Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma 1
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免疫学杂志 2005年第11期
Abstract
Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.
Introduction
lieved to be the principal effectors in an active immune surveillance network that protects immunocompetent individuals from expansion of neoplastic lesions (1). Accordingly, patients with advancing melanoma spontaneously develop cytotoxic T cell responses to multiple melanoma-associated Ags (MAA) 3 (2, 3), and these MAA-specific immune responses can be augmented by tumor vaccines (4, 5). However, these responses impart selective pressure on tumor cells, often leading to immune editing and selection of variant tumor cells that escape immune recognition by any of several mechanisms, including tumor Ag down-regulation (6), MHC down-regulation or loss (7, 8), defective Ag processing (9, 10, 11), and secretion of immunosuppressive cytokines (12). The finding of immune editing in cancer patients prompts concern that immunotherapy may ultimately fail as tumors undergo repeated rounds of selection and immune evasion. Extended survival after such immune editing would require the human immune system to respond spontaneously and to refocus responses against newly arising tumor variants in an adaptive manner. However, this phenomenon has not been well studied.
As mechanisms of immune escape are more clearly delineated, it is equally important to understand mechanisms by which the human immune system may overcome tumor immune editing. In the present report, we document a case of immune adaptation in a long-term survivor of metastatic melanoma. After the tumor evaded immune recognition through a combination of defects, including the ablation of presentation of an immunodominant MART-1 peptide, the host’s immune repertoire expanded to include recognition of a previously cryptic peptide from tyrosinase presented by the tumor cells in vivo. Significantly, the tyrosinase-derived epitope subsequently became the immunodominant Ag. This patient’s long-term survival, without specific immunotherapy, suggests that adaptive evolution of the immune response may be a protective mechanism that can overcome immune escape by tumor cells. These findings underscore the potential for active immunization as a tool to increase the magnitude of nonimmunodominant T cell responses and to broaden the tumor Ag-specific repertoire.
Materials and Methods
Expression of MART-1 and tyrosinase proteins was reconstituted in VMM5B-2m cells using recombinant vaccinia viruses expressing the respective full-length proteins. Cells infected in parallel with an identical vaccinia construct expressing influenza M1 protein were used as a negative control. Viruses were constructed as described (32), titered, and tested for appropriate expression using HLA-A2-restricted CTL (data not shown). VMM5B-2m cells were infected with 10 PFU/ml vaccinia virus for 30 min in HBSS supplemented with 0.1% BSA, 1.6 mM MgSO4, and 1.8 mM CaCl2, and then cultured for 6 h in RPMI 1640 medium supplemented with 10% FCS to allow transcription and expression of vaccinia-encoded proteins. Following vaccinia infection, targets were labeled in 100 μCi of Na51CrO4 for 2 h, and then standard 51Cr release assays were performed using MART-1 or tyrosinase-specific CTL to evaluate recognition of MART-127–35 or Tyr369–377D epitopes.
Results
The dominant immune response shifts from MART-1 to tyrosinase-derived epitopes in sequential metastatic recurrences
The patient VMM5 developed metastatic melanoma in LN 5 years after initial resection of primary disease, and metastatic disease reoccurred in regional LN a second time 6 years later. Tumor-involved nodes were collected at each occurrence of disease (TIN-A and TIN-B, respectively). To determine the endogenous responses against melanocyte differentiation protein (MDP)-derived Ags, lymphocytes from TIN-A and TIN-B were cultured for 14 days in the presence of tumor cells from the corresponding TIN and IL-2, and analyzed for peptide reactivity in an ELISPOT assay. The dominant reactivity from TIN-A was to the MART-127–35 peptide, AAGIGILTV, whereas the dominant response from TIN-B was against the Tyr369–377D peptide YMDGTMSQV (Fig. 1A). For further analysis, long-term CTL lines were generated from TIN-A and TIN-B (CTL-A and CTL-B, respectively). CTL-A recognized at least six peptide epitopes associated with the HLA-A2 Ag, including MART-127–35 (14, 16, 33), but did not recognize the Tyr369–377D epitope (Fig. 1B). CTL generated from PBL collected at the time of TIN-A resection, and for several years thereafter, likewise were lytic against targets bearing MART-127–35 but not the Tyr369–377D epitope (data not shown). In contrast to CTL-A, the CTL-B lines, which were restimulated with the VMM5A melanoma cell line, demonstrated significant reactivity against the Tyr369–377D epitope but significantly decreased reactivity against MART-127–35 (Fig. 1B). PBL harvested 1 year before detection of the second LN metastasis, and stimulated in the same way, recognized the Tyr369–377D epitope (Fig. 1C). Thus, whereas Tyr369–377D-specific CTLs are virtually absent from the first metastatic outgrowth, Tyr369–377D-specific CTLs are abundant in the second metastatic outgrowth and became apparent in the peripheral blood in advance of the second tumor recurrence. MART-127–35-specific CTLs, which were abundant in the initial metastatic lesion, are present in significantly smaller numbers in TIN-B and CTL-B. Collectively, these data demonstrate a significant change in immunodominance, suggesting that immune editing by the tumor may change the presentation of MDP-derived epitopes, leading the immune system to adapt dynamically by shifting the major antitumor CD8 response to a different Ag.
The shift in immunodominant CTL response is associated with a loss of MART-1 epitope-MHC complexes on tumors from the second LN metastasis (TIN-B)
The concurrent loss of MART-1 reactivity and development of tyrosinase reactivity within T cell populations infiltrating the TIN-B metastasis suggested an immune-editing event that would immunologically distinguish this tumor from the original LN metastasis (TIN-A). To test this possibility, we performed cytotoxicity assays using CTL with defined specificity to determine whether HLA-A2-MART-127–35 peptide complexes and HLA-A2-Tyr369–377D peptide complexes are differentially expressed on tumor cells isolated from TIN-A and TIN-B. Long-term in vitro-restimulated CTL-A, which had lost reactivity against other MDP epitopes but maintained strong, restricted reactivity to the MART-127–35 epitope, effectively lysed cultured tumor cells from TIN-A (VMM5A), but not TIN-B tumor cells ex vivo (Fig. 2A). CTL-A also lysed specific-epitope-pulsed MART-1-negative targets, MART-1-expressing melanoma cell line DM6, and MART-1-expressing melanoma cells from TIN of patient VMM162. These CTL failed to lyse the MART-1-negative melanoma cell line DM331 or MART-1-negative tumor cells from TIN of patient VMM87. Lysis of nonautologous ex vivo and cultured tumor cells that express HLA-A2 and MART-1 Ags suggests that the failure of CTL-A to lyse TIN-B tumor ex vivo was independent of culture conditions of the target cells. Collectively, these data are consistent with the loss of MHC-associated MART-127–35 epitope on the surface of TIN-B tumor cells.
To determine whether the failure of TIN-A CTL to lyse TIN-B tumor was Ag specific or was due to a more global defect in Ag presentation by TIN-B-derived tumor cells, lytic activity by HLA-A2-restricted Tyr369–377D-reactive CTL (VMM119 CTL) was evaluated (Fig. 2B). In contrast to VMM5-derived CTL-A, the VMM119 CTL lysed VMM5A tumor cells and TIN-B tumor cells ex vivo as well as ex vivo tumor from TIN of VMM87 and VMM162, and the HLA-A2+ Tyr369–377D-expressing melanoma cells DM6. These CTL failed to lyse the HLA-A2+ tyrosinase-negative DM331 melanoma cells. Thus, both TIN-A-derived VMM5A cells and TIN-B melanoma cells express HLA-A2-Tyr369–377D complexes, suggesting that the failure of CTL-A to recognize TIN-B melanoma cells may be due to loss of MHC-associated MART-127–35, and not simply loss of all HLA-A2 expression.
TIN-B melanoma cells express MDPs
The failure of MART-127–35-specific CTL to recognize and to lyse TIN-B-derived tumor could arise from loss of MART-1 expression at the protein level. However, immunohistochemistry demonstrates TIN-B melanoma cell expression of MART-1 protein, as well as tyrosinase and gp100 proteins (Fig. 3). Western blot analysis confirmed that MART-1 and tyrosinase are expressed both in VMM5A and TIN-B cells (Fig. 4). An additional high molecular mass band was present in TIN-B sample stained for MART-1 expression, but the significance of this finding is not clear.
We next evaluated the MART-1 gene in TIN-B-derived melanoma cells for mutations. Sequencing of RT-PCR-amplified products obtained from TIN-B ex vivo and from VMM5A cultured cells confirmed that the entire MART-1 coding sequence in TIN-B tumor matched the sequence obtained from VMM5A cells, as well as the published wild-type MART-1 sequence (GenBank accession no. NM 005511; data not shown). In summary, these data suggest the failure of MART-127–35-specific CTL to lyse TIN-B melanoma is not resultant to abrogated or altered expression of MART-1 protein.
Expression profiles of the Ag-processing machinery (APM) components in VMM5A and VMM5B melanoma cell lines
We next hypothesized that the failure of MART-127–35-specific CTL to lyse TIN-B melanoma may be the result of altered tumor cell presentation of the MART-127–35 epitope. Therefore, we evaluated the expression of APM components by TIN-A-and TIN-B-derived cell lines, VMM5A and VMM5B, respectively, to evaluate possible changes in the expression of these molecules. Intracellular flow cytometric analyses demonstrated that the major components of the APM are present in both cell lines, although the levels of low-molecular-weight protein (LMP)-7, LMP10, TAP1, tapasin, and HLA-A, -B, -C H chain expression was slightly lower in VMM5B cells than in VMM5A cells (Fig. 5). Additionally, there appears to be a subset of VMM5B cells with loss of TAP2. However, both lines express the necessary components to process Ag for presentation, and these data fail to explain the differential expression of MART-127–35 by VMM5B cells.
TIN-B melanoma cells down-regulate surface HLA-A2 expression
To evaluate whether loss of recognition of TIN-B melanoma cells by MART-1-specific T cells was associated with down-regulation of surface HLA class I Ag, we evaluated tumor cells by cell surface staining with HLA class I-specific Ab and also evaluated tumor cells by DNA typing to assess for allelic loss. Both the cultured melanoma cell lines VMM5A and VMM5B were negative for HLA-A11, B44, and Cw17 Ags, although PBL tested positive for these alleles, both by serologic and DNA typing (data not shown). Thus, HLA-A11, B44, and Cw17 allelic loss was an Ag escape mechanism that was acquired early in the course of the disease and may not be responsible for the described differential HLA-A2-peptide complex expression by tumor cells in the two lesions.
We next evaluated surface expression of HLA class I Ags by cell surface staining with HLA class I-specific mAb combined with cell surface staining for melanoma cells by their expression of the melanoma-specific HMW Ag (Ab 763.74) (29). Interestingly, two melanoma cell populations were identified in TIN-B (Fig. 6A). One included >80% of melanoma cells and was characterized by very low HLA class I Ag expression. This phenotype is likely to have been caused by loss of one 2m gene and by a point mutation in the other 2m gene copy (C.-C. Chang, unpublished results). The remaining 12% of melanoma cells maintain HLA class I Ag expression (Fig. 6A). The melanoma cell line VMM5B was derived from TIN-B and, thus, represents a subset of TIN-B cells. By surface staining, cells in this melanoma cell line have almost completely lost cell surface HLA class I expression (Fig. 6B); however, some HLA-A, -B, -C H chain expression was detected by intracellular staining of VMM5B melanoma cell line (see Fig. 5). The functional significance of this mutation is demonstrated by the restoration of surface HLA class I Ag on VMM5B cells transfected with a plasmid containing DNA for wild-type 2m under the control of a constitutive promoter (Fig. 6B). Furthermore, this mutation in 2m was present in a subpopulation of tumor cells in TIN-B, suggesting that some cells in TIN-B had reduced expression of HLA-A2 by the same mechanism. Recognition and lysis of tumor cells from TIN-B by tyrosinase-reactive CTL ex vivo (see Fig. 2) confirms that at least a portion of the cells in the tumor deposit retained expression in vivo of sufficient HLA-A2 Ag for T-cell recognition.
Cell surface presentation of HLA-A2-associated MART-1 epitope is limited by competition from tyrosinase-derived peptides
HLA class I Ag down-regulation provided many of the melanoma cells in the second metastasis of patient VMM5 with an escape mechanism from T cell recognition and destruction. However, a subset of melanoma cells retained low-level expression of HLA class I surface Ag. These latter cells appear to have lost their susceptibility to MART-127–35-specific CTL, but were susceptible to recognition by Tyr369–377D-specific CTL.
These findings raise the possibility that HLA class I Ag down-regulation influences the presentation of distinct Melanoma Ag-derived peptides, likely because of their differential binding affinity to HLA-A2 Ag. Studies of Valmori et al. (34) suggested that MART-127–35 binds to HLA-A2 Ag with relatively low affinity. Furthermore, Engelhard et al. (35) demonstrated that HLA-A2 Ag has a significantly lower binding affinity for MART-127–35 than for Tyr369–377D (IC50 of 950 and 74 nM, respectively), and mass spectrometry studies of peptides eluted from class I molecules of the human melanoma cell line DM6 indicated surface MART-127–35 to be present at 100-fold copies per cell less than Tyr369–377D.
We hypothesized that presentation of HLA-A2-MART-127–35 complexes may be functionally abrogated in cells with low HLA-A2 Ag expression, because other peptides with higher affinity for HLA-A2 compete for binding sites in a HLA class I molecule-limited environment. Limited availability for HLA-A2 Ag may arise either from decreased expression of HLA class I gene products, or from the failure to form stable peptide-HLA-A2 complexes in the absence of functional 2m (C.-C. Chang, unpublished results). Therefore, we evaluated the possibility that Tyr369–377D peptides compete for free HLA-A2 molecules and prevent sufficient cell surface presentation of MART-137–35 peptides for T cell recognition. To test this hypothesis, recombinant vaccinia viruses expressing full-length human MART-1 (MART-Vac) or tyrosinase (Tyr-Vac) were used to drive overexpression of these proteins, singly or in combination, in TIN-B melanoma cells stably transfected to express wild-type 2m (VMM5B 2m), which reconstituted functional HLA class I assembly and cell surface expression (see Fig. 6B). We then evaluated the lytic activity of Tyr369–377D- and MART-127–35-specific CTL (VMM119 and VMM5A, respectively) against uninfected or vaccinia-infected tumor cells. Expression of tyrosinase and MART-1 in infected cells was confirmed by intracellular staining and Western blot analysis using specific Abs (data not shown). Tyr-Vac infection led to processing and presentation of Tyr369–377D epitope by TIN-B-2m cells, as evidenced by specific lysis by VMM119 T cells (Fig. 7, A and B); likewise MART-127–35-specific VMM5-A CTL effectively lysed MART-1-Vac-infected tumor cells (C and D). However, whereas VMM119 T cells lysed either TyrVac-infected or Tyr-Vac/MART-Vac-coinfected melanoma cells with equal efficiency (Fig. 7A), VMM5-A CTL failed to lyse coinfected melanoma cells (C). Both CTL efficiently lysed HLA-A2+ DM6 melanoma cells, which express both tyrosinase and MART-1, but neither CTL mediated lytic activity against HLA-A2+ tyrosinase- and MART-1-deficient DM331 cells (Fig. 7, B and D). The absence of surface HLA-A2-MART-127–35 complexes consequent to the overexpression of tyrosinase is not unique to the TIN-B-derived tumor cell lines, because the phenomenon was also evident using vaccinia vectors to drive tyrosinase and MART-1 expression in DM331 cells (Fig. 7, B and D).
Discussion
We report here the adaptive evolution of a melanoma Ag-specific cellular immune response in a long-term survivor of melanoma who had two recurrences in regional LN. The immune response to the original tumor metastasis (TIN-A) was polyvalent, with immunodominance manifested against the MART-127–35 epitope (AAGIGILTV), which was expressed by TIN-A tumor cells. Disease progression was heralded by a change in the immune repertoire of the patient’s systemic CTL response to melanoma Ags. The CTL response in the second metastasis (TIN-B) was directed primarily against the Tyr369–377D epitope (YMDGTMSQV), to which there had been no detectable response in TIN-A. Response to the MART-127–35 peptide, which had dominated the TIN-A tumor recognition, was lost in TIN-B. This change in the repertoire of the CTL response to autologous melanoma cells in vivo corresponds temporally and spatially to a loss of MART-127–35 presentation in Ag expression by the melanoma cells in TIN-B.
Detailed evaluation of tumor cells from TIN-A and TIN-B revealed evidence for immune editing and selection of a variety of immune escape mutants during tumor progression. HLA-A11, B44, and Cw17 allelic loss was characteristic of this tumor at least since the time of the first metastasis. More recent phenomena were down-regulation of HLA class I expression in a subset of tumor cells, and loss of functional HLA class I Ag expression due to a point mutation in the 2m gene (C.-C. Chang, unpublished results). Down-regulation and loss of MDP expression was yet another ongoing process in at least one of the tumor cell subsets. Loss of expression of HLA class I molecules and of MAA presumably provided survival advantage for the tumor cells in the setting of a host MAA-specific response.
The second metastatic recurrence in this patient differed from the first in the loss of presentation of the MART-127–35 epitope, even though MART-1 protein was expressed at comparable levels in both metastases. Because TIN-B tumor cells were lysed by tyrosinase-reactive CTL restricted by HLA-A2, and because TIN-B tumor cells expressed unmutated MART-1, absence of recognition by MART-127–35-reactive CTL cannot be explained simply by loss of HLA-A2 expression or by lack of expression of the protein. Proteasome immunosubunits in dendritic cells can prevent presentation of the MART-127–35 epitope (36), but the VMM5B cell line derived from TIN-B does not appear to have a higher expression of proteasome immunosubunits (LMP2, LMP7, and LMP10), and most of the other components of the APM appear to be preserved in TIN-B cells. TAP2 down-regulation could potentially lead to changes in processing and presentation of selected peptides, and may well have been another immune escape mechanism for a subset of cells. However, transfection of cells with wild-type 2m and MART-1 restored recognition by MART-127–35 reactive T cells. Thus, changes in Ag processing alone do not explain the loss of MART-127–35 complexes.
We have explored an alternate hypothesis to explain a failure of TIN-B tumor cells to present the MART-1 peptide. We hypothesized that competition of peptides for HLA class I Ag binding may be a contributing factor to the selective loss of presentation of epitopes with low affinity for HLA-A2 molecules. The MART-127–35 epitope has low-affinity binding to HLA-A2 Ag, with an IC50 of 950 nM, whereas the Tyr369–377D peptide is a strong binder with IC50 of 74 nM (35). Furthermore, the epitope frequency on the cell surface of melanoma cells is much lower for MART-127–35 peptide than for Tyr369–377D peptide (35). In the setting of HLA class I Ag down-regulation on the tumor cells, the low number of MHC molecules limits the number of peptides that can be presented, such that peptides with low affinity for the MHC molecule may be available at too low a copy number to permit recognition by epitope-specific T cells.
This hypothesis was supported by our experiments in which tyrosinase and MART-1 proteins were re-expressed in the VMM5B-2m tumor cell line using vaccinia vectors (Fig. 7). When MART-1 or tyrosinase proteins were expressed in VMM5B-2M cells separately, each peptide was processed and presented in a context sufficient for recognition by peptide-specific CTL. However, recognition of HLA-A2/MART-127–35 by peptide-specific CTL was significantly diminished upon coexpression of MART-1 and tyrosinase proteins in the same cells. This result was not unique to the VMM5-2m cell line, but was repeated with DM331 cells with a similar result. Collectively, these data suggest that the number of HLA class I molecules usually is not limiting. However, in tumor cells with dysregulated HLA class I expression or disrupted HLA class I stability, the MHC molecules become limiting, and surface presentation of the weakly binding MART-127–35 is significantly diminished as more strongly binding epitopes compete for limited HLA class I docking sites. We propose a model in which down-regulation of surface HLA class I Ag expression does not simply decrease the total number of HLA class I Ag-peptide complexes, but also alters the proportion of specific epitopes presented on the cell surface according to peptide affinity for MHC. As far as we know, this novel mechanism of tumor escape and epitope selection has not been previously described.
Despite multiple immune escape mechanisms, there remains persistent expression of the Tyr369–377D epitope on tumor cells in TIN-B. Therefore, the immune response that has arisen in that node (and systemically) against that epitope is an adaptive response, suggesting an appropriate change in immune repertoire. The findings are 1) that tumor cells have evolved toward an Ag-loss phenotype in the setting of a T cell response to tumor Ags, 2) that the Ag-loss phenotype included loss of the immunodominant epitope of the host T cells but retained expression of a cryptic epitope (Tyr369–377D), and 3) that the CTL repertoire has changed simultaneously, in a compensatory and adaptive manner, with dominant targeting of the previously cryptic epitope.
The fact that tumor did not appear for 5 years after the original recurrence, and the fact that the patient remained disease-free after surgical resection, both suggest that this immune response may have had clinical relevance. However, the tumor did recur despite this adaptive response, and this suggests that the adaptive response was inadequate to control this particular metastasis completely. Multiple immune escape mechanisms occurred over time. A large subset of cells in TIN-B down-regulated expression of class I HLA molecules due to a point mutation in the 2m gene. This change would have permitted escape from recognition by HLA-restricted T cells. However, that change may also have made those cells more sensitive to NK-mediated lysis. The subsequent systemic control after surgery suggests that the tumor cells with the most complete immune escape phenotype either had not metastasized beyond this tumor deposit, or that other systemic mechanisms such as NK cells controlled micrometastases beyond this deposit. In any event, this presentation is a reminder that surgical intervention for isolated metastases can succeed in some cases.
Another example of what we consider an adaptive immune response has been described by Coulie and his associates (37) in a patient treated with tumor vaccines as well as surgery, where loss of MHC expression by a tumor was followed by development of a novel response by CTL bearing killer inhibitory receptors that targeted cells with loss of certain MHC molecules. By contrast, the present report shows evidence of an adaptive immune response in a patient treated only with surgery. Also, in this case, the adaptive response occurs by a different and more classic mechanism, responding to immune escape by retargeting to a CTL epitope still presented on the tumor after the selective loss of presentation of a previously immunodominant peptide. This observation also suggests the importance of developing immune therapy directed against multiple Ags simultaneously. As we learn more about mechanisms by which tumors may evade immune recognition, it is encouraging that the human immune system, even in this elderly patient, has the plasticity to evolve in an adaptive manner in response to immune escape by the tumor.
It is tempting to speculate that development of such an adaptive immune response may predict a favorable clinical outcome. In this regard, unraveling the cellular and molecular events that govern such changes will be worthy of investigation. The resulting information will not only improve our understanding of the delicate interactions between tumor cells and the immune system, but may also suggest relevant therapeutic strategies to prevent tumor progression in the setting of immune escape.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was funded by National Institutes of Health Grants R01 CA57653 (to C.L.S.) and RO1 CA67108 and P30 CA16056 (to S.F.). C.L.S. has been an Elaine Shepard Cancer Investigator supported by the Cancer Research Institute. C.-C.C. is supported by a Susan G. Komen Breast Cancer Foundation predoctoral fellowship. The work was supported also by the Cancer Center Support Grant (National Institutes of Health P30CA44579) at the University of Virginia (Tissue Procurement Facility, Flow Cytometry Core, Biomolecular Core), and by the Pratt Fund at the University of Virginia.
2 Address correspondence and reprint requests to Dr. Craig L. Slingluff, Jr., Department of Surgery, University of Virginia Health Sciences Center, P.O. Box 800709, Charlottesville, VA 22908. E-mail address: cls8h{at}virginia.edu
3 Abbreviations used in this paper: MAA, melanoma-associated Ag; LMP, low-molecular-weight protein; LN, lymph node; TIN, tumor-infiltrated node; 2m, 2-microglobulin; MDP, melanocyte differentiation protein; APM, Ag-processing machinery.
Received for publication October 25, 2004. Accepted for publication February 23, 2005.
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Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.
Introduction
lieved to be the principal effectors in an active immune surveillance network that protects immunocompetent individuals from expansion of neoplastic lesions (1). Accordingly, patients with advancing melanoma spontaneously develop cytotoxic T cell responses to multiple melanoma-associated Ags (MAA) 3 (2, 3), and these MAA-specific immune responses can be augmented by tumor vaccines (4, 5). However, these responses impart selective pressure on tumor cells, often leading to immune editing and selection of variant tumor cells that escape immune recognition by any of several mechanisms, including tumor Ag down-regulation (6), MHC down-regulation or loss (7, 8), defective Ag processing (9, 10, 11), and secretion of immunosuppressive cytokines (12). The finding of immune editing in cancer patients prompts concern that immunotherapy may ultimately fail as tumors undergo repeated rounds of selection and immune evasion. Extended survival after such immune editing would require the human immune system to respond spontaneously and to refocus responses against newly arising tumor variants in an adaptive manner. However, this phenomenon has not been well studied.
As mechanisms of immune escape are more clearly delineated, it is equally important to understand mechanisms by which the human immune system may overcome tumor immune editing. In the present report, we document a case of immune adaptation in a long-term survivor of metastatic melanoma. After the tumor evaded immune recognition through a combination of defects, including the ablation of presentation of an immunodominant MART-1 peptide, the host’s immune repertoire expanded to include recognition of a previously cryptic peptide from tyrosinase presented by the tumor cells in vivo. Significantly, the tyrosinase-derived epitope subsequently became the immunodominant Ag. This patient’s long-term survival, without specific immunotherapy, suggests that adaptive evolution of the immune response may be a protective mechanism that can overcome immune escape by tumor cells. These findings underscore the potential for active immunization as a tool to increase the magnitude of nonimmunodominant T cell responses and to broaden the tumor Ag-specific repertoire.
Materials and Methods
Expression of MART-1 and tyrosinase proteins was reconstituted in VMM5B-2m cells using recombinant vaccinia viruses expressing the respective full-length proteins. Cells infected in parallel with an identical vaccinia construct expressing influenza M1 protein were used as a negative control. Viruses were constructed as described (32), titered, and tested for appropriate expression using HLA-A2-restricted CTL (data not shown). VMM5B-2m cells were infected with 10 PFU/ml vaccinia virus for 30 min in HBSS supplemented with 0.1% BSA, 1.6 mM MgSO4, and 1.8 mM CaCl2, and then cultured for 6 h in RPMI 1640 medium supplemented with 10% FCS to allow transcription and expression of vaccinia-encoded proteins. Following vaccinia infection, targets were labeled in 100 μCi of Na51CrO4 for 2 h, and then standard 51Cr release assays were performed using MART-1 or tyrosinase-specific CTL to evaluate recognition of MART-127–35 or Tyr369–377D epitopes.
Results
The dominant immune response shifts from MART-1 to tyrosinase-derived epitopes in sequential metastatic recurrences
The patient VMM5 developed metastatic melanoma in LN 5 years after initial resection of primary disease, and metastatic disease reoccurred in regional LN a second time 6 years later. Tumor-involved nodes were collected at each occurrence of disease (TIN-A and TIN-B, respectively). To determine the endogenous responses against melanocyte differentiation protein (MDP)-derived Ags, lymphocytes from TIN-A and TIN-B were cultured for 14 days in the presence of tumor cells from the corresponding TIN and IL-2, and analyzed for peptide reactivity in an ELISPOT assay. The dominant reactivity from TIN-A was to the MART-127–35 peptide, AAGIGILTV, whereas the dominant response from TIN-B was against the Tyr369–377D peptide YMDGTMSQV (Fig. 1A). For further analysis, long-term CTL lines were generated from TIN-A and TIN-B (CTL-A and CTL-B, respectively). CTL-A recognized at least six peptide epitopes associated with the HLA-A2 Ag, including MART-127–35 (14, 16, 33), but did not recognize the Tyr369–377D epitope (Fig. 1B). CTL generated from PBL collected at the time of TIN-A resection, and for several years thereafter, likewise were lytic against targets bearing MART-127–35 but not the Tyr369–377D epitope (data not shown). In contrast to CTL-A, the CTL-B lines, which were restimulated with the VMM5A melanoma cell line, demonstrated significant reactivity against the Tyr369–377D epitope but significantly decreased reactivity against MART-127–35 (Fig. 1B). PBL harvested 1 year before detection of the second LN metastasis, and stimulated in the same way, recognized the Tyr369–377D epitope (Fig. 1C). Thus, whereas Tyr369–377D-specific CTLs are virtually absent from the first metastatic outgrowth, Tyr369–377D-specific CTLs are abundant in the second metastatic outgrowth and became apparent in the peripheral blood in advance of the second tumor recurrence. MART-127–35-specific CTLs, which were abundant in the initial metastatic lesion, are present in significantly smaller numbers in TIN-B and CTL-B. Collectively, these data demonstrate a significant change in immunodominance, suggesting that immune editing by the tumor may change the presentation of MDP-derived epitopes, leading the immune system to adapt dynamically by shifting the major antitumor CD8 response to a different Ag.
The shift in immunodominant CTL response is associated with a loss of MART-1 epitope-MHC complexes on tumors from the second LN metastasis (TIN-B)
The concurrent loss of MART-1 reactivity and development of tyrosinase reactivity within T cell populations infiltrating the TIN-B metastasis suggested an immune-editing event that would immunologically distinguish this tumor from the original LN metastasis (TIN-A). To test this possibility, we performed cytotoxicity assays using CTL with defined specificity to determine whether HLA-A2-MART-127–35 peptide complexes and HLA-A2-Tyr369–377D peptide complexes are differentially expressed on tumor cells isolated from TIN-A and TIN-B. Long-term in vitro-restimulated CTL-A, which had lost reactivity against other MDP epitopes but maintained strong, restricted reactivity to the MART-127–35 epitope, effectively lysed cultured tumor cells from TIN-A (VMM5A), but not TIN-B tumor cells ex vivo (Fig. 2A). CTL-A also lysed specific-epitope-pulsed MART-1-negative targets, MART-1-expressing melanoma cell line DM6, and MART-1-expressing melanoma cells from TIN of patient VMM162. These CTL failed to lyse the MART-1-negative melanoma cell line DM331 or MART-1-negative tumor cells from TIN of patient VMM87. Lysis of nonautologous ex vivo and cultured tumor cells that express HLA-A2 and MART-1 Ags suggests that the failure of CTL-A to lyse TIN-B tumor ex vivo was independent of culture conditions of the target cells. Collectively, these data are consistent with the loss of MHC-associated MART-127–35 epitope on the surface of TIN-B tumor cells.
To determine whether the failure of TIN-A CTL to lyse TIN-B tumor was Ag specific or was due to a more global defect in Ag presentation by TIN-B-derived tumor cells, lytic activity by HLA-A2-restricted Tyr369–377D-reactive CTL (VMM119 CTL) was evaluated (Fig. 2B). In contrast to VMM5-derived CTL-A, the VMM119 CTL lysed VMM5A tumor cells and TIN-B tumor cells ex vivo as well as ex vivo tumor from TIN of VMM87 and VMM162, and the HLA-A2+ Tyr369–377D-expressing melanoma cells DM6. These CTL failed to lyse the HLA-A2+ tyrosinase-negative DM331 melanoma cells. Thus, both TIN-A-derived VMM5A cells and TIN-B melanoma cells express HLA-A2-Tyr369–377D complexes, suggesting that the failure of CTL-A to recognize TIN-B melanoma cells may be due to loss of MHC-associated MART-127–35, and not simply loss of all HLA-A2 expression.
TIN-B melanoma cells express MDPs
The failure of MART-127–35-specific CTL to recognize and to lyse TIN-B-derived tumor could arise from loss of MART-1 expression at the protein level. However, immunohistochemistry demonstrates TIN-B melanoma cell expression of MART-1 protein, as well as tyrosinase and gp100 proteins (Fig. 3). Western blot analysis confirmed that MART-1 and tyrosinase are expressed both in VMM5A and TIN-B cells (Fig. 4). An additional high molecular mass band was present in TIN-B sample stained for MART-1 expression, but the significance of this finding is not clear.
We next evaluated the MART-1 gene in TIN-B-derived melanoma cells for mutations. Sequencing of RT-PCR-amplified products obtained from TIN-B ex vivo and from VMM5A cultured cells confirmed that the entire MART-1 coding sequence in TIN-B tumor matched the sequence obtained from VMM5A cells, as well as the published wild-type MART-1 sequence (GenBank accession no. NM 005511; data not shown). In summary, these data suggest the failure of MART-127–35-specific CTL to lyse TIN-B melanoma is not resultant to abrogated or altered expression of MART-1 protein.
Expression profiles of the Ag-processing machinery (APM) components in VMM5A and VMM5B melanoma cell lines
We next hypothesized that the failure of MART-127–35-specific CTL to lyse TIN-B melanoma may be the result of altered tumor cell presentation of the MART-127–35 epitope. Therefore, we evaluated the expression of APM components by TIN-A-and TIN-B-derived cell lines, VMM5A and VMM5B, respectively, to evaluate possible changes in the expression of these molecules. Intracellular flow cytometric analyses demonstrated that the major components of the APM are present in both cell lines, although the levels of low-molecular-weight protein (LMP)-7, LMP10, TAP1, tapasin, and HLA-A, -B, -C H chain expression was slightly lower in VMM5B cells than in VMM5A cells (Fig. 5). Additionally, there appears to be a subset of VMM5B cells with loss of TAP2. However, both lines express the necessary components to process Ag for presentation, and these data fail to explain the differential expression of MART-127–35 by VMM5B cells.
TIN-B melanoma cells down-regulate surface HLA-A2 expression
To evaluate whether loss of recognition of TIN-B melanoma cells by MART-1-specific T cells was associated with down-regulation of surface HLA class I Ag, we evaluated tumor cells by cell surface staining with HLA class I-specific Ab and also evaluated tumor cells by DNA typing to assess for allelic loss. Both the cultured melanoma cell lines VMM5A and VMM5B were negative for HLA-A11, B44, and Cw17 Ags, although PBL tested positive for these alleles, both by serologic and DNA typing (data not shown). Thus, HLA-A11, B44, and Cw17 allelic loss was an Ag escape mechanism that was acquired early in the course of the disease and may not be responsible for the described differential HLA-A2-peptide complex expression by tumor cells in the two lesions.
We next evaluated surface expression of HLA class I Ags by cell surface staining with HLA class I-specific mAb combined with cell surface staining for melanoma cells by their expression of the melanoma-specific HMW Ag (Ab 763.74) (29). Interestingly, two melanoma cell populations were identified in TIN-B (Fig. 6A). One included >80% of melanoma cells and was characterized by very low HLA class I Ag expression. This phenotype is likely to have been caused by loss of one 2m gene and by a point mutation in the other 2m gene copy (C.-C. Chang, unpublished results). The remaining 12% of melanoma cells maintain HLA class I Ag expression (Fig. 6A). The melanoma cell line VMM5B was derived from TIN-B and, thus, represents a subset of TIN-B cells. By surface staining, cells in this melanoma cell line have almost completely lost cell surface HLA class I expression (Fig. 6B); however, some HLA-A, -B, -C H chain expression was detected by intracellular staining of VMM5B melanoma cell line (see Fig. 5). The functional significance of this mutation is demonstrated by the restoration of surface HLA class I Ag on VMM5B cells transfected with a plasmid containing DNA for wild-type 2m under the control of a constitutive promoter (Fig. 6B). Furthermore, this mutation in 2m was present in a subpopulation of tumor cells in TIN-B, suggesting that some cells in TIN-B had reduced expression of HLA-A2 by the same mechanism. Recognition and lysis of tumor cells from TIN-B by tyrosinase-reactive CTL ex vivo (see Fig. 2) confirms that at least a portion of the cells in the tumor deposit retained expression in vivo of sufficient HLA-A2 Ag for T-cell recognition.
Cell surface presentation of HLA-A2-associated MART-1 epitope is limited by competition from tyrosinase-derived peptides
HLA class I Ag down-regulation provided many of the melanoma cells in the second metastasis of patient VMM5 with an escape mechanism from T cell recognition and destruction. However, a subset of melanoma cells retained low-level expression of HLA class I surface Ag. These latter cells appear to have lost their susceptibility to MART-127–35-specific CTL, but were susceptible to recognition by Tyr369–377D-specific CTL.
These findings raise the possibility that HLA class I Ag down-regulation influences the presentation of distinct Melanoma Ag-derived peptides, likely because of their differential binding affinity to HLA-A2 Ag. Studies of Valmori et al. (34) suggested that MART-127–35 binds to HLA-A2 Ag with relatively low affinity. Furthermore, Engelhard et al. (35) demonstrated that HLA-A2 Ag has a significantly lower binding affinity for MART-127–35 than for Tyr369–377D (IC50 of 950 and 74 nM, respectively), and mass spectrometry studies of peptides eluted from class I molecules of the human melanoma cell line DM6 indicated surface MART-127–35 to be present at 100-fold copies per cell less than Tyr369–377D.
We hypothesized that presentation of HLA-A2-MART-127–35 complexes may be functionally abrogated in cells with low HLA-A2 Ag expression, because other peptides with higher affinity for HLA-A2 compete for binding sites in a HLA class I molecule-limited environment. Limited availability for HLA-A2 Ag may arise either from decreased expression of HLA class I gene products, or from the failure to form stable peptide-HLA-A2 complexes in the absence of functional 2m (C.-C. Chang, unpublished results). Therefore, we evaluated the possibility that Tyr369–377D peptides compete for free HLA-A2 molecules and prevent sufficient cell surface presentation of MART-137–35 peptides for T cell recognition. To test this hypothesis, recombinant vaccinia viruses expressing full-length human MART-1 (MART-Vac) or tyrosinase (Tyr-Vac) were used to drive overexpression of these proteins, singly or in combination, in TIN-B melanoma cells stably transfected to express wild-type 2m (VMM5B 2m), which reconstituted functional HLA class I assembly and cell surface expression (see Fig. 6B). We then evaluated the lytic activity of Tyr369–377D- and MART-127–35-specific CTL (VMM119 and VMM5A, respectively) against uninfected or vaccinia-infected tumor cells. Expression of tyrosinase and MART-1 in infected cells was confirmed by intracellular staining and Western blot analysis using specific Abs (data not shown). Tyr-Vac infection led to processing and presentation of Tyr369–377D epitope by TIN-B-2m cells, as evidenced by specific lysis by VMM119 T cells (Fig. 7, A and B); likewise MART-127–35-specific VMM5-A CTL effectively lysed MART-1-Vac-infected tumor cells (C and D). However, whereas VMM119 T cells lysed either TyrVac-infected or Tyr-Vac/MART-Vac-coinfected melanoma cells with equal efficiency (Fig. 7A), VMM5-A CTL failed to lyse coinfected melanoma cells (C). Both CTL efficiently lysed HLA-A2+ DM6 melanoma cells, which express both tyrosinase and MART-1, but neither CTL mediated lytic activity against HLA-A2+ tyrosinase- and MART-1-deficient DM331 cells (Fig. 7, B and D). The absence of surface HLA-A2-MART-127–35 complexes consequent to the overexpression of tyrosinase is not unique to the TIN-B-derived tumor cell lines, because the phenomenon was also evident using vaccinia vectors to drive tyrosinase and MART-1 expression in DM331 cells (Fig. 7, B and D).
Discussion
We report here the adaptive evolution of a melanoma Ag-specific cellular immune response in a long-term survivor of melanoma who had two recurrences in regional LN. The immune response to the original tumor metastasis (TIN-A) was polyvalent, with immunodominance manifested against the MART-127–35 epitope (AAGIGILTV), which was expressed by TIN-A tumor cells. Disease progression was heralded by a change in the immune repertoire of the patient’s systemic CTL response to melanoma Ags. The CTL response in the second metastasis (TIN-B) was directed primarily against the Tyr369–377D epitope (YMDGTMSQV), to which there had been no detectable response in TIN-A. Response to the MART-127–35 peptide, which had dominated the TIN-A tumor recognition, was lost in TIN-B. This change in the repertoire of the CTL response to autologous melanoma cells in vivo corresponds temporally and spatially to a loss of MART-127–35 presentation in Ag expression by the melanoma cells in TIN-B.
Detailed evaluation of tumor cells from TIN-A and TIN-B revealed evidence for immune editing and selection of a variety of immune escape mutants during tumor progression. HLA-A11, B44, and Cw17 allelic loss was characteristic of this tumor at least since the time of the first metastasis. More recent phenomena were down-regulation of HLA class I expression in a subset of tumor cells, and loss of functional HLA class I Ag expression due to a point mutation in the 2m gene (C.-C. Chang, unpublished results). Down-regulation and loss of MDP expression was yet another ongoing process in at least one of the tumor cell subsets. Loss of expression of HLA class I molecules and of MAA presumably provided survival advantage for the tumor cells in the setting of a host MAA-specific response.
The second metastatic recurrence in this patient differed from the first in the loss of presentation of the MART-127–35 epitope, even though MART-1 protein was expressed at comparable levels in both metastases. Because TIN-B tumor cells were lysed by tyrosinase-reactive CTL restricted by HLA-A2, and because TIN-B tumor cells expressed unmutated MART-1, absence of recognition by MART-127–35-reactive CTL cannot be explained simply by loss of HLA-A2 expression or by lack of expression of the protein. Proteasome immunosubunits in dendritic cells can prevent presentation of the MART-127–35 epitope (36), but the VMM5B cell line derived from TIN-B does not appear to have a higher expression of proteasome immunosubunits (LMP2, LMP7, and LMP10), and most of the other components of the APM appear to be preserved in TIN-B cells. TAP2 down-regulation could potentially lead to changes in processing and presentation of selected peptides, and may well have been another immune escape mechanism for a subset of cells. However, transfection of cells with wild-type 2m and MART-1 restored recognition by MART-127–35 reactive T cells. Thus, changes in Ag processing alone do not explain the loss of MART-127–35 complexes.
We have explored an alternate hypothesis to explain a failure of TIN-B tumor cells to present the MART-1 peptide. We hypothesized that competition of peptides for HLA class I Ag binding may be a contributing factor to the selective loss of presentation of epitopes with low affinity for HLA-A2 molecules. The MART-127–35 epitope has low-affinity binding to HLA-A2 Ag, with an IC50 of 950 nM, whereas the Tyr369–377D peptide is a strong binder with IC50 of 74 nM (35). Furthermore, the epitope frequency on the cell surface of melanoma cells is much lower for MART-127–35 peptide than for Tyr369–377D peptide (35). In the setting of HLA class I Ag down-regulation on the tumor cells, the low number of MHC molecules limits the number of peptides that can be presented, such that peptides with low affinity for the MHC molecule may be available at too low a copy number to permit recognition by epitope-specific T cells.
This hypothesis was supported by our experiments in which tyrosinase and MART-1 proteins were re-expressed in the VMM5B-2m tumor cell line using vaccinia vectors (Fig. 7). When MART-1 or tyrosinase proteins were expressed in VMM5B-2M cells separately, each peptide was processed and presented in a context sufficient for recognition by peptide-specific CTL. However, recognition of HLA-A2/MART-127–35 by peptide-specific CTL was significantly diminished upon coexpression of MART-1 and tyrosinase proteins in the same cells. This result was not unique to the VMM5-2m cell line, but was repeated with DM331 cells with a similar result. Collectively, these data suggest that the number of HLA class I molecules usually is not limiting. However, in tumor cells with dysregulated HLA class I expression or disrupted HLA class I stability, the MHC molecules become limiting, and surface presentation of the weakly binding MART-127–35 is significantly diminished as more strongly binding epitopes compete for limited HLA class I docking sites. We propose a model in which down-regulation of surface HLA class I Ag expression does not simply decrease the total number of HLA class I Ag-peptide complexes, but also alters the proportion of specific epitopes presented on the cell surface according to peptide affinity for MHC. As far as we know, this novel mechanism of tumor escape and epitope selection has not been previously described.
Despite multiple immune escape mechanisms, there remains persistent expression of the Tyr369–377D epitope on tumor cells in TIN-B. Therefore, the immune response that has arisen in that node (and systemically) against that epitope is an adaptive response, suggesting an appropriate change in immune repertoire. The findings are 1) that tumor cells have evolved toward an Ag-loss phenotype in the setting of a T cell response to tumor Ags, 2) that the Ag-loss phenotype included loss of the immunodominant epitope of the host T cells but retained expression of a cryptic epitope (Tyr369–377D), and 3) that the CTL repertoire has changed simultaneously, in a compensatory and adaptive manner, with dominant targeting of the previously cryptic epitope.
The fact that tumor did not appear for 5 years after the original recurrence, and the fact that the patient remained disease-free after surgical resection, both suggest that this immune response may have had clinical relevance. However, the tumor did recur despite this adaptive response, and this suggests that the adaptive response was inadequate to control this particular metastasis completely. Multiple immune escape mechanisms occurred over time. A large subset of cells in TIN-B down-regulated expression of class I HLA molecules due to a point mutation in the 2m gene. This change would have permitted escape from recognition by HLA-restricted T cells. However, that change may also have made those cells more sensitive to NK-mediated lysis. The subsequent systemic control after surgery suggests that the tumor cells with the most complete immune escape phenotype either had not metastasized beyond this tumor deposit, or that other systemic mechanisms such as NK cells controlled micrometastases beyond this deposit. In any event, this presentation is a reminder that surgical intervention for isolated metastases can succeed in some cases.
Another example of what we consider an adaptive immune response has been described by Coulie and his associates (37) in a patient treated with tumor vaccines as well as surgery, where loss of MHC expression by a tumor was followed by development of a novel response by CTL bearing killer inhibitory receptors that targeted cells with loss of certain MHC molecules. By contrast, the present report shows evidence of an adaptive immune response in a patient treated only with surgery. Also, in this case, the adaptive response occurs by a different and more classic mechanism, responding to immune escape by retargeting to a CTL epitope still presented on the tumor after the selective loss of presentation of a previously immunodominant peptide. This observation also suggests the importance of developing immune therapy directed against multiple Ags simultaneously. As we learn more about mechanisms by which tumors may evade immune recognition, it is encouraging that the human immune system, even in this elderly patient, has the plasticity to evolve in an adaptive manner in response to immune escape by the tumor.
It is tempting to speculate that development of such an adaptive immune response may predict a favorable clinical outcome. In this regard, unraveling the cellular and molecular events that govern such changes will be worthy of investigation. The resulting information will not only improve our understanding of the delicate interactions between tumor cells and the immune system, but may also suggest relevant therapeutic strategies to prevent tumor progression in the setting of immune escape.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was funded by National Institutes of Health Grants R01 CA57653 (to C.L.S.) and RO1 CA67108 and P30 CA16056 (to S.F.). C.L.S. has been an Elaine Shepard Cancer Investigator supported by the Cancer Research Institute. C.-C.C. is supported by a Susan G. Komen Breast Cancer Foundation predoctoral fellowship. The work was supported also by the Cancer Center Support Grant (National Institutes of Health P30CA44579) at the University of Virginia (Tissue Procurement Facility, Flow Cytometry Core, Biomolecular Core), and by the Pratt Fund at the University of Virginia.
2 Address correspondence and reprint requests to Dr. Craig L. Slingluff, Jr., Department of Surgery, University of Virginia Health Sciences Center, P.O. Box 800709, Charlottesville, VA 22908. E-mail address: cls8h{at}virginia.edu
3 Abbreviations used in this paper: MAA, melanoma-associated Ag; LMP, low-molecular-weight protein; LN, lymph node; TIN, tumor-infiltrated node; 2m, 2-microglobulin; MDP, melanocyte differentiation protein; APM, Ag-processing machinery.
Received for publication October 25, 2004. Accepted for publication February 23, 2005.
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