Animals
Inbred female BALB/c and C3He mice, 6–10 weeks old, were obtained from the Institute's Animal House Facility, National Institute of Immunology, New Delhi and National Institute of Nutrition, Hyderabad. The animals were fed on standard pellet diet and water ad libitum. All the mice were housed under standard conditions at the Institute's Animal Facility.
Chemical and reagents
Fetal calf serum was purchased from Harlan Sera Lab (Crawley Down, GB), RPMI 1640, DMEM and HBSS from GIBCO (Grand Island, NY) and L-glutamine, L-pyruvate, penicillin and streptomycin were from Serva (Heidelberg, Germany). Recombinant IL-1 cytokine was procured from Genzyme (Cambridge MA) and recombinant IL-2, IL-4, IL-5, IL-12, and IFN-γ from Pharmingen (San Diego, CA). For ELISA of IL-4, IFN-γ and IL-5, the capture and detection (biotinylated) antibodies were obtained from Pharmingen (San Diego, CA). General chemicals used in the study were procured from the Sigma Chemical Co. (St. Louis, MO). 3H-thymidine was the product of Amersham Pharmacia Biotech AB (Uppsala, Sweden).
Cell lines and hybridomas
The Th2 clone D10G4.1 (TIB-224) was procured from American Type Culture Collection (ATCC), (Rockville, MD). Th1 clone (pGL-10) was a kind gift by Dr. V. M. Sanders, Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio and Th1 clone (AE7) was a kind gift by Dr. J. Rengarajan, Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA. Anti-CD3 Ab (145.2C11) hybridoma was kind gift from Prof. C. A. Janeway, Jr. (Yale University, New Haven, CT). The CHO-B7-1 transfectants were a kind gift from Dr. A. Ochi, John P. Robarts Research Institute, Ontario, Canada.
Medium
Cells were cultured in RPMI 1640 or DMEM medium supplemented with 10% FCS, L-glutamine (2 mM), penicillin (50 μg/ml), streptomycin (50 μg/ml), HEPES (100 mM) and 2-ME (0.05 mM).
Maintenance of antigen-specific Th1 and Th2 cells
The Th1 clones were maintained following the studied protocols [23] and Th2 clones were maintained according to ATCC protocol.
The cultures for Th cell proliferation were set using different concentrations (1 × 103–105 cells/well) of stimulator cells (macrophages untreated or treated with IFN-γ and LPS-activated B cells) and T cells (2 × 104/well) in the presence or absence of B7-1 Abs. The cultures were also set using HamIg as controls for anti-B7-1 Ab. On day 5 of MLR cultures, [3H]-thymidine was added and the cultures were harvested after 16 h and assayed for DNA synthesis by beta scintillation counting.
Preparation of antigen presenting cells (B cells, splenic and peritoneal macrophages)
A single cell suspension of mice spleens was prepared in a balanced salt solution (BSS). The RBCs were depleted by treatment with ACK lysis solution. The adherent splenic macrophages were removed by plating on the plastic Petri plates (Grenier, Germany) for two hour at 37°C and 7% CO2. The adherent cells were washed several times with BSS and then removed by gentle scraping with the rubber policeman from the plate and used as adherent splenic macrophages. The nonadherent cells were treated sequentially on ice for 45 min, with a mixture of anti-Mac-2, Mac-3, Thy1, CD4 and CD8 Abs followed by incubation with baby rabbit complement at 37°C for 45 min. The purity of cells stained with anti-IgM Ab by indirect immunofluorescence, were nearly 80%, as analyzed by flow cytometry (Becton Dickinson, Mountain view, CA).
The PECs (peritoneal exudate cells) were harvested from mice inoculated 4 days previously with 2 to 3 ml of Thioglycollate. The cells were washed with cold BSS. The macrophages were obtained by adhering on plastic Petri's dishes for 1 h at 37°C, followed by washing several times in cold BSS. The cells were then removed from Petri dishes by gentle scraping with the rubber policeman, washed with BSS and resuspended in medium.
The proliferation and cytokine secretion by Th1 and Th2 cells using different doses of first and second signals
T helper clones (pGL-10 and D10G4.1) were isolated after 7–9 days of activation with antigen-pulsed splenocytes. The dead cells were removed by ficoll-histopaque and washed twice with BSS. The cells (5 × 104/well) were added in 96w flat bottom microtitre plate, pre-coated with different doses of anti-CD3 Ab (0.01–0.1 μg/ml) for 2–3 h in PBS (pH 7.0) at 37°C. The wells were washed three times with BSS. Different concentrations of paraformaldehyde (0.5%) fixed transfectants (5 × 103–5 × 104/well) were then added to T helper cells.
Similar cultures were also set in combination with different cytokines [IL-2 (50 U/ml), IL-4 (100 U/ml), IL-12 (100 U/ml)] for Th1 cells and [IL-2 (50 U/ml), IL-4 (100 U/ml), IFN-γ (100 U/ml)] for Th2 cells. Both Th1 and Th2 cells were also stimulated with anti-CD3 Ab (0.01 μg/ml) and paraformaldehyde fixed transfectants (5 × 104/well). The cells were incubated at 37°C/7% CO2. After 48 h, the cultures were pulsed with 0.5 μCi of 3H-thymidine and harvested 16 h later by an automatic cell harvester. Radioactivity incorporated was measured by liquid scintillation counting and data were expressed as mean counts per minute (cpm). For the estimation of cytokines (IL-4, IL-5 and IFN-γ), 100 μl of culture supernatant was removed from each set of triplicate wells after 48 h and pooled for ELISA.
Estimation of cytokines
The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).
Proliferation and cytokine secretion by Th1 and Th2 cells in response to APCs (B cells, splenic and peritoneal macrophages) and cytokines (IL-1, IL-2, IL-4, IL-5, IL-12 and IFN-γ)
Th1 cells (5 × 104/well) were cultured with gamma-irradiated B cells, splenic macrophages (1 × 105/well) and peritoneal macrophages (5 × 104/well) as antigen presenting cells in 96 w flat-bottom plate. B cells were irradiated at 500 R and splenic and peritoneal macrophages at 3000 R. Those doses of APCs were chosen which induced the optimum proliferation of Th1 and Th2 cells. In the cultures, IL-2 (50 U/ml), IL-4 (100 U/ml), IL-12 (100 U/ml), IFN-γ (1000 U/ml) and ovalbumin (200 μg/ml) were also added. Th2 cells were cultured with IL-1 (1 ng/ml), IL-2 (50 U/ml), IL-4 (100 U/ml), IL-5 (100 U/ml), IFN-γ (1000 U/ml) conalbumin (100 μg/ml). Aminoguanidine (1 mM) was also added to the inhibit nitric oxide production in cultures where peritoneal macrophages were used as APC. The proliferation and cytokine secretion were estimated as described above.
Statistics
Statistical analysis was done using unpaired student's t-test. '◆' Represents p < 0.05, '†' p < 0.01, '*' p < 0.001.