A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development

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1 SUPPLEMENTARY INFORMATION A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development Isabelle Mothe-Satney, Joseph Murdaca, Brigitte Sibille, Anne-Sophie Rousseau, Raphaëlle Squillace, Gwenaëlle Le Menn, Akila Rekima, Frederic Larbret, Juline Pelé, Valérie Verhasselt, Paul A. Grimaldi, Jaap G. Neels SI Materials and Methods Animal models Lck-Cre mice (Cre recombinase under of the T cell specific Lck gene promoter were obtained from Jackson Laboratory (B6.Cg-Tg(Lck-cre)548Jxm/J, stock number 38). CAG-Stop-PPARβ mice, carrying a transgene containing the modified chicken β-actin promoter with the CMV/IE enhancer (CAG promoter) driving PPARβ-IRES-Hygromycin chimeric mrna expression under CRE-mediated recombination of a transcriptional Stop fragment, were described previously 1. Both strains are on the C57BL/6J background and were crossed to obtain double transgenic animals that overexpress PPARβ specifically in T cells (Lck-Cre/CAG-Stop-PPARβ mice). For convenience these double transgenic animals are referred to as mice. It has previously been reported that Cre expression in Lck-Cre mice results in off-target effects including a decrease in thymic cellularity (toxic to CD4+CD8+ cells). Therefore, the Lck-Cre mice were used as s throughout this study. Wild type C57BL/6J mice obtained from Charles River (France) were used for in vitro and in vivo GW74 treatment studies. For the latter in vivo studies, 15-week-old male C57Bl/6J mice were injected (.3 mg/kg/day I.P.) with the PPARβ agonist GW74 (Cayman Chemical) or vehicle (equivalent volume of DMSO) and tissues were harvested 48 hours later. Primary CD4+ T cell isolation Spleen and lymph nodes (cervical, brachial, mesenteric, and inguinal) were harvested from 1-weekold male wild-type C57BL/6J mice and converted into single cell suspensions in phosphate-buffered saline (PBS), ph 7., containing.5% fetal calf serum (FCS) and mm EDTA, using a gentlemacs Dissociator and appropriate gentlemacs C tubes by following the protocols provided by the manufacturer (Miltenyi Biotec). Single cell suspensions from both organs were pooled before continuing with the isolation of CD4+ cells by magnetic labeling and separation using CD4 (L3T4) microbeads and MACS Columns, respectively, following the protocols provided by the manufacturer (Miltenyi Biotec). Quantitative real-time PCR For standard quantitative real-time PCR reaction total RNA (1 μg) was reverse-transcribed using a QuantiTect Reverse Transcription Kit (Qiagen) on a QcyclerII. Quantitative PCR was done using SYBR Premix Ex Taq (Tli RNase H Plus) (Ozyme) on a StepOne machine (Life Technologies). The mrna levels of all genes reported were normalized to 36B4 transcript levels. Primer sequences are available upon request. For Mouse Fatty Acid Metabolism RT² Profiler PCR Arrays (SA 1

2 Biosciences), total RNA (.5 μg) was reverse-transcribed using the RT First Strand reagents as part of the array kit per the manufacturer s instructions and qpcr was performed on a StepOne machine following detection protocols recommended by SA Biosciences. Data was analyzed using the excel spreadsheet provided online on the manufacturer s website. Cell preparation and flow cytometry analysis Thymi, spleens and lymph nodes (cervical, brachial, mesenteric, and inguinal lymph nodes pooled together) were harvested from and Tg-T-PPARβ mice and converted into single cell suspensions in phosphate-buffered saline (PBS), ph 7., containing.5% fetal calf serum (FCS) and mm EDTA, using a gentlemacs Dissociator and appropriate gentlemacs C tubes by following the protocols provided by the manufacturer (Miltenyi Biotec). Splenocyte single cell suspensions were subsequently depleted of red blood cells with RBC lysis buffer (Sigma). The resulting single cell suspensions were incubated with Fc Block (anti-mouse CD16/CD3 monoclonal antibody, BD Biosciences) for 15 min at 4 C before staining with fluorescently labeled primary antibodies for min at 4 C in PBS,.5% BSA. CD3-fluorescein isothiocyanate, CD3-phycoerythrin, CD4- allophycocyanin, TCRβ-phycoerythrin-Cy7, TCRγδ-phycoerythrin, CD44-phycoerythrin-Cy7, CD6L-fluorescein isothiocyanate, CD5-phycoerythrin, and CD44-phycoerythrin FACS antibodies were purchased from ebioscience. CD8-Peridinin chlorophyll antibody was from BD Biosciences. For blood cell stainings 1 μl whole blood (with EDTA) was directly incubate 1:1 with antibody solutions (x concentrated) and incubated for min at room temperature followed by addition of PBS,.5% FCS, mm EDTA and centrifugation. The stained cell pellet was subsequently incubated with RBC lysis buffer and washed twice in PBS,.5% BSA. For BrdU stainings, we used the BrdU staining kit (FITC) from ebioscience according to the manufacturers instructions. Cells were gently washed twice and resuspended in PBS,.5% BSA with DAPI. Stained cell preparations were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences). DN/DN3 thymocyte isolation and OP9-DL1 co-cultures The DN/DN3 thymocytes used in co-cultures were isolated from thymocyte cell suspensions by labeling them using anti-cd5-phycoerythrin (PE) antibody and subsequently purifying them using an anti-pe multisort kit (Miltenyi Biotec). The resulting cell preparation still contained contaminating CD4+ cells, so the anti-pe microbeads were released from the cells using the MultiSort Release Reagent from the kit to allow for a second labeling with CD4 (L3T4) microbeads to deplete CD4+ cells. After CD4+ cell depletion, the purified DN/DN3 cell preparations were co-cultured on top of confluent monolayers of OP9-DL1 cells. DN/DN3 thymocytes isolated from and Tg T- PPARβ mice were added at a concentration of 1 4 cells/well to 4-well plates that were seeded the day before with 1 4 OP9-DL1 cells in Opti-MEM medium supplemented with GlutaMAX (Gibco), 1% FCS, 1 units/ml penicillin/streptomycin, and 5 μm -mercaptoethanol. Interleukin 7 (R&D Systems) was added to the co-cultures at ng/ml. After 3 days, half of the co-culture media was replaced by fresh media. At indicated times, the co-cultures were incubated for hrs with 1 μm BrdU before the cells were recovered and prepared for flow cytometry analysis as described above. References 1 Luquet, S. et al. Peroxisome proliferator-activated receptor delta s muscle development and oxidative capability. Faseb J. 17, (3). Shi, J. & Petrie, H. T. Activation kinetics and off-target effects of thymus-initiated cre transgenes. PLoS One. 7, e4659 (1).

3 Table S1: Mouse Fatty Acid Metabolism PCR Array data Gene Symbol RQ SEM P Acaa1a 1,,8,6779 Acaa 1,48,1,33 Acad1,93,9,761 Acad11 1,15,8,3781 Acad9,96,1,5756 Acadl 1,19,14,766 Acadm 1,11,8,1198 Acads 1,31,3,11 Acadsb,94,,6358 Acadvl,1,1,1 Acat1 1,,1,966 Acat,97,9,669 Acot1,1,37,57 Acot 1,54,68,758 Acot3 1,84 1,3, Acot6 3,59 4,41,411 Acot7 1,9,4,84 Acot8,91,11,1919 Acot9 1,3,14,787 Acox1 1,17,31,3761 Acox 1,51 1,5,5641 Acox3 1,38,57,3787 Acsbg1 1,,61,657 Acsbg,91,66,846 Acsl1,84,18,1856 Acsl3 1,3,7,4788 Acsl4 1,8,13,335 Acsl5 1,,7,9413 Acsl6,9,7,6755 Acsm,91,44,6998 Acsm3 1,7,65,886 Acsm4 1,56,95,379 Acsm5,93,4,6514 Aldh 1,7,7,698 Bdh1,86,9,418 Bdh 1,3,66,9354 Cpt1a 3,4,57,3 Cpt1b,98,18,835 Cpt1c 1,77,8,15 Cpt,95,14,554 Crat 1,54,34,716 Crot,9,14,46 Decr1 1,17,16,1867 Decr 1,19,45,47 Echs1 1,1,,3981 Eci 1,8,18,78 Ehhadh,97,1,7775 Fabp1 1,44 1,43,5848 Fabp,56,68,366 Gene Symbol RQ SEM P Fabp3 1,5,34,69 Fabp4 1,5,63,5467 Fabp5,94,1,378 Fabp6 1,17,93,749 Gcdh 1,6,3,6653 Gk 1,3,67,998 Gpd1 1,13,65,7484 Gpd,93,8,1757 Gyk,94,1,4 Hadha 1,,7,5618 Hmgcl 1,9,19,4389 Hmgcs1 1,15,15,56 Hmgcs 1,14,46,656 Lipe 1,,5,9819 Lpl 1,15 1,1,846 Mcee,94,3,6571 Mut 1,1,14,683 Oxcta,79,67,6433 Pecr,93,17,514 Ppa1,95,8,388 Prkaa1,96,1,486 Prkaa,69,36,444 Prkab1 1,74 1,,358 Prkab,89,7,44 Prkaca 1,,19,131 Prkacb 1,14,1,853 Prkag1 1,6,15,476 Prkag 1,7,5,6411 Prkag3,5,6,94 Slc7a1 1,33,3,1646 Slc7a 1,11,93,8593 Slc7a3,85,15,1559 Slc7a4 1,1,6,919 Slc7a5,6,35,145 Slc7a6,67,6,497 Actb 1,6,1,389 Bm,9,8,974 Gapdh 1,3,3,173 Gusb 1,3,1,6376 Hsp9ab1 1,,3,449 RQ=Relative Quantification of gene expression when comparing CD4+ T cells treated with 3 μm GW74 for 48 hrs compared to DMSO, SEM=Standard Error of the Mean, P=P value. N=5. Genes that are underlined are housekeeping genes. Less than 15% change from DMSO. Ct values >3 3

4 Figure S1: Generation of mice overexpressing PPARβ in T cells. Mice carrying a transgene containing a CAG promotor upstream of the PPARβ coding sequence, preceded by a transcriptional stop sequence flanked by LoxP sites, were crossed with mice expressing the Cre recombinase under of the T cell specific Lck gene promotor. In the resulting progeny, the action of the Cre recombinase results in removal of the transcriptional stop sequence specifically in T cells, allowing transcription of the PPARβ transgene and resulting in T cell-specific overexpression of PPARβ. 4

5 6 A 5 B c e lls (m illio n ) cells (million) c o n tr o l T g T -P P AR Figure S: Total cell counts are reduced in spleens and lymph nodes from mice. (A,B) Total cell counts in spleens (A) and lymph nodes (B) from (white bars) and Tg T- PPARβ (black bars) mice. Data is from tissues obtained from 4 mice per group (15-16 weeks of age) and are expressed as mean ± s.e.m. P<.5 when compared to (Mann-Whitney test). 5

6 A SPLEEN B LYMPH NODES C BLOOD 65.7±3.% count 3.4±.8% 17.8±3.% 43.5±6.5% 38.±4.6% 13.4±4.% CD3 D 33.5±1.7% 39.6±1.8% 41.8±1.3% 9.3±1.4% 56.9±.5% 4.8±.8% 55.±1.% 6.1±.6% 5.±1.4% 34.6±6.1% 37.5±5.3% 35.8±6.8%.9±5.1% 4.3±1.4% 14.7±4.% 47.4±.% 3.5±6.1% 4.7±6.7% CD8 CD4 E 1 F cells (million) CD3+ CD4-CD8- CD4+CD8- CD4-CD8- CD4-CD8+ CD3+ CD4+CD8- CD4-CD8+ Figure S3: Consequences of impaired T cell development in mice for peripheral T cell populations. (A-C) Flow cytometric analysis of T cell presence (CD3+ cells) in spleen (A), lymph nodes (B), and blood (C) of and mice. Relative percentages (mean ± s.e.m.) of CD3+ cells are indicated. (D) Flow cytometric analysis of CD4 and CD8 expression on CD3+ gated cells from spleen (left), lymph nodes (middle), and blood (right) of (upper flow plots) and (lower flow plots) mice. Relative percentages (mean ± s.e.m.) of DN (CD4-CD8-), SP4 (CD4+CD8-), and SP8 (CD4-CD8+) cells are indicated. (E,F) Quantification of various T cell populations (horizontal axis) in spleen (E) and lymph nodes (F) derived from data shown in (A,B,D) and Fig. S. Data is from tissues obtained from 4 mice per group, with flow histograms and plots shown in (A-D) representative of latter groups. Data shown in bar graphs (E,F) are expressed as mean ± s.e.m. P<.5 when compared to (Mann-Whitney test). 6

7 THYMUS Gated on TCRβ+ 17.±1.%.9±.3% 78.7 ±1.1% CONTROL Gated on TCRγδ+ Gated on TCRβ+ Gated on TCRγδ+ 1.5±.4% 15.9±.6% 1.9±1.% 8.7±1.% 6.6 ±1.% 4.±.5% 78.7 ±.9% 79.8±1.% 7. ±.6% 38.1±.5% 1.7±1.3% 3.6±1.5% 13.8±1.8% SPLEEN.3±.% 59.1 ±.6% 85.6±1.5% 1.4 ±.3%.3±.% 64.8 ±1.5% 84.3±1.6% 1.9 ±.3% LYMPH NODES 46.8±3.% 1.9±1.1% 51. ±3.% 16.8±.8% 78.3±.9% 4.9 ±1.1% 39.9±.9% 1.3±.% 58.7 ±.9% 15.1±1.5% 8.4±1.9% 4.4 ±.8% 31.3±5.5%.9±3.8% 44.4±.3% 18.8±1.1% BLOOD 67.3 ±5.7% 1.1 ±.7% 5.8 ±.9%.3 ±.3% 1.1±.5% 78.±3.9%.8±.9% 8.9±1.1% CD8 CD4 Figure S4: TCRβ+ and TCRγδ+ T cell subpopulations in lymphoid tissues from and Tg T- PPARβ mice. Flow cytometric analysis of CD4 and CD8 expression on CD3+TCRβ+ or CD3+TCRγδ+ gated cells from thymus, spleen, lymph nodes, and blood of and mice. Relative percentages (mean ± s.e.m.) of DN (CD4-CD8-), SP4 (CD4+CD8-), and SP8 (CD4- CD8+) cells are indicated. Data is from tissues obtained from 4 mice per group, with flow plots shown representative of latter groups. P<.5 when compared to (Mann-Whitney test). 7

8 re la tiv e P P A R m R N A le v e ls C D 4 + c e lls C o n tro l T g T -P P A R T c e lls Figure S5: Relative PPARβ mrna levels in CD4+ and γδ T cells from spleens from and mice. qpcr analysis of relative PPARβ mrna levels in CD4+ cells and γδ T cells isolated from spleens from and mice. Data are expressed as mean ± s.e.m. N=3. P<.5 when compared to (Mann-Whitney test). 8

9 A SPLEEN LYMPH NODES BLOOD CONTROL 6.5±.9% 9.3±.4% 7.4±.9% 65.9±.7% 16.8±.7% 73.6±1.5% 7.3±.6% 15.7± 1.3% 74.9±.% 1.6± 1.% 56.3±.7% 13.±.7% 19.4±.% 61.8±1.5%.4±.1% CD ±.8% 4.3±.5% 9.3±.9% CD ±4.7% 4.4±.4% 53.3± 4.7% 3.3±4.6% 19.± 3.8% 4.7±.5% 5.1±.3% 38.7±.6% 48.1±.1% 18.± 4.3% 4.8±9.% 11.± 4.5% 31.8± 7.9% 34.4±1.% 1.6± 6.9% CD4+ CD6L CD44 3.6± 4.5% 1.±.8% 39.3± 15.8% CD8+ cells (million) Gated on CD4+ naïve memory effector Spleen population Gated on CD8+ 3 B C 3 D 3 E 1 naïve memory effector Spleen population 1 Gated on CD4+ naïve memory effector Lymph node population 1 Gated on CD8+ naïve memory effector Lymph node population Figure S6: Changes in naïve, memory, and effector T cell populations in peripheral lymphoid tissues of mice. (A) Flow cytometric analysis of CD44 and CD6L expression on CD3+CD4+ or CD3+CD8+ gated cells from spleen, lymph nodes, and blood derived from and mice. Relative percentages (mean ± s.e.m.) of naïve (CD44-CD6L+), memory (CD44+CD6L+), and effector (CD44+CD6L-) cells are indicated. (B-E) Quantification of number of naïve, memory, and effector T cells in spleen CD3+CD4+ cells (B), spleen CD3+CD8+ cells (C), lymph node CD3+CD4+ cells (D), and lymph node CD3+CD8+ cells (E) from (white bars) and (black bars) mice. Data is from tissues obtained from 6 mice per group, with flow plots shown in (A) representative of latter groups. Data shown in bar graphs (B-E) are expressed as mean ± s.e.m. P<.5 when compared to (Mann-Whitney test). 9

10 Gated on DAPI- Gated on DN CD5+ CD5+CD4- CD5+ CD5+CD4-1.9%.5%.7%.% 1.% 17.9%.4%.7% 89.1% 99.1% 8.4%.% 8.5%.% 7.5% 74.9% CONTROL 95% 1.6%.% 3.1% 99.%.8%.%.%.4% 15.5% 9.6% 74.5%.3% 18.% 1.1% 8.4% CD8 CD4 CD44 CD5 Figure S7: Purification of DN/DN3 thymocytes from and mice. Thymocyte preparations from (upper flow plots) and (lower flow plots) mice were analyzed by flow cytometry after magnetic labeling and positive selection of CD5+ cells, or after subsequent magnetic depletion of CD4+ cells (CD5+CD4-). Flow plots on the left show flow cytometric analysis of CD4 and CD8 expression on DAPI- (alive) cells. Percentages of DN (CD4-CD8-), DP (CD4+CD8+), SP4 (CD4+CD8-), and SP8 (CD4-CD8+) thymocytes are indicated. Flow plots on the right show CD5 and CD44 expression on DN cells. Percentages of DN1 (CD5-CD44+), DN (CD5+CD44+), DN3 (CD5+CD44-), and DN4 (CD5-CD44-) cells are indicated. Data shown is from one purification of DN/DN3 thymocytes and is representative of purification efficiencies obtained. 1

11 cells (x1 4 /well) A Control B days Gated on DAPI- Gated on DN.9±.1% 18.4±1.5%.3±.6%.3±.% 58.±3.1% 41.±3.1% CONTROL 37.3±.7% 43.5±1.% 1.6±.% 16.4±.6%.4±.8% 1.9±.% 3.5±1.6% 74.± 1.4% 51.1±1.% 3.9±.6% CD8 cells (million/well) 3 1 CD4 Control C CD44 cells (million/well) DN DP SP4 SP8 CD5 Control TG-T-PPAR D DN1 DN DN3 DN4 11

12 Figure S8: Impaired T cell development observed in vivo in mice can be reproduced in vitro in the OP9-DL1 co-culture model. (A) Expansion of (open circles) vs (closed circles) DN/DN3 thymocytes after 3, 5, 7, and 1 days of co-culture with OP9-DL1 cells. Data are presented as number of cells per well (x1 4 ), with 1 4 DN/DN3 cells seeded on OP9-DL1 cells in a 4-well format at day. (B) Flow cytometric analysis of CD4 and CD8 expression gated on alive (DAPI-negative) cells (left) and CD5 and CD44 expression gated on DN (CD4-CD8-) cells (right) that were recovered after one week of co-culture of DN/DN3 thymocytes obtained from and mice. Relative percentages (mean ± s.e.m.) of DN (CD4-CD8-), DP (CD4+CD8+), SP4 (CD4+CD8-), and SP8 (CD4-CD8+) are indicated in the graphs on the left, and relative percentages (mean ± s.e.m.) of DN1 (CD5-CD44+), DN (CD5+CD44+), DN3 (CD5+CD44-), and DN4 (CD5-CD44-) cells are indicated in the graphs on the right. (C,D) Quantification of number of DN, DP, SP4 and SP8 cells (C) and number of DN1-4 cells (D) after one week of co-culture of DN/DN3 thymocytes obtained from and mice. Data were pooled from 3 independent experiments, with flow plots shown in (B) being from one representative experiment. Data shown in graphs (A,C,D) are expressed as mean ± s.e.m. P<.5 when compared to (Kruskal-Wallis test for (A) and Mann-Whitney test for (B-D)). 1