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PD-1 expression on Melan-A-reactive T cells increases during progression to metastatic disease†
Article first published online: 11 JAN 2012
Copyright © 2011 UICC
International Journal of Cancer
Volume 130, Issue 10, pages 2327–2336, 15 May 2012
How to Cite
Krönig, H., Julia Falchner, K., Odendahl, M., Brackertz, B., Conrad, H., Muck, D., Hein, R., Blank, C., Peschel, C., Haller, B., Schulz, S. and Bernhard, H. (2012), PD-1 expression on Melan-A-reactive T cells increases during progression to metastatic disease. Int. J. Cancer, 130: 2327–2336. doi: 10.1002/ijc.26272
This article was published online on 11 January 2012. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected on 9th February 2012.
Tel.: +49-89-4140-6223, Fax: +49-89-4140-4854
- Issue published online: 16 MAR 2012
- Article first published online: 11 JAN 2012
- Accepted manuscript online: 29 JUN 2011 09:58AM EST
- Manuscript Accepted: 6 JUN 2011
- Manuscript Received: 20 JAN 2011
- Research Council of Germany. Grant Numbers: SFB 456, DFG BE 1579/4-1
- Helmholtz Alliance (Immunotherapy of Cancer)
- malignant melanoma;
Programmed death 1 (PD-1) is known as an important factor for the development of tolerogenicity. This has been proven in chronic viral infections and different tumor models. To address the role of PD-1 and its ligand programmed death ligand 1 (PD-L1) in different stages of malignant melanoma, we investigated peripheral blood and tumor tissues in regard to overall survival (OS) and prognostic relevance. One hundred samples of peripheral blood mononuclear cells from HLA-A2+ patients with malignant melanoma (Stages I–IV) were analyzed in seven color FACS combined with multimer analyses for the immunodominant epitope of Melan-A (peptide A2/Melan-Ap26-35mod). Corresponding formalin-fixed paraffin-embedded tissues of primary tumor and distant organ metastases from 37 cases were analyzed by immunohistochemistry for Melan-A, PD-L1 and PD-1 expression. Compared to the total CD8+ T cell population, PD-1 expression by A2/Melan-A+ CD8+ T cells was over-represented in melanoma stages III and IV (p < 0.001). Although elevation of PD-1+ Melan-A+ CD8+ T cells had no significant influence on OS, a positive correlation was observed between PD-L1 expression on melanoma cells and OS (p = 0.05). Correlation of advanced tumor stage with increased A2/Melan-A-multimer+ PD-1+ T cells in the peripheral blood suggest that blocking of PD-1 could have therapeutic potential in advanced stage melanoma.
Among the numerous mechanisms of tumor-induced immunosuppression that cause resistance of tumors to cytotoxic T-lymphocyte (CTL) responses,1 a number of experimental animal studies2, 3 and in vitro experiments4 have suggested an important role of programmed death 1 (PD-1)/ programmed death ligand 1 (PD-L1/B7-H1) interactions in inhibiting the effector functions of tumor-specific CD8+ T cells. PD-1 is expressed by activated T and B cells5, 6 and binds to the ligands PD-L1 (B7-H1)7 and PD-L2 (B7-DC) that are widely expressed not only on cells such as macrophages, dendritic cells (DCs), T and B cells but also on many solid tumors.8 Recently, Fourcade et al. showed that combined upregulation of PD-1+ and Tim-3+ expression in advanced stage melanoma is associated with tumor antigen-specific CD8+ cell dysfunction.9
Virus-specific CD8+ T cells in mice chronically infected with lymphocytic choriomeningitis virus, HIV, hepatitis B or hepatitis C exhibit a reduced capability to proliferate and produce effector cytokines. These functionally “exhausted” T cells have been shown to upregulate PD-1. These preclinial studies indicated that PD-1 expression on virus-specific CD8+ T cells could inhibit the effectiveness of an immune response that was required to control a viral infection. Blockade of the PD-1/PD-L1 pathway led to increased cytokine production (TNF-α and IFN-γ) and proliferation capacity, resulting in a significant reduction of the viral load.10–15
Induction and expression of PD-1 on antigen-specific T cells are thought to regulate the immune responses not only for viral antigens but also for self antigens.6 The absence of PD-1 in a PDcd1−/− mouse model was associated with markedly improved tumor rejection using different melanoma cell lines.2 In this model, PD-1-deficient T cells caused tumor rejection in a setting in which even CTLA-4-deficient T cells failed.
One of the ligands of PD-1 is PD-L1 that is expressed by many solid tumors as for instance melanoma,16 breast cancer,17 ovarian cancer,18 renal cell carcinoma,19 lung cancer16 and even in hematological diseases like chronic myeloid leukaemia.20 Although increased PD-L1 expression on RCC significantly correlated with unfavorable prognosis,19–21 other malignancies showed differing expression patterns.22, 23
Previous studies on stage IV melanomas showed upregulation of PD-1 expression on ex vivo detectable CD8+ T cells.24, 25 To further clarify the role of PD-1/PD-L1 interactions in preventing CD8+ T cell responses against melanomas, we investigated PD-1 expression on CD8+ T cells in peripheral blood, which were specific for the immunodominant epitope of Melan-A/MART-1 (peptide A2/Melan-Ap26-35mod/ sequence ELAGIGILTV) in multimer stainings. We preferred the Melan-Ap26-35mod peptide analog because of its increased immunogenicity and the advantageous monitoring of Melan-A-specific CTLs in the peripheral blood of melanoma patients.26 In the collection of 100 HLA-A2+ patients, we found a significant increase of A2/Melan-A+ CD8+ T cells in the advanced tumor stages III and IV. Strikingly, PD-1 expression was upregulated on these cells. Whereas no correlation between PD-1 expression on A2/Melan-A+ CD8+ T cells in peripheral blood and PD-L1 expression on tumor cells was observed, there was a tendency to higher expression levels of PD-1 on A2/Melan-A+ CD8+ T cells in peripheral blood and PD-1 expression on tumor infiltrating lymphocytes (TILs). PD-1 expression on T cells did not show a significant influence on overall survival (OS), whereas statistical analyses revealed an association between OS and PD-L1 expression on melanoma cells.
Material and Methods
Patients, PBMC and tumor samples
Tumor tissues and peripheral blood samples were collected from HLA-A2+ patients with different histopathological subtypes of malignant melanoma in all four stages of disease from 2001 and 2007 at the department of hematology/oncology and the department of dermatology at the Klinikum Rechts der Isar, Technical University of Munich. The study was approved by the ethics committee for human research (Technical University of Munich) and was conducted in accordance with the precepts established by the Helsinki declaration. Peripheral blood mononuclear cells (PBMC) from patients with HLA-A2+ malignant melanoma (n = 100) were prepared over Ficoll-Hypaque (Biochrom GmbH, Berlin) gradient and cryopreserved until analyzed. Melanoma tissue specimen was collected from 37 primary tumors and 32 lymph nodes. All tumors were revised by the in-house pathologist according to the American Joint Committee (AJCC) staging system. Each tissue sample was fixed in 3,7-buffered formaldehyde and embedded in paraffin (FFPE material).
Flow cytometric analysis
The following monoclonal antibodies (mAbs) specific for human antigens were used: anti-human CD3 Pacific Blue (CD3-PB), anti-human CD8 APC (Dako, Glostrup, Denmark); anti-human CD8 PE A610, anti-human CD8 PE (Caltag/Invitrogen GmbH, Karlsruhe, Germany); anti-human CD8 PerCP, anti-human CD45RA PE Cy7 (BDPharmingen, Franklin Lakes, USA); anti-human CCR7(CD197) Fitc (R&D Systems, Minneapolis, USA); anti-human PD-1 APC (eBioscience, San Diego, USA) and the A2/Melan-Ap26-35-multimer-PE (A2/Melan-A+). Synthesis of PE-labeled HLA-A2/peptide multimer complexes and flow cytometric analyses were performed based on previous published methods.27, 28 For the detection of Melan-A26-35-specific T cells, the multimer consisting of the Melan-A26-35A27L analog (ELAGIGILTV)25 was used, which has a higher binding stability to the HLA-A2 and a higher immunogenicity than the natural Melan-A decapeptide (EAAGIGILTV).29 In brief, cells were resuspended in staining buffer (PBS containing 3% FBS) and blocked with mouse IgG mAbs (Caltag Labs) for 15–30 min at room temperature (RT). Cells were stained with mAbs against surface antigens for 30 min at 4°C in the dark. Cells were analyzed on the flow cytometer CyAn ADP (Dako), and data were analyzed using FlowJo software (Treestar, San Carlos, CA). The quadrants were set based on isotype control antibody staining, and the number in each quadrant represents the percentage of cells.
Immunohistochemical stainings for Melan-A/MART-1 Ab-3 Cocktail (Thermo Fisher Scientific, Fermont, USA), PD-L1 (rabit polyclonal Ab, ProSci, Poway, USA) and PD-1 were performed by using standardized protocols for paraffin embedded tissues. Tissue sections were cut into 3-μm-thick sections and dried at 70°C for 30 min. The sections were then deparaffinized in xylene and dehydrated through graded alcohols to water. Antigen retrieval was achieved by boiling the sections in citrate buffer, pH 6.0, using a microwave. Endogenous peroxidase activity was blocked by incubating the sections with peroxidase blocking reagent (DakoCytomation, Glostrup, Denmark) for 10 min. To reduce nonspecific binding, sections were incubated for 30 min at RT with 5% normal goat serum (Sanquin, Amsterdam, The Netherlands). Primary antibody (Ab) incubation was performed at RT in a humidified chamber. After PBS washing steps, the sections were incubated with a biotinylated secondary goat-anti-mouse or anti-rabbit secondary antibody (ImmunoVision Technologies, Brisbane, USA) for 30 min at RT. PD-L1 and Melan-A detection was performed using a streptavidin-alcalic phosphatase conjugate (30 min at RT) followed by chromogen red incubation (5 min at RT). PD-1 was detected by using a DAB substrate chromogen system (DakoCytomation), following preincubation with a streptavidin-horse radish peroxidase conjugate (30 min at RT). After counterstaining of the nuclei in hematoxylin (10 min), slides were permanently mounted with coverslips.
Validation of anti-human-PD-L1 specificity was performed by titrating the antibodies until the desired staining intensity was obtained as compared to an isotype control. Additional tests verified antibody specificity by preincubating anti-PD-L1 Ab with recombinant human PD-L1/FC chimera (R&D Systems, Minneapolis, USA).
Analysis of stained sections
All sections were scored in brightfield mode in 400× magnification (Fa. Olympus, BX40). On primary melanomas, PD-L1 expression was analyzed at the border of invasion, where signals were usually the strongest. Samples stained with anti PD-L1 (B7-H1) were scored in a three-tiered system according to the staining intensity, based on a publication of Hamanishi et al.18: Grade 3 represented strong expression, Grade 2 moderate, Grade 1 weak and Grade 0 no expression, in comparison to external controls. Cases with Grades 3 and 2 were summarized in the high expression group and cases with Grade 1 or 0 were defined as the low expression group. The same evaluation was performed for Melan-A. Infiltrating T lymphocytes (PD-1) were counted in absolute numbers for ten representative high power fields (labeled T cells per ten HPF).
All patients diagnosed with malignant melanoma in our hospital between 2001 and 2007 are included in this retrospective study. With 100 patients analyzed (70 with tumor stage I or II and 30 with stage III or IV), the study was sufficiently powered (about 90%) to detect a significant difference between both groups performing a Mann–Whitney U test on a two-sided level of significance of α = 5% if the probability for a higher value in one group compared to the other was 70% or higher.30 This translates to an effect size under normality of about 0.75 (difference in means divided by the common standard deviation).
Quantitative data are described by mean and standard deviation; qualitative data by absolute and relative frequencies. For the comparison of quantitative data between two groups, the Mann–Whitney U test was conducted. Quantitative measurements in the same subjects were investigated using the Wilcoxon signed-rank test. Fisher's exact test was performed to compare frequencies of binary data between two groups. Spearman's rank correlation coefficient was estimated to analyze the association between two quantitative measures. Survival curves were estimated for relevant groups by Kaplan–Meier method and compared by using the log-rank test. Survival time is defined as time of surgery to time of death. All tests were performed on a two-sided level of significance of α = 0.05. Since all tests were conducted in an explorative manner, level of significance was not adjusted for multiple comparisons. ***Statistical software R31 and PASW32 were used for statistical analysis.
Clinical patient profiles
The collection consisted of 100 HLA-A2+ patients (ratio of men to women 55:45) with an average patient age of 61.4 years (standard deviation 12.5 years). Thirty-six patients were diagnosed with nodular melanoma, 36 with superficial spreading melanoma, seven patients with acral lentiginous melanoma, one with lentigo malignant melanoma and 20 patients had malignant melanoma, which was not further specifiable. According to the AJCC version 7 staging system,33 12 patients (12%) had stage IA melanoma, 32 patients (32%) had stage IB melanoma, 18 patients (18%) had stage IIA melanoma, eight patients (8%) had stage IIB melanoma, 17 patients (17%) had stage IIIA melanoma, two patients (2%) had stage IIIC melanoma and 11 patients (11%) had stage IV melanoma. Lymph node metastases were observed in 20 patients.
PD-1 expression by CD8+ T cells in stages III and IV melanoma patients
We evaluated the HLA-A2/Melan-A specificity of CD3+ CD8+ T cells in all four stages of malignant melanoma by multimer staining. Representative flow cytometric data are shown in Figure 1. A significant increase of A2/Melan-A+ T cells was observed in the metastatic melanoma stages III and IV as compared to the local melanoma stages I and II (p < 0.001; Fig. 2a). We also observed a significant increase in PD-1 expression on the A2/Melan-A+ CD8+ T cell subpopulation in melanoma stages III and IV as compared to stages I and II (p = 0.035; (Fig. 2b). Even in the local melanoma stages I and II an elevated expression of PD-1 was observed on A2/Melan-A+ CD8+ T cells, but the effect was not statistically significant (p = 0.064). Importantly, a relevant difference of A2/Melan-A+ CD8+ T cells was observed in the metastatic stages III and IV (p < 0.001; Fig. 2c). The increased expression of PD-1 on A2/Melan-A+ CD8+ T cells had no significant effect on OS (Fig. 2d).
To investigate the potential association between CD8+ T cell differentiation/activation status, PD-1 expression and tumor stage, we compared the percentages of CD8+ T cells that express the surface markers CCR7 and CD45RA. There were no correlations between naïve (CD45RA+ CCR7+), effector (CD45RA+ CCR7−), central memory (CD45RA− CCR7+) and effector memory CD8+ T cells (CD45RA− CCR7−) and PD-1 expression and tumor stage (data not shown). There was a tendency of higher frequencies of effector memory RA+ T cells in all tumor stages than of the other CD8+ T cell phenotypes.
Melan-A and PD-L1 expression of primary melanomas
Primary tumor samples from 37 melanoma patients were stained immunohistochemically for Melan-A and PD-L1. Expression levels of both antigens were separated in low and high, with Grades 3 and 2 representing high expression and cases with Grade 1 or 0 representing low expression. There were 22 patients in the low expression group and 15 patients in the high expression group. Within primary tumors PD-L1 and Melan-A expression was usually strongest at the border of invasion, while centers were mostly weak to negative. Representative histopathological photographs are provided in Figure 3.
Survival times were significantly associated with tumor stage (p = 0.001) showing shorter survival times in higher tumor stages (Fig. 5a). According to Kaplan–Meier survival analyses and log-rank tests, there was a tendency to shorter survival times in the “low expression” than in the “high expression” (p = 0.05; Fig. 5b).
PD-1 expression of tumor infiltrating CD8+ T cells
The primary tumor samples from 37 melanoma patients were also stained immunohistochemically for PD-1 to investigate the PD-1 expression on TILs. There was no significant correlation between Melan-A-multimer+ PD-1+ CD8+ T cells and PD-L1 low and high expression on tumor cells (p = 0.31; Fig. 6a). Interestingly, highest frequencies of Melan-A-multimer+ PD-1+ CD8+ T cells in the blood were only found in cases that showed PD1+ TILs (p = 0.056; Fig. 6b). In contrast, cases that were negative for PD1+ infiltrating lymphocytes, were associated with frequencies below 50%, concerning Melan-A-multimer+ PD-1+ CD8+ T cells in the blood. PD-1 expression on tumor infiltrating CD8+ T cells though had no significant influence on OS (Fig. 6c).
The main focus of our studies was to characterize circulating A2/Melan-A+ CD8+ T cells from the blood of melanoma patients. Melan-A is enhanced in patients with HLA-A*0201-expressing melanoma in contrast to healthy individuals.34, 35 It was shown to be comparable with reactivity toward common pathogens like the Flu Matrix protein HLA-A*0201-associated epitope (FluM158-66).35 Although other hypotheses have been raised to explain the relative ease of identifying A2/Melan-A+ circulating T cells,36 it was reasonable to postulate that this phenomenon relates to the tumor microenvironment. We performed flow cytometric analysis on 100 HLA-A2+ melanoma patients, to analyze the role of tumor stages on the development of circulating A2/Melan-A+ T cells in the blood. Interestingly, A2/Melan-A+ CD8+ T cells increased significantly in metastasized melanoma stages (III/IV) as compared to locally confined tumor stages (I/II). A possible explanation might be an increasing concentration of Melan-A in the microenvironment during tumor progression.
The discrepancy between increased Melan-A-specific T cells in melanoma patients,37–39 and the low clinical response rate of malignant melanoma patients40–42 could be attributed to inhibitory mechanisms of the tumor microenvironment. We showed a significantly higher percentage of PD-1 expressing A2/Melan-A+ CD8+ T cells in the metastasized tumor stages III and IV as compared to the local tumor stages I and II. This finding indicates that tumor-antigen-specific immune activation is associated with increased inhibitory receptor expression on T cells. This is also supported by the observation that the population of PD-1+ HLA-A2/Melan-A multimer-negative CD8+ T cells showed no changes in relation to the tumor stage. Surprisingly, the increased expression of PD-1 on A2/Melan-A+ CD8+ T cells had no significant effect on OS. Possible explanations could be compensating immune mechanisms such as regulatory T cell-mediated T effector inhibition, TGF-ß, IL-10 and FAS-L overexpression, IDO induction, myeloid derived suppressor cells and stromal protection.43–46
In a recently published study Ahmadzadeh et al. detected lower frequencies of PD-1+ A2/Melan-A+ CD8+ T cells in the peripheral blood of patients with metastatic melanoma as compared to our population.47 However, the results of Ahmadzadeh et al. are based on a small patient population of three patients whereas our group analyzed 31 patients with malignant melanoma in the metastatic stages III and IV. Moreover, Ahmadzadeh et al. showed a significantly higher percentage of PD-1 expression on tumor-infiltrating A2/Melan-A+ CD8+ T cells compared to A2/Melan-A+ CD8+ T cells in the peripheral blood.
Both cell populations showed a reduced ability of IFN-γ production, which was even more pronounced in tumor-infiltrating cells with high PD-1 expression. Regarding our results, the extent of PD-1 expression in the advanced tumor stages III and IV should result in a significantly stronger functional impairment of PD-1+ A2/Melan-A+ CD8+ T cells in these stages compared to the local tumor stages I and II. This would be consistent with the poor prognosis in advanced tumor stages of malignant melanoma.
Furthermore, we investigated the expression of PD-L1 in tumor tissues from 37 melanoma patients. There was a tendency to higher PD-L1 expression in the tumor stages III and IV consistent with significantly higher PD-1 levels on CD8+ T cells in the peripheral blood. Interestingly, Kaplan–Meier analyses yielded a tendency toward longer survival times in patients that showed higher PD-L1 on their tumors. This finding was contrary to our own hypothesis and to a recent published study of Hino et al.25. This study reported high PD-L1 expression as an independent poor prognostic factor in melanoma patients. Similar results concerning T-cell exhaustion and disease progression were found in studies in renal cell carcinoma and chronic myeloid leukemia.18, 19
Our data are in line with the results of another group, which independently observed a trend toward a positive prognostic correlation of PD-L1 in stages III and IV melanoma patients.48 Although the latter group used the same antibody clone for PD-L1 detection as in our study, Hino et al. used another clone (clone 27A2; MBL; Nagoya, Japan) for PD-L1 detection.25 It is therefore possible that these contradictory findings are due to different antibodies, different techniques in readout or differences in the embedding process.
Recent publications indicate that PD-L1-mediated inhibition not only occurs during interaction of effector T cells and tumor cells but also during APC-T cell interaction and regulatory T cell expansion. In addition, PD-L1 expression was also described on monocytes and myeloid derived suppressor cells.49, 50 Our observation of PD-L1 expression mainly at the invasion front of melanomas might be a morphologic correlate of a preexisting inflammatory tumor environment with interferon-γ production. This could explain the improved prognosis, which we observed in cases with strong PD-L1 expression.2, 5 Interestingly, in patients that did not express PD1 on TILs, frequencies of PD-1+ A2/Melan-A+ CD8+ T cells in the peripheral blood were below 50%. In contrast, frequencies of PD-1+ A2/Melan-A+ CD8+ T cells in the blood above 50% were only found in melanomas that showed PD-1+ TILs. This finding suggests that the tumor microenvironment plays a pivotal role during induction of PD-1 on T cells that infiltrate or surround melanomas. Supporting our observation, Karim et al. recently described a group of PD-L1 positive cervical carcinomas with excess of infiltrating regulatory T cells. This group interestingly displayed an improved survival rate.51In vitro experiments of Karim et al. also showed that PD-1+ regulatory T cells become inactivated during interaction with PD-L1+ tumor cells. This in turn enabled development of an inflammatory reaction.
In conclusion, the elevated frequencies of PD-1+ CD8+ A2/Melan-A+ T cells in the blood of advanced stage melanoma patients suggest an increased immune dysfunction, but we could not observe any prognostic relevance of the PD-1/PD-L1 interaction when analyzing the tumor cell–T cell interaction, possibly due to the small sample size analyzed. However, PD-L1/PD-1 signaling may still be prognostically relevant when analyzing the involvement of other cellular compartments like the T cell–APC interactions. Targeting tumor-induced immune escape mechanisms will stimulate the tumor-specific immune responses and thereby support therapeutic approaches to induce T cell immunity, such as active immunization with cancer vaccines and infusion of competent T cells via adoptive T cell treatment.52
The authors thank Prof. Dirk H. Busch for HLA/peptide multimer production and Klara Fizi and Kathrin Hofer for excellent technical assistance. Additional support was provided by DFG grant SCHU 1518/2-1 and the Anton und Petra Ehrmann Stiftung (APES)
- 9Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients. J Exp Med 2010;207: 2175–86., , , , , , , , .
- 30Sample size determination for some common nonparametric tests. JASA 1987; 82: 645–7..
- 31R Development Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing, 2009.
- 37An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions. J Exp Med 1999; 190: 651–67., , , , , , , , , , .