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Author Notes:

Charles J. Dimitroff: cdimitroff@bwh.harvard.edu

NG and CD conceived the study; NG and AA performed the experiments and analyzed the data; NG, SB, and HW generated the ST3Gal1 cell lines; MK and RC provided technical assistance and expertise with CORA O-glycomics analysis; AA and SH performed O-glycomic analyses; GL assisted with tonsil tissue acquisition; NG, AA, JL, JG, SK, AD, SB, HW, SH, and CD contributed intellectually to the study; SH and AD supervised MS glycomics assessments; CD supervised the entire study. NG, AA, SH, and CD wrote the manuscript.

The authors thank Dr. Shiv Pillai (Ragon Institute of MGH, MIT, and Harvard) for providing Ramos and Raji B cell lines; Dr. Alan Epstein (USC Keck School of Medicine) for providing SUDHL-4 B cells; Dr. Ronald L. Schnaar for providing D,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl (PPPP) glycolipid inhibitor reagent; Chad Araneo at the Harvard Division of Immunology Flow Cytometry Core for assistance with cell sorting; and Drs. Galit Alter, W. Nicholas Haining, Michael Carroll, and Shiv Pillai for many helpful discussions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Research Funding:

This research was funded by an American Association of Immunologists Careers in Immunology Fellowship (to NG and CD); an Albert J. Ryan foundation fellowship (to NG); NIH grant NIH/NIAID R21AI125476 (to CD) ; and NIH/NCI R01 CA173610 (to CD); a Young Investigator Award from the Merck-Melanoma Research Alliance (to SB); a Research Scholar Grant from the V Foundation for Cancer Research (to SB); a Melanoma Research Scholar Award from the Rochester Melanoma Action Group/Outrun the Sun (to SB); a Biotechnology and Biological Sciences Research Council grant BBF0083091 (AD and SH) and BBK0161641 (AD and SH); and a Wellcome Trust grant (082098 to AD).

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Immunology
  • glycosylation
  • glycan
  • B cell
  • CD45
  • peanut lectin
  • PNA
  • ST3Gal1
  • GCNT1
  • PEANUT LECTIN BINDING
  • CD8(+) T-CELLS
  • CD45 ISOFORM
  • GERMINAL-CENTERS
  • MURINE B220
  • GLYCOSYLATION
  • ACTIVATION
  • EXPRESSION
  • MOUSE
  • SPECIFICITY

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

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Journal Title:

Frontiers in Immunology

Volume:

Volume 9

Publisher:

, Pages 2857-2857

Type of Work:

Article | Final Publisher PDF

Abstract:

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

Copyright information:

Copyright © 2018 Giovannone, Antonopoulos, Liang, Geddes Sweeney, Kudelka, King, Lee, Cummings, Dell, Barthel, Widlund, Haslam and Dimitroff.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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