Natural T Cell Immunity Against Cancer

4296 Vol. 9, 4296–4303, October 1, 2003 Clinical Cancer Research

Perspectives Natural T Cell Immunity against Cancer

Dirk Nagorsen, Carmen Scheibenbogen, Ref. 3). TAAs can be divided into various groups including Francesco M. Marincola, Anne Letsch, and differentiation antigens, e.g., melanoma-melanocyte antigens, Ulrich Keilholz1 shared antigens overexpressed on various cancers, such as CEA or telomerase, cancer germ-line antigens, mutated antigens, and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, NIH, Bethesda, Maryland 20892 [D. N., F. M. M.], viral antigens (reviewed in Ref. 3). These TAAs have facilitated and Medizinische Klinik III, Hematology, Oncology, and Transfusion the analysis of T cell responses to tumors and are promising Medicine, University Hospital Benjamin Franklin, Free University, targets for immunotherapeutic strategies. TAA-specific CD8ϩ 12200 Berlin, Germany [C. S., A. L., U. K.] T cells represent an important component of the host’s immune response against malignant diseases (4). Clinical studies using Abstract immunization with peptides derived from TAAs have shown that high levels of CD8ϩ T cells with antitumor activity can be It has long been a matter of debate whether tumors are raised in cancer-bearing patients (reviewed in Ref. 5). Several spontaneously immunogenic in patients. With the availabil- ity of sensitive methods, naturally occurring T cells directed recent studies have also shown that induction of specific T cells against tumor-associated antigens (TAAs) can be frequently is associated with clinical effects (6–8). detected in cancer patients. In this review, we summarize the At the same time, new methods have been developed to current data on T cell responses to TAAs in various malig- analyze T cell responses (9). The high sensitivity of modern nancies, including melanoma, colorectal cancer, leukemia, single cell assays allows direct analysis of antigen-specific T ex vivo in vitro and breast cancer. T cell responses against various antigens, cells , thus eliminating prior stimulation with including melanoma differentiation antigens, carcinoembry- cytokines, which could lead to major alterations in T cell state onic antigen, epithelial cell adhesion molecule, her-2/neu, (10). Functional T cell assays, such as the ELISPOT assay and Wilms’ tumor protein, proteinase 3, NY-ESO-1, and surviv- IC-FC, use antigen-specific induction of cytokines to detect ing, have been reported in a substantial number of patients. specific T cells on a single cell level (11, 12). Multimerized In contrast, other TAAs, including most antigens of the HLA class I molecules carrying a specific epitope-peptide and MAGE family, do not usually elicit spontaneous T cell re- labeled with a fluorescent marker (tHLA, tetramers) allow the ex vivo sponses. A distinction between direct ex vivo T cell responses most direct staining of specific T cells, but they do not and in vitro-generated T cell responses is provided because provide data about the functionality of the cells (13–15). In in vitro stimulation results in quantitative and functional addition, detailed phenotypic analysis of T cells is possible by changes of T cell responses. The possible role of TAA- using flow cytometric methods (IC-FC and tHLA). Several specific T cells in immunosurveillance and tumor escape and subpopulations of TAA-specific T cells have been defined using the implications for immunological treatment strategies are IC-FC or tHLA, and some studies have correlated them with T et al. discussed. Naturally occurring T cells against TAAs are a cell functions. Hamann (16) used CD27 and CD45RA ϩ common phenomenon in tumor patients. Understanding the expression to describe three subsets of CD8 T cells: ϩ ϩ mechanisms and behavior of natural TAA-specific T cells CD27 CD45RA T cells represent naive subpopulations; ϩ Ϫ could provide crucial information for rational development CD27 CD45RA T cells represent memory subpopulations; Ϫ ϩ of more efficient T cell-directed immunotherapy. and CD27 CD45RA T cells represent effector subpopulations. Sallusto et al. (17) further defined T cells based on the 2 expression of the lymph node-homing chemokine receptor TAAs and T Cell Assays CCR7 as CD45RAϩCCR7ϩ naive T cells, CD45RAϪCCR7ϩ The description of TAAs a decade ago was a groundbreak- central memory T cells, CD45RAϪCCR7Ϫ effector memory T ing step in cancer immunology (1, 2). During the last few years, cells, and CD45RAϩCCR7Ϫ differentiated cytolytic effector T effective strategies to identify TAAs recognized by specific T cells. A similar distinction of T cell subsets can be made using cells have been developed and have led to the characterization CD27/CD28 (18). These classifications represent a very helpful of various families of MHC class I-related TAAs, of which by tool to further characterize type and function of TAA-specific T now more than 60 have been identified (recently summarized in cell responses. Some investigators have performed in vitro restimulation of lymphocytes to increase the frequency of antigen-specific T cells. Although this procedure can result in substantial func- Received 2/20/03; revised 5/9/03; accepted 5/12/03. tional changes of the actual in vivo state of T cells (10), short- 1 To whom requests for reprints should be addressed, at University Hospital Benjamin Franklin, Medizinische Klinik III, Hematology, On- time in vitro expansion (not more than 2 weeks) usually does not cology and Transfusion Medicine, Hindenburgdamm 30, 12200 Berlin, generate specific T cells from naive precursors, and, thus, com- Germany. Phone: 49-30-8445-3906; Fax: 49-30-8445-4468; E-mail: parative quantitative analyses can be performed. Here we con- [email protected]. sider T cell responses found after short-time in vitro culture as 2 The abbreviations used are: CEA, carcinoembryonic antigen; Ep- CAM, epithelial cell adhesion molecule; IC-FC, intracellular cytokine reflecting in vivo stimulation. We labeled these T cell responses flow cytometry; TAA, tumor-associated antigen; tHLA, HLA class as “short-term in vitro stimulation” to distinguish them from T I/epitope tetrameric complex; IL, interleukin. cell responses analyzed directly ex vivo or after longer in vitro

stimulation. Studies in which tumor-directed T cell responses expressing tumor cells (26). In contrast, in another melanoma were generated in vitro after long-term culture and repeated patient analyzed by Lee et al. (21), tyrosinase368–376-specific T stimulation with tumor cells or antigens are not included in this cells were functionally unresponsive and unable to directly lyse review. melanoma target cells, although these cells also had many characteristics of effector T cells. Natural T cell responses Natural T Cell Responses against gp100 were found against 209-217-2M, a modified peptide, in one patient (22) and against gp100 after in vitro There is increasing evidence that CD8ϩ T cells directed 17–25 stimulation in another patient (28). against TAAs are spontaneously induced in various malignan- NY-ESO-1 is a germ-line antigen expressed in cancer, in cies, including melanoma, adenocarcinomas, and leukemias. testis, and, to a lesser degree, in placenta cells. T cell responses These studies were made possible by the development of the against NY-ESO-1 were reported in 3 of 10 (29) and in 7 of 22 sensitive and “high-throughput” techniques described above. melanoma patients (30). NY-ESO-1-reactive T cells in the study Mechanisms leading to spontaneous induction of specific T cell of Valmori et al. (29) were CD45RAϪCD28ϩ, representing a responses are not well understood. Here we review the occur- memory subset of T cells (16). CTLs responsive against another rence of TAA-directed CD8ϩ T cells in patients with various group of cancer germ-line antigens, the MAGE family, are very malignancies and in healthy subjects. This report excludes T cell rarely found, despite extensive studies (19, 22, 23, 31–35). responses induced by antigen-specific immunotherapy. Further- MAGE-A10-encoded nonapeptide , which is presented by more, we tried to differentiate between natural T cell responses 254–262 HLA-A2.1, could be an exception. CTL responses to this pep- and T cell responses in patients who had received previous tide were detected after short-time in vitro expansion in 8 of 12 cytokine therapy. However, this is sometimes difficult because, patients with a MAGE-A10-expressing melanoma using tetram- especially in melanoma studies, many patients have received ers of HLA-A2/peptide MAGE-A10 complexes (36). In- previous adjuvant IFN-␣, and the patient characteristics pro- 254–262 terestingly, samples from 3 of 10 patients whose tumors had no vided are often incomplete. Therefore, it cannot be excluded that detectable MAGE-A10 expression and 2 of 10 healthy donors the T cell response observed in some patients may have been also contained tHLAϩCD8ϩ T cells (36). CAMEL is a trans- elicited or altered by previous cytokine therapy. lational product of cancer germ-line antigen LAGE-1. The

CAMEL-derived peptide1–11 was detected by specific T cells Malignant Melanoma from peripheral blood of 3 of 33 melanoma patients using Cutaneous malignant melanoma is the most extensively tetramers after short-time culture (37). Another group confirmed investigated human malignancy in tumor immunology. Cutane- these results, detecting CAMEL1–11-directed T cell responses in ous melanoma is considered a highly immunogenic tumor, and 3 of 12 melanoma patients by ELISPOT assay after overnight several authors have described the presence of T cells with incubation with IL-2 (23). Thus, germ-line antigens show con- reactivity against TAAs. The most widely studied tumor antigen siderable heterogeneity in their immunogenicity. T cell re- is the melanoma differentiation antigen melanA/MART-1, sponses are found quite frequently against NY-ESO-1 and against which specific T cell responses were reported in 10– MAGE-A10, but not against other MAGE family members. One 75% of melanoma patients (19–24). Using tetramer staining, T possible explanation for this may be a difference in the fre- cells responsive against melanA/MART-1 were found in frequency of T cell precursors toward these antigens as suggested quencies of up to 0.4% of CD8ϩ T cells in peripheral blood of by Valmori et al. (36). Recently, T cell responses after short- melanoma patients (20). Up to 3.5% of melanoma draining term in vitro expansion were reported in various malignancies lymph node CD8ϩ cells were identified ex vivo as melanA/ against HLA-A2-binding peptide epitopes derived from sur- MART-1-specific T cells (after short-term in vitro expansion, vivin, a member of the inhibitor of apoptosis protein gene this percentage increased up to 21%; Ref. 25). Further analyses family. Using ELISPOT assays after in vitro stimulation, spe- have shown that peripheral melanA/MART-1-specific T cells cific T cells were detected against survivin peptides Sur196–104, ϩ high are mainly (about two-thirds) CD28 CD45RA or Sur995–104, and a modified Sur1 peptide in 2, 3, and 5 of 14 CD45RAhighCCR7ϩ representing naive T cells (16, 17, 20, 24). melanoma patients, respectively (38). However, one-third of melanA/MART-1-specific T cells are of However, the T cell responses against known TAAs de- effector memory type (CD45RAϪCCR7Ϫ). Interestingly, about scribed above may represent only a minor part of tumor-directed 95% of melanA/MART-1-specific T cells at the tumor site T cell immunity. Letsch et al. (39) have shown that T cells from represent this effector memory subtype (24). peripheral blood recognized autologous and/or HLA-matched Less frequently than responses against melanA/MART-1, allogeneic melanoma cell lines in 11 of 19 patients with meta- natural T cell responses are found against two other melanoma static melanoma with frequencies up to 0.81% of peripheral differentiation antigens, tyrosinase and gp100 (19, 21–23, 26). blood mononuclear cells. In a further study, T cell responses

Valmori et al. (26) analyzed a tyrosinase368–376-directed T cell against autologous melanoma cell lines were found in 5 of 7 response in a stage IV melanoma patient that reached a fre- patients with T cell frequencies of up to 2.7% of CD8ϩ T cells quency of Ͼ5% of CD3ϩCD8ϩ T cells. IC-FC and tetramer (26). Although they showed an overlap of T cell responses staining showed that most of these naturally occurring TAA- against autologous tumor and known antigenic epitopes in one directed T cells were CD45RAϩCCR7Ϫ granzyme Bϩ, which patient, these studies suggest a yet unknown occurrence and is characteristic of cytotoxic effector T cells (17, 26, 27). Con- magnitude of natural T cell responses, which are most likely sistent with their phenotype, tyrosinase-specific T cells were directed against a variety of yet-to-be-defined epitopes or TAAs directly lytic ex vivo and specifically recognized tyrosinase- (26). This hypothesis is also suggested by a study showing that

the majority of melanoma-reactive T cell clones do not recog- survivin peptides in 3 of 10 breast cancer patients and against

nize melanosomal antigens (40). NY-ESO-1157–165 in 1 breast cancer patient (30). Most studies have analyzed T cell responses to TAAs in Hoffmann et al. (53) analyzed patients with head and neck

peripheral blood, but this may underestimate the type and mag- cancer using wild-type p53264–272-specific tHLA. Twenty-three nitude of tumor-specific T cell responses in the tumor and other of 30 patients had a p53-specific T cell population of up to 0.1% anatomical compartments. Using intracellular cytokine and tet- of CD8ϩ T cells. However, the frequency of p53-specific T ramer assays, we detected up to 10-fold higher frequencies of cells was higher in patients whose tumors did not accumulate tyrosinase- and melanoma-reactive memory CD3ϩCD8ϩ T p53, whereas in patients whose tumors accumulated p53, lower cells in the bone marrow compared with the peripheral blood frequencies of predominantely naive T cells were found. Also, a (41). proportion of HLA-A2.1ϩ healthy donors had a low frequency of p53264–272-specific T cells. One possible explanation for these findings is that tumors accumulating p53 have induced Carcinomas apoptosis of p53-specific effector T cells. At least 10 TAAs and more than 35 MHC class I antigenic epitopes have been described for colorectal cancer (42). Re- Hematological Malignancies cently, we demonstrated the existence of naturally occurring T Recently, investigators have started to analyze T cell im- cells that recognized HLA-A2-binding epitopes of TAA Ep- munity in leukemia. Molldrem et al. (6) reported a remarkable CAM , her-2/neu , and CEA in 4, 5, and 6 of 263–271 654–662 571–579 immunogenicity of the leukemia-associated antigen proteinase 22 HLA-A2ϩ patients with colorectal cancer, respectively (43). 3, which is overexpressed in myeloid leukemias. Using tetram- Subsequently, specific T cell responses against at least one of ers, they detected a high frequency of T cells specific for this these antigens were observed in 25% of 49 patients (44). Pa- antigen in most chronic myelogenous leukemia patients who tients with lymph node metastases or distant metastases had achieved complete remission after allogeneic transplantation or specific T cell responses significantly more often. In three IFN-␣ therapy. However, in another recent study (54), T cell patients, a detailed analysis revealed that most of the TAA- responses to proteinase 3 could be detected in patients only after reactive CD3ϩCD8ϩ T cells detected by IC-FC express treatment with IFN-␣ but not after treatment with imatinib CD45RA. CD3ϩCD8ϩCD45RAϩ T cells immediately pro- (STI571), suggesting that these T cell responses have been ducing IFN-␥ when exposed to antigen were shown to belong to induced by treatment with IFN-␣. Scheibenbogen et al. (55) the effector-type T cell subset that should be able to directly studied acute myelogenous leukemia patients for T cell re- mediate cytotoxicity (45). In accordance with the above studies, sponses to proteinase 3 and the Wilms’ tumor protein (WT-1), T cell responses against a CEA-derived, modified peptide were a TAA overexpressed in several malignancies. Ex vivo T cell observed in approximately one-third of patients with CEA- responses to proteinase 3 and WT-1 were found in 2 and 3 of 15 expressing carcinomas using the IFN-␥ ELISPOT assay (46). patients with acute myelogenous leukemia using the IFN-␥ Several studies have sought T cell responses against TAAs ELISPOT assay. These results were confirmed by IC-FC show- in breast cancer patients. her-2/neu is a TAA that was identified ing a TAA-specific population in 2 and 4 of 12 patients with up as a target of cytotoxic T cell lines in patients with breast and to 0.7 of CD8ϩ T cells. TAA-specific T cells were determined ovarian tumors and is also the target for the monoclonal anti- as CD45RAϩCCR7Ϫgranzyme Bϩ in one patient representing body trastuzumab, which is effective in patients with metastatic differentiated effector T cells, according to the definition of breast cancer overexpressing her-2/neu (47–49). However, in Sallusto et al. (17). Specific T cell responses were also observed three studies, no natural ex vivo CD8ϩ T cell response against against survivin peptides after in vitro stimulation in patients her-2/neu was found in peripheral blood in 8, 7, and 19 patients, with chronic lymphatic leukemia (38, 56). Further promising respectively, using the IFN-␥ ELISPOT assay (44, 50, 51). targets for immunotherapy of leukemias (i.e., chronic myelog- Furthermore, no functional T cell responses were found against enous leukemia) are bcr-abl fusion peptides, but no ex vivo T Ep-CAM, CEA, and MUC1 from peripheral blood of breast cell response has been detected as of yet, despite the capability cancer patients (44, 51). Interestingly, although they did not of fusion region spanning peptides to bind to HLA molecules detect CD8ϩ T cell responses against a her-2/neu peptide, Disis and to be immunogenic in vitro (57–59). et al. (50) found CD4ϩ T cell responses against her-2/neu protein in 5 of 45 patients. In contrast to their functional ELIS- POT data, Feuerer et al. (51) detected up to 1% of CD8ϩ T cells T Cell Responses against Viral (Tumor-Associated) specific for her-2/neu and MUC1, respectively, in one-third of Antigens in Malignant Disease their patients using tHLA. Remarkably, the same group could Several viruses are known to cause malignant transforma- expand functionally active T cells directed against her-2/neu and tion of human cells. EBV is associated with Burkitt lymphoma, MUC1 from the bone marrow of about two-thirds of breast Hodgkin’s disease, and nasopharyngeal carcinoma. Human pap- cancer patients detected by IFN-␥ ELISPOT assay (51). These illoma virus infection is strongly associated with cervical can- data may suggest a preferential homing behavior of functionally cer. HTLV-1 causes adult T cell leukemia/lymphoma. HHV-8 active her-2/neu- and MUC1-specific T cells toward bone mar- (Kaposi’s sarcoma-associated herpesvirus) infection can lead to row in breast cancer patients. This is particularly important the development of Kaposi’s sarcoma or, less commonly, lym- because breast cancer often harbors minimal residual tumor phoma. These viruses encode viral antigens that can be pro- cells in the bone marrow (52). After short-term in vitro stimu- cessed by transformed cell and can therefore be considered lation, another group found specific T cell responses against TAAs. Although natural T cell responses are described against

antigens derived from these viruses in tumor patients [several With the exception of melanA/MART-1, T cell responses EBV antigens, including EBNA3 and LMP2 (60); human pap- against TAAs are rarely found in healthy individuals. Dhodap- illoma virus, antigens E6 and E7 (61, 62); HHV-8, antigens K12 kar et al. (22) reported low-frequency responses against tyro- and K8.1 (63)], their actual role in preventing/controlling ma- sinase and gp100 in 4 and 3 of 12 healthy donors. In contrast, lignant development remains to be elucidated. However, the others did not find tyrosinase- or gp100-directed T cell re- importance of immunological control of virus-associated tumor sponses in healthy subjects even after overnight incubation with growth is strongly underlined by the increased development of IL-2 (19, 23). Valmori et al. (36) found a T cell response against virus-caused malignancies during suppression or failure of cel- MAGE-10 in 2 of 12 normal subjects after 2 weeks of in vitro lular immunity, such as in AIDS (64) or after organ transplants stimulation with peptide, IL-2, and IL-7. Despite extensive (64). investigation in healthy donors, no T cell responses were found against MAGE-1 (19), MAGE-2 (23), MAGE-3 (19, 22), Private TAAs CAMEL (23), CEA (43, 44, 46), Ep-CAM (43, 44), her-2/neu Private TAAs are usually restricted to a single patient due (43, 44, 50, 51), MUC1 (51), proteinase 3, and WT-1 (55). In ϩ to a specific mutation or alteration. In one patient with long-term summary, natural CD8 T cell responses against melanA/ survival despite incompletely resected squamous cell lung car- MART-1 in healthy donors have been well demonstrated, but T cinoma, a specific T cell response against a mutated tumor- cell responses against other TAAs are very rare. specific antigen (coded by malic enzyme cDNA) was found by ϩ tetramers at a frequency of 0.4% of CD8 T cells (66). How- Discussion ever, it is unclear whether this specific T cell response occurred There is clear evidence that tumor patients are able to spontaneously because the patient had received prior vaccina- generate TAA-specific T cell immunity spontaneously. Whereas tion with autologous tumor cells. In another lung cancer (undif- the presence of tumor-specific T cells has been shown by many ferentiated, large cell) patient, specific T cells against an epitope groups and for various tumor types, much less is known about from a mutated ␣-actinin-4 gene product were detected after the function of TAA-specific T cells in vivo. Most of the TAAs short-time culture using tetramers (67). It seems probable that including differentiation, germ-line, and shared overexpressed more private TAA-directed T cell responses will be described, antigens are not tumor specific but are also expressed at low especially using new functional genomics approaches (68). Pri- levels in certain nonmalignant tissues. This should influence the vate TAA epitopes including MHC ligands derived from proto- type of T cell response because deletion of functional high- oncogenes or frameshift mutations might become targets for T avidity self-reactive T cells in the thymus as well as peripheral cell-based therapies in the future with an ongoing individual- deletion or anergy was shown in various animal models (re- ization of cancer immunotherapy (68). viewed in Ref. 74). There are a few recent studies analyzing the functional avidity of TAA-specific T cells in patients. In leuke- Healthy Donors mia patients, low-avidity T cells to proteinase 3, which are able T cell responses against TAAs rarely occur in healthy to kill leukemia cells, can readily be expanded. However, high- donors. An exception is melanA/MART-1, against which T cells avidity T cells can also be expanded from patients in cytogenetic are present in 8–60% of healthy donors (19, 20, 22). Pittet remission and from healthy subjects, suggesting incomplete et al. (20, 24) have determined these CD8ϩ T cells as self-tolerance to proteinase 3, which is expressed at low levels CD28ϩCD45RAhighCCR7ϩ representing naive T cells. Com- in normal myeloid cells (75). Similarly, the expansion of high- paring melanA/MART-1-directed T cell responses in healthy avidity T cells against the cancer germ-line antigen NY-ESO-1 donors with those found in melanoma patients, data are incon- by peptide vaccination could be demonstrated, although most sistent. Chen et al. (69) and Dhodapkar et al. (22) found specific T cells exhibited low avidity (76). In that study, only the melanA/MART-1-directed T cell responses more frequently in high-avidity T cells lysed tumor cells, whereas the low-avidity healthy donors than in melanoma patients, but in most other T cells failed to significantly recognize the tumor targets. On the studies, T cell responses against melanA/MART-1 were more other hand, it was shown in leukemia patients that high-avidity frequently observed in melanoma patients (19, 20, 23, 24, 70). proteinase 3-specific T cells, although killing leukemia cells Whereas healthy donors have 95% naive T cells responding more efficiently, underwent apoptosis when exposed to leuke- against melanA/MART-1, about one-third of peripheral mia cells, in contrast to the low-avidity T cells (75). Direct ex melanA/MART-1-specific T cells in melanoma patients are of vivo cytotoxic function of tetramer-sorted tyrosinase-specific T the effector memory type (20, 24). The antigen melanA/ cells has been demonstrated in one study, whereas in another MART-1 is unique thus far with its high number of specific T study, ex vivo sorted tyrosinase-specific T cells failed to lyse cell precursors in healthy individuals (24). The high frequency tumor cells (21, 26). Several other studies have analyzed the of melanA/MART-1-specific precursor T cells could be due to cytotoxic function of short-term in vitro-expanded TAA- abundant thymic presentation of potentially cross-reactive se- specific T cells, showing that the TAA-specific T cells expanded quences (71). Interestingly, T cell responses to the melanosomal from peripheral blood or lymph nodes can kill tumor cells (25, antigens melanA/MART, gp100, and tyrosinase were found in 29, 36, 77). In two studies, however, TAA-reactive T cells 35–75% of vitiligo patients, suggesting an important role of detected ex vivo by IFN-␥-ELISPOT assays could not be ex- these specific T cells in killing melanocyte lineage cells and panded in vitro and failed to recognize tumor cell lines (22, 23). emphasizing the tenuous balance between immune tolerance Taken together, these data clearly show that the T cell repertoire and immune defense (72, 73). to self-antigen TAAs has a potential role in antitumor immunity.

Little is known thus far about whether circulating T cells does not attract them anymore, due to tumor escape mecha- reactive with certain TAAs influence the clinical course of nisms. An alternate hypothesis is that the evasion of tumor cells, disease. Although data from selected patients suggest a favor- especially in lymph nodes, is a prerequisite for the induction of able clinical course in patients with natural TAA-directed T TAA-specific T cell responses. cells (26, 55, 66), no study has systematically compared patients A few reports describe differences in T cell responses with and without TAA-directed T cell responses. However, against the same TAAs between different tumor entities. One there is evidence that the presence of intratumoral T cells example is breast cancer and colorectal cancer, which share correlates with improved clinical outcome in various solid tu- various TAAs (44, 50, 51). Differences in tumor-directed im- mors (78–80). The assumption that a natural T cell immunity mune responses between these tumors may be explained either can prevent the development of tumors remains speculative. by differences in the antigen-presenting capacity of the tumor However, as a matter of fact, tumors can progress despite the cells, in the tumor microenvironment, in the local immune existence of TAA-specific T cell responses. Immune escape system, or in the migratory properties of the TAA-specific T mechanisms might hamper the effectiveness of natural antitu- cells (discussed in Ref. 44). There are few studies comparing T mor immunity. Possible mechanisms include immune tolerance, cell responses in peripheral blood and bone marrow. Feuerer et immune suppression, lack of tumor localization by T cells, al. (51) found functional T cell responses against TAAs in the Fas/Fas ligand interactions, inadequacy of tumor cells as targets, bone marrow but not in the peripheral blood of breast cancer HLA loss, antigen loss, or anergy induction (reviewed in Ref. patients. In melanoma patients, we found TAA-reactive 81). An example of antigen loss could be the report of natural CD3ϩCD8ϩ T cell responses in bone marrow in similar or MAGE-A10-specific T cells in the blood of three melanoma higher frequencies than in peripheral blood, and the subset of patients with MAGE-A10-negative tumors (36). TAA-reactive memory T cells was significantly increased in The analysis of natural immune responses in tumor patients bone marrow (41). Although it is too early to draw general has implications for the development of antigen-specific treat- conclusions from these studies, they suggest that bone marrow ment strategies. Because the majority of TAAs described thus may be an important compartment for tumor surveillance har- far are also expressed in some nonmalignant cells, although boring a tumor-specific memory T cell pool. mostly at lower levels, the problems of immune tolerance and Most studies analyzing T cell response to TAAs have autoimmunity are important issues to consider when attempting emphasized CD8ϩ T cells thus far. However, CD4ϩ T cells to elicit T cell responses against these antigens. Various animal may play a crucial role in both the induction and activation of models have shown that self-reactive T cells are frequently TAA-specific memory CD8ϩ T cells toward cytotoxic effector deleted in the thymus or anergized by cells expressing the T cells (86). CD4ϩ T cell responses against TAA-derived antigen in the absence of costimulatory signals. The demonstra- epitopes have been described for prostate-specific antigen in tion of functionally active T cell responses against TAAs indi- prostate cancer and for her-2/neu in breast cancer (50, 87). The cates that the T cell repertoire in adults frequently contains demonstration of antibody responses to various TAAs, including self-tumor antigen-reactive T cells. These specific T cell re- tyrosinase, NY-ESO-1, Ep-CAM, her-2/neu, and WT-1 (82–85, sponses may be activated and enhanced by cancer vaccination or 88), implies that specific CD4ϩ T-helper cells to these TAAs adoptive T cell therapies. One disadvantage of exploiting natural should also be present in these patients. T cell responses for immunotherapy may be that tumor escape In summary, natural peripheral T cell responses against variants have been selected already, as outlined above. Of various TAAs do exist in patients with melanoma, leukemias, further interest is that despite the presence of T cell responses to and carcinomas. Whether circulating TAA-specific T cells are TAAs, autoimmunity is rarely observed in patients with cancer. able to kill tumor cells in vivo remains unclear, as does their However, caution must be used in drawing conclusions from effect on the clinical course of disease. Additional studies are these data because most studies have not analyzed whether the necessary for a better understanding of the role of natural T cell TAA-specific T cells are able to lyse tumor cells. In one study, responses against TAAs. the direct ex vivo tumor lytic activity of tyrosinase-specific CD8ϩ T cells was shown in a melanoma patient who had no signs of skin depigmentation (26). References Most natural T cell responses are reported in patients with 1. van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De advanced disease. This may be simply because most of the Plaen, E., Van den Eynde, B., Knuth, A., and Boon, T. A gene encoding patients analyzed had advanced malignant disease. Only a few an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (Wash. DC), 254: 1643–1647, 1991. studies directly compare advanced-stage with early-stage dis- 2. Boon, T., and van der Bruggen, P. Human tumor antigens recognized eases. We found that T cell responses to TAAs occur more by T lymphocytes. J. Exp. Med., 183: 725–729, 1996. frequently in patients with metastatic colorectal cancer than in 3. Renkvist, N., Castelli, C., Robbins, P. F., and Parmiani, G. A listing those with limited disease (43, 44). Also, a higher frequency of of human tumor antigens recognized by T cells. Cancer Immunol. autoantibodies against TAAs (Ep-CAM, her-2/neu, tyrosinase, Immunother., 50: 3–15, 2001. and NY-ESO-1) was reported among patients with metastatic 4. Smyth, M. J., Godfrey, D. I., and Trapani, J. A. A fresh look at tumor stage of various tumors, including colorectal cancer, breast immunosurveillance and immunotherapy. Nat. Immunol., 2: 293–299, 2001. cancer, and melanoma (82–85). One hypothesis is that TAA- 5. Parmiani, G., Castelli, C., Dalerba, P., Mortarini, R., Rivoltini, L., specific T cells in patients with limited disease are at the tumor Marincola, F. M., and Anichini, A. Cancer immunotherapy with pep- site, keeping the tumor under control, whereas they are in the tide-based vaccines: what have we achieved? Where are we going? periphery in patients with metastatic disease because the tumor J. Natl. Cancer Inst. (Bethesda), 94: 805–818, 2002.

6. Molldrem, J. J., Lee, P. P., Wang, C., Felio, K., Kantarjian, H. M., 20. Pittet, M. J., Valmori, D., Dunbar, P. R., Speiser, D. E., Lienard, D., Champlin, R. E., and Davis, M. M. Evidence that specific T lympho- Lejeune, F., Fleischhauer, K., Cerundolo, V., Cerottini, J. C., and cytes may participate in the elimination of chronic myelogenous leuke- Romero, P. High frequencies of naive Melan-A/MART-1-specific mia. Nat. Med., 6: 1018–1023, 2000. CD8(ϩ) T cells in a large proportion of human histocompatibility 7. Banchereau, J., Palucka, A. K., Dhodapkar, M., Burkeholder, S., leukocyte antigen (HLA)-A2 individuals. J. Exp. Med., 190: 705–715, Taquet, N., Rolland, A., Taquet, S., Coquery, S., Wittkowski, K. M., 1999. Bhardwaj, N., Pineiro, L., Steinman, R., and Fay, J. Immune and clinical 21. Lee, P. P., Yee, C., Savage, P. A., Fong, L., Brockstedt, D., Weber, responses in patients with metastatic melanoma to CD34(ϩ) progenitor- J. S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P. D., Roede- derived dendritic cell vaccine. Cancer Res., 61: 6451–6458, 2001. rer, M., and Davis, M. M. Characterization of circulating T cells specific 8. Belli, F., Testori, A., Rivoltini, L., Maio, M., Andreola, G., Sertoli, for tumor-associated antigens in melanoma patients. Nat. Med., 5: M. R., Gallino, G., Piris, A., Cattelan, A., Lazzari, I., Carrabba, M., 677–685, 1999. Scita, G., Santantonio, C., Pilla, L., Tragni, G., Lombardo, C., Arienti, 22. Dhodapkar, M. V., Young, J. W., Chapman, P. B., Cox, W. I., F., Marchiano, A., Queirolo, P., Bertolini, F., Cova, A., Lamaj, E., Fonteneau, J. F., Amigorena, S., Houghton, A. N., Steinman, R. M., and Ascani, L., Camerini, R., Corsi, M., Cascinelli, N., Lewis, J. J., Srivas- Bhardwaj, N. Paucity of functional T cell memory to melanoma antigens tava, P., and Parmiani, G. Vaccination of metastatic melanoma patients in healthy donors and melanoma patients. Clin. Cancer Res., 6: 4831– with autologous tumor-derived heat shock protein gp96-peptide com- 4838, 2000. plexes: clinical and immunologic findings. J. Clin. Oncol., 20: 4169– 23. Griffioen, M., Borghi, M., Schrier, P. I., and Osanto, S. Detection 4180, 2002. and quantification of CD8ϩ T cells specific for HLA-A*0201-binding 9. Keilholz, U., Weber, J., Finke, J. H., Gabrilovich, D. I., Kast, W. M., melanoma and viral peptides by the IFN-␥-ELISPOT assay. Int. J. Disis, M. L., Kirkwood, J. M., Scheibenbogen, C., Schlom, J., Maino, Cancer, 93: 549–555, 2001. V. C., Lyerly, H. K., Lee, P. P., Storkus, W., Marincola, F., Worobec, 24. Pittet, M. J., Zippelius, A., Valmori, D., Speiser, D. E., Cerottini, A., and Atkins, M. B. Immunologic monitoring of cancer vaccine J. C., and Romero, P. Melan-A/MART-1-specific CD8 T cells: from therapy: results of a workshop sponsored by the Society for Biological thymus to tumor. Trends Immunol., 23: 325–328, 2002. Therapy. J. Immunother., 25: 97–138, 2002. 25. Romero, P., Dunbar, P. R., Valmori, D., Pittet, M., Ogg, G. S., 10. Monsurro, V., Nagorsen, D., Wang, E., Provenzano, M., Dudley, Rimoldi, D., Chen, J. L., Lienard, D., Cerottini, J. C., and Cerundolo, V. M. E., Rosenberg, S. A., and Marincola, F. M. Functional heterogeneity Ex vivo staining of metastatic lymph nodes by class I major histocom- of vaccine-induced CD8(ϩ) T cells. J. Immunol., 168: 5933–5942, patibility complex tetramers reveals high numbers of antigen-experi- 2002. enced tumor-specific cytolytic T lymphocytes. J. Exp. Med., 188: 1641– 11. Suni, M. A., Picker, L. J., and Maino, V. C. Detection of antigen- 1650, 1998. specific T cell cytokine expression in whole blood by flow cytometry. 26. Valmori, D., Scheibenbogen, C., Dutoit, V., Nagorsen, D., Asem- J. Immunol. Methods, 212: 89–98, 1998. issen, A. M., Rubio-Godoy, V., Rimoldi, D., Guillaume, P., Romero, P., 12. Scheibenbogen, C., Lee, K. H., Mayer, S., Stefanovic, S., Moebius, Schadendorf, D., Lipp, M., Dietrich, P. Y., Thiel, E., Cerottini, J. C., U., Herr, W., Rammensee, H. G., and Keilholz U. A sensitive Elispot Lienard, D., and Keilholz, U. Circulating tumor-reactive CD8(ϩ)T assay for detection of CD8ϩ lymphocytes specific for HLA class cells in melanoma patients contain a CD45RA(ϩ)CCR7(Ϫ) effector I-peptide epitopes derived from influenza proteins in the blood of subset exerting ex vivo tumor-specific cytolytic activity. Cancer Res., healthy donors and melanoma patients. Clin. Cancer Res., 3: 221–226, 62: 1743–1750, 2002. 1997. 27. Campbell, J. J., Murphy, K. E., Kunkel, E. J., Brightling, C. E., 13. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., Soler, D., Shen, Z., Boisvert, J., Greenberg, H. B., Vierra, M. A., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., and Davis, Goodman, S. B., Genovese, M. C., Wardlaw, A. J., Butcher, E. C., and M. M. Phenotypic analysis of antigen-specific T lymphocytes [erratum Wu, L. CCR7 expression and memory T cell diversity in humans. in Science (Wash. DC), 280: 1821, 1998]. Science (Wash. DC), 274: J. Immunol., 166: 877–884, 2001. 94–96, 1996. 28. Yamshchikov, G., Thompson, L., Ross, W. G., Galavotti, H., Aq- 14. Lee, K. H., Wang, E., Nielsen, M. B., Wunderlich, J., Migueles, S., uila, W., Deacon, D., Caldwell, J., Patterson, J. W., Hunt, D. F., and Connors, M., Steinberg, S. M., Rosenberg, S. A., and Marincola, F. M. Slingluff, C. L., Jr. Analysis of a natural immune response against tumor Increased vaccine-specific T cell frequency after peptide-based vacci- antigens in a melanoma survivor: lessons applicable to clinical trial nation correlates with increased susceptibility to in vitro stimulation but evaluations. Clin. Cancer Res., 7 (Suppl.): 909s–916s, 2001. does not lead to tumor regression. J. Immunol., 163: 6292–6300, 1999. 29. Valmori, D., Dutoit, V., Lienard, D., Rimoldi, D., Pittet, M. J., Champagne, P., Ellefsen, K., Sahin, U., Speiser, D., Lejeune, F., Cer- 15. Nielsen, M. B., Monsurro, V., Migueles, S. A., Wang, E., Perez- ottini, J. C., and Romero, P. Naturally occurring human lymphocyte Diez, A., Lee, K. H., Kammula, U., Rosenberg, S. A., and Marincola, antigen-A2 restricted CD8ϩ T cell response to the cancer testis antigen F. M. Status of activation of circulating vaccine-elicited CD8ϩ T cells. NY-ESO-1 in melanoma patients. Cancer Res., 60: 4499–4506, 2000. J. Immunol., 165: 2287–2296, 2000. 30. Jager, E., Nagata, Y., Gnjatic, S., Wada, H., Stockert, E., Karbach, 16. Hamann, D., Roos, M. T., and van Lier, R. A. Faces and phases of J., Dunbar, P. R., Lee, S. Y., Jungbluth, A., Jager, D., Arand, M., Ritter, human CD8 T cell development. Immunol. Today, 20: 177–180, 1999. G., Cerundolo, V., Dupont, B., Chen, Y. T., Old, L. J., and Knuth, A. 17. Sallusto, F., Lenig, D., Forster, R., Lipp, M., and Lanzavecchia, A. Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral Two subsets of memory T lymphocytes with distinct homing potentials and cellular immune responses. Proc. Natl. Acad. Sci. USA, 97: 4760– and effector functions. Nature (Lond.), 401: 708–712, 1999. 4765, 2000. 18. Appay, V., Dunbar, P. R., Callan, M., Klenerman, P., Gillespie, 31. Traversari, C., van der Bruggen, P., Luescher, I. F., Lurquin, C., G. M., Papagno, L., Ogg, G. S., King, A., Lechner, F., Spina, C. A., Chomez, P., Van Pel, A., De Plaen, E., Amar-Costesec, A., and Boon, Little, S., Havlir, D. V., Richman, D. D., Gruener, N., Pape, G., Waters, T. A nonapeptide encoded by human gene MAGE-1 is recognized on A., Easterbrook, P., Salio, M., Cerundolo, V., McMichael, A. J., and HLA-A1 by cytolytic T lymphocytes directed against tumor antigen Rowland-Jones, S. L. Memory CD8ϩ T cells vary in differentiation MZ2-E. J. Exp. Med., 176: 1453–1457, 1992. phenotype in different persistent virus infections. Nat. Med., 8: 379– 32. Gaugler, B., Van den Eynde, B., van der Bruggen, P., Romero, P., 385, 2002. Gaforio, J. J., De Plaen, E., Lethe, B., Brasseur, F., and Boon, T. Human 19. Scheibenbogen, C., Lee, K. H., Stevanovic, S., Witzens, M., Will- gene MAGE-3 codes for an antigen recognized on a melanoma by hauck, M., Waldmann, V., Naeher, H., Rammensee, H. G., and Keil- autologous cytolytic T lymphocytes. J. Exp. Med., 179: 921–930, 1994. holz, U. Analysis of the T cell response to tumor and viral peptide 33. van der Bruggen, P., Szikora, J. P., Boel, P., Wildmann, C., Som- antigens by an IFN␥-ELISPOT assay. Int. J. Cancer, 71: 932–936, 1997. ville, M., Sensi, M., and Boon, T. Autologous cytolytic T lymphocytes

recognize a MAGE-1 nonapeptide on melanomas expressing HLA- 49. Slamon, D. J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Cw*1601. Eur. J. Immunol., 24: 2134–2140, 1994. Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Pegram, M., 34. Chaux, P., Vantomme, V., Coulie, P., Boon, T., and van der Baselga, J., and Norton, L. Use of chemotherapy plus a monoclonal Bruggen, P. Estimation of the frequencies of anti-MAGE-3 cytolytic antibody against HER2 for metastatic breast cancer that overexpresses T-lymphocyte precursors in blood from individuals without cancer. Int. HER2. N. Engl. J. Med., 344: 783–792, 2001. J. Cancer, 77: 538–542, 1998. 50. Disis, M. L., Knutson, K. L., Schiffman, K., Rinn, K., and McNeel, 35. Zorn, E., and Hercend, T. A MAGE-6-encoded peptide is recog- D. G. Pre-existent immunity to the HER-2/neu oncogenic protein in nized by expanded lymphocytes infiltrating a spontaneously regressing patients with HER-2/neu overexpressing breast and ovarian cancer. human primary melanoma lesion. Eur. J. Immunol., 29: 602–607, 1999. Breast Cancer Res. Treat, 62: 245–252, 2000. 36. Valmori, D., Dutoit, V., Rubio-Godoy, V., Chambaz, C., Lie´nard, 51. Feuerer, M., Beckhove, P., Bai, L., Solomayer, E. F., Bastert, G., D., Guillaume, P., Romero, P., Cerottini, J. C., and Rimoldi, D. Frequent Diel, I. J., Pedain, C., Oberniedermayr, M., Schirrmacher, V., and cytolytic T cell responses to peptide MAGE-A10 254–262 in mela- Umansky, V. Therapy of human tumors in NOD/SCID mice with noma. Cancer Res., 61: 509–512, 2001. patient-derived reactivated memory T cells from bone marrow. Nat. Med., 7: 452–458, 2001. 37. Rimoldi, D., Rubio-Godoy, V., Dutoit, V., Lienard, D., Salvi, S., Guillaume, P., Speiser, D., Stockert, E., Spagnoli, G., Servis, C., Cer- 52. Diel, I. J., and Cote, R. J. Bone marrow and lymph node assessment ottini, J. C., Lejeune, F., Romero, P., and Valmori, D. Efficient simul- for minimal residual disease in patients with breast cancer. Cancer Treat. taneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary Rev., 26: 53–65, 2000. open reading frame-derived CTL epitopes in melanoma. J. Immunol., 53. Hoffmann, T. K., Donnenberg, A. D., Finkelstein, S. D., Donnen- 165: 7253–7261, 2000. berg, V. S., Friebe-Hoffmann, U., Myers, E. N., Appella, E., DeLeo, 38. Andersen, M. H., Pedersen, L. O., Capeller, B., Brocker, E. B., A. B., and Whiteside, T. L. Frequencies of tetramerϩ T cells specific for Becker, J. C., and thor Straten, P. Spontaneous cytotoxic T cell re- the wild-type sequence p53(264–272) peptide in the circulation of sponses against survivin-derived MHC class I-restricted T cell epitopes patients with head and neck cancer. Cancer Res., 62: 3521–3529, 2002. in situ as well as ex vivo in cancer patients. Cancer Res., 61: 5964–5968, 54. Burchert, A., Wolfl, S., Schmidt, M., Brendel, C., Denecke, B., Cai, 2001. D., Odyvanova, L., Lahaye, T., Muller, M. C., Berg, T., Gschaidmeier, 39. Letsch, A., Keilholz, U., Schadendorf, D., Nagorsen, D., Schmittel, H., Wittig, B., Hehlmann, R., Hochhaus, A., and Neubauer, A. Inter- A., Thiel, E., and Scheibenbogen C. High frequencies of circulating feron-␣, but not the ABL-kinase inhibitor imatinib (STI571), induces melanoma-reactive CD8ϩ T cells in patients with advanced melanoma. expression of myeloblastin and a specific T cell response in chronic Int. J. Cancer, 87: 659–664, 2000. myeloid leukemia. Blood, 101: 259–264, 2003. 40. Anichini, A., Mortarini, R., Maccalli, C., Squarcina, P., Fleisch- 55. Scheibenbogen, C., Letsch, A., Thiel, E., Schmittel, A., Mailaender, hauer, K., Mascheroni, L., and Parmiani, G. Cytotoxic T cells directed V., Baerwolf, S., Nagorsen, D., and Keilholz, U. CD8 T cell responses to tumor antigens not expressed on normal melanocytes dominate HLA- to Wilms tumor gene product WT1 and proteinase 3 in patients with A2.1-restricted immune repertoire to melanoma. J. Immunol., 56: 208– acute myeloid leukemia. Blood, 100: 2132–2137, 2002. 217, 1996. 56. Andersen, M. H., Pedersen, L. O., Becker, J. C., and Straten, P. T. 41. Letsch, A., Keilholz, U., Mailander, V., Assfalg, G., Thiel, E., and Identification of a cytotoxic T lymphocyte response to the apoptosis Scheibenbogen, C. The bone marrow contains melanoma-reactive inhibitor protein survivin in cancer patients. Cancer Res., 61: 869–872, CD8ϩ effector T cells and compared to peripheral blood enriched 2001. ϩ numbers of melanoma-reactive CD8 memory T cells. Cancer Res., 63: 57. Greco, G., Fruci, D., Accapezzato, D., Barnaba, V., Nisini, R., 5582–5586, 2003. Alimena, G., Montefusco, E., Vigneti, E., Butler, R., Tanigaki, N., and 42. Titu, L. V., Monson, J. R., and Greenman, J. The role of CD8(ϩ) Tosi, R. Two brc-abl junction peptides bind HLA-A3 molecules and T cells in immune responses to colorectal cancer. Cancer Immunol. allow specific induction of human cytotoxic T lymphocytes. Leukemia Immunother., 51: 235–247, 2002. (Baltimore), 10: 693–699, 1996. 43. Nagorsen, D., Keilholz, U., Rivoltini, L., Schmittel, A., Letsch, A., 58. Osman, Y., Takahashi, M., Zheng, Z., Koike, T., Toba, K., Liu, A., Asemissen, A. M., Berger, G., Buhr, H. J., Thiel, E., and Scheibenbo- Furukawa, T., Aoki, S., and Aizawa, Y. Generation of bcr-abl specific gen, C. Natural T cell response against MHC class I epitopes of cytotoxic T-lymphocytes by using dendritic cells pulsed with bcr-abl epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic an- (b3a2) peptide: its applicability for donor leukocyte transfusions in tigen in patients with colorectal cancer. Cancer Res., 60: 4850–4854, marrow grafted CML patients. Leukemia (Baltimore), 13: 166–174, 2000. 1999. 44. Nagorsen, D., Scheibenbogen, C., Schaller, G., Leigh, B., Schmit- 59. Wagner, W. M., Ouyang, Q., and Pawelec, G. Peptides spanning the tel, A., Letsch, A., Thiel, E., and Keilholz, U. Differences in T cell fusion region of Abl/Bcr are immunogenic and sensitize CD8(ϩ)T immunity towards tumor associated antigens between colorectal cancer lymphocytes to recognize native chronic myelogenous leukemia. Leu- and breast cancer. Int. J. Cancer, 105: 221–225, 2003. kemia (Baltimore), 16: 2341–2343, 2002. 45. Hamann, D., Baars, P. A., Rep, M. H., Hooibrink, B., Kerkhof- 60. Khanna, R., and Burrows, S. R. Role of cytotoxic T lymphocytes in Garde, S. R., Klein, M. R., and van Lier, R. A. Phenotypic and func- Epstein-Barr virus-associated diseases. Annu. Rev. Microbiol., 54: 19– tional separation of memory and effector human CD8ϩ T cells. J. Exp. 48, 2000. Med., 186: 1407–1418, 1997. 61. Bontkes, H. J., de Gruijl, T. D., van den Muysenberg, A. J., 46. Arlen, P., Tsang, K. Y., Marshall, J. L., Chen, A., Steinberg, S. M., Verheijen, R. H., Stukart, M. J., Meijer, C. J., Scheper, R. J., Stacey, Poole, D., Hand, P. H., Schlom, J., and Hamilton, J. M. The use of a S. N., Duggan-Keen, M. F., Stern, P. L., Man, S., Borysiewicz, L. K., rapid ELISPOT assay to analyze peptide-specific immune responses in and Walboomers, J. M. Human papillomavirus type 16 E6/E7-specific carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer cytotoxic T lymphocytes in women with cervical neoplasia. Int. J. Immunol. Immunother., 49: 517–529, 2000. Cancer, 88: 92–98, 2000. 47. Peoples, G. E., Goedegebuure, P. S., Smith, R., Linehan, D. C., 62. Youde, S. J., Dunbar, P. R., Evans, E. M., Fiander, A. N., Bo- Yoshino, I., and Eberlein, T. J. Breast and ovarian cancer-specific rysiewicz, L. K., Cerundolo, V., and Man, S. Use of fluorogenic histo- cytotoxic T lymphocytes recognize the same HER-2/neu-derived pep- compatibility leukocyte antigen-A*0201/HPV 16 E7 peptide complexes tide. Proc. Natl. Acad. Sci. USA, 92: 432–436, 1995. to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous 48. Fisk, B., Blevins, T. L., Wharton, J. T., and Ioannides, C. G. human papillomavirus antigens. Cancer Res., 60: 365–371, 2000. Identification of an immunodominant peptide of HER-2/neu protoonco- 63. Wilkinson, J., Cope, A., Gill, J., Bourboulia, D., Hayes, P., Imami, gene recognized by ovarian tumor-specific cytotoxic T lymphocyte N., Kubo, T., Marcelin, A., Calvez, V., Weiss, R., Gazzard, B., Boshoff, lines. J. Exp. Med., 181: 2109–2117, 1995. C., and Gotch, F. Identification of Kaposi’s sarcoma-associated herpes-

virus (KSHV)-specific cytotoxic T-lymphocyte epitopes and evaluation 76. Dutoit, V., Taub, R. N., Papadopoulos, K. P., Talbot, S., Keohan, of reconstitution of KSHV-specific responses in human immunodefi- M. L., Brehm, M., Gnjatic, S., Harris, P. E., Bisikirska, B., Guillaume, ciency virus type 1-infected patients receiving highly active antiretro- P., Cerottini, J. C., Hesdorffer, C. S., Old, L. J., and Valmori, D. viral therapy. J. Virol., 76: 2634–2640, 2002. Multiepitope CD8(ϩ) T cell response to a NY-ESO-1 peptide vaccine 64. Boshoff, C., and Weiss R. AIDS-related malignancies. Nat. Rev. results in imprecise tumor targeting. J. Clin. Investig., 110: 1813–1822, Cancer, 2: 373–382, 2002. 2002. 65. Hsieh, W. S., Lemas, M. V., and Ambinder, R. F. The biology of 77. Valmori, D., Pittet, M. J., Vonarbourg, C., Rimoldi, D., Lienard, D., Epstein-Barr virus in post-transplant lymphoproliferative disease. Speiser, D., Dunbar, R., Cerundolo, V., Cerottini, J. C., and Romero, P. Transpl. Infect. Dis., 1: 204–212, 1999. Analysis of the cytolytic T lymphocyte response of melanoma patients 66. Karanikas, V., Colau, D., Baurain, J. F., Chiari, R., Thonnard, J., to the naturally HLA-A*0201-associated tyrosinase peptide 368–376. Gutierrez-Roelens, I., Goffinet, C., Van Schaftingen, E. V., Weynants, Cancer Res., 59: 4050–4055, 1999. P., Boon, T., and Coulie, P. G. High frequency of cytolytic T lympho- 78. Naito, Y., Saito, K., Shiiba, K., Ohuchi, A., Saigenji, K., Nagura, cytes directed against a tumor-specific mutated antigen detectable with H., and Ohtani, H. CD8ϩ T cells infiltrated within cancer cell nests as HLA tetramers in the blood of a lung carcinoma patient with long a prognostic factor in human colorectal cancer. Cancer Res., 58: 3491– survival. Cancer Res., 61: 3718–3724, 2001. 3494, 1998. 67. Echchakir, H., Mami-Chouaib, F., Vergnon, I., Baurain, J. F., 79. Schumacher, K., Haensch, W., Roefzaad, C., and Schlag, P. M. Karanikas, V., Chouaib, S., and Coulie, P. G. A point mutation in the Prognostic significance of activated CD8(ϩ) T cell infiltrations within ␣-actinin-4 gene generates an antigenic peptide recognized by autolo- esophageal carcinomas. Cancer Res., 61: 3932–3936, 2001. gous cytolytic T lymphocytes on a human lung carcinoma. Cancer Res. 80. Zhang, L., Conejo-Garcia, J. R., Katsaros, D., Gimotty, P. A., 61: 4078–4083, 2001. Massobrio, M., Regnani, G., Makrigiannakis, A., Gray, H., Schlienger, 68. Weinschenk, T., Gouttefangeas, C., Schirle, M., Obermayr, F., K., Liebman, M. N., Rubin, S. C., and Coukos, G. Intratumoral T cells, Walter, S., Schoor, O., Kurek, R., Loeser, W., Bichler, K. H., Wernet, recurrence, and survival in epithelial ovarian cancer. N. Engl. J. Med., D., Stevanovic, S., and Rammensee, H. G. Integrated functional genom- 348: 203–213, 2003. ics approach for the design of patient-individual antitumor vaccines. Cancer Res., 62: 5818–5827, 2002. 81. Marincola, F. M., Jaffee, E. M., Hicklin, D. J., and Ferrone, S. Escape of human solid tumors from T cell recognition: molecular 69. Chen, Q., Jackson, H., Gibbs, P., Davis, I. D., Trapani, J., and mechanisms and functional significance. Adv. Immunol., 74: 181–273, Cebon, J. Spontaneous T cell responses to melanoma differentiation 2000. antigens from melanoma patients and healthy subjects. Cancer Immu- nol. Immunother., 47: 191–197, 1998. 82. Pupa, S. M., Menard, S., Andreola, S., and Colnaghi, M. I. Anti- 70. Marincola, F. M., Rivoltini, L., Salgaller, M. L., Player, M., and body response against the c-erbB-2 oncoprotein in breast carcinoma Rosenberg, S. A. Differential anti-MART-1/MelanA CTL activity in patients. Cancer Res., 53: 5864–5866, 1993. peripheral blood of HLA-A2 melanoma patients in comparison to 83. Fishman, P., Merimski, O., Baharav, E., and Shoenfeld, Y. Autoan- healthy donors: evidence of in vivo priming by tumor cells. J. Immu- tibodies to tyrosinase: the bridge between melanoma and vitiligo. Can- nother. Emphasis Tumor Immunol., 19: 266–277, 1996. cer (Phila.), 79: 1461–1464, 1997. 71. Loftus, D. J., Squarcina, P., Nielsen, M. B., Geisler, C., Castelli, C., 84. Mosolits, S., Harmenberg, U., Ruden, U., Ohman, L., Nilsson, B., Odum, N., Appella, E., Parmiani, G., and Rivoltini, L. Peptides derived Wahren, B., Fagerberg, J., and Mellstedt, H. Autoantibodies against the from self-proteins as partial agonists and antagonists of human CD8ϩ T tumour-associated antigen GA733-2 in patients with colorectal carci- cell clones reactive to melanoma/melanocyte epitope MART1(27-35). noma. Cancer Immunol. Immunother., 47: 315–320, 1999. Cancer Res., 58: 2433–2439, 1998. 85. Jager, E., Stockert, E., Zidianakis, Z., Chen, Y. T., Karbach, J., 72. Ogg, G. S., Rod Dunbar, P., Romero, P., Chen, J. L., and Cerun- Jager, D., Arand, M., Ritter, G., Old, L. J., and Knuth A. Humoral dolo, V. High frequency of skin-homing melanocyte-specific cytotoxic immune response of cancer patients against “cancer-testis” antigen T lymphocytes in autoimmune vitiligo. J. Exp. Med., 188: 1203–1208, NY-ESO-1: correlation with clinical events. Int. J. Cancer, 84: 506– 1998. 510, 1999. 73. Lang, K. S., Caroli, C. C., Muhm, A., Wernet, D., Moris, A., 86. Gao, F. G., Khammanivong, V., Liu, W. J., Leggatt, G. R., Frazer, Schittek, B., Knauss-Scherwitz, E., Stevanovic, S., Rammensee, H. G., I. H., and Fernando, G. J. Antigen-specific CD4ϩ T cell help is required and Garbe, C. HLA-A2 restricted, melanocyte-specific CD8(ϩ) T lym- to activate a memory CD8ϩ T cell to a fully functional tumor killer cell. phocytes detected in vitiligo patients are related to disease activity and Cancer Res., 62: 6438–6441, 2002. are predominantly directed against MelanA/MART-1. J. Investig. Der- 87. McNeel, D. G., Nguyen, L. D., Ellis, W. J., Higano, C. S., Lange, matol., 116: 891–897, 2001. P. H., and Disis, M. L. Naturally occurring prostate cancer antigen- 74. Stockinger, B. T lymphocyte tolerance: from thymic deletion to specific T cell responses of a Th1 phenotype can be detected in patients peripheral control mechanisms. Adv. Immunol., 71: 229–265, 1999. with prostate cancer. Prostate, 47: 222–229, 2001. 75. Molldrem, J. J., Lee, P. P., Kant, S., Wieder, E., Jiang, W., Lu, S., 88. Gaiger, A., Carter, L., Greinix, H., Carter, D., McNeill, P. D., Wang, C., and Davis, M. M. Chronic myelogenous leukemia shapes host Houghton, R. L., Cornellison, C. D., Vedvick, T. S., Skeiky, Y. A., and immunity by selective deletion of high-avidity leukemia-specific T Cheever, M. A. WT1 specific serum antibodies in patients with leuke- cells. J. Clin. Investig., 111: 639–647, 2003. mia. Clin. Cancer Res.; 7 (Suppl.): 761s–765s, 2001.

Dirk Nagorsen, Carmen Scheibenbogen, Francesco M. Marincola, et al.

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