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Int. J. Cancer: 76, 304?311 (1998)
r 1998 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l?Union Internationale Contre le Cancer
TUMOR-SPECIFIC PEPTIDES IN CUTANEOUS T-CELL LYMPHOMA: ASSOCIATION
WITH CLASS I MAJOR HISTOCOMPATIBILITY COMPLEX AND POSSIBLE
DERIVATION FROM THE CLONOTYPIC T-CELL RECEPTOR
Carole L. BERGER1*, B. Jack LONGLEY2, Suguru IMAEDA1, Inger CHRISTENSEN1, Peter HEALD1 and Richard L. EDELSON1
of Dermatology, Yale University School of Medicine, New Haven, CT, USA
2Department of Dermatology, Section of Dermatopathology, Columbia University, College of Physicians and Surgeons,
New York, NY, USA
1Department
We wished to identify and characterize tumor-associated
class I peptides which could potentially serve as immunogens
for an immunoprotective CD8 response in cutaneous T-cell
lymphoma (CTCL). Candidate idiotypic peptides were identified from the third complementarity determining region
(CDR3) of the clonotypic T-cell receptor (TCR) expressed on
malignant T cells and native class I peptides were identified
from CTCL cells. Idiotypic peptides were designed by sequencing of patients? CDR3 and identifying 9 amino acid peptides
that could be accommodated in the peptide-binding motif of
the class I alleles. Three candidate idiotypic peptides were
synthesized and tested by measuring release of tumor necrosis factor-a (TNF-a) from autologous CD8 cells. Native
peptides were acid-eluted from class I molecules on CTCL
lymphocytes, fractionated, tested in the TNF-a assay and
sequenced. Two unique idiotypic peptides were specifically
recognized by autologous CD8 cells from CTCL patients. In
addition, a native peptide eluted from class I molecules of
CTCL tumor cells was identified, in the protein data base, as a
novel molecule with partial sequence homology to the conserved portion of the patient?s TCR. This homology was used
to construct an extended native peptide sequence that was
immunogenic for CD8 cells from both CTCL patients. Our
results demonstrate that peptides derived from the TCR can
be used as tumor-specific immunogens that are recognized
by CD8 cells. Moreover, novel class I peptides isolated from
the tumor cell also serve as immunogens. These peptides
might form the basis of an anti-tumor vaccine for immunotherapy of CTCL. Int. J. Cancer 76:304?311, 1998.
r 1998 Wiley-Liss, Inc.
The goal of tumor immunotherapy is selective recognition and
destruction of malignant cells. For this to occur, distinctive
antigenic peptides must be expressed on the tumor cell surface in
association with the major histocompatibility complex (MHC)
class I or II molecules for presentation to CD4 and CD8 T
lymphocytes.
Malignant cells of cutaneous T-cell lymphoma (CTCL), a clonal
proliferation of CD41 (Edelson et al., 1979; Kung et al., 1981),
memory T cells (Picker et al., 1990) with an initial affinity for
epidermis, display tumor-specific antigens, which can be recognized by autologous CD8 T cells (Berger et al., 1996). Suggestions
that these tumor antigens can be targeted in vivo derives from 2 sets
of data. First, initial malignant infiltration of skin is often associated with a pronounced CD8 host response (Bagot et al., 1992),
which diminishes as the disease disseminates, and the intensity of
this CD8 response correlates with a good prognosis (Wood et al.,
1991). Second, remissions in leukemic CTCL patients treated with
photopheresis (Edelson et al., 1987), thought to immunize patients
against malignant cells (Edelson et al., 1994), appear to require an
intact CD8 T-cell compartment (Heald et al., 1992).
Tumor-specific antigens may be derived from viral gene products, point mutations of normal proteins and normal differentiation
antigens preferentially displayed on the malignant cells. A potential
source of immunogenic unique peptides in T-cell tumors is the
clone-specific T-cell receptor (TCR). Anti-tumor immune responses directed toward the idiotypic components of the CTCL
TCR would solely impact the malignant T-cell clone.
Therefore, we have investigated the possibility that the class I
MHC-associated clone-specific peptides identified on CTCL cells
may originate, at least in part, from degradation products of unique
components of the TCR protein. We report here our findings that
peptide components of the idiotypic region of the CTCL TCR beta
variable chain (Vb) are selectively recognized by autologous CD8
T cells and that a novel native peptide directly extracted from the
CTCL tumor cells is also a target for autologous CD8 T cells.
Together, these findings indicate that a single clone of CTCL cells
is characterized by multiple class I-associated antigenic peptides,
permitting an additive anti-tumor attack. These observations suggest that it may be possible to construct peptide vaccines, based on
the sequence of the TCR, for tumor immunotherapy of CTCL.
MATERIAL AND METHODS
Patient population
Leukapheresis blood was obtained from CTCL patients undergoing therapeutic photopheresis treatment in accordance with the Yale
University Human Investigational Review Board guidelines. The 2
patients studied had a clonal expansion of malignant T cells that
expressed a Vb8a TCR identified by flow cytometry using a
family-specific monoclonal antibody (MAb) (Table I; T Cell
Sciences, Cambridge, MA). The class I histocompatibility locus
alleles of the patients? peripheral blood lymphocytes were determined by histocompatibility typing (Columbia University, Histocompatibility Typing Laboratory, New York, NY).
Cell populations
Peripheral blood mononuclear cells were isolated from the
leukapheresis specimens by Ficoll-hypaque flotation. Neoplastic T
cells and CD8 T-cell lines were isolated and propagated using
magnetic bead technology and cytokine stimulation, as previously
described (Berger et al., 1996). The purity of the cell populations
was confirmed by flow cytometry and the neoplastic T cells were
generally 95% Vb8a1 and the CD8 populations were on average
84% pure. The long-term CD8 lines used in this report were the
same cell lines that had been assessed previously for cytotoxicity
and shown to be specifically cytolytic for autologous tumor cells
(Berger et al., 1996).
B lymphocytes were isolated from the peripheral blood of the
patients using CD19-conjugated magnetic beads (Dynal, Lake
Success, NY) and transformed by incubation with Epstein-Barr
virus obtained from the supernatant of a productively infected
marmoset B-cell line. The transformed B lymphoblasts were
phenotyped with the B-cell-specific CD19 MAb and were found to
Grant sponsor: NIH; Grant numbers: R01-CA43058 and P30-AR41942.
*Correspondence to: Department of Dermatology, Yale University
School of Medicine, 333 Cedar Street, New Haven, CT 06519-8059, USA.
Fax: (203) 785-7637. E-mail: [email protected]
Received 19 September 1997; Revised 15 December 1997
TUMOR-SPECIFIC PEPTIDES IN CTCL
TABLE I ? AMINO ACID SEQUENCE OF THE Vb8 TCR
Vb8 =
IYFNNNVPIDDSGMPEDRFSAK
Native peptide:
MPNASFSTLKIQPSEPRD
;Vb8 0 ;D = 0 J2.1 =
AR idiotypic peptide: SAVYFCASS LIGG SYNEQF
;Vb8 0 ;D= 0 J2.3 =
SS idiotypic peptide: SAVYFCASS FV
MRYTQY
0 C2=
FGPGTRLTVLEDLKNVFPPEV1
1Conserved sequence is presented in medium type and idiotypic and
native peptides are in bold.
be .95% CD191. The B-cell lines were maintained in RPMI
1640/10% fetal calf serum (FCS; GIBCO, Gaithersburg, MD) and
used to present peptides to CD8 T cells in the enzyme-linked
immunosorbent assay (ELISA).
Dendritic cells were isolated from the peripheral blood of the
patients by Ficoll-hypaque flotation and incubated overnight with
granulocyte-macrophage colony stimulating factor (GM-CSF; 0.05
ng/ml) and interleukin 4 (IL-4; 800 units/ml; R&D Systems,
Minneapolis, MN), in RPMI 1640/10% FCS. The floating cells
were removed and the adherent population was recultured in the
same media containing cytokines and used as peptide-presenting
cells after 8 days of in vitro culture. The dendritic cell lineage of the
cultured cells was confirmed by phenotyping with MAbs (.90%
reactive with MAbs identifying CD11c, class I and II, CD80;
non-reactive with T- or B-cell-specific antibodies).
Idiotypic peptides
The sequence of the patient?s TCR was determined by cDNA
sequencing, as previously described (Longley et al., 1995). Potential target CDR3 peptides were identified by examination of the
sequence of the b chain of the patient?s TCR CDR3 for protein
sequences that could be accommodated by the peptide-binding
motifs of the class I molecules expressed on the patient?s cells.
Motif-conforming CDR3 peptides were synthesized with a RANIN
Symphony peptide synthesizer, purified by reversed-phase high
performance liquid chromatography (RP-HPLC) and their purity
confirmed by mass spectrometry in the Yale University Keck
Protein Synthesis Facility.
Evaluation of synthetic idiotypic peptides
To determine if cytotoxic T cells recognized synthetic idiotypic
peptides, 2 3 104 autologous B lymphoblasts were g-irradiated
(2,000 rads, cesium source) and pulsed with 10 痞 of the optimal
concentration of peptide (predetermined by a dose-response curve)
in RPMI 1640/0.5% bovine serum albumin (BSA), containing 30
ng/ml b2-microglobulin, and incubated at 37蚓 for 1 hr. CD8 T-cell
lines (predetermined to be cytolytic for autologous tumor cells;
Berger et al., 1996) were added to the pulsed lymphoblasts at
varying effector to target ratios. The cultures were incubated at
37蚓 for 18 hr, centrifuged and the supernatant fluid was harvested
and tested for release of tumor necrosis factor-a (TNF-a) in a
colorimetric ELISA according to the manufacturer?s directions
(R&D Systems). The plates were analyzed in an ELISA reader
(Sigma, St. Louis, MO).
In class I blocking studies, the B-lymphoblast targets were
preincubated, on ice, with a murine MAb W6/32 (1:10 dilution of
culture supernatant, HB95; ATCC, Rockville, MD) or an isotype
control antibody of irrelevant specificity. The cells were washed
prior to peptide pulsing and the TNF-a ELISA was performed as
described above.
305
Extraction of class I peptides from neoplastic CTCL T
lymphocytes
Approximately 100 3 106 CTCL tumor cells were centrifuged
(900g), washed and the pellet was resuspended in 10 ml of 0.1%
trifluoroacetic acid (TFA). The pelleted cells were ground in a
tissue grinder and transferred to a Dounce homogenizer. After 20
passes of the homogenizer, the homogenate was transferred to a
sonicator and sonicated twice with 10 sec bursts. The pH of the
sonicate was adjusted to 2.0 by the addition of 1% TFA. The
suspension was incubated on ice for 30 min with periodic manual
agitation and then centrifuged (2,600g, 30 min, 4蚓) in an
ultracentrifuge (Beckman, Palo Alto, CA) and the supernatant fluid
lyophilized. The lyophilized supernatant was dissolved in 2 ml of
0.1% TFA and filtered through a Centricon-10 concentrator (,10
kDa filtrate; Amicon, Beverly, MA). The filtrate was fractionated
on an RP-HPLC unit (Gilson, Middleton, WI) with a stepwise
linear acetonitrile (Aldrich, Milwaukee, WI)-HCL gradient using a
Waters (Milford, MA) Delta-PAK 3.9 3 150 mm C-18 column.
Fractions (0.5 ml) were collected at 1 min intervals and the liquid
evaporated in a SpeedVac (Savant, Farmingdale, NY). The fractions were dissolved in RPMI 1640/0.5% BSA for testing in a
TNF-a ELISA.
TNF-a ELISA of HPLC fractions
To identify fractions containing peptides that were stimulatory
for autologous CD8 lines, 2 3 104 autologous B lymphoblasts were
g-irradiated (2,000 rads, cesium source), pulsed with 20 痞 of the
HPLC fractions in the presence of b2-microglobulin and incubated
at 37蚓 for 1 hr. The TNF-a ELISA conditions were identical to
those previously described for testing the synthetic idiotypic
peptides. Reactive fractions were refractionated by passage over a
VYDAC C-18 2.1 3 250 mm column (Hesperia, CA), retested and
consistently reactive subfractions were submitted for mass determination (Yale University, Keck Facility) by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI; Woods et al.,
1995). Positive subfractions, containing peptides of a size appropriate for class I binding, were sequenced by Edman degradation.
Identification and synthesis of native peptides
To identify the protein of interest, the native peptide sequence
was compared with known protein sequences (BLAST network
service search) in the data bases (Brookhaven Protein Data Bank,
Swiss Prot, PIR, Genpeptide, Kabat sequences of proteins of
immunological interest and transcription factor protein data base).
Since the highest degree of homology was obtained when the
native amino acid sequence was compared to a sequence contained
in the constant region of the Vb8 TCR, synthetic peptides were
constructed based on the known TCR DNA sequence. The identified 7-mer native peptide sequence was extended by the addition of
DNA-sequence predicted amino acids to provide the optimum
length for class I binding and to add class I allele, motif-fitting
anchor residues.
RESULTS
To identify immunogenic class I MHC-associated peptides on
CTCL cells from the 2 patients, both theoretical and direct
strategies were pursued. First, the tumor cell?s TCR Vb chains were
sequenced and peptides derived from the idiotypic region were
selected based on their predicted ability to provide anchor residues
that would theoretically fit into the class I alleles expressed on the
patient?s lymphocytes. The potential immunogenicity of these
peptides was evaluated by pulsing the peptides onto autologous B
lymphoblasts and testing their capacity to stimulate TNF-a release
from cytotoxic CD8 T-cell lines that had previously been shown to
be cytolytic for the patient?s tumor cells (Berger et al., 1996).
Sequence of the idiotypic region of the Vb chain of the TCR in
CTCL patients
The sequence of the idiotypic region of the Vb chain of the TCR
was determined in 2 CTCL patients. Studies of the neoplastic T
BERGER ET AL.
306
cells from these 2 patients were facilitated by the large majority
(83?92% Vb81 TCR; Berger et al., 1996) of lymphocytes being
clonally expanded malignant cells. The conserved portion of the
Vb chain was identical in both patients? TCR sequence, while a
different diversity and joining region was encoded in the DNA of
the two patients? cells (Table I).
Synthesis of motif-fitting idiotypic peptides
To identify the class I alleles expressed on the CTCL patient?s
cells, the patient?s peripheral blood lymphocytes were histocompatibility typed (Table II). Only 4 of the 6 class I alleles (B7, B44, Cw4
and Cw7) expressed on patient SS?s cells had known peptidebinding motifs. Three candidate tumor-specific antigenic peptides
were identified in the idiotypic CDR3 of the TCR b-chain sequence
of patient SS?s malignant T cells. These 3 idiotypic peptides could
be accommodated by the known peptide-binding motifs of SS?s
Cw4 and Cw7 class I alleles. The 3 idiotypic peptide sequences
synthesized for patient SS all contained a hydrophobic amino acid
at position 9 (Table II). This is in accordance with a preference for a
hydrophobic COOH anchor residue, which has been observed in
most class I HLA alleles (Davenport and Hill, 1996). The 3
idiotypic peptides differed at positions 1?3 and these differences
might influence optimal MHC-peptide amino-terminus interaction
and thus the antigenicity of these peptides (Bednarek et al., 1991).
Both Cw4 and Cw7 class I alleles have a preference for an aromatic
amino acid at position 2, as an auxiliary anchor (Jung et al., 1993).
The S2 and S3 peptides both contain aromatic amino acids at
position 2 (S2: tyrosine, S3: phenylalanine) that might serve as
secondary anchors. Position 6 in the binding groove of both Cw4
and Cw7 alleles has a preference for a hydrophobic amino acid and
this preference is fulfilled only in the sequence of peptide S1 by the
presence of an alanine residue. The internal sequence of amino
acids was identical in all 3 peptides, but shifted in position.
Therefore, differences in immunogenicity of these peptides might
be attributed to changes in the anchoring positions that could result
in reduced TCR access to potentially stimulatory internal amino
acids and the shifted alignment of the internal amino acid sequence.
Histocompatibility testing of patient AR?s lymphocytes revealed
that 4 class I alleles (A11, A31, B52 and Cw6) had known
peptide-binding motifs. Three segments of the CDR3 of the TCR b
chain of CTCL cells from patient AR coded for peptides that were
found to conform to the binding motif of his B52 or Cw6 class I
alleles and were therefore selected as potential candidate antigenic
peptides. The carboxy-terminal amino acids of 2 of the synthetic
idiotypic peptides (R1 and R2) were preferred hydrophobic residues. Peptide R3 had a hydrophilic amino acid, glycine, at the
carboxy terminus that might reduce the class I binding ability of
this peptide. The B52 class I allele has a preference for an aromatic
amino acid at position 3 (Rammensee et al., 1995) and 2 of the 3
idiotypic peptides fulfilled this criteria (R1: tyrosine, R2: phenylalanine). Peptide R1 differed from peptides R2 and R3 at the amino
terminus due to the presence of an alanine residue and this
difference might influence the binding and antigenicity of this
peptide. The internal sequence of amino acids was identical in the 3
idiotypic peptides but shifted in position. Any observed differences
in the antigenicity of these peptides might result from the shift in
internal sequence of amino acids or the influence of differences at
the amino or carboxy terminus on the ability of the peptides to bind
to the class I allele.
Control peptides were synthesized with appropriately positioned
amino acids to serve as anchor residues that fit the class I alleles of
the patients? cells. The intervening amino acid sequences, in the
control peptides, theoretically would not be recognized by antiCTCL CD8 T cells. Control peptide C1 retained allele-binding
anchor residues for the motifs of Cw4 and Cw7, but was considered
unlikely to be recognized by anti-CTCL CD8 T cells, due to
inversion of the internal peptide sequence. Control, peptide (C2),
was a peptide isolated from human immunodeficiency virus type 1
(HIV-1) and was known to bind to the Cw4 allele (Johnson et al.,
1993). Since this peptide was not derived from either patient?s TCR
b chain, it was considered unlikely to be recognized by autologous
CD8 T cells.
Evaluation of the idiotypic peptides for cytotoxic T-cell
recognition
We had previously demonstrated that cytotoxic T-cell lines
derived from these 2 patients were specifically cytolytic for
autologous tumor cells and that this cytolysis was class I-restricted
(Berger et al., 1996). We also demonstrated that the cytolytic CD8
T cells released TNF-a in response to their autologous tumor target
(Berger et al., 1996) and that the level of cytokine released reflected
the cytolytic capacity of the patient?s CD8 effector cells. The CD8
T-cell lines that demonstrated the highest percentage of cytolysis
also released the greatest amount of TNF-a (Berger et al., 1996).
Cytotoxicity assays, in which tumor cells are lysed by CD8 T cells,
commonly yield low results unless the target cells are blasts.
Because CTCL cells respond poorly to mitogens and do not
become blasts, we chose to use the TNF-a release assay that we had
shown to be as reliable as cytotoxicity in studying immunity in
CTCL (Berger et al., 1996). These same CD8 T-cell lines have been
in culture for more than 1 year and have subsequently lost cytolytic
capacity but retained the ability to secrete TNF-a when stimulated
with the appropriate tumor cells. Since we and others (Coulie et al.,
1994) have shown that TNF-a release is a highly sensitive and
reproducible assay that correlates well with cytotoxicity, we have
evaluated the CD8 T-cell response to TCR-derived peptides with
this assay system.
The 3 candidate idiotypic antigenic peptides were synthesized
based on the CDR3 sequence of patient SS?s Vb8 TCR (Table I)
and pulsed onto patient SS?s autologous B-lymphoblast line. When
a 10 然 concentration of each of the 3 idiotypic peptides was
tested, only peptide S2 increased the production of TNF-a over the
baseline response observed when CD8 cells were cocultivated with
B cells without peptide (Fig. 1). This finding demonstrated that
only peptide S2 was specifically recognized by the autologous CD8
T-cell lines.
TABLE II ? TCR IDIOTYPIC PEPTIDES
Patient
HLA phenotype
DNA predicted amino acid sequence
SS
A29/32, B7/44, Cw4/7
SAVYFCASSFV MRYT
Vb8
D Jb2.3
AR
A11/31, B52/57, Cw6/?6
SAVYFCASSLIGGSYNE
Vb8
D1.1 Jb2.1
Idiotypic peptide sequence
S1: AVYFCASSF(1,2,3)4
S2: VYFCASSFV1,2,3,4
S3: YFCASSFVM1,2(3,4)
C1: VYSSFCAFV1,2(3)4
R1: AVYFCASSL(1,2)3,4
R2: VYFCASSLI1,2,3,4
R3: YFCASSLIG1,2,3
C2: SFNCGGEFF1,2(4)5
Peptide contains allele-specific sequence motif for: 1HLA-Cw4; 2HLA-Cw7; 3HLA-B52; and 4HLA-Cw6.?
motif-fitting sequence (i.e., not all anchoring residues present).?5Known HLA-Cw4 binding
T-cell epitope from HIV-1 gp120 (380?388) that can also fit the motif of the other alleles tested.?6? Second
allele could not be identified with conventional typing sera.
(1,2,3,4)Partial
TUMOR-SPECIFIC PEPTIDES IN CTCL
FIGURE 1 ? Identification of the S2 peptide as being immunogenic.
Only the S2 idiotypic peptide stimulates the CD8 T-cell line S16,
isolated from patient SS, to produce levels of TNF-a that exceed the
background level found when CD8 cells are cultured with autologous B
lymphoblasts without peptide. All idiotypic peptides were tested at a 10
然 concentration and the effector to target ratio (E/T) of CD8 cells to B
lymphoblasts was 3:1.
The CD8 response to the S2 peptide was shown to be dosedependent (Fig. 2), with a peptide concentration as low as 1 nM
producing TNF-a levels that exceeded background release. The
response to the S2 peptide was not restricted to a single CD8 line
established from patient SS. Four independently isolated CD8 lines
tested at different time points consistently released increased levels
of TNF-a when stimulated with either the 10 然 or 1 nM
concentration of the S2 idiotypic peptide loaded on autologous B
cells (Figs. 1?3). Moreover, the response was shown to be specific
for the S2 peptide, with significantly elevated TNF-a release
demonstrated at both 10 然 and 1 nM concentrations of the S2
peptide in comparison to the levels found with 3 controls. The
controls tested were a control peptide, C1, which was predicted to
bind to patient SS?s class I alleles, but not stimulate due to an
internally inverted sequence of amino acids; patient AR?s idiotypic
peptide, R2, which was derived from his CDR3 region and differed
in sequence from the S2 peptide at the 2 carboxy-terminal amino
acids; and as a background control, CD8 cells admixed with B cells
without peptide (Fig. 3). These results, viewed in the context of the
non-recognition of the other S1 and S3 idiotypic peptides, indicate
the high level of specificity of the CD8 response for the S2
idiotypic peptide.
Three peptides derived from the idiotypic sequence of the Vb
region of the TCR of patient AR (Table II) were also tested by serial
dilution and pulsing onto autologous B lymphoblasts. One of the 3
peptides, R1, was identified as immunogenic by the R8 CD8 T-cell
line (effector to target ratio of 21:1). The concentration of TNF-a
released from AR?s CD8 T-cell line was significantly ( p # 0.001,
10 然, 1 nM; p # 0.01, 0.1 nM) increased when peptide R1 was
added to autologous B lymphoblasts in comparison to the level
found when a control motif-fitting peptide C2, or parallel concentrations of other idiotypic peptides that differed in sequence by only 2
amino acids (R2, R3; Table II) or CD8 T cells added to B
lymphoblasts without peptide were tested.
Class I blocking of idiotypic TCR peptides
To confirm that the CD8 response elicited in the TNF-a release
assay was related to class I binding of idiotypic TCR peptides, the
307
FIGURE 2 ? Dose-response curve of the S2 peptide. Concentrations
of the idiotypic S2 peptide ranging from 10 然 to 1 nM stimulated the
S5 CD8 T-cell line to release levels of TNF-a that exceed the
background level found when CD8 cells were cultured with B
lymphoblasts without peptide. The effector to target ratio (E/T) was
6:1.
FIGURE 3 ? Specificity of the S2 peptide. The S2 peptide was tested
at 10 然 and 1 nM concentrations with 2 independently isolated CD8
T-cell lines. The S2 peptide stimulated significantly more ( p # 0.03
and p # 0.004) TNF-a release from both CD8 lines than the control
peptide (C1) or an idiotypic peptide synthesized from the CDR3 of
patient AR (R2) or the baseline control of CD8 cells cultured with B
lymphoblasts without peptide. The effector to target ratios (E/T) were
5:1 for the S20 CD8 line and 3:1 for the S11 CD8 cell line.
capacity of the peptides to access the class I binding groove was
blocked by preincubation of the B lymphoblasts with an antibody
that recognizes all class I molecules (W6/32). Autologous B
lymphoblasts from patient SS or AR were preincubated with an
anti-class I antibody or an isotype-matched control murine ascites
of irrelevant specificity. Recognition of peptide S2 by a CD8 T-cell
line was reduced to control levels after the class I molecules on the
autologous B lymphoblasts were blocked with the anti-class I
308
BERGER ET AL.
FIGURE 4 ? Class I blocking of the response to the S2 idiotypic
peptide. Preincubation of the B lymphoblasts with the W6/32 MAb
inhibited the ability of peptide S2 to bind and stimulate release of
TNF-a from the CD8 T-cell line S21. Incubation of the B cells with
murine ascites (ASC) of the same isotype as W6/32 but with an
irrelevant specificity does not inhibit the binding of the S2 peptide,
resulting in elevated release of TNF-a above the background level
found when CD8 cells are cultured with B cells without peptide or a
control peptide (C1) or an idiotypic peptide synthesized from the
CDR3 of the other CTCL patient, AR (R2). The effector to target ratio
(E/T) was 5:1.
FIGURE 5 ? Evaluation of HPLC fractionated TFA-eluted class I
peptides. Class I peptides were eluted from CTCL patient AR?s tumor
cells with TFA and fractionated with a RP-HPLC. The fractions were
pulsed on autologous B lymphoblasts and tested with AR?s CD8 T-cell
line R8. Several reactive fractions (25, 26 and 29) reproducibly
stimulated increased release of TNF-a over that found when CD8 cells
were cultured with B cells without peptide. The effector to target ratio
(E/T) was 8:1.
antibody, W6/32 (Fig. 4). Incubation of the B lymphoblasts with
control murine ascites did not inhibit the binding or presentation of
the S2 peptide and resulted in enhanced release of TNF-a. Class I
blocking of the TNF-a response to the S2 peptide was confirmed
with 2 additional independently isolated CD8 T-cell lines (S3 and
S12; results not presented).
In a similar fashion, the R8 CD8 T-cell response to the R1
peptide was inhibited by class I blocking with W6/32, while
preincubation of the B lymphoblasts with murine ascites permitted
enhanced TNF-a release over control levels (C2 peptide or CD8
cells cocultivated with B cells without peptide; results not presented). These results demonstrate that recognition of idiotypic
TCR peptides is dependent on effective class I loading and
recognition.
Isolation and sequence determination of native CTCL peptides
The presence of an immune response in CTCL patients to
synthetic idiotypic TCR peptides suggests that the tumor cells
might present native peptides that could be recognized as immunogenic by CD8 anti-tumor cell lines. To pursue this possibility, we
isolated native peptides from class I molecules on the CTCL tumor
cells and determined their sequence. TFA-extracted peptides from
patient AR?s tumor cells were fractionated using a RP-HPLC and
the fractions tested for CD8 recognition by loading on autologous
B lymphoblasts and measuring TNF-a release. Several reactive
fractions were identified (Fig. 5) and fractions 21?25 were found to
contain peptides of an appropriate molecular mass by MALDI
which indicated that they could have been derived from 8?11
amino acid class I peptides. These fractions were subfractionated,
retested and several reactive subfractions identified (Fig. 6) that
contained peptides that were identical in mass to the peptides found
in the original fractions.
Subfraction 25-3 contained sufficient material for sequencing
and a 7 amino acid peptide sequence was identified (Table III). This
sequence, MPRASES, matched 5 of 7 amino acids encoded in the
FIGURE 6 ? Evaluation of subfractions of the original reactive
fraction 25. Fraction 25 was subfractionated by RP-HPLC and several
reproducibly reactive subfractions were identified (2, 3, 5 and 6).
Subfraction 25-3 stimulated increased TNF-a release above that found
when R8 CD8 cells were cultured with B cells without peptide at an
effector to target ratio (E/T) of 8:1. Subfraction 25-3 was found to
contain peptides that were identical in mass, by MALDI, to those found
in the original fraction 25 and was submitted for sequencing.
conserved portion of the TCR Vb8 chain. No known protein could
be identified in the protein data base that provided a better match
for this sequence, suggesting that the identified peptide antigen is
novel and may be unique to this particular set of CTCL cells. Since
the sequence lacked appropriate anchor residues and was shorter
than the optimum class I groove-fitting length of 8?10 amino acids,
additional residues based on the known DNA sequence for the Vb8
chain were added at the carboxy terminus.
TUMOR-SPECIFIC PEPTIDES IN CTCL
309
TABLE III ? NATIVE PEPTIDE SEQUENCE
Edman sequence:
DNA sequence:
Synthesized peptides
V-1:
V-2:
V-3:
1Divergent
MPRASES1
MPNASFSTLKI
MPNASFSTLKI
MPRASESLK
MPRASESTL
sequences are noted in bold.
CD8 T-cell recognition of native peptides
Three peptides were synthesized and tested to determine if they
were recognized as immunogenic by AR?s CD8 T-cell lines. The
synthesized V-1 peptide contains the entire 11 amino acid sequence
that was predicted by the DNA-encoded TCR constant region
sequence, with the amino acids asparagine and phenylalanine
replacing the amino acids argenine and glutamic acid that were
identified in the native peptide sequence. The carboxy-terminal
amino acids leucine, lysine and isoleucine provide anchor residues
that can be accommodated in AR?s A11, B52 or Cw6 class I alleles.
Leucine and isoleucine are hydrophobic residues and are therefore
preferred carboxy-terminal anchor residues. Lysine is a positively
charged amino acid and could represent a preferred anchor for the
motif of the class I A-11 allele (Falk et al., 1994). Although 11
amino acids exceeds the optimal 8?10 class I peptide size, the
internal amino sequence might be accommodated, particularly in
the A11 allele which is known to bind longer peptides, through
internal bulging of the peptide chain, between the anchor residues.
Peptides exceeding 11 amino acids in length have been isolated
from class I molecules (Falk et al., 1994).
The V-2 peptide contains the native peptide sequence with the
addition of 2 motif-fitting anchor residues, leucine and lysine. The
threonine residue was omitted to provide a length of 9 amino acids
while preserving the availability of the 2 strong anchor residues for
the A11, B52 or Cw6 class I alleles. Peptide V-3 extends the native
peptide sequence with the next 2 DNA predicted amino acids
threonine and leucine, providing one strong anchor residue for the
B52 or Cw6 alleles.
When these peptides were tested, the V-1 peptide was not
recognized, while both V-2 and V-3 provoked the CD8 lines to
release elevated amounts of TNF-a which were similar to the level
of TNF-a produced by the initial peptide pool (70?80 pg/ml; Fig.
7). In addition, the V-2 and V-3 peptides stimulated increased
release of TNF-a from AR?s CD8 T cells when compared to the
levels obtained when the synthetic R1 peptide was tested (20?35
pg/ml). These results imply but do not conclusively prove that
peptides partially derived from the conserved portion of the TCR in
CTCL patients may serve as immunogens for autologous CD8 T
cells and may be useful in dendritic cell-based vaccine protocols
designed to boost the immune response to the tumor cells.
Recognition of the native peptide sequence by CD8 cells from
other Vb81 CTCL patients
Since the native sequence isolated from tumor cells of patient
AR was homologous to the conserved sequence of the Vb8 chain,
this sequence was also present in the TCR of patient SS. We tested
whether CD8 cells from patient SS could recognize the native
peptides when they were presented by autologous dendritic antigenpresenting cells, a prerequisite for utility of this sequence as a
vaccine component. A CD8 cell line from patient SS recognized
peptide V-2 but not V-3 or a control peptide when the peptides were
presented on autologous dendritic cells (Fig. 8). The V-2 peptide
sequence contains at least 1 anchor residue consistent with motifs
found in patient SS?s B7, Cw4 and Cw7 class I alleles. These results
indicate that the native peptide is recognized by CD8 lines isolated
from 2 different CTCL patients whose malignant T cells express a
Vb8 TCR containing the conserved sequence from which the
native peptide was synthesized.
FIGURE 7 ? Evaluation of the synthesized native peptides. Three
peptides were synthesized based on the native peptide sequence
extended with DNA sequence-predicted amino acids. Peptides V-2 and
V-3 pulsed on AR?s B lymphoblasts stimulated increased release of
TNF-a from AR?s CD8 T-cell line R9 at an effector to target ratio (E/T)
of 9:1. The amount of TNF-a produced by the CD8 line when it was
presented with either the V-2 or V-3 peptide exceeded that produced by
presentation of the V-1 peptide or when CD8 cells were cultured with B
cells without peptide.
FIGURE 8 ? Recognition of the native peptides pulsed on dendritic cells
by SS?s CD8 cells. Native peptide V-2 isolated from patient AR?s tumor
cells also stimulated a CD8 T-cell line S18 from patient SS to produce
elevated levels of TNF-a in comparison to the level found with the V-3
peptide, a control peptide (C1) or CD8 cells cultured with dendritic
cells without peptide. The effector to target ratio (E/T) was 2:1. The
culture supernatants were diluted 1:2 to achieve values within the
reference range of the standard curve of the assay, due to the high
background level of TNF-a produced by the dendritic cells alone.
DISCUSSION
Our results reveal that synthetic peptides, designed to represent
segments of the CDR3 idiotypic region of the b chain of
clone-specific TCR of CTCL cells, are recognized by autologous
310
BERGER ET AL.
CD8 cells as immunogenic. This finding supports the possibility
that clonotypic TCR proteins are at least one source of tumorspecific antigenic peptides in CTCL.
We also report that an antigenic native peptide obtained directly
from CTCL cells is a novel protein, partially homologous in amino
acid sequence to a segment of the variable region of the clonal TCR
expressed on the same population of CTCL tumor cells. Although
this peptide is not definitively derived from TCR proteins, this is
direct evidence that CTCL cells display immunologically relevant
peptides.
The TCRs of malignant T cells provide a potential source of
tumor antigens in T-cell malignancy, since these protein heterodimers are clonotypically expressed at high levels on the tumor
cell surface and are likely to be abundantly represented in the
endoplasmic reticulum pool of candidate class I binding peptides.
Both murine and human cytotoxic CD81 T-cell clones can recognize peptides derived from the conserved region of the Vb of the
TCR of autologous CD41 T cells (Jiang et al., 1995; Ware et al.,
1995). Vaccination with peptides derived from the framework 3
region of the Vb8.2 TCR suppresses experimental autoimmune
encephalomyelitis and collagen II-induced arthritis (Kumar et al.,
1997). Extrapolation of these results to humans suggests that
TCR-derived peptides may be clinically relevant targets for
specific immunotherapy of both autoreactive and malignant T-cellmediated diseases.
The TCR is a heterodimer consisting of an a and b chain (Rowen
et al., 1996), made up of a constant and a variable region. The
variable portions are composed of the complementarity determining regions that contact the MHC/peptide bimolecular complex.
The third complementarity region (CDR3) encoded by the hypervariable region directly interacts with the MHC-held peptide
antigen. Diversity in this region generates a unique patient-specific
amino acid sequence that is clonotypically expressed by the TCR of
the malignant T cells in CTCL.
We have previously reported (Berger et al., 1996) that CD8 T
cells can recognize antigenic peptides on autologous CTCL cells in
the context of class I MHC glycoproteins. Our current results
extend these observations and suggest that clonotypic peptide
derivatives of the CTCL TCR are a source of such tumor-specific
antigens. TCR peptides constructed under theoretical constraints
imposed by MHC motif-binding requirements were able to sensitize autologous target B lymphoblasts, so that they could stimulate
release of TNF-a from anti-CTCL CD8 T cells from the same
individual.
In some individuals with advanced CTCL, despite the expansion
of malignant CD41 T cells, a functional cytotoxic CD8 T-cell
compartment persists (Berger et al., 1996). Although, this CD8
cells are unable to eradicate the malignancy, they are specifically
cytotoxic for the tumor cells and may play a role in ameliorating the
course of the disease.
Cytotoxic T lymphocytes recognize 8?10 amino acid peptides
derived from proteasomal degradation of endogenous proteins that
are transported into the endoplasmic reticulum by the TAP system,
where they associate with class I MHC molecules, prior to export to
the cell membrane. Class I MHC alleles have peptide-binding
motifs that designate preferred positional anchor residues (Rammensee et al., 1995). If the amino acid sequence of the peptide
contains appropriate amino acids that conform to the allele-specific
motif, the peptide will fit in the class I binding groove. Based on
pooled amino acid sequence data, it is possible to scan the sequence
of potentially immunogenic peptides and select those that are most
likely to be accommodated in a given class I allele (Rammensee et
al., 1995). Binding of these candidate peptides to the class I
molecules, and their capacity to sensitize target cells to attack by
selective CD8 T cells, can then be assessed. Using this approach,
we have demonstrated a high degree of specificity in the response
to a sequence-predicted idiotypic peptide, as shown by the
consistent recognition of a single peptide, by all the CD8 lines
tested from an individual patient. The other candidate idiotypic
peptides tested differed from immunogenic peptides by only 1?2
amino acids and were not effectively recognized by the CD8 cells.
Substitutions at the amino or carboxy terminus of these peptides
may have reduced their ability to bind to the class I allele or their
stimulatory capacity for the autologous CD8 cells. These results
suggest that immunization protocols should be tailored to optimally
select reactive peptides and that simple conformation to MHC
binding rules will not suffice for identification of relevant immunogenic epitopes.
In our second strategy, a native peptide was identified by direct
elution of class I molecules which bears a high degree of homology
to the known sequence of the constant portion of the Vb8 TCR. The
absence of total sequence homology suggests that this peptide
might be derived from a novel protein that is not represented in the
present data bases. Alternatively, the sequence divergence might
arise from errors in the sequencing process due to the low
concentration of the relevant peptide in the extracted pool.
While the derivation of the native peptide from the TCR remains
to be conclusively demonstrated, its recognition (after extension
with Vb8 TCR DNA sequence-predicted amino acids) by tumorreactive CD8 T-cell lines suggests that this peptide may be a
relevant epitope for therapy in CTCL. The specificity of the
immune response in CTCL for the native peptide is supported by
the restricted reactivity of the CD8 T-cell lines for CTCL tumor
cells (Berger et al., 1996). The same CD8 T-cell lines used in this
study were demonstrated to be cytotoxic for autologous tumor
cells, and non-lytic when stimulated with normal lymphocytes,
T-cell blasts or autologous B lymphoblasts. Therefore, the CD8
T-cell lines recognize peptides that are restricted to CTCL tumor
cells. While a small normal Vb8 population in the peripheral blood
of CTCL patients might be targeted by an anti-Vb tumor response,
depletion of this subset would be acceptable in the context of
reduction of the massively expanded tumor cell clone.
The ability of dendritic cells to present one of the native peptides
implies that peptide-pulsed dendritic cell vaccine immunotherapy
may be useful in the treatment of CTCL. Because of their display of
those accessory molecules which provide necessary second signals
to initiate immune responses, dendritic antigen-presenting cells
bearing tumor peptides in their class I molecules are efficient
components of experimental anti-tumor cellular vaccines.
To extend these findings, we are sequencing additional peptide
pools derived from CTCL tumor cells, to identify additional
candidate immunogenic peptides. In addition, murine studies in the
2B4.11 mouse model of T-cell malignancy are in progress to define
the optimal approach for peptide vaccination against TCR epitopes.
The existence of tumor-specific peptide antigens on malignant cells
in CTCL, a relatively common T-cell malignancy, may permit the
development of peptide-based immunotherapy for this disorder.
ACKNOWLEDGEMENTS
This work was supported by NIH grants R01-CA43058 and
P30-AR41942.
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