The B cell membrane expresses sialic-acid-binding immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis-ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes NEU1 and NEU3 on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca2+ levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.
© 2022 The Authors.

Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.
© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.

h4>ABSTRACT/h4> The B cell membrane expresses sialic acid binding Immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes, NEU1 and NEU3, on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca 2+ levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.

Breg cells, a regulatory cell subset that produces interleukin-10 (IL-10), play a significant role in suppressing autoimmune responses and preventing autoimmunity. This study was undertaken to examine the number and function of Breg cells in patients with systemic sclerosis (SSc), a disease with many autoantibodies.
Forty-five patients with SSc (12 with early SSc, 33 with established disease including 16 with SSc-associated pulmonary fibrosis [PF]), 12 healthy control subjects, and 10 patients with rheumatoid arthritis (RA)-associated PF were studied. The phenotypes of immature/transitional Breg cells (CD19+CD24(high) CD38(high) ) and memory Breg cells (CD19+CD27+CD24(high) ) were evaluated by flow cytometry. The function of Breg cells was assessed by measuring the production of IL-10 after B cell activation. In addition, activation of p38 MAPK and STAT-3 was measured following stimulation of the cells with B cell receptor (BCR) and Toll-like receptor 9 (TLR-9).
Percentages of memory Breg cells were decreased in patients with early SSc (mean ± SEM 1.85 ± 0.38%), those with established SSc (1.6 ± 0.88%), those with SSc-associated PF (1.52 ± 0.17%), and those with RA-associated PF (1.58 ± 0.26%), compared to healthy controls (6.3 ± 0.49%; each P < 0.001). Percentages of transitional Breg cells were also decreased. Expression of IL-10 by Breg cells after stimulation with TLR-9 was impaired in patients with SSc, particularly those with SSc-associated PF. Activation of STAT-3 and p38 MAPK was impaired in naive and memory B cells from patients with SSc after stimulation with BCR and TLR-9. Expression of the stimulatory CD19 receptor was increased in B cells and also increased, to a lesser extent, in Breg cells from patients with SSc compared to healthy controls. Percentages of memory B cells were decreased in patients with SSc, particularly in those with SSc-associated PF.
This is the first study to demonstrate that Breg cells are phenotypically and functionally impaired in patients with SSc. Furthermore, in SSc, B cells exhibit impaired p38 MAPK and STAT-3 activation upon stimulation with BCR and TLR-9. The findings of decreased numbers of Breg cells along with increased expression of CD19 support the idea of B cell autoaggression acting as an immunopathogenic mediator in SSc.
© 2016, American College of Rheumatology.