By
From the * Surgery Branch and the Metabolism Branch, National Cancer Institute, National Institutes
of Health, Bethesda, Maryland 20892; the § Department of Surgery, Veterans Administration Medical
Center, University of California San Francisco, San Francisco, California 94121; and the
Laboratory
of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892
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Abstract |
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Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation
antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T
cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both
normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6
mice with the rVV encoding the human gp100 elicited a specific CD8+ T cell response. These
lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma
upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db,
that was recognized by the T cells. The ability to induce specific T cells with human but not
mouse gp100 resulted from differences within the major histocompatibility complex (MHC)
class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced
by experiments in which mice were immunized with rVV containing minigenes encoding
these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability
of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability
to trigger interferon release by T cells. Thus, the fortuitous existence of a peptide homologue
with significantly greater avidity for MHC class I resulted in the generation of self-reactive T
cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.
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Introduction |
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The recent cloning of tumor antigens recognized by T cells has caused considerable interest in the development of antigen-specific cancer vaccines (1). Some antigens are especially attractive candidates for use in vaccines due to their shared nature between individuals, including the melanocyte differentiation antigens (MDA)1 gp100, melanoma antigen recognized by T lymphocytes (MART)-1, and tyrosinase (2), as well as several proteins in the MAGE family (1). However, as indicated by results from clinical trials thus far, inducing therapeutic T cells to these antigens has been difficult. One reason for the apparent hyporesponsiveness of the human immune system to many tumor antigens may be that they are normal, nonmutated "self"- proteins, expressed on normal tissues as well as on tumor cells. An incomplete understanding of the processes of central and peripheral tolerance has hampered the development of successful cancer vaccines targeting these autoantigens, limiting the use of the growing number of candidate tumor antigens.
The absence of an immune response to a defined autoantigen can be due to negative selection of self-antigen- specific T cells during maturation in the thymus, termed "central" tolerance (4). A low level of autoreactivity is required for positive selection in the thymus (5, 6).
T cells with low reactivity to autoantigens thus persist. Mature T lymphocytes with reactivity to self-antigens may remain in a functionally tolerant state, termed "ignorance", if they do not traffic to antigen-bearing cells, or if the target antigen is not processed and presented to a level that can trigger the specific TCR. Mature self-reactive T cells that encounter antigen on normal tissues in the absence of an activating costimulatory microenvironment can be functionally eliminated by anergization or physically by deletion, thus effecting extrathymic or peripheral tolerance (7, 8).
The mechanisms of breaking tolerance to self-antigens may be relevant for the induction of immune responses to tissue differentiation antigens expressed by tumors. To study requirements for activation of self-reactive, tumor-specific T cells specific for a naturally expressed antigen, we targeted gp100, a normal, nonmutated MDA. In humans, gp100 is expressed both by normal melanocytes and the majority of malignant melanomas tested (9). CD8+ T lymphocytes with reactivity to gp100 have been detected in patients with metastatic melanoma. The mouse homologue for gp100, also known as pmel 17, has been cloned previously (10, 11), and like its human counterpart is normally expressed in melanocytes in an unmanipulated C57BL/6 mouse as well as in mouse melanomas. We sought to determine the requirements to break T cell tolerance to a naturally expressed self-antigen. Furthermore, we evaluated the functional characteristics of autoreactive T cells in the recognition and destruction of a spontaneous mouse melanoma, B16, in vivo.
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Materials and Methods |
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Animals and Cell Lines.
Female C57BL/6 (H-2b) mice, 6-10 wk old, obtained from Frederick Cancer Research Center (Frederick, MD) and maintained in a barrier facility, were used for all experiments. EL-4 thymoma (H-2b) and the derivedPeptides.
All synthetic peptides were synthesized using regular F-MOC chemistry. The following synthetic peptides were all synthesized by Peptide Technologies (Washington, D.C.) to a purity >99% by HPLC and amino acid (AA) analysis: KVPRNQDWL, spanning AAs 25-33 of human (h)gp100; EGSRNQDWL, spanning AAs 25-33 of mouse (m)gp100; ASNENMETM, spanning AAs 366-374 of the nucleoprotein of influenza A (NP); DAPIYTNV, spanning AAs 96-103 of Escherichia coliRecombinant Viruses.
All recombinant vaccinia viruses (rVVs) used in this study were generated by insertion of the foreign genes by homologous recombination and subsequent purification of recombinant progeny as described by Earl et al. (14). rVVs encoding the human and mouse gp100 epitopes as minigenes were constructed using a recombination plasmid, pKT1401, containing an early promoter driving the signal sequence of the adenovirus E3/19k protein followed by an additional Ala using code GCA, resulting in a putative signal peptide cleavage site, and fused to oligonucleotides encoding the epitope sequences for mgp100, hgp100, orGeneration of gp100-reactive T Cell Line.
Mice were vaccinated twice at 3-wk intervals by a hand-held helium-driven device (Geniva Inc., Middletown, WI) with 1 µg of hgp100 DNA (23), and 3 wk after the second vaccination splenocytes were cultured with rVVhgp100-infected dendritic cells (DCs), generated as previously described (16) in CM with 30 IU/ml rhIL-2 (a gift from Chiron Corp., Emeryville, CA) for 7 d, and were subsequently restimulated every 7-10 d with 2 × 105 B16.B7-1 (60,000 rad) and 5 × 106 irradiated splenocytes (1,500 rad) in CM with 30 IU/ml rhIL-2. T cell clones were derived by limiting dilution using 0.3 T cells per well cocultured with 4 × 105 irradiated splenocytes and 2 × 104 irradiated B16.B7-1 cells in U-bottomed 96-well plates. The generation of theCytokine Release Assay.
To determine cytokine release, 5 × 104 target cells per well were added to effector T cells in round-bottomed 96-well plates at an E/T ratio of 1:1 for established T cell lines and 5:1 for secondary cultures, and were incubated for 24 h in CM. Supernatants were collected and tested using the mIFN-MHC Class I Stabilization Assay.
RMA/S cells were incubated at room temperature for 24 h, pulsed with peptide at the indicated concentrations for 45 h at room temperature, and then incubated at 37°C for 4 h to allow turnover of "empty" MHC class I molecules (13). Cells were then stained with FITC-conjugated Db-specific mAb KH95 (PharMingen, San Diego, CA) for 1 h at 4°C and staining was asserted using a FACScan® (Becton Dickinson, Sunnyvale, CA).Adoptive Transfer Treatment.
For adoptive transfer experiments, mice were injected with 2 × 105 B16 intravenously on day 0 followed by intravenous injection of the indicated number of T cells on day 3. rhIL-2 (3 × 105 IU) was given twice daily intraperitoneally for 5 d starting immediately after adoptive transfer. Alternatively, mice were injected intravenously with the indicated number of T cells, followed 5 d later by 2 × 105 B16 intravenously. Mice were killed on day 17 after tumor injection and pulmonary nodules were enumerated. All in vivo experiments were performed in a blinded, randomized fashion. ![]() |
Results |
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To generate gp100-specific T cells, mice were immunized with mgp100 encoded in plasmid DNA or rVV, two vectors with proven efficacy in the generation of specific T cells (3, 23, 24). Splenocytes from immunized mice were restimulated in
vitro using syngeneic DCs infected with appropriate rVV,
rFPV, or rAd, and subsequent restimulations were performed with irradiated splenocytes and irradiated B16.B7-1,
a clone of B16 retrovirally transduced to express the costimulatory molecule CD80 (B7-1). None of the cultures
exhibited gp100-specific reactivity when tested by IFN- release against gp100-positive targets (data not shown). Since xenogeneic antigen can, in some instances, induce immune
reactivity where the autologous antigen failed (25),
mice were immunized with pDNA encoding hgp100.
A CD4CD8+ lytic T lymphocyte line was generated by
gene-gun administration of pDNA encoding hgp100 followed by restimulation of splenocytes ex vivo with DCs
that had been infected with rVVhgp100. After two subsequent in vitro restimulations with B16.B7-1 melanoma, the T cells recognized B16 melanoma, as well as the immortalized normal melanocyte line Melan-A, with a high
degree of specificity (Fig. 1). The EL-4 thymoma and the
MC38 sarcoma were not recognized. However, recognition could be conferred upon 293 KbDb cells after infection
with rVV encoding either mgp100 or hgp100, but not after
infection with rVV containing any of the other known MDAs, tyrosinase, TRP-1, or TRP-2.
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We sought
to evaluate the relevance of mgp100 as a tumor rejection
antigen. To exclude the potential influence of T cells with
a specificity other than gp100, the bulk T cell line was cloned by limiting dilution. 12 clones were evaluated and
each had reactivities identical to the bulk T cell line shown
in Fig. 1. The gp100-reactive T cell clone (clone no. 9)
was tested in vitro (Fig. 2 A) and in vivo (Fig. 2 B). Mice
bearing 3-d-old B16 pulmonary nodules were infused with
clone no. 9 followed by rIL-2, resulting in a dramatic tumor destruction (P <0.0001, 4 × 106 -gal-specific T cells + rhIL-2 versus 4 × 106 clone no. 9 T cells + rhIL-2), whereas
treatment was ineffective using control
-gal-reactive T cells
or rIL-2 alone (P >0.5 versus no treatment) (Fig. 2 B).
Separate gp100-reactive T cells clones were comparable in
their ability to reject B16 in vivo (data not shown). These
results suggest that gp100 functions as a true tumor rejection antigen in established murine melanoma.
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We sought to understand why T cells
could only be induced by the xenogeneic, human form of
gp100 and not the self sequence. To determine whether
the difference in immunogenicity resided in the actual peptides from gp100 that were recognized by T cells, or in differences in sequences outside of those recognized, we characterized the epitopes recognized by the T cell clone. To identify the MHC class I molecule that restricted gp100
recognition, we used the human renal cell carcinoma line,
293, stably transfected with the mouse restriction elements,
Kb and Db. Both lines were transiently transfected with
plasmids encoding either mouse or human gp100, or with a
control plasmid encoding NP. Only 293 KbDb cells (and
not 293 Kb) cells expressing mgp100 or hgp100 triggered
the release of IFN- from gp100-reactive T cells, suggesting that Db was the dominant and perhaps only restriction
element for gp100 recognition (Fig. 3 A).
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At 626 AAs in length, many possible epitopes in gp100 could be recognized by gp100-reactive T cells. To reduce the number of candidate peptides, a set of progressively shorter versions of the gene encoding hgp100 was used (28). These shortened forms were generated by 3' exonuclease digestion or PCR amplification of the original hgp100 cDNA. Fragments were then transfected into 293 KbDb cells that were used as targets for T cell recognition (Fig. 3 B). Even the shortest fragment of the hgp100 cDNA, with a length of 300 bp by gel electrophoresis, conferred a high degree of recognition upon the 293 transfectants, implying that a major epitope was located in the first 100 AAs of the molecule.
To identify the sequence of the epitope, 9-AA-long peptides were evaluated for their potential to bind to Db using a computer-generated epitope forecast, based on previously published peptide binding data, that is designed to predict binding affinity for a variety of human and mouse MHC class I alleles (29, 30; and http://bimas.dcrt.nih.gov/molbio/hla_bind). Based on these predictions, sets of synthetic peptides were made from both hgp100 and mgp100, which although similar are not identical. When the peptides were added to cultures of gp100-reactive T cells, one peptide pair, gp10025-33, was recognized with a high degree of specificity in a GM-CSF release assay (Fig. 3 C).
T Cell Reactivity to gp100 Can Be Induced by Xenoimmunization.With the identification of a defined peptide epitope, we explored the immunological mechanism underlying the disparate ability of mgp100 and hgp100 to induce gp100-specific T cells. Mice were vaccinated with rVVmgp100, encoding mgp100, or rVVhgp100, encoding hgp100, and splenocytes were isolated after 3 wk and stimulated for 6 d in vitro with synthetic peptides corresponding to hgp10025-33, mgp10025-33, or OVA257-264, and then tested in a GM-CSF release assay (Table 1, first two columns). Splenocytes from mice vaccinated with rVVmgp100 and restimulated with mgp10025-33 or hgp10025-33 peptide failed to develop peptide reactivity. Conversely, splenocytes from mice vaccinated with hgp100 and restimulated with either mgp10025-33 or hgp10025-33 peptide developed reactivity to both peptides. Similar results were obtained using splenocytes from mice vaccinated with recombinant fowlpoxvirus or plasmid DNA (data not shown). These observations indicated that T cells recognizing mgp100 could be induced exclusively by xenoimmunization with the hgp100 molecule.
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Several mechanisms could account for the apparent immunological unresponsiveness to mgp100 and the ability of hgp100 to break it. Nonhomologous regions of the full-length hgp100 (which is 76% identical to mgp100 at the AA level; reference 11) could result in intramolecular epitope-spreading (31, 32) or facilitate antibody-mediated antigen capture by APCs (26, 33- 35). Alternatively, sequence differences in the relevant epitopes or their flanking sequences could result in differential proteolytic cleavage or transporter associated with antigen processing (TAP)-mediated transport across the endoplasmic reticulum (ER) membrane (36). To explore these possibilities, we constructed a series of rVV-containing minigenes encoding the relevant 9-AA T cell epitopes preceded by the E3/19K adenoviral ER-insertion signal sequence (ES), previously shown to result in TAP-independent transport of antigenic peptides (37), and followed by a double stop codon. These constructs eliminated differences in flanking sequences and other nonhomologous regions of the molecule.
Mice were vaccinated with rVVESmgp100, rVVEShgp10025-33, or rVVES-gal96-103, encoding the 9-AA mgp10025-33, hgp10025-33, and
-gal96-103 peptides, respectively (38). Splenocytes were cultured for 6 d with mgp10025-33 or hgp10025-33 peptides then tested for specificity in a cytokine release assay (Table 1). Reactivities were similar to those obtained with rVV encoding full-length gp100 molecules: immunization with the mouse minigene gp100 construct failed to induce T cells, whereas immunization with
the hgp100 construct induced T cells that were cross-reactive with both mgp10025-33 and hgp10025-33 peptides. These
results were not consistent with a major role for differential
peptide processing and transport, antibody-facilitated antigen
presentation, or intramolecular immunodominance in the
differential in vivo immunogenicity of mgp100 and hgp100.
To evaluate the relative avidity of the gp100-specific T cells for the mgp10025-33 and hgp10025-33 epitopes,
we pulsed the peptides onto EL-4 cells (H-2b) (Fig. 4). A
striking difference in the relative T cell activities to the
mgp10025-33 and hgp10025-33 peptides was observed. The
mgp10025-33 peptide was recognized at concentrations as
low as 109 M, with half-maximal recognition occurring at
about 10
8 M, whereas the hgp10025-33 peptide was recognized at concentrations as low as 10
11 M. Alanine-substitutions at positions 1, 2, or 3 in either the mouse or human
versions of the synthetic peptides did not substantially alter
recognition by gp100-reactive T cells (Fig. 5 A). Indeed, a
triple-substituted peptide, AAARNQDWL, fully retained
the ability to sensitize EL-4 for recognition. (Fig. 5 B). The
first three AAs could even be deleted and a substantial degree of recognition remained (Fig. 5 B). However, substitution of any of the AAs at positions 4 through 9 abrogated
recognition of both the hgp10025-33 and mgp10025-33 peptide variants. To determine the involvement of MHC class
I binding affinity, an MHC class I stabilization assay was
done on RMA/S cells, which lack activity of the TAP transporters (13). FACS® analysis revealed 50% stabilization
of Db by hgp10025-33 peptide at a concentration ~100-fold
lower than for mgp10025-33 (Fig. 6). This indicated that the
apparent avidity of gp100-reactive T cells for the hgp10025-33
peptide could be largely attributed to its greater ability to
stabilize the restricting MHC class I molecule, H-2Db.
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One major potential use for self-reactive T cells is
their application in cancer treatment. To evaluate the usefulness of altered antigen in the development of a synthetic
cancer vaccine capable of eliciting therapeutic T cells, mice
were vaccinated once with rVVhgp100 and restimulated
for three rounds with splenocytes pulsed with 1 µM
mgp10025-33 peptide. CD8+ T cells were tested for gp100
recognition by IFN- release (Fig. 7 A). T cells strongly
recognized both mgp10025-33 and hgp10025-33 peptides, but
not control OVA257-264 peptide, and recognized as well
melanoma B16.WT, B16.B7-1, and JB/MS, a chemically
induced C57BL/6 melanoma. Control H-2b, gp100
tumor lines were not recognized.
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The function of T lymphocytes generated using purely recombinant and synthetic forms of gp100 were evaluated by adoptive transfer of T cells to mice bearing tumors established for 3 d in vivo. Mice receiving mgp10025-33-specific T cells experienced a significant reduction in tumor burden compared with mice receiving control T cells (Fig. 7 B). The survival of these cells was evaluated in vivo by transfer 5 d before tumor challenge, with T cells retaining their antitumor activity for this period (Fig. 7 C). This indicated that tumor-specific T cells induced with an antigen-based cancer vaccine can survive in vivo and mediate efficient tumor destruction.
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Discussion |
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The nonhomologous sequences flanking a MHC-restricted epitope could influence the immunogenicity of the epitope through a variety of mechanisms. The full-length hgp100 and mgp100 molecules are 76% identical at the AA level (11). Xenoimmunization could induce antibodies to nonhomologous determinants on the xenoantigen. When expressed on the surface of B cells that produce them, these antibodies could capture the xenoantigen and make it available for B cell processing and presentation on MHC class II to activate CD4+ T cells (35). This mechanism has been postulated to play a role in the initiation of human autoimmune diseases such as SLE, which is largely mediated by CD4+ T cells and autoreactive antibodies (25, 34). Recent data suggest that B cells may cross-present antigen on MHC class I after capture (39). However, there is a vigorous debate over the ability of B cells to activate "virgin" T cells (i.e., T cells that have not previously been activated by antigen) (33, 40). DCs can also capture immune complexes containing xenoantigen through Fc receptors and present it through MHC class I and class II pathways, inducing de novo activation of autoreactive T cells (41). In this scenario, the completely autologous mgp100 would not induce such antibodies and thus would fail to be captured and presented. The involvement of extra-epitope sequences in the immunogenicity of the human gp100 molecules is not consistent with the results shown in Table 1 where the immunogenicity of human full-length and minimal determinant constructs are compared. In fact, the two constructs elicit comparable CD8+ T cell responses to the 25-33 epitope.
Another mechanism by which xenoimmunization could enhance immunogenicity is by the processing of a given epitope. For example, the hgp10025-33 peptide might be processed more efficiently than the mgp10025-33 peptide, since the two differ in the three NH2-terminal AAs, as well as in the AA sequences surrounding the peptides in the full-length molecule (36, 42). The differences between the processing of the human and mouse sequences is minimized and possibly eliminated by the use of minigene constructs preceded by ER-insertion signal sequences that bypass proteasome-mediated peptide liberation as well as TAP-mediated peptide transport (Table 1).
Indeed, the increased immunogenicity of human gp100 appeared to reside completely within the 9-mer peptide. The hgp10025-33 peptide differed from its mouse counterpart in three NH2-terminal AAs. In fact, these three differences were rather dramatic, with a positively charged lysine (K) replacing a negatively charged glutamic acid (E), a medium-sized valine (V) replacing a small glycine (G), and proline (P), which is a cyclic residue and known to reduce the number of possible conformations due to impaired hydrogen bonding, replacing serine (S), which may not induce such a structural distortion. Despite the presence in both peptides of optimal anchor residues at both dominant anchor positions, 5 (N) and 9 (L), the difference in the three NH2-terminal residues resulted in a dramatically increased affinity of the hgp10025-33 peptide for the mouse MHC class I allele, H-2Db, compared with the mgp10025-33 peptide (Fig. 6). Clearly, the ability of the peptide to stabilize Db molecules on the surface of RMA-S cells could reflect the amount of peptide presented on the surface of APCs, which in turn could determine the activation of T cell precursors. In retrospect, it was fortuitous that the interspecies differences in the gp100 sequences created such a high binder, whereas the similarities preserved sufficient T cell receptor contact residues to allow cross-recognition.
The efficacy of the mechanism described here through which xenoimmunization induced autoreactive T cells required that a number of criteria be satisfied. There must be an MHC-binding epitope in the autologous protein which is naturally processed and presented that can be recognized by the available T cell repertoire. Most importantly, a homologous epitope from the xenogeneic protein must also be naturally processed and presented, but must be presented in the context of MHC at a higher density on the surface of the APCs. The chance that such an epitope will be found in any give xenoantigen may be small. However, an understanding of this mechanism clearly points the way to the rational design of immunogens based on the enhancement of the stability of peptide-MHC complexes.
The data presented here suggest that the main difference between the mgp10025-33 peptide and its hgp10025-33 analogue resides in binding to MHC class I. In human in vitro studies, modification of the second AA of the hgp100 peptide epitope hgp100209-217 to methionine has been shown to significantly increase its affinity to HLA-A2, leading to dramatically increased ability to raise gp100-specific T cells from patient PBLs in vitro (43). The higher affinity peptide is also more effective in vaccinating patients in vivo, increasing gp100-specific T cell precursor levels, and possibly resulting in higher treatment response rates (43a). Similarly, other groups have reported increased immunogenicity of peptide epitopes with enhanced MHC class I binding (26, 44, 45).
Taken together, these data suggest that there is indeed unresponsiveness to gp100 in mice. However, the unresponsiveness is relative and can be broken by using a peptide homologue with higher affinity for MHC class I. One mechanism through which a peptide with higher MHC class I binding can break tolerance is based on the assumption that T cell tolerance exists to a level of antigen rather than to the identity of the antigen (44, 46, 47). In the case of gp100, CD8+ T cell precursors with the ability to recognize a certain amount of peptide in the context of MHC class I are inactivated, through either thymic and/or peripheral deletion or anergization. The remaining T cells have TCRs with an affinity that is too low to be triggered by the levels of gp100 peptide present on melanocytes or APCs in lymph nodes draining the skin. Therefore, these T cells are never activated, deleted, or anergized. Instead, they remain "ignorant" of MHC class I with mgp10025-33 peptide. A vaccination with autologous mgp100 will not trigger these T cells unless the vaccine is able to significantly raise the amount of peptide-MHC class I complex on professional APCs to a level high enough to surpass the TCR threshold. Only then do the T cells become activated. We are currently evaluating the clinical efficacy of antitumor effects of gp100-based cancer vaccines that contain epitopes with enhanced stability of peptide-MHC complexes.
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Footnotes |
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Received for publication 17 March 1998.
Correspondence to Nicholas P. Restifo, Senior Investigator, National Cancer Institute, Bldg. 10, Rm. 2B42, Bethesda, MD 20892-1502. Phone: 301-496-4904; Fax: 301-496-0011; E-mail: restifo{at}nih.govThe authors wish to thank Deborah R. Surman and Mr. Paul J. Spiess for expert technical assistance; Ms. Martha Blalock for assistance with graphics; Drs. Dennis Panicali and Linda Gritz (Therion Biologics) for providing rVVs; and Dr. Bruce Roberts for providing recombinant adenoviruses.
Abbreviations used in this paper
AA, amino acid;
-gal,
-galactosidase;
CM, complete medium;
DC, dendritic cell;
ER, endoplasmic reticulum;
FPV, fowlpox virus;
h, human;
m, mouse;
MART, melanoma antigen
recognized by T lymphocytes;
MDA, melanocyte differentiation;
NP, the
nucleoprotein from influenza A;
pDNA, plasmid DNA;
rVV, recombinant vaccinia virus;
TAP, the transporter associated with antigen processing;
TRP, tyrosinase-related protein.
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