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.

Patients with heterotaxy syndrome, commonly associated with complex congenital heart disease (CHD), exhibit a higher risk of severe bacterial infection (SBI). We sought to define the change of a novel immunologic marker, the immunoglobulin M (IgM) memory B-cell percentage, and its association with SBI.
We enrolled 46 (M/F 29/17) heterotaxy syndrome patients (42 right atrial isomerism (RAI) and 4 left atrial isomerism (LAI)) aged > 1 y during the period 2010-2012 in a tertiary care center. We analyzed IgM(+)CD27(+) memory B-cell percentages. Patients with simple and complex CHD served as controls.
The mean IgM memory B-cell percentages were the lowest in the heterotaxy syndrome group, compared with those in complex and simple CHD groups (1.8 ± 2.1 vs. 3.9 ± 3.2 vs. 5.1 ± 4.7, P < 0.001). In the heterotaxy syndrome group, 41.3% had low IgM memory B-cell percentages (<1% of B cells). Seven had a history of community-acquired SBI and 85.7% of these had low IgM memory B-cell percentages, which was the only significant factors related to community-acquired SBI (P = 0.028).
The memory B cell and IgM memory B-cell percentages are low in patients with heterotaxy syndrome, and the presence of IgM memory B-cell percentage < 1% correlates with community-acquired SBI.