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(a) CXCR4 antagonist AMD3100 specifically blocks SDF1-α induced chemotaxis of thymocytes. Thymocytes from C57BL/6 wild type mice were pre-treated with.

CXCR4 acts as a costimulator during thymic β-selection

Paul C. Trampont1, Annie-Carole Tosello-Trampont1, Yuelei Shen2, Amanda K. Duley3, Timothy P. Bender1,3, Ann E. Sutherland4, Dan R. Littman2 and Kodi S. Ravichandran1,3 1Carter

Immunology Center and the 3Department of Microbiology, 4Department of Cell Biology, University of Virginia, Charlottesville, VA 22908; 2Howard Hughes Medical Institute, Kimmel Center for Biology and Medicine, Skirball Institute, and New York University School of Medicine, New York, NY1001.

Trampont et al., Supplementary Figure 1

SCZ

CMJ

CD25 pixel intensity

SCZ

Cortex

Cortex CD25

CMJ

SDF-1α

Overlay

Supplementary Figure 1: Thymic distribution of CD25+ cells and SDF-1α. Localization of CD25+ cells (DN2 and DN3) and SDF-1α in the thymus of C57BL/6 mice were assessed by immunohistochemistry. Cryosections of thymi from 4 to 6 week old mice, were stained with antibodies to CD25 or SDF-1α. By scanning multiple serial sections from seven C57BL/6 mice, the average CD25 pixel intensity within the subcapsular zone (SCZ), cortex and cortico-medullary junction (CMJ) was quantified. Original magnification ×10. (n>5)

Trampont et al., Supplementary Figure 2

b

DN1

Input (%)

a

DN4 -

0.1

0.25

0.5

1

BSA

+ AMD3100 (µg/ml)

c Input (%)

DP Lck-Cre+ Cxcr4+/+ Lck-Cre+ Cxcr4fl/fl

MFI

DN2

MFI

120

405

205

852

214

524

MFI

DN3

MFI

197

131

439

607

236

296

MFI 70

Lck-Cre+ Cxcr4 +/+ Lck-Cre+ Cxcr4fl/fl

278

DN3

DN4

130

CXCR4 Supplementary Figure 2: SDF-1α- induced chemotaxis in murine thymocytes. (a) CXCR4 antagonist AMD3100 specifically blocks SDF1-α induced chemotaxis of thymocytes. Thymocytes from C57BL/6 wild type mice were pre-treated with increasing amount of AMD3100 for 15 minutes, and then tested in a Transwell chemotaxis assay (2 hours) for migration to SDF1α (n=4). (b) Flow cytometry analysis of CXCR4 expression deletion in thymic subsets from Lck-Cre+ Cxcr4+/+ and Lck-Cre+ Cxcr4fl/fl mice. Control isotype is shown as light grey histogram (n>5). (c) DN3 and DN4 thymocytes from Lck-Cre+ Cxcr4+/+ and Lck-Cre+ Cxcr4fl/fl mice were assayed for migration to SDF-1α.

Trampont et al., Supplementary Figure 3 Non-treated

Dexamethasone

SCZ Cortex

SCZ Cortex

Medulla CMJ CMJ

Medulla

Supplementary Figure 3: Diffuse distribution of single stranded DNA immunostaining in dexamethasone treated thymi. C57BL/6 mice were intraperitoneally injected with either PBS or dexamethasone for 6 hours. Thymic sections were stained for fragmented nuclei (with antibody F7-26) from apoptotic thymocytes (brown) and counterstained with hematoxylin. Images show regions of the cortex and medulla. Original magnification ×20. Cortical area within the insert is shown in the middle panels. Original magnification × 40. (n=7).

Trampont et al., Supplementary Figure 4

RNA from OP9-DL1 stromal cells

c Notch1 mRNA (fold induction)

SDF-1α (pg/ml)

Hprt1

Mock

Sdf1

b Mock

a

Growth medium

Growth medium

+ IL-7 + Flt3L

Cell supernatants from OP9-DL1

DN3e

DN3L

DN4

Lck-Cre+ Cxcr4+/+ Lck-Cre+ Cxcr4fl/fl

Supplementary Figure 4: Notch1 expression in thymic subsets and SDF-1α expression in the OP9-DL1 stromal cell line. (a) Sdf1 and Hprt1 transcripts were assessed by RT-PCR. Mock PCR reaction included the primers without the RT enzyme. (n=2). (b) OP9-DL1 were grown four days in MEM-α 20% FCS with or without 5ng/ml of IL-7 and Flt3-ligand (to mimic the situation of seeding DN3 thymocytes). Cell supernatants were then collected and assayed for SDF-1α by ELISA. Results are representative of two independent experiments (n=3). (c) Notch1 transcript expression in the thymic subpopulations. Total RNA was extracted from purified thymocytes and subjected to qPCR for Notch1 (n=3).

Trampont et al., Supplementary Figure 5

Cxcr4fl/fl 18%

10%

Lck-Cre+ Cxcr4fl/fl 16%

8%

Day 2 66%

10%

16%

70%

9%

9%

Day 4 63%

10%

60%

77%

12%

20%

CD8

Day 8 11%

60%

CD4

Supplementary Figure 5: DN3 to DN4 transition defect in CXCR4-deficient thymic progenitors seeded on OP9-DL1 cultures. DN3e immature thymocytes from indicated mice were sorted and seeded on OP9-DL1 cultures and analyzed as above on day 2, 4 and 8 for differentiation into DP thymocytes. (n=2).

mRNA fold induction

Trampont et al., Supplementary Figure 6

Il2ra Bcl2l1 (Bcl-xL) Bcl2 Bcl2a1 Non-stimulated

Saline buffer

anti-CD3ε

48 hours after intraperitoneal injection

Supplementary Figure 6: Pro-survival Bcl-2 family members regulation by pre-TCR signaling. Rag2-/- mice were either not treated or intraperitoneally injected with PBS or 150µg of anti-CD3ε for 48 hours. After thymi extraction, phenotypes were assayed by flow cytometry. Total RNA was then purified and quantitative PCR analysis for Bcl2l1, Bcl2, and Bcl2a1 mRNAexpression was performed. Il2ra expression (black filled bars) was used as a positive control to monitor the efficiency of CD3 stimulation in vivo. 18s and Gapdh were used to normalize gene expression. Error bars were obtained from variations of threshold cycle values (Ct) within each animal included in the experiment (n>3). Fold induction was measured using the Gapdh-normalized Ct values from the non-stimulated condition as 1.

Trampont et al., Supplementary Figure 7

SCIET27 Time (min)

0

SCB29

1 2 5 10 20 60 120

0

1

2

5 10 20 60 120

IB: p-Akt 1

7

8

7

7

9

1

9 10 10 9

11

IB: Akt

Supplementary Figure 7: Akt phosphorylation induced by SDF-1α. Akt phosphorylation (on Ser473) in SCIET27 and SCB29 pre-T cell lines upon 10nM SDF-1α stimulation. Fold induction of the Akt Serine phosphorylation was measured by densitometry and normalized to the non-stimulated condition and total Akt expression (red labels) (n=2).

Trampont et al., Supplementary Figure 8

b CD 3

DN3 from C57BL/6 No treatement

α-

AM D3 10 0

iE rk 5

hic le UO -1 26 iE rk 2u M

No ts tim ula te ve d

0u M

+ SDF-1α

a

p44 p42

IB: p-Erk1/2

SDF-1α AMD3100 Control peptide Erk inhibitory peptide

p44 p42

IB: Erk1/2

Annexin-V 1

2.5

0.6

2.4

1

0.9

2

Supplementary Figure 8: Effect of AMD3100 and inhibitory Erk peptides on SDF1-α induced Erk phosphorylation in thymocytes. (a) Thymocytes from C57BL/6 mice were pre-treated with either AMD3100 (1µg/ml) for 15 minutes, inhibitory Erk peptide (iErk) for 1 hour or with the MEK inhibitor UO-126 (25nM) for 15 minutes in serum free medium. After 5 min SDF-1α stimulation, cells were lysed and immunoblotted for Erk1/2 phosphorylation (top) or for total Erk protein (bottom). The densitometry values of the phospho-Erk1/2 signal intensity, normalized to the total Erk signal, is also shown in red. (n=2). (b) Viability of DN3 thymocytes is not affected by iErk peptide. Thymocytes from C57BL/6 mice were incubated with the iErk peptide, control peptide, AMD3100, or SDF-1α for 3 hours. Cells were then collected, stained for lineage markers and for phosphatidylserine exposure (indicative of apoptosis) using annexin-V. (n=2).

Trampont et al., Supplementary Figure 9

c Migrating DN3 (%)

a

iErk pep tide

cont pep rol tide AMD 3100

vehi cle

SDF -1α

LN

BSA

Time (min)

SDF-1α 0

2

5

10

SDF-1α + EGTA 10mM 20

60 120

0

2

5

10 20

60

120

IB: p-Erk1/2

p44 p42

IB: Erk1/2

p44 p42

1 17 16 11 7

5

1

1 19 22 11 8

6

2

SDF-1α + Laminin (LN)

b

+ SDF-1α

BA PT A

EG TA

iEr k

ve hic le AM 31 D 00

BS A

Input
(%)

SCB29

Supplementary Figure 9: Erk activation but not calcium release is required for SDF-1α dependent chemotaxis. (a) Purified DN thymocytes from C57BL/6 mice were treated with membrane permeable iErk, or the CXCR4 antagonist AMD3100 and migration to SDF-1α assessed on Transwells coated with laminin (see online methods). Bar graphs show percentage of migrated DN3 cells as a fraction of total cell input (n>5). (b) Migration of SCB29 to SDF-1α in the presence of calcium chelators EGTA and BAPTA, iErk, or AMD3100 (n=2) (c) CXCR4-mediated Erk phosphorylation in the presence of EGTA. SCB29 cells were stimulated for the indicated times with SDF-1α and assayed for phosphorylated Erk (pErk1/2) by immunoblotting.(n=2). Densitometry of the signal for Erk phosphorylation normalized to Erk expression is shown at the bottom in red.

Trampont et al., Supplementary Table 1

Primers used for quantitative PCR Ref Sequence 1 2 3 4 5 6 7 8 9 10 11 12

NM_009741.3 NM_009743.4 NM_008562.3 NM_007523.2 NM_007527.3 NM_007536 NM_008367.2 NM_008714.2 NM_053069.5 NM_028835.3 NM_008084

Name 18s Bcl2 Bcl-xL Mcl1 Bak1 Bax Bcl2A1d CD25 Notch1 Atg5 Beclin-1 GAPDH

ABI catalog number 4310893E 0802039 Mm 004776631-m1 Mm 00437783 m1 Mm 0072583251 Mm 00432045 m1 Mm 00432050 m1 Mm 00432050 m1 Mm 00434261 m1 Mm 00435249 m1 Mm 00504340 m1 Mm 00512209 m1 4352339E-0809019