2.1. Culture of αβ and γδ T cell clones
All studies were conducted with approval from the Institutional Human Studies Committee and synovial fluid samples were obtained with written informed consent by patients prior to inclusion in the study. Cultures of previously derived Borrelia
-reactive γδ T cell clones [10
] and αβ T cell clones [25
] were expanded as previously described [3
] at approximately 21-day intervals using 10 μg/ml of a sonicate of B. burgdorferi
, strain N40, grown in Barbour-Stoenner-Kelly II (BSKII) medium (Sigma, St. Louis, MO) or 1 μg/ml phytohemagglutinin (PHA) (Murex Biotech, Dartford, Kent, England). Clones were cultured in AIM-V (Invitrogen, Carlsbad, CA) containing 10% Hyclone characterized FBS (ThermoScientific, Rockford, IL) and 100 U/ml human recombinant IL-2 (Cetus, Emeryville, CA).
2.2. Proliferation assay by 3H-thymidine incorporation
Triplicates of 3×105 cells/well in 96 well plates were pulsed with 1 μCi/well 3H-thymidine (GE Lifesciences, Piscataway, NJ) and incubated for 18 h at 37° C. Cells were then lysed and DNA retained on nitrocellulose filters using Tomtec Harvester 96 (Tomtec, Hamden, CT). 3H-thymidine was subsequently counted using a Microbeta counter (PerkinElmer, Shelton, CT).
2.3. Antibodies, TUNEL, and flow cytometry
Antibodies used were to the following determinants: TCR-αβ (BMA 031; Invitrogen/BioLegend, San Diego, CA), TCR-γδ (5A6.E9; Invitrogen/BioLegend), CD25 (CD25-3G10; Invitrogen), Fas (DX2; BD Pharmingen, San Jose, CA/Invitrogen/BioLegend), and CD3 (HIT3a; BioLegend). Anti-CD3 antibody (TR66, gift of Dr. Antonio Lanzavecchia) at a final concentration of 10 μg/ml was used for short-term stimulation experiments. Cells were treated with DMSO (Sigma, St. Louis, MO) or benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone (z-VAD-fmk; zVAD) (50μM) (MP Biomedicals, Solon, OH) for 30 min prior to anti-CD3 stimulation for caspase inhibition studies. To assess the extent of apoptosis, DNA strand breaks were measured by TUNEL staining. Briefly, single-cell suspensions were treated with or without plate-bound anti-CD3 for 8 h, and then washed twice with cold PBS. Cells were then stained with surface antibodies and fixed with fresh 1% formaldehyde (Ted Pella, Redding, CA), and after two washes, permeabilized with ice-cold 70% ethanol for 15 min. DNA strand break labeling was achieved by incubation at 37°C in labeling mix (1× TdT buffer, 2.5 mM CoCl2, 1 U/5μl terminal deoxynucleotidyl transferase (TdT), and 0.1 nM FITC-dUTP (Roche Diagnostics, Mannheim, Germany)). Cells were washed twice in cold PBS containing 1% BSA and stored in PBS/BSA/1% formaldehyde. Samples were analyzed on an LSR II flow cytometer (BD Biosciences, San Jose, CA).
2.4. Immunoprecipitation of TCR complex
T cells were lysed in 20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM Hepes, 1 mM EDTA, 1 mM Na3VO4, protease inhibitors (Roche Diagnostics, Indianapolis, IN) and 1% Triton X-100 (Calbiochem, La Jolla, CA). Cleared lysates were then subjected to one round of immunoprecipitation with pan-TCRαβ and pan-TCRγδ monoclonal antibodies (ThermoScientific). Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to PVDF membranes and blotted with antibodies against CD3ε (Dako, Carpinteria, CA), TCRζ (BD Pharmingen), and FcRγ (Upstate Biotechnology, Lake Placid, NY).
2.5. Caspase activity assay
Overall caspase activities were determined using the Apo-ONE Assay (Promega, Madison, WI), which measures the cleavage of DEVD-rhodamine, according to the manufacturer's recommendations. Spectrophotometric readings were taken using a Fluorescence reader (Biotek Instruments, Winooski, VT).
2.6. Biotin-VAD-fmk active caspase precipitation assay
Cells were lysed in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2mM sodium orthovanadate, 10% glycerol, protease inhibitors, and 0.2% NP-40) containing 20 μM biotin-VAD-fmk (bVAD) (MP Biomedicals). 450 μg of protein lysate in 300 μl lysis buffer were precleared by rocking with 40 μl Sepharose 6B agarose beads (Sigma) at 4°C for 2 h. Supernatants were then rocked with 60 μl streptavidin-sepharose beads (Invitrogen) at 4°C overnight. Beads were washed 5 times in lysis buffer, then boiled in loading buffer. Beads were removed by centrifugation and immunoblot analysis was performed on supernatants.
2.7. Immunoblot analysis
Protein lysates from T cell clones were separated by SDS-PAGE using 10% or 12.5% gels, transferred onto PVDF membranes (BioRad Laboratories, Hercules, CA), and blocked using 4% milk in Tris-buffered saline plus 0.1% Tween-20 (American Bioanalytical, Natick, MA) at room temperature for 1 h as previously described [26
]. Membranes were incubated at 4°C overnight in milk/TBS-Tween containing mouse anti-human caspase-8 monoclonal antibody (3-1-9) (BD Pharmingen), anti-caspase-3 585 rabbit polyclonal antibody (the kind gift of Dr. Yuri Lazebnik), caspase-9 monoclonal antibody (5B4) (Stressgen Assay Designs, Ann Arbor, MI), affinity-purified goat anti-human DFF45/ICAD (R&D Systems, Minneapolis, MN), or affinity-purified goat anti-human/mouse BID (R&D Systems). Immunoreactive proteins were visualized using species-specific secondary antibodies conjugated to horse radish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; Southern Biotech, Birmingham, AL; Biomeda, Foster City, CA; Jackson Immunoresearch, West Grove, PA) and developed using LumiGLO (KPL, Gaithersburg, MD). Quantitative assessment of band densities on immunoblots were performed using the Quantity-One software (Bio-Rad) and are displayed as the sum of the intensities of the pixels inside the volume boundary × area of a single pixel (in mm2
) after correction for background signal.
2.8. Statistical Analyses
A random coefficients growth model was used to examine 3H-thymidine incorporation, cell death by TUNEL, CD25, Fas, and CD3 changes over time in the two groups of cells, with individual cell types within group (αβ versus γδ). Data from up to six experimental replications were used, and a random effect was included in the model to control for differences across experiments. Planned post-hoc tests were used to examine differences between cell groups on individual days. Group differences in caspase activity on day 14 were examined using a general linear mixed model, with cell groups defined as above and a random effect included to control for differences across experimental replications. An analysis of variance was used to examine the effect of restimulation on caspase activity. Data for TUNEL assays did not meet distributional assumptions; thus, all analyses were based on log-transformed data. A repeated-measures analysis of variance was used to examine differences in αβ versus γδ cells in human subjects. Because of lack of conformity to distributional assumptions as well as the small number of subjects involved, these analyses were based on ranked data, which allows a non-parametric test. All analyses were performed using SAS version 9.2. Student t-test was conducted to assess densitometry readings for γδ versus αβ T cells.