ARTICLE
Auteur(s) :, Michael Ramharter1,2,3, Heidi
Winkler1,3, Peter G Kremsner2,3, Ayola A
Adegnika2,3, Martin Willheim4, Stefan
Winkler1,3,*
1Department of Internal Medicine I, Division of
Infectious Diseases, Medical University of Vienna,
Waehringerguertel 18-20, A-1090 Vienna, Austria
2Department of Parasitology, Institute of Tropical
Medicine, University of Tübingen, Wilhelmstraße 27, 72074 Tübingen,
Germany
3Medical Research Unit of the Albert
Schweitzer-Hospital, Lambaréné, B.P. 118, Gabon
4Institute of Pathophysiology, Medical University of
Vienna, Waehringerguertel 18-20, A-1090 Vienna, Austria
In areas of Sub-Saharan Africa where Plasmodium falciparum is
hyperendemic, mortality is highest in infants younger than 6 years
of age [1]. During childhood and adolescence, natural immunity
develops slowly, resulting in fewer or even no clinical symptoms,
and only low-grade parasitemia in the adult. This kind of
protective immunity however, requires repeated exposure to the
parasite and wanes rapidly once this exposure ceases. The
mechanisms that lead to this protective immunity against
P. falciparum are under intense investigation, as they might
serve as models for the development of an efficient vaccine [2, 3].
The accumulation of a broad repertoire of antibodies against
different variant antigens such as the P. falciparum
erythrocyte membrane protein-1 or the rifin-proteins expressed on
the surface of P. falciparum-infected erythrocytes is thought
to contribute to the build-up of immunity observed in adults living
in hyperendemic areas [4-6]. In addition, age-dependent host
factors such as the gradual acquisition of immunity to
cross-reactive antigenic determinants might influence the immune
responsiveness towards malaria, as has been suggested for adult
transmigrants from malaria-free to malaria-endemic regions, who
were more quickly capable of limiting parasite densities than their
children [7, 8].T cells bearing the αβ chains of antigen receptors
are important components in the development of naturally acquired
immunity against malaria as a consequence of their ability to
provide help to B cells for antibody production and in regulating
antibody-independent protection. The CD8+ T cell subset
has been implicated predominantly in the inhibition of liver-stage
parasites, whereas CD4+ T cells are important both for
the induction of such CD8+ T cell responses as well as
the elimination of blood-stage malaria parasites
[9-12].TCRγδ+ T cells have been variously implicated in
host defenses against, and the pathophysiology of, malaria [13,
14]. Yet, a marked expansion early during infection with an
IFN-γ-biased cytokine profile has been shown in African children
[15]. Age-related differences in cytokine production of
TCRγδ+ T cells have, so far, not been reported from
malaria-endemic areas.The tight regulation of early IFN-γ responses
has been linked to the control of parasitemia and the absence of
clinical symptoms in naturally immune adults, which at the same
time presupposes the balanced secretion of the predominantly
counter-regulatory cytokines interleukin (IL)-10 and transforming
growth factor (TGF)-β [16, 17]. Previously, we have shown
age-dependent differences in the frequency of cytokine-producing T
cells in both healthy individuals and P. falciparum malaria
patients after mitogenic stimulation of PBMC [18, 19]. It appears
however, of importance to correlate the development of naturally
acquired immunity to the profile of P. falciparum-specific
cellular immune responses, as those responses might be a valuable
target for vaccination [2]. Therefore, the present study sought to
investigate the phenotypes and frequencies of both
P. falciparum-specific and non-specific cytokine-expressing T
cells in a cross-sectional study of healthy children and adults
living in a malaria-endemic area in Central Africa.
Participants and methods
Study site and participants
The study was conducted in the Albert Schweitzer Hospital in
Lambaréné, Gabon, where P. falciparum malaria is predominantly
hyperendemic, with an estimated annual entomological inoculation
rate of between 10 and 100 [20, 21]. A cohort of 14 children (5
males, 9 females; median age, 6 years, age range, 4-9 years) and 17
adults (7 males, 10 females; median age, 37 years, age range, 17-67
years) were included into the study. All participants were
recruited from the Lambaréné area and were clinically healthy.
Physical examination was unremarkable, leucocytes were within the
normal range and C-reactive protein (CRP)-levels were not
detectable in serum. Thick blood smears were used to assure that
participants were parasite-free. No precise data about the
frequency of HIV infection within the Lambaréné area are available,
the estimated HIV rate is, however, about 5-10%. CD4+ T
cell counts were within the normal range in all study participants.
Informed consent was obtained from the parents or guardians of
participating children. Human experimentation guidelines of the
authors’ institutions were followed in the course of clinical
research. Ethical clearance was given by the ethics committee of
the Albert Schweitzer Hospital in Lambaréné.
Parasite preparation
P. falciparum strain S007, originally isolated from a child
with severe malaria in Lambaréné, was cultured in human type 0
erythrocytes, adjusting the hematocrit to 5% and the parasitemia to
2-5%, in complete parasite medium (CPM: RPMI-1640 supplemented with
25mM Hepes, 2mM L-glutamine, 50 μg/mL of gentamicin, 0.5% Albumax
II [Gibco, Paisley, UK] with 200 μM of hypoxanthine and 2%
AB+ serum). All cultures and media were regularly tested
for Mycoplasma contamination by PCR amplification with
genus-specific primers (GPO-1 5´-ACT CCT ACG GGA GGC AGC AGT
A-3´and MGSO 5´- TGC ACC ATC TGT CAC TCT GTT AAC CTC-3´) of 16S
rDNA as described previously [22]. Nucleic acid was extracted with
the QIAamp DNA Blood Mini Kit (Quiagen, Hilden, Germany).
For synchronization and enrichment of
P. falciparum-infected erythrocytes (PFE), a magnet-activated
cell sorter (MACS) system was applied as described recently [23].
Parasite concentration could be increased up to 90% using CS
columns and VarioMACS (Miltenyi BioTec, Bergisch-Gladbach,
Germany). Yield and purity were assessed by microscopic examination
of Giemsa-stained eluate and flow-through. In addition, flow
cytometry, using staining with ethidium bromide, was employed for
the differentiation of uninfected from infected erythrocytes. The
MACS-eluate of P. falciparum strain S007 was frozen at -80°C,
thawed and immediately used for stimulation of the PBMC from all
study participants.
Detection of Plasmodium falciparum-specific T cell cytokine
expression by flow cytometry
PBMC were isolated from heparinized blood by ficoll-diatrizoate
centrifugation and plated out in 12-well plates at 2.5 x
106/well. Cells were cultured in Ultra Culture Medium
(UCM) (Bio Whittaker, Walkersville, Maryland, USA) supplemented
with L-glutamine (2 mM/L; Sigma, St. Louis, Missouri, USA),
gentamicin (170 mg/L; Sigma) and 2-mercaptoethanol (3.5 μl/L;
Merck, Darmstadt, Germany) for 18 hours at 37 °C in 5%
CO2, and stimulated with or without 50 μL of the
MACS-eluate containing the late-stage, schizont-rich parasite
preparation at a ratio of 10:1 PFE:PBMC as described previously
[24]. The co-stimulatory MAb CD28 (Pharmingen, San Diego, CA, USA)
was added at 10 μL to some wells (5 μg/mL final concentration) [25,
26]. Uninfected erythrocytes were processed as described above and
served as controls. Brefeldin A (1 μM; 10 μg/mL final
concentration, Sigma) was added after 6 hours, to block protein
secretion. Cells were then harvested on ice without scraping,
washed twice in phosphate-buffered saline (PBS), and fixed with 2%
formaldehyde (1 mL per 2x106 cells, Merck) for 20
minutes. After two additional washes in PBS, cells were then
resuspended in Hank’s balanced salt solution (HBSS, supplemented
with 0.3% bovine serum albumin [BSA] and 0.1% sodium-azide), and
stored at 4 °C in the dark until staining. Fixed cells were
washed twice with PBS and made permeable with saponin (0.1%,
Sigma). They were then resuspended with 50 μL of
saponin-buffer-diluted antibodies and incubated for 25 minutes at
room temperature, in the dark.
The following monoclonal antibodies were used: cytokine-specific
mouse anti-human monoclonal antibody (MAb) (IFN-γ [clone: B27],
fluorescein isothiocyanate [FITC]-labelled) and rat anti-human MAb
(IL-2 [MQ1-17H12], phycoerythrin [PE]-conjugated; IL-10 [JES3-9D7],
PE-labelled; TNF-α [MAb11], PE-labelled). All MAb were purchased
from Pharmingen. The anti-CD3-MAb was peridinin chlorophyll
(PerCP), the anti-TCRγδ Mab and the anti-CD69-MAb were
allophycocyanin (APC)-labelled (Becton Dickinson, Mountain View,
CA, USA). Four-colour staining was performed, and at least
105 cells were analyzed on a FACSCalibur (Becton
Dickinson) equipped with a two laser system (488 nm and 633 nm
wavelength, respectively). All cytokine combinations (IL-2/IFN-γ,
IL-10/IFN-γ, TNF-α/IFN-γ) were stained in conjunction with CD3 and
CD69, for the identification of P. falciparum-specific
cytokine-expressing TCRγδ+ T cells, the TNF-α/IFN-γ
combination was stained in conjunction with CD3 and TCRγδ.
T cells were defined by their side-scatter characteristics and
anti-CD3 MAb staining, the γδ T cell subset was additionally
defined by anti-TCRγδ Mab staining. The specificity of cytokine
staining in CD3+ cells was verified by counterstaining
with CD69 as a marker for activated lymphocytes. Only cells clearly
positive for CD69 were classified as cytokine-producing
CD3+ cells. The specificity of cytokine staining was
confirmed by the absence of significant background in controls with
isotype-matched, irrelevant MAbs. Data were analysed with CELLQuest
software (Becton Dickinson), and results were expressed as the
percentage of cytokine-producing cells in each CD3+ cell
(or TCRγδ+CD3+) population.
Detection of non-specific, mitogen-induced, T cell cytokine
expression by flow cytometry
Flow cytometric assessment of intracellular cytokine expression was
performed essentially according to the technique described
previously [19, 27]. PBMC were isolated from heparinized blood and
stimulated in UCM with phorbol 12-myristate 13-acetate (PMA, 10
ng/mL; Sigma) and ionomycin (1.25 μM; Sigma), in the presence of
brefeldin A (1 μM; Sigma) for 4 hours at 37 °C, in 5%
CO2. Cells were then harvested and fixed as described
above. For the staining procedure, the following monoclonal
antibodies (MAbs) were used: cytokine-specific mouse anti-human MAb
(IFN-γ [clone B27], fluorescein isothiocyanate [FITC]-conjugated);
rat anti-human MAb (IL-2 [MQ1-17H12], IL-4 [MP4-25D2], IL-10
[JES3-9D7], IL-13 [JES10-5A2], TNF-α [Mab11], all phycoerythrin
[PE]-conjugated), the anti-CD4 MAb and the anti-TCRγδ MAb were
allophycocyanin-conjugated, the anti-CD3 MAb and anti-CD8 MAb were
peridinin chlorophyll-conjugated; all cytokine-specific MAbs were
purchased from Pharmingen (San Diego, CA, USA), the surface
marker-specific MAbs from Becton Dickinson (Mountain View, CA,
USA). All cytokine combinations were stained in conjunction with
CD4 and CD8, as well as CD3 and TCRγδ. Data were analysed with
CELLQuest software (Becton Dickinson). Samples were gated on
lymphocytes according to their light scatter characteristics and
the results were expressed as the percentage of cytokine-producing
cells in the CD4+, CD8+ or
TCRγδ+CD3+cell population, respectively.
Statistical methods
Statistical analysis was performed using a standard statistical
package (SPSS 11.5 for Windows; SPSS Inc., Chicago, USA). The
Mann-Whitney U-test was applied for group differences (children
versus adults). Bivariate correlations were done by computing a
Spearman’s correlation coefficient. A p value of < 0.05 was
considered significant.
Results
Frequency of P. falciparum-specific CD3+ cells
expressing cytokines: differences between healthy children and
adults
Adults showed an overall increased frequency of activated
(CD69+) CD3+ cells expressing cytokines after
stimulation with MACS-separated, late stage parasites of
P. falciparum when compared with children. This was
significant for the type 1 cytokine IFN-γ and the pro-inflammatory
cytokine TNF-α, but not for IL-2 and IL-10 (table 1( Table 1 ) and figures 1, 2). Frequencies
of background events within the CD3+ cell population
(unstimulated cells; addition of MACS-processed, uninfected
erythrocytes) were always < 0.04%.
Table 1 Frequency of P. falciparum-specific
CD3+ cells expressing cytokine in malaria-exposed
healthy children and adultsa
|
Cytokines
|
Children (n = 14)
|
Adults (n = 17)
|
|
IL-2−/IFN-γ+
|
0.18 ± 0.05 (0.02-0.78)
|
0.18 ± 0.03 (0.04-0.51)
|
|
IL-2+/IFN-γ−
|
0.05 ± 0.01 (0.01-0.12)
|
0.07 ± 0.01 (0.00-0.26)
|
|
IL-2+/IFN-γ+
|
0.04 ± 0.01 (0.01-0.11)b
|
0.11 ± 0.02 (0.03-0.25)
|
|
TNF-α+/IFN-γ−
|
0.23 ± 0.08 (0.04-1.25)b
|
0.31 ± 0.04 (0.07-0.64)
|
|
TNF-α+/IFN-γ+
|
0.19 ± 0.06 (0.03-0.85)b
|
0.27 ± 0.04 (0.05-0.55)
|
|
IL-10+/IFN-γ−
|
0.03 ± 0.01 (0.01-0.08)
|
0.05 ± 0.01 (0.00-0.12)
|
|
IL-10+/IFN-γ+
|
0.04 ± 0.01 (0.01-0.15)
|
0.07 ± 0.01 (0.00-0.17)
|
aValues indicate mean percentages of CD3+
cells expressing cytokines ± SEM; respective ranges are given in
parentheses.
bSignificant different between groups, p < 0.05 as
calculated by the Mann-Whitney U-test.
Frequency of P. falciparum-specific
TCRγδ+CD3+ cells expressing TNF-α and IFN-γ:
differences between healthy children and adults
The frequency of TCRγδ+CD3+ cells expressing
TNF-α was significantly increased in adults when compared with
children (mean, 0.21%; range, 0-0.75% versus mean, 0.09%; range,
0-0.48%; p < 0.05). However, only 12 adults (71%) and 5 children
(36%) displayed frequencies of > 0.1% TNF-α-expressing
TCRγδ+CD3+ cells in response to PFE
stimulation. In addition, a frequency of > 0.1% IFN-γ-expressing
TCRγδ+CD3+ cells responsive to specific
stimulation was seen in only 5 adults and 4 children (29%,
respectively). Mean values for the frequencies of IFN-γ-expressing
cells were 0.08% for both adults and children. In the presence of
medium alone or when stimulated with uninfected erythrocytes,
frequencies of cytokine-responsive TCRγδ+CD3+
cells were always below 0.02%.
Frequency of P. falciparum-non-specific CD4+
and CD8+ cells expressing cytokines: differences between
healthy children and adults
The frequencies of cytokine-expressing CD4+ and
CD8+ cells after mitogenic stimulation with PMA and
ionomycin in the presence of brefeldin A, were significantly
different between adults and children with regard to the type 1
cytokines IFN-γ and IL-2, as well as for the pro-inflammatory
cytokine TNF-α (table 2( Table 2 )
and figures 3, 4). IL-4-expressing CD8+ cells were
more frequently observed in adults, most of them however, were
positive for IFN-γ (table 2).
Table 2 Frequency of CD4+ and
CD8+ T cells expressing cytokine in malaria-exposed
healthy children and adults from the Lambaréné area after
non-specific stimulation with PMA and ionomycin in the presence of
brefeldin Aa
|
Cytokines
|
% of CD4+
|
% of CD8+
|
|
Children (n = 14)
|
Adults (n = 17)
|
Children (n = 14)
|
Adults (n = 17)
|
|
IL-2¯/IFN-γ+
|
5.4 ± 0.5 (2.4-9.2)
|
5.8 ± 0.4 (2.8-9.8)
|
25.4 ± 2.5 (11.9-42.0)
|
34.9 ± 3.5 (8.7-63.9)
|
|
IL-2+/IFN-γ+
|
5.8 ± 0.4 (2.8-9.8)b
|
19.4 ± 1.5 (10.9-37.6)
|
5.3 ± 0.6 (3.1-10.3)b
|
12.2 ± 1.2 (4.7-20.4)
|
|
IL-2+/IFN-γ¯
|
29.7 ± 1.6 (21.2-43.1)b
|
44.1 ± 1.6 (32.8-57.9)
|
3.3 ± 0.4 (1.1-6.7)d
|
7.0 ± 1.3 (1.4-24.1)
|
|
IL-4+/IFN-γ¯
|
4.5 ± 0.5 (1.6-9.0)
|
5.2 ± 0.4 (2.0-8.4)
|
< 1
|
< 1
|
|
IL-4+/IFN-γ+
|
2.1 ± 0.2 (0.9-3.4)
|
3.3 ± 0.4 (0.4-6.6)
|
< 1c
|
1.7 ± 0.3 (0-4.4)
|
|
IL-10+/IFN-γ¯
|
< 1
|
< 1
|
< 1
|
< 1
|
|
IL-10+/IFN-γ+
|
1.1 ± 0.2 (0.1-3.5)
|
1.1 ± 0.3 (0.3-6.2)
|
< 1
|
< 1
|
|
IL-13+/IFN-γ¯
|
4.1 ± 0.5 (1.7-8.0)
|
4.3 ± 0.4 (1.6-7.2)
|
< 1
|
< 1
|
|
IL-13+/IFN-γ+
|
< 1
|
< 1
|
< 1
|
< 1
|
|
TNF-α+/IFN-γ¯
|
26.2 ± 1.8 (16.6-36.4)c
|
32.7 ± 149 (19.5-41.9)
|
1.8 ± 0.3 (0.3-5.4)
|
2.5 ± 0.3 (0.5-5.4)
|
|
TNF-α+/IFN-γ+
|
12.0 ± 1.2 (4.3-23.7)b
|
21.7 ± 1.9 (10.5-45.8)
|
21.1 ± 3.0 (6.8-47.5)
|
30.4 ± 3.1 (9.9-56.3)
|
aValues indicate mean percentages of cytokine expressing
CD4+ and CD8+ ± SEM and ranges (in
parentheses).
bsignificant different between children and adults, p
< 0.001 as calculated by the Mann-Whitney U-test.
csignificant different between children and adults, p
< 0.05 as calculated by the Mann-Whitney U-test.
dsignificant different between children and adults, p
< 0.01 as calculated by the Mann-Whitney U-test.
Frequency of P. falciparum-non-specific
TCRγδ+CD3+ cells expressing cytokines and the
relationship of T cell-cytokine responses after
P. falciparum-specific and non-specific stimulation
As shown for the CD4+ and CD8+ T cellular
subsets, a significant, age-related increase in the frequency of
TCRγδ+CD3+ cells expressing cytokine after
short-term stimulation with PMA and ionomycin in the presence of
brefeldin A was noted (table 3( Table
3 ) and ( figure 3 )). Again,
the differences were obvious for IFN-γ and TNF-α, but were also
significant for the classical, type 2 cytokines IL-4 and IL-13. As
seen for CD8+ cells, most type 2 cytokine-expressing
cells also stained positively for IFN-γ (IFN-γ/IL-4 or IFN-γ/IL-13
co-producers). Low percentages of TCRγδ+CD3+
cells expressed IL-10 (table 3 and ( figure 3 )).
Within both groups of study participants, the frequency of
cytokine-expressing CD4+ CD8+ and
TCRγδ+CD3+ T cells, after non-specific,
mitogenic stimulation, did not correlate with the frequency of
specifically activated, cytokine-expressing CD3+ and
TCRγδ+CD3+ T cells.
Table 3 Frequency of TCRγδ+ CD3+
T cells expressing cytokine after non-specific stimulation with PMA
and ionomycin in the presence of brefeldin Aa
|
Cytokines
|
% of TCRγδ+ CD3+
|
|
Children (n = 14)
|
Adults (n = 17)
|
|
IL-2¯/IFN-γ+
|
27.2 ± 3.4 (10.9-47.3)b
|
35.7 ± 3.8 (7.9-76.8)
|
|
IL-2+/IFN-γ+
|
3.7 ± 0.4 (0.6-7.5)
|
6.5 ± 0.9 (0.8-14.6)
|
|
IL-2+/IFN-γ¯
|
8.1 ± 1.8 (1.8-24.1)
|
8.1 ± 1.5 (0-27.9)
|
|
IL-4+/IFN-γ¯
|
< 1
|
1.4 ± 0.3 (0.1-5.8)
|
|
IL-4+/IFN-γ+
|
< 1c
|
2.2 ± 0.4 (0-7.2)
|
|
IL-10+/IFN-γ¯
|
< 1
|
< 1
|
|
IL-10+/IFN-γ+
|
< 1
|
< 1
|
|
IL-13+/IFN-γ¯
|
< 1
|
1.4 ± 0.4 (0-5.7)
|
|
IL-13+/IFN-γ+
|
< 1
|
< 1
|
|
TNF-α+/IFN-γ¯
|
7.5 ± 2.0 (0.6-25.9)c
|
15.4 ± 2.1 (2.9-38.5)
|
|
TNF-α+/IFN-γ+
|
23.2 ± 2.8 (6.9-47.9)b
|
35.9 ± 3.2 (7.7-62.6)
|
avalues indicate mean percentages of cytokine expressing
TCRγδ+ CD3+ T cells ± SEM and ranges (in
parentheses).
bsignificant different between children and adults, p
< 0.05 as calculated by the Mann-Whitney U test.
csignificant different between children and adults, p
< 0.01 as calculated by the Mann-Whitney U-test.
Discussion
This study clearly illustrates the significance of ageing in the
development of P. falciparum-specific and non-specific
cellular immune responses in individuals from a
malaria-hyperendemic area. The increased capacity of various T cell
subsets to produce cytokines in adults was especially seen after
mitogenic stimulation, but also when PBMC were incubated with a
late-stage, schizont-rich parasite preparation. According to their
intrinsic capabilities, CD3+, CD4+ and
CD8+ T cells as well as the TCRγδ+ subset
contributed to the impressive, age-dependent differences with an
emphasis on the type 1 cytokine IFN-γ and on pro-inflammatory
TNF-α. Although, a trend towards higher expression of IL-2 in adult
T cells was also noted after specific stimulation, differences
between children and adults were highly significant only in
CD4+ and CD8+ cells (not in TCRγδ+
T cells) after non-specific stimulation.
The role of IFN-γ and TNF-α as determinants in age-associated
cellular responses is especially notable, as the capacity to
produce the counteracting cytokine IL-10 appears to be independent
of age. These findings accord well with another study from the
Lambaréné area using the P. falciparum, liver-stage antigen
(LSA)-1-derived T cell epitope stimulation for the assessment of
cellular immune responses in healthy children and adults: an
age-related increase in the proportion of individuals capable of
producing IFN-γ, while the proportion of children and adults
producing IL-10 remained similar, was noted [28]. Once more, in
children from a malaria holoendemic area in Kenya, IFN-γ responses
to LSA-1 and the blood-stage antigen merozoite-surface protein
(MSP)-1, required increased age and/or repeated exposure, and IL-10
responses were again independent of age [29]. Such an age-dependent
decline in the influence of IL-10 was seen, even in
P. berghei-infected rats, when higher levels of
IFN-γ-dependent IgG2c antibodies and lower IL-10 serum levels were
associated with resistance of adult animals to a primary infection,
whereas young rats with high IL-10 levels succumbed to disease
[30].
Thus, there are several indications that naturally immune
residents from hyper-or holo-endemic areas generally possess an
increased capacity to mount an effective, antiparasitic, probably
type 1- and TNF-α-driven, host response when compared to more
disease-susceptible children, while the contribution of IL-10 is
maintained at a similar level throughout life and probably more
closely associated with clinical disease. Indeed, high initial
levels of IL-10 were strongly associated with less effective
clearance of P. falciparum parasites in African children [31],
which was attributed to its down-regulating activity on
antigen-presenting cells or the induction of suppressive,
regulatory T cells.
The importance of early type 1 cytokine production during
Plasmodium infection has been linked to successful control of
parasitemia in many animal models of malaria [30, 32-37], and
“appropriate” IFN-γ as well as TNF-α production appears to be
protective and necessary for limiting parasitemia in human malaria
too [38-42]. The increased capacity of IFN-γ and TNF-α production
in naturally immune individuals might be crucial in the rapid
mounting of antiparasitic effector mechanisms such as the synthesis
of nitric oxide and reactive oxygen intermediates, thus allowing a
more efficient control of parasitemia and subsequently the
prevention of clinical disease.
A substantial proportion of the early, innate cytokine responses
during malaria has been ascribed to TCRγδ+ T cells.
Here, this particular subset participated in the generally
observed, age-dependent increased capacity for IFN-γ and TNF-α
production, but also showed increased frequencies of IL-4 and IL-13
expression in adults after mitogenic stimulation. However, most
type 2 cytokine-expressing TCRγδ+ T cells also
co-produced IFN-γ, so these cells can not be referred to as
classical type 2 cells. The overall, low specific responsiveness of
TCRγδ+ T cells in this study might be due to the use of
freeze-thawed rather than live parasites in the field experiments,
which has been shown to make an important difference in the
cellular response to parasite-preparations [24, 43]. Nevertheless,
TNF-α expression was again more frequently observed in
TCRγδ+ T cells of adults when compared with children.
CD4+ T cell-derived IL-2 has been shown to play a role
in the activation of human TCRγδ+ T cells when
stimulated with freeze-thawed schizont extracts [44], which would
offer one possible explanation for the somewhat more frequent
cytokine responses to parasitic stimulation in the adult group.
However, whether TCRγδ+ T cells play a role in
cell-mediated immunity as has been postulated in rodent models, or
rather exert immune-regulatory activities during human
P. falciparum malaria has to be further elucidated.
Although a wealth of data confirm the importance of T
cell-derived cytokines in the pathophysiology and host defenses
against Plasmodium spp., the significance of cell-mediated immunity
to the overall development of naturally acquired immunity in
individuals from endemic areas is currently unknown, as the latter
appears to develop largely due to the acquisition of a broad
repertoire of antibodies to variant surface antigens [4-6].
Therefore, our data may be best seen as complementary to those many
studies trying to link humoral immune responses to the degree of
naturally acquired immunity, although more recently protection from
natural P. falciparum infection and disease was also
correlated with a strong CD4+ T cell response directed
to a conserved epitope in the circumsporozoite protein [45]. We are
well aware that the increased frequency of
P. falciparum-specific cytokine responses in adults may just
reflect the more frequent contact and increased cellular reactivity
to antigens expressed by both malaria parasites and commensal
organisms. It has been speculated, however, that even such periodic
exposure to cross-reacting antigens can maintain some degree of
immunity [46]. The induction of immunity appears unlikely as it has
been known for many years that malaria-specific T cells can be
detected, even in non-exposed and obviously non-immune individuals
[26, 47].
As lifetime cumulative exposure to P. falciparum evolves
co-linearly with ageing in malaria hyperendemic areas, it is
impossible to disentangle the effects of ageing from the
consequences of exposure. Age-related changes in antimalarial
immune responses, independent of prior exposure, have been reported
in migrant populations from Indonesia and from newly established
endemic areas in Africa [8, 48]. There, adults acquired clinical
immunity after a rather brief period of heavy exposure, whereas
children remained susceptible to disease manifestations, suggesting
constitutional and obviously malaria-relevant differences between
the immune system of children and adults. In line with this,
pubertal development associated with increased
dehydroepiandrosterone sulphate levels were found necessary for
maximal expression of antimalarial resistance in both females and
males from Kenya [49, 50]. Unfortunately, our data are not suitable
for resolving the fundamental question, as to whether ageing
itself, cumulative exposure with acquisition of antibodies/memory T
cells, or both, are implicated in the development of naturally
acquired immunity to P. falciparum. Likewise, it remains
speculative, whether our in vitro findings represent reliable
correlates of cell-mediated, protective immunity.
Nevertheless, we propose that immune interventions including
vaccines should consider the profound, age-related differences in
the capacity of T cell-cytokine production in areas where
P. falciparum malaria is hyperendemic. Our findings would
argue for a vaccine that efficiently induces “adult-like”, strong T
cell-derived IFN-γ and TNF-α (IL-2?) responses in order to be
protective in the recipient. Even if this response might not be
sufficiently efficient to prevent infection and the development of
blood-stages, it might reduce parasite densities and thus
complications of malaria.
Acknowledgements
The study was, in part supported by a grant from the
Medizinisch-Wissenschaftlicher Fonds des Bürgermeisters der Stadt
Wien (project 1931), from fortüne program, Medical Faculty,
University of Tübingen, Germany and from EU-INCODEV.
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