IL-4 Haploinsufficiency Specifically Impairs IgE Responses against Allergens in Mice [ALLERGY AND OTHER HYPERSENSITIVITIES]

Polymorphisms in genes involved in IL-4 responses segregate with allergic disease risk and correlate with IgE levels in humans, and IL-4 promotes IgE and IgG1 Ab production against allergens in mice. We report that mice with only one intact Il4 gene copy are significantly impaired in their ability to make specific IgE responses against allergens, whereas IgG1 responses to allergens remain unaffected. Il4-hemizygosity also resulted in a modest but detectable drop in IL-4 production by CD4⁺ T cells isolated from lymph nodes and prevented IgE-dependent oral allergen–induced diarrhea. We conclude that a state of haploinsufficiency for the Il4 gene locus is specifically relevant for IL-4–dependent IgE responses to allergens with the amount of IL-4 produced in the hemizygous condition falling close to the threshold required for switching to IgE production. These results may be relevant for how polymorphisms in genes affecting IL-4 responses influence the risk of IgE-mediated allergic disease in humans.

http://ift.tt/2ll5QOo

Antigen Processing in the Endoplasmic Reticulum Is Monitored by Semi-Invariant {alpha}{beta} TCRs Specific for a Conserved Peptide-Qa-1b MHC Class Ib Ligand [INFECTIOUS DISEASE AND HOST RESPONSE]

Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide–MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I–presented peptides, which elicit potent immune responses. An unusual subset of CD8⁺ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1^b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αβ TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRβ-transgenic mice were assessed for QFL-specific TCR α- and β-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3–TRAJ21 α-chain and TRBV5–TRBD1–TRBJ2-7 β-chain gene segments. Furthermore, the CDR3 regions of the α- as well as β-chains were required for QFL ligand recognition. Thus, the αβ TCRs used to recognize the peptide–Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

http://ift.tt/2l5uhwG

Modulation of Naive CD8 T Cell Response Features by Ligand Density, Affinity, and Continued Signaling via Internalized TCRs [ANTIGEN RECOGNITION AND RESPONSES]

T cell response magnitudes increase with increasing antigenic dosage. However, it is unclear whether ligand density only modulates the proportions of responding ligand-specific T cells or also alters responses at the single cell level. Using brief (3 h) exposure of TCR-transgenic mouse CD8 T cells in vitro to varying densities of cognate peptide-MHC ligand followed by ligand-free culture in IL-2, we found that ligand density determined the frequencies of responding cells but not the expression levels of the early activation marker molecule, CD69. Cells with low glucose uptake capacity and low protein synthesis rates were less ligand-sensitive, implicating metabolic competence in the response heterogeneity of CD8 T cell populations. Although most responding cells proliferated, ligand density was associated with time of entry into proliferation and with the extent of cell surface TCR downmodulation. TCR internalization was associated, regardless of the ligand density, with rapidity of c-myc induction, loss of the cell cycle inhibitor p27kip1, metabolic reprogramming, and cell cycle entry. A low affinity peptide ligand behaved, regardless of ligand density, like a low density, high affinity ligand in all these parameters. Inhibition of signaling after ligand exposure selectively delayed proliferation in cells with internalized TCRs. Finally, internalized TCRs continued to signal and genetic modification of TCR internalization and trafficking altered the duration of signaling in a T cell hybridoma. Together, our findings indicate that heterogeneity among responding CD8 T cell populations in their ability to respond to TCR-mediated stimulation and internalize TCRs mediates detection of ligand density or affinity, contributing to graded response magnitudes.

http://ift.tt/2l5Km5s

Periodic Fever with Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome Is Associated with a CARD8 Variant Unable To Bind the NLRP3 Inflammasome [INNATE IMMUNITY AND INFLAMMATION]

Periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a relatively common autoinflammatory condition that primarily affects children. Although tendencies were reported for this syndrome, genetic variations influencing risk and disease progression are poorly understood. In this study, we performed next-generation sequencing for 82 unrelated PFAPA patients and identified a frameshift variant in the CARD8 gene (CARD8-FS). Subsequently, we compared the frequency of CARD8-FS carriers in our PFAPA cohort (13.9%) with a healthy local population group (3.2%) and found a significant association between the CARD8-FS polymorphism and risk for PFAPA syndrome (p = 0.012; odds ratio: 4.96 [95% confidence interval, 1.33–18.47]). Moreover, CARD8-FS carriers display a distinct PFAPA phenotype that is characterized by a higher prevalence of symptoms out of flares and oral aphthosis (both p = 0.02 compared with PFAPA patients without the frameshift variant). CARD8 encodes a protein component of the NLRP3 inflammasome, which plays an important role in inflammation and contributes to the pathology of various autoinflammatory diseases. We found that the CARD8-FS variant led to a truncated CARD8 protein lacking the FIIND and CARD domains. As a result, the mutant CARD8 protein lost the ability to interact with the NOD domain of NLRP3. In summary, these results identify a new CARD8 variant associated with PFAPA and further suggest that disruption of the interaction between CARD8 and NLRP3 can regulate autoinflammation in patients.

http://ift.tt/2l5Kemo

Gliadin-Specific CD8+ T Cell Responses Restricted by HLA Class I A*0101 and B*0801 Molecules in Celiac Disease Patients [ANTIGEN RECOGNITION AND RESPONSES]

Initial studies associated the HLA class I A*01 and B*08 alleles with celiac disease (CD) susceptibility. Subsequent analyses showed a primary association with HLA class II alleles encoding for the HLA DQ2.5 molecule. Because of the strong linkage disequilibrium of A*01 and B*08 alleles with the DR3-DQ2.5 haplotype and a recent genome-wide association study indicating that B*08 and B*39 are predisposing genes, the etiologic role of HLA class I in CD pathogenesis needs to be addressed. We screened gliadin proteins (2α-, 2-, and 2-gliadin) using bioinformatic algorithms for the presence of peptides predicted to bind A*0101 and B*0801 molecules. The top 1% scoring 9- and 10-mer peptides (N = 97, total) were synthesized and tested in binding assays using purified A*0101 and B*0801 molecules. Twenty of ninety-seven peptides bound B*0801 and only 3 of 97 bound A*0101 with high affinity (IC₅₀ < 500 nM). These 23 gliadin peptides were next assayed by IFN- ELISPOT for recognition in peripheral blood cells of CD patients and healthy controls carrying the A*0101 and/or B*0801 genes and in A*0101/B*0801^– CD patients. Ten of the twenty-three peptides assayed recalled IFN- responses mediated by CD8⁺ T cells in A*0101/B*0801⁺ patients with CD. Two peptides were restricted by A*0101, and eight were restricted by B*0801. Of note, 50% (5/10) of CD8⁺ T cell epitopes mapped within the -gliadins. Our results highlight the value of predicted binding to HLA molecules for identifying gliadin epitopes and demonstrate that HLA class I molecules restrict the anti-gluten T cell response in CD patients.

http://ift.tt/2ll8bJi

Oxidized Low-Density Lipoprotein Immune Complex Priming of the Nlrp3 Inflammasome Involves TLR and Fc{gamma}R Cooperation and Is Dependent on CARD9 [INNATE IMMUNITY AND INFLAMMATION]

Oxidized low-density lipoprotein (oxLDL) is known to activate inflammatory responses in a variety of cells, especially macrophages and dendritic cells. Interestingly, much of the oxLDL in circulation is complexed to Abs, and these resulting immune complexes (ICs) are a prominent feature of chronic inflammatory disease, such as atherosclerosis, type-2 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. Levels of oxLDL ICs often correlate with disease severity, and studies demonstrated that oxLDL ICs elicit potent inflammatory responses in macrophages. In this article, we show that bone marrow–derived dendritic cells (BMDCs) incubated with oxLDL ICs for 24 h secrete significantly more IL-1β compared with BMDCs treated with free oxLDL, whereas there was no difference in levels of TNF-α or IL-6. Treatment of BMDCs with oxLDL ICs increased expression of inflammasome-related genes Il1a, Il1b, and Nlrp3, and pretreatment with a caspase 1 inhibitor decreased IL-1β secretion in response to oxLDL ICs. This inflammasome priming was due to oxLDL IC signaling via multiple receptors, because inhibition of CD36, TLR4, and FcR significantly decreased IL-1β secretion in response to oxLDL ICs. Signaling through these receptors converged on the adaptor protein CARD9, a component of the CARD9–Bcl10–MALT1 signalosome complex involved in NF-B translocation. Finally, oxLDL IC–mediated IL-1β production resulted in increased Th17 polarization and cytokine secretion. Collectively, these data demonstrate that oxLDL ICs induce inflammasome activation through a separate and more robust mechanism than oxLDL alone and that these ICs may be immunomodulatory in chronic disease and not just biomarkers of severity.

http://ift.tt/2l5KlhU

Cathepsin K Deficiency Ameliorates Systemic Lupus Erythematosus-like Manifestations in Faslpr Mice [AUTOIMMUNITY]

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Fas^lpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Fas^lpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Fas^lpr mice also showed elevated serum CatK levels. CatK deficiency in Fas^lpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Fas^lpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Fas^lpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4⁺CD25^highFoxp3⁺ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Fas^lpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

http://ift.tt/2l5KSk5