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,
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The Ben May Institute for Cancer Research and
Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL 60637;
Department of Internal Medicine, Division of Nephrology, Mayo Clinic and Foundation, Rochester, MN 55905;
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Department of Biochemistry, University of Illinois, Urbana, IL 61801;
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Genetics Institute, Cambridge, MA 02140; and
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National Cancer Institute, 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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and anti-CD28 scFvs on artificial APCs reduced the proliferation
and IL-2 production by resting and preactivated bulk T cells as well as
CD4+ and CD8+ T cell subsets. Importantly,
expression of anti-CTLA-4 scFv on the same cell surface as the TCR
ligand was essential for the inhibitory effects of CTLA-4-specific
ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of
components of the proximal TCR signaling apparatus was similarly
dependent on coexpression of TCR and CTLA-4 ligands on the same
surface. These findings support a predominant role for CTLA-4 function
in the modification of the proximal TCR signal. Using T cells from
DO11.10 and 2C TCR transgenic mice, negative regulatory effects of
selective CTLA-4 ligation were also demonstrated during the stimulation
of Ag-specific CD4+ and CD8+ T cells by
MHC/peptide complexes. Together these studies demonstrate that
selective ligation of CTLA-4 using a membrane-bound scFv results in
attenuated T cell responses only when coengaged with the TCR during T
cell/APC interaction and define an approach to harnessing the
immunomodulatory potential of CTLA-4-specific
ligation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of these immune regulatory pathways has generated interest in developing approaches to separately manipulate positive and negative regulation to alter the course of immune-mediated disease. The hypothesis that CTLA-4-mediated negative regulation can critically affect the course of disease processes such as neoplasia, infection, autoimmunity, and allograft rejection has been borne out by studies in which CTLA-4/B7 interactions have been blocked in vivo through administration of CTLA-4-specific mAbs (20, 21, 22, 23, 24). In addition, studies of Ag-specific T cell tolerance have shown that CTLA-4 is necessary for unresponsiveness to subsequent Ag exposure (25, 26, 27, 28). Although these results imply that enhancement of CTLA-4 function might be used to induce immune hyporesponsiveness or tolerance to disease-related Ags, the pursuit of such a strategy is complicated by the shared specificity of CTLA-4 and CD28 for their natural ligands. The therapeutic potential for manipulating CTLA-4 function will likely be best appreciated by progress in characterizing the intracellular signaling events that mediate its effects, the Ag presentation conditions that preferentially recruit CTLA-4, and the relative influence of CTLA-4 function on different T cell subpopulations.
With these concepts in mind, we generated a cell-surface,
CTLA-4-specific ligand with the dual purpose of allowing us to study in
isolation the functional and biochemical outcomes of CTLA-4 engagement
as well as that of exploring the immunomodulatory potential of such an
agent. The ligand was derived from a well-characterized mAb directed
against murine CTLA-4 (9) by modification of a
single-chain Ab
(scFv)3
(29, 30, 31). Along with the anti-CTLA-4 surface-linked
scFv, we have also generated similar surface-linked scFvs that interact
with murine CD28 and the CD3 component of the TCR. In the present
study, this panel of ligands was used in a flexible artificial APC
system to explore the consequences of isolated CTLA-4 engagement during
TCR stimulation under Ag-nonspecific and Ag-specific conditions as well
as in the presence and absence of additional selective engagement of
CD28. Our results lend further support to a mechanistic model of
negative regulation in which a physical association between CTLA-4 and
the TCR results in modified tyrosine phosphorylation of proximal
components of the TCR signaling apparatus. The results also suggest
that enhancement of CTLA-4 engagement during primary or subsequent
presentation of disease-related Ags represents a feasible approach to
attenuating destructive immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female mice between 6 and 12 wk of age were used for all experiments. Mice were maintained in the University of Chicago animal housing facility in a specific pathogen-free environment. BALB/c mice were purchased from Frederick Cancer Research and Developmental Center (National Cancer Institute, Frederick, MD). DO11.10 and 2C TCR transgenic mice were drawn from colonies maintained at the University of Chicago. The human embryonic kidney cell line 293 was provided by Dr. Craig Thompson (Department of Medicine, University of Pennsylvania). The murine B cell lymphoma cell line A20 was purchased from the American Type Culture Collection (Manassas, VA). The highly transfectable subclone of the murine mastocytoma cell line P815 (designated P1.HTR) has been described elsewhere (32). All cell cultures were conducted at 37°C, 10% CO2, in DMEM (Life Technologies, Grand Island, NY) supplemented with 5% or 10% FCS, 25 mM HEPES (Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO), 100 µg/ml streptomycin (Sigma-Aldrich), 2 mM nonessential amino acids (Life Technologies), and 5 µM 2-ME (Sigma-Aldrich). Adherent cell lines were lifted by incubation with a 0.02% solution of Na2EDTA (Sigma-Aldrich) in PBS. All tissue cultureware was purchased from Becton Dickinson (Franklin Lakes, NJ).
Generation of surface-linked scFv and I-Ad expression constructs
The strategy for generation of surface-linked scFv used for
these studies is outlined in diagrammatic form in Fig. 1
A.
PCR products corresponding to the variable regions of the H and L chain
transcripts were initially amplified using primers derived from the
amino terminus of the Ab chains (5') and from the constant or joining
region sequences (3'). The PCR products were then spliced together with
an intervening flexible peptide linker sequence. This basic scFv cDNA
was then further modified in a second PCR using tailed sense and
antisense primers to add a murine L chain leader peptide sequence at
the 5' terminus and a flexible spacer and surface linkage motif to the
3' terminus. The modified constructs (mem-scFvs) were ligated into a
mammalian expression vector for subsequent transfections of eukaryotic
cell lines. Using described techniques (31, 33, 34, 35), basic
scFv constructs were generated from RNA isolated from the hybridomas
145-2C11 (hamster anti-murine CD3; Ref. 36), PV1
(hamster anti-murine CD28; Ref. 37), and UC10-4F10
(hamster anti-murine CTLA-4; Ref. 9). The polypeptide
linkers incorporated between L chain variable
(VL) and H chain variable
(VH) region sequences were as follows: 2C11 scFv
and PV1 scFv, NH2-GGGGSGGGGSGGGGS-COOH; 4F10
scFv, NH2-SSADDAKKDAAKKDDAKKDDAKKDAS-COOH.
The surface-linked constructs (subsequently referred to as mem-scFvs)
were produced by a tailed-primer PCR strategy using the basic scFvs as
templates. For all amplifications, the DNA polymerase mixture eLONGase
(Life Technologies) and a Geneamp 9600 thermal cycler (Perkin-Elmer,
Norwalk, CT) were used. The primers used to amplify modified constructs
incorporated appropriate restriction enzyme sites for subsequent
ligations (5' HindIII, 3' AflII) and a 5' kozak
consensus sequence (GCCGCCACC) along with the following motifs: 5'
murine L chain leader peptide,
NH2-MRTPAQFLGILLLWFPGIKC-COOH; 3' flexible
spacer peptide,
NH2-AKTTAPSVYPLAPVSSGSGSL-COOH
(35). Complementary DNA cassettes encoding two
surface-linkage motifs were also prepared by tailed-primer PCR from
existing templates with appropriate in-frame restriction sites for
subsequent ligations (5' AflII, 3' XbaI). These
motifs consisted of the GPI anchor peptide from human LFA-3
(38) and the transmembrane domain and first 20 amino acid
residues of the intracytoplasmic portion of murine B7-1 (mCD80)
(39). The peptide sequences for these motifs are as
follows: GPI anchor,
NH2-SPSSGHSKHRYALIPIPLAVITTCIVLYMNVL-COOH;
mCD80-derived motif,
NH2-TKVKFGAGFGAVITVVVIVVIIKCFCKHRSCFRRNEASRETN-COOH.
The final mem-scFv constructs were generated by one-step ligations of
restriction endonuclease-digested modified scFv and surface-linkage PCR
products into the HindIII and XbaI sites of the
mammalian expression vector pCDNA3.1+
(Invitrogen, Carlisbad, CA). A fourth mem-scFv, constructed by the same
protocol from the mAb 5H7 (mouse anti-human class I MHC), was
provided by Dr. E .S. Woodle (Department of Surgery, University of
Cincinnati). As this Ab has no cross-reactivity with murine Ags, this
construct was employed as a control protein (40). Surface
linkage by the GPI anchor motif alone was employed for mem2C11 scFv,
memPV1 scFv, and mem5H7 scFv. For mem4F10 scFv, constructs
incorporating both the GPI anchor and the mB7-1-derived motif were
generated and tested. While similar functional results were achieved
for mem4F10 scFv with both surface-linkage strategies, high levels of
surface expression were more consistently achieved using the
B7-1-derived motif (data not shown), and it is this protein that was
used in the experiments reported.
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- and ß-chains of the murine
class II MHC protein I-Ad were generated by
RT-PCR. Total RNA was extracted from the BALB/c-derived B cell lymphoma
cell line A20 using Trizol reagent (Life Technologies) according to the
manufacturer’s instruction. Reverse transcription with an oligo(dT)
primer was conducted using the Superscript II first-strand cDNA
synthesis kit (Life Technologies) by recommended protocol and was
followed by PCR using primers derived from published sequence for the
coding regions of I-Ad and
I-Adß (41, 42, 43) and incorporating
appropriate restriction endonuclease sites (5' HindIII,
3' XbaI) for subsequent ligation into
pCDNA3.1+. Transient transfection of 293 cells
Cells were transiently transfected with one or more plasmid constructs by calcium phosphate precipitation (44). The constructs and amounts of DNA used in individual experiments are indicated in the relevant figure legends. Transfectants were harvested for flow cytometric analysis and for use in coculture experiments between 36 and 48 h after transfection.
Stable transfection of P1.HTR cells
The cDNA for mem4F10 scFv was ligated into the bicistronic vector LZRSpBMN-linker-internal ribosomal entry site (IRES)-enhanced green fluorescent protein (EGFP) (45), provided by Dr. Hergen Spits (Netherlands Cancer institute, Amsterdam, The Netherlands). This vector (subsequently referred to as LXIE) is derived from the MFG murine retroviral backbone. The cloning site for insertion of cDNAs is followed immediately by an IRES and the cDNA for EGFP. The resulting transcript allows for the independent translation of the protein of interest and of EGFP. The latter serves as a marker for transfection without functionally altering the product of the gene of interest. The vector also contains a puromycin resistance gene for the selection of stable transfectants. P1.HTR cells were transfected by calcium phosphate precipitation using the same protocol as described above for 293 cells. Cells were transfected separately with parent vector or with mem4F10 scFv-containing vector and cultured in the presence of 2 µg/ml puromycin. Following outgrowth of puromycin-resistant cells, populations of comparable EGFP expression were purified by flow cytometric cell sorting. Sorted, polyclonal populations of mem4F10 scFv- or vector-transfected cells were maintained in 2 µg/ml puromycin and periodically checked for EGFP expression.
Flow cytometric analysis
Transiently transfected 293 cells or stably transfected P1.HTR cells were suspended in FACS buffer (PBS, 0.1% BSA, 0.01% NaN3). Aliquots of 100 µl were incubated with and without fluorescein-coupled soluble murine fusion proteins (mCTLA-4-Ig or mCD28-Ig) for 30 min at 4°C, washed, resuspended in 250 µl of FACS buffer, and analyzed on a FACscan flow cytometer (Becton Dickinson) with the CellQuest software package. Dual expression of EGFP and mem4F10 scFv was conducted by incubation of transfectants with purified mCTLA-4-Ig (1 µg/105 cells for 30 min at 4°C) followed by incubations with biotinylated goat-anti-mouse IgG and PE-coupled streptavidin. FACS was conducted on a Vantage cell sorter (Becton Dickinson).
Purification and preactivation of murine lymph node T cells and 2C TCR transgenic splenic T cells
Purified T cells were prepared from the inguinal, axillary, and mesenteric lymph nodes of BALB/c mice by nylon wool column separation and complement-mediated lysis following incubation with the following hybridoma supernatants: anti-heat stable Ag (hybridoma J11d; Ref. 46) and anti-class II MHC (hybridoma MKD6; Ref. 47). For purification of CD4+ T cell or CD8+ T cell populations supernatants containing mAbs against CD4 (hybridoma RL172.4; Ref. 48) or against CD8 (hybridoma 3.155; Ref. 49) were also added. Ab binding cells were depleted by addition of an equal volume of rabbit complement (Pel-Freez Clinical Systems, Brown Deer, WI) diluted 1:5 in sterile PBS with incubation at 37°C for 45 min. Viable cells were then isolated by density-gradient centrifugation using Ficoll-Hypaque. Purity of the desired cell populations was between 95 and 99%. For studies in which preactivated T cells were employed, freshly purified T cells were added to six-well tissue culture plates that had been coated with goat anti-hamster IgG (10 µg/ml in PBS; ICN Pharmaceuticals, Costa Mesa, CA) followed by 145-2C11 and PV-1 mAb (2 µg/ml each in PBS). Between 60 and 72 h later, the cells were removed, washed in complete medium, and cultured at 37°C for a further 8 h before use in coculture studies. Freshly dissected spleens from 2C TCR transgenic mice were gently disrupted, filtered, depleted of RBC by a 5-min incubation in ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4), and then mixed with irradiated (2500 rad), RBC-depleted BALB/c splenocytes at 1:1 ratio in 24-well tissue culture plates. After 5 days of culture, activated 2C T cells were separated by density-gradient centrifugation with Ficoll-Hypaque, washed, resuspended in fresh medium, and cultured at 37°C for a further 8 h before use in coculture studies.
In vitro proliferation and cytokine assays
Transiently transfected 293 cells and P1.HTR transfectants were
treated with 50 µg/ml mitomycin C (Sigma-Aldrich) for 1 h at
37°C followed by extensive washing then added to wells of 96-well
tissue culture plates. The numbers of cells used per well for
individual experiments are indicated in the relevant figure legends. In
experiments where mixtures of 293 transfectants were used, cell
suspensions were premixed at 1:1 ratio just before their addition to
the wells. Resting or preactivated murine lymph node T cells were then
added to wells containing transfectants and incubated at 37°C. For
coculture experiments using 293 cells, flat-bottom plates were used,
while for experiments involving coculture with P1.HTR cells,
round-bottom plates were used. At defined time points, plates were
pulsed with 1 µCi of tritiated thymidine and incubated for a further
8–16 h. Plates were freeze-thawed and then harvested onto fiberglass
filters and analyzed using a Filtermate 196 cell harvester and Topcount
Scintillation Counter (Packard Instrument, Meridien, CT). Three or six
identical wells were analyzed for each condition, and results (in cpm)
are expressed as mean ± SD. In all cases, background thymidine
incorporation for mitomycin C-treated transfectants was determined and
was subtracted from final counts for experimental conditions. For
analysis of cytokine concentrations, culture supernatants were
withdrawn from wells at defined time points, and levels of the
cytokines IL-2, IL-4, and IFN-
were measured by commercial ELISA
kits according to the manufacturer’s recommendations (IL-2 and IL-4,
Endogen, Woburn, MA; IFN-, PharMingen, San Diego, CA).
Detection of T cell protein tyrosine phosphorylation
Aliquots of 5 x 106 preactivated lymph node T cells were mixed on ice in 1.5-ml tubes with aliquots of 2.5 x 106 transiently transfected 293 cells suspended in complete culture medium. The transfectants used in individual experiments are indicated in the relevant figure legends. Cell mixtures were then pelleted by brief centrifugation, transferred to a heating block prewarmed to 37°C, and incubated for 2 and 5 min followed by lysis in 1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, pH 8.0, 1 mM sodium vanadate, 10 µg/ml leupeptin, 10 µM aprotinin, 1 mM PMSF. Lysates were precleared once with protein A-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) and once with protein A-Sepharose beads coated with an irrelevant hamster mAb (UC3-10A6; Ref. 50). Immunoprecipitation was performed overnight at 4°C with the anti-phosphotyrosine mAb FB2 coated onto protein A-Sepharose beads. Immunoprecipitates were separated on a reducing 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and immunoblotted with mAb 4G10 to phosphotyrosine (Upstate Biotechnology, Lake Placid, NY). Bound proteins were detected by enhanced chemiluminescence according to the manufacturer’s instructions (Pierce, Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A panel of three surface-linked scFvs (mem-scFvs) directed against
the murine T cell proteins CD3, CD28, and CTLA-4 as well as a
control-scFv with no murine ligand was generated (see Fig. 1
, A and B). In
preliminary experiments using mem2C11 scFv and memPV1 scFv constructs
separately and together, varying amounts of plasmid construct per
transfection were tested to generate populations of 293 cells capable
of inducing significant T cell proliferation. We found that combined
transient transfection of low amounts of mem2C11 scFv (0.5–1.0 µg
per 2 x 106 cells) and memPV1 scFv
(1.0–2.0 µg per 2 x 106 cells) resulted
in a potent immunostimulatory effect (data not shown). Untransfected
cells and cells transfected with memPV1 scFv alone induced no T cell
proliferation, while mem2C11 scFv alone induced low-level
proliferation. Increased amounts of the transfected cDNA were
associated with qualitatively similar effects (data not shown).
Surface expression and ligand binding of mem4F10 scFv vs memPV1 scFv
were determined by flow cytometric analysis (Fig. 1C). Cells
expressing mem4F10 scFv bound soluble murine CTLA-4 (mCTLA-4-Ig) but
not soluble murine CD28 (mCD28-Ig). Those expressing memPV1 scFv bound
mCD28-Ig but not mCTLA4-Ig. Cotransfection of both constructs resulted
in binding of both soluble fusion proteins at levels comparable to
those of singly transfected cells. The functional properties of mem4F10
scFv were first characterized in experiments in which 293 cells,
transfected with combinations of the surface-linked scFvs, were
coincubated with resting murine T cells (Fig. 2). Transfectants expressing mem2C11 scFv
and memPV1 scFv were used as artificial APCs for purified resting T
cells. Coexpression of the control scFv was associated with a strong
proliferative response. In contrast, engagement of CTLA-4 by
coexpression of mem4F10 scFv resulted in substantial attenuation of T
cell proliferation and IL-2 secretion (Fig. 2A). A similar
result was observed in a repeated experiment (Fig. 2B). For
these and subsequent experiments, expression of mem4F10 scFv was
maximized. Lower levels of expression of the anti-CTLA-4 ligand
were associated with qualitatively similar but less potent inhibitory
effects (data not shown). As the proliferative response to combined
CD3 and CD28 engagement was found to be similar in the presence or
absence of control scFv, in subsequent experiments cells transfected
with equivalent amounts of empty vector or mem4F10 scFv were used.
Together these results confirmed that the various mem-scFvs are
expressed on the cell surface and mediate functional effects during
cell-cell interactions wherein anti-CD3 and anti-CD28 scFvs
function as immune activators while selective engagement of CTLA-4
exerts a negative regulatory effect on primary T cell activation.
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The concept of a protracted role for CTLA-4 in shaping the
magnitude, nature, and duration of an ongoing immune response is
compatible with its persistent expression in activated helper and
cytotoxic T cells (51, 52, 53, 54, 55), in memory T cells
(56), and in T cell-derived clones of a variety of
phenotypes (55, 57). With this in mind, experiments were
performed using preactivated T cells and T cell subsets (see
Materials and Methods). The activated T cells can be induced
to rapidly proliferate and secrete cytokines upon restimulation by a
TCR signal alone or combined with a CD28 signal as seen in Fig. 3. Artificial APCs expressing low levels
of mem2C11 scFv alone (upper graphs) or a combination of mem2C11 scFv
and memPV1 scFv (lower graphs) were cotransfected with either empty
vector or mem4F10 scFv. Peak proliferative responses and levels of
IL-2, IFN-, and IL-4 of the activated T cells are shown. While
untransfected 293 cells did not stimulate preactivated T cells, the
presence of a TCR ligand was sufficient to induce proliferation and
production of IL-2 and IFN- by the preactivated cells. No detectable
IL-4 was produced under these conditions. Coexpression of mem4F10 scFv
resulted in significant reductions in both T cell proliferation and
cytokine production. The addition of low-level expression of memPV1
scFv resulted in a 
10-fold increase in the measured responses as
well as inducing measurable secretion of IL-4. In the presence of
combined TCR and CD28 signals, the magnitude of the anti-CTLA-4
effect on proliferation and secretion of IL-2 and IFN- was similar
to that observed in the absence of additional CD28 engagement.
Attenuation of IL-4 secretion was also observed. Expression of mem4F10
scFv inhibited proliferation and IL-2 production by 80 and 67%,
respectively, with mem2C11 scFv alone and by 66 and 69% with combined
mem2C11 scFv and PV1 scFv. Fig. 4
illustrates the results of a similar experiment performed using
preactivated purified CD4+ T cells and
CD8+ T cells. CTLA-4 coengagement was associated
with significant reductions in peak responses of both subsets of T
cells. Negative regulation of T cell proliferation and secretion of
IL-2 and IL-4 were observed in the CD4+ subset,
while in the case of CD8+ T cells proliferation
and secretion of IFN- were negatively regulated by CTLA-4 ligation.
Thus, selective engagement of CTLA-4 upon restimulation of activated T
cells resulted in a substantial inhibition of their proliferative
capacity and cytokine production. Moreover, this effect could be
observed both in the presence and absence of CD28 ligation.
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We have previously reported that coengagement of TCR and CTLA-4
results in attenuation of tyrosine phosphorylation of components of the
proximal TCR signaling apparatus, thus providing a mechanistic basis
for negative regulation of T cell activation by CTLA-4
(58). To further ask whether physical proximity to the TCR
is essential for negative regulation to occur through CTLA-4, we
compared the effect of mem4F10 scFv expression on the same cell surface
("in cis") as mem2C11 scFv with that of expression on a
separate population of cells ("in trans"). Preactivated
T cells were employed and secondary stimulation was provided through
the TCR alone. Fig. 5A shows
that, while providing TCR and CTLA-4 engagement on the same cell
surface resulted in a significant attenuation of proliferation and IL-2
production, when the two ligands were provided in trans no
attenuating effects were observed. In fact, under these circumstances a
modest enhancement of the T cell responses was seen. Complementary
results were observed in biochemical studies in which preactivated T
cells were incubated in suspension with similar mixtures of
transfectants for defined time periods (Fig. 5B). As shown,
coexpression of mem4F10 scFv on the same cell surface as mem2C11 scFv
resulted in reduced levels of tyrosine phosphorylation of proteins of
approximate molecular masses of 36 and 23 kDa in preactivated T cells.
In contrast, the expression of the CTLA-4 ligand on a separate, admixed
population of cells did not attenuate tyrosine phosphorylation of these
proteins. We have previously identified these phosphoproteins by
immunoprecipitation with specific Abs (58) as linker for
activated T cells (LAT) (59) and hyperphosphorylated
TCR
, both integral components of the proximal TCR signaling
apparatus. These results are consistent with the hypothesis that a
physical association between CTLA-4 and the TCR underlies the primary
mechanism of negative regulation and imply that the interaction of
CTLA-4 with a cell-surface ligand may result in widely differing
outcomes depending on the point of ligation relative to the
TCR.
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Although multiple studies have been published demonstrating the
ability of CTLA-4 ligation to attenuate Ag nonspecific T cell
stimulation, limited data is available to support similar effects on
bona fide Ag-specific responses. Therefore, we used the mem-scFv system
to examine the effects of the membrane-bound anti-CTLA-4 scFv on
primary and secondary activation in a well-characterized model of
Ag-driven CD4+ T cell activation. T cells derived
from mice transgenic for the DO11.10 TCR (with specificity for the
OVA-derived peptide OVA323–339 presented by the
murine class II MHC I-Ad; Ref. 60)
were coincubated with 293 cells transfected with cDNAs encoding the
- and ß-chains of I-Ad and pulsed with
antigenic peptide. A second population of 293 cells, transfected with
memPV1 scFv, was admixed to provide CD28-mediated costimulation in
trans. In this way, the level of the TCR signal could be
accurately fixed by addition of exogenous Ag. Furthermore, by providing
the CD28 ligand on a discreet population of cells, it was possible to
observe separately the effects of CTLA-4 coligation with either the TCR
or with CD28 by coexpression of mem4F10 scFv on one or other cell
surface. Incubation of purified D011.10 T cells with class II-negative
293 cells did not induce proliferation even in the presence of a high
concentration (1.0 µg/ml) of peptide as well as the CD28 ligand (data
not shown). A mixture of peptide-pulsed I-Ad+ cells and
memPV1 scFv-expressing cells (Fig. 6)
induced proliferation of both resting and preactivated DO11.10 T cells.
Coexpression of mem4F10 scFv with the MHC/peptide complex resulted in
significantly reduced proliferation of resting and preactivated T
cells. In contrast, when CTLA-4 was engaged on the same cell surface as
CD28, the result was a modest increase of proliferation in both
experiments. These results demonstrate that CTLA-4 engagement by
mem4F10 scFv negatively regulated the Ag-specific activation of
CD4+ T cells. In addition, the regulatory
function of CTLA-4 was "colocalized" with the TCR, not CD28,
supporting a central regulatory role of CTLA-4 in TCR and not CD28
signal transduction.
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For studies performed using a transient transfection system, it
was not possible to ensure that the levels of the anti-CD3 scFv
were completely matched. Therefore, a second Ag-specific system was
employed using a stable population of P1.HTR transfectants generated
with dual expression of EGFP and mem4F10 scFv or with EGFP alone using
a bicistronic vector (Fig. 7A). Flow cytometric analysis
of these stable transfectants showed no difference in the levels of
Ld expression between the mem4F10 scFv and
control transfectants (data not shown). In a series of studies, the
ability of the Ld-expressing murine mastocytoma
P1.HTR to stimulate preactivated CD8+ 2C TCR
transgenic T cells in the absence of additional CD28 engagement was
analyzed (32). Fig. 7, B and C
demonstrate that expression of mem4F10 scFv by P815 cells was
associated with significant reductions in proliferation and production
of IL-2 and IFN- by 2C T cells. Similar results were observed in
four repeat experiments. Thus, the stable expression of mem4F10 scFv
negatively regulated the activation of Ag-specific
CD8+ T cells responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that transfection with small amounts of the anti-CD3
and anti-CD28 scFv constructs together provided a significant
mitogenic stimulus. In contrast, the combination of surface-linked
anti-CTLA-4 scFv with anti-CD3 and anti-CD28 during
primary and secondary activation of T cells demonstrated significant
reductions in proliferation and IL-2, IL-4, and IFN- production as
compared with conditions where no CTLA-4 ligand was provided. The level
of expression required to observe functional effects for the individual
surface-linked constructs reflects both signaling characteristics
of the targeted T cell proteins as well as properties of the engineered
ligands themselves such as binding affinity and stability of the
refolded Ag-binding domains. The functional effects mediated by each of
the surface-linked scFvs represent outcomes that had been predicted on
the basis of the functional properties of the parent mAb (9, 36, 37). Although some variability in level of expression of each
construct between transfectant populations is likely to occur in any
given experiment, we have observed qualitatively similar results in
multiple experiments. More importantly, we were able to demonstrate
similar inhibition of proliferation and cytokine production using
populations of stably transfected P1-HTR cells used as APCs for 2C TCR
transgenic T cells. These results are compatible with those of existing
reports, in which anti-CTLA-4 mAb has been used to co-crosslink
CTLA-4 with the TCR (9, 10, 11, 12, 15, 16, 17) and confirm that the
interaction of CTLA-4 with a surface ligand on an APC exerts a negative
influence on T cell activation events. In contrast to recent reports by
Wu et al. (63) and Zheng et al. (64), we did
not observe augmentation of T cell proliferation as a result of
selective CTLA-4 engagement except under circumstances in which the TCR
and CTLA-4 were ligated on separate cell surfaces (discussed in further
detail below).
One of the strengths of the current system was the ability to replace the nonspecific TCR mitogen with bona fide class I and class II MHC/peptide complexes. Under these conditions, selective CTLA-4 engagement suppressed both primary and secondary T cell activation of CD4+ DO11.10 T cells in a well-characterized Ag-driven system representing the first demonstration of CTLA-4-mediated attenuation of normal Ag-specific T cells responses. Of note, we could also clearly observe negative regulation of Ag-specific, preactivated CD8+ T cells following CTLA-4/TCR coengagement using the 2C T cells. Thus, although recent studies have shown that CLTA-4 may not exert significant effects during primary CD8+ T cell activation (52, 65), our results and those of others (53, 54) suggest that CTLA-4-mediated negative regulation of CD8+ T cells may occur both indirectly, through inhibition of CD4+ helper functions, and directly, through inhibition of expansion and cytokine production during secondary Ag encounters.
We have recently demonstrated that coligation of TCR and CTLA-4 results
in reduced tyrosine phosphorylation of early components of the TCR
signaling apparatus including p23 (hyperphosphorylated) TCR and p36
LAT (58). In this study, we have expanded this observation
by showing, both in proliferation and IL-2 assays as well as in
biochemical studies, that the negative regulatory effects of CTLA-4
engagement are abolished by expression of the TCR and CTLA-4 ligands on
separate cell surfaces. Indeed, moderate increases in proliferation and
IL-2 levels were frequently observed when CTLA-4 ligation was separated
from that of the TCR. Furthermore, by separating the TCR and CD28
ligands during activation of DO11.10 T cells by
I-Ad/OVA323–339, we were
able to demonstrate that the negative regulatory effects of CTLA-4
ligation were exerted exclusively upon coengagement with the TCR and
not upon the TCR-independent costimulatory effects of CD28 ligation.
Finally, the negative regulatory effect of our selective CTLA-4 ligand
was most clearly observed in the presence of low-level expression of
the TCR ligand, with or without coexpression of a CD28 ligand, and was
decreased or absent when anti-CD3 was expressed at high levels
(M. D. Griffin and J. A. Bluestone, unpublished
observations). These new observations of selective CTLA-4 engagement in
both Ag-nonspecific and Ag-specific systems suggest that proximity of
CTLA-4 to the TCR complex during activation is essential for negative
regulation, lending further support to a "modified proximal signal"
model of CTLA-4 function by which its effects are predominantly
mediated by modification of early phosphorylation events within the TCR
signaling complex following Ag/MHC engagement (58, 66, 67). One implication of this model is that the use of CTLA-4
engagement to reduce T cell-mediated responses to specific Ags in vivo
must allow for strict copresentation of Ag and CTLA-4 ligand on the
same cell surface and that CTLA-4 binding by non-Ag-bearing cells may
result in an enhanced immune response. In addition, the magnitude of
the CTLA-4 effect on proliferation and IL-2 production may depend on
the strength of the TCR signal (i.e., the amount of peptide/MHC
presented and its affinity for the TCR). Recent reports have continued
to identify potential physiological roles for CTLA-4 in shaping immune
responses. These include effects on peripheral T cell tolerance
(25, 26, 27, 28), thymic selection (68), Th
differentiation (55, 57), TGFß production
(69), the generation and response of memory T cells
(56), and the effector function of primed cytotoxic T
cells (53, 54). Although it is conceivable that each of
these influences could arise directly from modification of the TCR
signal in different subpopulations of T cells, the possibility of
additional mechanisms of action for CTLA-4 remains an important
unanswered question.
In conclusion, we have shown that positive and negative regulation of T cell activation can be separately manipulated through the generation of surface-linked ligands with specific affinity for CD28 and CTLA-4. Using this approach, it is possible to selectively recruit the inhibitory influence of CTLA-4 to T cell activation events delivered either by Ag-nonspecific signals or by antigenic peptide presented on MHC. Our extended observations of CTLA-4-mediated attenuation of proximal TCR signaling events and of the requirement for intimate coligation of TCR and CTLA-4 contribute to a growing understanding of how this essential protein functions. The outcomes of specifically engaging CTLA-4 during a variety of T cell/APC interactions suggest that a strategy of enhanced CTLA-4 recruitment by expression of such a ligand could be used to favor immune hyporesponsiveness or tolerance to selected Ags in the context of tissue transplantation or tissue-specific autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey A. Bluestone, The Ben May Institute for Cancer Research, University of Chicago, 5842 South Maryland Avenue, MC1089, Chicago, IL 60637.
3 Abbreviations used in this paper: scFv, single chain Ab; GPI, glycosylphosphatidylinositol; IRES, internal ribosomal entry site; EGFP, enhanced green fluorescent protein; LAT, linker for activated T cells.
Received for publication October 18, 1999. Accepted for publication February 9, 2000.
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