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Plasmodium falciparum schizonts induce proliferation of and cytokine production by peripheral blood mononuclear cells (PBMC) from humans with no prior exposure to malaria (1, 12, 16, 28, 37). The production by these cells of proinflammatory cytokines, such as gamma interferon (IFN-) (6, 9, 15), has led to the hypothesis that they may contribute to the pathology of the disease (5, 6, 9), which is characterized by release of macrophage-derived cytokines, such as tumor necrosis factor alpha (TNF-), interleukin-1 (IL-1), and IL-6 (23), and the consequent induction of fever. On the other hand, rapid activation of inflammatory responses may act to limit parasite growth and so be beneficial for the host (9). Identifying the parasite products responsible for polyclonal lymphocyte activation would represent a significant step forward in understanding the pathogenesis of malaria and in the development of a malaria vaccine.

Cells from unprimed donors which respond to malaria antigens are almost exclusively CD3⁺ T lymphocytes (1, 9, 14, 20, 37), but they have been variously described as predominantly CD4 CD8 TCR⁺ (2, 13, 14, 31) or predominantly CD4⁺ TCR⁺ (5, 6, 9, 30). The kinetics of the response and the requirement for antigen processing and presentation via HLA class II suggest that the effect is mediated by a classical antigen rather than a mitogen or superantigen (5, 9, 20), although some workers have proposed that a superantigen may be responsible for the activation of large numbers of V9V2⁺ T cells (2, 13, 25).

The in vivo expansion of the TCR⁺ subset of peripheral blood T cells during primary P. falciparum or Plasmodium vivax infections has been reported (4, 17, 18, 27, 31), and in vitro, preferential activation of TCR⁺ cells has been reported when PBMC from naive donors were incubated with live, intact P. falciparum-parasitized erythrocytes (14) or freeze-thawed merozoites and/or schizonts (2, 13, 31). In contrast, several groups (including our own) have reported that in vitro stimulation of naive PBMC with freeze-thawed schizonts leads almost exclusively to expansion of CD4⁺ TCR⁺ cells (5, 6, 9, 30). Interpretation of the existing data is complicated by differences in methodology between studies (e.g., the presence or absence of exogenous IL-2 or irradiated feeder cells) and made more difficult by the recent realization that many isolates of P. falciparum maintained in long-term culture are contaminated by mycoplasma and that this can lead to artifactual results with respect to cytokine induction (35).

In order to clarify the requirements for activation of naive human T cells by malaria antigens, we have compared the effects of (i) freeze-thawed P. falciparum schizont extract (PfSE), (ii) live (intact) schizont-infected erythrocytes, and (iii) supernatants from overnight cultures of mature P. falciparum schizonts (containing the products of rupturing schizonts) on the lymphoblastic response of naive PBMC and on the surface phenotype of the responding cells. In order to determine the effect of variations in experimental protocols, which might explain contradictory conclusions in the literature, cells were grown in the presence or absence of irradiated autologous feeder cells or recombinant IL-2 (rIL-2).

P. falciparum clone 3D7 (36) was maintained in continuous culture and periodically synchronized by sorbitol treatment (24). Cultures were used when parasitemia reached ~6 to 8% mature schizonts. Mature schizonts were separated on a 60% Percoll gradient (Pharmacia, Uppsala, Sweden) and adjusted to a concentration of ~10⁸ schizont-infected cells per ml. PfSE was prepared by two cycles of freeze-thawing in liquid nitrogen and was stored for up to 4 weeks at 70°C. Supernatant was collected from overnight cultures of rupturing schizonts and centrifuged at 10,000 × g for 60 min to remove cellular debris. Freeze-thaw preparations of uninfected erythrocytes (uRBC) (10⁸/ml) and supernatants from uRBC cultures were used as controls. Parasite and uRBC preparations were screened for mycoplasma contamination by using a commercial PCR kit (Stratagene, Cambridge, United Kingdom). PBMC were obtained from malaria-unexposed European blood donors by density centrifugation (Lymphoprep; Nycomed, Oslo, Norway) and resuspended at a concentration of 10⁶ cells/ml in complete medium (9). Autologous feeder cells were prepared by irradiating PBMC in a cesium source (7,500 rads).

PBMC (10⁵ cells/100 µl of complete medium) were cultured, in triplicate, with malaria antigens (10⁴ live schizont-infected erythrocytes or 10⁴ uRBC, or the equivalent mass of PfSE or culture supernatants diluted 1:2) or phytohemagglutinin (PHA) (2 µg/ml; Sigma) for 6 days and pulsed with tritiated thymidine (1 µCi/well; Amersham Life Sciences, Little Chalfont, United Kingdom) for 18 h; incorporation of [³H]thymidine was assessed by liquid scintillation counting. The change in counts per minute was calculated as the geometric mean (GM) counts per minute for triplicate antigen-stimulated wells minus the GM counts per minute of control wells.

Lymphocyte phenotyping was performed by two-color flow cytometry as described previously (9). PBMC (10⁶ cells/ml) were cultured for 7 days with or without antigen (10⁵ live schizonts or uRBC or an equivalent mass of PfSE) and with or without 5 × 10⁵ irradiated feeder cells or exogenous rIL-2 (20 U/ml; Genzyme Diagnostics, Cambridge, Mass.). Viable cells were counted by trypan blue exclusion. Fluorescein isothiocyanate-conjugated antibodies to CD20/H147 (Caltag, San Francisco, Calif.), CD4/S3.5 (Caltag), TCR/BW242/412 (T Cell Diagnostics, Woburn, Mass.), and TCR/5A6.E9 (T Cell Diagnostics) were used. Phycoerythrin-conjugated antibodies to CD3/S4.1(7D6) (Caltag), CD45RO/UCHL1 (Caltag), and CD8/3B5 (Caltag) were used.

The IL-2 concentration was measured by a two-site enzyme-linked immunosorbent assay (Human IL-2 Duoset; Genzyme Diagnostics).

Lymphoproliferative response to P. falciparum. Cells from all donors responded to live schizonts, parasite culture supernatant, and PfSE, with changes in counts per minute ranging from ~2,000 to 13,600 (Fig. 1). There was no significant difference in the magnitudes of the proliferative responses to the different malaria antigen preparations. There was no significant proliferative response to either uRBC or control culture supernatant (not shown).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Lymphoproliferative responses of naive human PBMC to P. falciparum. Each symbol represents data from a single donor (7-day cultures). Cells from all donors proliferated in response to PHA, showing that they were viable (data not shown).

Comparison of phenotypes of cells responding to live parasites and PfSE. Although the percentages of lymphoblasts induced by PfSE and live schizonts were not significantly different, the phenotypes of the responding cells were markedly and significantly different for the two stimuli (Table 1). In PfSE-stimulated cultures, 93% of the CD3⁺ blasts expressed the T-cell receptor (TCR) and 7% expressed the TCR; similar proportions of TCR⁺ and TCR⁺ cells were seen in the R1 population. This is in full agreement with our previous data (9) and that of others (5, 6, 30). However, in cultures stimulated with live schizonts, 49% of CD3⁺ blasts expressed the TCR and 51% were TCR⁺; the percentage of TCR⁺ cells in the resting lymphocyte population was also slightly increased.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Fluorescence-activated cell sorting plots showing proportions of TCR+ and TCR+ CD45RO+ lymphoblasts in cultures stimulated for 7 days with PfSE, live schizonts, or parasite culture supernatant for a single, representative donor. Only blasting cells were included in the gate. The numbers in each quadrant are the percentage of gated cells in that quadrant.

Effect of irradiated feeder cells or exogenous IL-2 on activation of naive T cells by P. falciparum. In previous studies of naive-T-cell responses to P. falciparum, exogenous rIL-2 had been added or cells had been cultured in the presence of autologous, irradiated feeder cells. Irradiated cells are frequently used, in vitro, as a source of antigen-presenting cells and as a nonspecific support mechanism, but there is little recent data on their precise function (26) and little information on the cytokines they secrete. However, their ability to support proliferating cells suggests that they are a useful source of cell growth factors. We therefore compared the effect of rIL-2 or irradiated feeder cells on the T-cell response to PfSE and live schizonts.

Is endogenous IL-2 present in cultures stimulated with live schizonts? As many human pathogens have acquired the ability to interact directly with the immune system by producing homologs of human cytokines or cytokine receptors (reviewed by Yao et al. [38]) and as extremely high levels of IL-2R in the plasma of malaria patients have been reported (8, 19, 21, 29), we investigated whether IL-2 or IL-2-like substances were secreted by live P. falciparum schizonts. We tested fresh P. falciparum culture supernatants (from cultures with 10 to 12% parasitemia that had undergone schizogony in the previous 12 h) for the presence of IL-2 by bioassay (33) but found no evidence of any IL-2-like activity (data not shown). This is consistent with the failure of culture supernatant alone to activate large numbers of T cells (Fig. 2).

View larger version (8K): [in this window] [in a new window]   FIG. 3.   IL-2 in culture supernatants of cells stimulated with PfSE () or live schizonts (). Control cultures (stimulated with uRBC) all contained <20 pg of IL-2 per ml at each time point. PHA-stimulated cultures all contained >650 pg of IL-2 per ml after 24 h of stimulation.