We are memory: B-cell responses in allergy and tolerance.

The significance of B-cell memory in sustaining IgE-mediated allergies but also ensuring the development of long-term allergen tolerance has remained enigmatic. However, well-thought murine and human studies have begun to shed more light on this highly disputed subject. The present mini review highlights important aspects, like the involvement of IgG1 memory B cells, the meaning of low- or high-affinity IgE antibody production, the impact of allergen immunotherapy, or the relevance of local memory established by ectopic lymphoid structures. Based on recent findings, future investigations should lead to deeper knowledge and the development of improved therapies treating allergic individuals.

Isolation of Lymphocytes from Human Skin and Murine Tissues: A Rapid and Epitope-Preserving Approach.

Tissue-resident immune cells have been shown to play an important role in skin health and disease. However, owing to limited access to human skin samples and time-consuming, technically demanding protocols, the characterization of tissue-derived cells remains challenging. For this reason, blood-derived leukocytes are frequently used as a surrogate specimen, although they do not necessarily reflect local immune responses in the skin. Therefore, we aimed to establish a rapid protocol to isolate a sufficient number of viable immune cells from 4-mm skin biopsies that can be directly used for a deeper characterization such as comprehensive phenotyping and functional studies of T cells. In this optimized protocol, only two enzymes, type IV collagenase and DNase I, were used to achieve both the highest possible cellular yield and marker preservation of leukocytes stained for multicolor flow cytometry. We further report that the optimized protocol may be used in the same manner for murine skin and mucosa. In summary, this study allows a rapid acquisition of lymphocytes from human or mouse skin suitable for comprehensive analysis of lymphocyte subpopulations, for disease surveillance, and for identification of potential therapeutic targets or other downstream applications.

Detection of rare autoreactive T cell subsets in patients with pemphigus vulgaris.

Analysis of T lymphocyte proliferation and activation after antigenic or mitogenic stimulation is a vital parameter used in the diagnosis of various immuno-deficiencies and during the monitoring of treatment responses. Most applied techniques are based on the incorporation of tritiated thymidine (3H-TdR) or ELISPOT analysis, both rely on rather time-consuming/-intensive ex vivo protocols or encompass inherent drawbacks such as the inability to distinguish specific cell populations (3H-TdR, ELISPOT) or focus on a single cytokine (ELISPOT). Here we aimed at characterizing the rapid expression of intracellular CD154 (CD40L) as a marker for rare antigen-specific CD4+ T cells in pemphigus vulgaris (PV). Upon stimulation with human desmoglein (Dsg) 3, the major autoantigen in PV, the expression of CD154 was significantly increased in PV patients compared to healthy controls (HC) and correlated with anti-Dsg3 IgG titers. Patients with active disease showed higher numbers of Dsg3-reactive CD4+ T cells in CXCR5+ T follicular helper cells. In remittent PV and HC, CXCR5+CD4+ T cells remained largely unaffected by Dsg3. IL-17 and IL-21 expression were significantly induced only in CD154+CD4+ T cells from PV patients, lending themselves as potential novel treatment targets. Additionally, stimulation with immunodominant Dsg3-derived epitopes strongly induced a CD4+ T cell response via CD40-CD154 interaction similar to the human Dsg3 protein. We here established a rapid ex vivo assay allowing the detection of Dsg3-reactive CD4+ T cells from activated systemically available PBMCs, which further supports the crucial concept of antigen-specific T cells in the pathogenesis of PV.

Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.

BACKGROUND: TH2 cells were thought to be a pivotal factor for initiation of the autoimmune blistering disease pemphigus. However, the role of other T-cell subsets in pemphigus pathogenesis remained unclear. OBJECTIVE: We aimed to characterize the exact phenotype of T cells responsible for the development of pemphigus. METHODS: Whole transcriptome shotgun sequencing was performed to determine differential gene expression in pemphigus lesions and skin of healthy individuals. The cutaneous cytokine signature was further evaluated by real-time quantitative PCR. In peripheral blood, the distribution of TH cell and folliclular helper (TFH) cell subsets was analyzed by flow cytometry. Finally, the capacity of TH and TFH cell subsets to induce desmoglein (Dsg)-specific autoantibodies by memory B cells was evaluated in coculture experiments. RESULTS: Transcriptome analysis of skin samples identified an IL-17A-dominated immune signature in patients with pemphigus, and Kyoto Encyclopedia of Genes and Genomes pathway analysis confirmed the dominance of the IL-17A signaling pathway. Increased expression of IL17A and associated cytokines was also detected by real-time quantitative PCR comparing lesional with perilesional or healthy skin. Interestingly, utilization of flow cytometry showed that patients with active pemphigus had elevated levels of circulating IL-17+, TH17, TFH17, and TFH17.1 cells. Notably, levels of TH17 and TFH17 cells correlated with levels of Dsg-specific CD19+CD27+ memory B cells, and patients with acute pemphigus showed higher levels of Dsg3-autoreactive TFH17 cells. Coculture experiments revealed TFH17 cells as primarily responsible for inducing Dsg-specific autoantibody production by B cells. CONCLUSION: Our findings show that TFH17 cells are critically involved in the pathogenesis of pemphigus and offer novel targets for therapeutic intervention.