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The Journal of Immunology, 2006, 176: 5504-5512.
Copyright © 2006 by The American Association of Immunologists

CD4 T Cells from Malaria-Nonexposed Individuals Respond to the CD36-Binding Domain of Plasmodium falciparum Erythrocyte Membrane Protein-1 via an MHC Class II-TCR-Independent Pathway1

Francis M. Ndungu*,{dagger}, Latifu Sanni*,{ddagger}, Britta Urban§, Robin Stephens*, Christopher I. Newbold, Kevin Marsh{dagger} and Jean Langhorne2,*

* Division of Parasitology, National Institute for Medical Research, London, United Kingdom; {dagger} Kenya Medical Research Institute, Centre for Geographic Medicine Research, Kilifi, Kenya; {ddagger} Department of Pathology, Leeds General Infirmary, Leeds, United Kingdom; § Nuffield Department of Clinical Medicine, Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom; and Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have studied the human CD4 T cell response to a functionally conserved domain of Plasmodium falciparum erythrocyte membrane protein-1, cysteine interdomain region-1{alpha} (CIDR-1{alpha}). Responses to CIDR-1{alpha} were striking in that both exposed and nonexposed donors responded. The IFN-{gamma} response to CIDR-1{alpha} in the nonexposed donors was partially independent of TCR engagement of MHC class II and peptide. Contrastingly, CD4 T cell and IFN-{gamma} responses in malaria-exposed donors were MHC class II restricted, suggesting that the CD4 T cell response to CIDR-1{alpha} in malaria semi-immune adults also has a TCR-mediated component, which may represent a memory response. Dendritic cells isolated from human peripheral blood were activated by CIDR-1{alpha} to produce IL-12, IL-10, and IL-18. IL-12 was detectable only between 6 and 12 h of culture, whereas the IL-10 continued to increase throughout the 24-h time course. These data strengthen previous observations that P. falciparum interacts directly with human dendritic cells, and suggests that the interaction between CIDR-1{alpha} and the host cell may be responsible for regulation of the CD4 T cell and cytokine responses to P. falciparum-infected erythrocytes reported previously.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There is long-standing evidence for the presence of T cells responding to Plasmodium falciparum Ags in most adults who have had no exposure to P. falciparum malaria (reviewed in Refs. 1 and 2). These T cells have been shown to respond to crude extracts of P. falciparum infected RBC (iRBC)3 ( 3), intact iRBC ( 4, 5), as well as a number of defined malarial Ags ( 6, 7, 8). The response of these T cells, often present at high frequencies ( 9), may be the result of mitogenic or superantigenic activity in the malaria parasite ( 10, 11, 12, 13, 14), or to previous exposure to environmental organisms that share common epitopes with P. falciparum ( 1, 2, 3, 15, 16). MHC class II-restricted T cells from nonexposed individuals responding to P. falciparum proteins or extracts have been shown to express the memory/activation marker CD45RO+ ( 4, 16, 17, 18), or the naive cell marker CD45RA+ ( 4, 17), suggesting that both memory and naive T cells may be stimulated by P. falciparum Ags, and that there may be more than one mechanism of T cell activation.

Because these responses occur in nonexposed individuals who can succumb to a malaria infection, they may not be protective, but may be preferentially expanded upon exposure to P. falciparum ( 19). It has been suggested that their presence may have the potential to skew the repertoire of P. falciparum-reactive T cells toward the cross-reactive epitopes and therefore limit the more protective responses ( 19). They may also be responsible for the initiation of the inflammatory response to P. falciparum in nonimmune individuals, which may in turn contribute to the pathology of disease ( 2, 16).

The active component of the iRBC that induces proliferation in memory CD45RO+CD4+ T cells is thought to be membrane bound ( 5). Candidate membrane-bound molecules are the variable surface Ags (VSA), which include those encoded by the var multigene family, such as P. falciparum erythrocyte membrane protein-1 (PfEMP-1). Interestingly, the cysteine interdomain region-1{alpha} (CIDR-1{alpha}) domain of PfEMP-1 has been shown to stimulate CD4 T cells from both exposed and nonexposed individuals ( 20). The CIDR-1{alpha} domain of PfEMP-1, which binds to CD36 ( 21), is thought to play a role in the adhesion of iRBC to the host endothelium ( 22, 23). It has been suggested that this region of PfEMP-1 could be used as an effective antiadhesive vaccine ( 24, 25). However, if CD4 T cells are activated nonspecifically by CIDR-1{alpha}, they may prevent the generation of specific and protective CD4 T cell responses. To determine whether pre-existing cross-reactive CD4 T cells would influence vaccine efficacy, it is necessary to understand how CIDR-1{alpha} interacts with CD4 T cells.

In addition to cross-reactive Ag, superantigen, and mitogenic effects of malaria parasite components on CD4 T cells, it is possible that CD4 T cells could be activated by cytokines independent of TCR engagement ( 26, 27, 28). IL-12 and IL-18 cytokines produced by dendritic cells (DCs) are able to induce IFN-{gamma} production in CD4 T cells. These two cytokines are produced by DCs that could interact with P. falciparum through pattern recognition receptors (PRRs) like TLRs or the scavenger receptor, CD36 ( 29). With this study presented here, we examined the nature of CIDR-1{alpha}-specific responses in malaria nonexposed and exposed individuals. We were able to show that in contrast to malaria nonexposed adults, exposed adults had MHC class II-restricted CD4 T cell and IFN-{gamma} in vitro responses to CIDR-1{alpha}, and that CIDR-1{alpha} directly activated DCs to produce IL-12, IL-10, and IL-18.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors and blood sampling

Nonexposed donors. Sixty healthy adult volunteers working at the National Institute for Medical Research (London, U.K.) were recruited. A detailed travel history was taken from each of these donors to confirm that they had never been exposed to P. falciparum (or never suffered from malaria). All experimental procedures followed the guidelines of good conduct in clinical research of the U.K. government, and were performed with the informed consent obtained from the volunteers. Ethical clearance was granted by the Barnet Ethical Committee in North London. From each of these donors, 20 ml of blood were collected in heparinized vaccutainers.

Exposed donors. Nineteen healthy adult volunteers, between 25 and 40 years of age, living in Ngerenya, a malaria endemic area on the Kenyan coast, were recruited. A detailed history was taken from each of these donors to ascertain that they had lived in Ngerenya most of their lives and that they were healthy at the time of blood donation. The area has prolonged seasonal P. falciparum malaria transmission following the short and long rains in the months of October to November and March through July, respectively. The Anopheles gambie sensu stricto mosquito complex is the main vector contributing to ~10 infective bites per person per annum ( 30). All experiments followed the guidelines of good conduct in clinical research of the Kenyan government, and were performed with informed consent from the volunteers. Ethical clearance was granted by the Kenyan national ethical committee. From each of these donors, 15 ml of blood were collected in heparinized vaccutainers.

Preparation and purification of PfEMP-1 fragments.

Two recombinant protein fragments of PfEMP-1 were expressed and purified as described elsewhere ( 20). CIDR1-{alpha} was obtained from a PfEMP-1 gene of the Malayan Camp laboratory isolate of P. falciparum (GenBank accession no. U27338), and a relatively conserved fragment of the intracellular exon 2 region of a PfEMP-1 gene was isolated from the A4 laboratory isolate (GenBank accession no. AJ413950). Before use, CIDR-1{alpha} and the negative control protein, exon 2, were taken through a thorough purification process involving gel filtration. To remove any residual LPS contamination, fractions corresponding to the recombinant protein of interest in the gel filtration elution profile were further purified by polymyxin B chromatography (Endo-trap; Profos Ag). The column was washed sequentially with 1% sodium deoxycholate (Sigma-Aldrich), water, and PBS before loading with the protein. The flow-through was collected and subjected to two additional rounds of polymyxin B affinity chromatography. Finally, PBS was replaced with endotoxin-free tissue culture-grade water using PD columns (Amersham Biosciences). The protein solutions were sterilized through a 0.2-µm filter, and stored at –80°C. The amount of endotoxin in each protein preparation was determined in a semiquantitative Limulus amoebocyte assay (E-Toxate; Sigma-Aldrich) using an endotoxin standard (Sigma-Aldrich), following manufacturer’s instructions. The endotoxin level was found to be <0.10 endotoxin units/mg (5 pg/mg protein), which is too low to induce cytokine production by human PBMC (at least 100 pg of LPS was required to induce cytokine production by human monocytes ( 31)).

P. falciparum cultures

The Malayan Camp P. falciparum parasites used for DC stimulations were cultured according to standard methods ( 32) using RPMI 1640 with albumax (Invitrogen Life Technologies) supplemented with L-glutamine until they reached the late trophozoite stage. All parasites were grown in the presence of 0.5 µg/ml mycoplasma removal agent (Serotec).

Isolation of PBMC from whole blood

PBMC were isolated as described previously ( 20). Blood was diluted 1/2 in RPMI 1640 without additives, and purified over Lymphoprep (Nycomed) by centrifugation at 800 x g for 20 min. The PBMC layer was then collected from the interphase, washed three times in RPMI 1640, and resuspended at the appropriate concentration in complete RPMI 1640 medium (RPMI 1640 containing 10% heat-inactivated human AB serum, 2 mM L-glutamine, 100 µg/ml streptomycin, 100 µg/ml penicillin, and 10 mM HEPES (Invitrogen Life Technologies)).

Determination of CD4 T cell and NK cell activation by CD69 expression and intracellular cytokine detection

PBMC (106) were resuspended in 250 µl of complete RPMI 1640 and plated out in 24-well microtiter plates. Costimulatory mAbs against CD28 and CD49d (BD Biosciences) were added to a final concentration of 1 µg/ml each, and either CIDR-1{alpha}, exon 2, or staphylococcal enterotoxin B (SEB; Sigma-Aldrich) was added at optimal concentrations (5 µg/ml for both CIDR-{alpha} and EXON2 and 1 µg/ml for SEB). The tubes were incubated in a humidified 37°C, 5% CO2 incubator for a total of 7 h. Brefeldin A (BFA; 10 µg/ml) was included for the last 4 h of culture. After 7 h, the cells were harvested and incubated with fluorochrome-labeled mAbs to CD4 (BD Biosciences clone RPA-T4), NK cells (anti-CD56-allophycocyanin Immunotec clone PNIM2474), CD69 (BD Pharmingen; clone FN50), and IFN-{gamma} (BD Pharmingen; clone B27), and IL-10 (BD Pharmingen; clone JES3-19F1). Four-color flow cytometric analyses were performed on the FACSCalibur flow cytometer (BD Immunocytometry Systems). Data were acquired using CellQuest (BD Immunocytometry Systems), collecting 5 x 104-gated CD4+ events. Data were displayed in two-color dot plots using CellQuest. Side scatter and FL3 (CD4 PerCP) gating were used to exclude any CD4+ monocytes during data analysis. Donors were considered positive if the proportion of CD4+ T or NK cells expressing CD69, or CD69 and cytokine production (separately) was at least 3-fold above the medium control.

PBMC proliferation assays

PBMC were labeled with CFSE (Molecular Probes) as described previously ( 33). Cells were washed three times with complete RPMI 1640, and dye incorporation was assessed by flow cytometry. CFSE-labeled PBMC were plated out at 2 x 105 cells/well in a 96-well U-bottom plate (Nunclon; Invitrogen Life Technologies), and cultured in a final volume of 200 µl of complete RPMI 1640. The CIDR-1{alpha} and exon 2 fragments of PfEMP-1 were used at a final concentration of 0.25 µg/ml. Purified protein derivatives (PPD) at 10 µg/ml were used as a positive control.

Each condition was set out in duplicate or triplicate. Plates were incubated at 37°C and 5% CO2 in a humidified atmosphere for 7 days. Supernatants were removed for the measurement of cytokines before cytometric analysis. The proliferative responses of the CFSE-labeled CD4 T cells were determined by flow cytometry. Cells were stained with the appropriate combinations of allophycocyanin-, PE-, and PerCP-conjugated Abs. Flow cytometry was performed either on a FACSCalibur using Cell Quest software (BD Biosciences) or on a Coulter EPICS XL with XL system II in London and Kilifi, respectively. Cell division was determined from the proportion of cells with reduced fluorescence intensity of CFSE as described previously ( 34) (see Fig. 2). The flow cytometer was set to count events for a fixed length of time (1 min) to permit the determination of the relative numbers of recovered viable cells within each well (a measure of the magnitude of the response to Ag) ( 34). Analysis using the allophycocyanin-, PE-, PerCP-conjugated Abs allowed the proportions of the different cell subsets within the dividing and nondividing populations to be determined. Data are presented as mean proportions or relative total numbers of dividing CD4 T cells. CD4 T cell proliferation was considered positive if the mean number of dividing cells in triplicate wells containing an Ag exceeded two times the mean value of triplicate wells without Ag by more than two stimulation indices.


Figure 2
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  		FIGURE 2. CD4 T cell proliferation, IFN-{gamma}, and IL-10 production in response to CIDR-1{alpha} in malaria nonexposed individuals. a, A representative example of CIDR-1{alpha}-specific CD4 T cell proliferative responses in nonexposed PBMC donors. Divided CD4 T cells were identified as those with reduced CFSE fluorescence intensity on the x-axis of similar histograms to those shown above. The numbers shown are the percentages of CD4 T cells in the respective gates. b, Proportions of malaria nonexposed donors responding to CIDR-1{alpha} by CD4 T cell proliferation (n = 34), IFN-{gamma} (n = 33), and IL-10 (n = 33) production. Exon 2 was used as a negative control and in all cases; the medium control value was subtracted from the Ag-specific value. Each dot represents data from a single individual. Cytokine responses that were 2-fold above the medium control, and proliferative responses above 2 stimulation indices were considered positive. Eight of 34 individuals had proliferative stimulation indices of over 2, 18 of 33 and 16 of 33 individuals had concentrations above 2-fold of the medium control for IFN-{gamma} and IL-10, respectively. c, Relationship between the numbers of divided CD4 T cells and the percentage of CD69+CD4 T cells after incubation of PBMC with CIDR-1{alpha} for 7 days and 7 h, respectively (n = 14). Generally, all those that responded by cell division also responded by CD69 up-regulation. However, more donors responded by CD69 up-regulation than cell division.

 
Inhibition assay with anti-MHC class II-blocking Ab

PBMC from normal healthy donors were incubated with PPD, and either the anti-MHC class II Ab L243 (directed against monomorphic determinants of HLA-DR; American Type Culture Collection), or the mouse IgG2a isotype control, at different concentrations; 0, 5 and 10 µg/ml, before determination of cytokine secretion or T cell proliferation as described above. For each positive response, the ability of the anti-MHC class II mAb to inhibit either CD4 T cell proliferation or IFN-{gamma} production in response to CIDR-1{alpha} was expressed as percentage inhibition (based on a corresponding response in the presence of an isotype control Ab at the same concentration as L243).

DC cytokine assays with whole PBMC

PBMC (106) were incubated with medium only, exon 2 (5 µg/ml), CIDR-{alpha} (5 µg/ml), or LPS (1 µg/ml) in a final volume of 500 µl in 48-well microtiter plates. BFA (10 µg/ml) was added 4 h before staining the cells for intracellular cytokine detection by flow cytometry. Two different subpopulations of DCs were identified in whole PBMC by surface staining of the cells with either anti-BDCA-1 (blood DC Ag-1) FITC and CD19 PerCP, or anti-BDCA-2 and CD123 PerCP ( 35, 36). The average percentages of BDCA-1- and BDCA-2-positive cells identified in PBMC were 0.43% (SD = 0.9) for seven individuals and 0.05% (SD, 0.046) for five individuals, respectively. Cells were fixed and then permeabilized for intracellular staining by using the Fix and Perm kit (Caltag Laboratories) according to the manufacturer’s instructions. Intracellular staining for IL-10 and IL-12 was done using allophycocyanin anti-human IL-10, and PE anti-human IL-12p70, respectively. A total of 4 x 105 cells were acquired per sample on the FACSCalibur.

Isolation of BDCA-1 (CD1C) positive myeloid DCs from whole PBMC

Positive selection of BDCA-1-positive myeloid DCs was done using a magnetic bead isolation system according to the manufacturer’s protocol (Miltenyi Biotec). Briefly, BDCA-1-expressing B cells were magnetically labeled with CD19 microbeads and subsequently depleted on a MACS column (Miltenyi Biotec), followed by positive selection of BDCA-1-positive blood DCs in the B cell-depleted fraction. This positive selection step was repeated to increase the purity of the BDCA-1 DCs collected. The purity of the isolated DCs was assessed by flow cytometry and was always ~95%. The enriched BDCA-1+ DCs contained <1% CD19+ B cells and <1.5% CD14+ monocytes (mean of 1.4% (SD, 0.4).

BDCA-1-DCs cytokine assays

A total of 104-105 purified BDCA-1 DCs were incubated with medium only, exon 2 (5 µg/ml), CIDR-{alpha} (5 µg/ml), Malayan camp P. falciparum schizonts (30 schizonts/DC), and LPS (1 µg/ml) in a final volume of 500 µl in 48-well microtiter plates. When cytokine production was assessed by flow cytometry, BFA (10 µg/ml) was added 4 h before harvesting and staining of cells. In this case, cells were examined for IL-12 and IL-10 production after 6- or 12-h stimulation, using a FACSCalibur as described above. Otherwise in cultures without BFA, supernatants for cytokine measurement by ELISA were harvested after 12 or 24 h poststimulation.

Determination of cytokine concentrations by ELISA

Supernatants from the PBMC or isolated DC cultures were tested for the presence of cytokines using the OTEIA sets (BD Pharmingen) for IFN-{gamma}, IL-10, and IL-12p70, and a human IL-18 ELISA kit (Medical and Biological Laboratories) following the manufacturer’s instructions. The concentration of the cytokines was determined from the averages of duplicate/triplicate wells. Donor responses were considered positive if the cytokine concentration was 2-fold over the medium control.

Data analysis

Data were stored, formatted, and analyzed with Microsoft Excel. Graphs were plotted in Prism-Graphics (GraphPad Software) and Stata version 8.0 computer softwares. Differences between groups were tested by Mann-Whitney U test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CIDR-1{alpha} activates CD4 T and NK cells from malaria nonexposed individuals to induce CD69 expression and IFN-{gamma} production

PBMC from healthy individuals were stimulated in vitro for 7 h with 5 µg/ml CIDR-1{alpha} protein. During this time, a proportion of CD4 T and NK cells up-regulated expression of CD69, and some of these cells produced IFN-{gamma}. The proportion of responding CD4 T and NK cells was determined by the combined flow cytometric analysis of CD69 expression, and intracellular staining of IFN-{gamma} or IL-10 in CD4 T and CD56 NK cells. Representative examples of typical responders are shown in Fig. 1a for CD4 T and NK cells. In these donors, 45% of CD4 T cells expressed CD69, and 4% produced IFN-{gamma} in response to CIDR-1{alpha} (~10% of the CD69+ cells). Similarly, CIDR-1{alpha} also activated over 60% of CD56+-positive NK cells and 12.3% (~20% of the CD69+ cells) produced IFN-{gamma}.


Figure 1
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  		FIGURE 1. CIDR-1{alpha} induce CD69 up-regulation and IFN-{gamma} production in CD4 T and NK cells from malaria nonexposed individuals. a, CIDR-1{alpha}-specific CD4 T and CD56 NK cells were detected directly from PBMC 7 h after stimulation with Ag (and controls) by flow cytometry. Cells were gated on CD4 (row 1) and CD56 (row 2) -positive cells. Specifically activated cells were identified as being CD69+. Some of the activated cells produced IFN-{gamma}. The percentages of the total number of cells responding by CD69 expression and IFN-{gamma} are shown in the upper left and right quadrants, respectively. b, Proportions of malaria nonexposed donors responding by CD4 T and NK cells to CIDR-1{alpha} by CD69 up-regulation and production of IFN-{gamma}. A response was considered positive when the proportion of CD69 or IFN-{gamma}-positive cells was 3-fold above the medium control (a 3-fold cutoff was preferred for use in this assay because of clustering around the 2-fold cutoff used with the 7-day proliferation assay). Each of the dots represents a single donor (n = 23). Twenty and 13 of 23 donors responded by CD69 up-regulation and IFN-{gamma} production in CD4 T cells, respectively. All of the 10 donors tested for NK cell activation responded by CD69 up-regulation, and 9 of them also responded by IFN-{gamma} production.

 
Results of CD69 and IFN-{gamma} expression on CD4 T and NK cells from 23 and 10 malaria nonexposed individuals, respectively, are summarized in Fig. 1b. PBMC from the majority of donors (89%) responded by up-regulating expression of CD69, and approximately half (56%) by both CD69 expression and IFN-{gamma} production on CD4 T cells. NK cells in PBMC from all of the 10 nonexposed individuals tested also expressed CD69 and produced IFN-{gamma}. All IFN-{gamma}-producing CD4 T and NK cells were CD69 positive. Intracellular IL-10 was not detectable in CD4 T cells stimulated by either CIDR-1{alpha} or SEB after 7 h of culture. Stimulation with exon 2 or SEB were conducted as negative and positive controls, respectively. Exon 2 did not induce CD69 expression or any significant cytokine induction, whereas the superantigen (SEB) readily stimulated both cell types. Taken together, these data demonstrate the presence of CD4 T and CD56 NK cells able to respond to CIDR-1{alpha} and produce IFN-{gamma} to this malaria protein in peripheral blood of P. falciparum nonexposed individuals.

Assessment of CD4 T cell division and cytokine production in response to CIDR-1{alpha} in nonexposed individuals

CFSE-labeled PBMC from healthy nonexposed adults were stimulated with CIDR-1{alpha} and control Ags for 7 days after which CD4 T cell proliferation was assessed by flow cytometry. A representative example of a positive CD4 T cell response is shown in Fig. 2a. The response to CIDR-1{alpha} was similar to that achieved with the positive control Ag (PPD), whereas little cell division took place in cultures of PBMC and the negative control Ag (exon 2). Fig. 2b summarizes the results of PBMC from 34 malaria nonexposed donors. Of these, 8 gave a positive proliferative response. The amounts of IFN-{gamma} and IL-10 were determined from PBMC culture supernatants harvested from the 7-day proliferation assays (Fig. 2b). Seventeen and 16 of 33 donors were positive for IFN-{gamma} and IL-10 production, respectively. As described previously ( 20), there was generally an inverse or no association between IL-10 production and CD4 T cell proliferation. Interestingly, some individuals responded by IFN-{gamma} production and no cell division, and we observed an unexpected positive association between IFN-{gamma} and IL-10 concentrations (Spearman’s {rho} coefficient = 0.62, p = 0001, data not shown). In donors (n = 14) where CD4 T cell responses were measured by both the CFSE-based proliferation and the 7-h activation assays, it was clear that while most of the individuals (10 of 14) responded by CD69 expression, only 4 of the 14 responded by cell division (Fig. 2c). Thus, up-regulation of expression of CD69 in response to CIDR-1{alpha} did not always lead to cell division.

IFN-{gamma} produced in response to CIDR-1{alpha} is not MHC class II-restricted in malaria nonexposed individuals

To determine whether the response to CIDR-1{alpha} in malaria nonexposed individuals is the result of an MHC class II/TCR-mediated interaction, PBMC and CIDR-1{alpha} were cultured in the presence of Ag and different concentrations of the anti-MHC class II Ab (L243). L243 is directed against HLA-DR, and inhibits classical MHC class II-restricted anti-PPD CD4 T cell proliferative and IFN-{gamma} production responses in PBMC obtained from individuals previously immunized with bacillus Calmette-Guérin (BCG) as shown in Fig. 3, left panel.


Figure 3
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  		FIGURE 3. Median percent inhibitions for the CD4 T cell and IFN-{gamma} responses to CIDR-1{alpha} by the anti-MHC class II mAb. PBMC from malaria nonexposed and exposed adults were incubated in the presence of CIDR-1{alpha} or PPD, and different concentrations of either the anti-MHC class II mAb, L243, or the mouse IgG2a isotype control; 0, 5, and 10 µg/ml. The cells were cultured for 7 days and thereafter analyzed for CD4 cell division by flow cytometry as described in Fig. 2. IFN-{gamma} concentrations were determined in culture supernatants by ELISA. Percent inhibitions were calculated by subtracting the L243 values from the corresponding values associated with the respective concentrations of the isotype control Ab. Each dot represents an individual donor and data is shown for 7 and 11 donors, for CD4 T cell division and IFN-{gamma} production, respectively. There was a significant dose dependent effect between 5 and 10 µg/ml for IFN-{gamma} (p = 0.002 Mann-Whitney U test) but not for the CD4 T cell response (p = 0.4).

 
PBMC from seven nonexposed individuals tested in this inhibition assay gave positive IFN-{gamma} responses but no CD4 T cell division in response to CIDR-1{alpha}, in contrast to the anti-PPD-specific CD4 T cell and IFN-{gamma} responses, which were inhibited by the anti-MHC class II Ab. The IFN-{gamma} response to CIDR-1{alpha} was not inhibited by anti HLA-DR Abs in five of seven nonexposed individuals, suggesting that the anti-CIDR-1{alpha} IFN-{gamma} response of the majority of nonexposed donors, unlike their response to PPD, does not require Ag presentation to the CD4 T-TCR in the context of the MHC class II-peptide complex (Fig. 3, middle panel).

Cross-reactivity between CIDR-1{alpha} epitopes with other Ags may explain the inhibition of IFN-{gamma} production when MHC class II molecules were blocked in two individuals. In this case, the anti-CIDR-1{alpha} response would present a classical memory response.

The majority of CD4 T cell and IFN-{gamma} responses to CIDR-1{alpha} in malaria-exposed individuals are MHC class II restricted

Despite the response of CD4 T cells to CIDR-1{alpha} and its apparent independence of HLA-DR presentation, it is possible that repeated exposure to malaria infection would result in the generation of an additional MHC class II-restricted response. If this is the case, it might be possible to use MHC class II-blocking Abs to distinguish memory responses from TCR-independent responses to CIDR-1{alpha} and possibly other malarial Ags.

Therefore, the dependence of CD4 T cells responding to CIDR-1{alpha} on MHC class II presentation was tested in 19 exposed adults. All responses (n = 7) where we observed proliferation to CIDR-1{alpha} (stimulation indices: 2 to 6.3) were inhibited by anti HLA-DR Abs, and those responses (n = 10) which resulted in IFN-{gamma} (54.80 and 422.40 pg/ml measured by ELISA) were also inhibited by the anti-HLA-DR Abs as shown in Fig. 3, right panel.

The median percentage inhibitions of CD4 T cell proliferation in response to CIDR-1{alpha} were 37 and 45%, for 5 and 10 µg/ml, respectively, and were significantly different from the L243-negative control (0% inhibition) (p = 0.001 and p = 0.001 (Mann-Whitney Test)).

The median percentage inhibition of the IFN-{gamma} responses was 19% for 5 µg/ml and 38% for 10 µg/ml Ab, and were significantly different from 0% (p = 0.002, p = 0.01, Mann-Whitney Test), suggesting that the IFN-{gamma} response to CIDR-1{alpha} in exposed individuals is, at least in part, MHC class II restricted. Together, these data suggest that both the CD4 T cell proliferative and IFN-{gamma} responses in exposed adults are MHC class II restricted and are therefore different from those observed in malaria nonexposed individuals, which may be mediated by various mechanisms including bystander T cell activation. In contrast, inhibition of both the CD4 and IFN-{gamma} responses of nonexposed and exposed individuals to PPD by the anti-MHC class II Ab was of similar magnitude.

Myeloid DCs from malaria nonexposed individuals produce IL-10, IL-12p70, and IL-18 in response to CIDR-1{alpha}

It is possible that DCs may directly interact with CIDR-1{alpha} through CD36 or PRRs such as TLRs (reviewed in Ref. 37) resulting in the production of IL-18 and IL-12p70, which in turn may activate and induce IFN-{gamma} transcription in the responding CD4 T and NK cells without engaging the TCR via the MHC class II peptide complex. It has been shown that the presence of these two cytokines is sufficient to induce IFN-{gamma} production in Th-1 T cells in mice ( 27, 28). Therefore, we investigated the cytokine response (IL-10, IL-12, and IL-18) of DCs to CIDR-1{alpha} from BDCA-1+ (myeloid DC) and BDCA-2+ (plasmacytoid DC) subpopulations in both whole PBMC and the isolated DC populations.

Whole PBMC were cultured with CIDR-1{alpha}, exon 2, LPS (positive control), or medium only (control). DCs from these cultures were then analyzed for intracellular IL-10 and IL-12p70 production by flow cytometry at 3, 6, 18, and 24 h poststimulation. CIDR-1{alpha} and LPS, but not exon 2 and the medium controls induced IL-10 and IL-12 production in the BDCA-1 subpopulation in all the individuals tested (Fig. 4). However, kinetics of IL-12 and IL10 production were different. Although the proportion of cells positive for IL-10 continued to increase throughout the 24-h culture period, those producing IL-12p70 reached a peak between 6 and 12 h. At 24 h, there were nearly no IL-12-positive cells. Clearly, these results suggest that similar to the response to LPS (Fig. 4), CIDR-1{alpha} induces IL-12p70 and IL-10 responses in DCs. By contrast, these cytokines were not detected in the plasmacytoid (BDCA-2+ cells) in response to CIDR-1{alpha} and LPS (data not shown).


Figure 4
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  		FIGURE 4. Kinetics of IL-10 and IL-12p70 production by BDCA-1+ DCs stimulated by CIDR-1{alpha}, LPS, and exon 2 (negative control protein). {square}, {blacksquare}, and {cjs2100} represent the percentages of cells positive for the cytokine after PBMC from malaria nonexposed were stimulated with exon 2, CIDR-1{alpha}, and LPS, respectively. The error bars are SEs of the means of five donors. Cells were harvested and stained for flow cytometry at 3, 6, 12, and 24 h. In each case, 4 x 105 cells were acquired and the percentage of BDCA-1+ cells positive cytokines determined.

 
To determine whether the BDCA-1 DC cytokine response to CIDR-1{alpha} was the result of a direct interaction between the DCs and CIDR-1{alpha} without the involvement of other cells, BDCA-1+ DCs were isolated using magnetic beads. A representative isolation is shown in Fig. 5a.


Figure 5
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  		FIGURE 5. Cytokine responses to CIDR-1{alpha} and control Ags in isolated DCs. a, Representative plots showing isolated BDCA-1+ DCs. DCs were isolated from PBMC obtained from buffy coats purchased from the National blood bank (U.K.) in two steps; depletion of CD19-positive B cells followed by positive selection of BDCA-1+ DCs. The data shown in these plots are gated on a live gate to exclude dead cells. A total of 1 x 105 cells were analyzed in each case. B cells were depleted before BDCA-1+ DCs were positively isolated from the flow-through fraction. Counterstaining with CD14 FITC shows that some of the isolated BDCA-1+ blood DCs were also CD14 positive. b, Left panel, IL-12p70 and IL-10 responses in BDCA-1+ isolated DCs at 6 ({square}) and 12 ({cjs2100}) hours after stimulation with Ag determined by flow cytometry. Isolated BDCA-1+ DCs were cultured at 1 x 105 cells/well in the presence of medium only, exon 2 (negative control), CIDR-1{alpha}, and LPS (positive control). A total of 5 x 104 cells were acquired on the flow cytometer and analyzed for cytokine production. The error bars represent the SEM for six donors. b, Right panel, Kinetics of IL-18 production by isolated BDCA-1-positive DCs stimulated by medium only, exon 2 (negative control), CIDR1-{alpha}, schizont-iRBC, RBC (negative control for iRBC), and LPS. Isolated BDCA-1+ cells were cultured at 100,000 cells/well and culture supernatants were harvested after 12 and 24 h of antigenic stimulation. IL-18 concentrations in the culture supernatants were determined by ELISA (appropriately labeled Abs for intracellular staining were unavailable). The concentrations for the medium controls were subtracted from those of the respective Ags. The error bars represent the SEMs of six donors.

 
To determine whether these enriched BDCA-1 DCs could make IL-18, IL-12, and IL-10 in response to CIDR-1{alpha} and P. falciparum schizont iRBC, DCs were cultured with CIDR-1{alpha}, iRBC, RBC, and control Ags for up to 24 h. Culture supernatants were harvested at 12 and 24 h and tested for the presence of IL-18 by ELISA (no appropriately labeled anti-IL-18 mAbs for intracellular staining were available). At these time points, IL-18 was present in the supernatants of DCs cultured with CIDR-1{alpha} or iRBC as shown in Fig. 5. To confirm that the isolated DCs also made IL-12 in response to CIDR-1{alpha}, cells were harvested after 6 and 12 h poststimulation and tested for IL-10 and IL-12p70 production by intracellular staining and flow cytometry. The purified BDCA-1 DCs produced both IL-10 and IL-12 in response to CIDR-1{alpha} and LPS, but not to medium or exon 2 controls (Fig. 5). However, the percentage of cytokine-positive cells from the isolated DCs were lower than those seen with the same BDCA-1+ DCs within whole PBMC in Fig. 4.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that the CD4 T cell response of P. falciparum nonexposed individuals to CIDR-1{alpha} (the CD36-binding domain of the variant Ag PfEMP-1 ( 21)), described earlier by us ( 20), is not dependent on engagement of the TCR with MHC class II. This contrasts strongly with the CD4 T cell response of immunized individuals to PPD, and more importantly is different from the response of exposed donors to CIDR-1{alpha}, where both responses are dependent on MHC class II (HLA-DR). It is highly unlikely that the difference between the response of the nonexposed and the exposed donors to CIDR-1{alpha} could be explained by racial differences, because the extents to which the PPD responses were inhibited in both groups were similar and the nonexposed groups were from a wide mix ethnic groups.

Activation of CD4 T cells from nonexposed donors by the CIDR-1{alpha} domain was unusual in that the IFN-{gamma} response was rapid and could be detected within 7 h of culture, and before cell division took place. Indeed, up-regulation of CD69 and IFN-{gamma} production were able to take place in the absence of subsequent cell division. Although CD69 is an accepted marker for a positive T cell response, our data would suggest that not all CD69+CD4 T cells become fully activated, and go on to proliferate. It may be that some of these T cells are activated in the absence of IL-2 and/or other costimulatory molecules. The early response of nonexposed individuals to CIDR-1{alpha} contrasts with conventional activation of CD4 T cells, where T cells first respond to their specific peptide-MHC class II complexes by making IL-2 and proliferating before differentiating into either Th1 or Th2 cells (reviewed in Ref. 38). CD4 Th1 cells then go on to produce IFN-{gamma}.

The possibility that the response of nonexposed donors to CIDR-1{alpha} may not be a classical CD4 T cell/MHC class II response is supported by the observation that in the majority of cases the response was not inhibited by blocking HLA-DR, in contrast to the response of these donors to PPD. The lack of inhibition by anti-class II Abs also rules out a superantigen-like response, which would involve binding to nonpolymorphic regions of MHC class II and TCR ( 39, 40).

CIDR-1{alpha} did not appear to stimulate CD4 T cells directly, and therefore was unlikely to act as a mitogen. CD4 T cell activation, proliferation, or cytokine production only took place when whole PBMC were cultured with CIDR-1{alpha}, suggesting that the effect on nonexposed CD4 T cells may be indirect.

It has previously been shown that the cytokines IL-12 and IL-18 can activate Th1 cells independently of TCR ligation ( 27, 28). In our case, the CIDR-1{alpha} domain of PfEMP-1 could stimulate myeloid (BDCA-1+) but not plasmacytoid (BDCA-2+) isolated directly from peripheral blood of nonexposed donors to produce both IL-12 and IL-18. In addition to these cytokines, IL-10 was also secreted, albeit with a slower kinetic. These effects of CIDR-1{alpha} on BDCA-1+ DCs could be reproduced by P. falciparum schizont-iRBC (data not shown). Our data therefore suggest that CIDR-1{alpha} can activate DCs to produce cytokines that allow a TCR-independent response. The mechanisms by which this takes place are not known. CIDR-1{alpha} can bind CD36 ( 21), and ligation of CD36 on DCs has been shown to lead to production of IL-10 ( 29). It is thus possible that this is the means of DC activation. It is also possible that ligation of PRR on DC, such as TLR, are involved ( 37). We are confident that the effects of CIDR-1{alpha} on DCs are not due to contaminant LPS or other bacterial products as the negligible levels of LPS measured were not able to stimulate DCs (our observation and Ref. 31), and another PfEMP-1 recombinant protein from the cytoplasmic tail of PfEMP-1 (exon 2) expressed in the same system and similarly purified had no stimulatory effect.

The slower and very significant production of IL-10 by the myeloid DCs in response to CIDR-1{alpha} is similar to earlier observations of Urban et al. ( 41), where they showed that iRBC expressing a PfEMP-1 on the surface, which bound CD36, inhibited the up-regulation of costimulatory molecules on human cultured DC, induced IL-10 production, and inhibited an allogeneic T cell response. Our results suggest that it is the CIDR-1{alpha} domain of PfEMP-1, known to bind CD36 ( 21), that is responsible for these down-regulatory responses of the DC, and further in our case the IL-10 response may be initially associated with a short and tightly regulated IL-12 response that is sufficient to activate NK cells and TCR-independent and TCR-dependent T cell IFN-{gamma} responses. It would be of interest to determine whether there is variability of this response among nonexposed donors, and whether those donors that do not make NK or T cell responses to CIDR-1{alpha}, do not make an initial IL-12 burst.

In stark contrast to the IFN-{gamma} response of nonexposed individuals, both CD4 T cell and IFN-{gamma} responses in malaria-exposed individuals were inhibited by the MHC class II Ab. It is likely that these differences between the CIDR-1{alpha} response in nonexposed and exposed individuals are due to the presence of memory CD4 T cells in the PBMCs from immune individuals. Because they have been exposed to P. falciparum infections throughout their lives, it is reasonable to expect that the malaria-exposed adults have accumulated memory CD4 T cells specific to P. falciparum Ags in their peripheral circulation. Our results raise the possibility that such anti-MHC class II-blocking Abs may provide a way of distinguishing normal Ag recall responses from the pre-existing TCR-independent T cell responses to malaria Ags found among unexposed donors, which would otherwise make it difficult to interpret T cells assays in nonimmune children or in vaccine studies with nonexposed volunteers.

The observation that malaria-immune adults have classical MHC class II-restricted CD4 T cell and IFN-{gamma} responses to CIDR-1{alpha} is encouraging and suggest that these individuals have CIDR-1{alpha}-specific memory T cells. Due to its functional conservation, surface location (accessible to Ab) and involvement with cytoadhesion of iRBC to endothelial cells (thought to mediate pathology) during P. falciparum infections, it is a potential vaccine candidate for malaria. It is not yet known how similar the Malayan camp var 1 CIDR-1{alpha} sequence used in these studies is to the CIDR-1{alpha} variants circulating in Kilifi (Ngerenya). Work to sequence var genes from field isolates is ongoing, and will allow us to compare the CIDR-1{alpha} variant used here with those expressed in field isolates. However, the fact that CD4 T cell and IFN-{gamma} responses observed in a large proportion of malaria-exposed individuals reacted with a single variant of CIDR-1{alpha} suggests that there might be cross-reactivity between CIDR-1{alpha} domains from different PfEMP-1 molecules. Similar cross-reactivity between CIDR-1{alpha} variants has been observed in studies where rodents were immunized simultaneously with several CIDR-1{alpha} variants ( 25, 42, 43). Together, these observations are encouraging in that it may be possible to overcome the immense antigenic diversity of PfEMP-1 in a CIDR-1{alpha}-based vaccine.

It is not entirely clear how the presence of pre-existing T cells will affect the development of CIDR-1{alpha}-specific vaccines. However, it seems unlikely that bystander activated CD4 T cells would protect the vaccinees from disease, because these cells may not give cognate help to B cells to make protective Ab. More studies will be needed to investigate whether the frequency of CIDR-1{alpha}-specific CD4 T cells is associated with pathology in nonexposed individuals being exposed to P. falciparum for the first time.


   Acknowledgments
 
We thank Cecile Voisine, Douglas Brown, Jane E. Blythe, and Anne-Marit Sponaas for their helpful criticism and advice. This paper is published with permission of the director of Kenya Medical Research Institute.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by European Union/BIOMALPAR, The Wellcome Trust, and the Medical Research Council (MRC), U.K. J.L., L.S., and F.M.N. are supported by the MRC; B.U. is a Wellcome Trust career development fellow. C.I.N. is funded by the Wellcome Trust and K.M. is a Wellcome Trust senior fellow. Back

2 Address correspondence and reprint requests to Dr. Jean Langhorne, Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, U.K. E-mail address: jlangho{at}nimr.mrc.ac.uk Back

3 Abbreviations used in this paper: iRBC, infected RBC; PfEMP-1, P. falciparum erythrocyte membrane protein-1; CIDR-1{alpha}, cysteine-rich interdomain region 1{alpha}; DC, dendritic cell; SEB, staphylococcal enterotoxin B; BFA, brefeldin A; PPD, purified protein derivative; PRR, pattern recognition receptor. Back

Received for publication December 5, 2005. Accepted for publication February 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Good, M. F.. 1990. Summary of the meeting on cellular mechanisms in malaria immunity. Immunol. Lett. 25: 1-10. [Medline]
  2. Good, M. F.. 1994. Immunological responses from non-exposed donors to malaria antigens: implications for immunity and pathology. Immunol. Lett. 41: 123-125. [Medline]
  3. Currier, J., H. P. Beck, B. Currie, M. F. Good. 1995. Antigens released at schizont burst stimulate Plasmodium falciparum-specific CD4+ T cells from non-exposed donors: potential for cross-reactive memory T cells to cause disease. Int. Immunol. 7: 821-833. [Abstract/Free Full Text]
  4. Goodier, M., P. Fey, K. Eichmann, J. Langhorne. 1992. Human peripheral blood {gamma}{delta} T cells respond to antigens of Plasmodium falciparum. Int. Immunol. 4: 33-41. [Abstract/Free Full Text]
  5. Dick, S., M. Waterfall, J. Currie, A. Maddy, E. Riley. 1996. Naive human {alpha}beta T cells respond to membrane-associated components of malaria-infected erythrocytes by proliferation and production of interferon-{gamma}. Immunology 88: 412-420. [Medline]
  6. Bilsborough, J., M. Carlisle, M. F. Good. 1993. Identification of Caucasian CD4 T cell epitopes on the circumsporozoite protein of Plasmodium vivax. T cell memory. J. Immunol. 151: 890-899. [Abstract]
  7. Ohta, N., K. Iwaki, M. Itoh, J. Fu, S. Nakashima, M. Hato, R. Tolle, H. Bujard, A. Saitoh, K. Tanabe. 1997. Epitope analysis of human T-cell response to MSP-1 of Plasmodium falciparum in malaria-nonexposed individuals. Int. Arch. Allergy Immunol. 114: 15-22. [Medline]
  8. Rzepczyk, C. M., R. Ramasamy, D. A. Mutch, P. C. Ho, D. Battistutta, K. L. Anderson, D. Parkinson, T. J. Doran, M. Honeyman. 1989. Analysis of human T cell response to two Plasmodium falciparum merozoite surface antigens. Eur. J. Immunol. 19: 1797-1802. [Medline]
  9. Zevering, Y., F. Amante, A. Smillie, J. Currier, G. Smith, R. A. Houghten, M. F. Good. 1992. High frequency of malaria-specific T cells in non-exposed humans. Eur. J. Immunol. 22: 689-696. [Medline]
  10. Ballet, J. J., P. Druilhe, M. A. Querleux, C. Schmitt, M. Agrapart. 1981. Parasite-derived mitogenic activity for human T cells in Plasmodium falciparum continuous cultures. Infect. Immun. 33: 758-762. [Abstract/Free Full Text]
  11. Greenwood, B. M., A. J. Oduloju, T. A. Platts-Mills. 1979. Partial characterization of a malaria mitogen. Trans. R Soc. Trop. Med. Hyg. 73: 178-182. [Medline]
  12. Gabrielsen, A. A., Jr, J. B. Jensen. 1982. Mitogenic activity of extracts from continuous cultures of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 31: 441-448. [Medline]
  13. Strickland, G. T.. 1978. Lymphocyte mitogenic factor in sera from patients with falciparum malaria. Tropenmed. Parasitol. 29: 198-203. [Medline]
  14. Wyler, D. J., H. G. Herrod, F. I. Weinbaum. 1979. Response of sensitized and unsensitized human lymphocyte subpopulations to Plasmodium falciparum antigens. Infect. Immun. 24: 106-110. [Abstract/Free Full Text]
  15. Chizzolini, C., L. Perrin. 1986. Antigen-specific and MHC-restricted Plasmodium falciparum-induced human T lymphocyte clones. J. Immunol. 137: 1022-1028. [Abstract]
  16. Currier, J., J. Sattabongkot, M. F. Good. 1992. ‘Natural’ T cells responsive to malaria: evidence implicating immunological cross-reactivity in the maintenance of TCR {alpha}beta+ malaria-specific responses from non-exposed donors. Int. Immunol. 4: 985-994. [Abstract/Free Full Text]
  17. Fern, J., M. F. Good. 1992. Promiscuous malaria peptide epitope stimulates CD45Ra T cells from peripheral blood of nonexposed donors. J. Immunol. 148: 907-913. [Abstract]
  18. Good, M. F., J. Currier. 1992. The importance of T cell homing and the spleen in reaching a balance between malaria immunity and immunopathology: the moulding of immunity by early exposure to cross-reactive organisms. Immunol. Cell Biol. 70: 405-410.
  19. Good, M. F., Y. Zevering, J. Currier, J. Bilsborough. 1993. ‘Original antigenic sin’, T cell memory, and malaria sporozoite immunity: an hypothesis for immune evasion. Parasite Immunol. 15: 187-193. [Medline]
  20. Allsopp, C. E., L. A. Sanni, L. Reubsaet, F. Ndungu, C. Newbold, T. Mwangi, K. Marsh, J. Langhorne. 2002. CD4 T cell responses to a variant antigen of the malaria parasite Plasmodium falciparum, erythrocyte membrane protein-1, in individuals living in malaria-endemic areas. J. Infect. Dis. 185: 812-819. [Medline]
  21. Baruch, D. I., X. C. Ma, B. Pasloske, R. J. Howard, L. H. Miller. 1999. CD36 peptides that block cytoadherence define the CD36 binding region for Plasmodium falciparum-infected erythrocytes. Blood 94: 2121-2127. [Abstract/Free Full Text]
  22. Newbold, C., A. Craig, S. Kyes, A. Rowe, D. Fernandez-Reyes, T. Fagan. 1999. Cytoadherence, pathogenesis and the infected red cell surface in Plasmodium falciparum. Int. J. Parasitol. 29: 927-937. [Medline]
  23. Baruch, D. I.. 1999. Adhesive receptors on malaria-parasitized red cells. Baillieres Best Pract. Res. Clin. Haematol 12: 747-761. [Medline]
  24. Baruch, D. I., X. C. Ma, H. B. Singh, X. Bi, B. L. Pasloske, R. J. Howard. 1997. Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: conserved function with variant sequence. Blood 90: 3766-3775. [Abstract/Free Full Text]
  25. Baruch, D. I., B. Gamain, J. W. Barnwell, J. S. Sullivan, A. Stowers, G. G. Galland, L. H. Miller, W. E. Collins. 2002. Immunization of Aotus monkeys with a functional domain of the Plasmodium falciparum variant antigen induces protection against a lethal parasite line. Proc. Natl. Acad. Sci. USA 99: 3860-3865. [Abstract/Free Full Text]
  26. Chakir, H., D. K. Lam, A. M. Lemay, J. R. Webb. 2003. "Bystander polarization" of CD4+ T cells: activation with high-dose IL-2 renders naive T cells responsive to IL-12 and/or IL-18 in the absence of TCR ligation. Eur. J. Immunol. 33: 1788-1798. [Medline]
  27. Yang, J., T. L. Murphy, W. Ouyang, K. M. Murphy. 1999. Induction of interferon-{gamma} production in Th1 CD4+ T cells: evidence for two distinct pathways for promoter activation. Eur. J. Immunol. 29: 548-555. [Medline]
  28. Yang, J., H. Zhu, T. L. Murphy, W. Ouyang, K. M. Murphy. 2001. IL-18-stimulated GADD45 beta required in cytokine-induced, but not TCR-induced, IFN-{gamma} production. Nat. Immunol. 2: 157-164. [Medline]
  29. Urban, B. C., N. Willcox, D. J. Roberts. 2001. A role for CD36 in the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98: 8750-8755. [Abstract/Free Full Text]
  30. Mbogo, C. N., R. W. Snow, C. P. Khamala, E. W. Kabiru, J. H. Ouma, J. I. Githure, K. Marsh, J. C. Beier. 1995. Relationships between Plasmodium falciparum transmission by vector populations and the incidence of severe disease at nine sites on the Kenyan coast. Am. J. Trop. Med. Hyg. 52: 201-206. [Abstract/Free Full Text]
  31. Matthews, J. B., A. A. Besong, T. R. Green, M. H. Stone, B. M. Wroblewski, J. Fisher, E. Ingham. 2000. Evaluation of the response of primary human peripheral blood mononuclear phagocytes to challenge with in vitro generated clinically relevant UHMWPE particles of known size and dose. J. Biomed. Mater. Res. 52: 296-307. [Medline]
  32. Trager, W., J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193: 673-675. [Abstract/Free Full Text]
  33. Parish, C. R.. 1999. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77: 499-508. [Medline]
  34. Allsopp, C. E., S. J. Nicholls, J. Langhorne. 1998. A flow cytometric method to assess antigen-specific proliferative responses of different subpopulations of fresh and cryopreserved human peripheral blood mononuclear cells. J. Immunol. Methods 214: 175-186. [Medline]
  35. Dzionek, A., A. Fuchs, P. Schmidt, S. Cremer, M. Zysk, S. Miltenyi, D. W. Buck, J. Schmitz. 2000. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J. Immunol. 165: 6037-6046. [Abstract/Free Full Text]
  36. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, et al 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon {alpha}/beta induction. J. Exp. Med. 194: 1823-1834. [Abstract/Free Full Text]
  37. Ndungu, F. M., B. C. Urban, K. Marsh, J. Langhorne. 2005. Regulation of immune response by Plasmodium-infected red blood cells. Parasite Immunol. 27: 373-384. [Medline]
  38. Seder, R. A., R. N. Germain, P. S. Linsley, W. E. Paul. 1994. CD28-mediated costimulation of interleukin 2 (IL-2) production plays a critical role in T cell priming for IL-4 and interferon {gamma} production. J. Exp. Med. 179: 299-304. [Abstract/Free Full Text]
  39. Muller-Alouf, H., C. Carnoy, M. Simonet, J. E. Alouf. 2001. Superantigen bacterial toxins: state of the art. Toxicon 39: 1691-1701. [Medline]
  40. Shoukry, N. H., P. M. Lavoie, J. Thibodeau, S. D’Souza, R. P. Sekaly. 1997. MHC class II-dependent peptide antigen versus superantigen presentation to T cells. Hum. Immunol. 54: 194-201. [Medline]
  41. Urban, B. C., D. J. Ferguson, A. Pain, N. Willcox, M. Plebanski, J. M. Austyn, D. J. Roberts. 1999. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400: 73-77. [Medline]
  42. Baruch, D. I., B. Gamain, L. H. Miller. 2003. DNA Immunization with the cysteine-rich interdomain region 1 of the Plasmodium falciparum variant antigen elicits limited cross-reactive antibody responses. Infect. Immun. 71: 4536-4543. [Abstract/Free Full Text]
  43. Gratepanche, S., B. Gamain, J. D. Smith, B. A. Robinson, A. Saul, L. H. Miller. 2003. Induction of crossreactive antibodies against the Plasmodium falciparum variant protein. Proc. Natl. Acad. Sci. USA 100: 13007-13012. [Abstract/Free Full Text]

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