The Journal of Experimental Medicine

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1 Supplemental Material Haines et al., The Journal of Experimental Medicine Subcloning of murine PTK7 cdna segments into pegfp-n3 and CHO cell transfection. A cdna segment encoding the entire extracellular region of murine PTK7 was PCR amplified using primers 5 -GGAATTCAGCCACCATGGGAGCCCGCCCGCTG-3 and 5 -TCCCCGCGGACTTC- CCGAACATCTCCACC-3 and reverse-transcribed mouse thymus total RNA as template. The final construct resulted in CMV I-E promoter driving transcription of the PTK7-EGFP fusion protein. The construct was sequenced (Bionexus) on both the sense and non-sense strands to ensure that no errors were introduced by PCR amplification, and was purified with an Endofree Plasmid Maxiprep kit (QIAGEN). 1.0 x 10 6 CHO cells were transfected by electroporation with 20.0 µg of the mptk7-egfp plasmid and then analyzed by flow cytometry 3 d later to verify EGFP fluorescence and PTK7 expression. PTK7 gene-disrupted mice and embryonic thymus isolation. Mice carrying null alleles of PTK7 were originally generated from embryonic stem cells in which the PTK7 gene had been disrupted as a result of insertional mutagenesis with a retrovirus containing a LacZ cassette. Because the PTK7 2/2 genotype results in embryonic lethality with neural tube defects, the mutant allele has been maintained by crosses of heterozygous PTK7 +/2 mice on a C57BL6/J background. Heterozygous mice were identified by incubating small biopsies of tail tissue with µl of PBS, ph 7.4, with 5.0 mm potassium ferricyanide, 5.0 mm potassium ferrocyanide, 2.0 mm MgCl 2, 2.5 mg/ml X-gal substrate, 0.01% sodium deoxycholate, and 0.02% NP-40 for 1 hour at 37 8 C. Mice were genotyped based on a blue (transgenic) or nonblue (wild type) appearance of the tissue. On embryonic day (E) 17 E18 of timed pregnancies from crosses between PTK7 +/2 females and males, the female mouse was sacrificed by cervical dislocation, embryos were extracted, and their fetal thymi were removed under a dissection microscope. PTK7 2/2 embryos were easily identified by the neural tube defect. Heterozygous and wild-type embryos were distinguished by the X-gal staining method described above using embryo tail sections. Real-time PCR assays for murine PTK7 transcripts. Total cellular RNA was purified from primary murine T-lineage cells, and real-time PCR was performed using the oligonucleotide primers 59-CAGGCATTGCTGAAGACTGG-39 and reverse 59-GGTTGTGGCGAAGAGAAACG-39, which amplify the exon 20 region. Samples were normalized and analyzed as for human PTK7. Murine thymocyte staining. C57BL6/J murine thymic tissue was disrupted using frosted slides into a single-cell suspension and filtered through a nylon mesh. Mouse thymocytes were stained as previously described for human thymocytes with the exception of using anti-cd16/32 Fc block (1:25; Invitrogen) instead of 10% heat-inactivated human AB serum for human cells. TCR Vb spectratyping. The CDR3 regions of 24 TCR Vb family members were PCR amplified using a unique TCR Vb family member 59 primer (1-24) and a conserved 39 TCR Cb primer. A nested PCR reaction was performed using a 39 FAM-labeled Cb primer to fluorescently label all lengths of the Vb chains within the repertoire from each Vb family. Labeled fragments were individually run out on a Genescan Sequencing analyzer, and the relative proportions of each length type were calculated and graphed to determine distribution at the Genome Core Facility at University of California, San Francisco (San Francisco, CA; courtesy of J. Woo). FoxP3 intracellular staining. Blood samples were obtained and prepared from adult donors, and PBMCs were stained for CD4-allophycocyanin (APC; Invitrogen), CD25-Tricolor (TC; Invitrogen), and PTK7-FITC. The cells were stained for fixed, permeabilized, and stained for FoxP3-PE or Isotype-PE according to a commercial staining protocol (ebioscience). In vitro proliferation of PTK7 + CD4 + RTEs using exogenous cytokines. PTK7 + CD4 + RTEs were purified by staining with antibodies for CD4 (PE- Cy5), CD45RA (APC), and PTK7 (detected using an unconjugated primary and a FITC-conjugated secondary antibody) and appropriate cell sorting. The sorted cells were then CFSE labeled and cultured in complete RPMI medium containing 25 ng/ml each of recombinant human IL-6, IL-7, IL-10, IL-15, and TNF-a (all from R&D Systems) for 7 d. The cells were then stained with an isotype control antibody or anti-ptk7, followed by detection using a secondary PE-conjugated antibody, or with a directly-conjugated PE-CD31 mab and analyzed by flow cytometry for CFSE versus PTK7, CD31, or CD4 expression.

2 Figure S1. PTK7 polyclonal antibody specifically binds native cell surface mouse PTK7 (mptk7) and reveals that mptk7 surface expression decreases with thymocyte maturation. (A) Flow cytometric analysis of CHO cells transfected with an mptk7-egfp PTK7 cdna construct encoding the extracellular and transmembrane domains of murine PTK7 fused to cytoplasmic enhanced GFP (representative results of two independent experiments). (B) PTK7 mrna and flow cytometric analysis of PTK7 surface expression. Real-time PCR analysis (mean 6 SD of triplicate wells with results representative of two independent experiments) of postnatal thymocytes (thymocytes), adult spleen CD4+ T cells (splenic CD4+), or EL-4 T-cell leukemia cells (EL-4). In NTC (no template control), no cdna template was added to the reaction mixture. For mptk7 antibody staining, tcd4 versus CD8 staining of E17 wild-type thymocytes are shown on the left, which demonstrates their expected lack of the mature CD3+CD4+CD82 subset at this stage of fetal development. A similar pattern of CD4 and CD8 surface expression was observed for E17 PTK72/2 thymocytes (representative results of three independent experiments). mptk7 surface staining of wild-type (solid blue line) and PTK72/2 (solid gray line) E17 murine fetal thymocytes and isotype-matched control antibody 2

3 Figure S2. Circulating numbers of PTK7 + naive CD4 + T cells decrease progressively and significantly with age. Healthy donors ranging in age between 4 and 55 yr (n = 18) were analyzed by staining for PTK7 and flow cytometric analysis. Spearman s correlation was used to evaluate the significance of the correlation between age and the concentration of circulating PTK7 + naive CD4 + T cells (Prism software). A two-phase exponential decay curve was fitted only for graphical purposes to provide an estimate of the shape of the relationship between the two variables. staining of wild-type thymocytes (dashed black line; representative results of three independent experiments) are shown in the middle. Staining of EL-4 cells with the mptk7 antibody (solid line) or the isotype-matched control antibody (dashed line; representative results of two independent experiments) is shown on the right. (C) Flow cytometric analysis of thymocytes from a 6-wk-old C57BL/6J mouse stained with antibodies for CD4 and CD8 and either mptk7 or isotype-matched control antibody. The left histogram shows the gates used to define the four major thymocyte subsets based on CD4 and CD8 expression. The other histograms show mptk7 (solid lines) or isotype-matched control antibody staining (dashed lines) of double-negative (CD4 2 CD8 2 ), double-positive (CD4 + CD8 + ), and single-positive (CD4 + CD8 2 or CD4 2 CD8 + ) thymocyte subsets. The PTK7 high cells within the CD4 2 CD8 + thymocyte gate (red arrow) are an immature single-positive subpopulation that does not express CD3 and is a transitional stage between double-negative and doublepositive thymocytes in the C57BL/6 strain. These plots are representative of three independent experiments using adult C57BL/6J (H-2 b ) thymi. Similar results were obtained for adult thymocytes from BALB/c (H-2 d ) and FVBN (H-2 q ) mice (thymocytes from each strain were analyzed once). 3

4 Figure S3. PTK7+ CD4+ RTEs and PTK7- naive CD4+ T cells have a similarly diverse ab-tcr repertoire. (A) PBMCs from a healthy adult donor were surface stained with antibodies to CD4, CD45RA, CD45RO, and PTK7 and were FACS purified into PTK7+ and PTK72 populations of CD45RAhighCD45ROlow CD4+ T cells using gating strategy indicated in the dot plots. (B) Spectratype analysis (performed once on a single donor) of the length distribution in base pairs of the CDR3 region of TCR-b transcripts encoded by the Vb4, Vb15, and Vb22 families for PTK7+ and PTK72 cells (y-axis is the relative arbitrary fluorescence units and x-axis is the number of base pairs of the CDR3 region). A similar Gaussian distribution of CDR3 lengths was obtained for both populations for an additional 18 Vb family members analyzed. 4

5 Figure S4. Circulating CD4 + CD25 + FoxP3 + regulatory T cells lack PTK7 expression. PBMCs from an adult donor were surface stained for CD4, CD25, and PTK7. The cells were fixed and permeabilized and then stained for Foxp3 and analyzed by flow cytometry. (A) Gating strategy for analysis of CD25 high and CD25 low CD4 + T cells. (B) PTK7 and Foxp3 expression by CD25 high and CD25 low CD4 + T cells. The results are representative of analysis of two different healthy adult donors. 5

6 Figure S5. PTK7+ T cells proliferating in vitro to cytokines progressively lose surface PTK7 expression with each successive cell division. Purified PTK7+ naive CD4+ T cells were isolated by antibody staining for PTK7 (FITC), CD4 (PE-Cy5), and CD45RA (APC) and cell sorting. The purified cells were CFSE labeled and incubated for 7 d in complete medium with a combination of cytokines. In pilot experiments, we found that the contribution of residual PTK7 surface staining detected by FITC to the much brighter CFSE signal analyzed by using the FL1 channel was negligible, as CFSE labeling intensity on day 7 was similar regardless of whether PE-conjugated or FITC-conjugated antibodies were used for PTK7 detection in the initial cell sorting step (not depicted). After 7 d of culture, aliquots of cells were either restained for PTK7 and a secondary antibody conjugated to a different fluorochrome (PE) than the one used for the initial sort purification (FITC), or for CD31 using a PE-conjugated antibody, and analyzed for the expression of the indicated markers versus CFSE. Cells were not restained with CD4-PE-Cy5 mab and were analyzed for CD4 surface expression. This provided a positive control for the loss of a surface marker with each cell division, as any new surface expression of CD4 that occurred following the initial staining was not included in the analysis. Two-color flow cytometric analysis of CFSE versus the indicated marker is shown on the left and single-parameter histogram overlays (derived from the indicated boxes) are shown on the right to better demonstrate the level of CD31, CD4, or PTK7 expression with each division (cell division 0, red; 1, blue; 2, green; 3, black). The median fluorescence intensity (MFI) is shown, as is the percentage of max median flourescence intensity for comparison between the markers. This experiment is representative of analysis from three different adult healthy donors. Note that the wider range of CD31 expression in PTK72 cells versus PTK7+ cells (Fig. 3 A) might account, at least in part, for the modest loss of percentage of max median flourescence intensity of CD31 expression of the PTK7+ cells as they divide to become PTK72 cells in vitro. Furthermore, an analysis of CD31 expression on purified PTK72 naive CD4+ T cells (not depicted) revealed a stable MFI with cell divisions 0 3 (318, 330, 316, and 315, respectively). 6