IL-21 limits peripheral lymphocyte numbers through T cell homeostatic mechanisms.

BACKGROUND: IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts to limit the size of the T cell pool. Furthermore, our data suggest IL-21 may contribute to the development of autoimmunity. CONCLUSIONS/SIGNIFICANCE: Taken together, our results suggest that IL-21 plays a global role in regulating T cell homeostasis, promoting the continuous adaptation of the T cell lymphoid space

A Hypermorphic Missense Mutation in PLCG2, Encoding Phospholipase Cγ2, Causes a Dominantly Inherited Autoinflammatory Disease with Immunodeficiency

Whole-exome sequencing was performed in a family affected by dominantly inherited inflammatory disease characterized by recurrent blistering skin lesions, bronchiolitis, arthralgia, ocular inflammation, enterocolitis, absence of autoantibodies, and mild immunodeficiency. Exome data from three samples, including the affected father and daughter and unaffected mother, were filtered for the exclusion of reported variants, along with benign variants, as determined by PolyPhen-2. A total of eight transcripts were identified as possible candidate genes. We confirmed a variant, c.2120C>A (p.Ser707Tyr), within PLCG2 as the only de novo variant that was present in two affected family members and not present in four unaffected members. PLCG2 encodes phospholipase Cγ2 (PLCγ2), an enzyme with a critical regulatory role in various immune and inflammatory pathways. The p.Ser707Tyr substitution is located in an autoinhibitory SH2 domain that is crucial for PLCγ2 activation. Overexpression of the altered p.Ser707Tyr protein and ex vivo experiments using affected individuals’ leukocytes showed clearly enhanced PLCγ2 activity, suggesting increased intracellular signaling in the PLCγ2-mediated pathway. Recently, our laboratory identified in individuals with cold-induced urticaria and immune dysregulation PLCG2 exon-skipping mutations resulting in protein products with constitutive phospholipase activity but with reduced intracellular signaling at physiological temperatures. In contrast, the p.Ser707Tyr substitution in PLCγ2 causes a distinct inflammatory phenotype that is not provoked by cold temperatures and that has different end-organ involvement and increased intracellular signaling at physiological temperatures. Our results highlight the utility of exome-sequencing technology in finding causal mutations in nuclear families with dominantly inherited traits otherwise intractable by linkage analysis

Typhoid conjugate vaccine effectiveness in Malawi: evaluation of a test-negative design using randomised, controlled clinical trial data

Background Typhoid conjugate vaccines are being introduced in low-income and middle-income countries to prevent typhoid illness in children. Vaccine effectiveness studies assess vaccine performance after introduction. The test-negative design is a commonly used method to estimate vaccine effectiveness that has not been applied to typhoid vaccines because of concerns over blood culture insensitivity. The overall aim of the study was to evaluate the appropriateness of using a test-negative design to assess typhoid Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT) effectiveness using a gold standard randomised controlled trial database. Methods Using blood culture data from a randomised controlled trial of Vi-TT in Malawi, we simulated a test-negative design to derive vaccine effectiveness estimates using three different approaches and compared these to randomised trial efficacy results. In the randomised trial, 27 882 children aged 9 months to 12 years were randomly assigned (1:1) to receive a single dose of Vi-TT or meningococcal capsular group A conjugate vaccine between Feb 21 and Sept 27, 2018, and were followed up for blood culture-confirmed typhoid fever until Sept 30, 2021. Findings For all three test-negative design approaches, vaccine effectiveness estimates (test-negative design A, 80·3% [95% CI 66·2 to 88·5] vs test-negative design B, 80·5% [66·5 to 88·6] vs test-negative design C, 80·4% [66·9 to 88·4]) were almost identical to the randomised trial results (80·4% [95% CI 66·4 to 88·5]). Receipt of Vi-TT did not affect the risk of non-typhoid fever (vaccine efficacy against non-typhoid fever –0·4% [95% CI –4·9 to 3·9] vs –1% [–5·6 to 3·3] vs –2·5% [–6·4 to 1·3] for test-negative design A, test-negative design B, and test-negative design C, respectively). Interpretation This study validates the test-negative design core assumption for typhoid vaccine effectiveness estimation and shows the accuracy and precision of the estimates compared with the randomised controlled trial. These results show that the test-negative design is suitable for assessing typhoid conjugate vaccine effectiveness in post-introduction studies using blood culture surveillance

Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis

Background: Impaired signaling in the IFN-g/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. Objective: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. Methods: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-g/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. Results: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-g–induced gene expression, but we found impaired responses to IFN-g restimulation. Conclusion: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-g–mediated inflammationFil: Sampaio, Elizabeth P.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Instituto Oswaldo Cruz. Laboratorio de Leprologia; BrasilFil: Hsu, Amy P.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Pechacek, Joseph. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Hannelore I.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Erasmus Medical Center. Department of Medical Microbiology and Infectious Disease; Países BajosFil: Dias, Dalton L.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Paulson, Michelle L.. Clinical Research Directorate/CMRP; Estados UnidosFil: Chandrasekaran, Prabha. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Rosen, Lindsey B.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Carvalho, Daniel S.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Instituto Oswaldo Cruz, Laboratorio de Leprologia; BrasilFil: Ding, Li. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Vinh, Donald C.. McGill University Health Centre. Division of Infectious Diseases; CanadáFil: Browne, Sarah K.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Datta, Shrimati. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Allergic Diseases. Allergic Inflammation Unit; Estados UnidosFil: Milner, Joshua D.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Allergic Diseases. Allergic Inflammation Unit; Estados UnidosFil: Kuhns, Douglas B.. Clinical Services Program; Estados UnidosFil: Long Priel, Debra A.. Clinical Services Program; Estados UnidosFil: Sadat, Mohammed A.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Host Defenses. Infectious Diseases Susceptibility Unit; Estados UnidosFil: Shiloh, Michael. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: De Marco, Brendan. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Alvares, Michael. University of Texas. Southwestern Medical Center. Division of Allergy and Immunology; Estados UnidosFil: Gillman, Jason W.. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Ramarathnam, Vivek. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: de la Morena, Maite. University of Texas. Southwestern Medical Center. Division of Allergy and Immunology; Estados UnidosFil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moreira, Ileana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; ArgentinaFil: Uzel, Gulbu. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Johnson, Daniel. University of Chicago. Comer Children; Estados UnidosFil: Spalding, Christine. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Zerbe, Christa S.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Wiley, Henry. National Eye Institute. Clinical Trials Branch; Estados UnidosFil: Greenberg, David E.. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Hoover, Susan E.. University of Arizona. College of Medicine. Valley Fever Center for Excellence; Estados UnidosFil: Rosenzweig, Sergio D.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Host Defenses Infectious Diseases Susceptibility Unit; Estados Unidos. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Primary Immunodeficiency Clinic; Estados UnidosFil: Galgiani, John N.. University of Arizona. College of Medicine. Valley Fever Center for Excellence; Estados UnidosFil: Holland, Steven M.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unido

IL-21 Limits Peripheral Lymphocyte Numbers through T Cell Homeostatic Mechanisms

IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents.To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts to limit the size of the T cell pool. Furthermore, our data suggest IL-21 may contribute to the development of autoimmunity.Taken together, our results suggest that IL-21 plays a global role in regulating T cell homeostasis, promoting the continuous adaptation of the T cell lymphoid space

IL-21 antagonizes the pro-survival effects of IL-7 <i>in vitro</i>.

<p>Analysis of the effects of IL-21 on T cell survival in the presence or absence of IL-7. Cells from the spleen and pancreatic lymph nodes of NOD or OT1 C57BL/6 mice were cultured with IL-7 (1 ng/ml) and/or IL-21 (1 ng/ml or 10 ng/ml) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003118#s4" target="_blank">Materials and Methods</a>. Graphical representation of Annexin V staining by FACS analysis for CD8+ T cell populations from (A) NOD and (B) OT1 C57BL/6 mice. Percentage of Annexin V+ cells cultured in media alone (no cytokine treatment) was set at 100 and Annexin V+ cells treated with cytokine are represented as fold change relative to media alone ±SEM. Percentage of Annexin V+ cells cultured with IL-21 by itself at 10 ng/ml is similar to cells cultured with IL-21 at 1 ng/ml (data not shown). The data shown is pooled from two independent experiments.</p

Increased T cell survival factors in IL-21R-deficient mice.

<p>(A) Representative histograms showing total Bcl-2 expression in indicated CD8 populations for individual NOD (dashed line) and IL-21R KO NOD (solid line) mice relative to isotype control staining (gray line). Below, the graph represents Bcl-2 flow cytometric data ±SEM for one of two independent experiments using n = 4 mice/group. Similar results were found in both experiments. Pancreatic lymph nodes of NOD mice with an average age of 13-weeks and age-matched IL-21R KO NOD mice were analyzed. (B) Levels of Bcl-2 expression in the indicated splenocyte subsets for individual C57BL/6 (dashed line) and IL-21R KO C57BL/6 (solid line) mice are shown in representative histograms relative to isotype control staining (gray line). Below, graphical representations of the percentage of Bcl-2 expression ±SEM pooled from three independent experiments with a total of n = 12 mice/group. 6-8-week-old C57BL/6 mice and aged-matched IL-21R KO C57BL/6 mice were used.</p

Reduced T cell proliferation in IL-21R-deficient mice.

<p>Representative histogram plots for individual NOD (thin line) and IL-21R KO NOD (thick line) mice showing flow cytometric analysis of BrdU incorporation in CD4+CD44^hi and CD8+CD44^hi populations after 5 days of BrdU treatment. The data, obtained from analyzing the spleens of 7 to 9-week-old NOD and age-matched IL-21R KO NOD mice, is presented as mean percentages of BrdU+ cells ±SEM from one experiment with a total of n = 4 mice/group. The experiment, using the same number and age of mice, was repeated twice with similar results. (B) Representative flow cytometric analyses of splenocytes from individual C57BL/6 (thin line) and IL-21R KO C57BL/6 (thick line) mice showing BrdU incorporation in CD4+CD44^hi and CD8+CD44^hi T cells after 5 days of BrdU treatment. Mean percentages of BrdU+ cells ±SEM are shown from one experiment with a total of n = 4 mice/group with an average age of 10–12 weeks. The experiment was repeated three times with similar results. For both A and B, the gate on the histogram plots represents isotype control staining. (C) Representative dot plot showing expression of CD44 and CD62L in the CD4+ population of individual NOD and IL-21R KO NOD mice. Pooled data ±SEM from a total of n = 8 mice/group, at 7 to 9-weeks of age, from two independent experiments is shown. Gates are based on isotype control staining.</p

Increased cell numbers in IL-21R-deficient mice.

<p>(A) Total splenocyte numbers in 6 to 8-week old C57BL/6 mice compared to age-matched NOD mice (n = 14 mice/group, p = 0.03). (B) Total cell numbers (n = 14 mice/group, p = 0.008), absolute CD4+ T cell numbers (n = 13 mice/group, p = 0.0002) and absolute CD8+ T cell numbers (n = 14 mice/group, p = 0.003) for the splenocytes of NOD mice, at 6 to 8-weeks of age, compared to those from age-matched IL-21R KO NOD mice. (C) Total cell numbers (n = 13 mice/group, p = 0.01), absolute CD4+ T cell numbers (n = 12 mice/group, p = 0.02), and absolute CD8+ T cell numbers (n = 19 mice/group, p = 0.04) from the spleens of C57BL/6 mice, average age of 8 weeks, and IL-21R KO C57BL/6 mice, average age of 9 weeks. (D) Absolute B cell numbers (n = 6 mice/group, p = 0.01) for mice described in C. Data shown are from three or four independent experiments. Results are presented as mean absolute numbers ±SEM.</p

Decreased T cell effector function in IL-21R-deficient NOD mice.

<p>Representative dot plots from individual NOD and IL-21R KO NOD mice showing frequency of (A) TNFα+/CD69+, TNFα+ /CD44+, (B) IFNγ+ /CD69+, IFNγ+/CD44+ populations in CD8+ T cells and (C) TNFα+/CD69+, TNFα+/CD44+, (D) IFNγ+/CD69+, IFNγ+/CD44+ populations in CD4+ T cells. Gates are based on isotype control staining. Graphs represent pooled flow cytometric data from pancreatic lymph nodes of 11-to-13 week old mice for two independent experiments with a total of n = 8 mice/group. Results are presented as the mean percentages ±SEM. (E) Percentages of the indicated cell populations from A–D were multiplied by the total number of cells in the pancreatic lymph nodes for each individual animal to give the absolute numbers for each indicated cell population. Data are shown as mean absolute numbers ±SEM. (F) Cumulative incidence of diabetes was monitored by measuring blood glucose levels in NOD (close circles) and IL-21R KO NOD (open squares) mice (n = 15 mice/group) at the indicated ages. (G) Paraffin sections of pancreata from NOD and IL-21R KO NOD mice stained with H&E. In total, four NOD and four IL-21R KO NOD mice were evaluated, with fifty individual islets examined per strain. The images shown in these panels were obtained from 2 different mice per strain; the NOD mice were 11 weeks of age; the IL-21R KO NOD mice were 13 weeks of age.</p