Multiple layers of B cell memory with different effector functions. Ismail Dogan, Barbara Bertocci, Valérie Vilmont, Frédéric Delbos,

Transcription

1 Multiple layers of B cell memory with different effector functions Ismail Dogan, Barbara Bertocci, Valérie Vilmont, Frédéric Delbos, Jérome Mégret, Sébastien Storck, Claude-Agnès Reynaud & Jean-Claude Weill 1

2 AID-Cre-EYFP WT IgM + PNA - IgM + PNA + IgG1 + PNA - IgG1 + PNA + IgM + PNA Number of mutations Number of sequences Number of unmutated sequences % of unmutated sequences Number of mutations per total sequences Number of mutations per mutated sequences Number of different VDJ junctions Mutation range Supplementary Table 1: Ig gene mutations in the B cell memory subsets. Mutations were determined at different time points after secondary immunization with SRBC in the memory subsets of AID-Cre-EYFP mice, and in the IgD IgM + GL7 CD73 + CD95 int subset from wildtype mice after secondary immunization with SRBC. 2

3 Flow cytometry clone dilution origine Primary antibodies B22-PB RA3-6B2 5 BectonDickinson CD138-biotin BecktonDickinson CD38-biotin 9 8 BecktonDickinson IgG1-PE X56 5μl/1 6 BecktonDickinson IgG1-APC X56 5 BecktonDickinson IgM-PECy7 R BecktonDickinson CXCR5-biotin 2G8 5 BecktonDickinson CD23-biotin B3B 2 BectonDickinson I-A b -PE AF BectonDickinson CXCR-PE 2B11 5 BectonDickinson CD73-PE TY/23 1 BectonDickinson CD95-PE Jo2 1 BectonDickinson CD8-APC 16-1A1 5 ebioscience CD69-PE H1.2F3 1 ebioscience CD62L-PE-Cy5 MEL-1 5 ebioscience B22-APC-A75 RA3-6B2 2 ebioscience GL7-biotin Ly-77 1 ebioscience PNA-biotin 2 Vector IgDbio/IgMbio Secondary reagents: SAV-PB 1 Molecular Probes SAV-PECy7 1 BectonDickinson Fc-block 2.G2 1 BectonDickinson Immunohistochemistry Primary antibodies CD-biot RM-5 5 BectonDickinson CD BectonDickinson Rabbit anti-gfp polyclonal 1 Clontech Chicken anti-gfp polyclonal 2 Abcam IgD-bio 1 B22-A67 RA3-6B2 1 BecktonDickinson Ki-67 B56 2 BecktonDickinson CD35-biotin 8C12 5 BecktonDickinson Secondary antibodies Goat anti-chicken-a88 polyclonal 5 Molecular Probes Goat anti-rabbit-a88/a555/a67 polyclonal 5 Molecular Probes Elispot Coating antibody IgG-A-M Goat-polyclonal 1 μg/ml Caltag-Invitrogen Secondary antibody: IgM-biotin Goat-polyclonal 1 μg/ml Southernbiotech IgG-biotin Goat-polyclonal 1 μg/ml Caltag-Invitrogen Tertiary reagent: 1 μg/ml HRP-Avidin D 5 μg/ml Vector Supplementary Table 2: Antibody list. Rat monoclonal antibodies were used if not specified 3

4 a) Exons ATG B2 STOP B2 Aicda locus probe 1 kb B2 B2 Cre- ERT2 Cre- ERT2 neo r Targeting construct B2 Targeted locus b) Kb WT neo r neo r Supplementary Figure 1: Generation of AID-Cre-ERT2 mice. (a) The Aicda locus was modified using a targeting construct in which exon 2 was replaced by the sequence coding for Cre-ERT2. Horizontal arrows indicate primers used to screen ES cells after homologous recombination, before and after neo r excision. Position of Bgl II (B2) restriction sites are indicated, as well as the probe used in (b) for Southern blot hybridization. (b) Southern blot analysis was performed after Bgl II digestion of DNA from the ES clone after (lane 1) and before (lane 2) neo r -excision and from WT ES cells (lane 3) using a 1 kb probe specific for the 3 untranslated region of AID.

5 Total EYFP + (x1 5 ) SRBC Control EYFP + IgM + PNA + (x1 5 ) EYFP + IgM + PNA - (x1 5 ) EYFP + IgG1 + PNA + (x1 5 ) EYFP + IgG1 + PNA - (x1 5 ) Supplementary Figure 2: Evolution of total EYFP + cells and EYFP + subsets in the spleen. Each point represents the number of cells x 1 5 per 1 8 spleen cells analyzed for one individual mouse. Black squares, SRBC immunized mice; red circles, control mice. Statistical significant differences are indicated (*, P <.5; **, P <.1). Means with standard deviations are included. 5

6 EYFP NIP EYFP + -gated CD95 CD69 I-Ab CD38 B22 GL7 GL7 NIP CD62L CD23 CXCR CXCR5 CD73 CD8 B22 + EYFP GL7 NIP B22 + EYFP + GL7 + NIP B22 + EYFP + GL7 NIP B22 + EYFP + GL7 NIP + Supplementary Figure 3: Surface phenotype of EYFP + subsets generated in a NP-CGG secondary response. The following subsets generated during NP-CGG immunization were analyzed for the surface markers indicated, 3 days after secondary antigenic challenge: B22 + EYFP GL7 (red), EYFP + GL7 + NIP (blue), EYFP + GL7 NIP (green), EYFP + GL7 NIP + (black). 6

7 Total cells EYFP +.15% 18% 51% Blood B22.6% 1%.5% IgG1 3% Bone marrow EYFP IgM Supplementary Figure : Presence of EYFP + IgM + and IgG 1 + PNA subsets in blood and bone marrow after secondary SRBC challenge. B22 + EYFP + cells, present in blood and bone marrow samples, 6 days after secondary SRBC immunization were analyzed according to surface IgM and IgG 1 criteria. A large fraction of B22 plasmocytes, showing brighter EYFP staining is visible in the bone marrow sample. Percentages of the gated populations are indicated. Data are representative of five experiments. 7

8 IgM secreting plasmocytes IgM + PNA + IgG + PNA + B cells IgG secreting plasmocytes Antigenic challenge Germinal center structures IgM layer IgM + PNA - IgM + IgG + PNA + PNA + Memory pool compartment IgG+ + PNA - IgG layer Supplementary Figure 5: Schematic representation of the splenic memory B cell response to SRBC. Four memory subsets are identified, located either in GC-like structures or in the memory B cell compartment. After a new SRBC challenge, most of IgM + B cells perpetuate the memory B cell pool with some differentiation into IgM-secreting plasmocytes, while IgG 1 + B cells mostly give rise to IgG 1 -secreting plasmocytes. 8

9 IgM + PNA IgM + PNA + IgG + PNA + IgG + PNA 6 (exp.1) 6 (exp.2) 9 18 (exp.1) 1/2 1/37 1/2 1/3 1/37 1/3 2/37 1/2 7/3 1/2 22/39 28/37 1/37 3/32 5/32 7/36 1/3 9/39 1/2 1/37 1/2 5/37 3/52 1/3 1/52 1/3 2/52 /3 nd 1/8 1/3 2/8 nd 18 (exp.2) 3/1 7/37 19/39 Supplementary Figure 6: Clonal relationships between EYFP + memory subsets in individual mice. Mice were analyzed 6, 9 and 18 days after secondary SRBC immunization. Rearranged J H sequences amplified from sorted EYFP + memory subsets were analyzed for the occurrence of shared VDJ junctions between subpopulations. Figures represents the number of sequences relative to the total number of sequences determined within a given subset that shows clonal relationship with another subset of the same mouse (marked by horizontal lines). nd means that no sequences were determined for this subset. Grey boxes highlight cases showing large clonal expansions, arbitrarily set at a value of 2% (i.e. one single clone identified by its VDJ junction and showing intraclonal diversity comprising more than 2% of sequences within this subset). Sequence data shown in Fig.7 and Supplementary Table 1 correspond to the first day 6-experiment and to the day 9 experiment analyzed here, while the day 18 experiment described in these Table and Figure did not show any clonal relationship between subsets, and is therefore not included. 9

10 a) NP-CGG day3 NP-CGG - day12 B22PNAEYFP b) μm μm c) 2 2 Tot IgG IgG-α-NP-BSA IgG-α-NP-CGG 25% 8% IgM + PNA IgG1 + PNA IgM + PNA + IgG1 + PNA + Total NIP+ Total NIP+ Total Total Supplementary Figure 7: NP-CGG immunization does not induce persistent germinal centers. a) Confocal analysis of spleen sections, 3 days and 12 days after NP-CGG immunization, using anti-b22 (blue), anti-eyfp (green) and PNA (red) labeling. (b) Hapten- vs. carrier-specific IgG secreting EYFP + B cells were determined by direct Elispot assay of sorted EYFP + cells, 8 days after a tertiary NP-CGG challenge, by analysis of total IgG, NP-CGG IgG or NP-BSA IgG secretion. Numbers of counted spots are indicated, with mean values when two wells were available for counting. Serial four-fold dilutions are represented. (c) Total numbers of EYFP + and NIP + EYFP + subpopulations per 1 8 spleen cells at 3 and 12 days after secondary immunization. Three mice were analyzed for each time point. 1

11 EYFP + cells per spleen (%) Number of tamoxifen ingestions Tamoxifen treatment 1 day Supplementary Figure 8: EYFP + labeling efficiency according to the timing of tamoxifen ingestion. The frequency of EYFP + cells arising after different protocols of tamoxifen ingestion was compared, according to the number of gavages performed during the primary (first figure) or the secondary immunization (second figure). The protocol used is squared in red. 11