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* Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, and
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction between T cells and APCs leads to tight conjugate formation and rapid segregation of supramolecular activation clusters at the T cell contact site (7, 8, 9). The formation of tight conjugates between T cells and APCs raises the question of how these cells subsequently disengage themselves, and whether the cells retain or exchange their respective cell surface molecules. Recently, studies in mouse systems demonstrated that T cell-APC interaction can cause APC-derived surface molecules to adhere to the surface of T cells and to be internalized through TCR endocytosis (10, 11). Moreover, data from our laboratory and others in mouse models demonstrated that CD80 molecules are physically acquired by naive as well as memory T cells from APCs shortly after T cell activation; this acquisition plays a role in the activation of bystander T cells (12). In the early 1970s, Hudson et al. (13, 14) also demonstrated that alloreactive murine T blast cells generated during a graft-vs-host reaction in vivo expressed Ig molecules. In addition, the same investigators showed that the Ig molecules were of donor origin. Later, other studies with murine bone marrow chimeras indicated that donor-derived thymocytes were able to acquire host MHC molecules (15, 16). Although the acquisition of costimulatory molecules by T cells in mice has now been established, no studies have addressed this phenomenon in humans. On the contrary, reports exist that upon stimulation, human T cells express CD80.
Early studies on the activation of T cells demonstrated that CD80 appears relatively late after T cell activation and can be detected on long-term activated human T cells isolated from PBMC, CD4+ T cell clones, and CD8 T cell clones (17). Moreover, studies performed on T cells from rheumatoid arthritis patients showed that 30% of synovial T cells express CD80 molecules on their surface, in contrast to synovial T cells from normal donors (18). In studies of patients with systemic lupus erythematosus, there was an increase in the amount of CD80 on PBLs, including T cells (19). The perception has thus been that the expression of CD80 on human T cells is attributable to de novo up-regulation of CD80 as the result of T cell activation. However, it should be pointed out that all of the studies mentioned above analyzed T cells at long intervals following activation; none of these reports analyzed the early events in human T cell activation.
In this study, we demonstrate for the first time that CD80 detection on T cells shortly after activation is a consequence of acquisition from APCs, and not from endogenous up-regulation of these molecules by human T cells; we also demonstrate that this phenomenon is perhaps mediated by the interaction of CD80 with CD28 receptors, and is enhanced if the APCs have increased CD80 expression. Furthermore, we demonstrate that purified CD4+ T cells that have acquired CD80 actually have the ability to enhance the proliferation of other human T cells and can thus, in essence, themselves act as APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was collected by apheresis from normal healthy donors. PBMC were isolated by standard Ficoll-Hypaque (ICN Biomedicals, Aurora, OH) density centrifugation. Cells were washed three times with 1x PBS, and were then resuspended at 20 x 106 cells/ml in a beads separation medium (1x PBS, 0.5% BSA, supplemented with 2 mM EDTA) (BioSource International, Camarillo, CA). CD4+ T cells were then selected by column purification using CD4+ beads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). CD4+ T cells were resuspended at 1 x 106 cells/ml in complete medium (RPMI 1640 medium; Life Technologies, Gaithersburg, MD) containing 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, and 100 U/ml penicillin-streptomycin, nonessential amino acids) for cell culture or they were resuspended in FACS buffer (PBS (Life Technologies) with 0.2% BSA (Sigma-Aldrich, St. Louis, MO)) for the FACS analysis.
Ag-presenting cells
In vitro generation of dendritic cells (DCs).2 CD14+ peripheral blood-derived monocytes were isolated from leukopaks from healthy donors by counterflow centrifugal elutriation. Fresh monocytes were cultured in six-well tissue culture plates at 2–3 x 106/ml (Costar, Cambridge, MA) in complete medium. At days 1, 3, and 5, recombinant human (rh)IL-4 and rhGM-CSF (PeproTech, Rocky Hill, NJ) were added to the culture at 50 ng/ml. DCs harvested at day 10 were repeatedly shown to be CD1a+, HLA-DR+, CD86+, CD40+, CD3-, CD14-/low, CD19-, and CD56- by flow cytometry (FACSCalibur; BD Biosciences, Mountain View, CA).
The B cell line C1R-A2 containing stable HLA-A2 construct was obtained from Dr. A. Tsang (Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD). Cells were grown in a complete medium, and were maintained at 37°C under a 5% CO2 humidified atmosphere. C1R-A2 cells were maintained under the pressure of 1 mg/ml selective agent G418 (Life Technologies).
Immunofluorescence and three-color immunofluorescent flow cytometry
CD4+ selected cells were stained with different Abs for 30 min on ice. The following Abs were used: CD4-FITC, CD45RA-FITC, CD45RO-FITC CD28-PE, CD80-PE, CD19-, and CD1a- PerCP (BD PharMingen, San Diego, CA). The three-color staining was analyzed on a FACSCaliber (BD Biosciences) using CellQuest software.
RNA extraction and PCR
RNA was isolated from CD4+ T cells and the
B cell line C1R-A2, using a RNA STAT-60 kit (Tel-Test, Friendswood,
TX). Five micrograms of total RNA were used in the cDNA synthesis
reaction using the SuperScript Preamplification System (Life
Technologies), according to the manufacturer’s instruction.
CD4+ T cells that acquired CD80 in contact with
APCs before the PCR were treated with 0.5% EDTA for 5 min. The cells
were then isolated using CD4+ beads and column
separation. The purity of these cells was verified by three-color FACS
analysis. Oligonucleotides specific for CD80 and 
-actin were
synthesized by Biosynthesis (Lewisville, TX). CD80 amplification was
performed by using 5' primer GAC CCT AAG CAT CTG AAG CCA TG and 3'
primer TGA TCC CCA CGA TCC ATG TAT C. Primers used for -actin
amplification (1-kb fragment) were: 5'-GCT CAC CAT GGA TGA TGA TAT CGC
and 3'-GGA GGA GCA ATG ATC TTG ATC TTC.
Platinum PCR Supermix (Invitrogen, Carlsbad, CA) was used for amplification reaction. Cycling conditions consisted of 94°C for 2 min and then 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by a finishing step at 72°C for 7 min. PCR products were analyzed on 1.5% agarose gels in 1x Tris EDTA acetate buffer, stained with ethidium bromide. The cDNA fragments were visualized by UV light.
CD28-CD80 interaction studies
Human CD152 (CTLA-4) Ig fusion protein. C1R-A2 cells (1 x 106) were incubated with 30 µg/ml CTLA-4 Ig fusion protein (Ancell, Bayport, MN) or purified IgG2a (BiosPacific, Emeryville, CA) for 30 min on ice. The cells were washed three times with 1x PBS and cocultured in a 1:1 ratio with CD4+ T cells for 24 h.
Human CD28 Ig fusion protein. C1R-A2 cells (1 x 106) were incubated with 30 µg/ml soluble CD28 Ig fusion protein (Ancell) or its isotype control, purified IgG2a (BiosPacific), for 30 min on ice. The cells were then cocultured with the selected CD4+ T cells, as described above.
Human CD80 Ig fusion protein. CD4+ T cells (1 x 106) selected from PBMC, as described below, were incubated with 30 µg/ml CD80 Ig fusion protein (Ancell) or its isotype control, purified IgG2a (BiosPacific), for 30 min on ice. Cells were then cocultured with an untreated B cell line, C1R-A2, in a ratio of 1:1 for a 24-h incubation.
Proliferation assay
Purified CD4+ T cells (5 x 106) were cocultured with 5 x 106 CIR-A2 in the presence of 1 µg/ml anti-CD3. Twenty-four hours postculture, CD4+ T cells that had acquired CD80 in the presence of APCs were treated with 0.5% EDTA for 5 min and were isolated by column separation and the purity of the cells was checked by three-color FACS analysis. Purified CD4+/CD80+ T cells (1.5 x 105) were added with or without 1 µg/ml anti-CD3, and cultured in 96-well plates (Costar) in a complete medium or a complete medium supplemented with 10% dextran for 48 h. Twenty-four hours before harvesting, cells were pulsed with 1 µCi [3H]thymidine to assess proliferative response.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the innate expression of CD80 on
CD4+ T cells upon short activation,
CD4+ T cells were purified from the PBMC of
normal healthy donors (purity >97–99%) and analyzed by two-color
FACS analysis. CD80 expression on CD4+ T cells
was shown to be a relatively rare event (Fig. 1
Aa), as was
similarly reported for murine T cells.
Specifically, 15 donors were analyzed and the level of CD80 detection
on 12 was between 1 and 3%, with the T cells of the remaining three
donors displaying between 3 and 7%.
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Ab). Activation of these cells for 24 h
in the presence of anti-CD3 and anti-CD28, respectively, did
not lead to a higher expression of CD80 (3–4%) (Fig. 1Ac).
However, when CD4+ T cells were stimulated for 3
days with anti-CD3 and anti-CD28 without any APCs, 30% of the
purified CD4+ T cells expressed CD80 on their
surface (Fig. 1Ad). These results demonstrated that the
innate level of CD80 expression on the CD4+ T
cells is extremely low. In addition, short-term activation (24 h) of T
cells did not lead to de novo expression of CD80 on
CD4+ T cells; however, long-term activation of T
cells (72 h) with high anti-CD3 (signal 1) and CD 28 (signal 2) can
lead to up-regulation of CD80 in CD4+ T cells.
All additional experiments were conducted with human T cells after
stimulation for 24 h or less. CD80 detection on CD4+ T cells activated with APCs
To investigate whether CD80 can be detected on
CD4+ T cells upon activation with APCs,
elutriated monocytes from healthy donors were cultured for 10 days in
the presence of rhGM-CSF and rhIL-4 (50 ng/ml) to generate DCs that
were used as APCs in these experiments. The phenotypes of the DCs were
checked for the cell surface markers; they were shown to be
CD1a+, HLA-DR+,
CD86+, CD80+,
CD3-, CD14-/low,
CD19-, and CD56-. The purified
CD4+ T cells (from healthy donors) were cultured
for 24 h with DCs expressing CD80 (Fig. 1Be). When DCs
were incubated with CD4+ T cells isolated from
the same individual (autologous CD4+ T cells),
CD80 was detected on 6–37% of purified T cells in the absence of
anti-CD3 (Fig. 2A, each
hatched bar is the representative of the percent of CD4 cells
expressing CD80 from one donor). However, when
CD4+ T cells were cultured in the presence of 1
µg/ml anti-CD3, CD80 was detected on 10–44.5% of
CD4+ T cells (Fig. 2A, black bars). Fig. 2A shows the results derived from autologous DCs and T cells
obtained from four different donors.
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B, each hatched bar is representative of CD4 cells from an
individual donor). In contrast, in the presence of anti-CD3 (as
signal 1), CD80 detection increased from 13.5 to 53% (Fig. 2B, black bars). These results demonstrated that upon
activation of T cells with autologous or allogeneic DCs, CD80 can be
detected on more CD4+ T cells upon TCR
engagement. To exclude the possibility that CD80 expression on T cells
is the result of doublet formation between DCs and T cells, samples
were simultaneously stained not only for CD4 and CD80, but also for
CD1a. As depicted in Fig. 2, C and D, CD4 cells
that express CD80 upon interaction with DCs did not express
CD1a.
To address the question of whether the level of CD80 detection on T
cells is related to the level of CD80 expression on APCs, we used a B
cell line (C1R-A2) stably transfected by the HLA-A2 gene. All C1R-A2
cells expressed a higher level of CD80 on their surface (Fig. 1Bf) as compared with human DCs. CD4+
T cells were incubated with C1R-A2 in the presence or absence of
anti-CD3 (1 µg/ml) for 24 h. Our results demonstrated that
in the absence of anti-CD3, CD80 was detected on 33–71% of
CD4+ T cells from different donors (Fig. 2E, hatched bars). CD80 was detected on a higher number of
CD4+ T cells (33–85%) in the presence of
anti-CD3 (Fig. 2E, black bars). All of the above
experiments (Fig. 2) were conducted with three-color FACS analysis for
CD4+, CD80, and either CD1a for DC detection or
CD19 for C1R2A detection. All
CD4+/CD80+ T cells were
<2% positive for either CD19 or CD1a (an example of this is also
shown in Fig. 6A).
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Effect of cyclohexamide (CHX) on CD80 acquisition
Studies were then conducted to determine whether the detection of
CD80 on human T cells shortly after activation with both APCs and
anti-CD3 is the result of de novo expression of CD80 by the T cell
or the acquisition of CD80 by the T cell from the APC. Thus, human T
cells were incubated with CHX (20 µg/ml for 12 h), an inhibitor
of protein synthesis, before incubation with APCs and anti-CD3.
Using three-color FACS analysis, T cells were analyzed for levels of
CD80 as well as the early activation marker CD69. As seen in Fig. 3A, untreated CD4+ T cells incubated
with anti-CD3 express high levels of CD69. In contrast, CHX-treated
CD4+ T cells express at least 1 log less CD69
after incubation with anti-CD3. When the same cells as seen in Fig. 3A were analyzed for CD80, both untreated and CHX-treated
CD4+ T cells were shown to contain similarly low
levels of CD80 after exposure to anti-CD3 (Fig. 3B).
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C). In contrast, when the CD4+ T
cells (Fig. 3C) were analyzed
for levels of CD80, prior incubation with CHX was shown to have no
effect on either the MFI or the percent of cells positive for CD80
(Fig. 3D). These studies thus provide evidence that the CD80
detected on T cells at times of early activation is not attributable to
de novo protein synthesis of CD80 by T cells, but rather its
acquisition from APCs. Analysis of CD80 mRNA by RT-PCR in activated CD4+ T cells
To demonstrate further that the CD80 detected on
CD4+ T cells after 24 h of activation with
APCs and signal 1 results from acquisition and not endogenous
up-regulation of CD80 mRNA, a RT-PCR study was performed. Purified
CD4+ T cells were incubated with or without APCs
(C1R-A2) in the presence or absence of anti-CD3 for 6 and 24
h. CD4+ T cells were separated from APCs, and
their purity as well as CD80 expression were detected by three-color
FACS analysis. The cells were >98% pure
(CD4+/CD19-). The B cell
line C1R-A2 was used as a positive control for the expression of CD80
mRNA. As shown in Fig. 4A, lane E, purified
CD4+ T cells expressed no mRNA for CD80. Upon
activation of CD4+ T cells with anti-CD3 for
24 h, there was no up-regulation in the expression of CD80 mRNA
(Fig. 4A, lane F). Moreover, there was no
expression of CD80 mRNA in purified T cells that were activated for
24 h with CIR-A2 either in the absence of anti-CD3 (Fig. 4A, lane G) or in the presence of anti-CD3
(Fig. 4A, lane H). In contrast, the mRNA from
C1R-A2 gave a positive amplification signal labeled "positive
control" (Fig. 4A). -actin expression demonstrated that
equal amounts of RNA were used in each PCR. Similar studies were also
conducted to analyze CD80 mRNA expression at 6 h of activation
with APCs and anti-CD3. As shown in Fig. 4A, lanes
A–D, no expression of CD80 mRNA was detected. To establish the
sensitivity of PCR for CD80 detection, a template dilution experiment
was performed on a C1R-A2 cell line. cDNA of C1R-A2 was serially
diluted (1/5- to 1/1200-fold dilution) and used for CD80 amplification
in RT-PCR. The CD80 message could be detected in C1R-A2 (C1R-A2
cDNA at a 0.5-ng level of RNA) diluted 800-fold. These experiments
demonstrated that there was no up-regulation of CD80 mRNA in
CD4+ T cells activated with APCs and anti-CD3
for 6 or 24 h, and that expression of CD80 on
CD4+ T cells is attributable to the acquisition,
and not the up-regulation, of endogenous CD80 mRNA.
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To investigate whether the interaction of CD80 on APCs with its
receptors on T cells is required for CD80 acquisition, a number of
studies were conducted to block CD80 on APC surfaces or CD28 on T cell
surfaces. Thus, to block CD80 on the surface of APCs, C1R-A2 cells were
treated with various concentrations of hCTLA-4 Ig fusion protein or Ig
control protein (as described in Materials and Methods).
These pretreated C1R-A2 cells were then cocultured with purified
CD4+ T cells and anti-CD3 for 24 h.
Using three color-FACS analysis, T cells were then analyzed for
acquisition of CD80. As seen in Fig. 5A, 
70% of T cells
incubated with C1R-A2 cells treated with Ig control acquired CD80 from
C1R-A2. In contrast, CD80 acquisition was significantly reduced by
purified CD4+ T cells that were cocultured for
24 h with anti-CD3 and C1R-A2 cells pretreated with hCTLA-4 Ig
fusion protein (30–40 µg/ml). Upon pretreatment of APCs with high
concentrations of hCTLA-4 fusion protein, the CD80 acquisition by T
cells became almost undetectable (Fig. 5A).
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B, incubation of CD4+ T cells
(pretreated with control Ig protein) with C1R-A2 cells in the presence
of anti-CD3 for 24 h again resulted in the acquisition of CD80
by 70% of T cells. However, when CD4+ T cells
were pretreated with a high concentration of CD80 fusion protein and
then cocultured with C1R-A2 cells in the presence of anti-CD3, CD80
acquisition by T cells was significantly inhibited. This inhibition was
directly related to the concentration of CD80 fusion protein that was
used to pretreat the T cells (Fig. 5B). These experiments
not only demonstrate that CD80 is acquired across the membrane from
APCs by T cells, but also suggest that this acquisition appears to be
possibly mediated through the CD28 receptor.
Further studies were then conducted to determine whether the transfer
of CD80 to T cells is indeed mediated through its receptors. As
summarized in Table I, only 1–2% of
purified CD4+ T cells expressed CD80 on their
surface; activation of these cells with anti-CD3 for 24 h did
not affect the CD80 expression. However, upon incubation of the
CD4+ T cells with C1R-A2 cells in the presence of
anti-CD3 for 24 h, >70% of these T cells acquired CD80 from
APCs. Incubation of CD4+ T cells with pretreated
CIR-A2 cells with control Ig protein in the presence of anti-CD3
also resulted in >70% of T cells acquiring CD80. However, when C1R-A2
cells were pretreated with hCD28 or hCTLA-4 fusion protein and then
cocultured with CD4+ T cells in the presence of
anti-CD3 for 24 h, only 0.3–2.6% of
CD4+ cells acquired CD80. Furthermore, when
purified CD4+ T cells were pretreated with 30
µg/ml CD80 fusion protein (to saturate the CD28 receptors) and
cocultured with C1R-A2 cells in the presence of signal 1 (anti-CD3,
1 µg/ml) for 24 h, the acquisition of CD80 by
CD4+ T cells was reduced from 70 to 12% of
the cells (Table I). To further investigate the role of CTLA-4 on
CD4+ T cells in the acquisition of CD80, we
examined CTLA-4 expression on the surface of the T cells before and
after their activation with APCs (24-h postactivation). CTLA-4
expression remained extremely low over the 24-h time period (2–3%,
data not shown). However, it should be mentioned that the failure to
detect the CTLA-4 on the surface of T cells does not exclude its role
in CD80 acquisition. These results (Fig. 5 and Table I) collectively
demonstrate that saturation of CD80 molecules on APCs by hCD28 or
hCTLA-4 fusion proteins or blocking of CD28 molecules on T cells by
CD80 fusion protein inhibits the CD80/CD28 interaction and results in a
significant reduction in the acquisition of CD80 by
CD4+ T cells. Moreover, these experiments further
demonstrate that CD80 acquisition is not a passive and/or random
process and is not due to de novo expression of CD80.
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To investigate the potential biological role of CD80 acquisition
by CD4+ T cells, freshly purified
CD4+ T cells from healthy donors (which express
very low levels or almost no CD80) were incubated with 1 µg/ml
anti-CD3 and CIR-A2 cells to acquire CD80. After 24 h,
CD4+ T cells were separated from APCs (by column
purification) and analyzed by three-color FACS analysis for
CD4+, CD80, and CD19. As shown in Fig. 6A, the purified
CD4+ T cells were CD19-,
which demonstrates their lack of APC contamination. Moreover, >70% of
the CD4+ T cells had acquired CD80 and expressed
MHC class II molecules (Fig. 6, B and C). To
establish the role of CD80 acquisition in T cell interactions,
CD4+ T cells that had acquired CD80, and were
purified as demonstrated in Fig. 6, were used in proliferation assays.
As demonstrated in Fig. 6D, lane A, purified
CD4+ T cells (that had not acquired CD80) showed
very little proliferation after 48 h. In comparison, when purified
CD4+ T cells that had acquired CD80 (as shown in
Fig. 6B) were cultured for 48 h in the absence of
anti-CD3, there was a significant proliferative response (Fig. 6D, lane B). This proliferation may be due to the
acquisition or up-regulation of peptide MHC complexes (as depicted in
Fig. 6C and Ref. 11) when CD80 acquisition
occurs; it may also be due to cross-presentation to neighboring cells.
The addition of anti-CD3 to the culture caused a slight enhancement
in their proliferative response (Fig. 6D, lane
C). When CD4+ T cells that had acquired CD80
were cultured in a medium containing 10% dextran (used to separate the
cells and reduce their cell-cell interaction without affecting their
characteristics), in the absence of anti-CD3, proliferative
responses of CD4+/CD80+ T
cells were significantly reduced (Fig. 6D, lane
D). Similarly, proliferative responses of purified
CD4+/CD80+ that were
cocultured with anti-CD3 in medium containing 10% dextran were
reduced significantly (Fig. 6D, lane E). By
adding dextran, a physical space between neighboring (bystander) cells
is created, thereby reducing the T cell to T cell interaction; this
leads to a decrease in proliferative response in CD80-acquired
CD4+ T cells.
These results demonstrated that CD4+ T cells, upon acquisition of CD80, are able to positively signal to themselves and other neighboring T cells in the presence or absence of signal 1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous studies, we had demonstrated that high affinity TCR-transgenic murine T cells were able to acquire CD80 (12). In this study, for the first time, we demonstrated that naive human CD4+ cells from normal donors can do the same. Moreover, we demonstrated that CD80 acquisition takes place in early activation stages of human T cells; however, long-term activation of T cells can lead to endogenous up-regulation of CD80. Using dextran studies, for the first time, we demonstrated that there is cross-presentation to other T cells. The CD4+/CD80+ cells that were treated with dextran also demonstrated a low level of proliferation, thereby suggesting that T cells may also continue to signal to activate themselves.
In the past few years, the mechanisms by which T cells migrate from the blood to secondary lymphoid organs, interact with APCs, and either become tolerant or acquire effector and/or memory function are now beginning to be understood. In this dynamic world of cognate cell-cell interaction, it is likely that, at several different stages, CD80 family members play crucial roles. The interaction of the CD80 molecules with CD28 generates costimulatory signals amplifying TCR signaling and preventing anergy, whereas interaction with CTLA-4 induces powerful inhibitory signals in T cells (20, 21, 22, 23, 24). Previously, it was reported that T cell-APC contact is followed by a down-regulation of TCR (25, 26) and that T cell-APC interaction can cause APC-derived surface molecules to adhere to the surface of T cells (27, 28). Recently, our laboratory as well as others have demonstrated the acquisition of CD80 molecules by naive and memory murine CD4+ T cells via direct transfer of these molecules at the site of interaction through CD28 receptors (10, 12). We further demonstrated that upon acquisition of CD80, CD4+ T cells from mice could act as an APC to stimulate other T cells, thus amplifying an immune response. This mechanism of T cell to T cell costimulation could facilitate the regulation of the immune response in murine models. The question then arises whether human T cells behave in a manner similar to that of their mice counterparts and, if so, what are the immunoregulatory consequences of acquiring costimulatory molecules in the human immune system? Indeed, all previous reports (17, 18, 19, 29, 30, 31, 32, 33) describing the presence of CD80 on human T cells reported the "expression" of CD80 on T cells, and none have described its acquisition from APCs.
The results of this study clearly demonstrated that human T cells are capable of acquiring CD80 from APCs in the early stages of T cell activation. A previous study by Azuma et al. (17) demonstrated that T cell clones coexpress both CD28 and CD80 simultaneously. Similarly, studies by Wyss-Coray et al. (34) demonstrated that cloned T cells activated with APCs and rested for various times express CD80 at 7–9 days postactivation. However, it should be pointed out that these T cells were stimulated in the presence of APCs for long period of times. Therefore, it is conceivable that CD80 expression on these cells was indeed due to the up-regulation of endogenous CD80, but it also could have been at least partially attributable to the acquisition of CD80 from APCs. To determine whether the freshly isolated CD4+ T cells (in the absence of APCs) were able to up-regulate CD80 expression, we stimulated purified CD4+ T cells with anti-CD3, or with a combination of anti-CD3 and anti-CD28, for 24 and 72 h. The FACS analysis showed no significant change in the level of CD80 expression on CD4+ T cells (<5%) at 24 h. Indeed, after 3 days of very potent stimulation, 30% of CD4+ T cells expressed CD80. These results taken together demonstrated that with a longer stimulation time, CD4+ T cells can indeed up-regulate CD80 to some degree; however, the level of expression was never as high as it was observed in the early stages of T cell activation in the presence of APCs and signal 1 (TCR engagement). Therefore, endogenous CD80 up-regulation on CD4+ T cells seems to be a late event during T cell activation. It should be pointed out that in our experiments we have used primary CD4+ T cells (as opposed to T cell clones) and examined early activation events (within the first 24 h) compared with longer time periods in previous studies (17, 18, 19). Therefore, our results in conjunction with these previous studies indicate two different mechanisms for the acquisition/expression of CD80 on the surface of T cells. One mechanism, the acquisition of CD80 molecules, may play a role during the early stages of activation of T cells and APCs; the second mechanism, endogenous up-regulation of CD80, may play a role at the late stages of activation (days after the activation of T cells) and involve up-regulation of endogenous CD80 in T cells.
In this study, the results of proliferation assays (using dextran to
create physical spaces between T cells and separate them from
neighboring cells) for the first time demonstrated that human T cells,
upon acquisition of CD80, can positively signal to themselves, thereby
directly inducing T cell proliferation. The data presented in Figs. 2 and 6 also rule out the possibility of contamination of purified T
cells by APCs. In addition, the study showed that
CD4+/CD80+ T cells, upon
acquisition of CD80, also expressed MHC class II and could positively
signal the neighboring T cells, causing a significant increase in the
proliferative activity of these cells. Moreover, it is important to
point out that data from proliferation assays clearly demonstrate that
CD80 molecules upon acquisition are still functional and acquisition of
these costimulatory molecules is not a static event. Previous studies
had suggested that T cells express CD80 and can function as APCs. In
our studies, for the first time, we demonstrate that
expression of CD80 in the human T cells—at least at early
activation stages—is due to the acquisition of CD80; this
acquisition is what leads to T cells functioning as APCs. These data
might suggest that CD80 acquisition by T cells may play a role not only
in self-sustained signal pathways, but perhaps in sending a positive
signal to adjacent T cells. Recent studies have demonstrated a similar
concept in HIV infection (35). It has been shown that the
newly formed HIV virions contain cellular host constituents. Indeed,
cell membrane proteins, such as MHC class II, ICAM-1, and LFA-1, were
found to be acquired by the retrovirus (35). Also, it was
demonstrated that several of these virion-acquired proteins have
functional effects on the biology of HIV-1 and play a key role in the
attachment process of HIV-1 to its target. Furthermore, Bounou et al.
(36) demonstrated that host-derived costimulatory
molecules, such as CD80 in virions, are not only functional but are
important in HIV-1 signal transduction pathways.
It has been speculated that CD80 expression on human T cells in PBMC of HIV patients, or on T cells from synovial fluid of rheumatoid patients or peritoneal fluid of cancer patients, is attributable to the up-regulation of CD80 (18, 19, 32, 37). In this study, for the first time, we demonstrated that acquisition of CD80 from APCs is indeed the mechanism for CD80 detection on T cells at the early stages of T cell activation. The question then arises: what is the physiological relevance of T cells acquiring costimulatory molecules in various pathological diseases? One may speculate that the T cells that had acquired CD80 may bring about unrestricted activation of bystander T cells, leading to a highly activated immune system. In HIV studies, this phenomenon may lead to HIV replication, which requires activated T cells, and the progression of the disease. In contrast, in cancer studies, the acquisition of CD80 by human T cells and their ability to activate bystander T cells may allow the potentiation of immune responses to tumor Ags. Therefore, depending on the immunological setting, CD80 acquisition may lead to different outcomes.
In conclusion, the studies reported here demonstrated for the first time that CD4+ T cells can acquire CD80 upon activation with autologous or allogeneic APCs. Furthermore, we demonstrated that CD80 acquisition by CD4+ T cells is mediated through CD28 receptors and plays an important role in the activation of the cells that have acquired these molecules, as well as adjacent T cells. The acquisition of CD80 by T cells might play a role in the regulation of immune responses to pathogens and/or in diseases of the immune system. Our laboratory is currently investigating the mechanism that may play a role in this phenomenon.
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2 Abbreviations used in this paper: DC, dendritic cell; CHX, cyclohexamide; MFI, mean fluorescence intensity; rh, recombinant human.
Received for publication May 13, 2002. Accepted for publication September 27, 2002.
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)
or presence (
) of 1 µg/ml anti-CD3 for 24 h.
B, Detection of CD80 on CD4+ T cells from
five different donors cultured with allogeneic DCs in the absence (
