mice on the BALB/c background were generated and screened as previously described (24
newborn mice were rescued by intraperiotneal (i.p.) transfer of 60×106
BALB/c splenocytes to generate naive Thy1.2+
T cells with the nonfunctional Foxp3ΔEGFP
mice were obtained from The Jackson Laboratory. The Animal Resource Committee at the Medical College of Wisconsin approved all animal experiments.
Cell purification and adoptive transfer
Pooled splenocytes and lymph node cells (axillary, brachial, inguinal, and mesenteric) were stained with either anti-CD4-allophycocyanin (RM4-5, BD Biosciences) or anti-CD4-Pacific Blue (RM4-5, Invitrogen), plus anti-CD90.1-PerCP (OX-7, BD Biosciences) and anti-CD45RB-allophycocyanin (C363.16A, eBioscience) as appropriate and sorted on the basis of Ab and EGFP fluorescence. All sorting was done on a FACSAria (BD Biosciences). The average purity and viability of the sorted CD4+ populations was 98.96 +/− 0.14 and 84.31 +/− 0.68 (n=120), respectively.
Colitis was induced in 6 to 8 week old Rag1−/− BALB/c mice by i.p. injection of 4×105 CD4+ CD90.1− EGFP− CD45RBhi cells. Mice were weighed twice weekly. In some experiments, when mice lost 2.5% (+/− 5.7%) of their initial body weight or began to exhibit symptoms of colitis (diarrhea, hunched posture), they were treated by i.p. injection of nTreg (Thy1.2+) plus iTreg (Thy1.1+) cells (5×105 each) purified by cell sorting. The nTreg cells were isolated on the basis of EGFP expression from the spleen and lymph nodes of Foxp3EGFP BALB/c mice or IL10−/−
Foxp3EGFP BALB/c mice. The iTreg cells were generated in culture (as described below) and isolated on the basis of EGFP expression.
For serial adoptive transfer experiments 1×105 CD4+ Thy1.1+ EGFP− ex-iTreg cells were isolated by sorting cells from the MLN and spleen of mice that were successfully treated with Thy1.1+ WT iTreg cells plus Thy1.2+ WT nTreg cells and injected i.p. into Rag1−/− hosts. In some experiments the mice were treated with 0.5×106 Thy1.2+ nTreg cells plus 0.5×106 Thy1.2+ iTreg cells and were co-injected with 15,000 Thy1.1+ EGFP− T cells (based on a 3% sort impurity, 3 times the calculated impurity). The Thy1.1+ EGFP− T cells were isolated from iTreg cells that had lost Foxp3 expression in culture.
TGF-β1-mediated in vitro conversion
Sorted CD4+ EGFP− cells from Foxp3EGFP or IL10−/− FoxpEGFP mice (1×106/mL) were cultured with anti-CD3 mAb (clone 14-2C11 at 2.5 µg/mL) coated dishes in the presence of soluble anti-CD28 mAb (1 µg/mL; clone 37.51), TGF-β1 (5 ng/mL; R&D Systems), and 100 U/mL IL-2. After 72h, cells were resorted based upon EGFP fluorescence and used for adoptive transfer or maintained in culture with IL-2.
IEL and LPL isolation
The entire colon and the distal 15 cm of the small intestine were used as the source of intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) (25
). IELs were removed by gentle shaking of 0.5-cm intestinal sections for 30 min in buffer containing 5% (vol/vol) FCS, 1 mM dithiothreitol (Sigma-Aldrich) and 5 mM EDTA. IELs were washed and isolated on a discontinuous Percoll gradient (67%, 44%). Washed intestinal sections were digested with collagenase D (1 mg/mL, Roche) in the presence of DNase I (Invitrogen). A discontinuous Percoll gradient (67%, 44%) was used to isolate washed LPLs.
Antibodies and flow cytometry
Cells were collected from the spleen, MLN, colon and small intestine and stained as indicated. The anti-mouse antibodies used were Pacific Blue-conjugated anti-CD4 (RM4-5, Invitrogen); PerCP-conjugated anti-CD90.1 (OX-7, BD Biosciences); Alexa Fluor-conjugated anti-CD44 (IM7, Biolegend), PE-conjugated anti-CD62L (MEL-14, BD Biosciences); Alexa Fluor 647-conjugated anti-CD103 (2E7, Biolegend); PE-Texas Red-conjugated anti-CD25 (PC61 5.3, Invitrogen); PE-Cy7-conjugated Klrg1 (2F1, eBioscience); APC eFluor 780-conjugated anti-TCRβ (H57-597, eBioscience); and APC-conjugated anti-GITR (DTA-1, eBioscience). A four-laser custom LSRII was used to collect the data, and FlowJo software was used for analysis.
Intracellular staining and cytokine analysis
Intracellular cytokine staining was performed after a 5 hour restimulation with phorbol 12-myristate 13-acetate (PMA, 5 ng/mL, Sigma-Aldrich) and ionomycin (0.5 µM, Sigma-Aldrich) in the presence of brefeldin A (1 μL/mL; BD Biosciences). Surface staining of cells was performed using a modified FACS buffer containing 10 μg/mL brefeldin A. Cells were stained on ice for 30 min with the primary anti-mouse antibodies PE-Cy7-conjugated anti-CD4 (RM4-5, BD Biosciences), PerCP-conjugated anti-CD90.1 (OX-7, BD Biosciences), and APC eFluor 780-conjugated anti-TCRβ (H57-597, eBioscience) then washed with the modified FACS buffer and fixed in 1% paraformaldehyde overnight at 4°C. After this incubation, cells were washed with 1mL PBS and then permeabilized with 1mL 0.1% Triton-X. Intracellular staining was performed for 30 minutes at room temperature with APC conjugated anti-IFN-γ (XMG1.2, BD Biosciences), Alexa Fluor 700 conjugated anti-TNF-α (MP6-XT22, BD Biosciences), Pacific Blue-conjugated anti-IL-17A (TC11-18H10.1, Biolegend) or with Pacific Blue-conjugated anti-Helios (22F6, Biolegend), and APC-conjugated anti-CTLA-4 (UC10-4B9, Biolegend). A four-laser custom LSRII was used to collect the data, and FlowJo software was used for analysis. Serum cytokines were measured using the eBioscience FlowCytomix kit following the manufacturer’s recommendations.
Complete colons were fixed in formalin, processed, and stained with H&E using a histology core facility. Blinded sections from the entire colon were examined by a pathologist (N.H.S.) and large intestine colitis scores were determined for the following inflammatory changes on a 4-point semiquantitative scale with 0 representing no change (26
). The following features were considered: severity, depth and chronic nature of the inflammatory infiltrate, crypt abscess formation, granulomatous inflammation, epithelial cell hyperplasia, mucin depletion, ulceration, and crypt loss.
RNA and cDNA isolation
Spleen and MLN cells of mice that were successfully treated with 0.5×106 Thy1.1+ WT iTreg cells plus 0.5×106 Thy1.2+ WT nTreg cells were stained with Pacific Blue-conjugated anti-CD4 (RM4-5, Invitrogen) and PerCP-conjugated anti-CD90.1 (OX-7, BD Biosciences) and were sorted on the basis of Ab and EGFP fluorescence. ex-iTreg (CD4+ Thy1.1+ EGFP−), iTreg (CD4+ Thy1.1+ EGFP+), and nTreg cells (CD4+ Thy1.1– EGFP+) were isolated by flow cytometry sorting on a FACSAria. Total RNA was extracted with the RNeasy Micro Kit for <100,000 cells or the RNeasy Mini Kit for >100,000 cells (Qiagen) according to the manufacturer’s protocol. cDNA was synthesized with the Superscript III First Strand Synthesis System and oligo(dT) primers (Invitrogen) according to the manufacturer’s protocol. Isolated cDNA was used for quantitative PCR, spectratype analysis, or CDR3 sequencing.
Quantitative PCR (qPCR)
qPCR was performed in an StepOnePlus PCR System (Applied Biosystems) using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and pre-developed specific Taqman primers for Il10 (Mm00439616_m1). GAPDH was used (Mm99999915_g1) as a housekeeping gene (Applied Biosystems). The standard curve for Il10 was developed by isolating bone marrow cells from C57BL/6J and culturing the cells at 4×104 cell/mL in DMEM/F12-10 media (DMEM/F12 (GIBCO), 10% (v/v) FBS (Atlantic Biologics), 10 mM L-glutamine (GIBCO), 100 IU penicillin/mL (GIBCO), 100 µg/mL streptomycin (GIBCO)) at 4×106 cells/mL in the presence of 10 U/mL MCSF (eBioscience, 14-8983-80). The cells were incubated at 37°C for 7 days (media was changed on day 3). Macrophages were isolated using Cellstripper (Cellgro), washed, and resusupended in DMEM/F12-10 at 2×105 cells/mL. The cells were plated for 8 hours at 37°C in a 24 well plate containing 50 ng/mL LPS (Sigma), 1 µg/mL OVA (323–339 peptide), and 15 µL/mL anti-Ova (AbD Serotec, 0220-1682). cDNA was isolated as described and the Il10 gene from the exon 2/3 boundary (forward primer: 5’–AATGCAGGACTTTAAGGGTTACTTGGG–3’) to the exon 4/5 boundary (reverse primer: 5’– CTTGTAGACACCTTGGTCTTGGAG–3’) was cloned into the pCR4-TOPO vector (Invitrogen) and transformed into DH5α cells. The plasmid was isolated with a mini prep (Promega) and then purified by agarose gel extraction. The concentration was determined using log-dilutions and measured with a NanoDrop (Invitrogen). The standard curve was created with 10 fold dilutions based on copy number.
Gene expression analysis
Total RNA for the iTreg, nTreg, and ex-iTreg sets was isolated with TRIzol (Invitrogen), according to the manufacturer’s protocol, from sorted cells pooled from 14 mice treated with 0.5×106
WT nTreg plus 0.5×106
WT iTreg cells. Labeled target was prepared and hybridized to Affymetrix 430 2.0 GeneChips in accordance with the manufacturer’s protocol. Two technical replicates were performed and the results were averaged. Probe sets that revealed a twofold difference (|log2
ratio| > 1.0) relative to Tconv cells were identified and used in subsequent analyses. The data was normalized with the Robust Mulit-array Analysis (RMA) algorithm derived by the Bioconductor group (http://www.bioconductor.org
). The mean fold change was calculated from 2 independent arrays for each cell type and was scored P
<0.05 with a false discovery rate (FDR) <10% by the non-parametric rank product test (28
). A false negative result is recovered for Foxp3+
Treg cells because the gene array probes for Foxp3
lie distal to the poly-A initiation site in the Foxp3EGFP
allele. The microarray data are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds
) under the accession number GSE35543. Tconv cell data was taken from GEO microarray data set GSE6875.
The methylation status of the Treg cell-specific demethylation region (TSDR) of Foxp3 in donor male nTreg, iTreg cells, and ex-iTreg cells purified from mice treated with WT nTreg plus WT iTreg cells by cell sorting, was assessed by bisulphite sequence analysis (29
). Briefly, genomic DNA was treated by bisulfite, to convert unmethylated cytosines into uracil leaving methylated cytosines unchanged. Bisulfite conversion of DNA was performed using EZ DNA methylation-Direct™ Kit (Zymo research) according to the manufacturer instructions. The TSDR of converted DNA was amplified by methylation-specific primer sequences 5'-TATTTTTTTGGGTTTTGGGATATTA-3' (forward) and 5'-AACCAACCAACTTCCTACACTATCTAT-3' (reverse). The PCR product was purified and inserted into a TOPO TA cloning vector (Invitrogen). The ligation product was used to transform competent bacteria (10–beta competent E. coli
, New England Biolabs) and clones were selected on kanamycin. Plasmid DNA was extracted by Qiagen miniprep kit and clones that present a 300bp fragment after EcoRI digestion were selected. Sequencing was done with M13R primer. Blast analysis was done by comparing the M13R sequence and converted FoxP3 gene sequence.
cDNA was amplified by PCR with a Cβ primer (5’ CTCAAACAAGGAGACCTTGGGTGG– 3’) and a Vβ primer from one of 22 Vβs (30
). An ABI 3100 Genetic Analyzer was used to analyze the length distribution of amplified cDNA products and Xplorer v2.4.2 (http://www.dnatools.com/download.html
) was used to create histograms.
Ion Torrent sequencing
cDNA was amplified by PCR with the C primer and a V 8.2 primer (5’ – GCTACCCCCTCTCAGACATCAGTG– 3’). PCR products were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s protocol and concentrated using ethanol precipitation. The DNA sample was purified using Agencourt AMpure beads. The purified samples were used to generate libraries for Next - Generation Sequencing using the Ion Torrent Personal Genome Machine (PGM) (Life Technologies, Carlsbad, CA) following the Ion Fragment Library Kit protocol. During preparation of the libraries, samples were size selected using the Sage Science Pippen Prep instrument and 2% agarose cassettes. Completed libraries were analyzed and quantified on the Agilent 2100 BioAnalyzer. The obtained libraries were processed for sequencing by dilution following the recommendations for the Ion Xpress Template Kit protocol that utilized an emulsion PCR, breaking and enrichment of each sample. Sequencing was performed using the Ion Torrent 314 or 316 chip and respective reagents. Sequence analysis and base calling was performed using the built in sequence software (v.1.9).
CDR3 repertoire analysis by subcloning
The purified PCR products were TA cloned into the pCR4-TOPO vector (Invitrogen) to create cDNA libraries. Individual colonies were subcloned and plasmids containing inserts were grown for 16 hours in LB media, frozen at -80°C in 50% glycerol and sent to Beckman Coulter Inc. for sequencing. The CDR3 regions were identified as the sequence between the second conserved cysteine encoded by the 5’ Vβ gene segment and the conserved phenylalanine encoded by the 3’ Jβ segment (IMGT).
The comparisons between groups for overall survival functions were done using the log-rank test. The random coefficient model was used to generate a quadratic fit of the weight change over time. For the colitis scores, cell frequencies and numbers, and serum cytokine levels a non-parametric Kruskal-Wallis test was used to compare the measurements between the groups. For the pairwise comparisons a Mann-Whitney U test was performed. The TCR repertoire data was analyzed using the Morisita-Horn Index, which was calculated using EstimateS software.