susceptible pneumococci belonged to serotype 19A. Between 2001 and 2011, antibiotic resistance among pneumococci responsible for AOM in France fell markedly, and PCV-7 serotypes were replaced by non-PCV-7 serotypes, especially 19A. We are continuing to assess the impact of PCV-13, introduced in France in 2010, on pneumococcal serotype circulation and antibiotic resistance.

Key requirements in type 1 diabetes (T1D) are in setting up new assays as diagnostic biomarkers that will apply to prediabetes, likely T-cell assays, and in designing antigen-specific therapies to prevent T1D development. New preclinical models of T1D will be required to help with advancing both aims. By crossing mouse strains that lack either murine MHC class I and class II genes and insulin genes, we developed YES mice that instead express human HLA-A*02:01, HLA-DQ8, and insulin genes as transgenes. The metabolic and immune phenotype of YES mice is basically identical to that of the parental strains. YES mice remain insulitis and diabetes free up to 1 year of follow-up, maintain normoglycemia to an intraperitoneal glucose challenge in the long-term range, have a normal β-cell mass, and show normal immune responses to conventional antigens. This new model has been designed to evaluate adaptive immune responses to human insulin on a genetic background that recapitulates a human high-susceptibility HLA-DQ8 genetic background. Although insulitis free, YES mice develop T1D when challenged with polyinosinic-polycytidylic acid. They allow the characterization of preproinsulin epitopes recognized by CD8 and CD4 T cells upon immunization against human preproinsulin or during diabetes development.

Immune system dysfunction is paramount in Coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in a cohort of 182 patients including patients at various stages of disease activity. A profound decrease of MAIT cell counts in blood of critically ill patients was observed. These cells showed a strongly activated and cytotoxic phenotype that positively correlated with circulating pro-inflammatory cytokines, notably IL-18. MAIT cell alterations markedly correlated with disease severity and patient mortality. SARS-CoV-2-infected macrophages activated MAIT cells in a cytokine-dependent manner involving an IFNα-dependent early phase and an IL-18-induced later phase. Therefore, altered MAIT cell phenotypes represent valuable biomarkers of disease severity and their therapeutic manipulation might prevent the inflammatory phase involved in COVID-19 aggravation.