Eberl and Littman

Science Supporting Online Material

Materials and Methods

Mice

The generation of gene-targeted Rorc(gt)+/GFP and Rorc(gt)GFP/GFP mice (S1), and of BAC-

transgenic Rorc(gt)-Bcl-xlTG mice (S2) have been described recently. The Rorc(gt)-CreTG

BAC-transgenic mice were generated following the same protocol. Id2-deficient (S3) and

R26R mice (S4) have been reported elsewhere. LTa- and Rag-2-deficient mice were

purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were bred and used

in our specific pathogen-free animal facility according to the New York University

School of Medicine Institutional Animal Care and Use Committee.

Antibodies

The following mAbs were purchased from Pharmingen (San Diego, CA): fluorescein

isothiocyanate (FITC)-conjugated Annexin V, phycoerythrin (PE)-conjugated anti-CD4

(RM4-5), anti-CD11c (HL3), anti-CD8b (53-5.8), anti-CD44 (IM7), anti-CD49b (DX5),

anti-ICAM-1 (3E2), anti-c-kit (2B8), anti-NK1.1 (PK136), anti-TCRb (H57-597),

allophycocyanin (APC)-conjugated anti-CD3e (145-2C11), anti-CD11b (M1/70), anti-

CD11c (HL3), anti-B220 (RA3-6B2), anti-Gr-1 (RB6-8C5), biotin-conjugated anti-

CD8a (53-6.7), anti-CD45.2 (104), anti-VCAM-1 (429), anti-TCRd (GL3), and purified

anti-CD16/32 (2.4G2). Rabbit anti-GFP, FITC-conjugated goat anti-rabbit and Alexa

Fluor 647-conjugated streptavidin were purchased from Molecular Probes (Eugene, OR).

Biotin-conjugated anti-IL-7Ra mAb was purchased from eBioscience (San Diego, CA).

Flow cytometry

Single cell suspensions were prepared from thymus, spleen and Peyer’s patches. Small

intestinal mononuclear cells were prepared as follows. Peyer’s patches were removed, the

intestine was cut into pieces less than 1 mm3, and incubated 1 hour at 37oC in 15ml

DMEM containing 1mg/ml collagenase D (Roche Diagnostics, Mannheim, Germany).

Fragments were then pressed through a 100µm mesh. Total intestinal cells were

resuspended in a 40% isotonic Percoll solution (Pharmacia, Uppsala, Sweden) and

underlaid with an 80% isotonic Percoll solution. Centrifugation for 20 min at 2000 rpm

yielded the mononuclear cells at the 40-80% interface. Cells were washed twice with

PBS-F (PBS containing 2% fetal calf serum, FCS), preincubated with mAb 2.4G2 to

block Fcg receptors, then washed and incubated with the indicated mAb conjugates for 40

min in a total volume of 100µl PBS-F. Cells were washed, resuspended in PBS-F and

analyzed on a FACScalibur flow cytometer (Becton-Dickinson, San Jose, CA). For cell

cycle analysis of thymocytes, cells were fixed in 70% ethanol 30 min at 4oC, washed with

PBS-F, and 5x105 cells were incubated 5 min at 37oC with 12.5 µg/ml of propidium

iodide (Sigma) and 50 µg/ml of RNAse A in 100 µl STE buffer (100 mM Tris base, 100

mM NaCl and 5 mM EDTA at pH7.5). Cells were then washed, resuspended in PBS-F

and analyzed.

Thymocyte survival assay

Thymocytes were isolated and cultured in DMEM medium supplemented with DMEM

containing 10 % FCS, 10 mM HEPES, 50 µM b-mercaptoethanol, and 1% glutamine.

After the indicated periods of time, cells were stained with Annexin V (Pharmingen) and

1µg/ml of propidium iodide to exclude dead cells, and analyzed by FACS.

Immunofluorescence histology

Intestines were washed several hours in PBS before being fixed overnight at 4oC in a

fresh solution of 4% paraformaldehyde (Sigma, St-Louis, MO) in PBS. The samples were

then washed 1 day in PBS, incubated in a solution of 30% sucrose (Sigma) in PBS until

the samples sank, embedded in OCT compound 4583 (Sakura Finetek, Torrance, CA),

frozen in a bath of hexane cooled with liquid nitrogen and stocked at -80oC. Blocs were

cut with a Microm HM500 OM cryostat (Microm, Oceanside, CA) at 8µm thickness and

sections collected onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). Slides

were dried 1 hour and processed for staining, or stocked at -80oC. For staining, slides

were first hydrated in PBS-XG, (PBS containing 0.1% triton X-100 and 1% normal goat

serum, Sigma) for 5 min and blocked with 10% goat serum and 1/100 of anti-Fc receptor

mAb 2.4G2 in PBS-XG for 1 hour at room temperature. Endogenous biotin was blocked

with a biotin blocking kit (Vector Laboratories, Burlingame, CA). Slides were then

incubated with primary polyclonal Ab or conjugated mAb (in general 1/100) in PBS-XG

overnight at 4oC, washed 3 times 5 min with PBS-XG, incubated with secondary

conjugated polyclonal Ab or streptavidin for 1 hour at room temperarture, washed once,

incubated with 4'6’-diamidino-2-phenylindole-2HCl (DAPI) (Sigma) 5 min at room

temperature, washed 3 times 5 min and mounted with Fluoromount-G (Southern

Biotechnology Associates, Birmingham, AL). Slides were examined under a Zeiss

Axioplan 2 fluorescence microscope equipped with a CCD camera and processed with

Slidebook v3.0.9.0 software (Intelligent Imaging, Denver, CO).

RORgto

41 8

RORgto BclTG

5.5

DNA content

BclTG

010

20

30

40

50

1 2 3 4 5

RORgto BclTG

BclTGRORgto

hours

Annexin V

Supplementary Figures and Tables

Figure S1. Normal cell cycle progression and in vitro survival of thymocytes fromRORgt-deficient, Bcl-xL BAC-transgenic mice. Cell cycle analysis was performed bypropidium iodide (PI) staining of fresh thymocytes isolated from Rorc(gt)-Bcl-xlTG mice(BclTG), Rorc(gt)GFP/GFP (RORgto) and from RORgto BclTG mice. Numbers indicate thepercent cells found in S+G2/M phase of the cell cycle. In vitro survival was evaluated bycultures of thymocytes for different periods of time and subsequent Annexin V stainingof live cells. Similar results were obtained with BclTG and wild-type mice. The datashown are representative of 3 individual experiments.

TCRab

EGFP

Wild-type RORgt-CreTG / R26R / Rag-2o

NK CD11b CD11c

D

AThymus

DN

SP8

SP4

DP

TCRgd

CD4-CreTG / R26R

EGFP

Spleen Intestine

TCRgd

TCRab TCRab

TK-Cre x R26R

TCRgdBIntestine

B

EGFP

8

565

22

CD4

Intestine lineage-

EGFP

C

Figure S2. Cell fate mapping of RORgt+ or CD4+ cells. (A) Cells from thymus, spleenand intestine of adult Rorc(gt)-CreTG / R26R (blue histograms) or control R26R mouse(red lines), were analyzed by flow cytometry for the expression of GFP. Cells were gatedas indicated. The data shown are representative of 3 individual experiments. (B)Expression of CD4 by intestinal lin- RORgt+ cells. Numbers indicate the percent cellspresent in each quadrant. The data shown are representative of 3 individual experiments.(C) To demonstrate that the Rosa26 promoter is also active in B cells and gd T cells,R26R mice were crossed to the ubiquitous deleter Tk-CreTG mouse line. Similar resultswere obtained with splenocytes. The data shown are representative of two independentexperiments. (D) Splenocytes from Rag-2-deficient Rorc(gt)-CreTG / R26R mice (bluehistograms) or Rag-2-deficient R26R mouse (red lines) were analyzed for the expressionof GFP. Cells were gated as indicated. The data shown are representative of 3 individualmice.

TCRab

EGFP

Wild-type RORgt-CreTG / R26R / Rag-2o

NK CD11b CD11c

D

LTao / RORgt-EGFPCD11c CD45 CD11c CD45

Figure S3. Absence of mature CPs and ILFs in LTa-deficient mice. Longitudinalsections of the small intestine of adult Lta-/- Rorc(gt)+/GFP mice were stained as indicated,as well as for GFP (green). In these mice, CP rudiments were found that consisted ofsmall clusters RORgt+ cells, but that contained very few CD11c+ dendritic cells. No ILFswere present. RORgt+ cells expressed low amounts of CD45, only apparent in thesepanels when the green fluorescence was removed. Magnifications are 100x (first twopanels) and 200x (last two panels). Sections shown are representative of at least 10individual sections and 3 individual mice.

day 2CD11c

day 7 day 14 day 21

Figure S4. RORgt+ cells in the postnatal intestinal lamina propria. Longitudinal sectionsof the small intestine of Rorc(gt)+/GFP mice at different times after birth were stained asindicated, as well as for GFP (green). Magnification is 40x. Sections shown arerepresentative of at least 5 individual sections and 2 independent experiments.

Table S1. The progeny of RORgt+ cells and CD4+ cells

1 Low levels of EGFP were also detected in CD4+ and CD8+ single positive (SP) thymocytes, even though Rorc(gt) mRNA and proteinwas not detected in these population. This may be due to the long half-life of EGFP (> 24hrs), present in SP thymocytes even aftercessation of Rorc(gt) transcription (S1).

Thymus Spleen IntestineDN DP SP4 SP8 B-T- B T4 T8 Tgd Tab

total 8- 8ab 8aackit+

IL-7R+

RORgt-EGFP - + +/-1 +/-1 - - - - - - - - - +RORgt-CreTG / R26R - + + + - - + + - + + + + +CD4-CreTG / R26R - + + + - - + + - + + + + -

Table S2. Phenotypic and developmental similarity of fetal RORgt+ LTi cells and

adult intestinal RORgt+ cells.

1 c-kit is expressed by CD3-IL-7Ra+ cells in PP anlagen (S5) and in low amounts by

CD3-CD4+ cells in newborn mesenteric LNs (S6).2 CD4 is expressed by 50% of LTi cells (S1, S5) and by 30-40% of intestinal RORgt+

cells (Fig. S2B).3 In LTa-deficient mice, LTi cells are present in LN and PP anlagen, but do not induce

activation of mesenchyma (S1); RORgt+ cells are present in the adult intestine, but do not

cluster into mature cryptopatches (Fig. S3).

Fetal LTi cells Intestinal RORgt+ cellsPhenotypeRORgt + +IL-7Ra + +c-kit +1 +CD44 + +CD45 + +ICAM-1 + +CD4 +/-2 +/-CD3 - -TCRab - -TCRgd - -B220 - -CD11b - -CD11c - -NK1.1 - -DX5 - -Gr-1 - -Gene dependenceRORgt + +Id2 + +LTa -3 -

RAG-2 - -

References

S1. G. Eberl et al., Nat Immunol 5, 64 (2004).

S2. T. Sparwasser, S. Gong, J. Y. H. Li, G. Eberl, Genesis 38, 39 (2004).

S3. Y. Yokota et al., Nature 397, 702 (1999).

S4. X. Mao, Y. Fujiwara, A. Chapdelaine, H. Yang, S. H. Orkin, Blood 97, 324 (2001).

S5. H. Yoshida et al., Int Immunol 11, 643 (1999).

S6. R. E. Mebius, P. Rennert, I. L. Weissman, Immunity 7, 493 (1997).