Download Human Invariant Natural Killer T cells possess

Medical Mycology, 2016, 54, 169–176
doi: 10.1093/mmy/myv074
Advance Access Publication Date: 18 October 2015
Original Article
Original Article
Human Invariant Natural Killer T cells possess
immune-modulating functions during
Aspergillus infection
Antonia Beitzen-Heineke1 , Maria Bouzani1 , Anna-Lena Schmitt1 ,
Oliver Kurzai2 , Kerstin Hünniger2 , Hermann Einsele1
and Juergen Loeffler1,∗
1
Medizinische Klinik und Poliklinik II, University Hospital Wuerzburg, Wuerzburg, Germany and 2 Septomics
Research Centre, Friedrich-Schiller-University Jena and Leibniz-Institute for Natural Products Research
and Infection Biology - Hans Knoell Institute, Jena, Germany
*To whom correspondence should be addressed. Juergen Loeffler, Universitätsklinik Würzburg, Josef-Schneider-Str. 2,
Haus C11, 97080 Würzburg. Tel: +49 931 201 36412; Fax: +49 931 201 36409; E-mail: [email protected]
Received 27 May 2015; Revised 11 July 2015; Accepted 12 July 2015
Abstract
Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted
invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact
the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined
by CD69 expression and cytokine production in response to distinct fungal morphotypes
in the presence of different CD1d+ antigen presenting cells using flow cytometry and
multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d+ subpopulations,
CD1d+ CD1c+ mDCs showed the highest potential to activate iNKT cells on a per cell
basis. The presence of A. fumigatus decreased this effect of CD1d+ CD1c+ mDCs on iNKT
cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1
and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating
function for human iNKT cells during A. fumigatus infection.
Key words: invariant natural killer T (iNKT) cells, Aspergillus fumigatus, CD1d+ cells, mDCs.
Introduction
Natural killer T (NKT) cells compose a small subset of T
lymphocytes that are restricted to recognition of glycolipids
presented by the MHC class I-like molecule CD1d and express a T cell receptor (TCR) as well as NK cell markers
(CD161/CD56). The human, predominantly studied type
I NKT cells, also called invariant natural killer T (iNKT)
cells, express an invariant TCR consisting of a Vα24-Jα18
α-chain paired with a Vβ11 β-chain.1
Their unique effector functions predispose iNKT cells
to be an important factor in linking innate and adaptive immunity and to impact a wide range of diseases including auto-immune diseases,2 allergic diseases,3 cancer,4
and microbial infections.5 Mostly due to their constitutive
C The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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169
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expression of mRNA for immune-regulatory cytokines,
iNKT cells are able to release high quantities of cytokines
very shortly after antigenic stimulation.6,7 iNKT cells produce a variety of cytokines, among them the Th1 cytokines
interferon (IFN)-γ and tumor necrosis factor (TNF)-α as
well as Th2-type cytokines like interleukin (IL)-4, IL-5, IL10, and IL-13, chemokines and growth factors.8,9 This cytokine secretion, as well as direct cytolytic activity of iNKT
cells through the expression of perforin, granzyme B, and
FasL10 plus direct cell to cell interactions through costimulatory molecules like CD40/CD40L and CD80/86 entail
a high potential for iNKT cells to modulate immune responses. The diverse effects of iNKT cells include maturation and increased cytotoxicity of natural killer (NK) cells,11
differentiation and maturation of dendritic cells (DCs),
cytokine production by DCs,12–14 promotion of CD4+
and CD8+ immunity14 and B cell proliferation and immunoglobulin production.11,15,16 These characteristics enable iNKT cells to play an important role during infection.
iNKT cell activation results either from direct recognition of microbial lipid antigens presented by the MHC I- like
protein CD1d on antigen presenting cells (APCs), or from
recognition of endogenous self-antigens presented by APCs
in combination with stimulation by cytokines.17 The lastmentioned pathway enables iNKT cells to amplify toll-like
receptor (TLR) derived signals which makes them responsive to a variety of pathogens. While there are numerous
studies about the function of iNKT cells in bacterial5 and
viral infections,18 knowledge about the iNKT cell response
to fungi remains limited.
The function of murine iNKT cells in infection with A.
fumigatus has been first described by Cohen et al.19 who
showed that CD1d-dependent iNKT cell activation played
a role in early pulmonary clearance of A. fumigatus. This
iNKT cell activation was postulated to be induced by the
recognition of fungal ß1,3-Glucans through Dectin-1 on
DCs and subsequent IL-12 secretion which then stimulated iNKT cells. Albacker et al.20 described a glycolipid
extracted from A. fumigatus that directly activated iNKT
cells via a CD1d dependent, Dectin-1 independent mechanism. This glycosphingolipid, asperamide B, was the first
fungal antigen shown to be presented by CD1d and to directly stimulate iNKT cells.
However, there is very limited data about the interaction
of human iNKT cells with A. fumigatus. Moreover, it is unclear whether all different CD1d expressing APCs interact
equally with iNKT cells. Thus, the aim of our study was to
analyze the in vitro interaction of human iNKT cells with A.
fumigatus. We established a co-culture system to incubate
expanded iNKT cells with different types of CD1d+ APCs
from human peripheral blood with distinct A. fumigatus
morphotypes. For the first time, this study presents data
Medical Mycology, 2016, Vol. 54, No. 2
on the influence of three main subpopulations of CD1d+
cells, namely CD14+ monocytes, CD19+ B-cells and CD1c+
myeloid dendritic cells (mDCs), on the interaction of iNKT
cells and A. fumigatus.
Material and methods
Cells
Mononuclear cells from peripheral blood (PBMCs) were
obtained from buffy coats of healthy volunteer donors by
Ficoll (Biochrom GmbH) density gradient centrifugation.
iNKT cells were expanded from PBMCs as previously described.21
CD1d+ cells were isolated from PBMCs, which were
frozen in liquid nitrogen during expansion of iNKT cells,
by MACS positive selection procedures using anti-CD1d-PE
antibodies coupled with PE Microbeads, CD14 Microbeads
(100 μl Microbeads / 1 × 108 PBMCs), CD19 Microbeads,
and CD1c Dendritic Cell Isolation Kit (all Miltenyi Biotec,
concentrations according to manufacturer’s protocol).
Fungal strains
A. fumigatus conidia and germ tubes were prepared as previously described.22 Briefly, to obtain germ tubes, conidia
were cultivated in RPMI at a concentration of 1 × 106 / ml
by room temperature overnight under continuous shaking
at 200 rpm. Temperature was then upregulated to 37◦ C until an even appearance of germ tubes with an approximate
length of 20 μm was achieved. Germ tubes and conidia
were inactivated using 100% ethanol.
In vitro co-cultures
For co-culture experiments, 1 × 106 expanded iNKT cells
were cultured with 1 × 105 CD1d+ cells in 1 ml medium
plus stimuli in 24-well-plates. Different stimuli per well
were 10 ng IL-12, 500 IU IL-2, 2 × 106 A. fumigatus germ
tubes and 2 × 106 A. fumigatus conidia (MOI = 2). Cells
were harvested after 16 hours at 37◦ C and analyzed by
flow cytometry, supernatants were analyzed by MultiplexELISA.
Flow cytometry
Purity of isolated CD1d+ cells was assessed by flow cytometry analysis. Purity of CD1d+ cells was >99% (CD14
FITC, CD1d PE [BD Pharmingen], CD3 PerCP, CD1c APC
[Miltenyi Biotec], CD19 PerCP [Biolegend]). For flow cytometric analysis of co-cultured iNKT cells, cells were stained
with CD69 FITC (BD Pharmigen), iNKT PE and CD3
PerCP (both Miltenyi Biotec) following a FcR blocking step.
Beitzen-Heineke et al.
171
For evaluation of CD69 expression on iNKT cells, cells
were gated on live, iNKT+ CD3+ cells, with an anti-iNKT
antibody reacting with the T-cell receptor Vα24-Jα18 combined with Vβ11. Flow cytometry analysis was conducted
using FACS Calibur (BD Biosciences), data analysis was
performed using FlowJo 7.6.5.
Multiplex ELISA Assays
The concentrations of cytokines within supernatants were
determined using multiplex technology (Bio-Plex Pro Human Cytokine 27-plex Assay, Bio-Rad). The analyses were
performed according to the manufacturer’s instructions.
Statistical analysis
Data were analyzed with Graph Pad Prism 5. Wilcoxon
matched pairs signed-ranked test was used for analysis
of ELISA data (n = 3 individual donors for CD14+ and
CD19+ , n = 7 individual donors for CD1c+ co-cultures)
and paired t-test was used for analysis of FACS data. Significant differences are indicated by ∗ (P < .05), ∗∗ (P <
.01), and ∗∗∗ (P < .001).
Ethics statement
Research with blood samples from healthy volunteer
donors was approved by the Ethics Committee of the University of Wuerzburg. Participants provided their written
consent to participate in this study, and the Wuerzburg
Ethics Committee approved this consent procedure.
Results
No direct activation of iNKT cells by A. fumigatus
To explore the influence of A. fumigatus on iNKT cells,
we first investigated their direct interaction by incubating
iNKT cells with different fungal morphotypes. No activation of iNKT cells as determined by the upregulation of
the activation marker CD69 on the iNKT cell surface was
observed for germ tubes (Fig. 1b) nor for conidia (Fig. 1c)
whereas the addition of cytokines (IL-2 and IL-12) resulted
in upregulated CD69 expression on iNKT cells. This stimulating effect of cytokines was observed for IL-2 (P < .05)
and the combination of IL-2 and IL-12 (P < .001), whereas
the cytokine cocktail led to a significantly higher activation
than IL-2 alone (P < .05). IL-12 alone had no stimulating
effect on iNKT cells (Fig. 1a).
Figure 1. No direct activation of iNKT cells by A. fumigatus. Graph shows
CD69 expression on iNKT cells. iNKT cells were cultured alone (unstim.)
or with (a) a cytokine cocktail of IL-2 (500 IU) and IL-12 (10 ng, n = 13),
(b) A. fumigatus germ tubes (A.f. GT, MOI = 1, n = 16) or (c) conidia
(A.f. Con., MOI 1, n = 5). CD69 expression on iNKT cells was determined
after 16 h of co-culture by flow cytometry, gating on iNKT+ CD3+ cells.
Mean and standard error of the mean (SEM) of the geometric mean
fluorescence intensity (geo MFI) from independent experiments with
n = number of individual donors is shown. Significant differences are
indicated by ∗ (P < .05), ∗∗∗ (P < .001) and NS (not significant).
The activating effect of CD1d+ cells on iNKT cells
is reduced by A. fumigatus
After analyzing the direct effect of cytokines and fungus,
we investigated the effect of APCs on iNKT cells. The presence of CD1d+ cells induced an activation of iNKT cells in
the absence of any further stimuli (P < .005). The presence
of IL-2 and the cocktail of IL-2 and IL-12 induced further
stimulation of iNKT co-cultured with CD1d+ cells (Fig. 2a).
In the presence of A. fumigatus germ tubes, CD1d+
cells stimulated iNKT cells, however to a lesser extent than
CD1d+ cells in the absence of fungal germ tubes (Fig. 2b).
A. fumigatus germ tubes decreased the stimulating effect of
CD1d+ cells on iNKT cells regarding their CD69 expression (Fig. 2b). In contrast, A. fumigatus conidia did not
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Medical Mycology, 2016, Vol. 54, No. 2
Figure 3. The CD1c+ subpopulation is primarily responsible for the effect
of CD1d+ cells on iNKT cells. Graph shows CD69 expression on iNKT
cells. iNKT cells were co-cultured with CD1d+ cells (n = 9) or different subpopulations of CD1d+ cells: CD14+ monocytes (n = 14), CD19+
B-cells (n = 15) and CD1c+ mDCs (n = 14). Graph 3a compares iNKT
cells cultured alone (iNKT alone) with iNKT cells cultured with different
CD1d+ subpopulations (CD14; CD19; CD1c), graph 3b shows comparison of iNKT cells in combination with all CD1d+ cells and iNKT cells cultured with the distinct subpopulations. CD69 expression on iNKT cells
was determined after 16 h of co-culture by flow cytometry, gating on
iNKT+ CD3+ cells. Mean and SEM of the geometric mean fluorescence
intensity (geo MFI) from independent experiments with n = number of
individual donors is shown. Significant differences are indicated by ∗ (P
< .05), ∗∗ (P < .01), ∗∗∗ (P < .001), and NS (not significant).
Figure 2. CD1d+ cells have activating effect on iNKT cells which is reduced by A. fumigatus. Graph shows CD69 expression on iNKT cells.
iNKT cells were cultured alone (no CD1d) and with CD1d+ cells (unstim.,
n = 9). Co-cultures of iNKT cells and CD1d+ cells were incubated with
(a) IL-2 (500 IU) and IL-12 (10 ng) (IL-2+IL12, n = 8), (b) A. fumigatus
germ tubes (A.f. GT, MOI = 1, n = 7), A. fumigatus germ tubes (MOI
= 1) plus IL-2 (500 IU) and IL-12 (10 ng) (IL-2+IL-12+GT, n = 5), and (c)
A. fumigatus conidia (A.f. Con., MOI = 1, n = 4), respectively. CD69 expression on iNKT cells was determined after 16 h of co-culture by flow
cytometry, gating on iNKT+ CD3+ cells. Mean and SEM of the geometric
mean fluorescence intensity (geo MFI) from independent experiments
with n = number of individual donors is shown. Significant differences
are indicated by ∗ (P < .05), ∗∗ (P < .01), ∗∗∗ (P < .001), and NS (not
significant).
influence the effect of CD1d+ cells on iNKT cells (Fig. 2c).
This effect of germ tubes on iNKT cells in the presence of
CD1d+ cells was reversed by the presence of IL-2 and IL-12
(P < .005) (Fig. 2b).
cells and CD1c+ mDCs. Although all three subpopulations
alone had a stimulating effect on iNKT cells as determined
by CD69 expression (Fig. 3a), the effect of monocytes and
B-cells was significantly lower than the effect of mDCs (P
< .05) (Fig. 3b). In conclusion, the mDC subpopulation
shows the highest potential for activating iNKT cells on a
per-cell basis.
The same co-cultures were run with A. fumigatus germ
tubes and conidia. As in the co-culture system with the
entire CD1d+ -population, A. fumigatus germ tubes reduced the stimulating effect of CD1c+ mDCs on iNKT cells
(Fig. 4a). The same effect toward iNKT cells was mediated
when CD1c+ mDCs were co-cultured with A. fumigatus
conidia (Fig. 4b). Neither germ tubes nor conidia in combination with monocytes or B-cells induced a similar effect
(data not shown).
CD1c+ subpopulation with highest potential for activating iNKT cells
CD1c+ mDCs increase cytokine production in the
absence of further stimuli
The three main subpopulations of CD1d+ cells are monocytes, B-cells, and myeloid dendritic cells. Peripheral blood
dendritic cells are composed of CD11c− plasmacytoid DCs
and CD11c+ myeloid DCs which are further subdivided in
CD1c (BDCA-1)+ , CD141 (BDCA-3)+ and CD16+ myeloid
DCs.23
To differentiate which subgroup of CD1d+ cells is accountable for the stimulating effects on iNKT cells, cocultures were set up with CD14+ monocytes, CD19+ B-
Multiplex ELISA analysis was performed with supernatants
from iNKT cell co-cultures with APCs and A. fumigatus
germ tubes. The co-incubation of CD14+ monocytes or
CD19+ B-cells with iNKT cells without further stimuli did
not cause production of any of the tested cytokines (data
not shown). Differently, the interaction of CD1c+ mDCs
with iNKT cells resulted in increased production of the Th1
cytokines IFN-γ , TNF-α, and IL-2, the Th2 cytokines IL4, IL-5, IL-6, IL-10, and IL-13, IL-1β, IL-1ra, the growth
Beitzen-Heineke et al.
173
Discussion
Figure 4. A. fumigatus germ tubes and conidia decrease the stimulating
effect of CD1c+ mDCs on iNKT cells. CD69 expression on iNKT cells is
shown. iNKT cells were cultured alone (iNKT alone) or with CD1c+ mDCs
(unstim.). iNKT cell / CD1c+ mDC co-cultures were incubated with (a) A.
fumigatus germ tubes (A.f. GT, MOI = 1, n = 14 individual donors) and (b)
A. fumigatus conidia (A.f. Con., MOI = 1, n = 4 individual donors). CD69
expression on iNKT cells was determined after 16 h of co-culture by flow
cytometry, gating on iNKT+ CD3+ cells. Mean and SEM of the geometric
mean fluorescence intensity (geo MFI) from independent experiments
with n = number of individual donors is shown. Significant differences
are indicated by ∗ (P < .05).
factors FGF, G-CSF, GM-CSF, and VEGF as well as the
chemokines IL-8 and RANTES (Fig. 5 and data not shown).
Reduced TNF-α and G-CSF production in CD1c+
mDC / iNKT cell co-culture with A. fumigatus
The presence of A. fumigatus did not affect the secretion
of IFN-γ , IL-1β, IL-1ra, IL-4, IL-5, IL-6, IL-8, IL-13, and
VEGF in iNKT cell co-culture with CD1c+ mDCs. However, the comparison of CD1c+ mDC / iNKT cell co-culture
with and without A. fumigatus germ tubes showed a reduced production of TNF-α, G-CSF, and RANTES when
fungal germ tubes were present (Fig. 5).
Surface expression
on CD1c+ mDC
of
CD1d
is
highest
The upregulation of CD69 expression on iNKT cells correlates positively with the CD1d expression on co-incubated
APCs (R square = 0.935, slope = 0.1026 ± 0.01104, P <
.0001; n = 8 [Fig. 6]). Differences in CD1d expression on
the surface of different subpopulations of CD1d+ cells may
explain the dominating effect of CD1c+ mDCs on iNKT
cells. On CD1c+ mDCs, surface expression of CD1d was
significantly higher than on CD14+ cells (P < .01; n =
6) and CD19+ cells (P < .001). This entails a higher potential for interaction with iNKT cells which may be one
reason for the greater impact of CD1c+ mDCs on iNKT
cells when compared to CD1d+ monocytes and B-cells
(Fig. 7).
Aspergillus species are a ubiquitous genus of mold with the
clinically most important species A. fumigatus, the most
frequent cause for invasive aspergillosis.24 Taking the high
mortality rate of immune-suppressed patients with invasive aspergillosis, further characterization of distinct mechanisms of the innate and adaptive immune responses is essential to develop new prevention strategies and treatment
options.
Interaction with CD1d+ cells is essential for the activation of iNKT cells. Our findings show that while the three
main subpopulations of CD1d+ cells all induce a stimulation of iNKT cells, it is the CD1c+ mDC subpopulation
which shows the highest potential for activating iNKT cells
on a per-cell basis. These results from flow cytometric analysis were confirmed by analysis of cytokine concentrations
in co-culture supernatants where B-cells and monocytes did
not cause an increase in cytokine production in iNKT cell
co-culture. In contrast, co-culture with CD1c+ mDCs resulted in augmented secretion of Th1 cytokines like IFN-γ
and TNF-α as well as various Th2 cytokines, growth factors
and chemokines. Although Th1 as well as Th2 cytokines
were produced, the considerably higher concentrations of
IFN-γ and TNF-α imply a predominance of Th1 cytokines
in the given context. However, considering the experimental setting and the bidirectional interaction between iNKT
and CD1d+ cells, it remains unclear whether cytokines were
secreted by iNKT cells or CD1d+ cells.
In previous studies, certain glycolipids that bind to CD1d
have been shown to also be presented by CD1c with the
capability to directly interact with NKT cells.25,26 Furthermore, co-expression of CD1c on CD1d+ APCs has been
demonstrated to enhance the stimulation of iNKT cells by
α-GalCer.26 The higher stimulating effect of CD1c+ mDCs
in this study might result from a direct interaction or costimulating effect of CD1c molecules and iNKT cells. Furthermore, a significant positive correlation of the CD69
upregulation on iNKT cells and the CD1d expression on
co-incubated APCs was observed. This suggests that the
potential of APCs to activate iNKT cells increases with the
level of CD1d expression. This study showed highest CD1d
surface expression on CD1c+ mDCs compared to other
CD1d+ APCs, resulting in the highest interaction potential
with iNKT cells on a per-cell basis. A role of cytokine secretion, further co-stimulatory molecules, or other factors
cannot be excluded at this point.
Previous studies have investigated the role of iNKT
cells during A. fumigatus infection in mice. Cohen et al.
suggested a defensive role of iNKT cells during pulmonary infection with A. fumigatus conidia. Analysis of
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Medical Mycology, 2016, Vol. 54, No. 2
Figure 5. A. fumigatus reduces secretion of G-CSF, TNF-α and RANTES in iNKT cell / CD1c+ mDC co-culture. Graph shows concentrations of (a)
G-CSF, (b) TNF-α, and (c) RANTES in culture supernatants. iNKT cells were co-cultured with CD1c+ mDCs with and without A. fumigatus germ tubes.
Supernatants were collected after 16 h of co-culture and cytokine concentrations were determined by ELISA. Mean and SEM of five independent
donors are shown. Significant differences are indicated by ∗ (P < .05), ∗∗ (P < 0.01) and NS (not significant).
Figure 6. Positive correlation of CD69 expression and CD1d expression.
Graph shows total upregulation of CD69 expression on iNKT cells correlated to CD1d expression of co-incubated APCs (R square = 0.935, slope
= 0.1026 ± 0.01104, P < .0001; n = 8).
bronchoalveolar lavage fluid of infected mice showed augmented iNKT cell numbers with increased CD69 surface expression and IFN-γ production.19 Albacker et al. described
IL-4 and IFN-γ secretion by murine iNKT cells in response
to the fungal lipid asperamide B. However, for human iNKT
cell lines, induction of IL-4 and IL-13 but not IFN-γ was
shown.20
Figure 7. CD1d expression is highest on CD1c+ mDCs. Graph exemplarily shows flow cytometric analysis of CD1d expression on isolated
CD19+ cells, CD14+ cells and CD1c+ cells of one donor. CD1c+ cells
show highest CD1d expression compared to CD14+ cells and CD19+
cells.
As to the role of iNKT cells in infection with A. fumigatus in humans, our data suggest that the fungus has an
impact on iNKT cells, which is mediated by CD1c+ mDCs.
This effect of fungal germ tubes and conidia on iNKT cells
Beitzen-Heineke et al.
was inhibitory, relating to a decrease in CD69 surface expression on iNKT cells and a reduction of TNF-α, G-CSF,
and RANTES concentrations in co-culture supernatants. At
the same time, secretion of IFN-γ and Th2 cytokines did
not change in iNKT cell co-cultures with CD1c+ mDCs
and A. fumigatus germ tubes. Since all experiments were
performed with ethanol-inactivated fungal morphologies,
this APC dependent suppressive effect of A. fumigatus on
iNKT cells was due to direct contact rather than soluble A.
fumigatus moiety.
CD69 has long been known as an early leucocyte activation marker.27 Recently, lack of CD69 in knockout mice
has been described to enhance Th17 pro-inflammatory responses.28
TNF-α, along with IFN-γ , has been ascribed a protective role in A. fumigatus infection29 and neutralization of
TNF-α by specific antibodies in a mouse model has been
shown to reduce neutrophil numbers in the lung while mortality increased.30 In parallel, G-CSF is an inducer of granulopoiesis and enhances the oxidative burst of neutrophils,
thus increasing the damaging effect of neutrophils against
A. fumigatus hyphae.31 The decreased CD69 expression
and the inhibited secretion of TNF-α and G-CSF by iNKT
cells in the presence of A. fumigatus germ tubes might eventually reflect an immunoregulatory function of iNKT cells
during infection with A. fumigatus. This confirms the multiply described role of iNKT cells as a fine-tuner of immune
responses.
These findings differ from the results of studies in mice.
Murine immune cells differ in their number and function from human immune cell populations, especially neutrophils which compose 50–70% of peripheral blood cells
in humans compared to only 10–25% in mice.32 Neutrophils play an essential role in the first line defense against
A. fumigatus33–35 and neutropenia is one of the most important risk factors for invasive aspergillosis. Furthermore,
humans express five different CD1 genes corresponding to
five different CD1 proteins, CD1a, CD1b, CD1c, CD1d,
and CD1e, whereas mice express only one CD1 protein
which is an ortholog to CD1d.36,37 The results of this study
as well as previous studies with human cells have predicted
an influential role for all CD1 proteins, especially CD1c, in
iNKT cell function.25,26 Taking these aspects, differences in
human and murine immune response to fungi appear quite
likely.
Analysis of cytokine concentrations in culture supernatants did not detect considerable amounts of IL-12. However, it is well known that cytokines like IL-12 and IL-18
play a major role in iNKT cell activation.17 Freshly isolated
CD1c+ mDCs, as used in our experiments, have previously
been ascribed a minimal to low capacity of IL-12 production,38–40 as well as poor production of IL-18.39 This low
175
ability of immature mDCs to produce IL-12 leaves room
for speculations about a different response of iNKT cells to
A. fumigatus if more potent IL-12 producers were used as
APCs.
It remains to be investigated if the effect of CD1d+ cells
on iNKT cells is mediated by CD1d molecule or by costimulatory molecules or cytokines. Further experiments
with mature CD1c+ mDCs will be essential to fully understand the potential of human mDCs to interact with
iNKT cells during A. fumigatus infection. Moreover, the
investigation of CD1c− DC subsets in this context will be
an interesting topic for further research.
In summary, our experiments do not demonstrate a direct interaction between A. fumigatus and human iNKT
cells. We provide first insights into the impact peripheral
blood CD1d+ cells have on iNKT cells in the presence of
A. fumigatus conidia and germ tubes by showing that in
this infectious context, the stimulating effect of peripheral
blood mDCs on iNKT cells is decreased, whereas iNKT
cell response to monocytes and B-cells is not affected by A.
fumigatus.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) and CRC/Transregio 124 “Pathogenic fungi and their human
host: Networks of interaction”, subproject A2.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
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