Lectins from opportunistic bacteria interact with acquired variable-region glycans of surface immunoglobulin in follicular lymphoma by Dunja Schneider, Marcus Dühren-von Minden, Alabbas Alkhatib, Corinna Setz, Cornelis A. M. van Bergen, Marco Benkißer-Petersen, Isabel Wilhelm, Sarah Villringer, Sergey Krysov, Graham Packham, Katja Zirlik, Winfried Römer, Christian Buske, Freda K. Stevenson, Hendrik Veelken, and Hassan Jumaa Blood Volume 125(21):3287-3296 May 21, 2015 ©2015 by American Society of Hematology

V-region sequences of FL receptors and B1-8 and Hy10 variants containing N-glycosylation sites. V-region sequences of FL receptors and B1-8 and Hy10 variants containing N-glycosylation sites. (A) V-region sequences of FL receptors are shown. The distinct framework region and CDR regions are indicated and positions of the acquired N-glycosylation sites are highlighted with arrowheads. (B) V-region sequences of B1-8 and Hy10 receptor variants containing FL-characteristic glycosylation sequons. N-glycosylation sites are indicated with arrowheads. All peptide sequence numbering without leader sequence. Figure generated using Geneious version 6 created by Biomatters. FR, framework region; VH, variable heavy, VL, variable light. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology

Mannose-terminating N-linked glycosylation of high-affinity receptor VH decreases antigen-affinity and antigen-induced BCR stimulation. Mannose-terminating N-linked glycosylation of high-affinity receptor VH decreases antigen-affinity and antigen-induced BCR stimulation. (A) Flow-cytometric measurement of surface expression of TKO cells expressing the indicated B1-8 receptor variants in comparison with empty-vector–transduced cells. (B-C) Surface binding profile of ConA (B) and NIP7-BSA (C) to B1-8 receptor and the indicated receptor variants compared with empty-vector–transduced TKO cells. Statistical analysis by nonparametric Mann-Whitney U test of NIP7-BSA binding is shown (mean; n = 5: B1-8 and N variants; n = 3: Q variants; **P < .01, *P < .05). (D-E) Kinetics of Ca2+ mobilization upon addition of OHT and the indicated stimuli after 1-minute baseline measurement in TKO cells expressing ERT2-SLP65 and the indicated B1-8 receptor variants measured in 1 tube after distinct eFluor670 labeling. (F) Flow-cytometric measurement of surface expression of TKO cells expressing the indicated Hy10 receptor variants. (G-H) Surface binding profile of ConA (G) and sHEL (H) to the indicated receptor variants compared with empty-vector–transduced TKO cells. Statistical analysis by nonparametric Mann-Whitney U test of sHEL binding is shown (mean; n = 5). (I-J) Kinetics of Ca2+ mobilization upon addition of OHT and the indicated stimuli after 1-minute baseline measurement in TKO cells expressing ERT2-SLP65 and the indicated Hy10 receptor variants or empty-vector–transduced cells measured in 1 tube after distinct eFluor670 labeling. Data are representative of >3 independent experiments. EV, empty vector. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology

FL receptors expressed in TKO cells are mannosylated on V-region glycosylation sites. FL receptors expressed in TKO cells are mannosylated on V-region glycosylation sites. (A) TKO cells were transduced with the respective HCs and LCs. Receptor surface expression of cYFP-positive cells expressing receptor FL A103, its V-region glycosylation-defective mutant FL A103gm, with glutamine replacing asparagine in the glycosylation sequon, the empty vector control, and control receptor BCR53 that was isolated from a healthy human mature B cell are shown. FACS analysis was performed using anti-human LC and anti-murine IgM antibodies. (B) FL receptor surface expression in TKO cells was compared with receptor surface expression on a primary FL sample. λ LC expression on IgM+-gated cells is shown. (C-D) Surface binding profile of indicated glycan-specific lectins to the designated receptors expressed on TKO cells. (E) ConA binding to FL receptor expressed on TKO cells in comparison with a primary FL sample. (F) Western blot analysis of total cell lysate and isolated surface proteins of TKO cells transduced with receptor FL A103 and FL A103gm subjected to glycosidase treatment as indicated. Size shift was analyzed by immunoblotting against anti-μHC. Data are representative of ≥3 independent experiments. EV, empty vector; TCL, total cell lysate. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology

DC-SIGN/Fc binding to FL receptor V-region mannosylation is not sufficient to induce Ca2+ influx. DC-SIGN/Fc binding to FL receptor V-region mannosylation is not sufficient to induce Ca2+ influx. (A-B) Lectin binding to transduced TKO cells in lectin buffer either in the absence (A) or in the presence (B) of Ca2+ was measured by FACS. (A) Gray shaded area represents binding to empty-vector–transduced cells in absence of Ca2+; dotted line represents binding to TKO cells expressing the indicated receptors in absence of Ca2+. (B) Gray shaded area shows binding to empty-vector–transduced cells in the presence of Ca2+; black line represents binding to receptor-positive cells in the presence of Ca2+. (C) FACS analyses of Ca2+ flux of TKO cells transduced with ERT2-SLP65 and FL receptors in lectin buffer containing 10 mM Ca2+. After 1-minute baseline measurement, cells were stimulated with OHT and DC-SIGN/Fc that was preincubated with anti-IgG for further multimerization. Anti-μHC stimulation served as control. (D-E) Lectin binding and Ca2+-flux measurements were carried out in medium supplemented with 1% FCS and physiological Ca2+-ion concentration. (D) Gray shaded area represents binding to empty-vector–transduced cells in medium; black line represents binding to TKO cells expressing the indicated receptors in medium (D) FACS analyses of Ca2+ flux of TKO cells transduced with ERT2-SLP65 and FL receptors in medium containing 1% FCS as described in (C). Results are representative of >3 independent experiments. EV, empty vector. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology

Binding of mannose-specific bacterial lectins stimulates FL receptors via V-region N-glycosylation. Binding of mannose-specific bacterial lectins stimulates FL receptors via V-region N-glycosylation. (A) Binding profile of bacterial lectins to FL-BCRs and the glycosylation-defective mutants expressed on TKO cells by FACS analyses. Empty-vector–transduced cells and BCR53+ cells served as controls. (B) Ca2+ mobilization of ERT2-SLP65+ TKO cells reconstituted with the indicated receptor variants upon stimulation with bacterial lectins or anti-BCR stimulation at the indicated concentrations in medium containing 1% FCS. (C) BCR surface expression on primary FL and HD PBMCs was acquired using anti-human CD19 and anti-human LC antibodies. Binding profile of Bc2L-A to CD19+- compared with CD19−-gated cells in FL sample from lymph node biopsy specimen or healthy donor control measured by FACS. (D) Ca2+-influx measurement of primary FL sample and healthy control PBMCs pregated on CD43− cells upon addition of the indicated stimuli after 1-minute baseline measurement. Experiments were performed in medium containing 1.5 mM Ca2+. (E) Mean fluorescence intensity values of Bc2L-A binding in CD19+ referred to CD19− cells revealed 2 FL subsets: binders and nonbinders (left). Statistical analysis of Ca2+-influx measurements of primary samples displayed as percentage of cells over threshold after Bc2L-A stimulation (FL binder, n = 4; FL nonbinder, n = 4; HD, n = 7; box and whiskers). *P = .0286 (binder vs nonbinder), P = .242 (binder vs HD), P = .5237 (nonbinder vs HD). Data are representative of >3 independent experiments. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology

Bacterial lectin binding induced Ca2+ flux in the human cell line WSU-FSCCL under physiological Ca2+ conditions. Bacterial lectin binding induced Ca2+ flux in the human cell line WSU-FSCCL under physiological Ca2+ conditions. (A) Flow-cytometric measurement of BCR surface expression on WSU-FSCCL and Ramos cell lines using anti-human IgM and anti-human κLC or λLC antibodies, respectively. (B) Surface binding profile of ConA to the indicated cells. (C) Western blot analysis of isolated surface proteins of the human cell lines subjected to glycosidase treatment as indicated. Size shift was analyzed by immunoblotting against anti-human μHC. (D) Binding profile of bacterial lectins to WSU-FSCCL and Ramos cell line cells measured by FACS. (E) Ca2+ mobilization of the indicated cell lines upon stimulation with bacterial lectins or anti-LC antibodies at the indicated concentrations. Data are representative of >3 independent experiments. Dunja Schneider et al. Blood 2015;125:3287-3296 ©2015 by American Society of Hematology