title: CD22 ligation inhibits downstream B cell receptor signaling and Ca(2+)  flux upon activation.
abstract: OBJECTIVE
  CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation.
  
  
  METHODS
  Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1).
  
  
  RESULTS
  Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling.
  
  
  CONCLUSIONS
  These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases.
authors.affiliation: Charité University Medicine Berlin, Berlin, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Sieger
authors.forename: S J
authors.initials: SJ
authors.lastname: Fleischer
authors.forename: H E
authors.initials: HE
authors.lastname: Mei
authors.forename: K
authors.initials: K
authors.lastname: Reiter
authors.forename: A
authors.initials: A
authors.lastname: Shock
authors.forename: G R
authors.initials: GR
authors.lastname: Burmester
authors.forename: C
authors.initials: C
authors.lastname: Daridon
authors.forename: T
authors.initials: T
authors.lastname: Dörner
mesh_terms: D000328:Adult; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D001327:Autoimmune Diseases / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051925:CD79 Antigens / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007074:Immunoglobulin G / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000276:immunology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000378:metabolism; D055815:Young Adult
chemical_list: D061067:Antibodies, Monoclonal, Humanized; C497867:CD22 protein, human; D051925:CD79 Antigens; C497881:CD79A protein, human; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D051918:Sialic Acid Binding Ig-like Lectin 2; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; C448700:epratuzumab; D020794:Receptor Protein-Tyrosine Kinases; C084944:TYRO3 protein, human; D051966:Phospholipase C gamma; D002118:Calcium