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The course of Plasmodium falciparum malaria is characterized by a complex interaction of host immune responses and parasite survival strategies. T lymphocytes and their products are essential both in regulating specific antibody formation and in inducing antibody-independent immunity to Plasmodium species (reviewed in reference 29). The CD4 T-cell subset is of major importance for the induction of blood-stage immunity, while the CD8 subset has been shown to be cytolytic against liver stages of the parasite. It has also been demonstrated, however, that the number of T cells as well as their response to antigenic stimulation is lower in the peripheral circulation during acute malaria (7, 11, 12). This cellular hyporesponsiveness has been associated with the sequestration of activated T cells expressing the adhesion molecule leukocyte function-associated molecule-1 (LFA-1) on their surfaces (11, 12). After the clearance of parasitemia, previously sequestered cells reemerge into the periphery, with subsequent restoration of immune responsiveness (7, 11). If this reallocation hypothesis is true, it would be of particular interest to identify the cytokine pattern displayed by these T cells during the course of disease, as Th1 cytokines, such as gamma interferon (IFN-) and interleukin-2 (IL-2), as well as Th2 cytokines, such as IL-4, appear to have substantial impact on disease outcome and the development of protection in infection due to Plasmodium spp. (10, 26).

By using flow cytometric analysis for the intracellular detection of cytokines, we sought to characterize the phenotypes and the frequency of cytokine-producing T cells during the course of drug-treated P. falciparum malaria from a region in Central Africa where the disease is hyperendemic.

Study population. The study was conducted in the Albert Schweitzer Hospital in Lambaréné, Gabon, where P. falciparum malaria is predominantly hyperendemic (32). Patients attending the outpatient clinic during November 1997 were enrolled if they met the following study criteria: infection with P. falciparum, no recent antimalarial treatment, and no other systemic infection. Fifteen patients (nine female) were included. The median age was 12 years (range, 4 to 35 years). The mean parasitemia was 32,100/µl (range, 1,500 to 158,500/µl) before treatment, decreased to 85/µl (range, 0 to 500/µl) on day 3, and was below the limit of detection in all patients on day 10. All patients were treated as outpatients with the combination of sulfadoxine and pyrimethamine, which has been shown to be curative in this area (22). The patients or their parents provided informed consent, and the study was approved by the Ethics Committee of the International Foundation of the Albert Schweitzer Hospital in Lambaréné.

Peripheral blood mononuclear cell cultures and intracellular cytokine detection by flow cytometry. Flow cytometric assessment of T-cell cytokine production was performed essentially according to the technique described by Jung et al. and modified by Willheim et al. (15, 33). Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-diatrizoate centrifugation. The cells were then cultured in Ultra Culture medium (BioWhittaker, Walkersville, Md.) supplemented with L-glutamine (2 mM/liter; Sigma, St. Louis, Mo.), gentamicin (170 mg/liter; Sigma), and 2-mercaptoethanol (3.5 µl/liter; Merck, Darmstadt, Germany) and were stimulated with phorbol 12-myristate 13-acetate (10 ng/ml; Sigma) and ionomycin (1.25 µM; Sigma) in the presence of monensin (1 µM; Sigma) for 4 h at 37°C in 5% CO₂. The cells were then harvested on ice, washed twice in phosphate-buffered saline (PBS), and fixed with 2% formaldehyde (1 ml per 2 × 10⁶ cells; Merck) for 20 min. After two additional washes in PBS, the cells were resuspended in Hank's balanced salt solution (supplemented with 0.3% bovine serum albumin and 0.1% sodium-azide) and stored at 4°C in the dark until they were stained. The fixed cells were washed twice with PBS and made permeable with saponin (0.1%; Sigma), resuspended with 50 µl of saponin buffer-diluted antibodies, and incubated for 25 min at room temperature in the dark. The following monoclonal antibodies (MAbs) were used: cytokine-specific mouse anti-human MAb (fluorescein-isothiocyanate [FITC]-labeled IFN- [clone B27]) and rat anti-human MAb (phycoerythrin [PE]-conjugated IL-2 [MQ1-17H12], PE-labeled IL-4 [MP4-25D2], FITC-labeled IL-4 [MP4-25D2], PE-labeled IL-5 [TRFK5], PE-labeled IL-10 [JES3-9D7], and PE-labeled IL-13 [JES10-5A2]). All MAbs were purchased from Pharmingen (San Diego, Calif.). The anti-CD4 MAb and the anti-CD8 MAb were allophycocyanin and peridinin chlorophyll labeled, respectively (Becton Dickinson, Mountain View, Calif.). Four-color staining was performed, and at least 10⁴ cells were analyzed on a FACSCalibur (Becton Dickinson) equipped with a two-laser system (488- and 630-nm wavelength, respectively). All cytokine combinations (IL-2-IFN-, IL-4-IFN-, IL-10-IFN-, IL-13-IFN-, and IL-4-IL-5) were stained in conjunction with CD4 and CD8. The data were analyzed with CELLQuest software (Becton Dickinson), and the results were expressed as the percentage of cytokine-producing cells in each CD4⁺- or CD8⁺-cell population (Fig. 1 and 2).

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Cytokine-specific anti-human MAbs (either FITC labeled or PE conjugated) were used in combination with allophycocyanin (APC)-labeled anti-CD4 MAb and peridinin chlorophyll (PerCP)-labeled anti-CD8 MAb for intracellular staining of cells within the lymphocyte scatter gate. The numbers in each quadrant represent the percentage of gated cytokine-producing cells within the CD4+- or CD8+-cell population. The dot plots from a representative child donor show that 46.8% of gated CD4+ cells were IL-2+-IFN-, 5.3% were IL-2+-IFN-+, and 3.1% were IL-2-IFN-+. Eleven percent of CD8+ cells produced IL-2+-IFN- cytokines, 2.3% produced IL-2+-IFN-+ cytokines, and 20% produced IL-2-IFN-+ cytokines.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Representative two-parameter dot plots displaying the kinetics of the frequency of cytokine-producing CD4+ cells during the course of antimalarial treatment. Each vertical row depicts the results of the cytometric analyses performed for one representative patient before treatment (day 0), during clearance of parasites (day 3), and after resolution of parasitemia (day 10). IFN- (IFNg; x axis) was stained in combination with IL-2, IL-4, IL-10, and IL-13 (all y axis). IL-4 (x axis) was also combined with IL-5 (y axis). The quadrant statistics were set on the basis of the corresponding negative controls. Corresponding data and statistics are displayed in Fig. 3 and its legend.

Statistical methods. Statistical analysis was performed with a standard statistical package (SPSS 7.5 for Windows; SPSS Inc., Chicago, Ill.). The general linear model repeated measures was applied for the analysis of dependent variables within the observation period. Bivariate correlations were done by computing Pearson's correlation coefficient. A P value of 0.05 was considered significant.

Patients. Fifteen patients with P. falciparum infection were studied. Sulfadoxine-pyrimethamine-induced clearance of parasitemia was rapid in eight patients, having eliminated their parasites on day 3. In all patients parasitemia was below the level of detection on day 10. Further clinical follow-up disclosed recrudescent P. falciparum malaria in the study participants; no side effects clearly attributable to the antimalarial treatment were observed.

Frequency of CD4⁺ and CD8⁺ T cells during parasite clearance. The percentage of cells within the lymphocyte scatter gate positively stained for CD4⁺ (mean ± standard error of the mean [SEM]: day 0, 50% ± 2%; day 3, 52% ± 1%; day 10, 49% ± 2%) or CD8⁺ (mean ± SEM: day 0, 19% ± 1%; day 3, 18% ± 1%; day 10, 19% ± 1%) did not change significantly during the observation period.

Frequency of Th1-cytokine-producing CD4⁺ and CD8⁺ T cells. The frequency of T cells producing IFN- or IL-2 or coproducing both cytokines was investigated (Fig. 1 to 3). With respect to the CD4⁺ subset, a gradually increased capacity for IL-2-IFN-⁺ cytokine production was found until day 10 (Fig. 2 and 3), and the frequency of IL-2⁺-IFN- cytokine-expressing cells within the same subset was significantly lower after parasite clearance. The frequency of T cells coproducing both cytokines (IL-2⁺-IFN-⁺) was not significantly different at any time (Fig. 2 and 3). Within the CD8⁺ subset a significantly higher frequency of IL-2-IFN-⁺ cytokine-producing cells and a significantly decreased frequency of IL-2⁺-IFN- cytokine-producing cells was found after parasite clearance. The percentages of IL-2-IFN-⁺ cells (mean ± SEM) were as follows: day 0, 29% ± 3%; day 3, 28% ± 2%; and day 10, 36% ± 3%; P < 0.05. The percentages of IL-2⁺-IFN- cells (mean ± SEM) were as follows: day 0, 7% ± 1%; day 3, 6% ± 0.5%; and day 10, 4% ± 0.3%; P < 0.01.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Frequency of cytokine-producing CD4+ cells following admission (day 0), during clearance of parasitemia (day 3), and after resolution of parasitemia (day 10) in 15 patients with uncomplicated P. falciparum malaria. The data points indicate the mean percentage of CD4+ cells ± SEM. The differences of mean frequencies throughout the observation period within the CD4+ subset were as follows: (A) IL-2-IFN-+, P < 0.001 and IL-2+-IFN+, not significant (n.s.); (B) IL-2+-IFN, P < 0.01; (C) IL-4+-IFN, P < 0.01 and IL-4+-IFN+, n.s.; (D) IL-13+-IFN, P < 0.01 and IL-13+-IFN+, n.s.; (E) IL-5+-IL-4, n.s. and IL-5+-IL-4+, n.s.; (F) IL-10+-IFN, P < 0.001 and IL-10+-IFN+, P < 0.001.

Frequency of Th2-cytokine-producing CD4⁺ and CD8⁺ T cells. The frequencies of IL-4-, IL-5-, and IL-13-producing CD4⁺ and CD8⁺ T cells were assessed (Fig. 2 and 3). In addition, the capacities of CD4⁺ and CD8⁺ T cells to coproduce IL-4 and IL-5 were studied (Fig. 2 and 3). While IL-4⁺-IFN- and IL-13⁺-IFN- cytokine-producing CD4⁺ cells decreased significantly during antimalarial treatment, the frequencies of IL-4-IL-5⁺ and IL-4⁺-IL-5⁺ cytokine-expressing T cells were not significantly different at any time (Fig. 2 and 3). No differences were seen in the IL-4⁺-IFN-, IL-13⁺-IFN-, IL-4-IL-5⁺, and IL-4⁺-IL-5⁺ CD8⁺ subpopulations, with each of them representing less than 1% of the CD8⁺ subset (data not shown).

Frequency of IL-10⁺-IFN- and of IL-10⁺-IFN-⁺ cytokine-producing CD4⁺ and CD8⁺ T cells. A marked increase was found in the IL-10⁺-IFN- and the IL-10⁺-IFN-⁺ cytokine-producing CD4⁺ subpopulations until day 10 (Fig. 2 and 3). IL-10⁺-IFN- and IL-10⁺-IFN-⁺ cytokine expression was found in less than 1% of CD8⁺ cells, and the frequency was not significantly different at any time.

Frequency of CD4⁺ and CD8⁺ T cells producing a Th0-type pattern of cytokines. The capacity of CD4⁺ cells to coproduce Th1 and Th2 cytokines as well as that of CD8⁺ cells to coproduce Tc1 and Tc2 cytokines (IL-4⁺-IFN-⁺ and IL-13⁺-IFN-⁺) was not significantly altered during the clearance of parasitemia (Fig. 2 and 3).