BCR-signaling synergizes with TLR-signaling to induce AID and

immunoglobulin class-switching through the non-canonical NF-κB pathway

Egest J Pone1,*, Jinsong Zhang

2,*, Thach Mai1, Clayton A White

1, Guideng Li

1, John K Sakakura

1, Pina

J Patel1, Ahmed Al-Qahtani

3, Hong Zan1, Zhenming Xu

1,* and Paolo Casali1

1 Institute for immunology and School of Medicine, University of California, Irvine, CA 92697-4120.

Correspondence should be addressed to Paolo Casali (email: pcasali@uci.edu).

* These authors contributed equally to this work.

2 Present address: Duke Human Vaccine Institute, Duke University, Durham, NC 27710.

3 Present address: UAEU Faculty of Medicine, Al Ain, UAE.

SUPPLEMENTARY INFORMATION

Supplementary Figure S1 BCR crosslinking potentiates TLR-induced CSR to IgG1, IgG2a, IgG3 and IgA.

Supplementary Figure S2 BCR crosslinking upregulates TLR9-dependent CSR to IgG1, IgG2a, IgG3, IgA and IgE, as assessed by IgH transcripts involved in CSR.

Supplementary Figure S3 BCR crosslinking increases TLR-dependent CSR to IgG1, IgG2a, IgG3, IgA and IgE, as assessed by titration of Ig secreted by B cells stimulated with CpG or LPS.

Supplementary Figure S4 BAFF or APRIL moderately enhances TLR-dependent CSR and synergizes with BCR to induce CSR to IgA.

Supplementary Figure S5 BCR potentiates TLR-dependent CSR, upregulates B cell expression of PNA-binding glycoproteins, and inhibits plasmacytoid differentiation.

Supplementary Figure S6 BCR enhances activation of the canonical NF-κB pathway induced by lipid A in a p85α-dependent fashion.

Supplementary Figure S7 Kinetics of induction of the non-canonical NF-κB pathway in p85α–/– and p85α+/+ B cells.

Supplementary Figure S8 CSR induced by dual BCR/TLR engagement or LPS, but not CD40, is

impaired in p85α–/– B cells.

Supplementary Figure S9 NP conjugated to LPS induces high titers of class-switched NP-specific IgG2b and IgG3.

Supplementary Figure S10 Cell preparations used for CSR experiments contained more than 99% B220+ sIgδ+ cells.

Supplementary Table S1 Antibodies.

Supplementary Table S2 Oligonucleotide sequences.

Supplementary Figure S1 BCR crosslinking potentiates TLR-induced CSR to IgG1, IgG2a, IgG3 and

IgA. (a-f) Proportions of class-switched sIgG1+, sIgG2a+, sIgG3+ or sIgA+ B cells after stimulation of sIgδ+

B cells by Pam3CSK4 (100 ng/ml), lipid A (1 µg/ml), R-848 (30 ng/ml), CpG (1 µM), LPS (3 µg/ml) or

mCD154 (1 U/ml) with appropriate cytokines (IL-4, 3 ng/ml; IL-5, 3 ng/ml; IFNγ, 25 ng/ml; and/or TGFβ, 2 ng/ml) and anti–δ mAb/dex (100 ng/ml), as indicated. Data are representative of three independent experiments.

Supplementary Figure S2 BCR crosslinking upregulates TLR9-dependent CSR to IgG1, IgG2a, IgG3, IgA and IgE, as assessed by IgH transcripts involved in CSR. Levels of germline IH-CH transcripts, post-

recombination Iµ-CH transcripts and mature VHDJH-CH transcripts in B cells stimulated by CpG (1 µM) in

the absence (for Iγ3-Cγ3, VHDJH-Cγ3 and Iµ-Cγ3 transcripts) or presence of appropriate cytokines, i.e., 3

ng/ml of IL-4 (for Iγ1-Cγ1, Iµ-Cγ1 and VHDJH-Cγ1 transcripts, and Iε-Cε, Iµ-Cε and VHDJH-Cε transcripts),

25 ng/ml of IFNγ (for Iγ2a-Cγ2a, Iµ-Cγ2a and VHDJH-Cγ2a transcripts), and 3 ng/ml of IL-4, 3 ng/ml of IL-

5 plus 2 ng/ml of TGFβ (for Iα-Cα, Iµ-Cα and VHDJH-Cα transcripts), in the absence (teal) or presence (plum) of anti–δ mAb/dex (100 ng/ml ). Data were normalized to the level of Cd79b and are depicted as the ratio to the expression levels in B cells stimulated in the absence of anti–δ mAb/dex (mean and s.e.m. data from three independent experiments).

Supplementary Figure S3 BCR crosslinking increases TLR-dependent CSR to IgG1, IgG2a, IgG3, IgA and IgE, as assessed by titration of Ig secreted by B cells stimulated with CpG or LPS. (a-f) Secreted IgM, IgG1 (in the presence of 3 ng/ml of IL-4), IgE (in the presence of 3 ng/ml of IL-4), IgG2a (in the

presence of 25 ng/ml of IFNγ), IgG3 (in the presence of 25 ng/ml of IFNγ, except not added for LPS) and

IgA (in the presence of 3 ng/ml of IL-4, 3 ng/ml of IL-5 and 2 ng/ml of TGFβ) in supernatants from

cultures stimulated with nil, CpG (1 µM), LPS (3 µg/ml) or mCD154 (1 U/ml), in the absence (teal) or presence (plum) of anti–δ mAb/dex (100 ng/ml) (mean and s.d. of data from three independent experiments; the level of IgG2a, IgG3, IgA and IgE in supernatants of B cells cultured with nil and that of IgE in supernatants of B cells cultured with CpG and IL-4 were below the detection limit of the specific assays used).

Supplementary Figure S4 BAFF or APRIL moderately enhances TLR-dependent CSR and synergizes

with BCR to induce CSR to IgA. (a) Proportions of sIgG1+ B cells after stimulation of Igδ+ B cells by lipid

A (1 µg/ml), LPS (3 µg/ml), CpG (1 µM) or mCD154 (1 U/ml) plus nil, BAFF (250 ng/ml), APRIL (250 ng/ml) or anti–δ mAb/dex (100 ng/ml) (all in the presence of 3 ng/ml of IL-4). (b) Proportions of sIgG1+ B

cells after stimulation of CFSE-labeled sIgδ+ B cells by lipid A, CpG, BAFF or APRIL, at indicated doses, in the absence or presence of anti–δ mAb/dex at indicated doses (all in the presence of 3 ng/ml of IL-4).

(c) Proportions of sIgA+ B cells after stimulation of CFSE-labeled sIgδ+ B cells by BAFF (250 ng/ml) or

APRIL (250 ng/ml) plus IL-4 (3 ng/ml), IL-5 (3 ng/ml) and TGFβ, (2 ng/ml) in the absence or presence of anti–δ mAb/dex (100 ng/ml). Data are representative of three independent experiments.

Supplementary Figure S5 BCR potentiates TLR-dependent CSR, upregulates B cell expression of PNA-binding glycoproteins, and inhibits plasmacytoid differentiation. (a) Proportions of sIgG1+ B cells

after stimulation of sIgδ+ B cells by CpG (1 µM), or the weak TLR9-agonist GpC (1 µM), and anti–δ mAb/dex (100 ng/ml), in the absence or presence of the endosomal TLR inhibitor chloroquine (all in the presence of 3 ng/ml of IL-4). (b) Proportions of CD138+ plasmacytoid cells/plasmacytes after stimulation

of CFSE-labeled sIgδ+ B cells by LPS (3 µg/ml) or mCD154 (1 U/ml) plus IL-4 (3 ng/ml) in the absence or presence of anti–δ mAb/dex (100 ng/ml) (top panels), and proportions of CD138+ cells at each cell

division (bottom panels). (c) Proportions of B220+ PNAhi B cells after stimulation of sIgδ+ B cells by nil,

CpG (1 µM), LPS (3 µg/ml) or mCD154 (1 U/ml) in the absence (teal) or presence (plum) of anti–δ mAb/dex (100 ng/ml), all in the presence of IL-4 (3 ng/ml). Data are representative of three independent experiments.

Supplementary Figure S6 BCR enhances activation of the canonical NF-κB pathway induced by lipid A

in a p85α-dependent fashion. (a,b) Levels of phosphorylated IκBα, total IκBα, phosphorylated p65, total

p65 and β-actin in p85α+/+ B cells stimulated by anti–δ mAb/dex (100 ng/ml, gray), or lipid A (1 µg/ml, plum) in the absence (open symbols) or presence (filled symbols) of anti–δ mAb/dex (100 ng/ml), plus IL-

4 (3 ng/ml) (a, left panels) or in p85α+/+ (plum) and p85α–/– (teal) B cells stimulated by lipid A (1 µg/ml)

and anti–δ mAb/dex (100 ng/ml) plus IL-4 (3 ng/ml) (b, left panels). The levels of phosphorylated IκBα

and p65 were normalized to the levels of total IκBα and p65, respectively, and data are depicted as the ratio to the levels in B cells prior to stimulation (0 m; set as 1, right panels).

Supplementary Figure S7 Kinetics of induction of the non-canonical NF-κB pathway in p85α–/– and

p85α+/+ B cells. (a) Induction of p52 and p100 in B cells stimulated with anti–δ mAb/dex (100 ng/ml, plum

color), lipid A (1 µg/ml, green diamond symbols) or CpG (1 µM, blue square symbols) in the absence

(open symbols) or presence (closed symbols) of anti–δ mAb/dex, LPS (3 µg/ml, red circle symbols) or mCD154 (1 U/ml, black triangle symbols) (all in the presence of 3 ng/ml of IL-4) for 0, 3, 24 and 48 h (top

panels). The levels of p52 and p100 were normalized to the level of β-actin and data are depicted as the ratio to the expression levels in B cells prior to stimulation (0 h, set as 1; bottom panels). Data are

representative of three independent experiments. (b) Levels of p52, p100 and β-actin in B cells

stimulated with anti–δ mAb/dex (100 ng/ml), lipid A (1 µg/ml) or CpG (1 µM) in the absence or presence

of anti–δ mAb/dex, LPS (3 µg/ml) or mCD154 (1 U/ml) (all in the presence of 3 ng/ml of IL-4) for 48 h in

p85α–/– (teal) and p85α+/+ (plum) B cells. Levels of p52 and p100 were normalized to the level of β-actin and data are depicted as the ratio to the expression levels in unstimulated (nil) B cells (set as 1; right panels).

Supplementary Figure S8 CSR induced by dual BCR/TLR engagement or LPS, but not CD40, is

impaired in p85α–/– B cells. (a-d) Proportions of sIgG1+, sIgG2a+, sIgG3+ or sIgA+ B cells after

stimulation of sIgδ+ p85α–/– B cells and their p85α+/+ counterparts by lipid A (1 µg/ml) or CpG (1 µM) plus

anti–δ mAb/dex (100 ng/ml), or LPS (3 µg/ml) or mCD154 (1 U/ml) in the absence or presence of anti–δ

mAb/dex (100 ng/ml), in the presence of indicated cytokines (IL-4 (3 ng/ml), IL-5 (3 ng/ml), IFNγ (25

ng/ml) and TGFβ (2 ng/ml)) . Data are representative of three independent experiments.

Supplementary Figure S9 NP conjugated to LPS induces high titers of class-switched NP-specific IgG2b and IgG3. (a, b) Titers (absorbance at O.D.492 nm) of NP-specific IgM, IgG1, IgG2a, IgG2b and IgG3 Abs in mice 6 d (a) and 12 d (b) after injection with NP-LPS, NP26 or LPS (n = 3; mean and s.d.; P values: t-test of titers of 1:1000 diluted IgM, IgG1, IgG2a, IgG2b and IgG3 in mice immunized with NP-LPS and in those immunized with NP26; *, P < 0.05). IgM, possibly with moderate affinity for NP, could be captured by NP3-BSA owing to their decavalency and high avidity.

Supplementary Figure S10 Cell preparations used for CSR experiments contained more than 99% B220+ sIgδ+ cells. B cells were purified from mouse lymph nodes using the Miltenyi “Mouse B cell isolation kit” following manufacturer’s instructions. Briefly, single B cell suspension in isolation buffer

(PBS, 0.5% BSA, 2mM EDTA) (40 µl per 107 cells). Cells were mixed with a cocktail of biotin-labelled

anti–CD43, anti–CD4 and anti–Ter119 mAbs (10 µl per 107 cells – in most experiments, the kit biotinylated mAbs were supplemented with biotinylated mAb to CD11c (clone N418, eBioscience) and to

IgG1 (clone A85-1, BD Biosciences)) for 15 m, followed by addition of isolation buffer (30 µl per 107 cells)

and anti–biotin magnetic MicroBeads (20 µl per 107 cells). After incubation for 15 m, cells were washed with isolation buffer (3 ml per 107 cells), pelleted by centrifugation at 300 g for 10 m and then

resuspended in isolation buffer (500 µl per 107 cells) for application to a midiMACS column. The column was then placed in a midiMACS magnet and non-bound cells were eluted by washing 3 times with 3 ml of isolation buffer. The eluted cells (i.e., negatively selected) were pelleted by centrifugation as above and resuspended in 3 ml of isolation buffer (for further analysis) or RPMI medium (for in vitro culturing). All steps were carried out on ice with pre-chilled buffers. Lymphocytes before and after purification were

analyzed for surface expression of B220, CD3, CD11c, Igδ and Igµ; in this preparation, 99.4% of the cells purified from lymph nodes were B220+ cells and 99.7% of the B220+ cells were sIgδ+ cells.