The autoimmune condition, Celiac Disease (CeD), displays broad clinical symptoms due to gluten exposure. Its genetic association with DQ variants in the human leukocyte antigen (HLA) system has been recognised. Monocyte-derived mature dendritic cells (MoDCs) present gluten peptides through HLA-DQ and co-stimulatory molecules to T lymphocytes, eliciting a cytokine-rich microenvironment. Having access to CeD associated families prevalent in the Czech Republic, this study utilised an in vitro model to investigate their differential monocyte profile. The higher monocyte yields isolated from PBMCs of CeD patients versus control individuals also reflected the greater proportion of dendritic cells derived from these sources following lipopolysaccharide (LPS)/ peptic-tryptic-gliadin (PTG) fragment stimulation. Cell surface markers of CeD monocytes and MoDCs were subsequently profiled. This foremost study identified a novel bio-profile characterised by elevated CD64 and reduced CD33 levels, unique to CD14++ monocytes of CeD patients. Normalisation to LPS stimulation revealed the increased sensitivity of CeD-MoDCs to PTG, as shown by CD86 and HLA-DQ flow cytometric readouts. Enhanced CD86 and HLA-DQ expression in CeD-MoDCs were revealed by confocal microscopy. Analysis highlighted their dominance at the CeD-MoDC membrane in comparison to controls, reflective of superior antigen presentation ability. In conclusion, this investigative study deciphered the monocytes and MoDCs of CeD patients with the identification of a novel bio-profile marker of potential diagnostic value for clinical interpretation. Herein, the characterisation of CD86 and HLA-DQ as activators to stimulants, along with robust membrane assembly reflective of efficient antigen presentation, offers CeD targeted therapeutic avenues worth further exploration.

Pimecrolimus (Elidel, SDZ ASM 981) is an anti-inflammatory and immunomodulatory 33-epichloro-derivative of macrolactam ascomycin, with low potential for affecting systemic immune responses compared with other calcineurin inhibitors, cyclosporin A and tacrolimus. Despite numerous studies focused on the mechanism of pimecrolimus action on mast cells, only the single report has addressed pimecrolimus effects on other typical FcεRI-expressing cells, the basophils. Patients allergic to birch pollen (n = 20), hymenopteran venoms (n = 23) and 10 non-allergic volunteers were examined. Primary human basophils pre-treated or not with 0.5–50 μMol pimecrolimus were exposed to various concentrations of recombinant Bet v 1a allergen, bee or wasp venom extracts and anti-IgE for 20 min, and then examined for the expression of CD45, CD193, CD203c, CD63 and CD164 using flow cytometry. The externalization of basophil activation markers (CD63 and CD164) was equally inhibited through pimecrolimus in cells activated by recombinant pollen allergen, hymenopteran venom extracts and anti-IgE. Although the individual response rate was subject to strong variation, importantly, pre-treatment with pimecrolimus lowered the number of activated basophils in response to any of the stimuli in the basophils from all patients. The inhibition was concentration-dependent; approximately half of the basophils were inhibited in the presence of 2.5 mMol pimecrolimus. Pimecrolimus is a valuable new tool for the inhibition of hyper-reactive basophils in patients with pollen allergy and a history of anaphylactic reactions to bee or wasp venoms. Further research should address short-term use of pimecrolimus in vivo in a wide spectrum of allergic diseases.

Summary Background CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FcεRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization. Objective We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides. Methods We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31‐8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT‐PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C. Results We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FcεRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides. Conclusions & Clinical Relevance The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16+ or CD16dim. Using CD16dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63.

Celiac disease (CeD) manifests with autoimmune intestinal inflammation from gluten and genetic predisposition linked to human leukocyte antigen class-II (HLA-II) gene variants. Antigen-presenting cells facilitate gluten exposition through the interaction of their surface major histocompatibility complex (MHC) with the T cell receptor (TCR) on T lymphocytes. This fundamental mechanism of adaptive immunity has broadened upon recognition of extracellular exosomal MHC, raising awareness of an alternative means for antigen presentation. This study demonstrates that conditioned growth media (CGM) previously exposed to monocyte-derived dendritic cells from CeD significantly downregulates the CD3+ lineage marker of control T cells. Such increased activation was reflected in their elevated IL-2 secretion. Exosome localization motif identification and quantification within HLA-DQA1 and HLA-DQB1 transcripts highlighted their significant prevalence within HLA-DQB1 alleles associated with CeD susceptibility. Flow cytometry revealed the strong correlation between HLA-DQ and the CD63 exosomal marker in T cells exposed to CGM from MoDCs sourced from CeD patients. This resulted in lower concentrations of CD25+ CD127− T cells, suggestive of their compromised induction to T-regulatory cells associated with CeD homeostasis. This foremost comparative study deciphered the genomic basis and extracellular exosomal effects of HLA transfer on T lymphocytes in the context of CeD, offering greater insight into this auto-immune disease.

BackgroundThe exact mechanism of systemic hypersensitivity to venom is not exactly understood. It is suggested T cells with regulatory potential can downregulate other T cell subsets and effector cells, ex. mast cell or basophils. We focused on relationship of specific basophil reactivity in relationship to proportion of regulatory T cells.MethodsForty-five patients with history of systemic symptoms of allergy to Hymenoptera venom were included. Basophil reactivity before the treatment and after one year of allergen immunotherapy (AIT) was measured by CD63 expression, CD203c marker used for basophil identification. Cells were stimulated by aqueous solution of allergen in concentration range 0.01 to 1 μg/mL. T regulatory cells were identified as CD4 positive, CD25 bright and CD127 negative at the same interval of treatment. Monoclonal antibodies conjugated with fluorochromes were used, measured by FACSCalibur. Paired t test and correlation analysis used for statistical evaluation.ResultsMedian Treg proportion before therapy was 2.160, after IT 1.960, basophil specific response (proportion of CD63 positive cells) at the same interval were 3.65 and 4.11 at 0.01 μg/mL, 13.1 and 16.1 for 0.1 μg/mL and 33.85 and 40.8 for 1 μg/mL. All differences were not statistically significant. Differences of basophil activation were not significantly related to proportion of T regulatory cells.ConclusionsIn our group of patients with HV allergy, treated by AIT, we did not found any relationship between basophil specific activation during allergen immunotherapy and proportion of T regulatory cells.