Chimpanzee CD4+ T cells are relatively
insensitive to HIV-1 envelope-mediated
inhibition of CD154 up-regulation
Eur J Immunol. 2008 Apr;38(4):1164-72
Abstract
CD40-CD154 interaction forms a key event in regulation of crosstalk between
dendritic cells and CD4 T cells. In human immunodeficiency virus (HIV)-1
infected patients CD154 expression is impaired, and the resulting loss of immune
responsiveness by CD4+ T cells contributes to a progressive state of
immunodeficiency in humans. Although chimpanzees are susceptible to chronic
HIV-1/SIVcpz infection, they are relatively resistant to the onset of AIDS. This
relative resistance is characterized by maintenance of CD4+ T cell populations
and function, which is highly compromised in human patients. In our cohort of
chronically HIV-1- and SIVcpz-infected chimpanzees, we demonstrated the
capacity to produce IL-2, following CD3/CD28 stimulation, as well as preserved
CD154 up-regulation. Cross-linking of CD4 with mAb was found to inhibit
CD3/CD28-induced up-regulation of CD154 equally in chimpanzees and humans.
However, specific cross-linking with trimeric recombinant HIV-1 gp140 revealed
reduced sensitivity for inhibition of CD154 up-regulation in chimpanzees,
requiring fourfold higher concentrations of viral protein. Chimpanzee CD4+ T
cells are thus less sensitive to the immune-suppressive effect of low-dose HIV-1
envelope protein than human CD4+ T cells.
76
Chapter 4
Introduction
Human immunodeficiency virus type 1 (HIV-1) infection in humans is characterized
by a precipitous loss of immune competence, eventually allowing the establishment of
opportunistic infections and cancers [1, 2]. This loss of immune function is ultimately
observed at later stages as a peripheral decrease in the number of CD4+ T cells [3, 4].
However, before the numerical loss of CD4+ T cells is apparent, HIV-1-infected
individuals already have reduced antigen-specific and mitogen-stimulated CD4+ T
cell responses [5–12]. These defects include diminished proliferative capacity and a
functional loss in the ability to produce key cytokines. Induction of these important
immuno-modulatory cytokines is strongly dependent on dendritic cell (DC)-T cell
interaction. Exposure to antigen will lead to DC activation [13–15], induce migration
to lymphoid tissues and result in antigen presentation to T cells via MHC-TCR/CD3
interaction [16]. Subsequent activation of antigen-specific T cells leads to CD154 upregulation which, via binding to CD40 on the DC, induces DC maturation, which is
accompanied by up-regulation of the costimulatory molecules CD80 and CD86 and
by IL-12 secretion [17–20]. Subsequent binding of these costimulatory molecules to
CD28 on the T cell in combination with IL-12 can then induce a Th1 response against
the antigen, mediated by IL-2, TNF-α and IFN-γ secretion [21–26]. Independently,
several groups have confirmed that the induction of CD154 is decreased in HIV-1infected persons [21, 27–32]. In addition, reduced capacity to produce IL-2 and
consequential reduced antigen-specific T cell proliferation were reported [8, 26],
which can be restored via addition of IL-2 or via CD28 co-stimulation [5, 33]. Crosslinking of CD4 with mAb or with HIV-1 envelope protein results in an inhibition of
the CD3-mediated up-regulation of CD154 via an impairment of the normal CD3
signal transduction cascade [34, 35]. This perturbation of the normal cell activation
and CD154 upregulatory pathway is thought to form a major contribution to immune
suppression [36], prior to the wholesale loss of CD4+ T-cells, thus facilitating HIV
evasion and ultimately immunodeficiency in chronically infected humans.
Chimpanzees, humankind's closest living relatives, are susceptible to chronic
infection by HIV-1 and closely related SIVcpz infections. However, despite 98%
genetic similarity, this species is relatively resistant to disease caused by lentiviruses.
During more than 20 years of observation of HIV-1 infection in this species, relatively
few animals have developed an AIDS-like disease, providing unique evolutionary
77
insight and the opportunity to investigate the key features in HIV-1 infection and (the
absence of) subsequent disease progression [37–40]. In most HIV-1-infected
chimpanzees, no reduction in CD4+ T-cell numbers, no decrease in response to recall
antigens, and no overt and sustained increase in expression of activation markers or
apoptosis is seen [40–43]. Furthermore, high proliferative responses against HIV as
well as other pathogens, like cytomegalovirus, were noted [44, 45]. This study
addressed the hypothesis that CD4+ T cell function is maintained in this species in the
face of persistent HIV-1 infection. Specifically, we examined levels of CD154
expression and its regulation in relation to binding of the HIV-1 envelope to the CD4
receptor. Our findings reveal reduced expression levels of CD4 in chimpanzees as
well as a reduced sensitivity for suppression of CD154 up-regulation if the natural,
trimeric HIV-1 gp140, the primary viral ligand for CD4, is used as a CD4 crosslinking agent.
Materials and Methods
Animal subjects and human donors
Peripheral blood mononuclear cells (PBMC) were obtained from adult HIV1/SIVcpz-infected as well as from uninfected chimpanzees housed at the BPRC in
Rijswijk, The Netherlands. All HIV-1/SIVcpz-infected animals belonged to a cohort
of animals that were exposed to various HIV-1 strains in several studies in the past, as
previously described in detail [38, 39, 43, 73]. Animals were housed in groups
according to the international guidelines for non-human primate care and use. PBMC
from fresh blood were isolated using density gradient centrifugation (lymphocyte
separation medium, ICN Biochemicals, Aurora, OH) and cryopreserved in FCS with
10% DMSO and stored at –135°C before use. Human peripheral blood was obtained
from informed healthy volunteer donors via The Netherlands Red Cross Blood Bank,
and, after obtaining informed consent, from anti-retroviral treatment naïve HIV-1infected patients (median CD4+ T cell count 380 X 106 cells/L (range 230–470) and
median plasma HIV-1 RNA 4.7 log10 copies/mL (3.4–5.2)). All human blood
samples were processed similar to the chimpanzee PBMC.
78
Chapter 4
CD154 and intracellular cytokine staining on PBMC of HIV-1 (un)infected subjects
PBMC were incubated with anti-CD3/CD28 antibody-coated microbeads (Dynal,
Oslo, Norway) added at a concentration of 2.5 µL/1 X 106 cells for 2 h, followed by
the addition of Golgiplug (BD Pharmingen, San Diego, CA) and overnight culture.
After culturing, cells were stained for CD3-PE (clone SK7), CD4-PERCP (clone
L200), CD8-APC-CY7 (clone SK1) and CD45RA-PE-CY7 (clone 5H9). After
permeabilization and fixation, using BD Pharmingen Cytofix/Cytoperm solution
according to the protocol, cells were stained for CD154-APC (clone TRAP1; all BD
Pharmingen) in the presence of 10% human AB serum (Sanquin reagents,
Amsterdam, The Netherlands). For the intracellular cytokine expression experiments,
cells were first surface-stained for CD3-APC (clone SK7), CD4-PERCP, CD8-APCCY7, CD45RAPE-CY7, followed by permeabilization and fixation and incubation
with IL-2-PE (clone MQ1–17H12) in the presence of 10% human AB serum. Cells
were fixed overnight with 2% paraformaldehyde in PBS and all samples were
analyzed on a flow cytometer (FACSaria; Beckton Dickinson, Mountainview, CA).
Inhibition of CD154 expression
In order to evaluate the inhibitory capacities of CD4 ligation, CD4+ Tcells were
isolated from PBMC using a CD4 negative selection kit (BD Pharmingen). Purified
CD4 cells were incubated with the following CD4-binding agents; anti-CD4 domain 1
antibody (clone QS4120; Ancell, Bayport, MN), anti-CD4 domain 2 anti-body (clone
M-T411; Ancell; negative control), recombinant gp120 and trimeric O-gp140 of HIV1 SF162 (Chiron, Emeryville, CA). After 1 h of incubation at 4°C, anti-CD3/CD28
antibody coated microbeads were added to the cells at a concentration of 2.5 µl/1 X
106 cells. The cultures were incubated overnight at 37°C. After stimulation, cells were
harvested and stained for CD154 (clone TRAP1) and CD69 (clone FN50; BD
Pharmingen) and fixed using 2% paraformaldehyde. CD154 expression was
determined on a flow cytofluorometer (FACSaria; Beckton Dickinson).
79
CD4 surface expression profiling and gp140 binding
In order to evaluate expression of CD4 on the surface of human and chimpanzee T
cells, PBMC were stained with a panel of antibodies directed against several epitopes
of the CD4 receptor, used in a concentration range made to reach saturation levels
(SK3-APC, L200-PE, QS4120 and M-T441). For the non-labeled antibodies, the cells
were further incubated with goat anti-mouse APC-conjugated secondary antibodies,
used at saturating levels. For assessing gp140 binding, CD4+ T cells were incubated
for 1 h at 4°C with increasing doses of O-gp140, followed by staining with a FITCconjugated anti-HIV-1gp120 mAb (clone BO1188; AALTO, Dublin, Ireland).
Samples were recorded using either a FACScan or FACSaria flow cytometer, both
from Becton Dickinson. All data were analyzed with Diva software (Becton
Dickinson).
80
Chapter 4
Results
IL-2 cytokine production and CD154 expression is not altered in chronically HIVinfected chimpanzees
Previous reports have demonstrated reduced expression of IL-2 as well as reduced
CD154 up-regulation in HIV-1-infected subjects following PMA/ionomycin
stimulation [34, 46]. Subsequently, a similarly impaired IL-2 expression was shown
following stimulation through the physiologically more relevant CD3/CD28 pathway
[10]. Previously, we have shown that PMA/ionomycin induced IL-2 expression was
maintained in a cohort of HIV-1- and SIVcpz-infected chimpanzees [41]. Here, we
applied stimulation through the CD3/CD28 pathway to investigate IL-2 as well as
CD154 expression, as a measure of T helper (Th) function, in uninfected versus HIV1- or SIVcpzANT-infected chimpanzees and compared this directly to uninfected
versus HIV-1-infected human subjects. As shown in Fig. 1A, CD3/CD28-triggered
production of IL-2 was highly reduced in CD4 T cells in the HIV-1-infected patients,
Figure 1. IL-2 and CD154 expression in CD4 T cells after stimulation with anti-CD3/CD28 antibodies
in healthy humans, HIV-1-infected persons, healthy chimpanzees and HIV-1/SIVcpz-infected
chimpanzees as measured by flowcytometry. Expression on all CD4 T cells, CD4CD45RA– and
CD4CD45RA+ cells is shown as percentage of the respective (sub)populations. Data represent averages
of at least five individuals per group with their standard deviation; * represents significant difference by
Student's t-test (p<0.005).
81
but maintained in HIV-1-infected chimpanzees. Further subdivision in CD45RA–
memory cells and CD45RA+ naive cells showed preferential expression of IL-2 in the
memory cell subset. However, the decrease in IL-2 production in HIV-infected
humans was seen to a similar extent in both the memory and naive subset, while high
levels of IL-2 production were maintained in both subsets in the HIV-1-infected
chimpanzees (Fig. 1A). Following CD3/CD28 ligation, only very low levels of
CD154 induction could be reached in human HIV-infected subjects (3%; Fig. 1B),
whereas in HIV-1-infected chimpanzees the expression of CD154 was induced in
29% of the CD4 Tcells, which is found to be in the same range as in uninfected
chimpanzees and humans (25 and 33%, respectively). No difference in CD154
expression was found between CD45RA– and CD45RA+ cells (Fig. 1B).
CD154 expression is inhibited by CD4 ligation.
As an upstream mechanism for the impaired CD154 expression in HIV-1-infected
humans, it was shown that cross-linking CD4 can inhibit CD3/CD28-triggered
expression of CD154 in humans. We therefore investigated whether the same effect
could be observed in chimpanzees. CD4+ T cells from healthy chimpanzees and
human donors were pre-incubated for 1 h with an anti-CD4 antibody directed against
domain 1 (functional part of the receptor) or domain 2 (non-functional). Subsequently,
cells were activated by culturing them in the presence of anti-CD3/CD28 antibodycoated microbeads. Both in human and chimpanzee CD4+ Tcells, the CD4 domain 1preligated group showed reduced expression of CD154 (Fig. 2), whereas the domain
2-ligated (data not shown) groups showed strong induction of CD154 by the
CD3/CD28 stimulus. In order to evaluate the effect of cross-linking of CD4 with the
HIV-1 envelope, the experiment was repeated with recombinant monomeric gp120 as
well as trimeric gp140 at a relatively high concentration (10 µg/ml).
82
Chapter 4
Figure 2. Inhibitory effect of CD4
cross-linking on CD3/CD28-induced
CD154
expression.
(A)
Representative example of CD154
expression
on
unstimulated,
CD3/CD28-stimulated CD4 T cells
and CD4 T cells stimulated with antiCD3/CD28 antibodies in the presence
of anti-CD4 domain 1 blocking mAb.
Expression is shown in uninfected
human cells (top row) and uninfected
chimpanzee cells (bottom row). (B)
Expression of CD154 (top graphs) or
CD69
(bottom
graphs)
on
CD3/CD28-stimulated human (left
graphs) or chimpanzee cells (right
graphs), without blocking agents, in
the presence of anti-CD4 domain 1
blocking mAb (10 lg/mL), or in the
presence of trimeric gp140 (10
lg/mL). Average inhibition " standard
deviation from three human and three
chimpanzee donors is shown; *
represents significant difference by
Student's t-test (p<0.005).
As shown in Fig. 2, both chimpanzee and human CD4+ T cell activation was reduced
by trimeric gp140 binding to CD4, resulting in a strong decrease of CD154
expression, which was of similar magnitude as ligation of CD4 with the domain 1specific antibody. Monomeric gp120 did not result in a significant decrease of CD154
expression (data not shown), indicating that the trimeric envelope complex is required
for functional consequences of cross-linking of the CD4 receptor. As shown in Fig.
2B, expression of the activation marker CD69 was not effected by cross-linking of
CD4, either by the anti-CD4 domain 1 mAb or the trimeric gp140.
CD4 expression differs between species
While chimpanzee and human CD4+ T cells seemed to be equally susceptible to the
immune-suppressive effect of CD4 cross-linking, subsequent mAb titration studies
with a library of CD4 antibodies to several epitopes of the receptor showed a 30%
lower expression of CD4 on chimpanzee cells at saturation level (Fig. 3A).
83
Figure 3. CD4 expression
on
human
versus
chimpanzee T cells. (A)
Mean fluorescence intensity
(MFI) is shown measured at
different concentrations of
added anti-CD4 mAb; clone
SK3, clone QS412 (domain
1), clone L200 and clone
M-T441 (domain 2). (B)
MFI of clone L200 and
SK3, at saturating antibody
concentration, determined
in four different human and
chimpanzee donors.
Further analysis of the expression of CD4 in at least four different humans and
chimpanzees showed similar results (Fig. 3B), leading to the conclusion that the
differences were not due to a different binding capacity of the antibody, but to the
expression of CD4 on the membrane of chimpanzee T cells. Chimpanzee CD4+ T
cells are less sensitive to gp140 mediated suppression of CD154 up-regulation.
In order to evaluate the functional relevance of this difference in CD4 expression, a
CD154 induction experiment was performed in the presence of decreasing
concentrations of anti-CD4 domain 1 antibody. This titration did not expose any
differences between humans and chimpanzees with respect to the inhibitory capacity
of the CD4 antibody, i.e. both human and chimpanzee CD4+ T cells showed
impairment at a concentration of approximately 0.01 µg/mL of anti-CD4 antibody
(Fig. 4A), which is far below the saturation level of the CD4 receptor (Fig. 3).
84
Chapter 4
Figure 4. Dose-response curves of the
inhibitory effects of CD4-binding agents on
CD3/CD28-induced CD154 expression on
CD4+ T cells of healthy humans and in
chimpanzees. Percentage of inhibition of
expression is
plotted against the
concentration of anti-CD4 antibody (A) or
recombinant HIV-1 gp140 (B). Data are
represented as averages with SEM
calculated from experiments on four (A) or
five (B) separate individuals; * represents
significant difference by Student's t-test
(p<0.005).
The dose-dependent effects of viral envelope protein were tested in a similar manner
as the anti-CD4 antibody. The results of this experiment revealed that in human CD4+
T cells 0.2 µg/ml gp140 sufficed to inhibit CD154 up-regulation, whereas in
chimpanzees 0.8 µg/ml was required (Fig. 4B). These results seem to indicate that in
an environment with relatively low concentrations of viral envelope protein,
chimpanzee CD4+ Tcells can function normally, in contrast to human CD4+ T cells.
The concentration of trimeric gp140 at which these divergent effects were detected
was far below the saturation level (Fig. 5). However, it must be noted that at these low
gp140 concentrations, a slightly reduced binding to chimpanzee CD4+ T cells could
be seen in comparison to human CD4+ T cells.
50
Human
Chimp
45
Figure 5. O-gp140 binding to
chimpanzee and human CD4+ T
cells. Cells were incubated with
increasing concentrations of Ogp140 and subsequently stained
for bound protein using FITCconjugated anti-HIV-1 gp120
mAb. Shown is the MFI average
± standard deviation of two
chimpanzees and two human
donors.
40
35
MFI
30
25
20
15
10
5
0
0,01
0,1
1
10
concentration O-gp140 (ug/ml)
85
100
Discussion
Generation of effective immune responses to pathogens or cancer cells requires
closely coordinated crosstalk between Th cells and DC, as one of the first steps in
orchestrating a strong cellular immune response. CD4+ T cells that have been
activated via their T-cell receptor express CD154, which via binding to its ligand
CD40 leads to the necessary subsequent activation of DC. Subsequently, upregulation of CD80 and CD86 provides additional T cell stimulation signals via CD28
binding, inducing T cell proliferation and IL-2 production. Both CD154 expression on
CD4+ T cells and IL-2 production have been reported to be reduced in HIV-1infected persons [8, 10, 30, 47–52]. Here, we showed that, in addition to the
previously published maintenance of PMA/ionomycin-induced IL-2 expression [37,
40, 42, 53, 54], stimulation with a more physiological CD3/CD28 cross-linking agent
similarly generated high IL-2 expression in CD4+ T cells of HIV-1-and an
SIVcpzANT-infected chimpanzee. This preservation was seen in the IL-2 highexpressing CD45RA– memory cell subset as well as in CD45RA+ cells, while IL-2
expression was lost in both subsets in HIV-1-infected humans.
In view of the central regulatory role of CD154, we then proceeded to study its
expression in HIV-1-infected chimpanzees.
In contrast to its low expression in HIV-1-infected humans, we found strong
expression of CD154 following CD3/CD28 stimulation in HIV-1-infected
chimpanzees. There was no difference observed between CD45RA– and CD45RA+
cells, which is probably due to the overnight stimulation period, which has been
reported to result in similar levels of induction on both subsets after PMA/ionomycin
stimulation [55].
Several groups have found evidence that the binding of HIV-1 envelope protein to the
CD4 receptor can lead to reduced CD154 expression [34, 35]. Cross-linking of CD4
was shown to interfere with signal transduction cascades initiated by CD3/CD28
stimulation [56], resulting in inhibition of up-regulation of CD154. Using CD4
blocking agents we showed that, as in humans, chimpanzee CD4+ T cells are
susceptible to CD4-induced inhibition of CD3/CD28-induced CD154 expression.
Both an antibody to domain 1 of the CD4 receptor as well as recombinant trimeric
gp140 were able to inhibit CD154 up-regulation. Importantly, when we used trimeric
86
Chapter 4
gp140 for cross-linking of CD4, a reduced sensitivity for inhibition of CD154
expression was found on chimpanzee compared to human CD4 T cells. These
differences were masked when trimeric gp140 was used at a high concentration or
when a relatively strong cross-linking agent like anti-domain 1 mAb was used.
Strikingly, we observed that chimpanzee CD4+ T cells express 30% lower amounts of
the CD4 receptor on their membrane. This cannot be explained by reduced binding
capacity of the antibodies, since we have observed this effect with four different antiCD4 mAb and know that at least the SK3 binding site is identical between human and
chimpanzee CD4 [57]. An explanation for this lower expression of CD4 is at present
unknown. HIV-1 Nef was reported to induce similar cell surface down-regulation of
CD4 on human and chimpanzee CD4+ T cells [58]. While it could be speculated that
the lower expression of CD4 on chimpanzee cells is responsible for their reduced
sensitivity to CD4-mediated CD154 down-modulation, it must be stressed that these
inhibitory effects have been observed below receptor saturation levels (Fig. 3, 5). Also
at these lower antibody orgp140 concentrations, a slight difference in fluorescence
signal is seen between human and chimpanzee cells. On the other hand, the data do
imply that the reduced sensitivity of chimpanzee CD4 T cells to the CD154 downmodulating effect of gp140 cross-linking is not an indirect consequence of the number
of CD4 molecules that need to be cross-linked, as chimpanzees have less CD4 but
require more gp140 for CD154 inhibition. Importantly, Fomsgaard et al. [57] have
identified four amino acid changes in the V1J1 loop of chimpanzee CD4 that are all
part of the HIV-1 binding site, which could possibly explain why we observed a
reduced sensitivity of chimpanzee cells to cross-linking with gp140, but not with antiCD4 mAb. Especially, a difference in net charge induced by the change from glutamic
acid to glycine at position 77 could affect CD4 conformation and binding of gp120.
While it has been described that chimpanzee T cells express high levels of sialic acidbinding immunoglobulin-like lectin (SIGLEC) 5 and respond poorly to TCR/CD3
stimulation [59], we obtained similar levels of CD69 expression, varying between 80
and 90%, in both species. Thus, an intrinsic difference in susceptibility to CD3/CD28
stimulation does not seem to play a role. Although the full implications of this
difference in sensitivity to gp140-mediated down-modulation of CD154 between
chimpanzees and humans are not yet clear, it could imply that low plasma virus levels
would contribute to the relative resistance of chimpanzees to immunosuppressive
effects of HIV-1. This should become especially apparent several months after
87
infection, when virus replication reaches relatively low steady-state levels. It is
striking that initially in most infected humans strong CTL responses are formed,
which are known to precede control of virus replication. However, subsequently, Th
responses become impaired, leading to the formation of a relatively dysfunctional
CTL response characterized by elevated PD-1 expression, decreased IFN-γ and
perforin expression, resulting in a poor control of virus replication [60–71].
Maintenance the CD154-CD40 costimulatory pathway may be an important factor in
the preservation of adequate CD4+ T cell help during this critical phase of the
infection. Besides preservation of Th function, nonpathogenic infection of HIV-1 or
SIVcpz in chimpanzees is also accompanied by low levels of activation marker
expression [41]. However, since cross-linking of CD4 was found to have no effect on
CD69 expression, other mechanisms may be involved in preserving this low state of
cell activation. Additionally, the low-level expression of viral receptors on target cells
in the chimpanzee host, especially in active sites of viral replication such as the
intestinal mucosa [72], may provide this host with a selective advantage.
Statistical analysis
Statistical analysis was performed on the data as indicated (*) in the figures by
Student's t-test. A non-parametric Mann-Whitney test was used to compare CD4
expression levels between human and chimpanzee peripheral blood T cells shown in
Fig. 3B.
Acknowledgements
We would like to thank Dr. T. B. H. Geijtenbeek and M. de Jong from the Free
University Medical Center (Amsterdam, The Netherlands) for reagents and support.
Recognition of personal assistance: We are very grateful to the HIV infected patients
and healthy volunteers, who cooperated in this study and who generously provided
blood samples. We would like to thank the veterinary and animal care staff for
excellent and kind animal care. This work was funded by the Biomedical Primate
Research Centre
88
Chapter 4
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