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Surgery Branch, National Cancer Institute, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the emphasis on CD8+ T cell responses, there is growing evidence to support an important role for CD4+ T cells in antitumor immunity. Murine studies have demonstrated that CD4+ T cells exert helper activity through the induction and maintenance of CD8+ T cells and B cells and have direct and indirect effects on tumor cells, including those deficient in MHC class II (reviewed in Ref. 6). In humans, CD4+ T cells play a crucial role in the induction of several autoimmune diseases (7) and in the resistance to pathogens (8, 9). The evidence for their role in human cancer is less substantial and limited to studies that demonstrate both tumor-specific CD4+ and CD8+ T cells at the tumor site (10, 11). In addition, tumor-specific IgG Abs have been detected in cancer patients, thus implying the active role of CD4+ T cells in isotype switching (12).
There is accumulating evidence that the combined application of class I and class II epitopes derived from the same tumor Ag may potentiate antitumor effector function and long-term immunity (12A, 13, 14, 15, 16). CD4+ T cells activate dendritic cells (DC),2 principally through the interaction of CD40 and its ligand. "Conditioned" DC can, in turn, cross-present Ag to cognate CD8+ T cells at the tumor site or in remote locations (13, 17, 18, 19, 20). Therefore, a need exists for the development of techniques useful in the identification of tumor-associated MHC class II-restricted epitopes in human cancers. While the targets of several CD4+ T cells with potential anti-tumor activity have been identified (21, 22, 23, 24, 25, 26), no reliable, repeatable method for class II-restricted epitope identification has been established.
We sought to identify an epitope derived from the melanocyte differentiation Ag, human (h) gp100. More than 75% of melanomas express h-gp100 (27) and CD8+ T cells derived from tumor-infiltrating lymphocytes (TIL) recognize h-gp100 presented on the surface of melanoma cells (28). The adoptive transfer of TIL that have specificity for h-gp100 are significantly more efficacious at mediating tumor regression than TIL with specificities for other Ags (29). Thirteen different MHC class I-restricted epitopes from h-gp100 have now been identified (2). Vaccination with a modified, "anchor-fixed" h-gp100 CD8+ T cell epitope in combination with IL-2 has been reported to result in a 42% response rate in patients with metastatic melanoma (3, 30). However, only a minority of patients responded clinically to this vaccine regimen, and most of the responses observed were transient in nature (3).
To increase the immunogenicity and therapeutic efficacy of vaccines comprised of h-gp100 CD8+ T cell epitopes, we sought to provide Ag-specific CD4+ T cell help. To identify a shared DR-restricted epitope from h-gp100 of potential clinical value, we targeted an MHC class II allele with the highest prevalence in patients with metastatic melanoma (31). Our strategy involves the immunization with recombinant h-gp100 protein of DR4-IE transgenic (Tg) mice that express a chimeric mouse-human MHC class II molecule from HLA-DRB1*0401. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes derived from a computer-assisted prediction algorithm.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine class II-deficient, DR4-IE Tg mice express HLA-DRA-IE-
and HLA-DRB1*0401-IE-ß chimeric genes containing the Ag-binding 1
and ß1 domains from the human HLA-DRA and HLA-DRB1*0401 molecules,
respectively, with the remaining domains comprised of murine
IEd-2 and IEd-ß2
chains (32). Female DR4-IE Tg mice 6- to 10-wk-old were
used for all experiments. Founder mice were obtained through Paul
Lehmann (at Case Western Reserve University, Cleveland, OH) and
maintained and bred in a barrier facility (Biocon, Rockville, MD). Mice
were confirmed to have two allelic copies of the DR4-IE chimeric
transgene using PCR amplification of genomic DNA (oligonucleotide
primers previously described (32)). Eighty microsatellite
markers (Research Genetics, Huntsville, AL), evenly distributed across
the murine genome, were used to assess genetic similarity to C57BL/6n
mice (33). Purebred DR4-IE Tg mice were matched at 77 of
80 markers and accepted the normal growth of murine melanoma B16
(H-2b) (data not shown).
HLA typing
HLA serotypes and DNA genotypes of fresh human PBMC and tumor cell lines were determined by the National Institutes of Health HLA Laboratory, as described (34). The MHC class II genotype of patient "Te" was HLA-DRB1*0401, 1501, DQB1*0302, 0602, DRB4*01, DRB5*0101; the genotype of patient "Th" was HLA-DRB1*0401, 1501, DRQB1*0302, 06, DRB4*01, DRB5*0501; and the genotype of patient "Wa" was HLA-DRB1*0401, 1501, DRQB1*0301, 06, DRB4*01, DRB5*0501. The HLA-DRB1* genotypes of tumor lines used in the following experiments included 697 Mel and EBV-B (0401,1501); 888 Mel (1502,1601); 1102 Mel and EBV-B (0401,1502); 1359 Mel and EBV-B (0401,0301); and 1498 Mel (0401,0802).
Peptides
The 21 h-gp100 peptides used in the initial screening of candidate epitopes were synthesized using a solid-phase method based on fluorenylmethoxycarbonyl (F-moc) chemistry on a multiple peptide synthesizer (Model AMS 422; Gilson, Worthington, OH). The molecular masses of peptides were verified by laser desorption mass spectrometry (Biosynthesis, Lewisville, TX). All other peptides were synthesized by Macromolecular Resources (Fort Collins, CO) to a purity >99% as assessed by HPLC and laser desorption mass spectrometry.
Recombinant h-gp100 protein
The gene encoding h-gp100 (28) was amplified by PCR with primers (forward: AGG,CGC,AGA,CTT,ATG,AAG,CA; reverse: CTG,CCC,AAG,GCC,TGC,TTC,TTG) designed to delete the N-terminal 23 amino acids (probable signal sequence) and the C-terminal 66 amino acids (probable transmembrane region). The truncated gene was then cloned into the PET28a+ expression vector (Novagen, Madison, WI) and transformed into BL21(DE3) Escherichia coli (Novagen). E. coli were grown to OD600 0.6, then protein expression was induced with isopropyl ß-D-thiogalactoside 1 µg/ml for 3 h. The bacteria were harvested; inclusion bodies were isolated and lysed in 6 M urea, then proteins were purified by preparative scale SDS-PAGE in a Prep cell (Bio-Rad, Hercules, CA) electrophoresis chamber. Protein fractions at 61.5 kDa (theoretical molecular mass of truncated h-gp100) were collected, dialyzed, and precipitated, and a purity of >80% was estimated based on SDS-PAGE with Colloidal Blue (Novex, San Diego, CA) staining.
DRB1*0401 computer-assisted algorithm
To generate a list of candidate h-gp100 epitopes capable of
binding HLA-DRB1*0401, we selected an algorithm based on work by
Alessandro Sette and his colleagues (35), which uses
numerical values corresponding to the relative strength of individual
amino acids along a nine-residue core region critical for MHC binding.
By multiplying each value from P1 through P9, an algorithm value is
obtained. A computer program was written that would permit the input of
protein sequences of any length, generating a list of nine-residue
peptides in order of decreasing predicted binding affinity down to an
algorithm score that predicts 75% of all binders (<2.617; an
arbitrary cut-off point) (35). The full-length h-gp100
amino acid sequence (GenBank, accession no. M77348; 661 aa) was
analyzed with this program, yielding 92 predicted candidate epitopes.
The 24 peptides with the highest MHC binding affinities (Table I
) were selected for further study, and
two amino acids based on the native sequence were added to both the N
terminus and C terminus. Twenty-one 13-residue candidate epitopes were
then synthesized. Peptides ranked 5, 20, and 24 (start positions 614,
613, and 608, respectively) were omitted from analysis because their
start positions were distal to the C terminus of the truncated
recombinant h-gp100 protein used for immunization experiments (see
above). The computer-assisted algorithm is currently available on the
Surgery Branch, National Cancer Institute web site:
http://surgery.nci.nih.gov.
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DR4-IE Tg mice were immunized once with either 50 µg of the truncated, recombinant h-gp100 protein or with 100 µg of h-gp10046–58 peptide to initiate T cell lines A9895 and C1056, respectively. Both recombinant protein and peptide h-gp10046–58 were emulsified in CFA (final volume, 100 µL), divided equally, and administered s.c. into the rear footpads. On day 12, the animals were sacrificed and bilateral hindlimb popliteal and inguinal lymph nodes were harvested. Cells were cultured in 24-well plates at 5 x 106 cells per well with peptide h-gp10046–58 at 100 µg/ml. Twelve days after the first ex vivo stimulation, both lines were restimulated with peptide-pulsed, irradiated, syngeneic DR4-IE splenocytes. Splenocytes were pulsed with h-gp10046–58 at 600 µg/ml for 3 h at 37°C, washed, irradiated with 3000 rad, then added to each T cell culture at a 10:1 ratio (5 x 106 APCs per well). Culture medium (CM) containing IL-2 (Chiron, Emeryville, CA) at 7.5 CU/ml was added 2 days after the stimulation (day 14). Thereafter, lines were stimulated and maintained using the same methods every 13–15 days. Both lines were found to be >95% CD4+ by flow cytometry, following three in vitro stimulations (data not shown).
Generation of human CD4+ T cell lines
Fresh PBMC from three HLA-DRB1*0401-positive patients (Te, Th, Wa) with documented h-gp100 expressing metastatic melanoma were used for ex vivo peptide sensitization (see above). For patient Te, PBMC were cultured at 1.5 x 105 cells per well in a flat-bottom 96-well plate in CM containing h-gp10044–59 at 50 µM and the mAb MAR4 (anti-CD29; IgG1; PharMingen) at 2.5 µg/ml (36). On day 9, cells were restimulated with autologous, peptide-pulsed, irradiated (3000 rad) PBMC. PBMC were pulsed with h-gp10044–59 at 100 µM for 3 h at 37°C, then washed and added at 1.5 x 105 cells per well in CM containing IL-2 at 50 CU/ml. On day 23, all wells from the original parental 96-well plate were tested for specific peptide reactivity using HLA-DRB1*0401-matched EBV-B cells pulsed with h-gp10044–59. The most reactive wells were selected and stimulated with autologous, irradiated, pulsed (as above) PBMC in 24-well flat-bottom plates at a 10:1 ratio (5 x 106 APCs per well). Two weeks after restimulation, each subline was screened for specific peptide and tumor reactivity. One line, Te-22, was selected for further analysis. For patients Th and Wa, fresh PBMC were initially depleted of CD8+ T cells with mAb-coated magnetic beads (Dynal, Lake Success, NY). Remaining cells were added to 96-well flat-bottom plates at 3 x 105 cells per well and cultured with GM-CSF 200 U/ml, IL-4 100 U/ml, and h-gp10044–59 at 50 µM. IL-2 at 25 CU/ml was added on day 4, and each microculture was restimulated on day 8 with pulsed (100 mM), irradiated (3000 rad), allogeneic DRB1*0401-matched EBV-B cells (106 per well). On day 22, individual wells were pooled as single lines and tested for peptide and tumor reactivity. All T cell lines were found to be >95% CD4+ by flow cytometry, following three in vitro stimulations (data not shown).
Other cell lines
EBV-B cell lines 697 EBV-B, 1102 EBV-B, 1359 EBV-B (and 1359 EBV-B transfectants); melanoma lines 697 Mel, 888 Mel, 1102 Mel, 1300 Mel, 1498 Mel; breast cancer line MDA-231; and stable transfectant Myeloma #25 (expressing chimeric DR4-IE Tg MHC; a gift from K. Ito) were maintained in the appropriate CM, as previously described (murine (37) or human (38)). The human CD8+ T cell clone (K4/H5) reactive against h-gp100209–217 and human melanoma in the context of HLA-A*0201 (M. E. Dudley; unpublished observation) was maintained in CM with IL-2 (50 CU/ml). To generate 1359 EBV-B transfectants expressing h-gp100 or green fluorescence protein (GFP), the episomal vector pEAK-8 (Edgebiosystems, Gaithersburg, MD) containing the appropriate inserts was electroporated into B cells. Autologous human DCs were generated from fresh PBMC by culturing in CM with 500 ng/ml of both GM-CSF and IL-4 for 7 days.
Assessment of T cell responses
To assess ex vivo reactivity to either candidate epitopes or to
the peptide h-gp10046–58, lymphocytes from lymph
nodes were harvested from Tg mice 12 days after vaccination (as
described above) and plated at 5 x 105
cells per well in 96-well U-bottom plates. Protein or peptides were
added to each microculture well at 100 µg/ml, then cultured for
24 h. To assess the reactivity of the CD4+
murine T cell lines A9895 and C1056, DR4-IE splenocytes or EBV-B cells
were incubated for 12–18 h with peptide, protein, or with freeze-thaw
lysates of tumor cells (105 cell equivalents per
well). APCs were then cocultured (105 cells per
well) with T cells (105 cells per well) in
U-bottom 96-well plates for 24 h. To assess recognition of whole
tumor cells, individual human or murine CD4+ T
cell lines were cultured in U-bottom 96-well plates at
105 cells per well in the presence of irradiated
tumor cells (12,000 rad) at 105 cells per well
for 24 h. All melanomas, except 1102 Mel (constitutive expression
of MHC class II), were pretreated with IFN-
500 U/ml for 48 h
to up-regulate MHC class II expression. Culture supernatants were
assayed for IFN-, GM-CSF, IL-4, IL-5, and TNF- using commercially
available ELISA kits (Endogen, Woburn, MA). In addition, commercially
available ELISA kits (Endogen) were used to assess chemokines
macrophage inflammatory protein (MIP)-1, MIP-1ß, and RANTES.
ELISAs for chemokines MIP-3 and MIP-3ß were prepared by coating
96-well plates at 2 µg/ml with primary Abs (PeproTech, Rocky Hill,
NJ) and were detected with biotinylated secondary Abs at 2 µg/ml (R&D
Systems, Minneapolis, MN). In blocking experiments, mAbs were
used to inhibit specific Ag recognition by T cells. Abs
included L243 (against HLA-DR; IgG2a; PharMingen), W6/32 (against
HLA-A, B, C; IgG2a; American Type Culture Collection,
Manassas, VA), and H129.19 (against murine CD4; IgG2a;
PharMingen). For cytolysis experiments, target cells were pulsed with
200 µCi of 51Cr for 60 min, washed, and
distributed onto U-bottom 96-well plates at 104
cells per well with effector T cells added at varying E:T ratios.
Percent lysis was calculated using the standard formula:
[(experimental 51Cr release - spontaneous
release)/(total release - spontaneous release)] x 100. All
experiments were performed between two and four times with similar
results using different but comparable targets, thus demonstrating the
generalizability of the experimental findings. For example, a variety
of DR4+ or DR4- and
gp100+ or gp100- targets
were used to address the specificity of T cell responses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the predictive capacity of the computer-assisted algorithm described above (35), we selected protein sequences from pre-proinsulin and OspA. HLA-DRB1*0401-restricted autoimmune epitopes for both proteins have been described and identified by separate methods (39, 40). The algorithm predicted the correct epitope in the number 3 and number 1 ranked positions, respectively (data not shown), thus demonstrating the potential for accurately forecasting candidate HLA-DRB1*0401 epitopes. In each case, the program forecasted >90 candidate epitopes.
To identify a shared HLA-DRB1*0401-restricted epitope for h-gp100, we
screened 21 of the top 24 candidate epitopes for h-gp100
(Table I
) for ex vivo peptide reactivity using DR4-IE Tg mice
previously vaccinated with recombinant h-gp100 protein. IFN-,
GM-CSF, and IL-4 cytokine levels were measured by ELISA 24 h after
the initial stimulation with candidate peptide epitopes. Only one
peptide, h-gp10046–58 (ranked 1 of 92), elicited
significant amounts of IFN- secretion (168 pg/ml; Table I) and
GM-CSF (10 pg/ml; data not shown), while IL-4 production was not
detected with any of the peptides, except that induced with a control
anti-CD3 mAb (data not shown). Importantly, a longer version of
this peptide, h-gp10044–59, was previously
eluted from MHC II-peptide complexes from DR4+
melanoma cells (41, 42). This peptide was included
in subsequent experiments and compared with peptides
h-gp10046–58 and
murine-gp10046–58 (see Table II).
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To characterize the specific reactivity of murine
CD4+ T cells to peptides
(h-gp10044–59 and
h-gp10046–58), melanoma lysate processed by
APCs, and intact melanoma cells, two distinct lymphoid lines (A9895 and
C1056, as described above) were generated. Using DR4-IE Tg
splenocytes as APCs, specific reactivity was detected to peptides
h-gp10044–59 and
h-gp10046–58, as well as to the recombinant
h-gp100 protein and a lysate of a h-gp100 expressing human melanoma
(1300 Mel) (Fig, 1, A and
B). As seen previously, no recognition was detected to the
murine variant of gp10046–58, the control
peptide HA306–318, or the irrelevant tumor
lysate (breast carcinoma; MDA-231). Comparable reactivity to peptides
h-gp10044–59 and
h-gp10046–58 was seen when using a human myeloma
line expressing the DR4-IE chimeric MHC as a source of Ag presentation
(data not shown). In both sets of experiments, recognition was markedly
stronger using the longer peptide h-gp10044–59,
indicating that amino acids outside the nine-residue core were
important in enhancing T cell recognition. To assess the recognition of
endogenously processed h-gp10044–59, A9895 and
C1056 CD4+ T cell lines were cultured for 24
h with various human tumor targets (Fig. 1, C and
D). HLA-DRB1*0401-matched tumors expressing h-gp100 (697
Mel, 1498 Mel, 1102 Mel) were recognized by both T cell lines. Control
tumors 888 Mel and 697 EBV-B, mismatched for the appropriate
restriction element and Ag expression, respectively, were not
recognized by either murine CD4+ T cell
line.
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, IL-2, GM-CSF, TNF-, but not IL-5, consistent with a Th1
cytokine profile, and, in addition, produced significant amounts of the
chemokine RANTES (Fig. 2A). To
verify that recognition of melanoma by line A9895 was CD4 directed and
HLA-DR restricted, mAbs directed against murine CD4, HLA-DR, and human
class I were used to inhibit T cell recognition (Fig. 2B). A
human CD8+ T cell clone (K4/H5), previously
shown to recognize h-gp100209–217 in the
context of HLA-A*0201 (see Materials and Methods), was used
to test for nonspecific or deleterious effects of either mAb on target
cell interaction. Monoclonal Abs L243 (anti-HLA-DR) and H129.19
(anti-murine CD4) inhibited recognition of 697 Mel and 1102 Mel by
line A9895. In the control group, recognition by the
CD8+ T cell clone K4/H5 of 697 Mel and 1102 Mel
(both HLA-A*0201) was inhibited by mAb W6/32 (anti-class I) and
unaffected by the mAb directed against murine CD4. A9895
CD4+ T cell line was also tested for cytolytic
activity against two h-gp100-expressing melanoma lines (Fig. 2C). Specific lysis of 1102 Mel (DR4+
melanoma) at 61% was observed at an E:T ratio of 30:1, while no
evidence of lysis was observed to 888 Mel (DR4-
melanoma). To confirm that lysis of 1102 Mel was indeed DR restricted,
the mAb L243 (anti-DR) was shown to specifically block cytolysis by
line A9895 (data not shown).
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To compare T cell recognition of the algorithm-predicted
h-gp10046–58 to the naturally processed
h-gp10044–59, 1102 EBV-B cells were pulsed with
decreasing concentrations of each peptide (Fig. 3A). The naturally processed
16 amino acid peptide, h-gp10044–59, was capable
of inducing significant recognition (>1000 pg/ml of IFN-) at
concentrations of 0.3 µM or higher. The 13 amino acid peptide
h-gp10046–58 required concentrations of 3–10
µM to induce significant reactivity.
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B). Optimal P1 binding peptides,
characterized by large nonpolar residues, have been based on the x-ray
crystal structure of DRB1*0401 complexed with human collagen
II1168–1180 (43), as well as
sequence motifs based on phage display and synthetic peptide libraries
(44, 45, 46). A nonconservative substitution involving
glutamine (Q) for leucine (L) in position 48 substantially reduced T
cell recognition, while the substitution Y49Q had no effect, suggesting
L48 as the P1 anchor. Sequential truncations of
h-gp10046–58 demonstrated a dramatic step-off in
T cell recognition with the loss of L48 (P1) and abrogation of T cell
recognition with the removal of Q56 (P9 position). The core
nine-residue sequence (LYPEWTEAQ) based on this analysis represents the
precise sequence predicted by the computer-assisted DRB1*0401 algorithm
(Table I). The importance of amino acids outside the core nine-residue
sequence were also determined by altering T cell recognition by making
sequential truncations of h-gp10044–59 (Fig. 3C). The removal of glutamine (Q) in position 47 and
arginine (R) in position 57 markedly reduced T cell recognition from
peptides h-gp10046–56 and
h-gp10048–58, respectively. Human lymphocytes sensitized ex vivo to h-gp10044–59 recognize melanoma
The peptides h-gp10044–59 and
h-gp10046–58 differ from their murine
counterpart at several positions, particularly those centered over
putative TCR contact points (P2 and P8 positions; see Table II), and
are thus foreign Ags in mice. To test if this self-Ag could be of
equivalent immunogenicity in humans, fresh PBMC from three patients
with confirmed h-gp100-expressing metastatic melanoma were sensitized
ex vivo to h-gp10044–59 (as described above).
Bulk CD4+ T cell lines from patients Th, Wa, and
line 22 from patient Te (Te-22) were screened for peptide and tumor
reactivity. In Fig. 4, A and
B, lines from patients Th and Wa are shown to react against
DR4+ EBV-B cells (1102 EBV-B) pulsed with peptide
h-gp10044–59 and DR4+
intact melanoma line (1102 Mel) expressing h-gp100 endogenously. There
was no reactivity detected against control targets 888 Mel
(DR4-) and 1102 EBV-B pulsed with the irrelevant
peptide (HA306–318). In Fig. 4C,
CD4+ T cell line Te-22 is shown to react with
DR4+ EBV-B cells pulsed with peptide
h-gp10044–59, recombinant h-gp100 protein pulsed
onto autologous DCs, DR4+ EBV-B cells transfected
with h-gp100, and DR4+ intact melanoma lines
expressing h-gp100 endogenously. As before, there was no reactivity
against the control peptide (HA306–318),
autologous DCs pulsed with the irrelevant recombinant GFP protein,
EBV-B cells transfected with GFP, or against the
DR4- melanoma line (888 Mel). Importantly, these
experiments demonstrate that the epitope
h-gp10044–59 can be processed through both
exogenous and endogenous pathways and deliberately imitate the design
of the first experiment in which the recombinant protein was used to
begin the search for CD4+ T cell reactivity with
candidate epitopes.
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,
GM-CSF, IL-2 but not IL-4 (Fig. 4D, experiment 1) and
producing significant amounts of chemokines MIP-1, MIP-1ß,
MIP-3ß, and RANTES (Fig. 4D, experiment 2) when stimulated
with the specific Ag (1102 Mel). This pattern of cytokine and chemokine
secretion parallel that of the murine CD4+ T cell
line A9895 (see Fig. 2A). In a 12-h chromium release assay,
lytic activity was observed at titrating E:T ratios with 15.3%
specific lysis seen at an E:T of 10:1 (Fig. 4E). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although our initial screening of candidate peptides demonstrated reactivity to h-gp10046–58, we elected to study in subsequent experiments the naturally processed epitope h-gp10044–59. This peptide was previously shown by reversed-phase HPLC to be on the surface of DRB1*0401-matched human melanoma after eluting MHC-peptide complexes (41, 42). When compared with h-gp10046–58, the naturally processed, endogenously presented ligand induced nearly 50% greater reactivity from the murine CD4+ T cell line A9895 at a peptide concentration of 100 µM when pulsed onto DRB1*0401-matched EBV-B cells. These results are consistent with the specific binding affinity of peptide h-gp10044–59 to isolated HLA-DRB1*0401 molecules, previously measured at IC50 = 7 µM using an in vitro binding and competition assay (41). The ability to detect by reversed-phase HPLC endogenously processed peptides from eluted MHC class II-peptide complexes represents a technically challenging, but potentially useful, method for identifying other immunodominant Ags.
CD4+ T cells generated from DR4-IE Tg mice were found to react against specific peptides and unexpectedly against HLA-DRB1*0401-matched human melanomas. Residues 110 and 139 within the ß2 domain of the murine MHC class II molecule have been shown to be critical for optimal interactions with the murine CD4 molecule (52). Despite differences within the ß2 domain of human MHC class II, murine CD4+ T cell lines described above were capable of specific xeno-interactions with human melanoma targets. This finding suggested a high-avidity interaction between murine T cell and human tumor, limiting the contribution of species-specific CD4 molecules required for T cell recognition. The ability to test the reactivity of murine CD4+ T cell lines against human melanoma may permit for the potential identification of endogenously presented MHC class II-restricted epitopes derived from other melanoma differentiation Ags (tyrosinase, TRP-1, TRP-2, MART-1, OA1, and P. polypeptide). We are currently investigating these Ags within the context of DR4-IE Tg mice.
Computer-assisted algorithms based on the peptide binding motifs for individual MHC class I alleles have been used to predict tumor-associated epitopes (53, 54). Such an approach is more difficult for MHC class II molecules because they have an "open" peptide-binding grove. The majority of MHC class II molecules bind peptides between 10 and 20 residues in length, with sizes between 13 and 16 amino acids being the most frequently observed (55, 56). Thus, binding epitopes have no definitive amino or carboxyl termini, making prediction of the primary anchor positions more difficult. In the absence of allele-specific epitope forecasts, gene truncations or overlapping peptides covering the full length of the protein were required for the identification of MHC class II-restricted epitopes. Computer-assisted algorithms, such as the one described here, can be used to forecast the most avid binding candidate epitopes for proteins of any length. Indeed, the DRB1*0401 algorithm described by Southwood et al. (35) accurately predicted immunodominant epitopes from pre-proinsulin and OspA in the number 3 and number 1 ranked positions, respectively. In both cases, overlapping peptide libraries were used in the original studies to identify the epitopes described (39, 40).
We sought to link class I- and II-restricted epitopes capable of
binding individual MHC alleles with maximal prevalence in patients with
metastatic melanoma (HLA-A*0201 and DRB1*0401). The coadministration of
immunodominant, MHC class I- and II-restricted epitopes derived from
the same Ag could potentially increase the immunogenicity and
therapeutic efficacy of CTL through the activation or
"conditioning" of a common, intermediary APC (17, 19, 20). Following immunization with the full-length Ag, or specific
class I and II epitopes, APCs within draining lymph nodes or the spleen
present Ag to cognate CD4+ T cells principally
through the interaction of CD40 and its ligand. Activated APCs secrete
IL-12, up-regulate costimulatory molecules CD54, CD80, and CD86, and
cross-present Ag through MHC class I to cognate
CD8+ T cells either within the same draining
lymph node basin or in remote locations. The secretion of
chemokines such as RANTES, MIP-1, MIP-1ß, and MIP-3ß (see
Fig. 4D) from activated CD4+ T cells
could potentiate the immune response further still through the
attraction, amplification, and differentiation of NK cells, monocytes,
T cells (CD4+ and CD8+),
and DCs (57, 58, 59).
Although we were able to detect significant reactivity to peptide
h-gp10046–58 during the initial screening of
candidate HLA-DRB1*0401 epitopes, more than one potential epitope is
likely to exist (see Table I). The detection of cytokine production by
ELISA within 24 h after ex vivo stimulation may be ineffective at
detecting low-level T cell precursor frequency, a problem potentially
circumvented with the addition of the enzyme-linked immunospot
techniques, [3H]thymidine proliferation assays,
and quantitative RT/PCR assays. However, a more conservative and
probably more reliable method would involve serial restimulation with
all candidate epitopes. T cell cultures that proliferate can then be
tested for specific peptide reactivity as well as the recognition of
endogenously processed epitopes.
This simple strategy emphasizes the value of Tg mice and allele-specific epitope forecasts used to identify and characterize candidate MHC class I- and class II-restricted epitopes. In retrospect, we would have arrived at similar results had we initiated our search for T cell reactivity using ex vivo-sensitized human lymphocytes similar to that of investigators who have identified MHC class II-restricted epitopes for MAGE-3 (23, 24). By using DR4-IE Tg mice, we circumvented the need to sensitize human lymphocytes to a large number of peptides, allowed for the naturally processed, immunodominant epitope to be presented in vivo, and established the basis for a workable tumor treatment model. The data from this study has led to the implementation of a clinical trial at the Surgery Branch, National Cancer Institute. Following the coimmunization with immunodominant class I- and class II-restricted epitopes from h-gp100, we plan to evaluate both clinical response as well as the role of Ag-specific T cell help in antitumor immunity.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; h, human; TIL, tumor-infiltrating lymphocytes; CM, culture medium; GFP, green fluorescence protein; Tg, transgenic; MIP, macrophage inflammatory protein.
Received for publication October 25, 1999. Accepted for publication January 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-herpesvirus in the absence of CD4+ T cells. J. Exp. Med. 184:863.This article has been cited by other articles:
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  V. Robila, M. Ostankovitch, M. L. Altrich-VanLith, A. C. Theos, S. Drover, M. S. Marks, N. Restifo, and V. H. Engelhard MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes J. Immunol., December 1, 2008; 181(11): 7843 - 7852. [Abstract] [Full Text] [PDF]
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K. Kawamura, K. Yao, J. A. Shukaliak-Quandt, J. Huh, M. Baig, L. Quigley, N. Ito, A. Necker, H. F. McFarland, P. A. Muraro, et al. Different Development of Myelin Basic Protein Agonist- and Antagonist-Specific Human TCR Transgenic T Cells in the Thymus and Periphery J. Immunol., October 15, 2008; 181(8): 5462 - 5472. [Abstract] [Full Text] [PDF]
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 C. M. Paulos, A. Kaiser, C. Wrzesinski, C. S. Hinrichs, L. Cassard, A. Boni, P. Muranski, L. Sanchez-Perez, D. C. Palmer, Z. Yu, et al. Toll-like Receptors in Tumor Immunotherapy Clin. Cancer Res., September 15, 2007; 13(18): 5280 - 5289. [Abstract] [Full Text] [PDF]
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 S. Sugita, H. Takase, C. Taguchi, Y. Imai, K. Kamoi, T. Kawaguchi, Y. Sugamoto, Y. Futagami, K. Itoh, and M. Mochizuki Ocular Infiltrating CD4+ T Cells from Patients with Vogt-Koyanagi-Harada Disease Recognize Human Melanocyte Antigens. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2547 - 2554. [Abstract] [Full Text] [PDF]
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 S. Lepage and R. Lapointe Melanosomal Targeting Sequences from gp100 Are Essential for MHC Class II-Restricted Endogenous Epitope Presentation and Mobilization to Endosomal Compartments Cancer Res., February 15, 2006; 66(4): 2423 - 2432. [Abstract] [Full Text] [PDF]
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A. Paschen, M. Song, W. Osen, X. D. Nguyen, J. Mueller-Berghaus, D. Fink, N. Daniel, M. Donzeau, W. Nagel, H. Kropshofer, et al. Detection of Spontaneous CD4+ T-Cell Responses in Melanoma Patients against a Tyrosinase-Related Protein-2-Derived Epitope Identified in HLA-DRB1*0301 Transgenic Mice Clin. Cancer Res., July 15, 2005; 11(14): 5241 - 5247. [Abstract] [Full Text] [PDF]
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A. Bonehill, C. Heirman, S. Tuyaerts, A. Michiels, K. Breckpot, F. Brasseur, Y. Zhang, P. van der Bruggen, and K. Thielemans Messenger RNA-Electroporated Dendritic Cells Presenting MAGE-A3 Simultaneously in HLA Class I and Class II Molecules J. Immunol., June 1, 2004; 172(11): 6649 - 6657. [Abstract] [Full Text] [PDF]
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M. Ruiz, H. Kobayashi, J. J. Lasarte, J. Prieto, F. Borras-Cuesta, E. Celis, and P. Sarobe Identification and Characterization of a T-Helper Peptide from Carcinoembryonic Antigen Clin. Cancer Res., April 15, 2004; 10(8): 2860 - 2867. [Abstract] [Full Text] [PDF]
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H. L. Hanson, S. S. Kang, L. A. Norian, K. Matsui, L. A. O'Mara, and P. M. Allen CD4-Directed Peptide Vaccination Augments an Antitumor Response, but Efficacy Is Limited by the Number of CD8+ T Cell Precursors J. Immunol., April 1, 2004; 172(7): 4215 - 4224. [Abstract] [Full Text] [PDF]
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R. Schroers, L. Shen, L. Rollins, C. M. Rooney, K. Slawin, G. Sonderstrup, X. F. Huang, and S.-Y. Chen Human Telomerase Reverse Transcriptase-Specific T-Helper Responses Induced by Promiscuous Major Histocompatibility Complex Class II-Restricted Epitopes Clin. Cancer Res., October 15, 2003; 9(13): 4743 - 4755. [Abstract] [Full Text] [PDF]
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R. Schroers, L. Shen, L. Rollins, Z. Xiao, G. Sonderstrup, K. Slawin, X. F. Huang, and S.-Y. Chen Identification of MHC Class II-restricted T-cell Epitopes in Prostate-specific Membrane Antigen Clin. Cancer Res., August 1, 2003; 9(9): 3260 - 3271. [Abstract] [Full Text] [PDF]
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 P. Guan, I. A. Doytchinova, C. Zygouri, and D. R. Flower MHCPred: a server for quantitative prediction of peptide-MHC binding Nucleic Acids Res., July 1, 2003; 31(13): 3621 - 3624. [Abstract] [Full Text] [PDF]
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R. Lapointe, A. Bellemare-Pelletier, F. Housseau, J. Thibodeau, and P. Hwu CD40-stimulated B Lymphocytes Pulsed with Tumor Antigens Are Effective Antigen-presenting Cells That Can Generate Specific T Cells Cancer Res., June 1, 2003; 63(11): 2836 - 2843. [Abstract] [Full Text] [PDF]
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M. R. Parkhurst, C. DePan, J. P. Riley, S. A. Rosenberg, and S. Shu Hybrids of Dendritic Cells and Tumor Cells Generated by Electrofusion Simultaneously Present Immunodominant Epitopes from Multiple Human Tumor-Associated Antigens in the Context of MHC Class I and Class II Molecules J. Immunol., May 15, 2003; 170(10): 5317 - 5325. [Abstract] [Full Text] [PDF]
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C. E. Touloukian, W. W. Leitner, R. E. Schnur, P. F. Robbins, Y. Li, S. Southwood, A. Sette, S. A. Rosenberg, and N. P. Restifo Normal Tissue Depresses While Tumor Tissue Enhances Human T Cell Responses In Vivo to a Novel Self/Tumor Melanoma Antigen, OA1 J. Immunol., February 1, 2003; 170(3): 1579 - 1585. [Abstract] [Full Text] [PDF]
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 G. Consogno, S. Manici, V. Facchinetti, A. Bachi, J. Hammer, B. M. Conti-Fine, C. Rugarli, C. Traversari, and M. P. Protti Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3 Blood, February 1, 2003; 101(3): 1038 - 1044. [Abstract] [Full Text] [PDF]
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P. F. Robbins, M. El-Gamil, Y. F. Li, G. Zeng, M. Dudley, and S. A. Rosenberg Multiple HLA Class II-Restricted Melanocyte Differentiation Antigens Are Recognized by Tumor-Infiltrating Lymphocytes from a Patient with Melanoma J. Immunol., November 15, 2002; 169(10): 6036 - 6047. [Abstract] [Full Text] [PDF]
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C. E. Touloukian, W. W. Leitner, P. F. Robbins, Y. F. Li, X. Kang, R. Lapointe, P. Hwu, S. A. Rosenberg, and N. P. Restifo Expression of a "Self-"Antigen by Human Tumor Cells Enhances Tumor Antigen-specific CD4+ T-Cell Function Cancer Res., September 15, 2002; 62(18): 5144 - 5147. [Abstract] [Full Text] [PDF]
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 M. Sugaya, M. Takenoyama, T. Osaki, M. Yasuda, A. Nagashima, K. Sugio, and K. Yasumoto Establishment of 15 Cancer Cell Lines From Patients With Lung Cancer and the Potential Tools for Immunotherapy* Chest, July 1, 2002; 122(1): 282 - 288. [Abstract] [Full Text] [PDF]
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G. Zeng, Y. Li, M. El-Gamil, J. Sidney, A. Sette, R.-f. Wang, S. A. Rosenberg, and P. F. Robbins Generation of NY-ESO-1-specific CD4+ and CD8+ T Cells by a Single Peptide with Dual MHC Class I and Class II Specificities: A New Strategy for Vaccine Design Cancer Res., July 1, 2002; 62(13): 3630 - 3635. [Abstract] [Full Text] [PDF]
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 H. Y. Wang, J. Zhou, K. Zhu, A. I. Riker, F. M. Marincola, and R.-F. Wang Identification of a Mutated Fibronectin As a Tumor Antigen Recognized by CD4+T Cells: Its Role in Extracellular Matrix Formation and Tumor Metastasis J. Exp. Med., June 3, 2002; 195(11): 1397 - 1406. [Abstract] [Full Text] [PDF]
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C. E. Touloukian, W. W. Leitner, P. F. Robbins, S. A. Rosenberg, and N. P. Restifo Mining the Melanosome for Tumor Vaccine Targets: P.polypeptide Is a Novel Tumor-associated Antigen Cancer Res., November 1, 2001; 61(22): 8100 - 8104. [Abstract] [Full Text] [PDF]
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R. Lapointe, R. E. Royal, M. E. Reeves, I. Altomare, P. F. Robbins, and P. Hwu Retrovirally Transduced Human Dendritic Cells Can Generate T Cells Recognizing Multiple MHC Class I and Class II Epitopes from the Melanoma Antigen Glycoprotein 100 J. Immunol., October 15, 2001; 167(8): 4758 - 4764. [Abstract] [Full Text] [PDF]
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H. Kobayashi, J. Lu, and E. Celis Identification of Helper T-Cell Epitopes That Encompass or Lie Proximal to Cytotoxic T-Cell Epitopes in the gp100 Melanoma Tumor Antigen Cancer Res., October 1, 2001; 61(20): 7577 - 7584. [Abstract] [Full Text] [PDF]
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 T. Matsutake and P. K. Srivastava The immunoprotective MHC II epitope of a chemically induced tumor harbors a unique mutation in a ribosomal protein PNAS, March 27, 2001; 98(7): 3992 - 3997. [Abstract] [Full Text] [PDF]
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L. Qi, J.-M. Rojas, and S. Ostrand-Rosenberg Tumor Cells Present MHC Class II-Restricted Nuclear and Mitochondrial Antigens and Are the Predominant Antigen Presenting Cells In Vivo J. Immunol., November 15, 2000; 165(10): 5451 - 5461. [Abstract] [Full Text] [PDF]
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B. Cochlovius, M. Stassar, O. Christ, L. Raddrizzani, J. Hammer, I. Mytilineos, and M. Zoller In Vitro and In Vivo Induction of a Th Cell Response Toward Peptides of the Melanoma-Associated Glycoprotein 100 Protein Selected by the TEPITOPE Program J. Immunol., October 15, 2000; 165(8): 4731 - 4741. [Abstract] [Full Text] [PDF]
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G. Zeng, C. E. Touloukian, X. Wang, N. P. Restifo, S. A. Rosenberg, and R.-F. Wang Identification of CD4+ T Cell Epitopes from NY-ESO-1 Presented by HLA-DR Molecules J. Immunol., July 15, 2000; 165(2): 1153 - 1159. [Abstract] [Full Text] [PDF]
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G. Zeng, X. Wang, P. F. Robbins, S. A. Rosenberg, and R.-F. Wang CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production PNAS, March 27, 2001; 98(7): 3964 - 3969. [Abstract] [Full Text] [PDF]
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