Lars Blom

IL-33 induces IL-9 production in human CD4+ T cells and basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4+CD45RA+CD45RO−CD25− T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

IL-1 Family Members IL-18 and IL-33 Upregulate the Inflammatory Potential of Differentiated Human Th1 and Th2 Cultures

The IL-1 family members IL-1β, IL-18, and IL-33 are potent cytokines in relationship to amplifying the CD4+ T cell cytokine production. To evaluate their impact on in vitro-differentiated human Th1 and Th2 cultures, such cultures were established from naive T cells, purified from healthy blood donors, and reactivated in the presence of IL-1β, IL-18, or IL-33. Interestingly, we observe modifying responses in Th1 and Th2 cultures induced by IL-18 or IL-33 but not by IL-1β, both contributing to amplify production of IL-5, IL-13, and IFN-γ. IL-18 or IL-33 stimulation of Th1 cultures resulted in increased IFN-γ and IL-13 production concurrent with reduced IL-10 gene transcription and secretion even though Th1 cultures, in contrast to IL-18Rα, had low ST2L expression. Furthermore, adding IL-18 to Th1 cultures promoted Tbet mRNA expression and production. Th2 cultures stimulated with IL-18 or IL-33 had an increased IL-5 secretion. Interestingly, E4BP4 gene expression and the percentage of E4BP4+ cells of the recently shown IL-10 transcriptional regulator E4BP4 correlated with IL-10 gene expression and protein secretion in Th1 cultures. Taken together, we report that the IL-1 family “alarmins” IL-18 and IL-33 in addition to amplifying both Th1- and Th2-associated cytokines block production of the regulatory cytokine IL-10 in Th1 cultures.

IgE‐mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase‐9 from monocytes

IgE‐mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF‐α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase‐9 (MMP‐9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF‐α/MMP‐9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE‐mediated activation of human basophils induces TNF‐α and MMP‐9 release.

The immunoglobulin superfamily member CD200R identifies cells involved in type 2 immune responses

The pathology of allergic diseases involves type 2 immune cells, such as Th2, ILC2, and basophils exerting their effect by production of IL‐4, IL‐5, and IL‐13. However, surface receptors that are specifically expressed on type 2 immune cells are less well documented. The aim of this investigation was to identify surface markers associated with type 2 inflammation.

Circulating allergen-specific TH2 lymphocytes: CCR4+ rather than CLA+ is the predominant phenotype in peanut-allergic subjects

To the Editor: Early exposure to peanut through a dysfunctional skin barrier may result in sensitization to peanut while oral exposure is suggested to promote tolerance induction. A study on circulating peanut-specific Th cells from peanut-allergic (PA) subjects has supported this hypothesis by demonstrating stronger proliferation of skin-homing cutaneous lymphocyte-associated antigen (CLA) expression compared with gut-homing (a4b7) Th cells. Moreover, high levels of the TH2 subtype-associated skinand lunghoming C-C motif chemokine receptor 4 (CCR4) have been described on Ara h 1–specific Th cells of subjects with PA. It is well recognized that TH2 cells dominate peanut-responsive Th cells in patients with PA. To better understand the mechanism of food allergen sensitization and the importance of exposure route, we aimed to investigate the gutand skin-homing phenotype of circulating human peanut-specific Th cells of PA and nonallergic (NA) subjects. PA subjects all had IgE to Ara h 2 and the median year of latest allergic episode to peanuts was 3 (1-5 years). All PA subjects had concomitant atopic dermatitis (AD) and allergic rhinoconjunctivitis. Moreover, 8 of the 9 subjects with PAwere diagnosed with asthma (see Table E1 in this article’s Online Repository at www. jacionline.org). Circulating Th cells of PA and NA subjects showed similar expression of the investigated skin markers CLA, CCR4, and CCR10 as well as the gut-homing antigen integrin a4b7 (b7) (see Fig E1, A and C, and this article’s Methods section in the Online Repository at www.jacionline.org). Moreover, no differences were found in the frequency of circulating memory (CD161) Th cells and conventional (CRTH2CD161) or pathogenic (CRTH2CD161) TH2-cell subpopulations in PA and NA subjects (Fig E1, D and E). Peanut-responsive Th cells were identified by increased CD154 expression after short-term ex vivo stimulation with whole peanut extract of PBMCs from PA and NA subjects. PA subjects showed stronger peanut-specific CD154Th-cell responses than NA subjects (P 5 .0252, mean frequencies of 113 vs 35 per million Th cells and ratios compared with the unstimulated control of 15 vs 8, Fig 1, A and B). In line with our findings, one study reported comparable frequency of peanut-specific Th cells (100-200 per million Th cells) in PA subjects. Peanut-responsive Th cells of PA subjects had increased expression of the TH2 cell type–associated skinand airwayhoming chemokine receptor CCR4 compared with NA subjects (P 5 .0042, 24% vs 8%), but neither the signature skin-homing antigen CLA (16% vs 14%) nor the other skin-homing chemokine receptor CCR10 (13% vs 7%, Fig 1, D and E). In agreement with our data, another study characterizing Ara h 1–specific Th cells of PA subjects reported expression of CLA in approximately 10% of the cells. Furthermore, no difference in expression of b7 was found in the peanut-responsive Th cells of PA and NA subjects (2.5% vs 3.1%, Fig 1, D and E), an indication of nongut priming. As expected, the largest subpopulation of the peanut-responsive Th cells of PA but not NA subjects was TH2 cells (CRTh2 ) (44% vs 9%, Fig 1, E). Also, most CRTH2-positive peanutspecific Th cells of PA subjects coexpressed CD161, indicative of a pathogenic TH2 cell profile with production of IL-5 (Fig 1, F and G). Finally, peanut-responsive Th cells of PA but not of NA subjects were characterized by high expression of the newly described type 2 immune response–associated receptor CD200R (median fluorescence intensity, 1602 vs 633, Fig 1, H and I). We next characterized the cytokine profile of the peanut-responsive Th cells (see Fig E2, A, in this article’s Online Repository at www.jacionline.org). The peanut-specific Th cells of PA subjects had a larger fraction of conventional (IL-4IL-5, P 5 .0002, 20% vs 1.6%) and pathogenic (IL-4IL-5, P 5 .0002, 29% vs 1.5%) TH2 cells compared with the NA subjects (Fig E2, B and C). This is in agreement with a previous publication, reporting the 2 largest subpopulations of peanut-responsive TH2 cells as IL-5 TH2 (IL-4IL-5) and IL-5TH2 (IL-4 IL-5) cells in PA subjects. Furthermore, the peanut-specific Th cells of PA subjects had a greater percentage of single-positive IL-5IL-4 cells (9.6% vs 1.4%) as well as IL-31, another TH2 subtype-associated cytokine (2% vs 0.6%) than NA subjects (Fig E2, B and C). In contrast, compared with the PA subjects, most peanut-reactive Th cells of NA subjects showed a TH1 profile with more IFN-g– positive cells (10% vs 45%, Fig E2, B and C). Having established that most peanut-responsive Th cells of PA subjects were of a TH2 profile, we failed to find different expression of the canonical skin-homing marker CLA in the PA compared with the NA subjects. Chan et al showed that the isolated CLATh cell fraction of PA subjects primarily produced TH2 cytokines while a TH1 profile was the main population observed of isolated b7 cells in peanut-tolerant subjects. However, the same authors later reported in an elegant transcriptomic study, of sorted CD69 peanut-specific CLA and b7 Th cells, that TH2and TH9-associated cytokines were equally expressed in the CLAand b7-homing Th cells. By analyzing the skinand gut-homing potential of the largest cytokine cell populations of IL-4IL-5, IL-4IL-5, and IFN-g peanut-specific Th cells of PA subjects, unbiased grouping analysis using t-SNE (t-distributed stochastic neighbor embedding) revealed an association of CCR4 but not of CLA to peanut-specific TH2 cells in the PA subjects (Fig 2, A). Interestingly, even though all PA subjects had AD, the main subpopulation of peanut-specific pathogenic (IL-4IL-5) TH2 cells lacked CLA (<1%) and weakly (11%) coexpressed CCR4 (Fig 2, B-D). These findings were unexpected, because the main (>90%) skin-homing T-cell population in subjects with AD is characterized by CLA expression, a surrogate marker for cutaneous T cells. More studies are needed to verify whether the phenotype of circulating peanut-specific Th cells mirrors peanut-specific Th cells located in the airway and skin. Furthermore, CCR4 but not CLAwas in particular expressed by the conventional IL-4IL-5 TH2 cells, with 21% expressing CCR4 (CCR4CLA) in the PA subjects (Fig 2, B-D). However, it would be interesting to study PA subjects without asthma to elucidate whether the CCR4 expression by the conventional TH2 cells is asthma related. On an allergen-specific level, we confirm the link between CCR4 expression of IL-4IL-5 Th