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SUMMARYIn recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8 ¹ T cells, IL-2 and interferon-gamma (IFN-g) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8 þ T cells IL-2 was optimally induced after 10 h and IFN-g after 6 h. The levels of IL-2 and IFN-g in CD8 þ and CD8 ¹ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2 þ (range 9 . 7-41 . 3) and CD3/IFN-g þ cells (10 . 1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n ¼ 5) there was a significantly decreased percentage of CD3 þ /CD8 þ peripheral blood T cells expressing IFN-g and an increased percentage of CD3 þ /CD8 ¹ T cells expressing IL-4 compared with non-atopic dermatitis controls (n ¼ 5). Possible applications of this technique are discussed.

SummaryChronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)−17 and IL‐22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex‐vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in‐vitro techniques, such as intracellular cytokine staining and enzyme‐linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6+CXCR3–CCR4+CD161+ T helper cells generates results that correlate with intracellular cytokine staining for IL‐17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6+CXCR3–CD4+ T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause.

SUMMARYThis study describes an in vitro model of peripheral blood mononuclear cell (PBMC) migration through human endothelial cells, held on polycarbonate inserts, which allows automatic differential counting of migrated cells as lymphocytes and monocytes. Using this system it was found that treatment of PBMC with the phosphodiesterase (PDE) inhibitors theophylline (at 1 and 10 ¹ g/ml) and RO-20-1724 inhibited the migration of the lymphocyte component to 64 . 2 6 16 . 4%, 48 . 9 6 3 . 0% and 47 . 5 6 5 . 8% of the control values, respectively, while the migration of the monocytes component was largely unaffected. The PDE inhibitors needed to be present during the assay to inhibit migration, whereas pre-treatment of either the endothelium or the PBMC did not consistently effect lymphocyte migration. The drugs also inhibited the migration of lymphocytes through control inserts, either uncoated or coated with fibronectin, suggesting that some of the inhibition is an effect on lymphocyte motility rather than lymphocyte-endothelial interactions. Lymphocyte migration through fibronectin-coated filters was significantly enhanced compared with uncoated filters. Activation of the PBMC by anti-CD3 MoAb increased motility and migration by up to 300%. This migration appeared to be greatly inhibited by the PDE inhibitors, although the effect was complicated by problems of lymphocyte aggregation. This study provides a novel method of measuring mononuclear cell transendothelial migration, and suggests a possible role of PDE inhibitors in reducing this process.

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