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Immunology |
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, 1005 Lausanne, Switzerland [D. V., V. D., V. R-G., P. R., J-C. C., D. L.]; University Hospital Benjamin-Franklin, Medizinische Klinik III, Hematology, Oncology and Transfusion Medicine, Free University of Berlin, 12200 Berlin, Germany [C. S., D. N., A. M. A., E. T., U. K.]; Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland [D. R., P. G., J-C. C.]; Skin Cancer Unit, German Cancer Research Center, University Hospital Heidelberg, 68135 Mannheim, Germany [D. S.]; Max-Delbrueck-Center for Molecular Medicine, 13092 Berlin, Germany [M. L.]; Division of Oncology, Laboratory of Tumor Immunology, University Hospital, 1211 Geneva 14, Switzerland [P-Y. D.]; and Multidisciplinary Oncology Center, University Hospital, 1005 Lausanne, Switzerland [D. L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA+CCR7-, a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer+ CD8+ T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of melanoma patients using the ELISPOT IFN--secretion assay (7)
. To additionally characterize tumor-reactive T cells ex vivo, in this study, we have performed a multiparametric flow cytometry-based phenotypical analysis of circulating CD8+ T cells producing IFN- in response to stimulation with the autologous tumor line (8
, 9)
. The results of these analyses revealed that these tumor-reactive populations mostly lacked the expression of CCR7, a chemokine receptor that mediates homing to secondary lymphoid tissue. CCR7 is expressed by naive T cells and by a subset of memory cells (called central memory cells) but is down-regulated in the so-called effector memory T-cell fraction, characterized by the ability to migrate into inflammatory tissue (10)
. However, in contrast to most antigen experienced CD8+ T lymphocytes, characterized by the expression of the CD45RO isoform, a considerable fraction of tumor-reactive CD8+ T cells producing IFN- ex vivo on stimulation with the autologous tumor expressed the CD45RA isoform and contained elevated levels of the serine protease granzyme B. The CD45RA+CCR7- phenotype has been specifically identified among CD8+ but not CD4+ T lymphocytes (10
, 11)
, and because of the prominent serine protease expression it has been proposed to characterize terminally differentiated effector T cells (10)
. Whereas the antigen specificity of tumor-reactive CD8+ IFN- producing T cells was unknown for most patients, in the case of an HLA-A2-expressing patient, by using A2/peptide multimers incorporating peptide tyrosinase368–376 (tyrosinase multimers thereafter), we identified a large proportion (>5%) of CD8+CD45RA+CCR7- tyrosinase multimer+ T cells that were detectable for at least 3 years in the circulating T-cell compartment. Consistent with their phenotypic characteristics, tyrosinase multimer+ T cells displayed ex vivo-specific lytic activity on HLA-A2+ tyrosinase-expressing targets. These results provide the first direct evidence that high proportions of circulating tumor-specific T cells that phenotypically and functionally are lytic effectors ex vivo can be found in cancer patients. |
MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. After informed consent, PBMCs were drawn at different points of time before or after resection of the metastatic lesions from which the cell lines were established. Before analysis, frozen samples were thawed and cultured overnight in CTL medium (Iscove’s medium supplemented with 10% human serum). This investigation had been approved by the Institutional Ethics Committee. The melanoma cell line Me 275 (expressing HLA-A2 and tyrosinase) was established at the Ludwig Institute for Cancer Research, Lausanne, from melanoma patient LAU 50. Tyrosinase 368–376-specific CD8+ T-cell clone LAU132 1G4/1 was described previously (12)
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-producing cells, a minimum of 250,000 events were acquired for each analysis. Fluorescence-conjugated mouse immunoglobulins were used as isotype control reagents.
Stimulation and Detection of Cytokine Production.
Analysis of specific T-cell responses by intracellular staining for IFN- was performed as described previously (9)
. For intracellular staining of IFN- and granzyme B, and costaining with mAbs, PBMC were stimulated during 6 h with synthetic peptide at 10 µg/ml or with melanoma cells at a 10:1 lymphocytes to tumor cell ratio in the presence of Brefeldin A (Sigma Chemical Co., Steinheim, Germany; 20 µg/ml final, added 1 h after the beginning of the stimulation) to inhibit cytokine secretion. At the end of the incubation cells were stained with cell surface mAb for 20 min at 4°C, washed once, and then fixed and permeabilized using lysing and permeabilizing solution (Becton Dickinson, San Jose, CA) according to the manufacturer’s instructions. Cells were then stained by incubation with anti-IFN-FITC for 30 min at 4°C. For experiments designed to FACS sort IFN--producing T cells, staining was performed by using a IFN--secretion assay kit (Miltenyi Biotec; Bergisch, Gladbach, Germany), which avoids cell permeabilization, thus allowing for the sorting of life IFN--producing cells. Briefly, after being cocultured during 6 h with melanoma cells, PBMCs were labeled for 5 min at 4°C with a bispecific antibody-antibody conjugate directed against CD45 and IFN-. Cells were then incubated in medium at 37°C during 45 min to allow surface capture of secreted IFN-, washed, and stained for 30 min at 4°C with anti CD8FITC mAb and anti IFN-PE mAb.
Cytotoxicity Assay.
CTLs were assayed for specific lysis of target cells alone or prepulsed with synthetic peptide using a chromium release assay. Briefly, target cells were labeled with 51chromium during 1 h at 37°C and washed three times. Where indicated, synthetic peptide (1 µg/ml) was added during this incubation period. Labeled target cells (1000 cells/well) were then incubated with various numbers of effector cells at 37°C in V-bottomed microwells for the indicated period. Chromium release was then measured in the supernatant of the cultures using a gamma counter. The percentage of specific lysis was calculated as: 
.
CDR3 Size Analysis of TCR BV Transcripts.
The complementary determining region 3 (CDR3) region of the PCR-amplified TCR BV1–24 transcripts was analyzed using a run-off procedure, as described previously (17
, 18)
. Briefly, total RNA was prepared from sorter purified CD8+ tyrosinase multimer+ (7500 cells) and multimer- (2 x 105 cells) T-cell fractions using TRIzol (Life Technologies, Inc., Pasley, United Kingdom) and converted to cDNA by standard methods using reverse transcriptase and an oligo(dT) primer. These cDNAs were then amplified using a panel of validated 5' sense primers specific for the 24 ß variable (BV) subfamilies and one 3' antisense primer specific for the ß constant (BC) gene segment (19)
. The run off products were then run on an automated sequencer in the presence of fluorescent size markers. The length of the DNA fragments and the fluorescence intensity of the bands were analyzed with Immonoscope software (developed by C. Pannetier, Paris, France).
Western Blot Analysis.
Lysates from cultured cells were prepared in 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), added with protease inhibitors (MiniComplete mixture; Roche). For the tumor sample, thick sections were cut from the frozen block in a cryostat and lysed in the same buffer. Aliquots of extracts (50 µmg) were boiled in Laemmli buffer and analyzed by SDS-PAGE (9% and 15% acrylamide for tyrosinase and Melan-A/MART-1, respectively), followed by transfer to a membrane (Hybond ECL; Amersham). Immunodetection was performed with antityrosinase T311 and anti-Melan-A/MART-1 M2–7C10 monoclonal antibodies (gifts from E. Stockert, Ludwig Institute for Cancer Research, New York, NY and from S. A. Rosenberg, NIH, Bethesda, MD, respectively; Refs. 20
, 21
) and an horseradish peroxidase-conjugated sheep antimouse immunoglobulin (Amersham) followed by a chemiluminescence detection system (ECL; Amersham).
mRNA Expression Analysis.
Quantitative real-time reverse transcription-PCR was performed to assess the expression of tyrosinase in tumor tissue and cell lines as described in detail elsewhere (22)
. Briefly, RNA extraction was carried out using the High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Random hexamers and avian myeloblastosis virus reverse transcriptase were used for cDNA synthesis. Tyrosinase and porphobilinogen deaminase as housekeeping gene were amplified using published primer sequences and conditions using a LightCycler equipment (Roche). A standard curve was established using artificial plasmids for both RNA species. The results are expressed as copy number of tyrosinase per copy of porphobilinogen deaminase.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The melanoma cell lines were analyzed for the expression of MHC antigens by flow cytometry (for cell line description see Table 1
). As compared with unstimulated samples used as internal controls and after a 6 h incubation of PMBCs with autologous tumor cells, specific IFN--producing CD3+CD8+ T cells were identified in five of seven patients and ranged from 0.19% to 2.73% of CD3+CD8+ T cells (Fig. 1A
; Table 2
). In contrast, no significant CD3+CD4+ T-cell response was detected. T cells specifically producing IFN- in response to stimulation with autologous tumor were additionally phenotypically characterized for expression of CD45RA, CCR7, and granzyme B by staining with the corresponding mAb simultaneously to the measurement of IFN- production. As illustrated in Fig. 1B
for patient 6 and summarized in Table 2
for all of the analyzed patients, a large proportion of CD3+CD8+ IFN--secreting T cells expressed CD45RA but were mostly CCR7-. Although no direct costaining of CCR7 and CD45RA could be performed in this particular experimental setting (because of limiting number of channels simultaneously available for FACS analysis), from the results reported in Table 2
it could be deduced that a substantial proportion of IFN--producing T cells were CD45RA+CCR7-, a phenotype proposed recently to characterize effector cells able to exert direct lytic activity (10)
. In line with this, a considerable fraction of these cells ranging from 38% in patient 1 to 87% in patient 6 expressed granzyme B. In the case of patient 6 the expression of CD45RA and CCR7 was simultaneously assessed by omitting from the staining protocol the anti-CD3 mAb. The results of this analysis (Fig. 1C)
confirmed the phenotype deduced in the previous experiments.
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were available for analyses. For both patients CD3+CD8+ T cells specifically producing IFN- in response to stimulation with autologous tumor cells were clearly detected at each time analyzed (Table 2A)
. However, whereas in the case of patient 2 the percentage of the cells remained quite constant over time, it clearly increased (10-fold in a 2-year period) in patient 6. In both patients large progressively growing metastatic lesions confined to a single site had been resected 9 and 6 months before the blood samples 9/00 and 1/01 were drawn from patients 2 and 6, respectively. Both patients had been free of disease since then. Whereas the antigen specificity of tumor-reactive CD8+ IFN--producing T cells was generally unknown, in the case of patient 2 (HLA-A2 expressing) a high proportion of CD8+ T cells producing IFN- in response to stimulation with an HLA-A2-restricted peptide (tyrosinase 368–376; Ref. 15
) was detected (Table 2B)
. Interestingly, this population displayed phenotypical characteristics (CD45RA+CCR7-) similar to that observed for a substantial proportion of the population producing IFN- in response to stimulation with the autologous tumor line (Table 2A)
. These results prompted us to more directly analyze this population by using A2/peptide fluorescent multimers.
Ex Vivo Detection and Phenotypic Characterization of Tumor-Antigen-specific T-Cell Populations in Melanoma Patient 2 by Staining with A2/Peptide Multimers.
CD8+ T-cell response of patient 2 to two A2-restricted epitopes widely recognized by A2 melanoma patients, namely Melan-A26–35 and Tyrosinase368–376 was analyzed by ex vivo staining of circulating lymphocytes (sample 9/00) with A2/peptide multimers containing the corresponding peptides. A relatively high frequency of Melan-A-multimer+ CD8+ T cells was clearly detectable (0.2% of gated CD8+ T cells; Fig. 2A
). Interestingly, Melan-A-multimer+ CD8+ T cells displayed a mixed CD45RAbright/CD45RAlow phenotype, which we have detected previously in PBMCs from a fraction (30%) of melanoma patients, whereas in the remaining melanoma patients and in healthy donors Melan-A-multimer+ CD8+ T cells represent an average 0.07% of total circulating CD8+ T cells and uniformly display a CD45RAbright phenotype (3)
. Melan-A-multimer+ CD45RAbright cells expressed high levels of CCR7, whereas Melan-A-multimer+ CD45RAlow cells mostly had down-regulated the latter consistently with a naive and antigen experienced phenotype of these populations, respectively.
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, we detected elevated proportions of tyrosinase multimer+ T cells (5.6% of CD8+ T cells), which exhibited a uniform CD45RA+ CCR7- phenotype. Tyrosinase multimer+ T cells accounted for 16% of the total CD45RA+CCR7- circulating T cells in this patient (data not shown). In addition, they were clearly persistent in the circulation along the time period analyzed as confirmed by analysis of additional times (5.8% in sample 3/99 and 6.7% in sample 3/01, not shown). It is of note that in previous studies using A2/tyrosinase multimers (3
, 12)
we found that A2/tyrosinase multimer+ CD8+ T cells were below detection limits, ex vivo, in both A2 healthy donors and in melanoma patients analyzed before this study. However, in a proportion of individuals belonging to both groups tyrosinase368–376-specific CD8+ T cells become clearly detectable after in vitro stimulation with the corresponding synthetic peptide (12
, 23)
. Thus, the high proportion of A2/tyrosinase multimer+ CD8+ T cells detected ex vivo in patient 2 was remarkable, as was their phenotype. However, this patient is not a unique case as, in a recent survey of tumor antigen-specific circulating CD8+ T cells from additional metastatic melanoma patients we found a clearly detectable albeit lower level of CD8+ multimer+ T cells displaying a similar phenotype in another patient (LAU 337, 0.2% of total circulating CD8+ T cells, data not shown).
To additionally assess the phenotypic characteristics of the A2/tyrosinase multimer+ CD8+ T -ell population in patient 2, cells from times 9/00 and 3/01 were costained with A2/tyrosinase multimers, anti-CD8 mAbs, and with a panel of mAbs directed against several T-cell surface or intracellular markers. As illustrated in Fig. 3
for time 9/00, the large majority of tyrosinase multimer+ cells expressed CD57, CD69, intracellular perforin, and granzyme B but were CD45RO-, CD27-, CD28-, HLA-DR-, and CLA-. This phenotype was maintained over time, as similar results were obtained with a sample from time 3/01 (not shown).
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, bottom panel).
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(top panel) tyrosinase multimer+ cells were able to lyse T2 cells (A2+ tyrosinase-) only when loaded with peptide tyrosinase368–376 but efficiently lysed Me 275 cells (A2+ tyrosinase+) both in the presence or in the absence of peptide. No significant tyrosinase-specific nor tumor-specific lysis was detected for the A2/tyrosinase multimer- CD8+ T-cell fraction. Similar results were obtained with a sample from time 3/99 (not shown).
The ability of tyrosinase multimer+ T cells to secrete IFN- ex vivo on stimulation with tyrosinase-expressing tumors was also assessed. As illustrated in Fig. 4B
1.7% of CD8+ cells in PBMCs (sample 9/00) from patient 2 specifically secreted IFN- in response to 6-h stimulation with Me 275 and were mostly tyrosinase multimer+ (Fig. 4C)
. The total proportion of IFN--secreting T cells that were tyrosinase multimer+ was, in this experimental setting, roughly comparable with that of tyrosinase-specific clone LAU 132 1G4/1 (Fig. 4C)
.
The clonal composition of tyrosinase multimer+ and multimer- sorted fractions was analyzed by spectratyping (analysis of TCR-ß chain V segment usage and CDR3 length) as described recently (24)
. The typical bell-shaped pattern generally observed for polyclonal T-cell populations was obtained for all of the different BV subfamilies in the case of the multimer- population (Fig. 5
, bottom line and data not shown). In contrast, for tyrosinase multimer+ T cells, single peaks that indicated the accumulation of recurrent transcripts of identical size were detected in the case of 4 BV subfamilies (Fig. 5
, top line), whereas no signal was detected for the remaining ones (not shown). The major peak was detected for BV4, whereas minor peaks were detected for BV2, BV3, and BV14. The proportion of BV4-expressing tyrosinase multimer+ T cells could not be directly assessed by costaining with multimers and anti-BV mAbs (25)
, because no anti-BV4-specific mAb is commercially available. On the other hand, no significant levels of tyrosinase multimer+ T cells expressing BV2, BV3, or BV14 were detected using the corresponding mAbs (not shown). Altogether, these data indicate that the TCR repertoire of CD8+ T cells of the tyrosinase multimer+ population ex vivo is highly restricted and mostly composed of a single BV4 using clonotype or of different BV4 using clonotypes displaying a CDR3 region of 10 amino acids.
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. The phenotypic characteristics of the cultured population were additionally assessed at day 21 (Fig. 6B)
. It is of note that at this time the cells had already partially re-expressed CD45RA (compare Fig. 6, A and B
) and expressed intermediate levels of CD45RO. In addition, HLA-DR was clearly positive. As for the remaining markers analyzed, the expression pattern was mostly maintained as compared with ex vivo analysis (Fig. 3)
, although variations in the expression levels were measurable.
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. The multimer+ T-cell line displayed high avidity of antigen recognition (50% maximal lysis required 200 pM of antigenic peptide). Me 275 cells were efficiently lysed both in the presence and in the absence of peptide tyrosinase 368–376, whereas T2 cells (A2+, tyrosinase-) were efficiently lysed only in the presence of peptide. Interestingly, only a modest level of lysis (comparable with the one obtained with the control clone) was obtained on the patient autologous tumor cell line in the absence of exogenously added peptide, whereas high levels of specific lysis (although slightly lower than on Me 275) were obtained on loading with peptide. This tumor line also failed to be significantly lysed by a Melan-A26–35 polyclonal monospecific CD8+ T-cell line in the absence of peptide Melan-A26–35, whereas it was efficiently lysed on peptide loading. In contrast, Me 275 was efficiently lysed by Melan-A-specific T cells both in the presence and in the absence of peptide Melan-A26–35. It is of note that, in this experiment, a later cell passage of the autologous tumor cell line was used as compared with the early one used in the initial IFN- secretion experiments described in Table 2
, which was not available anymore because of the initial very slow growing rate. Indeed, analysis of the expression of both Melan-A and tyrosinase tumor antigens by the later tumor cell passage revealed that the expression levels of the corresponding proteins were low to undetectable as compared with Me 275 used as positive control. However, this finding is likely to reflect an in vitro selection phenomenon not necessary relevant to the in vivo situation in the tumor lesion, because normal levels of expression of both Melan-A and tyrosinase were detected on fresh patient tumor samples (Fig. 6D)
. Accordingly, levels of tyrosinase mRNA were 20-fold lower in the later tumor cell passage (1.3 x 104 copies of tyrosinase/105 copies of porphobilinogen deaminase [PBGD]) as compared with the patient fresh tumor sample (3.2 x 105), but only 2-fold lower (1.5 x 105) in a very early tumor cell passage (after 14 days in culture). In vitro selection most likely occurred relatively early during cell culture; indeed, the percentages of IFN--secreting CD8+ T cells detected on stimulation with the autologous tumor line in the experiments where an earlier tumor cell passage was used (Table 2A)
were significantly lower than that obtained on stimulation of samples from the same time with tyrosinase peptide (Table 2B)
or with Me 275 (Fig. 4B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in response to the autologous tumor in five of seven patients analyzed. In contrast, we failed to detect significant levels of responder cells among CD4+ T cells despite the fact that tumor cell lines from three of these five patients expressed MHC class II molecules. Albeit the present study focused on the analysis of ex vivo tumor-reactive T cells producing IFN-, the assessment of the production of other cytokines (i.e., IL-4) would also be of interest, because it could unveil additional and possibly functionally distinct subsets of ex vivo tumor-reactive T cells. An important and yet unresolved question is whether self-tumor-reactive CD8+ T cells are able to home to metastatic sites and destroy tumor cells in vivo. An insight into this question is given by the phenotypical characteristics of ex vivo tumor-reactive T cells identified here, mostly CD45RA+CCR7-. On the basis of the expression of CD45RA and CCR7, four subsets of CD8+ T cells with different functional capacities and differentiation stages have been defined recently (10)
. Whereas naive T cells express both CD45RA and CCR7, memory T cells are characterized by down-regulation of CD45RA and can be additionally subdivided, according to the expression of CCR7, into the so-called effector memory and central memory subsets. The fourth T-cell subset, CD45RA+ but CCR7-, is exclusively found among CD8+ but not CD4+ T cells (10
, 11)
. Several recent reports have defined CD45RA+ CCR7- T cells or corresponding CD45RA+ T cell populations, which lack the expression of CD27 and or CD28, as terminally differentiated effector T cells able to migrate to inflamed tissues and exert vigorous ex vivo effector functions including target cell lysis (10
, 11 , 26)
.
The results obtained with patient 2, for which the antigen specificity of CD45RA+CCR7- T cells secreting IFN- ex vivo in response to antigen-expressing tumor cells could be directly assessed using MHC/peptide multimers, clearly show that these cells are indeed effector T cells able to specifically kill tumor cells. This is, to the best of our knowledge, the first example showing direct ex vivo cytotoxicity of tumor antigen-specific T cells in a cancer patient. Consistent with the results of the above mentioned reports, circulating tyrosinase multimer+ T cells in patient 2 contained high levels of granzyme B and perforin, expressed CD57, and lacked expression of two major costimulatory molecules, CD27 and CD28. In addition, in agreement with the limited usage of Vß elements by CD8+ CD45RA+ CD27-, CD28-, and CD57+ T cells (27)
, spectratyping analysis of the sorted tyrosinase multimer+ T-cell fraction revealed that this population is most likely composed of a single T-cell clone.
FACS sorted tyrosinase multimer+ T cells could be efficiently expanded in vitro on stimulation with mitogen in the presence of IL-2. Cell growth resulted in down-regulation of CD45RA expression (which was at least partially re-expressed later in the culture), expression of HLA-DR, and of intermediate levels of the CD45RO isoform. Thus, despite the fact that their phenotype has been defined previously as terminally differentiated, this population conserved some, at least in vitro, growing potential and the ability of modulating the expression of naive/memory T-cell markers, suggesting that similar changes could take place in vivo.
Recently, monoclonal CD8+ T-cell populations directed against two distinct T-cell epitopes derived from mutated tumor antigens and accounting for 0.4% and 1.2% of specific circulating CD8+ T lymphocytes have been detected by staining with multimers in a melanoma (28) and a lung cancer (29) patient, respectively. It is of note that, in the case of patient 2 the CTL population analyzed here was directed against a nonmutated sequence derived from the melanocyte lineage differentiation antigen tyrosinase. Thus, genetic mutation does not appear to be a prerequisite for the vigorous clonal expansion of tumor-reactive CTL populations. Interestingly, the CD8+ population specific for a mutated sequence from malic enzyme (29) was mostly CD45RA+ before treatment and showed a decreased CD45RA expression after vaccination with irradiated autologous tumor cells.
A proportion of Melan-A-multimer+ CD8+ T cells, which accounted for 0.2% of CD8+ T lymphocytes, was also detected in patient 2. These Melan-A multimer+ T cells displayed two distinct phenotypes, CD45RA+CCR7+ and CD45RA-CCR7-, which correspond to naive and effector memory T cells, respectively. Whereas Melan-A multimer+ T cells expressing CD45RA+ are detectable in the large majority of HLA-A2-expressing individuals including a large fraction of melanoma patients, CD45RA- Melan-A multimer+ T cells have been detected previously in 30% of melanoma patients (3)
and most likely represent spontaneous immune responses to melanoma. According to a recently proposed pathway of T-cell lineage differentiation (30)
the CD45RA-CCR7- stage would represent a differentiation stage that precedes the CD45RA+CCR7- stage. Thus, populations of CD8+ T cells specific for different tumor antigens and at distinct stages of differentiation can be found at the same moment of the disease evolution in this patient, suggesting that, for yet unknown reasons, these antigens could differently drive the differentiation of tumor-reactive CD8+ T lymphocytes.
In a previous study (31) a circulating tyrosinase multimer+ T-cell population specific for the same 368–376 epitope and accounting for 2% of total CD8+ T cells was found in a melanoma patient. However, this population was unable to exert effector functions including both cytokine secretion and target cell lysis. The anergic state appeared to be profound and irreversible, because effector functions could not be rescued on in vitro culture in the presence of IL-2. In contrast with those findings, the results reported here brighten the scenario of spontaneous immune responses against cancer, as they clearly indicate full functionality of circulating tumor-specific CTL. Thus, although T-cell anergy of tumor-specific T lymphocytes could occur in some patients, it does not appear to be a general feature of spontaneous immune responses against cancer. In addition, although direct tumor-specific lytic activity could only be assessed for patient 2 because of the known antigen specificity, the fact that tumor-reactive T cells with a similar phenotype could be detected in four of seven randomly selected stage IV melanoma patients suggests that tumor-reactive effector T cells could be present in a significant fraction of cancer patients and are not a rare finding.
Tumor-specific response in patient 2 was most likely spontaneously induced by the tumor, because it was found before any treatment and did not appear to be significantly affected by various treatments including administration of IFN-
and chemotherapy, vaccination with MAGE-3 protein, and irradiation. Remarkably, neither the frequency nor the phenotype of the tyrosinase multimer+ population in this patient showed any significant modification during the 3-year period analyzed, including 15 months after resection of progressively growing metastatic lesions confined to a single site that left the patient free of detectable disease up to now. Persistency of such a large population of antigen-specific effector-type T cells in the circulation in the apparent absence of antigen is in contrast with previous findings on virally induced diseases, which indicate that effector T cells develop relatively early after virus infection and rapidly decline thereafter (32)
. Although tyrosinase is expressed in some normal tissues such as in the melanocytes of the skin, this is unlikely to be the source of antigen involved in the survival of the population of study because, despite the presence of >5% ex vivo lytic tyrosinase-specific CTLs, the patient showed no signs of vitiligo nor of other autoimmune disease. Consistently with this observation, tyrosinase multimer+ T cells did not express the skin-homing receptor CLA, of which the expression by melanoma-specific CD8+ T cells has been suggested to be associated with the development of vitiligo (33)
. Persistency of tumor-reactive effector type T cells could be because of persistence of minimal residual disease in this patient resulting in continuous stimulation of the population of study or to altered trafficking into the periphery after a reduction in tumor burden after surgery. Alternatively, a difference in the kinetics of the immune response to virally derived versus tumor-associated antigens or the presence of residual antigen sequestered for example by professional antigen presenting cells or of tyrosinase cross-reactive sequences contributing to the long term maintenance of the population of study can be imagined. However, because of the lack of reported tumor-specific responses of effector type before this one, additional speculations must await a more systematic and comparative longitudinal analysis in additional patients.
Factors involved in the development of tumor-specific responses in the remaining responder patients (patients 1, 4, and 6) are more difficult to evaluate, because they had all received various treatments before analysis. Although the occurrence of ex vivo detectable tumor immune responses to cancer in patients at different stages of the disease is yet unknown, it is likely that such responses would spontaneously develop only at a relatively late stage, when a large antigen load is present. Therefore, the role played by spontaneous immune responses to cancer on the clinical course of the disease is difficult to evaluate, because various escape mechanisms could intervene at that stage. Thus, to thoroughly evaluate the potential of tumor-reactive CD8+ T cells of the effector type, it is crucial to devise vaccination protocols, which would be able to stimulate and maintain such responses earlier in the course of disease when they may be much more effective to eradicate minimal residual disease and prevent relapses. The goal for future monitoring of T-cell responses in vaccination studies should, therefore, be aimed not only at quantitating specific CD8+ T-cell responses but also at additionally characterizing them ex vivo on the basis of CD45RA/CCR7 antigen expression and direct lytic potential.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Deutsche Forschungsgemeinschaft Grant Sche 478/1-3 and Dr. Mildred-Scheel-Stiftung Grant 1072-Ke2. 
2 These two authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Universitaetsklinikum Benjamin-Franklin, Medizinische Klinik III, Hindenburgdamm 30, D-12200 Berlin, Germany. Phone: 49-30-8445-3906; Fax: 49-30-8445-4468; E-mail: ulrich.keilholz{at}ukbf.fu-berlin.de
4 The abbreviations used are: CTL, cytotoxic T cell; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; TCR, T-cell receptor; FACS, fluorescence-activated cell sorter; IL, interleukin; CLA, cutaneous lymphocytes-associated antigen.
Received 9/ 4/01. Accepted 1/ 9/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 C. Maccalli, V. Di Cristanziano, V. Fodale, D. Corsi, G. D'Agostino, V. Petrangeli, L. Laurenti, S. Guida, A. Mazzocchi, F. Arienti, et al. Induction of Both CD8+ and CD4+ T-Cell-Mediated Responses in Colorectal Cancer Patients by Colon Antigen-1 Clin. Cancer Res., November 15, 2008; 14(22): 7292 - 7303. [Abstract] [Full Text] [PDF]
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O. C. Goodyear, G. Pratt, A. McLarnon, M. Cook, K. Piper, and P. Moss Differential pattern of CD4+ and CD8+ T-cell immunity to MAGE-A1/A2/A3 in patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma Blood, October 15, 2008; 112(8): 3362 - 3372. [Abstract] [Full Text] [PDF]
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 P. Zhang, A. L. Cote, V. C. de Vries, E. J. Usherwood, and M. J. Turk Induction of Postsurgical Tumor Immunity and T-Cell Memory by a Poorly Immunogenic Tumor Cancer Res., July 1, 2007; 67(13): 6468 - 6476. [Abstract] [Full Text] [PDF]
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 J. M. Curtsinger, M. Y. Gerner, D. C. Lins, and M. F. Mescher Signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor J. Immunol., June 1, 2007; 178(11): 6752 - 6760. [Abstract] [Full Text] [PDF]
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M. Grube, S. Moritz, E. C. Obermann, K. Rezvani, A. Mackensen, R. Andreesen, and E. Holler CD8+ T cells Reactive to Survivin Antigen in Patients with Multiple Myeloma Clin. Cancer Res., February 1, 2007; 13(3): 1053 - 1060. [Abstract] [Full Text] [PDF]
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A. M. Asemissen, U. Keilholz, S. Tenzer, M. Muller, S. Walter, S. Stevanovic, H. Schild, A. Letsch, E. Thiel, H.-G. Rammensee, et al. Identification of a Highly Immunogenic HLA-A*01-Binding T Cell Epitope of WT1 Clin. Cancer Res., December 15, 2006; 12(24): 7476 - 7482. [Abstract] [Full Text] [PDF]
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S. M. Walton, M. Gerlinger, O. de la Rosa, N. Nuber, A. Knights, A. Gati, M. Laumer, L. Strauss, C. Exner, N. Schafer, et al. Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients J. Immunol., December 1, 2006; 177(11): 8212 - 8218. [Abstract] [Full Text] [PDF]
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H. Takata and M. Takiguchi Three Memory Subsets of Human CD8+ T Cells Differently Expressing Three Cytolytic Effector Molecules J. Immunol., October 1, 2006; 177(7): 4330 - 4340. [Abstract] [Full Text] [PDF]
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D. E. Speiser, P. Baumgaertner, C. Barbey, V. Rubio-Godoy, A. Moulin, P. Corthesy, E. Devevre, P.-Y. Dietrich, D. Rimoldi, D. Lienard, et al. A Novel Approach to Characterize Clonality and Differentiation of Human Melanoma-Specific T Cell Responses: Spontaneous Priming and Efficient Boosting by Vaccination J. Immunol., July 15, 2006; 177(2): 1338 - 1348. [Abstract] [Full Text] [PDF]
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M. Griffioen, J. H. Kessler, M. Borghi, R. A. van Soest, C. E. van der Minne, J. Nouta, S. H. van der Burg, J. P. Medema, P. I. Schrier, J.H. F. Falkenburg, et al. Detection and Functional Analysis of CD8+ T Cells Specific for PRAME: a Target for T-Cell Therapy. Clin. Cancer Res., May 15, 2006; 12(10): 3130 - 3136. [Abstract] [Full Text] [PDF]
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U. Keilholz, P. Martus, and C. Scheibenbogen Immune monitoring of T-cell responses in cancer vaccine development. Clin. Cancer Res., April 1, 2006; 12(7): 2346s - 2352s. [Abstract] [Full Text] [PDF]
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B. Kubuschok, F. Neumann, R. Breit, M. Sester, C. Schormann, C. Wagner, U. Sester, F. Hartmann, M. Wagner, K. Remberger, et al. Naturally Occurring T-Cell Response against Mutated p21 Ras Oncoprotein in Pancreatic Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1365 - 1372. [Abstract] [Full Text] [PDF]
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V. Monsurro, E. Wang, Y. Yamano, S. A. Migueles, M. C. Panelli, K. Smith, D. Nagorsen, M. Connors, S. Jacobson, and F. M. Marincola Quiescent phenotype of tumor-specific CD8+ T cells following immunization Blood, October 1, 2004; 104(7): 1970 - 1978. [Abstract] [Full Text] [PDF]
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E. Xystrakis, P. Cavailles, A. S. Dejean, B. Cautain, C. Colacios, D. Lagrange, M.-J. van de Gaar, I. Bernard, D. Gonzalez-Dunia, J. Damoiseaux, et al. Functional and Genetic Analysis of Two CD8 T Cell Subsets Defined by the Level of CD45RC Expression in the Rat J. Immunol., September 1, 2004; 173(5): 3140 - 3147. [Abstract] [Full Text] [PDF]
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M. Di Nicola, C. Carlo-Stella, R. Mortarini, P. Baldassari, A. Guidetti, G. F. Gallino, M. Del Vecchio, F. Ravagnani, M. Magni, P. Chaplin, et al. Boosting T Cell-Mediated Immunity to Tyrosinase by Vaccinia Virus-Transduced, CD34+-Derived Dendritic Cell Vaccination: A Phase I Trial in Metastatic Melanoma Clin. Cancer Res., August 15, 2004; 10(16): 5381 - 5390. [Abstract] [Full Text] [PDF]
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D. Atanackovic, A. Block, A. de Weerth, C. Faltz, D. K. Hossfeld, and S. Hegewisch-Becker Characterization of Effusion-Infiltrating T Cells: Benign versus Malignant Effusions Clin. Cancer Res., April 15, 2004; 10(8): 2600 - 2608. [Abstract] [Full Text] [PDF]
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E. Mallard, F. Vernel-Pauillac, T. Velu, F. Lehmann, J.-P. Abastado, M. Salcedo, and N. Bercovici IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity J. Immunol., March 15, 2004; 172(6): 3963 - 3970. [Abstract] [Full Text] [PDF]
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R. H. Vonderheide, S. M. Domchek, J. L. Schultze, D. J. George, K. M. Hoar, D.-Y. Chen, K. F. Stephans, K. Masutomi, M. Loda, Z. Xia, et al. Vaccination of Cancer Patients Against Telomerase Induces Functional Antitumor CD8+ T Lymphocytes Clin. Cancer Res., February 1, 2004; 10(3): 828 - 839. [Abstract] [Full Text] [PDF]
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E. B. Walker, D. Haley, W. Miller, K. Floyd, K. P. Wisner, N. Sanjuan, H. Maecker, P. Romero, H.-M. Hu, W. G. Alvord, et al. gp100209-2M Peptide Immunization of Human Lymphocyte Antigen-A2+ Stage I-III Melanoma Patients Induces Significant Increase in Antigen-Specific Effector and Long-Term Memory CD8+ T Cells Clin. Cancer Res., January 15, 2004; 10(2): 668 - 680. [Abstract] [Full Text] [PDF]
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A. Gati, N. Guerra, C. Gaudin, S. Da Rocha, B. Escudier, Y. Lecluse, A. Bettaieb, S. Chouaib, and A. Caignard CD158 Receptor Controls Cytotoxic T-Lymphocyte Susceptibility to Tumor-Mediated Activation-Induced Cell Death by Interfering with Fas Signaling Cancer Res., November 1, 2003; 63(21): 7475 - 7482. [Abstract] [Full Text] [PDF]
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L. Rivoltini, C. Castelli, M. Carrabba, V. Mazzaferro, L. Pilla, V. Huber, J. Coppa, G. Gallino, C. Scheibenbogen, P. Squarcina, et al. Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells J. Immunol., October 1, 2003; 171(7): 3467 - 3474. [Abstract] [Full Text] [PDF]
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D. Nagorsen, C. Scheibenbogen, F. M. Marincola, A. Letsch, and U. Keilholz Natural T Cell Immunity against Cancer Clin. Cancer Res., October 1, 2003; 9(12): 4296 - 4303. [Abstract] [Full Text] [PDF]
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A. Letsch, U. Keilholz, G. Assfalg, V. Mailander, E. Thiel, and C. Scheibenbogen Bone Marrow Contains Melanoma-reactive CD8+ Effector T Cells and, Compared with Peripheral Blood, Enriched Numbers of Melanoma-reactive CD8+ Memory T Cells Cancer Res., September 1, 2003; 63(17): 5582 - 5586. [Abstract] [Full Text] [PDF]
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M. Ayyoub, D. Rimoldi, P. Guillaume, P. Romero, J.-C. Cerottini, D. Valmori, and D. Speiser Tumor-reactive, SSX-2-specific CD8+ T Cells Are Selectively Expanded during Immune Responses to Antigen-expressing Tumors in Melanoma Patients Cancer Res., September 1, 2003; 63(17): 5601 - 5606. [Abstract] [Full Text] [PDF]
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A. Anichini, A. Scarito, A. Molla, G. Parmiani, and R. Mortarini Differentiation of CD8+ T Cells from Tumor-Invaded and Tumor-Free Lymph Nodes of Melanoma Patients: Role of Common {gamma}-Chain Cytokines J. Immunol., August 15, 2003; 171(4): 2134 - 2141. [Abstract] [Full Text] [PDF]
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V. Mazzaferro, J. Coppa, M. G. Carrabba, L. Rivoltini, M. Schiavo, E. Regalia, L. Mariani, T. Camerini, A. Marchiano, S. Andreola, et al. Vaccination with Autologous Tumor-derived Heat-Shock Protein Gp96 after Liver Resection for Metastatic Colorectal Cancer Clin. Cancer Res., August 1, 2003; 9(9): 3235 - 3245. [Abstract] [Full Text] [PDF]
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D. Valmori, M. Ayyoub, C. S. Hesdorffer, U. Keilholz, and C. Scheibenbogen A monoclonal melanoma-specific T-cell population phenotypically indistinguishable from CD3+ LGL-leukemia Blood, June 1, 2003; 101(11): 4643 - 4644. [Full Text] [PDF]
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C. Scheibenbogen, A. Letsch, E. Thiel, A. Schmittel, V. Mailaender, S. Baerwolf, D. Nagorsen, and U. Keilholz CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia Blood, August 28, 2002; 100(6): 2132 - 2137. [Abstract] [Full Text] [PDF]
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