immunology.sciencemag.org/cgi/content/full/4/39/eaax4453/DC1

Supplementary Materials for

Lin28b controls a neonatal to adult switch in B cell positive selection

Stijn Vanhee, Hugo Åkerstrand, Trine Ahn Kristiansen, Sebak Datta, Giorgia Montano, Stefano Vergani, Stefan Lang,

Jonas Ungerbäck, Alexander Doyle, Karin Olsson, Giulia Beneventi, Christina T. Jensen, Cristian Bellodi, Shamit Soneji, Mikael Sigvardsson, Elin Jaensson Gyllenbäck, Joan Yuan*

*Corresponding author. Email: joan.yuan@med.lu.se

Published 27 September 2019, Sci. Immunol. 4, eaax4453 (2019)

DOI: 10.1126/sciimmunol.aax4453

The PDF file includes:

Fig. S1. Gating strategies for developing B cells. Fig. S2. Cell cycle analysis of tet-Lin28b ABM pre-B and ImmB subsets. Fig. S3. Determination of the minimal DOX treatment window during B cell maturation required for efficient CD5+ B-1 cell output from tet-Lin28b ABM. Fig. S4. Barcode filtering and analysis strategy. Fig. S5. Differentially expressed genes within top enriched Hallmark gene sets. Fig. S6. Genetic evidence implicating Lin28b in the CD19/PI3K/c-Myc pathway. Fig. S7. Transient tet-Lin28b expression of ABM progenitors promotes the output of functionally competent CD5+ B-1 cells. Table S2. Antibodies used in this study.

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/4/39/eaax4453/DC1)

Data file S1 (Microsoft Excel format). Table S1: Raw data supplement. Data file S2 (.gz format). VDJseq PerC B-1 CD5 slices. Data file S3 (Microsoft Excel format). DESeq2 normalized counts for adult tet-Lin28b and WT ImmB RNA-seq. Data file S4 (Microsoft Excel format). DESeq2 normalized counts for neonatal Lin28b+/+, Lin28b–/– ImmB RNA-seq.

Supplementary figures

Fig. S1. Gating strategies for developing B cells. (Related to Figure 2 and Figure 3) (A)

Gating strategy for pro-B, pre-B and ImmB cells in bone marrow of neonatal and adult mice.

Lineage panel: Ter119–CD11b

–Gr1

–CD3e

–. (B-C) FACS analysis of CD5 levels on splenic T1

cells (Lin–CD19

+CD93

+CD23

–IgM

+) in 2-day-old neonatal or adult mice of the indicated

genotypes (D) Adult mice of the indicated genotypes upon 4 weeks of DOX diet treatment.

Lineage panel: Ter119–CD11b

–Gr1

–CD3e

–.

Fig. S2. Cell cycle analysis of tet-Lin28b ABM pre-B and ImmB subsets. (related to

Figure 3) Data were obtained from mice kept on a DOX diet for 4 weeks. (A) Edu uptake

FACS analysis of the indicated cell types 2 hours following one single dose of intraperitoneal

EdU injection. (B) Cell cycle analysis using Ki-67 of the indicated cell types. (C)

Quantification of data in B. (D) Absolute cell numbers of the indicated populations per 2 hind

legs. Data from 3 independent experiments. ns= not significant, *=P≤ 0.05.

Fig. S3. Determination of the minimal DOX treatment window during B cell maturation

required for efficient CD5+ B-1 cell output from tet-Lin28b ABM. (Related to Figure 3)

(A) Schematic of adoptive transfer experimental set up. (B) Readout of PerC B-1 cell CD5

expression of recipients adoptively transferred with the indicated cell types FACS sorted from

the indicated donor sources with the indicated DOX treatment regimens at 3 weeks post

transfer. (C) Schematic summarizing the data presented, showing that DOX treatment

between the pro-B and immB stages is required for robust CD5+ B-1 cell output by tet-Lin28b

ABM.

Fig. S4. Barcode filtering and analysis strategy. (Related to Figure 4) (A) Schematic of

Barcode analysis strategy for data shown in Figure 4. (B) Stacked bar graphs represent read

frequencies retrieved from the indicated populations by deep sequencing. Results from

technical PCR replicates are shown side by side. Number on top of the bars indicate the

number of unique high confidence barcodes detected in both replicates. (C) The absolute

number of barcoded B cells sorted from recipients in Figure 4 by FACS. (D) The total number

of unique high confidence barcodes detected. (E) Equation showing calculation used to

enumerate barcode complexity in Figure 4C.

Fig. S5. Differentially expressed genes within top enriched Hallmark gene sets. (Related

to Figure 5) Heatmaps showing row z-scored expression values of genes in the indicated

hallmark gene sets obtained from RNAseq of WT and tet-Lin28b immB cells.

Fig. S6. Genetic evidence implicating Lin28b in the CD19/PI3K/c-Myc pathway. (Related

to Figure 6) (A) Representative FACS plots depicting Kappa and Lambda light chain usage in

ABM ImmB cells of the indicated genotypes. (B) Quantification of data in A. (C)

Quantification of relative FSC of ABM ImmB cells of the indicated genotypes. Data are

shown relative to a representative WT sample in each experiment. (D) Histogram of CD5

expression on ABM ImmB cells of the indicated genotypes. (E) Quantification of data shown

in D. Data are shown relative to a representative WT sample in each experiment. (F) CD19

surface levels of ABM ImmB cells as determined by FACS. (A – F) n=5, 2 independent

experiments. Data are shown relative to a representative WT sample in each experiment. (G)

Representative western blot showing murine Lin28b levels in lineage depleted (Ter119–

CD11b–Gr1

–CD3e

–) spleen cells from the indicated genotypes. Human ESC and murine ESC

lysates were used as negative and positive controls for the antibody. (H) qPCR showing

relative expression of c-Myc and murine Lin28b in immB cells of the indicated genotypes. (I)

Frequency and (J) absolute number of peritoneal cavity B-1 cells of mice fed DOX food for 3

weeks. (n=4-7 from 3 independent experiments). (K) Representative FACS plots of data in I.

(L) Frequency of ABM ImmB cells from mice fed DOX food for 3 weeks (n=4-5, from 3

independent experiments). ns= not significant, ***=P≤ 0.001, ****=P≤ 0.001. nd= not

detected.

Fig. S7. Transient tet-Lin28b expression of ABM progenitors promotes the output of

functionally competent CD5+ B-1 cells. (Related to Figure 7) (A) End-point western blot

analysis for tet-Lin28b transgene expression in total splenocytes of recipient mice receiving

the indicated DOX treatment regimens. Lysates from two representative recipient mice are

shown for each condition. (B) Left panel: Frequency CD5+ T1 B cells out of total splenic B

cells from the indicated recipients as assessed by FACS. Right panel: Frequency of CD5+ B-1

cells out of total peritoneal cavity B cells from the indicated recipients as assessed by FACS.

Data points represent individual mice. Colors represent experimental replicates. (C)

Representative FACS analysis of donor derived B cells from Rag1KO recipient at the

endpoint of the DOX-STOP set-up (Figure 7A). (D) CD5 levels of total B-1 populations gated

in C. (E) Compilation of CD5 surface levels on total B-1 cells as gated in C. Levels are shown

as the relative fluorescence intensity (RFI), when compared to control B-1 cells from an

untransplanted WT adult mouse (n=8-14 biological replicates, from 3 independent

experiments). (F) CD5 levels on the indicated populations following 36hr 10ug/ml LPS

stimulation in vitro. (G) TdT levels in ABM pro-B cells from WT or tet-Lin28b mice fed

DOX diet for 7 days. (H) IL-10 production by the indicated populations as determined by

ELISA following 36hr of 10ug/ml LPS stimulation (n=2-3 biological replicates for LPS

treated samples, from 2 independent experiments). As a reference, IL-10 production from

non-stimulated samples is shown. (I) Spontaneous IgM production by splenic CD5+ B-1 and

B-2 populations from the indicated mice as determined by ELISPOT (n=3-5 biological

replicates, from 2 independent experiments). (J) Graphical abstract. Ns = not significant,

*=P≤ 0.05, **=P≤ 0.01, ***=P≤ 0.001. Error bars show the standard deviation of the mean.

Table S2. Antibodies used in this study.

Antigen Fluorochrome Clone Manufacturer Reference #

7-AAD 7-AAD - Sigma-Aldrich

SML1633-

1ML

αTubulin Purified 11H10

Cell Signaling

technologies 2125

B220 APCCy7 ra3-6B2 Biolegend 103224

B220 APC ra3-6B2 Biolegend 103212

B220 Biotin ra3-6B2 Biolegend 103204

B220 PeCy5 ra3-6B2 Biolegend 103210

B220 PE-CF594 RA3-6B2 BD biosciences 562313

β-Actin purified AC-15 Sigma-Aldrich A1978

CD11b APC M1/70 BioLegend 101212

CD11b Biotin M1/70 Biolegend 101204

CD11b PeCy5 M1/70 Biolegend 101210

CD150 BV605

TC15-

12F12.2 Biolegend 115927

CD19 BV786 1D3 BD biosciences 563333

CD19 BV650 6D5 Biolegend 115541

CD19 APC 1D3 BD Pharmingen 550992

CD1d PE 1B1 Biolegend 123509

CD1d PerCP-Cy5.5 1B1 Biolegend 123514

CD23 PECy7 B3B4 Biolegend 101613

CD23 APC 2G8 Abcam ab25457

CD24 PECy7 M1/69 Biolegend 101821

CD25 PE eFluor610 PC61.5 eBioscience 61-0251-80

CD3e Bio 145-2C11 Biolegend 100304

CD3e PeCy5 145-2C11 Biolegend 100310

CD3e BV711 145-2C11 BD biosciences 563123

CD43 FITC S7 BD Pharmingen 553270

CD43 BV605 S7 BD biosciences 563205

CD43 PE S7 BD biosciences 561857

CD45.1 FITC A20 Biolegend 110706

CD45.2 APC 104 BD Pharmingen 561875

CD48 APCCy7 HM48-1 Biolegend 103431

CD5 BV421 53-7.3 BD biosciences 562739

CD5 PE 53-7.3 Biolegend 100607

CD93 APC AA4.1 Biolegend 136509

CD93 BV421 AA4.1 BD biosciences 562739

CD93 Biotin AA4.1 eBioscience 13-5892-82

cMYC purified Y69 Abcam ab32072

ckit BV421 2B8 BD biosciences 562609

ckit APC 2B8 Biolegend 105812

DAPI DAPI - Invitrogen D1306

F(ab')2-Goat anti-

Rabbit IgG (H+L)

Cross-Adsorbed

Secondary Antibody

Alexa Fluor

594 Polyclonal Invitrogen A-11072

Flt3 BV421 A2F10.1 BD biosciences 562898

Gr1 Bio RB6-8C5 Biolegend 108404

Gr1 PeCy5 RB6-8C5 Biolegend 108410

human Lin28b Purified Polyclonal

Cell Signaling

technologies 4196

IgM BV711 R6-60-2 BD biosciences 564026

IgM FITC II/41 BD biosciences 553437

IgM PE RMM1 SONY Biotech 2632540

Ig Kappa light chain PerCP-Cy5.5 187.1 BD biosciences 560668

Ig Lambda1, 2 & 3

light chain Biotin R24-46 BD biosciences 553433

murine Lin28b Purified Polyclonal Abcam ab71415

PtC Marina blue

DOPC/CHOL

Liposomes Formumax

F60103F-

MB

PtC

Fluorescein

DHPE

DOPC/CHOL

Liposomes Formumax F60103F-F

Phospho-GSK-3β

(Ser9) Purified D85E12

Cell Signaling

technologies 5558

Phospho-PDK1

(Ser241) Purified C49H2

Cell Signaling

technologies 3438

pS6 (Ser235/236) PE D57.2.2E

Cell Signaling

technologies 5316

pS6 (Ser235/236) AlexaFluor647 D57.2.2E

Cell Signaling

technologies 4851

SA-APCCy7 APCCy7 streptavidin Biolegend 405208

SA-FITC FITC streptavidin BD biosciences 554060

SA-Texas Red Texas Red streptavidin BD biosciences 551487

SA eFluor® 450 eFluor® 450 streptavidin eBioscience 48-4317-82

Sca1 PeCy7 D7 Biolegend 108114

Tdt PE 19-3 eBioscience 12-5846-80

Ter119 Bio TER-119 RB6-8C5 116204

Ter119 PeCy5 TER-119 Biolegend 116210

VeriBlot for IP

Detection Reagent

(HRP) Purified - Abcam ab131366