Analysis of T-Cell Receptor Vβ Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays

padlock probes and oligonucleotides

All 96mer padlock probes (Table 1⇓ ) were analyzed for secondary structures by use of the web-based mfold software (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/) and were purchased 5′-phosphorylated from Thermo Hybaid. Oligonucleotide targets, amplification primers, and spot oligonucleotides were purchased from Thermo Hybaid and DNA Technology. Spot oligonucleotides were complementary to the tag sequences and had a spacer sequence [5′-NH₂-T(CTT)₅-3′] connected to their 5′ ends. Tag sequences were selected from the GeneFlex™ Tag Array collection (Affymetrix).

seb stimulation

Peripheral blood mononuclear cells (PBMCs) from a healthy blood donor were obtained by density gradient centrifugation using Ficoll Hypaque (Amersham Biosciences), washed twice in phosphate-buffered saline, and then resuspended in RPMI 1640 (Invitrogen) supplemented with 100 mL/L human serum (Sigma-Aldrich), 2 mmol/L l-glutamine, 100 kIU/L penicillin, and 100 mg/L streptomycin. The cells were incubated at 37 °C in 5% CO₂ at 1–2 × 10⁶ cells/mL. Cells were stimulated with 5 mg/L SEB (Sigma-Aldrich) for 72 h, washed, and incubated for an additional 5 days in the absence of superantigen. Cell concentration was maintained at 1–3 × 10⁶ cells/mL throughout the culture period. Activation was assessed by visual examination and by flow cytometry using forward and side scatter characteristics. Samples for flow cytometry and quadruplicate samples for padlock probe analysis were obtained before cultures were supplemented with SEB and after 48 h, 72 h, 6 days, and 8 days. Four samples of RNA from each time point were collected for cDNA synthesis. Only two cDNA samples were prepared from the 48-h time point.

patients and specimens

Tumor tissue and lymph nodes were obtained from patients undergoing surgery for malignant melanoma or urinary bladder cancer at the University Hospital of Uppsala, Sweden. The local ethics committee approved the study, and all patients gave informed consent.

Tumor-draining lymph nodes were identified by peritumoral injection of Patent Blue Dye (Guerbet) as described previously (19)(20). Slices ∼1 mm thick were obtained from primary tumors and from tumor-draining and non-tumor-draining lymph nodes. Specimens were processed immediately to minimize degradation of RNA.

purification of t cells

Single-cell suspensions of lymph node cells were obtained by grinding in a loose-fit glass homogenizer. Cell suspensions were washed twice in phosphate-buffered saline containing 50 mL/L fetal calf serum, 2 mmol/L EDTA, and 0.1 g/L sodium azide. TCRαβ+ or CD4+ cells were magnetically separated over MACS LS columns (Miltenyi Biotech), by positive selection using fluorescein isothiocyanate (FITC)-conjugated anti-TCRαβ or anti-CD4 antibody (Becton Dickinson), respectively, and anti-FITC Microbeads (Miltenyi Biotech). Alternatively, the cells were separated by negative selection with the CD4+ T Cell Isolation Kit (Miltenyi Biotech), according to the manufacturer’s recommendations. Negative selection purities commonly reached 50–70%, but all TCR+ cells were also CD4+ by fluorescence-activated cell sorting (FACS) analysis. Positively selected cell suspensions were >95% purity, as assessed by FACS analysis.

flow cytometric analysis

PBMCs and lymph node cell suspensions at 1 × 10⁵ cells/sample were washed in phosphate-buffered saline supplemented with 20 mL/L fetal calf serum and 0.5 g/L sodium azide, followed by staining with fluorophore-conjugated antibodies. The following antibodies were used: anti-TCRαβ FITC, anti-CD4 FITC, anti-CD69 phycoerythrin (PE), anti-CD3 peridinin chlorophyll-α protein (PerCp), and anti-CD4 allophycocyanin (Becton Dickinson). Flow cytometric analysis of the Vβ repertoire was performed with the IOTest Beta Mark Kit (Beckman Coulter), according to the manufacturer’s instructions. Briefly, 1 × 10⁶ cells/sample were stained with a panel of 24 Vβ family-specific antibodies, combined in groups of 3 in eight tubes, one antibody being conjugated to FITC, another to PE, and the third to both FITC and PE. Costaining was performed with anti-CD3 PerCp (Becton Dickinson). A lymphocyte gate was established on the basis of forward and side scatter characteristics. The relative representation of a given Vβ family was expressed as the percentage of cells stained with the family-specific antibody among CD3+ (PerCP+) cells in the lymphocyte gate. Immunofluorescence was detected by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed with the Cellquest software (Becton Dickinson).

rna extraction and cdna synthesis

Cells were pelleted and lysed in 1 mL of TRIzol (Sigma-Aldrich), and total RNA was isolated according to the manufacturer’s protocol. RNA concentrations and quality were determined on an Agilent 2100 Bioanalyzer (Agilent Technologies). We used 4 μg of RNA from SEB-stimulated PBMCs or total RNA from patient samples for random hexamer (Gibco BRL)-primed double-stranded cDNA synthesis according to the manufacturer’s protocol (Invitrogen). Samples were finally purified on a G-50 Sepharose Spin Column (Amersham Bioscience).

padlock probe ligation, exonuclease treatment, and amplification

We combined 3 μL of ligation mixture with 7 μL of cDNA sample (H₂O in the background control reaction) for a final composition of 14 mM Tris-HCl (pH 8.3), 100 mM KCl, 7 mM MgCl₂, 0.35 mM NAD, 0.07 mL/L Triton X-100, 1.5 U of Ampligase (Epicentre), and 90 pM each padlock probe. Reaction mixtures were placed in a thermal cycler at 95 °C for 5 min and then cycled four times between 46 °C for 3 h, 60 °C for 10 min, and 95 °C for 2 min; after the cycling, they were kept at 8 °C until use. We added 10 μL of exonuclease mixture to the reactions for final concentrations of 67 mM Tris-HCl (pH 9.0), 50 mM KCl, 3.7 mm MgCl₂, 0.1 μg/μL bovine serum albumin, and 0.5 U/μL exonuclease I and exonuclease III (New England Biolabs). Reactions were incubated at 37 °C for 1 h, followed by 95 °C for 5 min. We then transferred 6 μL to 24 μL of PCR mixture for final concentrations of 30 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.9 mM MgCl₂, 200 μM deoxynucleotide triphosphates, 400 nM each PCR primer, and 33 mU/μL Platina Taq Polymerase (Invitrogen). Reactions were placed in a thermal cycler at 94 °C for 2 min and then cycled 25 times between 95 °C for 30 s and 56 °C for 10 s.

oligonucleotide arrays and hybridization

Microarray slides and reusable silica masks were prepared as described by Banér et al. (18). For hybridization, 30-μL PCR reactions were combined with 20 μL of 5× standard saline citrate (SSC), 1 mL/L Triton X-100, 11 mmol/L EDTA, and 0.1 nmol/L Hyb oligonucleotide (Table 1⇑ ) as hybridization control. Samples were heated at 95 °C for 1 min and immediately placed on ice. We then transferred 40 μL to individual subarrays and incubated the hybridization cassette at 55 °C for 1.5 h. The silica mask was removed in a 0.02× SSC washing solution, and the slide was transferred to a 0.02× SSC wash for 10 min, followed by 10 s in 1 mmol/L MgCl₂, and was finally dried with pressurized air. Microarrays were scanned in a Genepix 4000B (Axon Instruments), and images were analyzed with QuantArray 2.0 software (GSI Lumonics).

Local background on the microarray was subtracted from recorded signals, and a mean was calculated from triplicate spots. Negative results were set to zero. Sample signals (S_Vβx) were compared with the corresponding background signals (bc_Vβx; padlock probe ligation reaction with no sample added) and set to zero if they were lower than 2(bc_Vβx). The representation of each Vβ subfamily was finally calculated as S_Vβx/Σ(S_Vβx S_Vβn). All calculations were made in an Excel spreadsheet using a macro.

padlock probe design and evaluation of assay performance

We designed padlock probes for 23 Vβ subfamilies, including two probes for the Vβ5 family (Table 1⇑ ), positioning the ligation site within or in immediate proximity of previously described Vβ-specific PCR primer sequences (21)(22)(23). We excluded the Vβ10-family-specific probe from the panel because the single member of that family has been reported to be a pseudogene (8). We chose the sense strand as target, which allowed ligation either templated by RNA (16) or by second-strand cDNA. The segment connecting the target-complementary arms of each padlock probe contained sequences recognized by two general amplification primers, along with a specific sequence assigned to a Vβ subfamily (referred to as subfamily-specific tag sequence; Fig. 1A⇓ ). The experimental procedure is described in Fig. 1B⇓ . A pool of all padlock probes was combined with a cDNA sample and ligase. After ligation and exonucleolytic degradation of unreacted probes, all circularized padlock probes were amplified in parallel by the same PCR primer pair, one of which was fluorescently labeled. Amplification products were then hybridized to a microarray slide printed with oligonucleotides complementary to the subfamily-specific tag sequences and analyzed in a fluorescence scanner.

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Figure 1.

Parallel analysis of TCR Vβ family expression by padlock probes.

(A), each subfamily-specific probe has target-complementary sequences at both the 5′ and 3′ ends, and the segment connecting these includes a general amplification sequence and a tag sequence (red) assigned to the subfamily. When the probe ends hybridize to a target sequence, the ends are brought in close proximity, allowing a DNA ligase to join them, converting the probe to a circular molecule. The illustration exemplifies a padlock probe for the Vβ1 subfamily hybridizing to TCR Vβ1 cDNA. For simplicity, only parts of the hybridizing sequences are shown (see Table 1⇑ ). (B), schematic workflow of the assay. A pool of 24 padlock probes, each with a subfamily-specific tag sequence (color coded), is combined with a cDNA sample along with a ligase. After ligation, unreacted probes are degraded by exonuclease treatment, followed by PCR amplification of the circularized probes with a general primer pair, one of which is fluorescently labeled. The PCR products are applied to a microarray, where the tag sequences hybridize to complementary oligonucleotides deposited at predefined positions. Finally, the array is scanned for simultaneous readout of the Vβ distribution in the sample.

We investigated the repeatability of the method by assaying a pool of synthetic oligonucleotide targets several times. These data were used to determine thresholds for experimental variation of each individual Vβ subfamily. We defined a change in Vβ representation as significant if it deviated by more than 3 SD from the corresponding representation obtained with the oligonucleotide targets (data not shown). Repeated analysis of the same samples on different occasions gave very similar results, demonstrating the robustness of the method. The amount and quality of RNA from patient samples were determined on an Agilent Bioanalyzer before cDNA synthesis. Most of the patient samples contained RNA in the range of nanograms per microliter, which seemed to be the lower detection limit of the assay. We estimate that the ligation reactions required cDNA from ∼50 ng of total RNA to generate reliable microarray signals (data not shown).

analysis of seb-stimulated pbmcs reveals expected changes in vβ distribution

The TCRs also bind a class of microbial proteins, known as superantigens, that stimulate T cells bearing particular Vβ elements (24). To demonstrate the ability of the method to detect changes in a Vβ distribution, we incubated PBMCs from healthy individuals with SEB, after which the superantigen was removed and cultures were continued for an additional 5 days. The Vβ repertoire was analyzed before stimulation and after 48 h, 72 h, 6 days, and 8 days. A fivefold increase in cell numbers was evident at 48 h, followed by a plateau and a slight decrease at day 8 (data not shown). The results of flow cytometry analysis at each time point is presented in Fig. 2A⇓ . We observed a decrease in the percentage of cells in the CD3+ population expressing Vβ3, Vβ12, Vβ14, and Vβ17 at 48 and 72 h, followed by a dramatic increase in the same Vβ families at day 6, consistent with data from the literature (23)(25). Measurements of Vβ distributions with the set of 24 padlock probes revealed that SEB stimulation significantly increased the representations of Vβ3, Vβ12, Vβ14, and Vβ17 (Fig. 2B⇓ , left) by 48 h. We observed a minor expansion of Vβ15, which has also previously been reported to increase on stimulation by SEB, whereas the representation of Vβ20 decreased slightly, in contrast to its previously reported response to SEB. Flow cytometry data confirmed that no expansion of Vβ20 had occurred. In addition, Vβ18 representation increased slightly, whereas representation of the remaining Vβ gene families did not increase significantly during the investigated time period (Fig. 2B⇓ , right).

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Figure 2.

SEB stimulation of T cells in vitro.

PBMCs were collected from a healthy donor and incubated in the presence of SEB for 48 h ( ), 72 h (▪), 6 days (□), and 8 days ( ). (A), flow cytometric analysis of CD3⁺ cells. Representations of TCR Vβs are expressed as ΔVβ (Vβ_SEB − Vβ_unstimulated). Cells expected to respond to SEB are shown on the left, separated by a vertical line. ND, no antibodies were available. (B), padlock probe-based analysis of Vβ distributions expressed as ΔVβ (Vβ_SEB − Vβ_unstimulated). Vβ families expected to respond to SEB are shown on the left, separated by a vertical line. Error bars indicate SD for four samples, except for 48 h, for which two samples were analyzed (see Materials and Methods).

vβ representations in patients with malignant melanoma or urinary bladder cancer

cDNA was prepared from total αβ T cells or CD4+ Th cells from tumors, and from tumor-draining and non-tumor-draining lymph nodes of patients undergoing surgery for malignant melanoma or urinary bladder cancer. In one patient with a squamous cell carcinoma of the bladder, the tumor-draining lymph node contained metastatic tumor cells. Predominant Vβ2 and Vβ23 representation, along with minor overrepresentations of Vβ13 and Vβ21, was detected among TILs and in the draining lymph node compared with the corresponding Vβ families in a non-tumor-draining lymph node (Fig. 3A⇓ ). In the second patient, pathology examination revealed an adenocarcinoma confined to the bladder wall. In the TILs, Vβ13 dominated the expression profile, and the same subfamily dominated expression in the draining lymph node. On the other hand, Vβ13 expression was not detectable in a non-tumor-draining lymph node compared with the corresponding Vβ subfamilies in peripheral blood (Fig. 3B⇓ ). Two patients with malignant melanoma, from whom the primary tumors had been removed on previous occasions, underwent extended lymph node dissection. The expression profile of a draining lymph node from one of these patients (Vβ7, Vβ11, and Vβ13) differed from that of the corresponding non-tumor-draining lymph node (Fig. 3C⇓ ). Interestingly, two tumor-draining lymph nodes in the second patient had similar Vβ profiles, with strong representation of Vβ9 and Vβ12 and a much less prominent signal from Vβ13, compared with a non-tumor-draining lymph node (Fig. 3D⇓ ).

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Figure 3.

Expression of Vβ families in samples from tumor patients.

(A), Vβ repertoire in TCRαβ⁺ cells from a patient with squamous cell carcinoma of the bladder. ▪, TILs; ▦, tumor-draining lymph nodes (TDLN); □, non-tumor-draining lymph nodes (NLN). (B), distribution of Vβ subfamilies in CD4⁺ cells from a patient with adenocarcinoma of the bladder. ▪, TILs; □, non-tumor-draining lymph nodes; , tumor-draining lymph nodes; , PBMCs. (C and D), Vβ repertoires in CD4⁺ cells from two patients with previously resected malignant melanoma. Symbols for tumor-draining (TDLN) and non-tumor-draining lymph nodes (NLN) are as in A. Columns represent the mean of two measurements; error bars indicate maximum values.