Supplementary Figure 1. Markedly decreased numbers of marginal zone B cells in DOCK8 mutant mice Supplementary Figure 2.

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Marian Holmes
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1 Supplementary Figure 1. Markedly decreased numbers of marginal zone B cells in DOCK8 mutant mice. Percentage of marginal zone B cells in the spleen of wild-type mice (+/+), mice homozygous for cpm or pri mutations, and cpm/pri compound heterozygous animals. The latter show the failure of the two alleles to complement one another to form marginal zone B cells. Columns are arithmetic means, error bars standard error of the mean, and dots individual mice. Results of three independent experiments. MZP MZ ns * * ** * ns ** ns Supplementary Figure 2. Marginal zone B cell defect not corrected by overexpression of BAFF. Absence of marginal zone B cells is not corrected in bone marrow chimeras constructed with bone marrow and irradiated BAFF-transgenic mice. Note that the overexpression of BAFF increases the marginal zone subset in chimeras with wildtype bone marrow. Columns are arithmetic means, error bars standard error of the mean, and dots individual mice. Statistical analysis by unpaired, two-tailed t test with Welch s correction, ns, not significant;*,p<0.05, **,P<0.01. Experiment performed once.

2 Supplementary Figure 3. Mutation maps to chromosome 19. Key recombinant haplotypes, SNP markers, chromosomal locations, and corresponding phenotypes of informative (B6xCBA)F 2 intercross cpm animals.

3 B220+ B cells CD4+ CD8+ T cells thymus Supplementary Figure 4. DOCK8 is highly expressed in cells of the immune system. Relative expression of DOCK8 mrna in the indicated mouse tissues, as determined by GNF SYMATLAS Affymetrix microarray profiling data (Su AI et al 2002 Proc Natl Acad Sci USA 99: ;

4 d2.5 d3.5 d4.5 +/+ * Supplementary Figure 5. Location of SW HEL B cells day after adoptive transfer. Histochemical staining for SW HEL B cells of the indicated genotypes in spleen on day 2.5, 3.5 or 4.5 after adoptive transfer and immunization with HEL 2x - SRBC or unconjugated SRBC (*). Blue, HEL binding; Brown, IgD.

5 +/+ * srbc Supplementary Figure 6. Adoptive transfer spleen or lymph node SW HEL cells. The primurus mutation results in a comparable decrease in SW HEL B cell accumulation in GCs on day 4.5 after HEL 2X -SRBC immunization, when either spleen or lymph node cells are adoptively transferred.

6 wildtype SWHEL GC B cells: average # mut silent mutations Mutations in CDR2: r 31 total r s # sequences Total mutations CDR2 D V Q L Q E S G P S L V K P S Q T L S L T C S V T G D S I T S D Y W S W I R K F P G N R L E Y M G Y V S Y S G S T Y Y N P S L K S R I S I T R D T S K N Q Y Y L D L N S V T T E D T A T Y Y C A N W D G D Y W G Q G T L V T V S A Single cell product total r s WT3-C R N S C WT3-C F WT3-C F F T D WT3-E N N WT3-E WT4-A F C N F A n WT2-B A D l H I WT2-B P WT2-B Y q S WT2-B C N N I WT2-E C WT4-A R - WT4A WT4-B g s I F C WT1-D WT1-D s F a WT1-D K r F N k WT1-D e N F A WT1-D WT1-E WT1-E N WT2-E N S G H t y WT2-E WT2-E WT2-E WT2-E D R y WT3-B WT3-B N D T C P WT3-D T D D S WT3-D L R WT3-D s C WT3-D K e H D WT3-D T T S g G Y WT4-A WT4-D N P WT4-E N WT4-E Y WT4-E Sums: # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Mutations per residue: r s C 82B 82A SWHEL GC B cells: total r s s 3 # sequences Total mutations average # mut CDR2 D V Q L Q E S G P S L V K P S Q T L S L T C S V T G D S I T S D Y W S W I R K F P G N R L E Y M G Y V S Y S G S T Y Y N P S L K S R I S I T R D T S K N Q Y Y L D L N S V T T E D T A T Y Y C A N W D G D Y W G Q G T L V T V S A Single cell product total r s P4-A P4-A P4-A P4-C P3-A s P3-A P3-A P3-C P3-C P3-E P N F P1-D V i F g N y t L P1-D s D s V P1-E p S P1-F P2-B2 * P2-B g P2-B V t I I C P2-C s Y P1-A v T S P1-B G R H t P1-B s N T p S y H P1-B F P1-B P2-G D P2-F H I N s M A P2-F F C S Y P2-F P2-E R C s t P2-E P2-D s F P3-E P3-E L g N N S P3-E I v F F S P3-G P3-G P4-D C e v R T H l G P4-D D T P4-E i P4-E N N S P4-E P4-E A N R T P4-G P P4-G V v Y Y P1-G Sums # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Mutations per residue: r s C 82B 82A Mutations in CDR2: r 24 s 10 Supplementary Figure 7. Heavy chain VDJ-region mutations in flow-sorted single germinal center wildtype or SWHEL B cells. SWHEL GC cells were sorted on day 9 after HEL2x-SRBC immunization from four different recipients of wildtype SWHEL cells (prefix WT1 - WT4) and four different recipients of SWHEL cells (prefix P1-P4). The SWHEL heavy chain V region was amplified by nested PCR from single cells, and the PCR products directly sequenced without cloning. Silent (s) and replacement (r ) mutations are shown. Heavy chain residues 50, 52, 53, 54, 56 and 58 make contacts with HEL on or next to D101. At this stage of the response the recurrent Y53D mutation observed on day 15 has yet to become dominant in wildtype SWHEL GC cells. Recurrent Y58F mutations are observed prior to and independently of Y53D in normal SWHEL cells (R Brink et al unpublished data), and these are evident in six wildtype SWHEL GC cells at day 9. X-ray structural comparisons show that the Y58F substitution increases the buried apolar surface between antibody and antigen, explaining increased HEL-affinity in the related antibody HyHEL8 compared to HyHEL10 SwHEL (Li Y et al Nat Struct Biol :482. X-ray snapshots of the maturation of an antibody response to a protein antigen).

+/+ 10 5 Counts 10 4 10 3 Stimulus: Anti-IgM (!g/ml) IC10 (!g/ml)! 1 10 10!!! 10 Supplementary Figure 8. Assessment of proliferation by DNA synthesis.

7 +/ Counts Stimulus: Anti-IgM (!g/ml) IC10 (!g/ml)! !!! 10 Supplementary Figure 8. Assessment of proliferation by DNA synthesis. DNA synthesis by cultured lymph node cells from four individual wild-type and four homozygous animals. The cells from each mouse were cultured in triplicate wells, either unstimulated (US), or with the indicated concentrations of antibody to IgM and/or antibody to CD40 (1C10). Each culture was pulsed with 3H-thymidine after 36 hours, harvested, and the mean and standard error of incorporated radioactivity (counts) in triplicates from individual animals expressed by columns and error bars, respectively.

8 Stimulus: Anti-IgM (!g/ml): cpm/cpm cpm/cpm LPS CFSE 1.0 Supplementary Figure 9. Proliferation assessed by CFSE. Mutant or cpm/cpm cells (red histograms) were CFSE labelled and cultured with wild-type, congenically cells (black histograms) in the same wells to control for experimental variation. Each panel shows an independent pair of mutant and wild-type samples co-cultured. Each independent sample was from separate mice, and typically pooled from two or more animals. In 6 of the 8 samples the CFSE dilution profiles of mutant and wild-type were superimposable, and in 2 there was a subtle decrease in CFSE dilution in mutant cells.

9 Supplementary Figure 10. Expression of activation markers. Median and s.e. of cell surface fluorescence staining for activation markers CD25, CD69 and CD86 on lymph node B cells cultured from four individual wild-type and four homozygous animals. The cells from each mouse were cultured either unstimulated (US), or with the indicated concentrations of antibody to IgM and CD40 (1C10). Each culture was harvested after 18 hr, stained for B220 and the indicated markers, analyzed by flow cytometry, and median fluorescence intensity calculated. There was no statistical difference in median fluorescence between mutant and wildtype mice for any stimulus condition. Supplementary Figure 11. Assessment of B:T conjugation. Enumeration of B cell: T cell conjugates formed in vitro after addition of separately cultured OT-II T cells (with and without peptide stimulation) and B cells (stimulated with LPS or CD40L and IL-4). Results show decreased conjugation formation with SAP KO OT-II T cells (as expected), while conjugation formation with B cells was comparable to +/+ B cells. Columns are arithmetic means, error bars standard error of the mean, and dots individual results. Representative of two independent experiments.

Each black bar shows the percentage contribution of CD45.2 cells in individual mice (numbered 1-4) to the follicular B cell subset in the spleen and the peripheral lymph nodes (pln).

10 CD45.2 (%) Spleen pln Supplementary Figure 12. Assessment of LN B cells in bone marrow chimeras. Mutant B cells competitively accumulate in peripheral lymph nodes in mixed bone marrow chimeras reconstituted with wild-type CD45.1 bone marrow and CD45.2 marrow. Each black bar shows the percentage contribution of CD45.2 cells in individual mice (numbered 1-4) to the follicular B cell subset in the spleen and the peripheral lymph nodes (pln). Note that there is no consistent difference in the percentage of B cells that are derived from CD45.2 in pln compared to the percentage of these cells in the spleen (P=0.83, unpaired, two-tailed t-test with Welch s correction). Similar results are found in cpm/cpm mixed bone marrow chimeras (data not shown). Stimulus: nil S1P (mm) CXCL13 CXCL /+ cpm/cpm Migrating cells (% input) Supplementary Figure 13. B cell chemotaxis. Migration of wild-type and mutant CD19 + B cells to S1P, CXCL12 (0.3 µg/ml) and CXCL13 (1 µ g/ml) in vitro. Data from 3 independent experiments. Columns are arithmetic means, bars s.e.m, and dots individual mice.

11 Primers for amplification of DOCK8 cdna cdna Primers Primer sequence Spanning exons 1F GGTGGAAATGCGGAAGTTT 1-5 1R AGCACGTTTAAGGGATGACG 2F ACCTTGGAGTGCAGTGAACC R TTGGCAGAAGGATTCAGCTT 3F AAAGAAAGCGATGGTGGAAA R TGGGGGTATACGTACAGAAGG 4F GCGTCCACACAAGGAGATTT R CGGTTTGAAGACGTTCGTTTA 5F CAGCTGTCAGCAGAAGCAAG R GTAGGAGGCCAGAAGGCAGT 6F GCATCATCTGCCTCAACTCC R GGCAAGGCTGATGTTGATCT 7F GGTCACCTCGGAGATAGCAG R TCATGATCCACAGGAAGCAA 8F TGAAGACCAGTGGAGCAATG R CTCCTCGAACAGCAGGTCTC 9F ACCAGAGCACCACCTACCTG R CTGCAGACTCCTCAAGCACA 10F ATGCTGGAGGACCACAGCTA R CACCCGGTCTTTCATCTCAT 11F TGTTTCGGTGCAGAGTTTGT R GATTTTCCGCTCAATCATGG 12F CAAGCTGAGGTTGTGCTTCA R TGCTGGACAGTGTTCCACTC Primers for amplification of genomic DOCK8 DNA spanning introns Genomic Primers Primer sequence D8-Intron1F CTGTTTCTTCCGTCCACACC D8-Intron1R GAGTGGCAAAGGGTGAAGAA D8-Intron2F CTTCCTCCACCTGGTGCTT D8-Intron2R AGTACATCTGCCTGCCTGCT D8-Intron3F AGCAGGTGGGTAGGCAGTC D8-Intron3R CTTTCGGGATAGCATTTGAA D8-Intron4F TGTGCATCTCCAGTTTAAAGG D8-Intron4R AGGGCCCACAATGGAGTAG D8-Intron5F TAGGGTTGCAGACCCCTTC D8-Intron5R CTAATTCATGGCATCGCTCA D8-Intron6F GCAAGTAAAGATGCTTGTCTCCA D8-Intron6R GTGTACGTGCGTGCCTGTAT D8-Intron7F AGCATCTGGCCTCTGCATAC D8-Intron7R AGCCAACTTTGTGCCTGTTT D8-Intron8F AGAATGGGATGAGCTGATGG D8-Intron8R CATGCCTGGCTTACACCTTT D8-Intron9F CCCAGTTTTCAGCTGAGTTT D8-Intron9R TTCCTCAGGTCCTTGCTGTT Supplementary Table 1: Primer sequences spanning DOCK8 cdna and introns 19-21

Nature Genetics: doi: /ng Supplementary Figure 1. The phenotypes of PI , BR121, and Harosoy under short-day conditions.

Nature Genetics: doi: /ng Supplementary Figure 1. The phenotypes of PI , BR121, and Harosoy under short-day conditions. Supplementary Figure 1 The phenotypes of PI 159925, BR121, and Harosoy under short-day conditions. (a) Plant height. (b) Number of branches. (c) Average internode length. (d) Number of nodes. (e) Pods