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*
Research Institute, Palo Alto Medical Foundation, Palo Alto, CA 94301;
Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and
DNAX Research Institute of Molecular and Cellular Biology, Inc., Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to T.
gondii by T cells from T. gondii-seronegative
individuals occurred only after stimulation with monocytes that
exhibited increased expression of CD80 and CD86 (monocytes infected
with viable parasites) and was almost completely ablated by the
combination of anti-CD80 plus anti-CD86 mAb. Moreover,
proliferation and IFN- production by CD4+
CD45RA+ T cells from unexposed individuals were dependent
on both CD80 and CD86. These data indicate that pathogen-monocyte
interaction influences the ensuing T cell response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Data accumulated to date suggest that the interaction between CD28 expressed on T cells and its counter-receptors CD80 (B7-1) and CD86 (B7-2) expressed on specialized APC provides the most important costimulatory signal (5, 6). Both CD80 and CD86 can provide costimulation to T cells for proliferation and IL-2 production (7, 8). However, these molecules differ in their expression on APC. Whereas dendritic cells express both costimulatory ligands, monocytes/macrophages constitutively express only CD86 (9). Resting B cells have low levels of expression of CD86 and no significant expression of CD80 (9).
It has been reported that the expression of CD80 and CD86 on
APC can be regulated. Incubation with IFN- induces the
expression of CD80 and up-regulates the expression of CD86 on monocytes
(10, 11). In addition, activation of B cells results in induction of
the expression of CD80 and up-regulation of the expression of CD86
(9, 12). Regulation of the expression of CD80 and CD86 on APC may be an
important feature of the biology of these molecules with potential
implications in self/nonself discrimination.
The outcome of infections with certain organisms has been shown to
correlate with the type of cytokines produced by T cells (13). In this
regard, there is ample evidence of the pivotal role that IFN- plays
in the induction of protective immunity against intracellular
micro-organisms (14). Thus, the identification of factors that
influence cytokine production triggered by infectious organisms are
crucial to our understanding of the mechanisms that determine whether a
protective immune response is elicited. Toxoplasma gondii,
an obligate intracellular protozoan that infects all nucleated cells,
provides an example of a micro-organism against which cell-mediated
immunity with resulting IFN- production plays a critical role in
controlling infection (15, 16). Indeed, this parasite has become a
major opportunistic pathogen in immunocompromised individuals (17). The
demonstration that presumably unprimed human T cells respond in vitro
to this parasite (18) made T. gondii well suited for study
of the roles of CD80 and CD86 in the early events of the T cell
response against an intracellular organism. Our results indicate that
human monocytes discriminate between noxious and innocuous preparations
of T. gondii, which translates into the induction of the
expression of CD80 and the up-regulation of the expression of CD86 only
when these cells encounter viable (noxious) organisms. Our results
reveal that, in turn, T cell proliferation is dependent on CD80 and
CD86, and that the induction/up-regulation of expression of these
molecules is associated with the generation of an IFN- response by T
cells from unexposed individuals. These data provide evidence of the
importance of pathogen-monocyte interaction, especially through
pathogen-mediated induction/up-regulation of costimulatory ligands, in
shaping the ensuing T cell response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mAbs were used for cell purifications: anti-CD2, anti-CD3, anti-CD8, anti-CD14, and anti-CD56 (all from Becton Dickinson, San Jose, CA); anti-CD16 (Medarex, Inc., Annandale, NJ); anti-CD19 (Coulter, Hialeah, FL); anti-CD45RA, anti-CD45RO, and anti-CD66b (all from Immunotech, Westbrook, MA); and anti-glycophorin A (10F7 MN, American Type Culture Collection, Rockville, MD).
Anti-CD14 (RM052, Immunotech), anti-CD80 (L307, Becton Dickinson),
anti-CD86 (Fun-1, PharMingen, San Diego, CA), CTLA-4-Ig (gift from
Dr. Peter Linsley) (19), and neutralizing mAb against the cytokines
GM-CSF,3 IFN- (R&D
Systems, Minneapolis, MN), and IFN-
(BioSource International,
Camarillo, CA) were used in functional assays (all at 10 µg/ml).
Isotype-matched mAbs and human IgG1 were obtained from PharMingen and
Sigma Chemical Co. (St. Louis, MO), respectively. IFN-, purchased
from R&D Systems, was used at 100 U/ml.
The following conjugated Abs were used for flow cytometry (purchased from Caltag, South San Francisco, CA, except when indicated): FITC-anti-CD3 (Becton Dickinson), FITC-anti-CD14, FITC-anti-CD19, FITC-anti-CD45RA (Immunotech), FITC-anti-CD45RO (Immunotech), FITC-anti-CD66b (Immunotech), phycoerythrin (PE)-anti-CD11a, PE-anti-CD16 (Becton Dickinson), PE-anti-CD54, PE-anti-CD56 (Becton Dickinson), PE-anti-CD58 (Becton Dickinson), PE-anti-CD80 (Becton Dickinson), PE-anti-CD86 (PharMingen), or PE-anti-HLA-DR.
Cell purifications
PBMC were isolated from buffy coats of heparinized blood of healthy volunteers donors obtained from the Stanford Blood Bank (Stanford, CA). Serologic tests for detection of anti-T. gondii IgG and IgM were performed in all samples of blood (20). This allowed for division of the donors into T. gondii-seronegative (negative anti-T. gondii IgG and negative anti-T. gondii IgM) and T. gondii chronically infected individuals (positive anti-T. gondii IgG and negative anti-T. gondii IgM).
To obtain purified monocytes, PBMC were incubated with the following mAb: anti-CD2, anti-CD3, anti-CD8, anti-CD19, anti-CD56, anti-CD66b, and anti-glycophorin A. After addition of magnetic beads coated with anti-mouse IgG (Dynal, Great Neck, NY), rosetting cells were removed with a magnet (Dynal). This resulted in populations that were >96% pure for monocytes by microscopic examination of Giemsa-stained cytocentrifuge preparations. In addition, cytofluorometric analysis indicated that >92% of the cells were CD14+, with <0.5% CD3+, <0.5% CD19+, <0.5% CD56+, and <2% CD66b+ cells. In certain experiments monocytes were purified further by incubation with FITC-conjugated anti-CD14 mAb (Becton Dickinson) followed by FACS sorting. This procedure resulted in populations of highly purified CD14+ monocytes (>99% by flow cytometry). For some experiments purified monocytes were incubated for 2 days in complete medium (CM) consisting of RPMI 1640 with 10% dye test-negative human AB serum (Irvine, Santa Ana, CA) before infection with T. gondii. This resulted in populations of cells that acquired macrophage morphology (21).
Resting T cells (>99% CD3+) were obtained from nylon
wool-nonadherent PBL that were incubated with anti-CD16 plus
anti-CD56 mAb and subjected to depletion using immunomagnetic
beads. To obtain purified populations of CD4+
CD45RA+ TCR-ß+, anti-CD8 (OKT8,
American Type Culture Collection), anti-
TCR
(anti-TCR1, gift from Dr. Michael Brenner), anti-CD19
(Coulter Cytometry, Hialeah, FL), and anti-CD45RO (UCHL-1,
Immunotech) were added to the combination of mAb mentioned above.
Addition of magnetic beads was repeated once. The populations obtained
(>98% CD4+ CD45RA+ TCR-ß+)
did not proliferate in response to a recall Ag (tetanus toxoid),
whereas unseparated CD4+ TCR-ß+ and
CD4+ CD45RO+ TCR-ß+ cells
exhibited significant proliferation in response to this Ag.
T. gondii and infection
T. gondii tachyzoites were obtained from both infected human foreskin fibroblasts (American Type Culture Collection) and peritoneal cavities of infected mice and exposed to UV light as previously described (22). Neither uninfected human foreskin fibroblasts nor tachyzoite-free peritoneal lavage fluids from infected mice (after passage through a 0.45-µm filter) mediated changes in expression of the surface molecules tested. In certain experiments, tachyzoites were killed by incubation in 1% paraformaldehyde in PBS (18). To obtain T. gondii lysate Ags (TLA), tachyzoites were harvested from infected human foreskin fibroblasts and lysed in H2O by three cycles of freezing and thawing followed by reconstitution with 10x PBS. Antigenic preparations were devoid of detectable levels of endotoxin (<10 pg/ml) using a Limulus amebocyte lysate assay (Sigma).
Unless stated otherwise, PBMC were incubated with tachyzoites of T. gondii at a ratio of 1:1 for 24 h, and purified monocytes were incubated with four tachyzoites per cell for 48 h before cytofluorometric analysis. Cells were cultured in Teflon vessels in CM. The percentage of cells with intracellular parasites was determined by light microscopy (22). In some experiments, Transwell inserts (Corning Costar Corp., Cambridge, MA) were used to separate monocytes plus T. gondii tachyzoites from monocytes alone by a membrane with pores 0.4 µm in diameter.
Flow cytometry
Freshly isolated and cultured cells were stained with either
FITC-conjugated anti-CD14 or FITC-conjugated anti-CD19 mAb and
one of the following PE-conjugated mAb: anti-CD11a, anti-CD54,
anti-CD58, anti-CD80, anti-CD86, or anti-HLA-DR. When
analyzing PBMC, an electronic gate was set on FITC-stained
CD14+ or CD19+ cells to identify monocytes
and B cells, respectively. For surface molecules whose levels of
expression followed a bimodal distribution (CD80 and CD86 on
monocytes), the percentages of CD80+ and
CD86high cells were calculated by determining the
percentage of cells that stain above the value of fluorescence obtained
with isotype control mAb (CD80+) or the value of
fluorescence of CD86int cells (CD86high). For
surface molecules whose levels of expression followed a unimodal
distribution, data are expressed as the corrected mean fluorescence
intensity (MFI). The corrected MFI for PE-conjugated mAb were
calculated by subtracting the MFI of the appropriate isotype control
from the MFI of each specific mAb. In some experiments, the T.
gondii-induced increase in the levels of expression of CD80 and
CD86 is expressed as 
MFI, which were calculated by subtracting
corrected MFI for CD80 and CD86 of uninfected monocytes from corrected
MFI for CD80 and CD86 on CD80+ and CD86high
monocytes, respectively. Sorting of monocytes into CD80-,
CD80+, CD86int, and CD86high
populations was performed 18 h after incubation with T.
gondii.
T cell proliferation assays
T cells (5 x 105/ml) were incubated with
either T. gondii-infected or uninfected, -irradiated,
autologous PBMC with or without TLA (10 µg/ml) as previously
described (18). In certain experiments, monocytes were used instead of
PBMC. Infected cells were added to T cells at a ratio of five T cells
per one infected cell (18). Concentrations of uninfected PBMC or
monocytes matched those of infected cells. CTLA-4-Ig, anti-CD80
(L307), and anti-CD86 (Fun-1) mAb or control Abs were added to
either PBMC or monocytes 30 min before incubation with T cells. Unless
indicated, these reagents were used at 10 µg/ml. Cells were cultured
in 96-well plates for 7 days, labeled for the final 18 h with
[3H]thymidine, and harvested (18). Radioactivity was
measured in a beta scintillation counter (18). Results are expressed as
the mean counts per minute of [3H]thymidine incorporation
of triplicate wells ± SEM. Data are also presented as stimulation
indexes (counts per minute of culture with T. gondii/counts
per minute of culture without T. gondii).
Cytokine assays
Purified, resting peripheral blood T cells (1 x
106/ml) were incubated with purified monocytes
(2–5 x 105/ml) that were uninfected, infected with
viable UV-attenuated tachyzoites of T. gondii (8 x
105/ml to 2 x 106/ml), or incubated with
TLA (10 µg/ml) in 96-well plates. Supernatants were collected at 24,
48, and 72 h and stored at -70°C. Concentrations of IL-2, IL-4,
and IFN- were measured by ELISA (18) in supernatants collected at
24, 48, and 72 h, respectively. Data are presented as the mean of
triplicate wells ± SEM. None of the cytokines tested was detected
in supernatants obtained from wells that lacked T cells and contained
only monocytes with or without T. gondii antigenic
preparations.
Statistical analysis
Statistical significance was assessed by unpaired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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APC express accessory molecules that can enhance Ag-driven T cell
responses (23, 24). The effects of T. gondii on the
expression of accessory ligands such as CD11a, CD54, CD58, CD80, and
CD86 and on the expression of HLA-DR molecules on monocytes and B cells
were analyzed by cytofluorometry. Incubation of PBMC with T.
gondii tachyzoites consistently resulted in a striking induction
of expression of CD80 and up-regulation of expression of CD86 on
monocytes. The levels of expression of these molecules followed a
biphasic distribution, giving rise to a subpopulation of monocytes with
remarkable levels of expression of CD80 (CD80+) as well as
a subpopulation of monocytes with increased levels of expression of
CD86 (CD86high; see Fig. 1
).
The levels of expression of CD54 and HLA-DR on monocytes and of CD54,
CD86, and HLA-DR on B cells were also increased in seven of 10
experiments in which PBMC were incubated with the parasite (Table I). However, the levels of expression of
these latter molecules remained in a unimodal distribution. It is
interesting to note that the expression of CD80 on B cells was never
affected by T. gondii. Whether PBMC originated from
individuals with or without Abs to T. gondii did not
influence the magnitude of change in the levels of expression of any of
the above mentioned molecules (p 
0.2).
T. gondii did not induce nonspecific up-regulation of
expression of surface molecules on monocytes and B cells, since the
levels of expression of CD11a and CD58 on these cells were not
increased after incubation with the parasite. Tachyzoites used in these
experiments were exposed to UV light to inhibit their intracellular
multiplication and prevent destruction of infected cells (18). Changes
in the levels of expression of accessory ligands were not caused by
treatment with UV light, since unexposed tachyzoites exerted similar
effects on the expression of these molecules (data not shown). Thus,
T. gondii induced changes in the levels of expression of
costimulatory ligands that were particularly striking on monocytes,
since there was both induction of expression of CD80 and up-regulation
of expression of CD86.
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Given the remarkable changes observed in the expression of
costimulatory ligands on monocytes and the importance of macrophages to
the immune response against intracellular pathogens (25), our
additional studies concentrated on the effects of T. gondii
on monocytes/macrophages. Since T cell-mediated cognate (cell
contact-mediated) signals can induce the expression of CD80 on B cells
(26, 27), experiments were conducted to determine whether changes in
the expression of costimulatory ligands on monocytes that were mediated
by T. gondii occurred in the absence of lymphocytes.
Incubation of purified monocytes with tachyzoites resulted in the
appearance of subpopulations of monocytes with remarkable levels of
expression of CD80 molecules (CD80+; Fig. 1
A) as well as subpopulations of cells with increased
levels of expression of CD86 molecules (CD86high; Fig. 1B). Although the changes in expression of CD80 were
striking, levels of expression of CD86 on CD86high
monocytes (MFI = 1579.2 ± 70.1) were, on the average, 11.7
times higher than those of CD80 on CD80+ monocytes
(MFI = 139.5 ± 15.4; n = 7). The increase in
the levels of expression of CD80 and CD86 were not caused by
nonspecific effects mediated by phagocytosis of foreign particles,
since incubation with latex beads did not alter the expression of any
of these molecules (data not shown). Incubation of purified monocytes
with increasing numbers of tachyzoites per monocyte resulted in a
progressive increase in the percentages of CD80+ and
CD86high monocytes (Fig. 1C). T.
gondii tachyzoites also induced up-regulation of the expression of
CD80 and CD86 molecules on monocyte-derived macrophages (data not
shown).
Experiments were conducted to determine whether preparations of dead
tachyzoites of T. gondii as well as of parasite soluble Ags
could also induce changes in the expression of CD80 and CD86 on
purified monocytes. Whereas preparations containing viable
(UV-attenuated) tachyzoites induced the appearance of CD80+
and CD86high monocytes, neither paraformaldehyde-killed
tachyzoites nor TLA affected the levels of expression of these
molecules (Fig. 1D). Similar results were obtained
regardless of whether monocytes originated from individuals with or
without Abs to T. gondii.
Role of infection of monocytes with T. gondii and of
IFN-, IFN-, and GM-CSF on induction of expression of CD80 and
up-regulation of expression of CD86 molecules
Experiments were conducted to determine whether induction of the expression of CD80 and up-regulation of the expression of CD86 caused by T. gondii were due to infection of monocytes and/or to the secretion of cytokines known to affect the expression of these molecules on monocytes/macrophages. Purified monocytes incubated with the parasite were sorted by FACS into CD80+, CD80-, CD86high, and CD86int populations. Microscopic examination of these populations revealed that whereas at least 50% (59 ± 8%) of CD80+ and CD86high monocytes had evidence of intracellular tachyzoites (mostly degenerated organisms), the percentages of CD80- and CD86int monocytes with intracellular tachyzoites were never more than 2% (1.5 ± 0.5%; n = 3).
Since IFN-, IFN-, and GM-CSF have been reported to induce the
expression of CD80 and/or to up-regulate the expression of CD86 on
monocytes/macrophages (9, 10, 26, 27, 28, 29), we performed experiments to
determine whether the effects of T. gondii on the levels of
expression of these costimulatory ligands were mediated by these
cytokines. Incubation of purified monocytes with saturating
concentrations of neutralizing mAb against IFN- or IFN- did not
affect either the T. gondii-mediated induction of
CD80+ and CD86high monocytes (Fig. 2A) or the increase in
the levels of expression of CD80 and CD86 on CD80+ and
CD86high cells, respectively (Fig. 2, B and
C; n = 3). Although a neutralizing mAb
against GM-CSF did not significantly affect the percentages of
CD80+ and CD86high monocytes, this mAb induced
a modest, but consistent, inhibition of parasite-mediated increase in
the levels of expression of CD80 (30.6 ± 5% inhibition) and CD86
(18.8 ± 2% inhibition) on CD80+ and
CD86high monocytes, respectively (p
0.04; Fig. 2, A–C). At the concentration used,
anti-GM-CSF mAb completely ablated the GM-CSF-mediated induction of
expression of CD80 on monocytes (data not shown). Even though our
results suggest that GM-CSF is involved in the T.
gondii-mediated induction of expression of CD80 and the
up-regulation of expression of CD86, experiments performed with
Transwell inserts indicate that it is unlikely that soluble factors
(such as GM-CSF) alone are sufficient to mediate these effects.
Incubation of monocytes with T. gondii tachyzoites separated
from monocytes alone by a membrane permeable to particles 0.4 µm in
diameter resulted in induction of the expression CD80 and up-regulation
of the expression of CD86 only on monocytes directly exposed to the
parasite (data not shown).
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Since IFN- is consistently produced upon exposure of human PBMC to
T. gondii (31), experiments were conducted to determine
whether, in the presence of lymphocytes, IFN- modulated the T.
gondii-induced changes in the expression of CD80 and CD86 on human
monocytes. As shown in Figure 3,
A and B, addition of anti-IFN- mAb to PBMC
incubated with T. gondii did not result in a significant
inhibition of the induction of CD80+ and
CD86high monocytes or a significant decrease in the levels
of expression of these molecules on CD80+ and
CD86high monocytes (p 0.2;
n = 4). Similar results were obtained regardless of the
serologic status of the donor. At the concentration used,
anti-IFN- ablated the IFN--mediated induction of expression
of CD80 and up-regulation of expression of CD86 on monocytes (Fig. 3C). Thus, these results indicate that, even in the
presence of lymphocytes, the early induction of CD80 and up-regulation
of CD86 on human monocytes triggered by T. gondii are not
mediated by IFN-.
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The presence of costimulatory ligands during the early phases of T
cell-APC interaction would appear to be required to affect T cell
responses. Thus, experiments were conducted to determine the kinetics
of the T. gondii-mediated changes in the levels of
expression of CD80 and CD86 on purified monocytes. Figure 4A demonstrates that a
significant increase in the percentages of CD80+ and
CD86high monocytes was detected 12 h after incubation
with T. gondii tachyzoites and reached a peak at 24 h.
Similarly, when the MFI of CD80+ and CD86high
cells were analyzed, maximum levels of expression of these molecules on
the CD80+ and CD86high populations were
observed at 24 h (Fig. 4B). Thus, these data
indicate that T. gondii triggers a rapid induction of
expression of CD80 and up-regulation of expression of CD86 molecules on
monocytes. It is interesting to note that CD80+ and
CD86high cells underwent a progressive decrease in the
levels of expression of CD14 (Fig. 1, A and
B). In contrast to dendritic cells derived by
incubation of monocytes with GM-CSF plus IL-4, T.
gondii-induced CD80+ and CD86high
monocytes remained CD1a- (data not shown).
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We have previously demonstrated that both peripheral blood resting
T cells from healthy T. gondii chronically infected donors
as well as those from T. gondii-seronegative donors
proliferate when incubated with either parasite-infected cells or
T. gondii-soluble Ags (18). We used this experimental system
to assess the functional significance of CD80 and CD86 molecules on the
response of resting T cells to T. gondii. T cells from
T. gondii-seronegative donors were stimulated with
autologous T. gondii-infected PBMC in the presence of
CTLA-4-Ig, a chimeric protein with high affinity for CD80 and CD86 that
blocks the interaction between these molecules and CD28 (19). Figure 5A shows that CTLA-4-Ig
induced a dose-dependent inhibition of the T.
gondii-mediated T cell proliferation. To determine the individual
roles of CD80 and CD86 in the proliferative response of T cells,
anti-CD80 and/or anti-CD86 mAb were added to T cells stimulated
with the parasite. Incubation with either anti-CD80
(p 0.03) as well as anti-CD86 mAb
(p 0.008) resulted in a statistically
significant inhibition of the T cell proliferative response to T.
gondii-infected autologous PBMC (Fig. 5B).
Anti-CD80 mAb reduced the proliferative response by 30.1 ±
13.0%; anti-CD86 induced a 74.1 ± 5.4% inhibition of this
response (n = 4). Furthermore, when
anti-CD80 was used in combination with anti-CD86 mAb, the
proliferative response was abrogated (96.0 ± 2.1% inhibition;
p 0.01; Fig. 5B; n = 4).
In contrast, anti-CD86 mAb (p 0.02), but
not anti-CD80 mAb (p 0.4), significantly
inhibited T cell proliferation in response to TLA (Fig. 5C). Anti-CD86 mAb induced a 76.8 ± 17.4%
inhibition of T cell proliferation in response to TLA, and incubation
with the combination of anti-CD80 and anti-CD86 did not result
in any further inhibition (76.8 ± 19.4% inhibition;
p 0.008) of TLA-mediated T cell proliferation
(n = 4). Similar results were obtained when T
cells were stimulated with monocytes, including T.
gondii-infected highly purified CD14+ monocytes
(n = 3; data not shown). These results
demonstrate that both CD80 and CD86 are critical costimulatory ligands
for T. gondii-mediated proliferation of T cells from
seronegative individuals, and that whereas CD80 plays a role only in
the T cell proliferation mediated by infected cells, CD86 plays a role
in both proliferation mediated by cells infected with T.
gondii and that mediated by cells pulsed with
parasite-soluble Ags.
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0.001), was almost completely
inhibited by anti-CD86 mAb (83.1 ± 6.8% inhibition;
p 0.0001), and was ablated by the combination of
these two mAb (98.1 ± 1.8% inhibition; p
0.0001; Fig. 5D). Thus, these results indicate that
the proliferation of presumably unprimed T cells in response to
T. gondii-infected cells is dependent on both CD80 and CD86
molecules.
To determine the role of costimulation through CD28 in the response of
T cells from individuals previously exposed to T. gondii,
experiments similar to those described above were performed using T
cells from healthy, chronically infected donors. As shown in Figure 6A, anti-CD86 mAb
significantly inhibited the proliferation of T cells in response to
T. gondii-infected PBMC (58.5 ± 14.0% inhibition;
p 0.02; n = 4). Incubation with
anti-CD80 mAb resulted in variable partial inhibition (17.7 ±
7.4% inhibition; p = 0.01–0.1) of the T cell
proliferation in response to T. gondii-infected PBMC. The
combination of anti-CD80 plus anti-CD86 mAb resulted in
additive inhibitory effect (84.4 ± 4.1% inhibition;
p 0.01), leading to a dramatic reduction in T cell
proliferation. Figure 6B shows that anti-CD86 mAb
inhibited proliferation of T cells in response to TLA (67.5 ±
11.4% inhibition; p 0.001), and there was no
inhibitory effect exerted by anti-CD80 mAb (2.7 ± 2.7%
inhibition; p 0.1) and no additive effect observed
after combining anti-CD80 plus anti-CD86 mAbs (76.1 ±
13.3% inhibition; p 0.0003). Thus, CD80 and CD86
are also important costimulatory ligands in the in vitro proliferative
response to T. gondii by T cells from previously exposed
individuals.
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We studied whether the T. gondii-induced changes in the
levels of expression of CD80 and CD86 on monocytes modulated cytokine
production by T cells in response to the parasite. Whereas there was no
significant production of IFN- when resting T cells from T.
gondii-seronegative individuals were incubated with uninfected,
untreated monocytes, T cells secreted significant amounts of this
cytokine when incubated with T. gondii-infected monocytes
(Fig. 7A; n =
3). Interestingly, stimulation of T cells with monocytes incubated with
paraformaldehyde-killed tachyzoites or TLA did not result in the
production of significant amounts of IFN-. This was observed despite
the fact that T cells proliferated not only in response to T.
gondii-infected monocytes but also in response to monocytes that
had phagocytosed killed parasites and to monocytes incubated with TLA
(Fig. 7A; n = 3). The lack of production of
IFN- in response to incubation with monocytes and either killed
parasites or TLA was not caused by an inherent inability of these
stimuli to trigger IFN- production, since T cells from healthy
individuals chronically infected with T. gondii produced
considerable amounts of IFN- after stimulation with monocytes plus
either killed tachyzoites (945 pg/ml) or TLA (582 pg/ml;
n = 3).
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by T cells from seronegative individuals. In this
regard, production of IFN- by these T cells in response to T.
gondii-infected monocytes was significantly inhibited by
anti-CD86 (40.8 ± 5.2% inhibition; p
0.04). Incubation with anti-CD80 mAb resulted in variable partial
inhibition (7.5 ± 7.0% inhibition; p =
0.02–0.1) of IFN- production; the combination of CD80 plus
anti-CD86 mAb resulted in an additive inhibitory effect (80.2
± 1.4% inhibition; p 0.002), leading to almost
complete ablation of IFN- production (n = 3;
Fig. 7B). Similar results were obtained using highly
purified CD14+ monocytes (data not shown). In addition,
production of IFN- by CD4+ CD45RA+ T cells
from seronegative individuals was inhibited by either anti-CD80
(69.47 ± 6.1% inhibition; p 0.001) or
anti-CD86 mAb (53.0 ± 5.1% inhibition) and was ablated by
the combination of mAbs (100% inhibition p 0.001;
n = 2; Fig. 7C). Taken together,
these results indicate that the early production of IFN- by
presumably unprimed T cells is dependent on the expression of CD80 and
CD86 on monocytes. We attempted to determine whether T. gondii-mediated changes in expression of costimulatory ligands affect T cell production of IL-2 and IL-4 in response to the parasite. Stimulation of T cells with monocytes plus PHA resulted in the secretion of measurable concentrations of IL-2 and either low or undetectable concentrations of IL-4. However, neither of these cytokines was detected in supernatants from T. gondii-stimulated T cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by T cells from unexposed individuals. Our results are of
particular relevance to immunity against intracellular organisms, since
production of IFN- as a result of the interaction between infected
monocytes/macrophages and T cells would lead to activation of these
phagocytic cells, enabling them to act as major effectors of
antimicrobial defense.
It is well recognized that cognate signals and certain cytokines can
induce or up-regulate the expression of CD80 and CD86 on APC. We
demonstrate that induction of the expression of CD80 and up-regulation
of the expression of CD86 on human monocytes caused by T.
gondii occurred in the absence of significant concentrations of
lymphocytes and was not mediated by IFN- or IFN-, which are
cytokines capable of inducing the expression of CD80 and up-regulating
the expression of CD86 on monocytes/macrophages (9, 10, 29). In these
same experiments, GM-CSF was found to be involved in the process of
induction of the expression of CD80 and up-regulation of the expression
of CD86 triggered by T. gondii. These latter results suggest
that, similar to what has been reported for dendritic cells (33),
GM-CSF may have immunomodulatory activity on infected monocytes that,
through promoting up-regulation of costimulatory ligands, may lead to
an enhanced immunostimulatory function of these APC. In addition to the
cytokines mentioned above, we have recently studied TNF-, IL-1,
and IL-12 and demonstrated that neutralizing Abs against these
cytokines failed to inhibit the T. gondii-mediated changes
in the expression of CD80 and CD86 on monocytes (C. S. Subauste,
unpublished observations). Despite the results that we obtained after
neutralization of GM-CSF, cell-sorting experiments as well as data
obtained with Transwell inserts indicated that infection of monocytes
with viable tachyzoites and not soluble factors appeared to play the
primary role in induction of the expression of CD80 and up-regulation
of the expression of CD86. Although not all the CD80+ and
CD86high monocytes contained intracellular tachyzoites
18 h after incubation with T. gondii, our microscopic
examination of monocytes incubated with the parasite is consistent with
this conclusion. Monocytes can rapidly eliminate intracellular
tachyzoites so that the percentage of infected monocytes 18 to 24
h after challenge with T. gondii will be remarkably lower
than the percentage of infected monocytes 1 h after challenge
(34).
T. gondii-mediated up-regulation of expression of CD86 on
human monocytes has been described recently (35). The authors reported
that the expression of this molecule followed a unimodal distribution,
and that this up-regulation appeared to be inhibited by an
anti-IFN- polyclonal Ab. However, in our studies, double
staining with anti-CD14 and anti-CD86 mAb allowed us to
demonstrate that up-regulation of CD86 occurred only in a subpopulation
of monocytes. Furthermore, we demonstrated that IFN- did not play an
important role in this up-regulation, since an increase in the levels
of expression of CD86 occurred in populations of highly purified
monocytes and was not affected by a neutralizing anti-IFN-
mAb.
Except in experimental models using cells transfected with CD80 or transgenic mice expressing CD80 on pancreatic ß cells (36, 37), it has been difficult to demonstrate significant costimulation by CD80 molecules (12, 38). The predominant costimulatory activity of CD86 molecules may be explained, at least in part, by the fact that after APC activation, CD86 is expressed earlier and at higher levels than CD80 (39, 40). In contrast, our results demonstrate that infection of monocytes with T. gondii results in a rapid induction of CD80 and up-regulation of CD86, indicating that both costimulatory ligands can be present in the early phases of the immune response to an intracellular pathogen. Moreover, our studies indicate that CD80 molecules provided significant costimulation under conditions in which the expression of CD80 was induced in human monocytes (infection with viable tachyzoites).
One of the hallmarks of the immune response elicited by T.
gondii is the production of IFN-. Indeed, this cytokine is a
major mediator of protection against the parasite (16, 41). The
critical role of IFN- in the immune response to intracellular
pathogens makes identification of factors required for production of
this cytokine one of the most important questions in the study of
immunity to infectious organisms. We have recently demonstrated that
both human ß and T cells from T.
gondii-seronegative individuals secrete significant amounts of
IFN- after in vitro stimulation with the parasite (18) (see Footnote
4). Our data indicate that IFN- production by human T cells from
unexposed individuals is associated with a T. gondii-induced
increase in the levels of expression of costimulatory ligands on
infected monocytes. IFN- was produced only when T cells were
incubated with monocytes that displayed high levels of expression of
CD80 and CD86 (monocytes infected with viable tachyzoites). Moreover,
anti-CD80 plus anti-CD86 mAb almost completely inhibited T cell
production of IFN-. Of particular relevance to the events in the
initiation of the immune response to intracellular pathogens are our
results with CD4+ CD45RA+ cells, which indicate
that presumably naive CD4+ T cells require CD80 and CD86
for the production of IFN- in response to T.
gondii-infected monocytes. These data are of importance to the
induction of protective immunity to the parasite, since the early
production of IFN- may confer protection to the host not only
because of direct effects of this cytokine on the growth of
intracellular tachyzoites (41), but also because IFN- appears to
play a role in promoting the generation of a Th1 cytokine pattern
(42).
It is well established that proliferation and IL-2 secretion by naive T cells are dependent on the presence of costimulatory signals (32). Indeed, we have demonstrated that the proliferation of CD4+ CD45RA+ T cells in response to T. gondii is ablated by anti-CD80 plus anti CD86 mAb. However, our results indicate that these costimulatory ligands also play a central role in the parasite-triggered proliferation of T cells from chronically infected individuals. In this regard, human CD45RO+ memory T cells undergo optimal anti-CD3-mediated proliferation in the presence of CD80-transfected cells (43).
Our model provides a clear example of the capacity of monocytes to
discriminate among different microbial preparations and illustrates
that the T cell cytokine response is affected by the type of microbial
preparation that elicits the immune response. In the case of T.
gondii, this pathogen-monocyte-T cell interaction would result in
IFN- production in situations (noxious stimulus; i.e., infection
with viable tachyzoites) where an IFN--dependent cell-mediated
response would be appropriate, whereas no such a response would be
triggered when encountering nonviable parasite preparations (harmless
stimulus). A probable in vivo correlate to our observations can be
drawn from the demonstration that infection with viable T.
gondii bradyzoites, rather than immunization with TLA, is
necessary for acquisition of resistance to tachyzoites of a virulent
strain of the parasite (44).
There is increasing evidence of the importance of innate immunity in
host defense against intracellular organisms (45, 46). It is well
established that this arm of the immune system can promote the
generation of a protective immune response through IFN- production
by NK cells (47). It has also been proposed that the mechanisms used by
the innate immune system to recognize pathogens would determine the
type of adaptive immunity elicited (46). Our results support this
proposal, since they provide evidence that nonclonal recognition of a
micro-organism by monocytes can affect the nature of the T cell
response to the offending pathogen. In addition, given that IFN-
production by NK cells can be enhanced by costimulation through CD28
(48, 49), it is likely that microbially mediated
induction/up-regulation of costimulatory ligands can further promote
the generation of protective immunity through stimulation of secretion
of IFN- by NK cells.
The results of our present study illustrate the importance of a
microbially induced increase in the levels of expression of
costimulatory ligands for the production of protective cytokines (i.e.,
IFN-). Of interest in this regard is the evidence that certain
intracellular organisms, such as Leishmania donovani and
Mycobacterium tuberculosis, either fail to up-regulate or
actually decrease the expression of these molecules (50, 51). These
effects on costimulatory ligands may represent strategies used by the
pathogens to avoid recognition, induce anergy, or cause
immunosuppression. Thus, intracellular organisms can have a major
influence on antimicrobial immunity through regulation of
costimulation.
Our demonstration that tachyzoites, but not parasite-soluble Ags, are
capable of inducing these changes and that the tachyzoites have to be
viable to affect the levels of expression of CD80 and CD86 on monocytes
suggest that the process of induction of CD80 and up-regulation of CD86
in monocytes may be multifactorial. Signals triggered within monocytes
by the presence of viable intracellular organisms, factors released by
viable intracellular parasites and/or interactions between the intact
pathogen and molecules on the surface of monocytes may be involved in
this process. Identification of the mechanisms by which microbes induce
the expression of CD80 and up-regulate the expression of CD86 are of
paramount importance, since they may tie molecular events that occur
during the interaction of pathogens with APC with the outcome of
infections. Comparison of the molecular events triggered in monocytes
by infection with T. gondii with those triggered by
pathogens that fail to increase levels of expression of costimulatory
ligands may provide an understanding of the mechanisms by which
monocytes can direct T cell responses. Finally, our results have
important implications for the efforts to establish a vaccine against
intracellular pathogens, since they suggest that vectors that
induce/up-regulate the expression of costimulatory ligands on APC will
help generate a protective (IFN--dependent) immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carlos S. Subauste, Division of Infectious Diseases, Department of Medicine, University of Cincinnati College of Medicine, P.O. Box 670560, Cincinnati, OH 45267–0560. E-mail address:
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; PE, phycoerythrin; CM, complete medium; TLA, Toxoplasma lysate Ags; high, high level; int, intermediate level; MFI, mean fluorescence intensity.
4 Subauste, C. S., R. de Waal Malefyt, and J. S. Remington. 1998. ß T cell response to Toxoplasma gondii in previously unexposed individuals. J. Immunol. In press.
Received for publication July 14, 1997. Accepted for publication October 28, 1997.
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