Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.
© 2023 The Authors.

The epithelium-associated cytokine thymic stromal lymphopoietin (TSLP) can induce OX40L and CCL17 expression by myeloid dendritic cells (mDCs), which contributes to aberrant Th2-type immune responses. Herein, we report that such TSLP-induced Th2-type immune response can be effectively controlled by Dectin-1, a C-type lectin receptor expressed by mDCs. Dectin-1 stimulation induced STAT3 activation and decreased the transcriptional activity of p50-RelB, both of which resulted in reduced OX40L expression on TSLP-activated mDCs. Dectin-1 stimulation also suppressed TSLP-induced STAT6 activation, resulting in decreased expression of the Th2 chemoattractant CCL17. We further demonstrated that Dectin-1 activation was capable of suppressing ragweed allergen (Amb a 1)-specific Th2-type T cell response in allergy patients ex vivo and house dust mite allergen (Der p 1)-specific IgE response in non-human primates in vivo. Collectively, this study provides a molecular explanation of Dectin-1-mediated suppression of Th2-type inflammatory responses and suggests Dectin-1 as a target for controlling Th2-type inflammation.
Copyright © 2021 Gu, Upchurch, Horton, Wiest, Zurawski, Millard, Kane, Joo, Miller and Oh.

Polychromatic flow cytometry enables the detection and characterization of markers which are helpful in defining phenotype of various cell subsets. Here we describe flow cytometry-based method to characterize phenotype of naïve, memory, and effector T cells. Being able to differentiate these cells is crucial in understanding immune response, and immune profiling. Naïve T cells enable the body to fight off new, unrecognized infections and diseases, and memory T cells are enriched for response to recall antigens. Furthermore, the antigen-experienced T cell populations can be broadly divided into effector and memory cell compartments, both of which are needed for sustaining a responsive immune system. Simplistically, the effector T cells require active antigenic stimulation to eliminate pathogens. On the other hand, memory T cells are described as cells which remain present in the absence of antigenic stimulation and have the capacity to expand rapidly upon secondary challenges. Recently, with the identification of central and effector memory T cell subsets, tremendous efforts have been devoted to characterize markers on the surfaces of these cells. Though, various markers have been used to identify the subsets, no single marker that segregates one subset from the other has been described. Thus, multiple markers are needed to subset the cells in order to characterize them. Here we report the verification of a nine-color panel (CD3, CD4, CD8, CD45RO, CD28, CD95, CCR7, Live/Dead Aqua, dump channel-CD19, CD14, CD56, CD16) that can successfully identify six distinct CD4 and CD8 T cell populations within the naïve and effector cell subsets from human donors.

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4 + Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.
We conducted a longitudinal study of 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2-6 consecutive monthly infusions. Among these 15 patients, 10 responded to ivIg therapy with clear clinical improvement. We characterized Tregs and determined TCR spectratypes of four Vβ families with reported oligoclonality. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vβ-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points.
For 11 out of 15 patients, average Vβ1/Vβ2/Vβ11/Vβ14 repertoires were less perturbed under than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vβ11 and Vβ14 perturbations measured under ivIg therapy.
This indicates a role of active Tregs in the therapeutic effect of ivIg.