| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Signaling and CD28 Costimulation Are Simultaneously Required for Efficient IL-2 Secretion and Can Be Integrated Into One Combined CD28/CD3 Signaling Receptor Molecule1
*
Klinik I für Innere Medizin, Labor Tumorgenetik, Universität zu Köln, Köln, Germany;
St. Elisabeth-Krankenhaus Köln-Hohenlind, Köln, Germany; and
Johannes Gutenberg-Universitat, III. Medizinische Klinik, Mainz, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(BW431/26-scFv-Fc-CD3), or
Fc
RI
(BW431/26-scFv-Fc-). By retroviral gene transfer, we
grafted activated T cells from the peripheral blood with these
immunoreceptors. T cells that express the FcRI or CD3
signaling receptor lysed specifically CEA+ tumor cells and
secreted high amounts of IFN- upon receptor cross-linking, whereas
anti-CEA-CD28 receptor-grafted T cells did not, indicating that
CD28 signaling alone is not sufficient for efficient T cell activation.
CD28 costimulation did not affect cytolysis by T cells equipped with
- or -signaling receptors, but enhanced both IFN- secretion
and proliferation. CD28 costimulation, however, was required for
efficient IL-2 secretion of anti-CEA- receptor-grafted T cells.
Both purified CD4+ and CD8+ T cells grafted
with immunoreceptors required CD28 costimulation for complete T cell
activation. We integrated both CD28 and CD3 signaling domains into
one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3)
with dual signaling properties. T cells grafted with the combined
CD28/CD3 signaling receptor secreted high amounts of IL-2 upon Ag
binding without exogenous B7/CD28 costimulation, demonstrating that
both MHC-independent cellular activation and CD28 costimulation for
complete T cell activation can be delivered by one recombinant receptor
molecule. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI receptor -chain or the CD3 chain. T
cells engrafted with the recombinant immunoreceptor induce an
Ag-specific, MHC-independent immune response upon Ag-mediated receptor
cross-linking (for review, see Refs. 4 and
5). According to the dual signal model of T cell activation, a costimulatory signal in addition to signaling through the TCR/CD3 complex is required for efficient activation of resting T cells, resulting in cellular proliferation, cytokine secretion, CTL-mediated target cell lysis, and prevention of activation-induced anergy (for review, see Refs. 6 and 7). Resting T cells, however, can be alternatively activated via B7-independent pathways or even without any costimulation (8, 9). Accordingly, analyses of CD28-deficient mice suggest that CD28 preferentially amplifies and sustains a primary T cell response (10) and lowers the amount of Ag required to achieve full cellular activation (11). In contrast to resting T cells, in completely activated T cells Ag-specific cytolysis via the TCR/CD3 complex appears to be independent of CD28/B7 costimulation. The role of CD28 costimulation in preactivated T cells, however, is not yet completely resolved. Particularly, proliferation of CD8+ T cells was demonstrated to be uncoupled from their cytolytic activity, but was substantially enhanced by B7 costimulation (12), indicating that each T cell activation parameter seems to be differentially affected by B7/CD28 costimulation.
From the viewpoint of adoptive immunotherapy, a long lasting antitumor
response of completely activated T cells grafted with the Ag-specific
immunoreceptor will be crucial for the therapeutic efficacy that
requires, in addition to highly efficient target cell lysis,
proliferation and cytokine secretion of grafted T cells. To address
this issue, we explored the role of CD28 costimulation for recombinant
immunoreceptor-mediated T cell signaling for use in adoptive
immunotherapy. Preactivated T cells were retrovirally grafted with a
panel of chimeric receptors that display Ab-like specificity for
carcinoembryonic Ag (CEA) and harbor intracellularly the signaling
moieties of either 1) CD28 (BW431/26-scFv-Fc-CD28), 2) CD3
(BW431/26-scFv-Fc-CD3), 3) FcRI (BW431/26-scFv-Fc-), or 4)
both CD28 and CD3 combined in one receptor molecule
(BW431/26-scFv-Fc-CD28/CD3). Utilizing T cells grafted with these
recombinant receptors and CEA+ colorectal tumor
cells that express both B7-1 and B7-2, we demonstrate in this study
that 1) cellular proliferation and, moreover, Ag-induced IL-2 secretion
of grafted T cells require CD28 costimulation, and 2) CD28
costimulation can be delivered together with CD3 signaling in a
combined immunoreceptor molecule to induce complete cellular
activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LS174T (ATCC CCL 188) is a CEA-expressing colon carcinoma cell
line. The anti-CEA mAb BW431/26, the anti-HRS3 idiotypic mAb
9G10, and the anti-idiotypic mAb BW2064/36 with specificity for the
anti-CEA mAb were described elsewhere (13, 14, 15). The
anti-CD3 mAb OKT3 was obtained from American Type Culture
Collection (ATCC, Manassas, VA; ATCC CRL 8001). Hybridoma cells 15E8
that produce an anti-CD28 mAb were kindly provided by R. van Lier
(NCB, Amsterdam, The Netherlands). The cell lines were cultured in RPMI
1640 medium supplemented with 10% (v/v) FCS (all from Sigma,
Deisenhofen, Germany). Abs were purified from murine ascites and cell
culture supernatants utilizing an agarose-coupled anti-mouse IgG Ab
(Sigma). The anti-CEA mAb CEJ065, the FITC-conjugated anti-B7-1
mAb MAB104, and the PE-conjugated anti-B7-2 mAb HA5.2B7 were
purchased from Coulter-Immunotech (Hamburg, Germany). The PE-conjugated
anti-CD3 mAb UCHT-1, the PE-conjugated anti-CD4 mAb MT310, and
the PE- and FITC-conjugated anti-CD8 mAb C8/144B, respectively,
were purchased from Dako (Hamburg, Germany). FITC-conjugated
F(ab')2 anti-human IgG1 and anti-mouse
IgG1 Abs were purchased from Southern Biotechnology (Birmingham, AL).
The anti-human IFN- mAb NIB42 and the biotinylated
anti-human IFN- mAb 4S.B3 were purchased from PharMingen (San
Diego, CA).
Generation of B7 transfectants
The bicistronic expression plasmid pCB/neo contains the coding sequences for the B7-1 molecule and the B7-2 molecule, linked by an internal ribosomal entry site sequence, for simultaneous expression of both B7-1 and B7-2 under control of the CMV early promoter/enhancer (16). The colorectal carcinoma cell line LS174T was transfected with the pCB/neo DNA utilizing FuGENE transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. After culture for 2 days, transfected cells were selected in the presence of G418 (2 mg/ml; Sigma) and subcloned by limiting dilution techniques. Simultaneous expression of B7-1 and B7-2 on the surface of transfected cells was determined by flow cytometry analysis, as described below.
Generation of chimeric receptors and transduction of peripheral blood T cells
The generation and expression of the CEA-specific
BW431/26-scFv-Fc- (438) and -CD3 (439) receptors in
peripheral blood T cells were recently described in detail
(17, 18, 19, 20). The recombinant BW431/26-scFv-Fc-CD28 (637) and
BW431/26-scFv-Fc-CD28/CD3 (607) receptors were generated as follows:
Briefly, the cDNA coding for the transmembrane and intracellular part
of CD28 (aa 135–202) was amplified by PCR utilizing CD28 cDNA
as template (21) and flanked with BamHI (5')
and XhoI (3') restriction sites using the primer
oligonucleotides 1-CD28-BamHI-sense and
2-CD28-XhoI-antisense (Table I
). The PCR product was digested and
inserted into the Moloney murine leukemia virus-derived retroviral
expression vector pBULLET (22) at the BamHI and
XhoI sites. To generate the cDNA coding for the chimeric
CD28/CD3 signaling domain, the cDNA of the transmembrane and
intracellular part of CD28 and the intracellular part of CD3
comprising aa 135–202 and 29–142, respectively, were amplified
by PCR utilizing CD28 and anti-CEA- receptor (439) cDNA as
templates and oligonucleotides 1-CD28-BamHI-sense,
3-CD28-CD3-antisense, 4-CD28-CD3-sense, and
5-CD3-XhoI-antisense as primers (Table I). Herewith, the
CD28 and CD3 sequences were flanked by overlapping sequences. The
rCD28/CD3 cDNA sequences were assembled by a PCR reaction,
reamplified utilizing the primer oligonucleotides
1-CD28-BamHI-sense and 5-CD3-XhoI-antisense
introducing BamHI and XhoI restriction sites, and
inserted into the retroviral expression vector pBULLET, as described
above. The sequences coding for the extracellular scFv binding and IgG1
Fc C domains were amplified by PCR utilizing the anti-CEA-
receptor (439) cDNA as template and flanked by NcoI and
BglII restriction sites by the oligonucleotides
6-L
-NcoI-sense and 7-hIgG1Fc-BglII-antisense
(Table I). The PCR product was digested with NcoI and
BglII and inserted into the NcoI and
BamHI restriction sites of the retroviral expression vector
pBULLET containing the cDNA sequences for the transmembrane and
intracellular part of CD28 and CD28/CD3, respectively. The final
chimeric receptor cDNAs were designated BW431/26-scFv-Fc-CD28 (637) and
BW431/26-scFv-Fc-CD28/CD3 (607), respectively. To generate gibbon
ape leukemia virus-pseudotyped retrovirus for infection of peripheral
blood T cells, the retroviral expression vector DNA (6 µg DNA) was
cotransfected with the retroviral helper plasmid DNAs pHIT60 and pCOLT
(each 6 µg DNA) into 293T cells by calcium phosphate coprecipitation.
pHIT60 DNA encodes the murine leukemia virus gag and
pol genes, whereas pCOLT DNA encodes the gibbon ape leukemia
virus-envelope gene under control of the CMV promotor/enhancer
(22). Cotransfection results in transient production of
high titers of infectious retrovirus. PBLs from healthy donors were
isolated by density centrifugation and cultured for 48 h in RPMI
1640 medium supplemented with 10% FCS in the presence of IL-2 (400
U/ml; Endogen, Woburn, MA) and OKT3 mAb (100 ng/ml). Cells were
harvested, washed, resuspended in medium with IL-2 (400 U/ml), and
cocultured for 48 h with transiently transfected 293T cells. T
cells were harvested and receptor expression was monitored by flow
cytometric analysis.
|
CD4+ and CD8+ T cells were isolated from the peripheral blood by MACS utilizing magnetic beads-conjugated anti-CD4 and anti-CD8 mAbs, respectively (both Miltenyi, Bergisch Gladbach, Germany). Briefly, PBLs from healthy donors were isolated by density centrifugation, and monocytes were depleted by plastic adherence. Nonadherent lymphocytes were washed with cold PBS containing 0.5% (w/v) BSA, 1% (v/v) FCS, and 0.1 M EDTA, and incubated for 15 min on ice with either magnetic beads-conjugated anti-CD4 or anti-CD8 mAbs, according to the manufacturer’s recommendations. The cells were washed twice with cold PBS, 0.5% (w/v) BSA, and 0.1 M EDTA, and separated on magnetic columns in a mini-MACS separator (Miltenyi). The number of positively enriched CD4+ and CD8+ T cells was determined by two-color flow cytometry utilizing FITC- and PE-conjugated anti-CD4 and anti-CD8 mAbs. The number of contaminating CD8+ and CD4+ cells was lower than 2% in the population of enriched CD4+ and CD8+ cells, respectively. MACS-enriched T cells were washed, cultured for 48 h in RPMI 1640 medium supplemented with 10% (v/v) FCS, 400 U/ml IL-2, and 100 ng/ml anti-CD3 mAb OKT3, and grafted with recombinant receptors, as described above.
Immunofluorescence analysis
Receptor-grafted T cells were identified by two-color immunofluorescence utilizing a FITC-conjugated F(ab')2 anti-human IgG1 Ab (2 µg/ml) and a PE-conjugated anti-CD3 mAb (UCHT-1, 1:200). Expression of B7-1 and B7-2 was determined using FITC-conjugated anti-B7-1 (MAB104) and a PE-conjugated anti-B7-2 mAb (HA5.2B7). CEA expression was monitored by incubation with the anti-CEA mAb CEJ065 (10 µg/ml) that was detected by a FITC-conjugated F(ab')2 anti-mouse IgG1 Ab (2 µg/ml). Immunofluorescence was analyzed using a FACScan cytofluorometer equipped with the CellQuest research software (Becton Dickinson, Mountain View, CA).
Stimulation of receptor-grafted peripheral blood T cells
Microtiter plates were coated with several combinations of the
anti-CD28 mAb 15E8 (2 µg/ml), the anti-CD3 mAb OKT3 (2
µg/ml), the anti-BW431/26 idiotypic Ab BW2064/36 (4 µg/ml), and
an IgG1 control mAb (4 µg/ml) (PharMingen). Transduced or
nontransduced peripheral blood T cells (1 x
105 cells/well) were incubated for 48 h at
37°C in coated microtiter plates. Alternatively, receptor-grafted and
nontransduced T cells (0.016–10 x 104/well) were
cocultivated for 48 h with B7-transfected
CEA+ colon carcinoma cells that express both B7-1
and B7-2 (LS174T-B7) and nontransfected CEA+
colon carcinoma cells (LS174T) (5 x
104/well), respectively. The culture supernatants
were analyzed by ELISA for the presence of IFN- and IL-2.
Briefly, IFN- was bound by a solid-phase anti-human IFN- mAb
(1 µg/ml) and detected by a biotinylated anti-human IFN- mAb
(0.5 µg/ml). IL-2 was bound by a solid-phase anti-human IL-2 Ab
(1:250) and detected by a biotinylated anti-human IL-2 Ab (1:250)
(OptEIA-Set; PharMingen). The reaction product was visualized by a
peroxidase-streptavidin conjugate (1:10,000) and ABTS (both purchased
from Roche Diagnostics) as a substrate.
Cell proliferation of PKH26-labeled cells
The membrane of receptor-grafted and nontransduced blood lymphocytes was labeled with the red fluorescent dye PKH26 (Sigma), as recently described (23, 24). PKH26-labeled, receptor-grafted, and nontransduced lymphocytes, respectively, were cocultured for 72 h with B7 transfected and nontransfected CEA+ colon carcinoma cells (5 x 104 cells/well), respectively. Nonadherent PBLs were harvested, stained with a FITC-conjugated anti-human IgG1 Ab, and analyzed by two-color flow cytometry. The lymphocyte population was defined by setting forward and side scatter parameters; receptor-grafted T cells were defined by green fluorescence. Dead cells were excluded from analysis by staining with propidium iodide. Cell division results in reduced intensity of the membrane dye PKH26. Proliferating cells were monitored by PKH26 fluorescence intensity, and histogram markers were set with >97.5% of freshly labeled viable lymphocytes laying outside the defined histogram region.
2,3-Bis(2-methoxy-4-nitro-5-sulfonyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) (XTT)-based cytotoxicity assay
Specific cytotoxicity of receptor-grafted T cells to target
cells was monitored by a XTT-based colorimetric assay, according to the
method of Jost et al. (25). Briefly, receptor-grafted and
nontransduced T cells were cocultivated in round-bottom microtiter
plates with B7-transfected (LS17T-B7) and nontransfected (LS174T)
CEA+ tumor cells, respectively, as described
above. After 48 h, cells were incubated with XTT reagent (1 mg/ml;
Cell Proliferation Kit II; Roche Diagnostics) for 90 min at 37°C.
Reduction of XTT to formazan by viable tumor cells was monitored
colorimetrically at an adsorbance wavelength of 450 nm and a reference
wavelength of 630 nm. Maximal reduction of XTT was determined as the
mean of six wells containing tumor cells only, the background as the
mean of six wells containing RPMI 1640, 10% FCS. The nonspecific
formation of formazan due to the presence of effector cells was
determined from triplicate wells containing effector cells alone, in
the same number as in the corresponding experimental wells. The
viability of tumor cells (%) was calculated as
follows: % viability
 |
To test for specificity of receptor-mediated lysis of CEA+ tumor cells, the assay was additionally performed in the presence of the anti-BW431/26 idiotypic mAb BW2064/36 and in the presence of the anti-HRS3 idiotypic mAb 9G10 (each 2 µg/ml) as a control. All assays were done in triplicate.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We generated a panel of recombinant immunoreceptors that harbor
extracellularly the same Ag binding domain with specificity for CEA,
but intracellularly different transmembrane and signaling domains
derived from the CD3 chain, the FcRI chain, and the CD28
coreceptor, respectively (Fig. 1). To
combine the CD28 and CD3 activation motifs in one receptor molecule,
we fused the DNA sequences for the transmembrane and intracellular part
of CD28 to the intracellular part of the CD3 chain. Peripheral blood
T cells were preactivated and retrovirally grafted with the recombinant
receptors, as described in Materials and Methods. Transduced
T cells that express the anti-CEA receptor were identified by
two-color fluorescence utilizing anti-CD3 mAb and anti-human
IgG Fc Ab that detects the extracellular CH2CH3 C domain of the
receptor. As demonstrated in Fig. 2, the
recombinant receptors were expressed in peripheral blood T cells with
nearly similar efficiency.
|
|
R-grafted T cells upon
coculture with B7-positive tumor cells
We asked whether B7 expression on target cells modulates 1)
cytokine secretion of receptor-grafted T cells, 2) specific
cytolysis of target cells, and 3) Ag-driven T cell proliferation upon
receptor cross-linking. We transfected the CEA+
colorectal cancer line LS174T with pCB7neo DNA that contains the
expression cassette for both B7-1 and B7-2. Transfected cells,
designated LS174T-B7, simultaneously express B7-1 and B7-2, as
demonstrated by flow cytometry (Fig. 3).
To monitor Ag-driven cellular activation, we cocultured nontransduced T
cells and T cells grafted with the BW431/26-scFv-Fc- (438) receptor
together with B7-expressing and nontransfected LS174T cells and
recorded the cytolytic activity and the amount of IFN- and IL-2
secretion of grafted T cells. Coincubation of receptor-grafted T cells
with CEA+ LS174T tumor cells resulted in highly
efficient lysis of CEA+ tumor cells, whereas T
cells lacking the CEA-specific receptor were poorly cytolytic (Fig. 4A). Coincubation with
LS174T-B7 tumor cells that express B7-1 and B7-2 did not alter the
cytolytic efficacy of receptor-grafted T cells. IFN- secretion,
however, is dramatically increased after coculture of receptor-grafted
T cells with B7+ tumor cells compared with
coincubation with B7- tumor cells (Fig. 4A). Notably, receptor-grafted T cells secrete high amounts
of IL-2 upon coincubation with B7+ LS174T-B7
cells, but no IL-2 was detected after coincubation with
B7- LS174T cells (Fig. 4A). Coculture
of nontransduced lymphocytes with B7+ and
B7- tumor cells, respectively, did not induce
IL-2 secretion, indicating that B7-mediated signaling alone is not
sufficient for IL-2 induction. Induction of cytokine secretion by
grafted T cells and cytolysis against B7+ and
B7- tumor cells, respectively, are specifically
induced by signaling through the BW431/26-scFv-Fc- (438) receptor
because coculture in presence of the anti-BW431/26 idiotypic mAb
BW2064/36 inhibited specific cytokine secretion and tumor cell lysis,
whereas incubation with an isotype-matched control Ab (9G10) did not
(Fig. 4B).
|
|
R-grafted T cells upon incubation
with B7+ CEA+ tumor cells
To monitor specific proliferation of grafted T cells in the
context of B7-CD28 costimulation, we labeled the cell membrane of
lymphocytes, grafted with the BW431/26-scFv-Fc- (438)
immunoreceptor, with the red fluorochrome PKH26, as described in
Materials and Methods. Labeled lymphocytes were cocultured
with B7-positive and B7- LS174T tumor cells.
After 72 h, nonadherent cells were harvested and stained with
a FITC-conjugated anti-human IgG1 Ab to monitor cells that express
the recombinant immunoreceptor. Flow cytometric analysis revealed that
incubation with LS174T cells induced proliferation of
immunoreceptor-grafted lymphocytes (Fig. 5). Recombinant receptor-triggered T cell
proliferation was substantially enhanced upon coincubation with
B7+ LS174T-B7 cells. As controls, lymphocytes
with and without specific receptor, respectively, did not
proliferate significantly in the absence of CEA+
tumor cells. Proliferation is predominantly restricted to the cell
compartment that expresses the immunoreceptor, indicating that 1)
proliferation of grafted lymphocytes is specifically mediated by the
anti-CEA receptor, and 2) T cells without CEA-specific
immunoreceptor are not induced to proliferate upon incubation with
CEA+ tumor cells, although the tumor cells
express B7.
|
receptor, upon
cocultivation with B7-expressing tumor cells
To analyze the impact of CD28/B7 costimulation on the
immunoreceptor-mediated activation of T cell subsets, we isolated
CD4+ and CD8+ T cells from
peripheral blood cells by MACS utilizing magnetic beads conjugated with
anti-CD4 and anti-CD8 Abs, respectively. By this procedure, we
obtained highly enriched CD4+ and
CD8+ T cell populations (purity >98% each; data
not shown). Both T cell populations were retrovirally transduced to
express the anti-CEA BW431/26-scFv-Fc- (438) receptor on the
cell surface at similar levels (46% transduced
CD4+ and 50% CD8+ T
cells), as revealed by FACS analysis (Fig. 6). We coincubated receptor-grafted
CD4+ and CD8+ T cells in
increasing numbers with CEA+ LS174T and LS174T-B7
and CEA- A375 tumor cells, respectively, and
monitored target cell lysis and IFN- and IL-2 secretion. Both
receptor-grafted CD4+ and
CD8+ T cells specifically lysed
CEA+ tumor cells with high efficiency, whereas
nontransduced T cells did not (Fig. 7, A–C). Cytolysis of
CEA+ target cells by receptor-grafted
CD4+ and CD8+ T cells,
respectively, is Ag specific because CEA- target
cells were not lysed, and nontransduced CD4+ and
CD8+ T cells without expression of the
recombinant anti-CEA- receptor did not lyse
CEA+ target cells. Corresponding results were
obtained with transduced lymphocytes from different
blood donors (not shown). The cell culture supernatants of these
experiments were additionally tested by ELISA for the presence of
IFN- (Fig. 7, D and E) and IL-2 (Fig. 7, G–I). Both receptor-grafted CD4+ and
CD8+ T cells secreted high amounts of IFN-
upon cocultivation with LS174T target cells. IFN- secretion of
grafted CD4+ T cells was enhanced by B7
costimulation, whereas IFN- secretion of grafted
CD8+ T cells was not affected by B7
costimulation. In contrast, receptor-mediated IL-2 secretion of grafted
CD4+ and CD8+ T cells
strictly required B7 costimulation in addition to Ag-mediated receptor
signaling because no IL-2 secretion was monitored upon receptor
cross-linking in the absence of B7. These data moreover indicate that
in receptor-grafted CD4+ and
CD8+ T cells, IL-2 secretion is similarly
modulated by B7-CD28 costimulation.
|
|
signaling and CD28 costimulation can be integrated into a
single recombinant receptor molecule
The experiments described above indicate that CD28 costimulation
is a prerequisite for complete T cell activation via recombinant TCR
molecules. We therefore tested whether the costimulatory
signal can be delivered additionally via the recombinant receptor
independently of signaling by endogeneous CD28. We stimulated
receptor-grafted T cells via the recombinant receptor by binding to the
immobilized ligand with or without CD28 costimulation and monitored the
IFN- and IL-2 content in the culture supernatants. Thus, the
anti-CD3 mAb OKT3 (2 µg/ml), the anti-CD28 mAb 15E8 (2
µg/ml), the anti-BW431/26 idiotypic mAb BW2064/36 (4
µg/ml) that is directed toward the scFv domain of the receptor, and
an isotype-matched IgG1 control mAb (4 µg/ml) were coated alone or in
combination onto microtiter plates and were incubated with T cells
grafted with the anti-CEA- (438), anti-CEA- (439),
anti-CEA-CD28/CD3 (607), and anti-CEA-CD28 (637)
immunoreceptor, respectively. The results of these
experiments are summarized in Fig. 8. T
cells grafted with the anti-CEA- (438), anti-CEA- (439),
and anti-CEA-CD28/CD3 (607) receptor, respectively,
secrete IFN- upon stimulation by the receptor ligand BW2064/36 mAb
as well as upon stimulation by the anti-CD3 mAb. IFN- secretion
of anti-CEA- (438)- and anti-CEA- (439)-grafted T cells
was furthermore enhanced by CD28 costimulation, whereas IFN-
secretion of anti-CEA-CD28/CD3 (607)-grafted T cells was not.
Specific IL-2 secretion of anti-CEA- (438)- and anti-CEA-
(439)-grafted T cells was only observed upon CD28 costimulation in
addition to Ag stimulation. In contrast, T cells grafted with the
anti-CEA-CD28/CD3 (607) receptor secreted high amounts of IL-2
without exogenous CD28 costimulation. IL-2 secretion by these T cells
could not further be increased by additional CD28 costimulation. These
data indicate that both signals for T cell activation are delivered
through the same receptor with a chimeric CD28/CD3 signaling domain.
T cells grafted with the anti-CEA-CD28 (637) receptor did not
secrete significant amounts of IFN- or IL-2 upon specific receptor
stimulation without the CD3 signal. Cytokine secretion of
anti-CEA-CD28 (637) receptor-grafted T cells, however, could be
enhanced by additionally signaling via the CD3/TCR complex, indicating
that the anti-CEA-CD28 (637) receptor can transmit CD28 signaling
upon specific receptor cross-linking. In summary, this set of
experiments furthermore demonstrates that simultaneous signaling via
CD28 and CD3/TCR is required for complete T cell activation.
|
(439), anti-CEA-CD28/CD3 (607), and
anti-CEA-CD28 (637) receptor, respectively, with
CEA+ B7- (LS174T) and
CEA+ B7+ (LS17T-B7) tumor
cells and, for control, with CEA- A375 tumor
cells. We recorded specific target cell lysis (Fig. 9A–C) and IFN- (Fig. 9D–F) and IL-2 (Fig. 9G–I) secretion of grafted
T cells. T cells grafted with the anti-CEA- (439) and
anti-CEA-CD28/CD3 (607) receptor were highly cytolytic against
LS174T cells. The efficiency of cytolysis of LS174T and LS174T-B7 cells
was nearly similar, once again demonstrating that the
efficiency of Ag-driven cytolysis is independent of CD28 costimulation.
Both anti-CEA- (439) and anti-CEA-CD28/CD3 (607)
receptor-grafted T cells secreted IFN- upon specific receptor
cross-linking. IFN- secretion of anti-CEA- (439)
receptor-grafted T cells was furthermore enhanced by B7 expression on
LS174T cells, whereas additional B7 costimulation did not enhance
IFN- secretion of anti-CEA-CD28/CD3 (607) receptor-grafted T
cells. In contrast to IFN- secretion, induction of IL-2 secretion by
anti-CEA- (439) receptor-grafted T cells requires CD28
costimulation, whereas anti-CEA-CD28/CD3 (607) receptor-grafted
T cells secrete high amounts of IL-2 without exogenous CD28 signaling.
IL-2 secretion, however, was furthermore enhanced by additional
exogenous CD28 costimulation. Remarkably, anti-CEA-CD28/CD3
(607) receptor-grafted T cells secreted much more amounts of IL-2
without exogenous CD28 costimulation than anti-CEA- (439)
receptor-grafted T cells did upon coculture with
B7+ LS174T cells. Nontransduced T cells and
anti-CEA-CD28 (637) receptor-grafted T cells neither lysed
CEA+ tumor cells nor secreted IFN- and IL-2,
respectively, demonstrating again that CD28 signaling alone is not
sufficient for complete T cell activation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
signaling through the
same immunoreceptor molecule that harbors the signaling moieties of
both CD28 and CD3. We demonstrate in this work that 1)
receptor-mediated target cell lysis does not require CD28
costimulation, 2) receptor-mediated induction of cytokine secretion and
T cell proliferation is substantially modulated by CD28 costimulation,
and 3) CD28 costimulation can be combined with CD3 signaling in a
single recombinant receptor molecule. These results have substantial
impact on the concept of cellular targeting by recombinant receptors:
specific target cell lysis by receptor-grafted T cells is independent
of CD28 costimulation, al-lowing efficient cytolysis of
B7-, Ag-expressing tumor cells. Other activation
parameters such as cytokine secretion and proliferation, however, are
uncoupled from the lytic capacity of receptor-grafted T cells and are
substantially affected by CD28 costimulation.
The therapeutical efficacy of T cells grafted with immunoreceptors is
expected to depend on a long lasting antitumor response. A prolonged
antitumor reactivity, however, requires, in addition to short-term
tumor cell lysis, sustained proliferation of grafted T cells and
secretion of high amounts of IL-2. Since IL-2 plays a key role for T
cell proliferation and Th1-based cellular immunity (26),
targeting of tumor cells by receptor-grafted T cells without additional
CD28 signaling is expected to end in a limited immune response despite
high IFN- secretion levels. Particularly, the acquisition of
additional effector cells at the tumor site, e.g., NK cells, will
depend on the presence of IL-2, whose induction requires CD28
costimulation of tumor-specific T cells. Moreover, CD28 costimulation
in addition to IL-2 secretion synergistically prevents
activation-induced T cell death by up-regulation of the antiapoptotic
proteins bcl-xL and bcl-2,
respectively (27, 28, 29). Accordingly, targeting of
receptor-grafted T cells without CD28 costimulation is likely to be
accompanied by enhanced T cell apoptosis, thus further limiting the
therapeutic efficacy. On the other hand, CD28 signaling alone without
additional signaling via the endogenous CD3/TCR complex or via a
recombinant anti-CEA- and anti-CEA- immunoreceptor,
respectively, is not sufficient for complete T cell activation,
indicating that both signaling pathways must be simultaneously switched
on to induce the plethora of T cell activation functions. CD28
costimulatory signals are physiologically delivered by professional
APCs, e.g., dendritic cells, to activate CD4+ and
CD8+ T cells at the onset of an immune response.
Other cell surface molecules with costimulatory activity, e.g., ICAM-1
(30, 31), may only partially substitute B7 costimulation.
This will be of physiological significance because the majority of
tumor tissues do not express costimulatory molecules of the B7 family.
Once activated, tumor-specific CD8+ CTLs are only
triggered by peptide-loaded MHC class I molecules or specific
tumor-associated Ags on the cell surface. Because the immunoreceptor
bypasses MHC molecule-restricted target cell recognition, the
recombinant TCR strategy allows also the recruitment of both
CD4+ and CD8+ T cell
subpopulations for highly efficient target cell lysis
(17). However, other cellular effector functions,
especially those of grafted CD4+ T cells, will
still require efficient costimulation.
To overcome the limitations of appropriate CD28 costimulation in T
cell-based adoptive immunotherapy, we generated a recombinant
immunoreceptor that harbors the signaling domains of both CD28 and
CD3. T cells grafted with this type of immunoreceptor were found to
be highly cytolytic and to secrete high amounts of IL-2 upon receptor
cross-linking without additional costimulation. These data suggest that
both activation pathways are successfully integrated, at least in part,
into the anti-CEA-CD28/CD3 receptor that combines MHC class I-
and class II-independent target cell recognition with dual signaling.
We expect that multiple effector functions of both
CD4+ and CD8+ T cells
engrafted with this type of immunoreceptor will be specifically
activated at the tumor site even in the absence of APCs or exogenous
costimulation. Accordingly, the combined signaling receptor
anti-CEA-Fc-CD28/CD3 will be superior to recombinant receptors
that activate a single activation pathway only, and is therefore
expected to enhance substantially the efficacy of the recombinant
receptor approach for use in the cellular immunotherapy of malignant
diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Dipartimento di Biologia Cellulare e Moleculare, Università degli Studi di Perugia, I-06123 Perugia, Italy.
3 Address correspondence and reprint requests to Dr. Hinrich Abken, Laboratory of Tumor Genetics, Department I of Internal Medicine, University of Cologne, Josef-Stelzmann-Strasse 9, D-50931 Cologne, Germany. E-mail address: hinrich.abken{at}medizin.uni-koeln.de
4 Abbreviations used in this paper: scFv, single-chain Ab fragment; CEA, carcinoembryonic Ag; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfonyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide.
Received for publication October 31, 2000. Accepted for publication September 20, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or subunits of the immunoglobulin and T-cell receptors. Proc. Natl. Acad. Sci. USA 90:720.g-chain signalling receptor that directs peripheral blood T cells to specific lysis of carcinoembryonic antigen (CEA) positive tumor cells. Int. J. Cancer 88:115.[Medline]
eRI -chain immune receptors: an extracellular spacer domain impairs antigen dependent T cell activation but not antigen recognition. Gene Ther. 7:1067.[Medline]
receptor-mediated T-cell lysis of tumor cells is coregulated by adhesion and accessory molecules. Int. J. Cancer 77:181.[Medline]This article has been cited by other articles:
|
|
 |
|
  K. Dobrenkov, M. Olszewska, Y. Likar, L. Shenker, G. Gunset, S. Cai, N. Pillarsetty, H. Hricak, M. Sadelain, and V. Ponomarev Monitoring the Efficacy of Adoptively Transferred Prostate Cancer-Targeted Human T Lymphocytes with PET and Bioluminescence Imaging J. Nucl. Med., July 1, 2008; 49(7): 1162 - 1170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Brentjens, E. Santos, Y. Nikhamin, R. Yeh, M. Matsushita, K. La Perle, A. Quintas-Cardama, S. M. Larson, and M. Sadelain Genetically Targeted T Cells Eradicate Systemic Acute Lymphoblastic Leukemia Xenografts Clin. Cancer Res., September 15, 2007; 13(18): 5426 - 5435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Koehler, D. Kofler, A. Hombach, and H. Abken CD28 Costimulation Overcomes Transforming Growth Factor-{beta}-Mediated Repression of Proliferation of Redirected Human CD4+ and CD8+ T Cells in an Antitumor Cell Attack Cancer Res., March 1, 2007; 67(5): 2265 - 2273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Kowolik, M. S. Topp, S. Gonzalez, T. Pfeiffer, S. Olivares, N. Gonzalez, D. D. Smith, S. J. Forman, M. C. Jensen, and L. J.N. Cooper CD28 Costimulation Provided through a CD19-Specific Chimeric Antigen Receptor Enhances In vivo Persistence and Antitumor Efficacy of Adoptively Transferred T Cells. Cancer Res., November 15, 2006; 66(22): 10995 - 11004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Hombach, C Schlimper, E Sievers, S Frank, H H Schild, T Sauerbruch, I G H Schmidt-Wolf, and H Abken A recombinant anti-carcinoembryonic antigen immunoreceptor with combined CD3{zeta}-CD28 signalling targets T cells from colorectal cancer patients against their tumour cells Gut, August 1, 2006; 55(8): 1156 - 1164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Schaft, B. Lankiewicz, J. Drexhage, C. Berrevoets, D. J. Moss, V. Levitsky, M. Bonneville, S. P. Lee, A. J. McMichael, J.-W. Gratama, et al. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFN{gamma} production Int. Immunol., April 1, 2006; 18(4): 591 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. L. Teng, M. H. Kershaw, Y. Hayakawa, L. Cerutti, S. M. Jane, P. K. Darcy, and M. J. Smyth T Cells Gene-engineered with DAP12 Mediate Effector Function in an NKG2D-dependent and Major Histocompatibility Complex-independent Manner J. Biol. Chem., November 18, 2005; 280(46): 38235 - 38241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Moeller, N. M. Haynes, M. H. Kershaw, J. T. Jackson, M. W. L. Teng, S. E. Street, L. Cerutti, S. M. Jane, J. A. Trapani, M. J. Smyth, et al. Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection Blood, November 1, 2005; 106(9): 2995 - 3003. [Abstract] [Full Text] [PDF] |
