GATA2 (n 5 1), and 22q11.2 (n 5 4) mutations and deletions, suggesting improved clinical utility of our comprehensively designed target capture.CVID is the most common PID, representing a heterogeneous group of hypogammaglobulinemias of largely unknown molecular defects. Our system established a genetic diagnosis of 22q11.2 deletion syndrome (22q11.2DS) in a patient with CVID in accordance with a previous study, reporting that a subgroup of patients with adult 22q11.2DS could have hypogammaglobinemia. 9 Moreover, the incidence of this syndrome is relatively high compared with that of other PIDs. Therefore patients with CVID should always be evaluated for the possibility of 22q11.2DS. In this context our customized design to capture this chromosomal lesion is useful.Recent studies revealed that newborn screening for T-cell receptor excision circles can efficiently detect infants with severe combined immunodeficiency and complete DiGeorge syndrome (mainly because of 22q11.2DS). 10 Our comprehensive genetic diagnostic system covering virtually all of the PID genes and 22q11.2DS, would provide an effective genetic confirmation test for infants with positive results on T-cell receptor excision circle screening tests, who require precise and rapid genetic diagnoses for appropriate clinical management.In summary, we developed an NGS-based comprehensive, rapid, and efficient PID diagnostic system that could become a first-line genetic analysis of PID-suspected patients, including infants with positive newborn screening results.We thank the patients and family who made this study possible by providing clinical samples. We also thank Ms Yoshie Miura, Ms Yuko Imanishi, and Ms Hiroe Namizaki for their valuable assistance. We acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for technical support for NGS. Finally, we thank Enago (www.enago.jp) for the English-language review.

Therefore our findings identify PKC-θ as a critical factor for ILC2 activation that contributes to T2 cell differentiation, which is associated with IRF4 and NFAT1 expression in allergic lung inflammation.

Background:We previously showed that patients with severe allergic asthma have high numbers of circulating ILC2s expressing CCR10. Method:Herein, CCR10 + ILC2s were further analyzed in the blood of healthy individuals or patients with allergic and non−allergic asthma. Characteristics of human CCR10 + and CCR10 − ILC2s were assessed by flow cytometry as well as single-cell multiplex RT-qPCR. The role of CCR10 + ILC2s in asthma pathophysiology was studied in allergen-treated mice.Results: When compared to healthy controls, CCR10 + ILC2s are enriched in the blood of both allergic and non-allergic severe asthmatic patients, and these cells are recruited to the lungs. Plasma concentrations of the CCR10 ligand CCL27 are significantly increased in severe asthmatics when compared to non-asthmatic patients.CCR10 + ILC2s secrete little T H 2 cytokines, but exhibit ILC1-like properties, including a capacity to produce IFN-γ. Also, single-cell analysis reveals that the CCR10 + ILC2 subset is enriched in cells expressing amphiregulin. CCR10 + ILC2 depletion, as well as blocking of IFN-γ activity, exacerbates airway hyperreactivity in allergen-challenged mice, providing evidence for a protective role of these cells in allergic inflammation. Conclusions:Frequencies of circulating CCR10 + ILC2s and CCL27 plasma concentrations represent candidate markers of asthma severity. The characterization of CCR10 + ILC2s in human samples and in mouse asthma models suggests that these cells downregulate allergic inflammation through IFN-γ production.

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BackgroundT-cell engagers have proved to be a promising therapeutic strategy in immunotherapy, for redirecting T cells activity against tumor cells. To facilitate the preclinical assessment of novel T-cell engagers and their translatability, we have developed an immunocompetent CD3 epsilon N-terminal epitope humanized mouse model.MethodsThis model was developed to express the human epitope of the CD3 epsilon chain, which is recognized by approximately 70% of the T-cell engagers (clone SP34). The rest of the extracellular domain was kept from mouse origin to preserve the amino acids involved in the interaction with CD3 gamma and delta. Similarly, the transmembrane domains and the intracellular domains where kept murine to enable salt bridges interaction, interaction with the CD3 zeta and the signaling into mouse cells.ResultsT cells from CD3 epsilon epitope humanized mice are found in comparable frequency in spleen, blood and bone marrow from WT mice. B cells, monocytes, dendritic cells and NK frequencies are also similar to the frequencies of these cell types in WT mice, suggesting that the humanization of the epitope of CD3 epsilon did not alter the immune cells distribution in these mice. Activation of T cells with antibodies targeting human CD3 (clone SP34) induced CD4 and CD8 T cell proliferation, as well as production of IL-2 and IFN-gamma. The CD3 functionality was demonstrated in vitro by the ability of B cells to produce IgM upon activation of T cells, suggesting a proper cooperation between T and B cells. Additionally, a first class of T-cell engagers targeting both human CD3 and a tumoral antigen, induced tumor cell lysis of MC38-Ag in a concentration-dependent manner. A second class of T cell engagers, also targeting CD3 and a tumoral antigen, showed an anti-tumor effect in vivo, and this effect was also shown to be dose-dependent.ConclusionsThese data suggest that the CD3 epsilon N-terminal epitope humanized mouse model enables the assessment of efficacy and mechanism of action of T-cell engagers.This model is currently being intercrossed with immunostimulatory humanized mouse models to provide new opportunities for assessment of bi-specific antibodies targeting the CD3 and immunostimulatory molecules. This model is the first generation of a broader program aiming at developping a Pan CD3 humanized model, where the gamma, delta and epsilon chains of the CD3 complex will be humanized. The Pan CD3 humanized mice are currently being investigated for immune responses and would provide a broader tool for assessment of T-cell engagers.