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*
Laboratory of Tumor Immunology and Biology, Division of Basic Sciences, National Cancer Institute;
Metabolism Branch, Division of Clinical Sciences, National Cancer Institute; and
Laboratory of Immunology, 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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Various studies have reported that CD80 can also be detected on activated human T cell clones, T cells activated repeatedly in vitro, and T cells that have infiltrated skin lesions and are in rheumatoid synovial fluid (6, 7, 8, 9, 10, 11). Studies by Kochli et al. that analyzed the expression of CD80 and MHC complex class II molecules on circulating T cells of HIV-infected individuals have suggested that these T cells develop an Ag-presenting phenotype by up-regulating expression of HLA and CD80 (12). Moreover, Wolthers et al. (13) reported increased levels of CD80 expression on both CD4+ and CD8+ cells in HIV-infected patients and revealed that these T cells with accessory cell properties can costimulate proliferation in purified responder T cells from healthy control donors. Further studies of lupus patients have demonstrated that these individuals have increased amounts of CD80 on PBLs, including T cells (14). However, although all of these studies reported increased levels of CD80 molecules on T cells, none of the studies have actually demonstrated an endogenous up-regulation of CD80 molecules on T cells (6, 7, 8, 9, 10, 11, 12, 13, 14).
In parallel murine studies, the amount of CD80 on T cells was reported to vary with the state of activation. Resting T cells displayed little or no CD80; however, upon activation, CD80 levels on the cell surface increased concomitantly, peaking at 72 h (15). In a recent study, Weintraub et al. (16, 17) demonstrated that although splenic T cells from normal B6 mice were negative for CD80, freshly isolated T lymphocytes from the spleens of autoimmune B6/gld and B6/lpr mice showed higher levels of CD80. This phenomenon was age dependent and may have contributed to lymphoproliferation and autoimmunity. Although these studies mentioned "increases in CD80 expression in T cells," no data were actually presented in these studies as to whether these increases were due to up-regulation of CD80 in T cells or acquisition of CD80.
Previous studies have demonstrated that T cells and thymocytes can acquire MHC class I and class II determinants from other cells present in the local environment (18, 19). Furthermore, it has been demonstrated that double-negative thymocytes can passively acquire a CD4+/CD8+ determinant from other cells in the thymus that are actively expressing their CD4+/CD8+ genes (20). A recent study by Huang et al. (19) demonstrated that when peptide-specific T cells interacted with APCs, MHC molecules on APCs formed clusters at the site of T cell contact. Afterward, these clusters were acquired by T cells and internalized through TCR endocytosis. These studies also showed that the presence of APC-derived peptide-MHC molecules on the T cell surface could make these T cells susceptible to lysis by neighboring T cells. Moreover, it has been reported recently that the activation of T cells with Drosophila cells as APCs may result in the rapid transfer of MHC molecules and other costimulatory molecules, such as CD80, from APCs to T cells (21).
Although the concept of molecule acquisition by T cells originated 20 years ago, the actual interactions of receptor ligands and the factors affecting ligand acquisition still remain unclear. In this regard, we analyzed CD80 levels on murine T cells upon stimulation with various APCs expressing different levels of CD80 on their surface. Although CD80 was previously thought to be endogenously up-regulated, results of studies of CD80/CD86 double-knockout (B7dKO) mice reported here demonstrate that CD80 is physically acquired by T cells from APCs shortly after T cell activation. We also demonstrate here that CD80 acquisition by T cells is mediated by the TCR and its CD28 ligand (confirming the recent data by Hwang et al. 21) and that the level of CD80 acquisition by T cells is related to both the level of CD80 expression on APCs and the strength of signal 1. Moreover, our data for the first time suggest that acquisition of CD80 by T cells might be of importance for the Ag-presenting capacity of T cells. Thus, these findings might have important implications for a further understanding of immune regulation and the pathogenesis of immunopathological diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6, B10.A, and CD28KO mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. B7dKO mice were provided by R. Hodes (National Institute on Aging, National Institutes of Health, Bethesda, MD) and were originally obtained from A. Sharpe (Harvard University, Boston, MA). Pigeon cytochrome c88–104-transgenic (PCC/TCR-Tg) (5CC7 TCR-Tg) mice, which are TCR transgenic for PCC, were obtained from Taconic Farms (Germantown, NY) and bred in the National Cancer Institute animal facility under pathogen-free conditions. PCC/TCR-Tg IL-2 green fluorescence protein (GFP)ki (PCC/I-Ek-specific) mice were provided by M. Naramura (National Institute of Allergy and Infectious Diseases, National Institutes of Health).
mAbs and flow cytometry
To evaluate CD80 expression on T cells, cell suspensions were stained with directly conjugated mAbs (anti-CD4PE; anti-CD80 FITC). The proportion of CD4+ cells expressing CD80 was determined by gating on CD4+ T cells, excluding the dead cells. Fluorochrome mAb Abs to CD4, CD80, MHC class II I-Ek, CD28, and purified anti-CD3, anti-CD80, hamster IgG, and anti-CD28 Abs were purchased from PharMingen (San Diego, CA), and samples were analyzed with a FACSCaliber (Becton Dickinson, Mountain View, CA) using CellQuest software for the Macintosh.
APCs
DCs were prepared as described previously (22). Briefly, bone marrow cells from 6- to 8-wk-old mice were depleted of lymphocytes using a mixture of magnetic beads specific for CD4, CD8, MHC class I, and MHC class II (MiniMACS, Miltenyi Biotec, Auburn, CA). Cells were incubated in six-well plates (5 x 106 cells/well) with medium supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4 (R&D Systems, Minneapolis, MN). Cells were replated in fresh cytokine-supplemented medium on days 2 and 4. On day 6, cells were harvested for infection and analysis. The following cell lines were provided by R. Germain (National Institute of Allergy and Infectious Diseases, National Institutes of Health): fibroblast cell line DCEK, which expressed a low level of CD80; MHC class II I-Ek and 13.9 fibroblast cell line, which expressed a high level of CD80; and COS (monkey-kidney) cells, which expressed MHC class II I-Ek. Murine colonic adenocarcinoma cells (MC38) and the retrovirally transduced MC38/CD80 cell line have been described previously (23). A20 cells are murine B cell lymphoma cells expressing 37% CD80 on their surface.
Separation of APCs by beads
Fibroblast cells (20 x 106) were cultured with 250 µl of goat anti-mouse Dynabeads (Dynal, Lake Success, NY) in the presence of various concentrations of peptide. After 2 days, the fibroblasts that had absorbed the beads were separated by a magnet. These cells were then used as APCs (2 x 105 cells) and cultured with 1 x 106 effector/memory T cells. The APCs were separated from T cells 24 h later by a magnet. This procedure yielded a preparation of T cells devoid of any APCs.
Recombinant fowlpox viruses and infection of DCs
Recombinant fowlpox virus expressing CD80 (rF-CD80) and wild-type fowlpox (FP-WT) have been described previously (24). DCs were harvested on day 6 of culture and washed with OptiMem (Life Technologies, Gaithersburg, MD). The cells (1.2 x 106/ml) were infected with FP-WT or rF-CD80 at 50 MOI (multiplicity of infection; PFU/cells) for 2 h at 37°C. Cells were first washed and then incubated in condition medium at 37°C overnight. After 18 h, DCs were harvested for in vitro assays.
Peptide
The I-Ek-restricted PCC peptide was synthesized and HPLC-purified by American Peptide (Sunnyvale, CA).
Cell preparation and culture
CD4+ cells were purified using microbeads conjugated to anti-CD4 mAb, according to the manufacturer’s instructions (MiniMACS, Miltenyi Biotec), on MACS columns. Isolated CD4+ cells (5 x 105 cells/ml) were cultured with APCs (1 x 105 cells/ml) with or without signal 1, with either anti-CD3 Ab or PCC peptide (at concentrations indicated in each figure legend), in RPMI 1640 supplemented with the following: 10% heat-inactivated FBS, 5 x 10-5 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 µM/ml streptomycin, and 10 mM HEPES (all obtained from Life Technologies, Gaithersburg, MD), and 15 µg/ml gentamicin (BioWhittaker, Walkersville, MD).
Effector/memory CD4+ T cells
Effector/memory CD4+ T cells were generated by activating naive splenic CD4+ T cells from PCC/TCR-Tg mice in culture with 10 µg/ml PCC peptide for 3 days. Following centrifugation with Ficoll gradient, the live cells were washed and then rested for 4–6 days in six-well plates with 5 IU/ml of murine IL-2 medium. The cells were washed and given IL-2 every 3 days following the 3-day PCC activation. The effector/memory CD4+ cells were analyzed for markers as follows: FITC-conjugated CD62 ligand, PE-conjugated CD44, FITC-conjugated CD45RB, and FITC-conjugated CD69. Appropriate isotype control was used for all of the markers.
Apoptosis assay
Effector/memory CD4+ cells were cultured for 24 h in the presence of various concentrations of peptide as described in Results. Apoptosis was assessed using the TUNEL kit (PharMingen) according to manufacturer’s instructions.
RNA extraction and PCR
RNA was prepared using an RNA STAT-60 kit (Tel-Test,
Friendswood, TX), and 2 µg of total RNA was used to synthesize cDNA
with the SuperScript Preamplification System (Life Technologies,
Gaithersburg, MD), using oligo(dT) according to the manufacturer’s
instructions. Oligonucleotides specific for CD80 and 
-actin were
prepared by Cruachem (Dulles, VA) and Invitrogen (Carlsbad, CA).
Primers for CD80 (517-bp fragment) were 5'-ATCCAGGATACACCACTCC-3'
(sense) and 5'-TCCAACCAAGAGAAGCGAGG-3' (antisense). Primers for
-actin (1-kb fragment) were 5'-GCTCACCATGGATGATGATATCGC-3' (sense)
and 5'-GGAGGAGCAATGATCTTGATCTTC-3' (antisense). cDNA samples were
subjected to amplification using PCR (model 9600; Perkin-Elmer Cetus,
Norwalk, CT). Samples were initially denatured for 2 min at 94°C.
CD80 and -actin-specific amplification was conducted for 25 cycles,
where each cycle consisted of denaturation at 94°C for 30 s,
annealing at 55°C for 30 s, and polymerization at 72° for
30 s, followed by a finishing step at 72° for 10 min. Samples
(20 µl) were resolved on 1% agarose gels in Tris-borateEDTA
(TBE), electrophoresed at 100 V, and stained with ethidium bromide; the
cDNA fragments were visualized directly by UV light.
GFP-CD80 fusion protein
A cDNA clone of murine CD80 was cloned and sequenced as
described previously (25). Restriction endonuclease
digestion of this clone with Kpni and Apai released a 1-Kb fragment
that was gel purified and ligated into the Kpni/Apai sites of pEGFP-C1,
the C-terminal protein-fusion expression vector (Clontech Laboratories,
Palo Alto, CA). DH5
Escherichia coli cells were
transformed with this construct (Life Technologies, Rockville, MD), and
kanemycin-resistant colonies were selected and sequenced for CD80
expression. pEGFP-CD80 DNA was CsCl-banded and subsequently used to
transfect COS cells for confocal studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the innate expression of CD80 on naive T cells, CD4+ cells from four strains of mice were analyzed by two-color FACS analysis. T cell expression of CD80 is a relatively rare occurrence in these strains, as determined by immunofluorescence and flow cytometry. CD80 expression levels on T cells varied among the different strains of mice, ranging from 2 to 9% in C57BL/6 mice, to very small (1–2%) or undetectable levels in B7dKO, CD28KO, PCC/TCR-Tg mice.
Previous studies have reported that activated T cells express CD80
molecules on their surface 3–7 days after activation
(15). However, none of these studies used B7dKO mice as
the source of T cells. CD4+ naive T cells from
B7dKO mice were used to investigate whether CD80 expression on T cells
shortly after activation is induced endogenously or acquired from APCs.
Splenocytes from B7dKO mice lack functional genes for both CD80 and
CD86; upon activation, these cells are unable to express CD80 or CD86
on their surface (26). CD4+ cells
from B7dKO mice were incubated with 10 µg/ml of anti-CD3 as
signal 1 and A20 cells (a B cell line expressing 37% CD80) as APCs.
The T cells were monitored for 24 h. As shown in Fig. 1
A, CD4+
cells from B7dKO mice did not express CD80; levels were similar to
isotype control. Activation of B7dKO CD4+ cells
with 10 µg of anti-CD3, in the absence of APCs (A20 cells), did
not change CD80 expression (Fig. 1B). As a result of
incubating B7dKO CD4+ cells with A20 cells in the
absence of signal 1, 4% of CD4+ cells acquired
CD80 (Fig. 1C). However, when CD4+
cells were incubated with A20 cells as APCs in the presence of
anti-CD3 (10 µg/ml) as signal 1, 30% of the
CD4+ cells from the B7dKO mice acquired CD80
(Fig. 1D).
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). However, in the presence of
signal 1 (10 µg/ml anti-CD3), 55–57% of
CD4+ cells acquired CD80 on their surface (Table I). When B7dKO CD4+ cells were incubated with
rF-CD80-infected DCs, the CD4+ cells acquired
more CD80 in both the absence (53%) and presence (87%) of signal 1
(Table I). These results using T cells from B7dKO mice further
demonstrate that CD80 levels on T cells after a short period of
activation are due to the acquisition of CD80 from APCs, and not the
constitutive expression and/or up-regulation of endogenous CD80.
Moreover, these results demonstrate that both the presence of signal 1
and the level of CD80 expression on APCs influence this phenomenon.
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CD80 acquisition by T cells raises the question of the role of
CD28 in this process. To examine the role of the CD28 molecule in
acquiring CD80, CD4+ cells from CD28KO
(CD28-/-) mice and normal C57BL/6 mice were
incubated for 24 h with MC38/CD80 cells; MC38/CD80 are
retrovirally transduced murine carcinoma cells expressing CD80. These
experiments were conducted either in the presence or absence of
anti-CD3 (10 µg/ml) as signal 1. As shown in Table II, T cells from CD28KO mice did not
acquire CD80 upon incubation with MC38/CD80 in the absence or presence
of signal 1 (1–2% CD80 acquisition). However, 19% of
CD4+ cells from C57BL/6 mice (expressing
constitutively high levels of CD28) did acquire CD80 upon stimulation
with MC38/CD80 in the presence of signal 1. These studies demonstrate
that the acquisition of CD80 by T cells is mediated through the CD28
ligand and not by way of a random process. Experiments were also
conducted to show that the ligand on resting T cells for CD80
acquisition is not CTLA-4. T cells from PCC/TCR-Tg mice were incubated
with fibroblasts expressing CD80 in the presence and absence of
anti-CTLA-4 Ab. In both cases, there was no reduction in the
acquisition of CD80 by T cells (data not shown).
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To investigate the physical nature of the CD80 binding to the
surface of T cells, COS (monkey-kidney) cells expressing
I-Ek MHC class II were transfected with a
construct containing GFP-CD80. As determined by FACS analysis, 30% of
COS cells expressed membrane-bound GFP-CD80 fusion protein (data not
shown). Confocal microscopy studies of COS cells demonstrated that when
these cells were transfected with GFP-CD80 construct, CD80 was
expressed on cell surfaces (Fig. 2A). To further demonstrate
cell surface expression of CD80, these cells were double-stained with
PE-anti-CD80. Superimposition of red PE on GFP led to the
appearance of a yellow/orange color on cell membranes (Fig. 2, B–D). When PCC/TCR-Tg CD4+ cells were
incubated with transfected COS cells in the presence of PCC peptide (10
µg/ml) as signal 1, T cells were shown to acquire CD80 molecules
(Fig. 2, E–H). These results further demonstrate the
physical acquisition of CD80 by T cells.
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Recent studies have demonstrated the existence of a new homolog of the B7 family of molecules (27). This observation raises the possibility that there might be a CD80 homolog that can be endogenously up-regulated rapidly on T cells upon stimulation, and that the molecule might interact with the anti-CD80 Ab. To exclude this scenario and further rule out the de novo generation of CD80 in short-term activated T cells, CD4+ cells from PCC/TCR-Tg mice were treated with the protein synthesis inhibitor CHX (20 µg/ml) for 2 h. After CHX treatment, CD4+ cells were incubated with irradiated fibroblast cells expressing high levels of CD80 and I-Ek MHC class II molecules for 4 h and for 24 h. There were no significant differences in CD80 acquisition between the CHX-treated cells and the untreated CD4+ cells at either 4 h (22% for CHX-treated and 30% for untreated cells) or 24 h (99% acquisition of CD80 for both treated and untreated cells). These results further indicate that CD80 expression on T cells upon activation is indeed due to acquisition, rather than up-regulation, of CD80 or any other homolog of the B7 family.
Analysis of naive and activated CD4+ T cells for CD80 mRNA by PCR
To further demonstrate that CD80 is acquired by T cells upon
activation, and not by the endogenous up-regulation of CD80 mRNA, PCR
studies were performed (Fig. 3). Purified
CD4+ cells of PCC/TCR-Tg mice were incubated with
fibroblast cells expressing high levels of CD80 (Fig. 4C) and 10 µg/ml of PCC
peptide as signal 1. After 24 h of incubation,
CD4+ cells were carefully removed from
supernatant fluid and cultured two additional times (each time
recovering cells in the supernatant) to ensure that no fibroblast (an
APC that expresses CD80) was contaminating the T cell population.
Purity of CD4+ cells, as well as the expression
of CD80, was checked by FACS analysis. The cells were 100%
CD4+, and 79–80% of these cells had acquired
CD80 on their surface (Fig. 4D). Simultaneously, as
controls, naive PCC/TCR-Tg CD4+ cells were also
purified from PCC/TCR-Tg animals and were checked for CD80 expression
by FACS. These cells had very little or no CD80 on their surface. Using
RT-PCR, we then analyzed the expression of CD80 mRNA on unstimulated
PCC/TCR-Tg CD4+ cells or on
CD4+ cells that were stimulated for 24 h. As
shown in Fig. 3, a 517-bp CD80 fragment could readily be amplified from
the APC fibroblast cell line that expresses CD80 (positive control,
lane B). However, CD80 could not be amplified from two
different preparations of unstimulated CD4+ cells
(Fig. 3, lanes C and D) or
CD4+ cells that were stimulated for 24 h
(Fig. 3, lane E). -actin expression demonstrated that
equal amounts of RNA were added in each PCR. To establish the
sensitivity of detection of CD80 in PCR, a template dilution experiment
was performed on a CD80-transfected fibroblast cell line. cDNA encoding
CD80 was synthesized using 2 µg of RNA from transfected fibroblasts;
serial dilutions of cDNA (1:10- to 1:320-fold dilutions) were used as a
template for PCR amplification. Analysis of the product on an agarose
gel revealed that the DNA bands could not be detected when the template
was diluted >1:160-fold, which is 
1 ng of RNA. Therefore, we can
conclude that the T cells that have been activated and acquired CD80
express 1 ng of CD80 RNA. These results illustrate that upon
activation of CD4+ cells, there is no
up-regulation of detectable levels of mRNA for CD80 at the 24-h time
point; a 24-h time point was used in the above studies to detect the
acquisition of CD80.
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Studies were undertaken to investigate whether increased density
of CD80 on APCs might further stabilize the interaction and, therefore,
lead to more acquisition of CD80 by CD28 on T cells. To address this
question, purified CD4+ cells of PCC/TCR-Tg mice
were incubated for 24 h with fibroblast cells expressing either
relatively low levels of CD80 (Fig. 4
A) or high levels of
CD80 (Fig. 4C) and PCC peptide (10 µg/ml) as signal 1.
CD80 acquisition on T cells was proportional to CD80 expression by
APCs. Although CD4+ cells stimulated with
fibroblast cells expressing a low level of CD80 acquired much less CD80
on their surface (26%, Fig. 4B), 79% of
CD4+ cells that were incubated with fibroblast
cells expressing a high level of CD80 acquired CD80 (Fig. 4D).
To further determine whether this level of CD80 on APCs influenced CD80 acquisition by T cells, purified CD4+ cells from PCC/TCR-Tg mice were incubated in the presence of PCC peptide (10 µg/ml) with either uninfected or rF-CD80-infected DCs. Normal DCs express very high levels of CD80 (75%); DCs infected with rF-CD80 showed 90% CD80 expression. Incubation of CD4+ cells with uninfected DCs led to 35% of CD4+ cells acquiring CD80 on their surface. Moreover, CD4+ cells incubated with rF-CD80 DCs obtained markedly higher levels of CD80 (81%) on their surface.
Studies were then conducted to investigate T cell selectivity in the acquisition of CD80 molecules; toward this goal, the fate of another surface marker, CD40, on APCs was investigated in terms of acquisition by T cells. CD4+ cells were incubated with either normal DCs or DCs infected with FP-WT or rF-CD80 in the presence or absence of various concentrations of PCC peptide as signal 1. FACS analysis was performed on gated T cells, based on size and cell surface markers for T cells. An average of 30–40% of DCs express CD40 on their surface, whereas only 3–4% of T cells express CD40. Incubation of CD4+ cells with APCs in the presence of different concentrations of signal 1 PCC peptide led to little or no increase in the acquisition of CD40 by CD4+ cells, which remained 3–4% positive for CD40. These results further demonstrate that CD80 expression on T cells is not due to contaminating APCs, and that CD4+ cells show selectivity in the acquisition of molecules from APCs.
To further investigate the selectivity in CD80 acquisition, naive
CD4+ cells from PCC/TCR-Tg mice were incubated
with a fibroblast cell line expressing high levels of CD80 and PCC
peptide (10 µg/ml) as signal 1 for 24 h (as described
previously) in the presence of CD8+ cells from
non-Tg mice (B10.A mice). As shown in Fig. 4E, non-Tg
CD8+ cells acquired relatively little CD80 (18%)
above their 6–7% normal level of expression, whereas PCC/TCR-Tg
CD4+ cells acquired high levels of CD80 (77%,
Fig. 4F). These results further demonstrate that CD80
acquisition by T cells is specific and not due to random acquisition or
incorporation.
CD80 acquisition by T cells is directly related to the strength of signal 1
Naive CD4+ cells from PCC/TCR-Tg mice were
incubated with fibroblast cells expressing low and high levels of CD80
in the presence of various concentrations of PCC peptide to investigate
the effect of the concentration of signal 1 on T cell acquisition of
CD80. Acquisition of CD80 by CD4+ cells upon
stimulation with fibroblast cells expressing a low level of CD80 was
shown at the high level of signal 1 (Fig. 5A). When PCC/TCR-Tg
CD4+ cells were stimulated with fibroblast cells
expressing high levels of CD80, a direct relationship between the
strength of signal 1 and the acquisition of CD80 by T cells could be
more clearly defined (Fig. 5B). These results further
demonstrate that the acquisition of CD80 by T cells can be influenced
by the level of signal 1.
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To investigate the biological consequence of CD80 acquisition by
effector/memory T cells, effector/memory CD4+
cells were generated from naive CD4+ cells, as
described above, and then cultured with fibroblasts expressing high
levels of CD80 as APCs in the presence of various concentrations of PCC
peptide for 24 h. Before incubation with APCs, 8–9% of effector
T cells were positive for CD80 expression, as compared with 1–2% of
naive T cells (data not shown). When effector/memory
CD4+ cells were cultured with fibroblasts
expressing high levels of CD80 as APCs, 
40% of the cells acquired
CD80. This is in contrast to the relatively low percent CD80
acquisition by naive CD4+ cells when cultured
with the same APCs expressing high levels of CD80 (Fig. 6). Based on our observations, the
probable reason for this difference between effector/memory and naive T
cell acquisition is that effector/memory T cells express higher levels
of CD28, the CD80 ligand, than naive T cells. Indeed, the
effector/memory CD4+ cells from PCC/TCR-Tg mice
used here expressed much higher levels of CD28 than naive T cells from
these mice (70% for memory vs 32% for naive). When both naive and
effector/memory CD4+ cells were cultured with
APCs (expressing high amounts of CD80) and various concentrations of
peptide, the effector/memory cells always acquired more CD80 at a given
peptide concentration (Fig. 6). At a peptide concentration of 0.1
µg/ml, 90% of naive T cells acquired CD80; no cell death was
observed in these T cells.
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To determine whether the cell death in effector/memory T cell
populations (as shown in Fig. 6) was due to apoptosis, effector/memory
CD4+ T cells were generated by activating naive
cells with PCC peptide for 3 days and then resting them with IL-2 for 4
days. These effector/memory cells were then divided into two groups.
Cells in the first group were cultured alone with various
concentrations of PCC peptide for 24 h. As shown in Fig. 7A–C, these cells
demonstrated minimal apoptosis when cultured with or without peptide.
The second group consisted of effector/memory T cells that were
cultured with APCs (fibroblasts expressing high amounts of CD80) in the
presence of 0.001 µg/ml of PCC peptide for 24 h. Approximately
80% of these T cells acquired CD80 (as shown in Fig. 6), and the T
cells were then separated from the fibroblast APCs by magnetic beads
and cultured further in the presence of various concentrations of PCC
peptide (i.e., 0, 0.001, or 0.1 µg/ml) for 24 h. Upon
acquisition of CD80, 34% of these effector/memory T cells underwent
spontaneous apoptosis (Fig. 7D) in the absence of exogenous
peptide. Although the addition of 0.001 µg/ml PCC peptide had minimal
effect (Fig. 7E), the addition of 0.1 µg/ml of PCC clearly
augmented the occurrence of apoptosis of CD4+ T
cells (Fig. 7F). To further ascertain that the acquisition
of CD80 caused the effector/memory cells apoptotic death, CD80 Ab was
included in the assay, with an even higher concentration (1 µg/ml) of
peptide as signal 1. As seen in Fig. 8A and B, isotype
control Ab showed no inhibitory effects on the apoptotic death of these
CD4+ T cells, whereas effector/memory
CD4+ T cells treated with anti-CD80 Ab
demonstrated a meaningful decrease in their apoptosis (46%, Fig. 8C). Based on these results, we propose that CD80
acquisition can in part lead to apoptosis of effector/memory cells
and the level of apoptosis is enhanced with increased levels of
signal 1.
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Studies were then conducted to determine the biologic consequence that results when naive T cells acquire CD80. We asked specifically whether T cells that have acquired CD80 can now function as APCs to naive CD4+ T cells. To this end, IL-2 production was measured as an indicator of T cell activation. However, one cannot simply measure IL-2 levels in the supernatant, because IL-2 can be produced not only from T cells functioning as APCs but also from T cells receiving the costimulatory signal, if any. We took advantage of an IL-2-GFPki mouse (28) in which the IL-2 exon from one of the IL-2 alleles was replaced by GFP. In these mice, IL-2 production can be monitored by cell-associated (of the producer cell) fluorescence from GFP expression driven by the endogenous IL-2 promoter.
PCC/TCR-Tg CD4+ cells that have acquired CD80
were prepared in the manner described above. Fibroblasts were used as
APCs of choice to minimize the possibility of contamination of
CD4+ cells that had acquired CD80 from
professional APCs because they can easily be separated from T cells by
magnetic bead separation. Isolated CD4+ cells
(2–3% expressing CD80 spontaneously) were irradiated and used as
control APCs. In the absence of PCC peptide, these control APCs were
unable to activate the responding T cells (Fig. 9A); in the presence of PCC
peptide (10 µg/ml), they could activate 16% of
PCC/TCR-Tg/IL-2-GFPki CD4+
cells, possibly due to the spontaneous expression of CD80 (Fig. 9B). In contrast, CD4+ cells that had
acquired CD80 from fibroblasts were much more potent as stimulators of
PCC/TCR-Tg/IL-2-GFPki cells to produce IL-2 at 24 h in
the absence (Fig. 9C) or presence (Fig. 9D) of
peptide. The possible reason for this will be discussed below. To
further demonstrate that CD80 acquisition allows naive
CD4+ cells to become APCs, anti-CD80 Ab was
included in the assay. As seen in Fig. 9F, APCs treated with
anti-CD80 Ab led to a marked decrease of GFP-IL-2
CD4+ cells becoming activated (Fig. 9F), when compared with APCs treated with control Ab (Fig. 9E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table I). Because the CD80 and CD86 genes are deleted in B7dKO mice,
neither one of these molecules can be expressed (26). In
these studies, we also used normal DCs and a B cell line as APCs to
eliminate the possibility that CD80 acquisition might be due to some
abnormal phenomenon on vector-transfected APC membranes. The PCR
studies (Fig. 3) and the studies with CHX-treated T cells further
demonstrated that the expression of CD80 on T cells is not de novo. In
view of the recent discovery of B7h (a new homolog of CD80 and CD86),
these results also ruled out the possibility of interaction between
anti-CD80 Ab and this hom-olog. The confocal microscopy studies
using GFP/CD80 fusion protein further strengthened the notion that CD80
molecules are actually acquired upon T cell/APC interaction (Fig. 2).
Moreover, we demonstrated here that the level of acquisition by T cells
is related to the strength of signal 1 (Fig. 5). It has been demonstrated that shortly after T cell/APC interaction, immunological synapses or supramolecular activation clusters form at the contact site (29). Formation of these immunological synapses is associated not only with TCRs and MHC molecules, but also with various costimulatory molecules such as LFA/ICAM, CD2/CD48, and CD28/B7 receptor ligands (30, 31, 32). Wade et al. (33) have demonstrated that truncation of the intracytoplasmic tail of MHC molecules enhances lateral movement of these complexes but decreases immunogenicity. Presentation of MHC/peptide complexes in a cross-linked form can lead to overt T cell activation (33). It has been suggested that the accumulation and interaction of costimulatory molecules with their receptors and, perhaps, cytoskeletal proteins may promote the stabilization of TCR/MHC interactions. In addition, costimulatory molecules at the early stages of T cell activation may function by enhancing TCR cross-linking, therefore intensifying signal 1. In light of the above-mentioned studies, it is possible that increased CD80 expression on APCs, along with increased signal 1 in our models, may stabilize TCR/MHC interactions, contribute to the overall avidity of T cell/APC interactions, and thus lead to the increased transfer of CD80 to T cells. The data reported here on CD80 acquisition by CD28-/- cells support the hypothesis by Hwang et al. (21) that a high enough affinity for adhesion between CD28 and CD80 may actually result in the transfer of CD80 molecules from APCs to T cells.
Previous studies have shown that varying the strength of costimulation dramatically reduces the Ag dose required for T cell activation (34). Our observations indicate that increasing the density of CD80 on APCs may facilitate a strong interaction with CD28, which would lead to stabilization of the T cell/APC interaction and, therefore, acquisition of higher levels of costimulatory molecules in the presence of less signal 1.
Several groups have previously reported the expression of CD80 on T
cell clones as well as on T cells from patients with autoimmune
diseases or HIV infection (6, 7, 8, 9, 10, 11, 12, 13). Interestingly, there
has been no study to address the up-regulation of CD80 message in T
cells. T cells in these studies have always been in the presence of
APCs either in vitro or in vivo; therefore, it is reasonable to infer
that reported CD80 expression on T cells in these studies might have
actually been due to acquisition, rather than up-regulation of
expression, of CD80. The studies reported here demonstrate that at the
time of acquisition of CD80 by CD4+ cells (24 h),
no CD80 mRNA could be detected by PCR (Fig. 3).
Although two different biological consequences of CD80 acquisition by T
cells are demonstrated here, this phenomenon will most likely be the
subject of numerous investigations in the future. During an initial
encounter with Ag, naive Ag-specific lymphocytes proliferate and
differentiate to become activated effector/memory T cells. The current
dogma states that most of these activated effector T cells die after a
brief life span (35, 36, 37). Mechanisms underlying the
regulated expansion of effector/memory T cells are not well understood.
However, two of the findings reported here may indicate negative
regulatory consequences upon acquisition of CD80 by memory cells: 1)
effector/memory T cells can acquire significantly higher levels of CD80
in the presence of low levels of signal 1; and 2) these cells undergo
apoptosis upon acquisition of CD80 and increased levels of signal 1.
The apoptosis observed in the results of Figs. 7 and 8 is most likely
not due to a consequence of the way cells were handled because the
apoptosis was dependent on the amount of peptide present (including no
apoptosis with no peptide), and all cells were handled in the same way.
Also, it should be mentioned that our data do not exclude the
possibility that effector/memory CD4+ cells could
also acquire the MHC/peptide complex; therefore, these MHC complexes
might play a role in the apoptosis of these cells. It has been
demonstrated that activated T cells can process and present Ags to
other T cells in vitro (38). The studies reported here
using CD4+ cells from PCC/TCR-Tg mice that have
acquired CD80 show not only that these cells expressed MHC class II
molecules, but also that they were able to present soluble PCC peptide
to T cells from PCC/TCR-Tg/IL-2-GFPki mice more efficiently
than CD4+ cells from PCC/TCR-Tg mice that had not
acquired CD80; this led to activation and production of IL-2.
Interestingly, the CD4+ cells that had acquired
CD80 were able to activate the
PCC/TCR-Tg/IL-2-GFPki CD4+
cells without adding additional peptide. One possible explanation of
this is that, because these CD4+ cells had been
incubated with fibroblast cells in the presence of peptide and had
up-regulated their MHC class II molecules (data not shown), these T
cells actually also acquired peptide-MHC and were able to activate
PCC/TCR-Tg/IL-2-GFPki CD4+
cells in the absence of the addition of exogenous PCC peptide. These
results suggest that activated T cells that have acquired CD80
molecules have all the characteristics of professional APCs. However,
these functions are not expressed constitutively; instead, they occur
only after T cell activation, when class II synthesis is up-regulated
and T cells have acquired the CD80 ligand. Studies are now in progress
investigating whether the APC function of CD4+
cells that had acquired CD80 as reported here is dependent on other
acquired molecules such as MHC/peptide complexes or other costimulatory
molecules. However, the data presented here provide preliminary
evidence that upon acquiring CD80, T cells can more efficiently
activate bystander T cells.
There are at least two possible reasons why T cells become APCs in vivo. One potential advantage is that activated T cells would deliver both signals 1 and 2 to other T cells, thus amplifying an immune response; the other is that T cells could deliver only signal 2 to T cells that have received signal 1 from nonprofessional APCs (38). Consequently, although the initiation of the T cell response is dependent on professional APCs, it is possible that T cells may costimulate each other by acquiring CD80 under certain conditions (e.g., crowding of many T cells around the same DC). This would allow the APCs to interact further with other T cells. This mechanism of "T cell to T cell" costimulation could facilitate the regulation of the immune response.
In conclusion, the studies reported here demonstrate for the first time that naive CD4+ cells acquire CD80 upon activation by syngeneic APCs. This acquisition was shown to be directly related to both the strength of signal 1 and the level of signal 2 on APCs. Moreover, we demonstrate that CD80 acquisition is CD28 mediated. The specificity of the process of CD80 acquisition was demonstrated using both CD80 and CD28KO mice. Moreover, the acquisition of high levels of CD80 by effector/memory CD4+ cells and the resulting increased apoptosis of these cells together indicate a negative regulatory function. In contrast, when naive T cells acquire CD80, these cells themselves can act as more efficient APCs. Therefore, CD80 acquisition by T cells might play an important role in activating and/or regulating the immune response. The specific consequences of CD80 acquisition by T cells in the regulation of the immune response to pathogens, in anti-tumor responses, and in diseases of the immune system will require further investigation.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; KO, knockout; B7dKO, CD80/CD86 double-knockout; PCC/TCR-Tg, TCR transgenic for pigeon cytochrome c88–104; rF-CD80, recombinant fowlpox virus expressing CD80; FP-WT, wild-type fowlpox; GFP, green fluorescence protein; CHX, cyclohexamide.
Received for publication August 24, 2000. Accepted for publication December 4, 2000.
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) or effector/memory
CD4+ T cells (
) (5 x 105) were
cultured with 1 x 105 fibroblasts expressing high
levels of CD80 as APCs with varying amounts of PCC peptide (0–0.1
µg/ml) as signal 1 in 48-well plates for 24 h. CD4+
cells were carefully removed without disturbing the adherent fibroblast
layer, and were analyzed for the presence of surface CD4 and CD80 by
two-color FACS analysis. *, Denotes that a high number of
effector/memory T cells were dead when analyzed by FACS analysis and
trypan blue assay (as shown in Fig. 7
