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Experimental Therapeutics, Preclinical Pharmacology |
Department of Hematology/Oncology, Freiburg University Medical Center, D-79106 Freiburg, Germany [M. O., U. M., D. B., A. L., A. M.]; Institute of Molecular Medicine, Nuffield Department of Clinical Medicine, Oxford OX3 9DS, United Kingdom [J-L. C., V. C.]; Department of Hematology/Oncology, University of Regensburg, D-93042 Regensburg, Germany [A. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These cells revealed a very potent
monospecific CTL response, even at low E:T ratios, against
Melan-A-pulsed and Melan-A-expressing target cells. Altogether, our
study demonstrated that we have developed an efficient method for
generating large numbers of peptide-specific T cells in
vitro that may be used for adoptive T-cell transfer in tumor
immunotherapy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that APCs play a dominant role in induction of tumor-specific CTLs in vivo and in vitro (7 , 8) . The availability of large numbers of DCs, generated from hematopoietic progenitor cells or monocytes in vitro, has profoundly changed preclinical research as well as the clinical evaluation of these cells (9, 10, 11) . When pulsed with the relevant peptide, DCs are attractive for in vitro induction and activation of antigen-specific tumor-reactive CTLs (12) .
It has been shown recently that melanoma-reactive CTL lines and clones
can be isolated and purified using peptide-MHC tetramers
(13, 14, 15)
. Because cytokines are the major parameter of
specific T-cell effector function, Manz et al.
(16)
have developed a technology that allows the analysis
and separation of activated T cells according to secreted cytokines. An
affinity matrix for the secreted cytokine is generated by attaching a
specific antibody to the cell surface. Subsequently, the cells are
allowed to secrete the cytokine under defined conditions. Cells can
then be stained for the secreted molecule, which is now bound to the
affinity matrix with specific fluorochrome-labeled detection antibodies
(16)
. This method permits an assessment of the total
number of antigen-specific T cells that can be used for purification
approaches. In the present study, we used this technique to analyze and
purify Melan-A-specific CTLs, generated in vitro from
CD8-purified T cells after stimulation with Melan-A-pulsed autologous
DCs, according to secreted IFN-. Purified IFN--secreting T
cells showed Melan-A-specific cytotoxic activity even at very low E:T
ratios. Our results demonstrate an alternative strategy for generating
and purifying antigen-specific CTL from peripheral blood
lymphocytes, which can be used for adoptive transfer in tumor
immunotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells.
Tumor cell lines and T2 cells were maintained in RPMI 1640 supplemented
with 200 mM L-glutamine, 50 µM
ß-mercaptoethanol, 100 mM sodium pyruvate, MEM vitamins,
40 µg/ml streptomycin, 40 units/ml penicillin (standard medium M'),
and 10% FCS (PAN Systems GmbH, Aidenbach, Germany). Melanoma
cell lines MeE384, MeT413, and MeI493 were established from surgically
excised melanoma metastasis. Expression of HLA-A2 and Melan-A was
assessed by FACS analysis using an anti-HLA-A2-specific mAb (BB7.2;
ATCC, Rockville, MD) and by reverse transcription-PCR, respectively, as
described previously (22)
: MeE384 is
HLA-A2-, Melan-A+; MeT413
is HLA-A2+, Melan-A+;
MeI493 is HLA-A2+,
Melan-A+. T2 cells are HLA-A*0201 human lymphoid
cells that are defective in antigen processing but effectively present
exogenously supplied peptides (23)
.
Preparation of TCGF.
The preparation of TCGF was described previously (24)
.
TCGF was produced by stimulating 2.5 x
106/ml PBMCs for 2 h with 5 µg/ml
phytohemagglutinin (Murex, Dartford, England), 5 ng/ml phorbol
myristate acetate (Sigma Chemical Co, St. Louis, MO), and 5,000
rad-irradiated EBV-transformed B cells. The cells were then washed to
remove the mitogens and resuspended in RPMI 1640 supplemented with
2.5% human AB serum. After 40 h of incubation, supernatants were
harvested, passed through 0.2 µm filters, and stored at -70°C.
mAbs and Flow Cytometry.
Surface marker analysis of in vitro-cultured cells was
performed using a FACScalibur (BD, Mountain View, CA) and the
CellQuest software. We used the following mAbs conjugated to
FITC or PE for direct fluorescence: anti-CD1a (Coulter
Immunology, Hialeah, FL), anti-CD3, anti-CD4, anti-CD8, anti-CD14,
anti-CD16, anti-CD45RA, anti-CD45RO, anti-CD56, anti-CD80, anti-CD83,
and anti-CD86 (all from BD).
Generation of Peptide-specific T-Cell Lines.
This study was approved by the institutional ethics committee. All
donors and patients gave written informed consent before enrolling in
the study. HLA typing was carried out at the Freiburg University HLA
Typing Laboratory. CD8+ T lymphocytes were
enriched from PBMCs by depletion of CD4+,
CD11b+, CD16+,
CD19+, CD20+, and
CD56+ cells with magnetic cell sorting using a
midiMACS device (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The
resulting population consisted of >90% CD8+ T
cells and was used as the responder cell population. DCs were generated
from monocyte-enriched cell fractions as described previously
(10)
. Briefly, monocytes were isolated from PBMCs by
plastic adherence. Adherent cells were cultured with standard medium
(M') plus 2% autologous serum, supplemented with 1000 units/ml human
granulocyte-macrophage colony-stimulating factor (Essex Pharma,
Novartis Pharma, Basel, Switzerland), 100 units/ml IL-4 (CellGenix
GmbH, Freiburg, Germany) and 150 units/ml transforming growth
factor-ß1 (CellGenix). After 5–7 days of culture 100 ng/ml
lipopolysaccharide (from Escherichia coli, serotype 0111:B4;
Sigma) was added for 24 h. Cells displayed typical surface markers
of DCs (CD1a+, CD14-,
CD80+, CD83+, and
CD86+). The monocyte-derived DCs were then
harvested and pulsed for 2 h at 37°C with 30 µg/ml of the
appropriate peptide and human ß2-microglobulin
(10 µg/ml) in serum-free M'-medium. Peptide-pulsed DCs were washed
twice and resuspended in M' medium, and 104
responder cells/well and 5 x 103
peptide-pulsed autologous DCs/well were cocultured in 96-well
round-bottomed plates in 200 µl M' medium/well supplemented with 5%
autologous plasma and 3% TCGF. Medium and TCGF were replenished twice
a week. On day 7, T cells were harvested, counted, and replated at
104 T cells/well together with 5 x
103 peptide-pulsed autologous DCs/well in
complete medium supplemented with 3% TCGF. Subsequent restimulations
with peptide-pulsed DCs were performed once a week; a total of three to
four stimulation cycles were conducted before functional analysis.
Responder cells were tested for their specificity in a conventional
chromium release assay.
Expansion of Antigen-specific CTLs.
For expansion of antigen-specific CTLs, T cells were transferred to
25-cm2 flasks coated with anti-CD3/anti-CD28
mAbs. Briefly, 25-cm2 flasks (Falcon, Heidelberg,
Germany) were coated with anti-human CD3 mAb (OKT3; Ortho
Pharmaceutical Corp., Raritan, NJ) and anti-human CD28 mAb (L293; BD)
at a concentration of 1 µg/ml in PBS-100 mM HEPES buffer
(pH 9) After incubation overnight at 4°C, coated flasks were washed
twice with PBS. CD8+ T cells were placed on the
precoated and washed flasks at 5 x 105
cells/ml in 10 ml of M' medium supplemented with 2% autologous serum
and 100 IU IL-2/ml (EuroCetus, Amsterdam, the Netherlands). Cells were
cultured for 2 weeks; culture medium and IL-2 (100 IU/ml) were changed
twice a week.
Chromium Release Assay.
The cytotoxic activity of T-cell lines was measured by a conventional
4-h 51Cr release assay using triplicate
cultures in V-bottomed plates. Target cells analyzed included
HLA-A2+, Melan-A+, or
HLA-A2-, Melan-A+ melanoma
cell lines, peptide-pulsed or nonpulsed T2 cells, and the natural
killer target K562. For the peptide recognition assay, T2 target
cells were preincubated with 30 µg peptide /ml overnight at 37°C in
serum-free M' medium. E:T ratios were 25:1, 5:1, and 1:1 on 2000 target
cells/well. Triplicate wells were averaged and the percentage of
specific cytotoxicity was calculated as [(sample - spontaneous
release)/(maximum release - spontaneous release)]. For
spontaneous release, targets were plated without T cells in M' medium
plus 5% autologous plasma. For maximum release, targets were plated
with 5% NP40 (IGEPAL; Sigma) detergent.
HLA-Peptide Tetrameric Complexes and Flow Cytometry.
Tetrameric peptide-HLA-A*0201 complexes were synthesized as described
previously (25
, 26)
. In brief, purified HLA heavy chain
and ß2-microglobulin were synthesized using a prokaryotic expression
system (pET; Novagen, Milwaukee, WI). The heavy chain was modified by
deletion of the transmembrane/cytosolic tail and COOH-terminal addition
of a sequence containing the BirA enzymatic biotinylation site. The
expressed heavy chain and ß2-microglobulin were solubilized and
refolded together in vitro in the presence of a modified
Melan-A peptide 26-35 ELAGIGILTV. The 45-kDa refolded product was
isolated using fast protein liquid chromatography and biotinylated by
BirA (Avidity, Denver, CO) in the presence of biotin (Sigma), ATP
(Sigma), and Mg2+ (Sigma). The biotinylated
product was separated from free biotin by gel filtration and ion
exchange using fast protein liquid chromatography. Streptavidin-PE
conjugate (Sigma) was added in a 1:4 molar ratio, and the tetrameric
product was concentrated to 1 mg/ml. Analysis of cells for the
expression of cell surface markers was performed using FACScan (BD) and
CellQuest software (BD). Frozen CTLs were thawed and cultured in RPMI
1640 supplemented with 5% human serum. After 24 h of incubation,
cell viability was assessed. For tetramer staining,
106 PBMCs or in vitro-cultured T cells
were centrifuged at 300 x g for 5 min and resuspended
in 50 µl of cold PBS. Tetramer and anti-CD8-Tricolor (Caltag
Laboratories, Burlingame, CA) were added, and the cells incubated for
another 30 min. The samples were washed twice with PBS before
formaldehyde fixation. Double-color analysis was performed with
tetramer-PE and anti-CD8-Tricolor.
Detection and Purification of Antigen-specific T Cells by Staining
for Secreted IFN- with the Affinity Matrix Technology.
Antigen-specific CTLs were detected and purified from T-cell lines
using the IFN- secretion assay (Miltenyi Biotec GmbH) according to
the manufacturer’s instructions. This method has been described by
Manz et al. (16)
and Assenmacher et
al. (27)
. Briefly, an antibody matrix is
attached to the cell surface. Cells, stimulated with the specific
antigen, secrete cytokines for a defined period of time, allowing the
secreted cytokine to bind to the affinity matrix. The bound cytokine is
then stained as an artificial surface molecule. To induce IFN-
secretion in peptide-specific CTLs, T-cell lines were stimulated
in vitro for 6 h with T2 cells pulsed with the
appropriate peptide (30 µg/ml). Cells were then labeled with an
affinity matrix for the secreted IFN- (catch reagent). After 45 min
of incubation at 37°C, the secreted cytokine, bound to the catch
reagent, was stained with a PE-conjugated IFN--specific antibody
(detection reagent). IFN-+ T cells were then
isolated by magnetic cell sorting using anti-PE MicroBeads.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, that
three stimulation cycles with antigen-pulsed DCs provided the most
efficient conditions for the generation of antigen-specific T cells. T
cells exhibited a high cytotoxic activity against FluM1 peptide-pulsed
T2 cells but did not lyse unlabeled T2 cells or T2 cells labeled with
an irrelevant peptide (see Fig. 1C)
. In contrast, T cells
stimulated for only 1 or 2 weeks with peptide-pulsed DCs revealed a
nonspecific cytotoxic potential against unlabeled T2 cells (Fig. 1, A and B)
.
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,
CD8+ T cells demonstrated Melan-A peptide
specificity by lysis of Melan-A-pulsed T2 cells but not by lysis of
FluM1 peptide-pulsed or nonpulsed T2 cells. Melan-A-specific T cell
lines also showed specific killing of the
HLA-A2+, Melan-A-expressing melanoma cells with
no reactivity against HLA-A2- melanoma cells,
indicating that Melan-A-specific CTLs recognize the endogenously
processed and presented Melan-A antigen. Peptide titration revealed
that a half-maximal lysis for Melan-A-specific CTLs was obtained at a
peptide concentration of 
50 ng/ml (data not shown).
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, functional analysis of
CD8+ T cells after 4 weeks of antigen-specific
stimulation revealed a mean specific lysis of 38.9% (range,
15–65.8%) against Melan-A-pulsed T2 cells, whereas the mean cytotoxic
activity against an irrelevant peptide was only 7.9% (range,
1.8–22.9%). T cells derived from PBMCs of melanoma patients showed
similar results (data not shown).
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Maximal Expansion of Melan-A-specific CTLs in Vitro
Using Antigen-independent Stimulation with Anti-CD3 and Anti-CD28 mAbs.
Our goal was to define optimal culture conditions for obtaining large
numbers of antigen-specific CTLs for an adoptive transfer in melanoma
immunotherapy. It has been shown that antigen-specific T cells can be
expanded by secondary anti-CD3/anti-CD28 mAb activation (15
, 28
, 29)
. After 3–4 weeks of weekly stimulation with peptide-pulsed
DCs, Melan-A-specific CD8+ T cells were further
expanded with anti-CD3/anti-CD28 mAbs supplemented with IL-2 (100
IU/ml). Culture flasks (25 cm2) were
coated with anti-CD3 mAb (1 µg/ml) and anti-CD28 mAb (1 µg/ml).
Medium and IL-2 (100 IU/ml) were replenished twice a week. A
representative experiment is shown in Fig. 3
, demonstrating significant expansion of
CD8+ T cells under these culture conditions.
After 2 weeks of antigen-independent stimulation, we could obtain as
many as 6 x 108 CD8+
T cells from an initial number of 4 x 107
cells, corresponding to a mean expansion of 15-fold.
CD8+ T cells were tested before and 2 weeks after
antigen-independent stimulation for their cytotoxic activity against
HLA-A2+ Melan-A-expressing melanoma cells and
Melan-A-pulsed T2 cells. As shown in Fig. 4
, expanded CD8+ T
cells exhibited the same specific cytotoxic activity against
Melan-A-pulsed T2 cells before and after antigen-independent
stimulation. In addition, Melan-A-specific T-cell lines exhibited
specific killing of HLA-A2+, Melan-A-expressing
melanoma cells (Fig. 4)
. These results demonstrate that
antigen-independent stimulation with anti-CD3/anti-CD28 mAbs together
with IL-2 provides an efficient strategy for secondary activation and
expansion of tumor-specific CD8+ T cells that
maintains their antigen-specific cytotoxic activity.
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Secretion.
. After 5 weeks of in
vitro stimulation, the number of Melan-A-specific
CD8+ T cells increased from 0.01% to
0.04–0.07% and from 0.48% to 3.42%, respectively (Fig. 5B)
.
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-secreting T cells after
stimulation with Melan-A-pulsed target cells was measured by two-color
FACS analysis. Nonstimulated Melan-A-specific CTLs revealed no IFN-
secretion (data not shown). The frequency of
CD8+/IFN-+ cells that
had been stimulated with Melan-A-pulsed DCs once a week for a total of
3 weeks and expanded with anti-CD3/anti-CD28 mAbs for another 2 weeks
varied between 2.3% and 6.6%. Enrichment of
IFN-+ T cells was performed with one cycle of
purification using anti-IFN- mAb-labeled immunomagnetic beads and
subsequent antigen-specific restimulation for 1 week. As shown in Fig. 6
, cells could be purified from 3.7%
(Fig. 6A)
to 51.8% (Fig. 6B)
CD8+/IFN-+ T cells after
one round of immunomagnetic separation. The T-cell fraction that did
not bind to anti-IFN- mAb-labeled beads (negative fraction) and
consisted of 1.24%
CD8+/IFN-+ cells (Fig. 6C)
. Functional characterization of these three different
T-cell lines demonstrated that
CD8+/IFN-+-purified T
cells exhibited a high level of specific cytotoxicity against
Melan-A-pulsed T2 cells and to a lesser extent against
HLA-A2+ Melan-A-expressing melanoma cells (Fig. 6B)
. In contrast, the negative fraction displayed lower
levels of cytotoxic activity against Melan-A-pulsed T2 cells but higher
levels against nonpulsed T2 cells (Fig. 6C)
. These results
demonstrate that T cells, purified by magnetic separation according to
secreted IFN-, exhibit a specific CTL response against
Melan-A-pulsed and Melan-A-expressing target cells. In addition,
similar to the unsorted T-cell population, purified Melan-A-specific
CTLs can be further expanded in vitro (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability to induce tumor-reactive T cells ex vivo using peptide-pulsed DCs as APCs suggests an important role for DCs in adoptive T-cell immunotherapies (12) . In the present study, we demonstrate that repetitive stimulation with Melan-A-pulsed autologous DCs is an essential component for in vitro induction of Melan-A-specific CD8+ CTLs. A requirement for achieving effective adoptive therapy is the ability to obtain large numbers of tumor-specific CTLs from circulating CTL precursors after short-term in vitro stimulation. The present study indicates that antigen-specific CTLs can expand up to 600-fold after antigen-specific and subsequent nonspecific stimulation. Our results also demonstrate that CD8+ peptide-specific CTLs, induced by in vitro stimulation with antigen-pulsed DCs, can be expanded by coculture with immobilized mAbs against the CD3 and CD28 molecules without losing their specificity. Anti-CD3 plus anti-CD28 mAbs have been used by other groups to expand T-cell lines and T-cell clones (15 , 28 , 29) . Riddell and Greenberg (28) demonstrated long-term culture (3 months) of CMV-specific CD8+ T-cell clones that maintained their antigen-specific activity.
Replicative senescence may have important implications for adoptive immunotherapy with antigen-specific T cells. Our results demonstrate that 4–5 weeks are required for induction and expansion of antigen-specific CTL lines to reach sufficient numbers of CTLs suitable for adoptive transfer. In contrast, isolation and expansion of peptide-specific CTL clones, as described by Yee et al. (14) , requires more than 2 months of in vitro culture.
The induction and expansion of T lymphocytes recognizing Melan-A within
healthy donors, as demonstrated by our study and by those of other
investigators (12)
, indicates that the T-cell repertoire
is capable of generating efficient immune responses against these
differentiation antigens. The frequency of circulating Melan-A-specific
CTLs measured by peptide-MHC tetramer staining varied from 0.01% to
0.48% (Fig. 5A)
. These results are comparable to the
frequencies of circulating CD8+ T cells detected
in melanoma patients (13)
. One limitation of our approach
concerns the fact that induction and expansion of T cells in melanoma
patients may fail because of defects in antigen presentation or in
functional responsiveness of the T cells. Our strategy, which allows
the rapid preparation of antigen-specific T cells from healthy donors,
may also be used for the generation of donor lymphocytes specific for
hematopoietic system-restricted minor histocompatibility antigens after
allogeneic bone marrow transplantation (32)
.
The isolation and purification of Melan-A-specific CTLs according to
secreted IFN- allowed us to separate them from the rest of the bulk
cultures at an early phase of specific in vitro stimulation.
These findings confirm and extend observations of other investigators
who succeeded in generating and purifying peptide-specific CTLs
(13
, 14) . Those investigators have recently demonstrated
that Melan-A-specific CTLs can be generated in vitro from
the patient’s peripheral blood lymphocytes by stimulation with
autologous peptide-pulsed APCs and then isolated using peptide-MHC
tetramers. Dunbar et al. (15)
have obtained
rapid cloning of melanoma-specific CTLs from different tissues by
tetramer-guided sorting. CTLs were not restimulated with peptides, but
with solid-phase mitogenic signals. These results demonstrate that
peptide-specific CTLs can be purified before further expansion. A novel
approach used in our study consists of cytokine-guided purification of
a specific lymphocyte subpopulation. It has been demonstrated by Manz
et al. (16)
and Assenmacher et al.
(27)
that T cells activated with an antigen of interest
can be analyzed and sorted according to expression of cytokines like
IFN- or IL-2, using the cellular affinity matrix technology. It has
been demonstrated that expression of surface IFN- is precisely
linked to secretion of IFN- (27)
. Our results
demonstrate that surface IFN-+ cells purified
from polyclonal Melan-A-specific CD8+ T cells
exhibit a higher cytotoxic activity against Melan-A-pulsed and
Melan-A-expressing target cells than the IFN--negative fraction or
unseparated T cells. Whether this may be due to up-regulation of MHC
molecules on target cells induced by IFN- is not yet clear. An
additional advantage of this strategy is its applicability to other
tumors, such as renal cell carcinoma, where tumor antigens are mostly
unknown. There are some limitations to the adoptive transfer of
antigen-specific CTLs. One important limiting factor of this method is
the lack of CD4+ T-cell help that may be
necessary for an efficient antitumor immune response induced by
CD8+ T cells (33)
. The addition of
cytokines such as IL-2 and IL-12, however, might be suitable to
overcome this problem (34)
. An alternative strategy
implies the co-administration of melanoma-antigen-specific
CD4+ T-cell lines (35
, 36)
. Again,
the novel strategy of cytokine-guided isolation and purification of
specific CD4+ helper T-cell populations would be
helpful for adoptive transfer regimens. A second limitation of our
strategy relates to the generation of monospecific CTLs. Tumor escape
mechanisms include immunoselection of antigen-loss variants. Thus, a
therapeutic strategy should optimally be based on adoptive transfer of
CTL lines specific for a broader range of antigens.
In summary, we have shown that a sequential approach based on initial antigen-specific activation with autologous peptide-pulsed DCs, followed by secondary antigen-independent stimulation with anti-CD3/anti-CD28 mAbs, represents an efficient tool for generation and expansion of antigen-specific CTLs that can be used for adoptive transfer in tumor immunotherapy. In addition, cytokine-driven purification is a new method for selection of antigen-specific CTLs after a short period of specific in vitro stimulation. Further studies are needed to compare the efficiency of this approach with other techniques such as tetramer-guided selection (13, 14, 15) or T-cell receptor ß-chain variable region-driven selection (37) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the Deutsche
Forschungsgemeinschaft (Sonderforschungsbereich 364). 
2 To whom requests for reprints should be
addressed, at Department of Hematology/Oncology, University of
Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany.
Phone: 49-941-9445580; Fax: 49-941-9445502; E-mail: andreas.mackensen{at}klinik.uni-regensburg.de
3 The abbreviations used are: APC,
antigen-presenting cell; DC, dendritic cell; FluM1, influenza
matrix protein FluM158-66 peptide GILGFVFTL; mAb,
monoclonal antibody; TCGF, T-cell growth factor; PBMC, peripheral blood
mononuclear cell; BD, Becton Dickinson; PE, phycoerythrin; IL,
interleukin.
Received 9/29/99; revised 2/ 7/00; accepted 2/16/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or presence of the relevant (FluM1; 
) or
irrelevant (tyrosinase; 
) peptide. The cytotoxic
activity of one representative CTL line is shown. Values represent
triplicates at E:T ratios of 25:1, 5:1, and 1:1.
)
peptide. Values represent triplicates at E:T ratios of 25:1, 5:1, and
1:1. The results shown are representative of one experiment.
