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Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (Fc domain of human IgG1) fusion
protein to cells precoated with palmitated protein A, fine titration of
the B7-1 extracellular domain was achieved. The
B7-1 · Fc1 reincorporated into cell membranes by
this method retained costimulator function, as measured by an in vitro
proliferation assay. The degree of proliferation was dependent on the
surface density of B7-1 · Fc1. Significantly, the
threshold B7-1 · Fc1 density required for cytokine
production differed between IFN- and IL-2 and mirrored the hierarchy
(IFN- < IL-2) described previously for the TCR activation
threshold. Hence, this study invokes a novel protein transfer strategy
to establish that the levels of surface costimulator on APCs can
dictate both the magnitude and the quality of evoked T cell responses.
The notion of costimulator receptor activation thresholds
emerges. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little is known about quantitative aspects of costimulator signaling, and the possibility of "costimulator activation thresholds," that might parallel TCR activation thresholds, has not been systematically evaluated. There are two related questions in this regard. First, at any given Ag density, are there a threshold number of costimulator receptors that must be triggered to activate T cells? Second, if costimulator thresholds do indeed exist, is there a hierarchy of such thresholds within single cells, with different T cell outputs pegged to different costimulator inputs? Evidence in favor of such costimulator thresholds comes from recently reported studies by several groups that point to the importance of APC costimulator levels on elicited T cell responses (8, 11, 12).
The quantitative study of costimulation has been limited by technical factors. Gene transfer strategies do not permit fine control of costimulator densities at APC surfaces. As an alternative, cell-free systems have been invoked by some investigators. For example, differing concentrations of costimulators can be immobilized on plastic substrates (13, 14, 15) or on cell-sized latex microspheres (16). Such cell-free systems, however, by definition ignore contextual molecular determinants at the APC surface.
Protein transfer offers an alternative means for titrating costimulator
densities at APC surfaces. We (17, 18) and others
(19, 20) have described one costimulator protein transfer
method that invokes chimeric GPI-modified costimulators as
membrane-reincorporable "protein paints." However, scaling up the
production of these protein derivatives is complicated by the need to
purify them from transfectant cell membranes. In the present study, we
report a unique protein transfer strategy that bypasses the need to
work with recombinant membrane proteins. This strategy builds upon work
by Kim and Peacock (21) who described a two-step approach
for binding Abs to cell surfaces. According to their approach, protein
A, after derivatization with palmitate, is first incorporated into cell
membranes, and in turn, this membrane-associated palmitated protein A
(pal-prot A)4 is used
as a trap for secondarily added IgG molecules. Building upon this
finding, the present study demonstrates that Fc fusion proteins can be
similarly delivered to pal-prot A-precoated cell surfaces with
preservation of trans-signaling protein function. Moreover,
we proceed to utilize costimulator · Fc1
(Fc domain of human IgG1) protein transfer to establish the existence
of costimulator activation thresholds within T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant protein A (Calbiochem, La Jolla, CA) was derivatized with the N-hydroxysuccinimide ester of palmitic acid (Sigma, St. Louis, MO) as described (21). The lipid-derivatized protein A was purified as described (22) on a 30-ml Sephadex G-25 (Sigma) column. The protein product was quantitated using a bicinchoninic acid kit (Bio-Rad, Richmond, CA), filter sterilized, and stored at 4°C until use.
Membrane incorporation of pal-prot A
Cells (3–7 x 106/ml) were resuspended in RPMI 1640 medium (BioWittaker, Walkersville, MD) after three washes with this same medium. Varying concentrations of pal-prot A (or nonderivatized protein A as negative control) were added to the cell suspension, and the mixture was incubated at 4°C for 2 h with constant mixing. To assess the incorporation of pal-prot A onto cell surfaces, cells were washed twice in buffer (0.25% BSA/0.01% sodium azide/PBS) and then incubated on ice for 1 h with 100 µl of 100 µg/ml FITC-human IgG (Sigma) diluted with the same buffer. Cells were washed twice in the buffer and analyzed on a FACStar (Becton Dickinson, Mountain View, CA).
Preparation of recombinant B7-1 · Fc1
The expression plasmid pCDM8/B7Ig, encoding the complete human
B7-1 extracellular domain linked in-frame to the
Fc1, was obtained from the American Type
Culture Collection (Manassas, VA). The sequence encoding
B7-1 · Fc1 was mobilized from pCDM8/B7Ig
by digesting with XbaI, filling-in with Klenow fragment, and
subsequently digesting with HindIII. The mobilized fragment
was subcloned into the EBV episomal expression vector pREP7
(Invitrogen, San Diego, CA) with HindIII and filled-in
BamHI sites. The plasmid was transfected into 293 cells
(human kidney cell line; American Type Culture Collection), and
hygromycin B-resistant colonies were selected in serum-free
UltraCulture medium (BioWhattaker) supplemented with 10 mM glutamine,
penicillin/streptomycin, and 200 µg/ml hygromycin B. Secreted
B7-1 · Fc1 was purified from conditioned
medium by protein A-agarose (Life Technologies, Germantown, MD)
affinity chromatography and verified by SDS-PAGE.
B7-1 · Fc1 protein transfer
Cells precoated with pal-prot A were washed once and resuspended in RPMI 1640 medium (3–7 x 106 cells/ml). The mixture was incubated at 4°C for 1 h with constant mixing. To monitor protein delivery, 106 cells were washed twice with the same buffer as above, incubated on ice for 1 h with 1 µg of human B7-specific mAb BB-1 (PharMingen, San Diego, CA) in 100 µl of buffer. Cells were washed once and immunostained (on ice for 1 h) with 100 µl of 1:100 diluted FITC-conjugated goat F(ab')2 anti-mouse Ig (Boehringer Mannheim, Indianapolis, IN) as secondary Ab. Cells were washed once, resuspended in PBS, and analyzed on a FACStar.
Quantitation of exogenously incorporated
B7-1 · Fc1 at cell surfaces
Human B7-1 · Fc1 was iodinated
using Iodo-beads (Pierce, Rockford, IL) according to the
manufacturer’s protocol, and the labeled protein was purified on a
Sephadex G-25 column (Pharmacia, Piscataway, NJ). The specificity was
adjusted to 2.1 x 106cpm/µg by addition
of unlabeled B7-1 · Fc1. Protein transfer
was performed as described earlier, substituting the labeled protein.
All experiments were performed in duplicate. To control for nonspecific
binding, excess amounts of unlabeled human IgG (Sigma) were added to
specifically block the binding of
B7-1 · Fc1 to protein A. After repeated
washing, counts in cell pellets were determined using a gamma counter
(1272 Clinigamma; LKB Instruments, Gaithersburg, MD). Counts resulting
from specific binding of B7-1 · Fc1 were
calculated by subtracting nonspecific counts obtained with human IgG.
The average number of molecules on a single cell was calculated
according to the formula A x
B-1 x
C-1 x
NA, where A is the
determined radioactivity (cpm) in the cell pellet, B is the
specific activity of the labeled protein expressed as cpm/mol,
C is the number of cells in the cell pellet, and
NA is Avogadro’s constant.
Proliferation assays
PBMC were isolated from fresh whole blood by Ficoll density
centrifugation. T cells were purified by two rounds of treatment with
Lympho-kwik (One Lambda, Canoga Park, CA). T cell purity was verified
by lack of a proliferative response to PHA or PMA in the absence of
accessory cells. The human CD3-specific mAb HIT3a (PharMingen) was
bound to 96-well plates at the indicated concentrations and used in
this form to provide a first activating signal to T cells.
Alternatively, PHA was used in soluble form as a source of a first
signal. K562 cells transfected with the negative control vector
pREP7 (K562/pREP7) were precoated with pal-prot A and secondarily
coated with B7-1 · Fc1. For each
proliferation assay, 1 x 105 T cells were
incubated with 4 x 104
B7-1 · Fc1-coated and mitomycin C-treated
K562/REP7 cells for 60 h at 37°C. Wells were pulsed with 1
µCi [3H]thymidine for the last 16 h of
the incubation period. Cells were harvested and counted on a Betaplate
liquid scintillation counter (Wallac Oy, Turku, Finland).
ELISA measurement of secreted cytokines
A total of 106 T cells was incubated with
5 x 105 processed K562/REP7 cells
(B7-1 · Fc1 positive or negative) in
48-well plates using either plate-bound HIT3a or PHA as a source of
first signal. Supernatants were collected after 48 h, and ELISAs
for human IFN- and IL-2 were performed using a commercial ELISA kit
according to manufacturer’s protocol (Genzyme, Cambridge,
MA).
Analysis of intracellular cytokine production
A total of 106 T cells was incubated with
5 x 105
B7-1 · Fc1-coated K562/REP7 cells in
48-well plates for 48 h. Again, either plate-bound HIT3a or PHA
was used as a source of first signal. Monensin (Sigma) was added to a
final concentration of 3 µM, and the mixture was incubated for an
additional 6 h to accumulate cytokine within the cells. Cells were
then collected, fixed by incubating them in 100 µl of fixation
solution (4% paraformaldehyde/PBS (pH 7.4)) on ice for 20 min, and
then washed twice with staining buffer (0.1% saponin/1%
heat-inactivated FCS/0.1% sodium azide/Dulbecco’s PBS).
Immunostaining for intracellular cytokines was performed by incubating
the cells on ice for 1 h with 100 µl of the staining buffer
containing 0.5 µg of FITC-anti-IFN- and 0.5 µg of
PE-anti-IL-2 Abs (PharMingen). Cells were subsequently washed once
with staining buffer without saponin. T cells were gated using forward
light scatter/side light scatter parameters, and 2–5 x
104 cells were analyzed in each run.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 protein transfer
A two-step strategy was developed for quantitatively
"painting" costimulators onto cell surfaces. This approach was
adapted from a method originally designed by Kim and Peacock
(21) for delivering intact IgG to cell surfaces. According
to our modification, Fc fusion proteins are substituted for intact IgG.
The first step involves precoating cells with a chemically palmitated
prot A derivative (pal-prot A; see Materials and Methods).
In a first set of optimization experiments, efficient incorporation of
palmitated prot A was documented in four cell lines (Fig. 1
A) as detected with
FITC-conjugated human IgG. As a negative control, nonderivatized
protein A lacked the capacity to bind to the same cells. Membrane
incorporation was dose dependent and started to plateau at 33 µg/ml
pal-prot A (Fig. 1B). Pal-prot A incorporation was rapid,
appearing immediately after addition to the cells and reaching a
plateau at 
1 h (Fig. 1C).
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1
fusion protein, incorporating the Fc region of human IgG1, was produced
(see Materials and Methods) and visualized by SDS-PAGE as a
dominant single band of 80 kDa under both reducing and nonreducing
conditions (Fig. 2A). Its
identity was confirmed by ELISA, with the recombinant protein binding
strongly to the human B7-1-specific mAb, BB-1, but not to control Ab
(data not shown).
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1 onto pal-prot A precoated cells
was established. When K562 cells were precoated with pal-prot A,
secondarily applied B7-1 · Fc1 attached to
the cell surface, as detected by immunostaining of the cells with
anti-B7-1 BB-1 mAb and FITC-conjugated goat anti-mouse IgG
(Fig. 2
B). When a control Fc fusion protein
(CD28 · Fc1) was substituted for
B7-1 · Fc1, no BB-1 binding was observed,
substantiating BB-1 mAb’s B7-1 specificity (Fig. 2B). When
nonderivatized protein A was substituted for pal-prot A, no BB-1
binding was observed, indicating the dependence of Fc protein
attachment on protein A lipid anchoring (Fig. 2B). Next, the
feasibility of quantitatively painting
B7-1 · Fc1 onto pal-prot A precoated cells
was established. When K562 cells were precoated with excess amounts of
pal-prot A (33 µg/ml), surface levels of
B7-1 · Fc1 were dependent on the
concentrations of applied B7-1 · Fc1 (Fig. 2C). Surface B7-1 epitope levels started to plateau at 33
µg/ml, and the epitope density was similar to that on
B7-1-transfected K562 cells (data not shown). The average number of
B7-1 · Fc1 painted per cell was determined
using 125I-labeled
B7-1 · Fc1. Again, K562 cells incorporated
increasing amounts of B7-1 · Fc1 as the
reagent concentration was increased during the painting process (Table I). At the lowest concentration used
(0.033 µg/ml), 460 molecules became anchored onto each K562 cell.
At the highest concentration used (33 µg/ml), about 460,000
B7-1 · Fc1 molecules became incorporated.
Taken together, these data establish that
B7-1 · Fc1 can be applied to pal-prot
A-coated cells in a quantitative fashion.
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1 costimulates T cells
In vitro proliferation assays were performed to determine whether
membrane-tethered B7-1 · Fc1 can
costimulate T cells. In these assays, PHA and
B7-1 · Fc1-coated K562/REP7 cells
(i.e., K562 cells stably transfected with the pREP7 EBV episomal
expression vector) were used to provide first and second signals,
respectively, to T cells. K562/REP7 cells lack detectable B7-1 (data
not shown) and provide a suitable negative control for experiments with
K562/B7-1 cells (i.e., K562 cells stably transfected with a pREP7
vector containing human B7-1 cDNA sequence). Surface B7-1 levels on
K562/B7-1-transfected cells and
B7-1 · Fc1-coated K562/REP7 cells were
determined by immunostaining, and the mean fluorescence intensities
were 550 and 450, respectively. As shown in Fig. 3A, in the presence of
suboptimal PHA concentrations (<0.5 µg/ml),
B7-1 · Fc1-coated K562/REP7 cells, but
not K562/REP7 cells, significantly enhance T cell proliferation. The
costimulatory effect was comparable to that achieved with
K562/B7-1-transfected cells. The
B7-1 · Fc1/pal-prot A-dependence of the
observed costimulation was verified by showing that cells treated with
a combination of (nonderivatized) protein A and
B7-1 · Fc1, or with a combination of
pal-prot A and control CD8 · Fc1, did not
enhance T cell proliferation. In the presence of higher PHA
concentrations (> 1 µg/ml), K562/REP7 cells also costimulate T cell
proliferation, although to a lesser extent than the B7-1-positive
cells.
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1, proliferation assays were
performed in which plate-bound anti-human CD3 mAb was substituted
for PHA as a more physiological first signal. In this setting, in the
presence of suboptimal concentrations of anti-CD3 mAb (< 10
µg/ml), cell-associated B7-1 · Fc1
costimulated even more effectively than native B7-1 expressed at
equivalent levels on transfected cells (Fig. 3B). Again,
CD8 · Fc1, as a negative control Fc fusion
protein, did not costimulate under the same conditions. Taken together,
these results establish that B7-1 · Fc1,
tethered to membranes via pal-prot A, effectively costimulates T cell
proliferation.
Concentration-dependence of cell-associated
B7-1 · Fc1’s costimulatory activity
With an effective costimulator protein transfer method in hand,
quantitative aspects of B7-1 costimulation were evaluated. To this end,
T cell proliferation assays were performed using K562/REP7 cells
painted with variable concentrations of
B7-1 · Fc1. The concentration dependence
of B7-1 · Fc1-mediated costimulation could
be readily demonstrated when a fixed suboptimal concentration of PHA
(0.25 or 0.5 µg/ml) was used as a source of first signal (Fig. 4A). For example, in the
presence of 0.5 µg/ml PHA, T cell proliferation was observed once a
threshold B7-1 · Fc1 concentration (0.1
µg/ml) was reached, and the level of proliferation continued to rise
with increasing B7-1 · Fc1 concentrations
until reaching a plateau at 3.3 µg/ml. In the presence of a lower
concentration of PHA (0.25 µg/ml), T cell proliferation was observed
when a higher threshold B7-1 concentration (1 µg/ml) was reached,
indicating that costimulator thresholds can be modulated by the
strength of the first signal.
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B). Again, in
the presence of a fixed suboptimal concentration of plate-bound
anti-CD3 mAb (0.37 or 1.1 µg/ml), costimulation was observed only
after a threshold B7-1 · Fc1 concentration
was reached, and a further dose-dependent increase in proliferation was
also seen. Hence, in the presence of a suboptimal first signal (whether
PHA or anti-CD3 mAb), a threshold B7 level is required for T cells
to proliferate and the extent of T cell proliferation is dictated by
the costimulator level. A hierarchy of B7-1 costimulator thresholds for distinct cytokine responses
Having documented that B7-1 levels can modulate the extent of T
cell proliferative responses, we next determined whether B7-1 levels
can also dictate the quality of immune responses by altering the ratios
of cytokines produced by activated T cells. ELISA was used to measure T
cell cytokine secretion in response to varying painted
B7-1 · Fc1 concentrations and fixed
suboptimal primary stimulus concentrations. At a fixed PHA dose, the
B7-1 · Fc1 concentrations eliciting
minimal and maximal cytokine responses differed for IFN- and IL-2
with the general hierarchy being IFN- < IL-2 (Fig. 5A). A similar hierarchy for
the cytokine responses was observed when anti-CD3 mAb (3.3 µg/ml)
was used as a source of first signal (Fig. 5B). For
instance, at a B7-1 · Fc1 concentration of
0.33 µg/ml, IFN- output was 60% of the maximal response, whereas
IL-2 output showed no increase above basal levels (Fig. 5B).
This observed IFN- > IL-2 hierarchy for B7-1 costimulator
thresholds matches the order described for TCR activation
thresholds.
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To substantiate the ELISA findings with bulk T cell
populations, multiparameter flow cytometric analyses were performed to
assess intracellular IFN- and IL-2 levels within individual cells.
At low B7-1 · Fc1 concentrations, the T
cell response is dominated by IFN--only producers; however, at
higher B7-1 · Fc1 concentrations,
substantial numbers of IFN- and IL-2 double producers emerge (Fig. 6). Relatively few IL-2-only producers
were observed, even at the highest
B7-1 · Fc1 concentrations. These findings
are consistent with the bulk T cell cytokine response data, showing
that an IFN- response requires less B7-1 costimulation than does an
IL-2 response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1
fusion protein was "painted" onto tumor cells that had been
precoated with chemically lipidated protein A, and the influence of
varying levels of painted protein on T cell activation was monitored.
Key findings included the following: 1)
B7-1 · Fc1 can be quantitatively titrated
onto protein A-precoated cell surfaces. 2) Painted
B7-1 · Fc1 retains costimulator function.
3) At a specified level of TCR triggering, there is a threshold level
of costimulator trans-signaling required for T cell
activation. 4) There are distinct costimulator thresholds at the
single-cell level that are each coupled to different T cell outputs,
and the hierarchy of observed T cell cytokine responses parallels that
previously documented for TCR activation thresholds. Previous studies bear upon the question of costimulator thresholds. Itoh and Germain (8) reported that at a given Ag density, APC expressing high levels of B7-1 and ICAM-1 stimulated more IL-2-producing cells than did APC with low levels of the costimulators. In another study, increasing LFA-3 density on melanoma cells was shown to enhance cytokine production by melanoma-specific CTL clones (11). Recently, Murtaza et al. (12) reported that the level of costimulation has a significant effect on responses to an Ag, and that a strong costimulatory signal can convert a weak agonist into a full agonist and an agonist into a superagonist. Although these studies point to the importance of APC costimulator levels on elicited T cell responses, and thus support the conclusions of the present study, they were limited in two ways by the experimental systems employed. First, all of these studies used transfected and/or supertransfected sublines that were selected on the basis of low vs high costimulator expression. Although the sublines were carefully examined to ensure comparable expression of several immunoregulatory molecules, such as I-EK (8), HLA-A2, and ICAM-1 (11), it remains possible that observed functional differences might stem from variable expression of yet other immunoregulatory molecules. Second, the costimulator levels on the transfected sublines could not be tightly controlled, and, consequently, the functional impact of low vs high, but not graded, expression of costimulators was examined in those studies. In contrast, the protein transfer approach of the present study enabled titration of costimulators at the cell surface, while avoiding the confounding effects of subline selection and consequent molecular heterogeneity.
Protein transfer offers a number of advantages over gene transfer for
engineering APC. These include the suitability of the former to poorly
transfectable cells (for example, biopsy-derived tumor cells), the
simplicity of expressing multiple proteins on the same cell surface,
and the relative ease and rapidity of the procedure (reviewed in Ref.
23). Previously, we (17, 18, 24, 25) and
others (19, 20) have reported the successful application
of protein transfer to costimulator and MHC proteins using their
recombinant GPI-modified derivatives. However, one limitation of the
GPI protein transfer strategy is in scaling up the purification of GPI
proteins from membranes of the transfected host cells. The protein
transfer approach presented here circumvents this problem, since Fc
fusion proteins are soluble, are secreted at reasonably high levels
from transfectants (1–3 mg/l spent culture medium for
B7-1 · Fc1), and are amenable to
single-step purification by protein A/G chromatography.
The Fc fusion protein transfer procedure reported here builds upon the Ab protein transfer method first reported by Kim and Peacock (21). However, in contrast to their method, which invoked an 18-h dialysis step for precoating the cells with lipidated protein A, our method reduces this step to 1 h by directly incubating the lipidated protein A with the cells. The extension of Ab protein transfer to anti-CD28 mAbs, and hence costimulation, was reported recently (26). In that report, an alternative multistep approach was employed in which cells were first chemically biotinylated, protein G-biotin was linked to the biotinylated cells using avidin as a bridge, and, finally, mAb was attached to the anchored protein G. The limitation of this latter protein transfer procedure, as compared with the original one advocated by Kim and Peacock and our own, is in its dependence on chemical biotinylation of cells and the associated perturbation of resident cell surface molecules.
The principle finding of the present study is that levels of costimulators can not only tune the magnitude of an evoked immune response, but can also dictate the quality of the immune response. Taken together with the TCR activation threshold model (4, 5, 6, 7, 8, 9, 10), this argues that T cell responses at the single-cell level are quite plastic and can be driven in qualitatively different ways by altering the levels of both antigenic and costimulator inputs. The interplay between these antigenic and costimulatory inputs is likely to be of considerable interest and physiological significance, especially once insights into costimulator cooperativity emerge.
One class of tumor cell vaccines consists of immunogenic tumor cells engineered via gene or protein transfer to express lymphoid costimulators (27). Given its simplicity and effectiveness, our protein transfer method offers an attractive new option for tumor vaccine engineering. Flexible use of costimulator · Fc proteins, invoking diverse costimulator combinations that are tailored to individual tumor types, may provide significant therapeutic advantages.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark L. Tykocinski, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 6 Gates Building, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail address:
3 J. Rachmilewitz, J.-H. Huang, A. Chen, and M. L. Tykocinski, Submitted for publication.
4 Abbreviations used in this paper: pal-prot A, palmitated protein A; Fc1, Fc domain of human IgG1.
Received for publication July 12, 1999. Accepted for publication November 4, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or pal-prot A (•). Cells
were then incubated with 100 µg/ml of FITC-conjugated human IgG and
analyzed by FACS. C, EL-4 cells were incubated with 33
µg/ml of pal-prot A for the indicated periods of time and processed
as above.
