All experiments were performed in accordance with UK laws and with the approval of the University of Birmingham Ethics Committee. Normal, RAG1−/− and T-cell-deficient mice14 were bred and maintained in our animal facility. T-cell-deficient mice generated by disturbing mouse CD3-ε expression with over 30 copies of the human CD3e gene have neither T cells nor natural killer cells.14
Preparation of CD4+ and CD4− LTi cells
Cell suspensions for isolation of CD4+ and CD4− LTi cells were made from the spleens of adult RAG−/− or T-cell-deficient mice. Briefly, CD11c+ cells were positively enriched by using CD11c-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Thy-1+ cells were enriched from CD11c+-depleted populations using Thy-1.2-coated magnetic beads (Miltenyi Biotec), and the resulting CD4+-enriched populations were sorted into CD4+ Thy-1+ CD3− CD27− B220− CD11c− (CD4+ LTi) and CD4− Thy-1+ CD3− CD27− B220− CD11c− (CD4− LTi) populations.
For the preparation of embryonic CD4+ and CD4− LTi cells, spleens were organ cultured in medium with or without 100 ng/ml interleukin-7 (IL-7; PeproTech EC, London, UK) on a 0·8-μm sterile nucleopore filter (Millipore, Watford, UK) in a 10% CO2 incubator for 7 days.
Fluorescence-activated cell sorting staining
Phycoerythrin (PE) -conjugated or biotinylated monoclonal antibodies (mAbs) against CD3 (145-2C11), CD8 (53-6.7), CD11c (HL3), CD27 (LG.3A10) and B220 (RA3-6B2) were used as exclusion markers, and allophycocyanin-conjugated anti-CD4 (GK1.5) and fluorescein isothiocyanate (FITC)-conjugated anti-Thy-1.2 FITC (30-H12) mAbs were used to detect CD4+ and CD4− LTi cells. The cells were stained with mAbs against α4/β7 integrin (DATK32), IL-7 receptor α (IL-7Rα; A7R34), CD69 (H1.2F3), OX40L (RM134L), CD30L (RM153), c-Kit (2B8) (BD Biosciences, Palo Alto, CA), and TRANCE (IK22/5) (eBiosciences, San Diego, CA).
Expression of DR3 was detected using polyclonal immunoglobulin G (IgG) goat anti-mouse DR3 (R&D Systems, Minneapolis, MN) and donkey anti-goat IgG Cy2 (Jackson Immunoresearch Laboratories, West Grove, PA).
To stain with lymphotoxin β-receptor–immunoglobulin (LTβR-Ig) or control-Ig fusion protein, cells were cultured in the presence of 100 ng/ml IL-7 for 7 days before staining with LTβR-Ig (a kind gift of Dr Jeff Browning; Biogen Idec, Cambridge, MA). The second-step reagent was goat anti-human IgG-FITC (Jackson Immunoresearch Laboratories). Prepared E15 cells were cultured with 100 ng/ml recombinant mouse TL1A (R&D Systems) for 2 days and then stained for flow cytometry analysis.
TaqMan low density array analysis
TaqMan primer sets are designed to work with an efficiency approaching 100%, enabling the quantitative comparison of messenger RNA (mRNA) expression for different genes not only within a cell type but also between cells of different lineages. Housekeeping genes (β-actin in these experiments) were used to correct for total mRNA (the level to which the β-actin signal was corrected in all mRNA samples).
TaqMan low-density real-time polymerase chain reaction (PCR) arrays (Applied Biosystems, Foster City, CA) were designed with a 96-gene format. A list of all the genes measured is as follows: chemokines (CCL19, CXCL12, CXCL13), chemokine receptors (CCR7, CXCR3, CXCR5), cytokines [IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, IL-15, IL-18, TSLP, interferon-α1 (IFN-α1), IFN-β, IFN-γ, transforming growth factor-β1), cytokine receptors (IL-2Rα, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10Rα, IL-10Rβ, IL-12Rβ1, IL-12Rβ2, IFN-γR1, IFN-γR2), costimulatory molecules (CD80, CD86, CTLA4, ICOS, ICOSL), dendritic cell marker (DC-SIGN, cathepsinS, integrin alpha x), house-keeping (CD4, β-actin, 18S), MHC class [β2-microglobulin (β2m), CD74], Toll-like receptor (TLR; MyD-88, TLR2, TLR3, TLR4, TLR5, TLR7, TLR9), TNF family (LTα, TNFα, LTβ, OX40L, CD40L, FASL, CD70, CD30L, 4-1BBL, TRANCE, TWEAK, APRIL, BAFF, LIGHT, TL1), TNFR family (TNFR1, TNFR2, LTβR, OX40, CD40, FAS, CD30, 4-1BB, RANK, TWEAKR, BAFFR, HVEM, GITR, DR3), transcription factor (Bcl-2, Bcl-6, Bcl-xL, RORγ, GATA3, foxP3, T-bet), others (perforin, granzymeB).
Complementary DNA (cDNA) was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems). This was added to the TaqMan Low Density array, and PCR was performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer’s recommendations.
The relative signal per cell was quantified by setting a threshold within the logarithmic phase of the PCR and determining the cycle number at which the fluorescence signal reached the threshold (Ct). The Ct for the target gene was subtracted from the Ct for β-actin. The relative amount was calculated as 2−ΔCt × 106.
Single-cell PCR analysis
Using an automatic cell deposition unit of a FACStar Plus (BD Biosciences) single cells were sorted into 384-well PCR plates containing 1 μl nuclease-free water. After sorting, the plates were stored frozen at −80°. Expression of CXCR5, CCR7 and β2m RNA was detected by real-time PCR. The following primers were used at limiting concentrations: CXCR5: forward ATATGGATGACCTGTACAAGGAACTG (60 nm), reverse CAGGCATGAATACCGCCTTAA (400 nm), CCR7: forward GGTGGCTCTCCTTGTCATTTTC (40 nm), reverse GTGGTATTCTCGCCGATGTAGTC (400 nm), β2m: forward CTGCAGAGTTAAGCATGCCAGTAT (100 nm), reverse ATCACATGTCTCGATCCCAGTAGA (100 nm).
The probe for CXCR5 was CCTTCTACAGTAACAGCACGGAGATTCCCCTAC labelled with CAL Fluor Orange 560 and Black Hole Quencher 1 (Biosearch Technologies, Novato, CA), for CCR7 it was TGCTTCTGCCAAGATGAGGTCACCG labeled with tetrachlorofluorescin (TET) and Black Hole Quencher 1 (Biosearch Technologies), and for β2m it was a NED labelled minor groove binder (MGB) probe CGAGCCCAAGACC (Applied Biosystems). Primers and probes were added to the lysate together with QuantiTect Multiplex reverse transcription-PCR buffer (Qiagen, Hilden, Germany) in a total volume of 5 μl. Reactions were run for 45 cycles in a 7500 Real-Time PCR System (Applied Biosystems). Relative quantification for gene expression in single cells was achieved by setting thresholds within the logarithmic phase of the PCR and determining the cycle number at which the threshold was reached (Ct). The relative amount of CXCR5 or CCR7 message to β2m in wells that showed a β2m product was plotted.
Tissue samples for immunofluorescence were embedded in Tissue-Tek OCT compound (Bayer Healthcare, Newbury, UK) and 6-μm thick sections were cut and fixed in acetone. The mAbs used were anti-mouse Thy-1.2 biotin (clone 53-2.1), CD4 (clone GK1.5), and vascular cell adhesion molecule-1 (VCAM-1)-FITC (clone 429) (BD Biosciences). Anti-CD4 mAbs were directly conjugated using an Alexa Fluor 647 Monoclonal Antibody Labelling Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Biotinylated antibodies were detected using mouse anti-biotin-tetramethyl rhodamin isothiocyanate mAbs (Stratech Scientific Ltd, Newmarket, UK). The FITC-conjugated antibodies were detected using rabbit anti-FITC antibodies (Sigma, Welwyn Garden City, UK), then goat anti-rabbit-FITC antibodies (Southern Biotech, Birmingham, AL). Sections were mounted using Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Confocal images were obtained at room temperature on a laser scanning microscope 510 Meta microscope (Zeiss, Welwyn Garden City, UK) equipped with either a 10 × and 40 × 1·4 numerical aperture water lens and 488, 543 and 633 lasers using Zeiss lsm software.