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Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that the in vitro reactivity of g9-209 2M-stimulated PBMC cultures from some of the g9-209 2M-vaccinated patients was consistently higher against g9-209-pulsed target cells than against melanoma cells. This observation raised the possibility that immunization with g9-209 2M can stimulate a group of T cells with fine specificities capable of recognizing the native g9-209 peptide with a broad range of avidities. To investigate this possibility, we generated limiting dilution cloids from three patients, vaccinated with g9-209 2M, whose PBMC were further stimulated in vitro with g9-209 2M before cloning. We also prepared cloids from g9-209-reactive TIL that consistently reacted as well with melanoma cell lines as with g9-209-pulsed target cells. These results showed that the majority of the T cell cloids generated from PBMC, in contrast with those generated from TIL, reacted with the native g9-209 peptide or its modified g9-209 2M form but not HLA-A2+, gp100+ melanoma cells. These observations may have important implications for therapeutic strategies that use peptide epitopes in vaccines or use peptide-stimulated PBMC rather than TIL for adoptive immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TIL cultures were grown as previously described (14). Briefly, tumor samples were enzymatically digested to yield a single cell suspension, and the cells were grown in AIM V medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated pooled human AB serum (Sigma, St. Louis, MO) and 6000 IU/ml rhIL-2 (Cetus Oncology, Emeryville, CA).
Bulk peptide-stimulated PBMC cultures
Stage IV melanoma patients were vaccinated with 1 mg g9-209 2M
peptide emulsified in IFA, delivered s.c. at 3-wk intervals for at
least four treatments. PBMC from g9-209 2M-vaccinated patients were
obtained by apheresis 3 wk following their final treatment. On day 0,
1.5 x 106 or 3 x 106 PBMC per well
were plated in 24-well plates in 2 ml per well of Iscove’s medium
(Biofluids, Rockville, MD) containing 10% heat-inactivated human
pooled AB serum (Sigma), penicillin (100 U/ml)-streptomycin (100
µg/ml)-glutamine (2.92 mg/ml) (Life Technologies), and 1 µg/ml
g9-209 2M peptide. Plates were incubated at 37°C in a humidified
incubator containing 5% CO2. On day 2 and 6, rhIL-2 was
added to yield a concentration of 300 IU/ml, and, if the cell density
was greater than 2 x 106 cells/ml on day 6, the
cultures were split 1:2. On day 12, the cultures were restimulated by
coculturing 5 x 105 PBMC with 4 x
106 irradiated (100 Gy) autologous PBMC that had been
pulsed with 1 µg/ml g9-209 2M peptide for 2–3 h at 37°C in a
humidified incubator containing 5% CO2. On day 13, rhIL-2
was added to a final concentration of 300 IU/ml, and the cultures were
split 1:2 if they exceeded 2 x 106 cells/ml. Cultures
were restimulated as described above on days 18 and 25. On day 32, the
cultures were harvested, and their reactivity was tested in hIFN-
or
hGM-CSF release assays.
Limiting dilution cloning of bulk PBMC
PBMC cultures from day 12 bulk cultures were plated at 100, 10, and 1 cells per well in 96-well microtiter plates in 0.2 ml per well. Cells were expanded using anti-CD3 stimulation following the method of Walter et al. (15). Briefly, PBMC were plated in RPMI 1640 (Biofliuds) containing 11% heat-inactivated human pooled AB serum (Sigma), penicillin (100 U/ml)-streptomycin (100 µg/ml)-glutamine (2.92 mg/ml) (Life Technologies), 25 mM HEPES, 25 µM 2-ME, with 5 x 104 irradiated allgoneic PBMC (100 Gy), 1 x 104 irradiated allogeneic EBV-B cells (100 Gy) per well, and 30 ng/ml anti-CD3 mAb (OKT3; Ortho Biotech, Raritan, NJ). The following day, 120 IU/ml rhIL-2 was added. On day 5, the medium was exchanged, and fresh medium containing 120 IU/ml rhIL-2, without OKT3, was added. On day 8, fresh rhIL-2 was added to yield a final concentration of 120 IU/ml. Cells were tested for reactivity on day 12 in cytokine release assays, and peptide-specific cloids were restimulated as described above. To expand the reactive cloids, the culture volume was increased, and the numbers of PBMC and EBV-B were adjusted accordingly. The clonality of some of these cultures has not been established, so they are referred to as cloids.
TIL clones/cloids
All TIL cultures were derived from metastatic melanoma lesions
from different patients as described (14). Two g9-209-reactive T cell
clones were generated from day 33 TIL cultures by limiting dilution
cloning; clone CO3 was derived from TIL 620, and clone JH1A3 was
derived from TIL 1520. These clones were obtained from 100 cell/well
cultures grown in AIM-V medium (Life Technologies) containing 10%
heat-inactivated pooled human AB serum (Sigma), L-glutamine
(2.92 mg/ml) (Life Technologies), and 6000 IU/ml rhIL2, in 96-well
microtiter plates. Irradiated allogeneic PBMC (2.5 x
105 cells/ml; 100 Gy) were added weekly in fresh medium
containing 6000 IU/ml rhIL-2. The cultures were expanded to larger
volumes, and the number of irradiated allogeneic PBMC were adjusted
accordingly. The cultures were split 1:2 when cell density was greater
than 2 x 106 cells/ml. Clonality of CO3 and JH1A3 was
confirmed by TCR 
ß sequence analysis (T. Clay, manuscript
in preparation).
TIL cloid VM62 was generated by limiting dilution cloning from a TIL culture from melanoma patient VM. The lesion was enzymatically digested as described (14), and 106 cells per well were cultured in 24-well plates in RPMI 1640 medium supplemented with 10% heat-inactivated pooled AB serum (Sigma) and L-glutamine (2.92 mg/ml) (Life Technologies). On day 2, rhIL-2 was added to a final concentration of 120 IU/ml. On day 7 and weekly thereafter, resulting TIL were restimulated by culturing 5.0 x 105 TIL with 1.5 x 105 irraditated (300 Gy) HLA-A2+ allogeneic melanoma cells, 2 x 106 irradiated (30 Gy) autologous PMBC, and 120 IU/ml rhIL-2. On day 36, TIL were cloned at 0.5 cells/well and expanded using anti-CD3 mAb as described above.
Tumor cell lines
HLA-A2 positive and negative human melanoma cell lines expressing gp100 were established in the Surgery Branch (National Cancer Institute) from resected tumor lesions as previously described (16). 501 Mel (HLA-A2+), 1088 Mel (HLA-A2+), 397 Mel (HLA-A2-), 624-28 (HLA-A2-), and 624-38 (HLA-A2+) were maintained in RPMI 1640 medium supplemented with heat-inactivated 10% FBS (Biolfuids), penicillin (100 U/ml)-streptomycin (100 µg/ml)-L-glutamine (2.92 mg/ml) (Life Technologies).
Peptides
Peptides were synthesized on a model 422 peptide synthesizer (Gilson, Worthington, OH) using solid phase methods, as previously described (12). The sequences of the peptides used in this study are as follows: g9-209 (parent peptide) ITDQVPFSV (11); g9-209 2M IMDQVPFSV (12); g9-209 2I IIDQVPFSV (12); g9-209 2L ILDQVPFSV (12); g9-209 2A IADQVPFSV; g9-280 YLEPGPVTA (12); MART-1 m9-27 AAGIGILTV (17).
Assessment of culture reactivity
Peptide-stimulated PBMC were tested for reactivity in hIFN-
or hGM-CSF release assays against T2 cells either alone or pulsed with
peptide (2 µg/ml) in RPMI 1640 medium for 2–3 h at 37°C. Cells
were also tested for reactivity with gp100-expressing melanoma lines.
Responder cells (5,000) and 10,000 stimulator cells were cocultured in
a 0.2-ml volume for 24 h. hGM-CSF and hIFN- secretion was
measured in culture supernatants using commercially available ELISA
kits (R&D Systems, Minneapolis, MN; and Endogen, Cambridge, MA,
respectively). We have previously found that hGM-CSF and hIFN-
secretion have comparable sensitivity for the assessment of culture
reactivity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Peptide-stimulated bulk PBMC cultures
from all three patients reacted with T2 cells pulsed with the g9-209
peptide and also reacted with HLA-A2+,
gp100+-expressing melanoma cell lines (Table I). The
melanoma cell lines, however, appeared to stimulate significantly lower
levels of cytokine secretion than T2 cells pulsed with the g9-209
peptide. In contrast, g9-209 peptide-pulsed T2 cells and melanoma cell
lines stimulated comparable levels of cytokine secretion from a TIL
culture derived from patient JH (TIL 1520; Table I). Control TIL 1235
cells recognized m9-27 but not g9-209 peptide-pulsed T2 cells as
previously described (11). Similar results have been observed
previously, when comparing the recognition of tumor to the recognition
of peptide-pulsed T2 cells by TIL (11).
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shows representative data for in vitro
peptide-stimulated bulk PBMC cultures from each patient after one
peptide stimulation. Bulk PBMC cultures from these patients stimulated
in vitro with g9-209 2M peptide consistently recognized T2 cells pulsed
with g9-209 peptide and also recognized the HLA-A2+,
gp100+ melanoma cell line 624–38. Data from representative
cloids is shown in Table III. PBMC cloids
generated from a bulk g9-209 2M-stimulated PBMC culture from patient SW
showed three distinct patterns of reactivity (Table III). Some
cloids recognized the target pulsed with the g9-209 2M peptide but
failed to recognize g9-209-pulsed targets or HLA-A2+
melanoma cells (Table III: SW1D2 and SW1E6). Another group of cloids
recognized both the parental peptide and the g9-209 2M peptide but did
not recognize HLA-A2+ melanoma tumor cell lines (Table III:
SW195). Finally, some cloids recognized the parental and g9-209 2M
peptides and also recognized HLA-A2+ melanoma cell lines
(Table III: SW1A2 and SW268).
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Although 3 cloids from these two patients recognized g9-209 2M alone
and 11 cloids recognized g9-209 and g9-209 2M, none of them recognized
the three HLA-A2+, gp100 expressing melanomas tested. In
fact, of all of the PBMC cloids analyzed from the three patients (11
from KS, 3 from JH, and 6 from SW), only three cloids from one patient
(SW) recognized HLA-A2+ melanomas (SW1A2, SW268, and SW201;
data not shown). In contrast, g9-209-reactive T cells generated from
TIL obtained from three melanoma patients (VM, CO and JH), specifically
released GM-CSF in response to stimulation by HLA-A2+
melanoma cell lines that express gp100 (Table III; JH1A3, VM62, CO3).
Therefore, we observed a distinct difference in the specificity of
peptide-stimulated PBMC cloids in comparison with TIL cloids,
suggesting that immunization with the modified peptide g9-209 2M may
have resulted in a TCR repertoire skewed toward T cells with low
avidity for the native peptide as presented on the surface of melanoma
cells.
The fine structure reactivity of the PBMC and TIL cloids was examined
further by evaluating the recognition of additional peptides modified
at position 2 by substituting amino acids other than methionine (Fig. 1). For PBMC cloids that recognized
g9-209 and g9-209 2M peptides, position 2 substitutions did not appear
to alter peptide recognition (Fig. 1A). The PBMC cloids that
recognized g9-209 2M but not the native g9-209 peptide either did not
respond (SW1D2) or responded weakly (JH85 or KS72) to peptide
containing leucine or isoleucine substitutions at position 2 (Fig. 1B). The TIL clones CO3 and JH1A3 and the TIL cloid VM62
tolerated some position 2 substitutions (2M, 2I, 2L; Fig. 1C). However, the 2A substitution was generally not
recognized to the same level as the parental g9-209 peptide by the TIL
clones based upon the levels of GM-CSF released (Fig. 1C).
Clone CO3 secreted lower levels of GM-CSF when stimulated with targets
pulsed with the 2A, 2L, and 2I peptides compared with targets pulsed
with the parental (2T) and 2M peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The apparent differences in tumor recognition between peptide-stimulated PBMC and TIL were further studied by analyzing the reactivity of limiting dilution cloids. The g9-209-reactive TIL cloids obtained from three different patients reacted with both peptide-pulsed T2 cells and HLA-A2+ melanoma lines. These results are typical for the MART-1 (18, 19, 20, 21), gp100 (Ref. 22;, M. Dudley, unpublished observations) and MAGE-1 (23)-reactive TIL clones that have been isolated. In contrast, cloids derived from peptide-stimulated PBMC cultures exhibited distinctly different patterns of reactivity from the TIL cloids. The majority of PBMC cloids generated from the three patients were not tumor reactive. Twelve of fifteen PBMC cloids that recognized both the g9-209 peptide and g9-209 2M-modified peptide did not recognize melanoma lines. Only three cloids that were obtained from a single patient recognized melanoma tumor cell lines. This may reflect a lower level of MHC expression, concentration of antigenic peptides, or accessory molecules on the surface of tumor cells relative to T2 cells (24, 25, 26). An alternative explanation may be a difference in the TCR affinity of the three tumor-reactive cloids, compared with the cloids that did not recognize tumor. Also, it is possible that some of these cloids did not recognize tumor cells due to the expression of NK-inhibitory receptors (27).
Several of these cloids and others derived from the same patients were tested for recognition of g9-209 peptide-pulsed melanoma lines. Some cloids that did not recognize unpulsed melanoma lines did recognize g9-209 peptide-pulsed melanoma lines, suggesting that, for these cloids, the surface density of antigenic peptide on the melanoma lines is below their threshold for recognition (data not shown; R. Hurst, manuscript in preparation).
Based upon TCR ß sequence analysis, some of the PBMC cloids are in
fact clones, and others contain more than one clonotype (T.
Clay, manuscript in preparation). However, the fact that some cloids
are not clonal does not alter the interpretation of our results. The
majority of peptide-reactive cloids do not recognize tumor,
irrespective of whether they are clonal or consist of several
clonotypes. It is possible that the cloids that do recognize tumor may
contain multiple clonotypes, but we have never previously found a
tumor-reactive clone that does not recognize the native g9-209 or
g9-209 2M modified peptides.
Five of the PBMC cloids were g9-209 2M specific and therefore would not
be expected to be tumor reactive because they do not recognize the
parental g9-209 peptide. For these cloids, threonine at position 2
abrogates peptide recognition (Fig. 1). Position 2 is an HLA-A*0201
anchor residue that is not considered a TCR contact residue. However,
our data suggested that an HLA-A2 molecule containing the g9-209 2M
peptide had a different conformation than an HLA-A2 molecule containing
the parent g9-209 peptide, and T cells were capable of detecting these
differences. Other position 2 substitutions were also not recognized by
these cloids, even though leucine and isoleucine at position 2 enhanced
the binding affinity of the peptide to the HLA-A*0201 molecule (12).
Thus, conformational changes induced by MHC binding anchor residues
that do not contact the TCR could influence T cell recognition. Before
immunization with the g9-209 2M peptide, PBMC cultures from these
patients were not reactive with peptide-pulsed targets (4). Therefore,
the reactivities of cloids described in this study are the result of
immunization with the modified gp100 peptide and are not due to
preexisting reactivities.
Studies using T cell clones that are specific for the same parental peptide have shown that altered peptide ligands can result in differences in the T cell activation state among the different T cell clones (28). Changing one amino acid in a known agonist peptide sequence may result in a peptide that is an agonist for some CTL clones, while for other CTL clones the altered peptide may act as an antagonist or partial agonist and prevent full T cell activation. Chen et al. have reported that, following single amino acid changes at the MHC anchor residues of a murine OVA/Kb peptide, four CD8+ T cell hybridomas specific for the normal determinant showed different responses to the altered peptides (29). More recently, Chen et al. described a human CD4+ T cell clone that is specific for an HLA-DR4-restricted streptococcal peptide and examined its response to a large panel of modified peptides. They found modified peptides that acted either as agonists, or as antagonists causing partial T cell activation, or antagonists that inhibited T cell activation (sometimes referred to as null peptide ligands) (30). It is also possible that a modified peptide may not be recognized at all by some clones. Hsu et al. have reported that a single amino acid substitution at an anchor residue in a murine hemoglobin peptide Hb (64–76) recognized by I-Ek restricted T cell clones, did not affect recognition by some Hb(64–76)-specific T cell clones, but other Hb(64–76)-specific clones did not recognize the modified peptide (31).
We have previously shown that there is diverse TCR V gene usage among melanoma-reactive T cells (17, 32). This diversity may have been magnified by the in vivo and/or in vitro stimulation with the modified g9-209 2M peptide. The 2M modification appeared to have pronounced effects on the T cell repertoire to g9-209, expanding T cells with reactivities not normally represented in anti-g9-209 immune responses. This was most evident by the appearance of cloids specific for the g9-209 2M peptide and cloids that failed to recognize tumor cells. However, this analysis of our cloids does not necessarily reflect the relative proportion of tumor-reactive vs non-tumor-reactive g9-209-specific T cells in vivo.
The g9-209 2M peptide is a more potent immunogen than the parent g9-209
peptide despite the deleterious effects we report here on the T cell
repertoire (4). However, 2M-vaccinated patients fail to respond despite
having an elevated frequency of g9-209-reactive T cells in their
peripheral blood. This study has demonstrated that the 2M modification
can expand low avidity g9-209-reactive T cells that poorly recognize
tumor cells. There could be a genetic or physiologic explanation for
why these cells fail to mediate tumor regression in vivo. Cells bearing
low affinity TCRs for the g9-209/HLA-A2 ligand may be the majority of
the T cells effectively stimulated by the g9-209 2M/HLA-A2 ligand.
Since tumor-reactive T cells are present in the peripheral blood of
patients vaccinated with the modified peptide g9-209 2M, boosting these
patients with the parental g9-209 peptide may promote expansion of
those T cells bearing higher affinity receptors for g9-209, resulting
in enhanced anti-tumor responses. Alternatively, it is possible
that the g9-209 2M peptide stimulates and expands T cells bearing high
affinity receptors yet the tumor environment renders these T cells
nonresponsive. The down-regulation of CD3 
-chain in tumor-bearing
mice and patients could account for these low avidity T cells (33, 34, 35).
CD3 -chain expression can be restored by cytokines such as IL-2 (36, 37). Therefore, cytokines that can influence T cell function may lead
to enhanced anti-tumor responses. This could explain the
observation that patients treated with a combination of g9-209 2M
peptide and IL-2 had a 42% response rate, which was higher than
response rates seen in patients treated with g9-209 2M peptide or IL-2
alone (4). However, if both TCR affinity and T cell physiology are
important, then the best strategy may be to expand individual
tumor-reactive cloids in vitro and use them individually or in pools in
adoptive immunotherapy protocols to treat patients as described (15).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: g9-209, gp100 native peptide (amino acids 209–217); g9-209 2M, gp100 peptide (amino acids 209–217) with methionine substitution at the second position; gp100+, cell line expressing gp100; hGM-CSF, human granulocyte-macrophage CSF; TIL, tumor-infiltrating lymphocytes.
Received for publication August 13, 1998. Accepted for publication October 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain and p56lck in T-cells infiltrating human renal cell carinoma. Cancer Res. 53:5613. chains in tumor-infiltrating T-cells and NK cells of patients with colorectal carcinoma. Cancer Res. 53:5610. chain expression but fails to generate tumor-specific lysis activity in tumor-infiltrating lymphocytes derived from human colorectal hepatic metastases. Br. J. Cancer 77:1072.[Medline]
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T. N. J. Bullock, T. A. Colella, and V. H. Engelhard The Density of Peptides Displayed by Dendritic Cells Affects Immune Responses to Human Tyrosinase and gp100 in HLA-A2 Transgenic Mice J. Immunol., March 1, 2000; 164(5): 2354 - 2361. [Abstract] [Full Text] [PDF]
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 M. I. Nishimura, D. Avichezer, M. C. Custer, C. S. Lee, C. Chen, M. R. Parkhurst, R. A. Diamond, P. F. Robbins, D. J. Schwartzentruber, and S. A. Rosenberg MHC Class I-restricted Recognition of a Melanoma Antigen by a Human CD4+ Tumor Infiltrating Lymphocyte Cancer Res., December 1, 1999; 59(24): 6230 - 6238. [Abstract] [Full Text] [PDF]
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G. E. Peoples, B. W. Anderson, T. V. Lee, J. L. Murray, A. P. Kudelka, J. T. Wharton, and C. G. Ioannides Vaccine Implications of Folate Binding Protein, a Novel Cytotoxic T Lymphocyte-recognized Antigen System in Epithelial Cancers Clin. Cancer Res., December 1, 1999; 5(12): 4214 - 4223. [Abstract] [Full Text] [PDF]
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 A. K. Sharma, J. J. Kuhns, S. Yan, R. H. Friedline, B. Long, R. Tisch, and E. J. Collins Class I Major Histocompatibility Complex Anchor Substitutions Alter the Conformation of T Cell Receptor Contacts J. Biol. Chem., June 8, 2001; 276(24): 21443 - 21449. [Abstract] [Full Text] [PDF]
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