Mice, Parasite and experimental infection
The 4-week-old and 8-week-old female BALB/c mice were purchased from Beijing Animal Institute. P.y17XL and P.y17XNL strains were provided by Dr. Motomi Torii (Department of Molecular Parasitology, Ehime University Graduate School of Medicine, Ehime, Japan). Infections were initiated by intraperitoneal (i.p.) injection of 1×106 P.y 17XL or 1×106 P.y 17XNL parasitized erythrocytes in BALB/c mice. All animal procedures were conducted in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1), and humanely treated. The experimental mice were matched for age and sex. Parasitemia was examined by light microscopy of Giemsa-stained, tail blood smears. Mortality was monitored daily. All experiments were performed in compliance with local animal ethics committee requirements. The animals were not submitted to euthanasia during the process of plasmodium infection. Other mice were submitted to euthanasia during detecting the relative index in indicated time points,the way to do it is posterior cervical dislocation after eyeball blood extraction.
Spleen cell culture
Spleen cell culture was prepared as previously described [16]. Briefly, we aseptically removed spleen from each mouse, and then passed through a sterile fine-wire mesh with 10 ml of RPMI1640 including 5% heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, Inc.), 25mM Hepes (Life Technologies), 0.12% gentamicin (Schering, Montreal, Quebec, Canada) and 2mM glutamine (Life Technologies). Cell suspensions were centrifuged at 350×g for 10 min at room temperature (RT). Using cold 0.17M NH4Cl to lysed Erythrocytes. Following the cells were washed twice with fresh medium, and then viability of the spleen cells was confirmed by trypan blue exclusion, and was always >90%. Spleen cells were adjusted to a final concentration of 107cells/ml in RPMI1640 supplemented with 10% heat-inactivated FCS. Aliquots (500μl/well) of the cell suspension were incubated in 24-well flat-bottom culture plates (FALCON) in triplicate for 48 hours at 37°C in a humidified 5% CO2 incubator. Then, the plates were centrifuged at 350×g for 10 min at RT, supernatants were collected and stored at -80°C until they were assayed for the levels of IFN-g, IL-4, IgG, IgG1, IgG2a and P. y MSP-1-specific IgG.
Cytokine analysis
Commercial enzyme-linked immunosorbent assay (ELISA) kit smeasured levels of IFN-γ and IL-4 according to the manufacturer’s protocols (R&D Systems, Minneapolis, MN). Using a microplate reader read the OD values at 450 nm. The concentrations of cytokines in samples were calculated against the standard curve generated using recombinant IFN-g and IL-4, respectively.
Multiplex assay for antibody determination
Levels of total serum IgG, IgG1, IgG2a and P.y MSP-1-specific IgG were measured by ELISA as previously described with some modifications [17]. Briefly, Maxisorp flat-bottomed, 96-well microplates were coated overnight at 4°C with 50 μg of P.y MSP-1 antigens in a carbonate-bicarbonate buffer (pH 9.6). The plates were washed with PBS-Tween (PBS-T) and blocked with 0.05% bovine serum albumin (BSA)-PBS-T. Next, 100 μl of plasma dilutions in 0.05% BSA-PBS-T (1:50 for P.y MSP-1 IgG) were added in duplicate and incubated at RT for 2 h. After washing with PBS-T, the plates were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, USA) at a dilution of 1:5000. The OD values were read in a microplate reader at 490 nm.
Cell surface/intracytoplasmicstaining and flow cytometry
To assess the function of CD4+ T cells, we detected Tfh (CD4+CXCR5+cells), CD4+PD-1+cells and CD4+CD62-PD-1+cells, spleen cells from BALB/c mice infected with P.y17XL/P.y17XNL at different time points were double-stained with FITC-conjugated anti-CD4 (clone GK1.5, BD), BV421-conjugated anti-PD-1 (clone J43, BD), PE-conjugated anti-CXCR-5 (clone 2G8, BD) and APC-conjugated anti-CD62L (MEL-14, BD), followed by two washes, staining and analysis by flow cytometry.
To assess dynamics of Th1(CD4+T-bet+IFN-γ+) cells and Th2 (CD4+GATA3+IL-4+) cells, spleen cells from BALB/c mice infected with P.y17XL/P.y17XNL at different time points were triple-stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (clone GK1.5), PE-conjugated anti-T-bet (clone eBio4B10, eBioscience), APC-conjugated anti-IFN-γ (XMG1.2, BD) for Th1 cells, and FITC-conjugated anti-CD4 (clone GK1.5), PE-conjugated anti-GATA-3 (clone L50-823, BD), APC-conjugated anti-IL-4 (clone 11B11, BD) for Th2 cells. After stimulation for 2 hours with PMA and ionomycin at 37°C, Golgi Stop (BD Bioscience) was added to each reaction (1:500, vol/vol). After co-culture for 4 hours at 37°C, the cells were washed with 3% FCS and then resuspended in 100μl of 3% FCS. FITC-anti-CD4, PE-anti-T-bet and PE-anti-GATA3 were added for surface staining. Then, the cells were fixed and permeabilized, and intracytoplasmic staining was performed using allophycocyanin (APC)-anti-IFN-γ. We used the isotype control antibodies as follows: Table 1. All antibodies were purchased from BD Pharmingen.
Statistical analysis
All analyses were performed using GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA). Data are presented as mean ± standard error of the mean (SEM). Survival analysis was performed using the Kaplan-Meier log-rank test. Statistical significance of differences between the two groups was assessed by unpaired Student’s t-tests. P-values were calibrated using Bonferroni correction, and were considered statistically significant if they were less than 0.05.