Blood -- Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti-factor VIII immune responses

Blood, Vol. 114, Issue 7, 1423-1428, August 13, 2009

Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses
Blood Ettinger et al. 114: 1423

Supplementary materials for: Ettinger et al

T-cell clone expansion method
Peripheral blood mononuclear cells (PBMCs) were isolated from an HLA-mismatched individual, resuspended at 10⁷ cells/ml in T-cell medium (RPMI 1640 with 25 mM HEPES RPMI-HEPES, 15% human serum MP Biomedicals, LLC, Solon, Ohio, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin) and irradiated at 5500R. The PBMC concentration was adjusted to 5 × 10⁶ cells/ml, phytohemagglutinin (PHA) (Remel, Lenexa, KS) was added to 2 µg/ml final concentration, and 500 µl aliquots were transferred to wells of a 48-well plate. The cryopreserved T-cell clones were then thawed and slowly diluted 1:10 with 10% FBS in RPMI-HEPES. Cells were washed twice with 5% FBS in RPMI-HEPES and resuspended in T-cell medium at 0.5–1.5 × 10⁶ cells/ml. 500 µl aliquots were added to the PBMC-PHA mixtures and the plates were incubated at 37°C. Twenty-four to 48 hours later, 500 µl supernatant was removed from each well and replaced with a 1:10 dilution of human IL-2 (Hemagen Diagnostics, Inc., Columbia, MD) in T-cell medium. IL-2 was administered every two days over 12–14 days, at which time cell growth slows and cell size decreases. The resting clones were then either re-stimulated or cryopreserved. Clonality of the expanded cells was confirmed routinely by staining with the DR0101-FVIII_2194–2213 tetramer.

Cytokine sandwich ELISA method
NUNC Maxisorp 96-well ELISA plates were coated with 100 ∝l of 2–4 ∝g/ml purified coating antibody (eBioscience) in 1× coating buffer (eBioscience), incubated overnight at 4°C, and washed with 1× phosphate buffered saline containing 0.05% Tween 20 (PBS-T). Coating antibodies were anti-human IFN-γ antibody clone MD-1; anti-human IL-4 clone 8D4-8; anti–anti-human IL-17A clone eBio64CAP17; and anti-human/mouse TGF-β₁ clone eBioTB2F. Plates were blocked with 1× ELISA diluent solution (eBioscience) for 1 hour at room temperature and washed with PBS-T. Recombinant cytokine standard (100 ∝l) (Cell Sciences or eBioscience) or 20–50 ∝l undiluted cell supernatant was added to the wells, which were incubated overnight at 4°C, and the plates were then washed with PBS-T. When measuring TGF-β₁ latent TGF-β₁ was activated to an immunoreactive form by treatment with 1N HCl (4 ∝l/20 ∝l cell supernatant) for 10 minutes followed by neutralization with 1N NaOH. A biotin-labeled detection antibody (100 ∝l at 2 ∝g/ml) (eBioscience) was added to the wells, incubated at room temperature for 1 hour, and plates were washed with PBS-T. Detection antibodies were biotin anti-human IFN-γ antibody clone 4S.B3, biotin anti-human IL-4 clone MP4-25D2, biotin anti-human IL-17 clone eBio64DEC17, and biotin anti-human/mouse TGF-β1 clone eBio16TFB. One hundred ∝l of avidin horseradish peroxidase (eBioscience) (diluted 1:1000) was added to the wells, incubated at room temperature for 30 minutes, and plates were washed with PBS-T. Super Aquablue ELISA substrate (100 ∝l) (eBioscience) was added to the wells and A₄₀₅ was measured at various time points between 10 minutes and 1 hour using a Bio-Rad Model 550 microplate reader (Hercules, CA). The cytokine concentrations were interpolated from standard curves. The linear concentration ranges of the human cytokine standard curves were: human IFN-γ (Cell Sciences, Canton, MA), 125 to 8000 pg/ml; human IL-4 (Cell Sciences), 8 to 500 pg/ml; human IL-17A (eBioscience), 62 to 4000 pg/ml; and human TGF-β₁ (eBioscience), 300 to 20,000 pg/ml.