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Immunology |
1 Immunology/Institut National de la Santé et de la Recherche Médicale ERM0208 and Immunotherapy Unit, Department of Pathology, Dermatology Unit, Institut Gustave-Roussy, Villejuif, France; 2 Laboratory of Immunological Research, SPRI, Schering-Plough, Dardilly, France; 3 Division of Clinical OncoImmunology, Ludwig Institute for Cancer Research, Lausanne Branch, and Department of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 4 Institut National de la Santé et de la Recherche Médicale U277, Institut Pasteur, Paris, France
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Mature dendritic cells (DCs) are the most potent antigen-presenting cells (5) capable of priming tumor-specific T cells, and their use in cancer immunotherapy appears to be a promising way to elicit and expand efficient antitumor immune responses (6 , 7) . However, little is known about the role of DCs in the natural immunosurveillance of cancer. It is generally postulated that spontaneous immunity against tumor antigens is elicited by DCs maturing in response to various inflammatory signals, but the sites and conditions in which DCs capture and/or encounter tumor antigens remain largely unknown. Indeed, most studies have addressed this question by use of nonspecific antibodies (8) , whereas other reports describe the localization and phenotypes of DCs in tumor samples without clinical correlates (9) .
Melanoma has become a paradigm for studying the interactions between tumor and host immune cells for the following reasons. First, the primary lesion occurs in the skin, the main residency compartment for Langerhans cells, which have high potential to migrate to skin-draining lymph nodes (LNs; Refs. 10 , 11 ). Second, sentinel LNs (SLNs) are privileged sites of both T-cell priming and initial metastases, the extent of SLN involvement representing an independent predictive factor for survival (12 , 13) . Third, cutaneous and LN metastases appear to be the tumor sites most sensitive to vaccine-based immunotherapies (6 , 14) .
Using a panel of antibodies recognizing DC subsets and maturation markers, we addressed the clinical relevance of DC infiltration on regressing tumor lesions and SLNs in melanoma patients. Here we demonstrate, in a case report involving spontaneous regression of metastatic melanoma, dramatic infiltration of DC-Lamp+ DCs in close contact with effector memory Melan-A/Mart-1-specific CTLs in situ. In addition, in a series of 19 metastatic SLNs, we also show that the extent of DC-Lamp+ DC infiltration is significantly associated with a high incidence of tumor-free status after lymphadenectomy and a favorable clinical outcome. Altogether, our results strongly suggest that mature DCs play a key role in the local expansion and differentiation of protective tumor-specific immune responses at the first sites of melanoma metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Case Report
The patient was a 28-year-old male with an atypical nevus syndrome who in December 2000 developed a right axillar lymphadenopathy corresponding to a melanoma metastasis. The patient reported in the previous months a pigmented nodular lesion in the right arm that had regressed spontaneously. This residual lesion was removed, and a lymphadenectomy in the right axillar basin was performed. Histologically, the cutaneous lesion displayed features typical of regression: absence of associated melanoma cells, a flat epidermis and a fibrotic dermis containing ectasic vessels, heterogeneous melanin pigmentation, and a dense lymphocytic infiltrate. The lymphadenectomy revealed 8 metastatic LNs from a total of 24. After a 10-month disease-free period, a right subclavicular mass developed that corresponded, at surgical examination, to a LN 2 cm in diameter. Histologically, this LN was free of tumor cells and appeared almost completely necrotic with extensive peripheral fibrosis. LN samples were kept frozen for further analyses, and the primary cutaneous site was paraffin-embedded only. This patient remains disease-free 34 months after diagnosis without any adjuvant therapy.
Series of 19 Melanoma-Positive SLNs
All patients underwent surgical SLN resection at the time of or a few weeks after excision of the primary cutaneous melanoma, according to the recommended procedures (12)
. For this study, we selected a consecutive series of 19 patients with a melanoma-positive SLNs followed in our institution between June 1999 and March 2002. A few weeks after the SLN procedure, all underwent a regional completion lymphadenectomy to detect metastatic extensions in non-SLNs. The materials used consisted of paraffin-embedded sections from recorded paired SLN and non-SLN samples.
Characterization of Melan-A/Mart-1-Specific CTLs from Peripheral Blood
Immunophenotyping Using Phycoerythrin (PE)-Labeled HLA-A*0201/Peptide Tetramer Complexes.
HLA-A*0201/Melan-A/Mart-126–35 A27L analog (ELAGIGILTV) and control HLA-A*0201/HIVgag77–85 (SLYNTVATL) were synthesized as described previously (15)
. Total peripheral blood mononuclear cells or immunopurified CD8+ T cells, obtained after magnetic cell sorting with a MiniMACS device (CD8+ T Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany), were stained with the PE-labeled tetramers (5 nM) at room temperature for 45 min in PBS containing 2% BSA, washed once, stained for cell surface differentiation antigens with monoclonal antibodies (mAbs) for 30 min at 4°C, washed again, and analyzed by flow cytometry. Conjugated anti-CD3, -CD8, and -CD45RA mAbs were purchased from BD Biosciences (Le Pont de Claix, France), and anti-TCRVß2 and -TCRVß3 were purchased from Beckman Coulter (Villepinte, France). Samples were also incubated with anti-CCR7 rat IgG2a mAb 3D12 (kindly provided by M. Lipp, Berlin, Germany) for 30 min at 4°C, washed, and additionally stained with an antigen-presenting-cell-labeled goat antirat IgM+IgG (Southern Biotechnology, Birmingham, AL). Fluorescent isotype-matched mAbs were used as controls. Direct ex vivo analysis was performed after acquisition of >106 events on a FACSCalibur (BD Biosciences) with Cellquest software.
Sorting, Cloning, Expansion, and Characterization of Anti-Melan-A/Mart-1 CTLs.
CD3+CD8+HLA-A*0201/Melan-A/Mart-126–35+ cells were sorted by flow cytometry with FACS Vantage (BD Biosciences) and cultured in V-bottomed 96-well plates either in bulk or after direct cloning in limiting dilution on feeder cells consisting in irradiated allogeneic peripheral blood mononuclear cells, EBV-transformed LAZ509 B cells, and MEL-FON tumor cells, a HLA-A2+ melanoma tumor cell line expressing the Melan-A/Mart-1 antigen (15)
. Growing clonal colonies were expanded at 37°C and 5% CO2 in RPMI 1640 (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 10% human AB serum (Institut Jacques Boy, Reims, France), 50 IU/ml recombinant human interleukin-2 (Proleukin; Chiron, Rattingen, Germany), and 3% T-cell growth factor, a supernatant from phytohemagglutinin/phorbol 12-myristate 13-acetate-allostimulated T cells. T-cell clones were characterized for immunophenotype by use of HLA-A*0201/Melan-A/Mart-126–35 tetramers and mAbs directed against CD3 and CD8 and by TCR-Vß repertoire analysis with a panel of 21 anti-BV mAbs (IOTest ß Mark; Beckman Coulter Immunotech, Marseille, France), and for cytotoxic activity in a standard 4-h 51Cr-release assay against MEL-FON tumor cells and Transporter associated with Antigen Processing (TAP)-deficient EBV-transformed T2 cells loaded with 10 µg/ml Melan-A/Mart-126–35 synthetic peptide. IFN-
secretion in response to the same targets was also assessed by use of the high-affinity capture matrix assay according to the manufacturer’s instructions (IFN- secretion assay; Miltenyi Biotec).
In Situ Tetramer Staining Analyses
Tissue samples were snap-frozen immediately after collection and cryopreserved in liquid nitrogen until use. Serial multiple 10-µm cryostat tissue sections were used for in situ tetramer staining. To block nonspecific binding sites, tissue sections were first recovered with 200 µl of 1x PBS containing 0.5% BSA and 10% FCS for 15 min at room temperature. After elimination of the buffer, PE-labeled HLA-A*0201/Melan-A/Mart-126–35 or peptide-irrelevant tetramer complexes were incubated at 2.6 µg/ml for 45 min at room temperature in a dark humidified atmosphere. The sections were washed with PBS and incubated with direct fluorescence or biotin-conjugated mAbs (anti-CD3, -CD8, -TCRVß2, and -TCRVß3) for an additional 30 min at 4°C. For triple staining, the biotin-coupled antibody was detected with streptavidin-Cy5.5 conjugate. After washing, sections were fixed with PBS-buffered 1% formaldehyde for 15 min at room temperature, mounted in antifading medium, and covered with a coverslip. Slide examination and micrography were performed with a fluorescence microscope (DMRXA; Leica, Weitzlar, Germany), and/or slides were evaluated with a Leica SP1 confocal microscopy system equipped with an Ar/Kr laser for the 488 and 568 nm lines and a HeNe633 laser (Leica Laser Technik; Mannheim, Germany) to excite the Cy5.5 label. Simultaneous acquisition of signals for the FITC-, PE-, and Cy5.5-labeled antibodies was performed at low illuminating laser power and with spectrophotometer-based separation of the fluorescence emissions. The fields of interest were selected on the basis of CD8- or TCRVß2-FITC-labeled infiltrates to avoid bias in scoring of tetramer-PE staining before confocal image acquisition.
Immunohistochemistry of Infiltrating T Cells and Antigen- Presenting Cells
Sections 4-µm thick were prepared on glass slides from paraffin-embedded formaldehyde-fixed material and were deparaffinized and rehydrated. For all antibodies except for anti-CD123, -CCR7, and -CD68, slides were pretreated in sodium citrate at 68°C (pH 6) for 20 min. After a blocking step (Blocking agent, Ultra-tech HRP kit; Beckman-Coulter Immunotech), slides were incubated with the primary antibodies at the following dilutions: 1:1 for anti-CD1a and anti-CD68 (Immunotech); 1:20 for anti-MIP3
/CCL20 (Schering-Plough, Dardilly, France); 1:50 for anti-CD3, anti-CD4 (Dako, Trappes, France), and anti-MIP3ß/CCL21 (R&D Systems, Abingdon, United Kingdom); 1:60 for anti-p55 (Dako); 1:100 for anti-CD8 (Dako) and TIA-1 (Beckman Coulter); 1:200 for anti-Langerin (Schering-Plough) and anti-CCR7 (BD PharMingen); 1:300 for anti-DC Lamp and anti-CD123 (Schering-Plough); and 1:500 for anti-CCR6 and anti-6CKine/CCL19 (R&D Systems) for 1 h at room temperature, followed by incubation with secondary antibodies and detection (Ultra-tech HRP kit). The slides were examined by two independent observers. Semiquantitative evaluation of DC-Lamp+ DC infiltration was made as follow: (a) the slide was examined at intermediate magnification (x100) to select the three areas containing the maximum number of DC-Lamp+ DCs; (b) the number of DC-Lamp+ cells per mm2 was then counted at high magnification (x400) in 3-mm2 sections per hot spot; (c) the final count used for DC-Lamp quantitative evaluation was the average number of the nine countings made on the slide. Statistical analyses comparing the groups according to DC-Lamp+ DC infiltration used Fisher’s exact method, and statistically significant differences were given at 95% confidence.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, direct ex vivo staining of peripheral CD3+CD8+ T lymphocytes with HLA-A*0201/Mart-126–35 tetramers revealed 0.15% specific T lymphocytes, which produced four tetramer+ T-cell clones. Two expressed TCR-Vß2+ with specificities for BV2J2.6 and BV2J2.7 gene segments, one expressed Vß3+ (BV3J1.5), and one expressed Vß13+ (BV13; Fig. 1B
). All of these CTL clones recognized not only TAP-deficient T2 cells pulsed with Melan-A/Mart-126–35 peptide, but also the naturally processed Melan-A/Mart-1 antigen presented by the HLA-A2+ MEL-FON melanoma cell line, as assessed by cytotoxicity (Fig. 1B)
and IFN- secretion assays (data not shown). Interestingly, half-maximal lysis of peptide-pulsed T2 cells was observed with peptide concentrations ranging from 5 x 10-9 to 5 x 10-10 M, demonstrating the high T-cell receptor (TCR) avidity of these clones (data not shown), which were all brightly stained with the tetramers (Fig. 1A)
. Such high-avidity TCRs have been reported to be expressed by melanoma-reactive CTLs, whereas CD8+ T cells with impaired proliferative and effectors functions mostly display low-avidity TCRs (16)
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, we confirmed the predominance of naive (CD45RAbrightCCR7+) over memory (CD45RA-/dimCCR7-) anti-Melan-A/Mart-1 T cells (18
, 19)
. However, although the majority of tetramer+Vß3+CD8+ T cells appeared to be naive, most te-tramer+Vß2+ cells displayed a memory phenotype (Fig. 1C)
. We thus addressed the relevance of such a melanoma-reactive memory T-cell response with high-avidity TCRVß2+ cells in the spontaneously regressing lesions.
In Situ Characterization of Tumor-Infiltrating Melan-A/Mart-1-Specific CTLs.
The lymphocytic infiltrate of the metastatic LNs contained a majority of CD3+CD8+ lymphocytes displaying a dominant Vß2 specificity (data not shown). Importantly, a high proportion of lymphocytes were stained with the TIA-1 mAb, which recognizes perforin/granzyme-containing granules that are preferentially associated with memory/effector cytotoxic functions (Fig. 2A)
. To determine the Melan-A/Mart-1 specificity of tumor-infiltrating CTLs in the first metastatic LN, we performed a double staining with PE-labeled HLA-A*0201/Mart-126–35 tetramer and FITC-labeled anti-CD8 or anti-Vß2 mAbs. We observed significant numbers of tetramer+CD8+ T lymphocytes (e.g., >10 cells at high magnification) in the tumor areas (Fig. 2, B and C)
, approximately half of which were Vß2+ cells (Fig. 2E)
. However, a more precise quantification of specific T cells by flow cytometry analysis could not be performed because fresh LN fragments were not available at the time of lymphadenectomy. The specificity of tetramer staining was assessed with a peptide-irrelevant PE-labeled HLA-A*0201/HIVgag77–85 tetramer on serial sections of the same LN (Fig. 2D)
and with the relevant PE-labeled HLA-A*0201/Mart-126–35 tetramer on a tumor-free alternative LN from the same axillary area (Fig. 2F)
. Confocal microscopy revealed in double-positive cells an overlay of FITC and PE labeling concordant with the colocalization of TCR signals obtained with tetramers and anti-TCR mAbs (Fig. 2E)
. Of note, tetramer staining was frequently polarized toward the T-cell membrane directly in contact with adjacent tumor cells with occasional intracellular vesicles, suggesting capping and/or recycling of TCR-containing vesicles. These features may also correspond to dense accumulation in lipid rafts and engagement of TCRs, an important process for T-cell activation; lipid raft integrity is also required for efficient MHC class I tetramer binding (20)
.
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Accumulation of Maturing DCs Interacting with Tumor Cells in LN T-Cell-Rich Areas.
The availability of a panel of mAbs directed against DC surface molecules enabled staining of paraffin-embedded tissue sections and allowed us to characterize the infiltrating DCs in the different samples collected from this patient. We first examined two Langerhans cell markers, CD1a and the lectin Langerin/CD207, which are both expressed by immature Langerhans cells. As for the noninvaded adjacent skin, numerous intradermal CD1a+ and Langerin+ cells were detected in the epidermis of the primary regressing cutaneous lesion (data not shown), but very few were observed in the draining metastatic LNs. Such findings contrasted with the detection of significant numbers of CD1a+ and Langerin+ cells in the subcapsular areas of normal LNs collected from other donors (data not shown). In addition, in contrast to what is also seen in normal LNs, we found no CD123/IL-3R+ plasmacytoid DCs in the LNs of our patient (not shown).
We also examined the expression of the actin-bundling protein p55/fascin and the lysosomal protein DC-Lamp, both of which are associated with a mature DC phenotype. In the metastatic LN (Fig. 3A)
as well as in the residual lymphocytic regions of the necrotic LN (not shown), we observed dense infiltration of p55+/DC-Lamp+ double-positive cells within the T-cell-rich regions and peritumoral areas, which contrasted with the scattered and mostly subcapsular localization in a nonmetastatic LN (Fig. 3B)
. These cells exhibited the typical morphology of "mature" DCs and were surrounded by T lymphocytes forming clusters (Fig. 3, C, E, and G)
that have been described as characteristic of ongoing immune reactions and have been observed at the periphery of human breast tumor cell clusters (9)
. DC-Lamp+ (not shown) or p55+ DCs (Fig. 3G)
frequently also accumulated around neomicrovessels within tumor areas. We made similar observations in the primary skin site, where p55+ DCs were often surrounded by lymphocytes in the regression area but not in the normal adjacent dermis (not shown), suggesting local recruitment and/or differentiation of DC precursors from the peripheral blood. Interestingly, in a metastatic LN (Fig. 3C)
, the cellular localization of DC-Lamp was diffuse, including staining of peripheral dendrites, in contrast to that observed in a nonmetastatic LN (Fig. 3D)
, in which the DC-Lamp staining appeared mostly concentrated in a "dot-like" juxtanuclear region of the cells. Considering that DC-Lamp has previously been reported to accumulate in juxtanuclear late lysosomal compartments during maturation (23)
, these findings suggest that an efficient immune response is more likely associated with a maturing DC phenotype rather than with a fully mature differentiation stage. Although the function of DC-Lamp has not been elucidated, our results indicate that its subcellular localization and/or its abundance on DC membranes in close apposition with tumor cells could be beneficial for tumor antigen capture and presentation or is associated with the presence of highly active antigen-presenting cells.
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, reminiscent of data from experimental in vitro systems in which DCs ingested tumor cell bodies and processed tumor-associated antigens to elicit specific immune responses (10)
. Because these DCs also interacted with several surrounding lymphocytes, one can postulate that such interactions may trigger in situ activation, expansion, and effector functions of antigen-specific T cells, culminating, as in this patient, in tumor regression.
Expression of CCR6/CCR7 and Their Ligands in the Metastatic LN.
Considering their critical role in DC chemoattraction, we assessed the expression of the CC chemokine receptors (CCR) CCR6 and CCR7 and their respective ligands, MIP-3/LARC (CCL20), 6CKine/SLC (CCL19), and MIP-3ß/ELC (CCL21). In the metastatic LN, we found that CCR6 was expressed predominantly within tumor areas by cells exhibiting a dendritic morphology (Fig. 4A)
. Accordingly and as described previously (9)
, tumor cells and macrophages strongly expressed the CCR6 ligand CCL20 and clearly colocalized with CCR6+ DCs (Fig. 4B)
. CCR7 was expressed by a few cells in tumor areas, i.e., tumor cells and lymphocytes, but not by infiltrating DCs (Fig. 4C)
. In contrast, large dendrite-shaped CCR7bright cells were detected occasionally in the T-cell-rich areas without tumor cell infiltration (Fig. 4D)
, and the CCR7 ligands CCL19 and CCL21 were slightly positive on DCs and stromal and endothelial cells of microvessels (not shown). During their migration from blood to peripheral tissues, CCR6+ DC precursors appear to be attracted by a CCL20 gradient that is established by local inflammation (5
, 10
, 24)
. Peripheral immature DCs may then undergo a maturation process characterized by several modifications, including the reciprocal loss of CCR6 and acquisition of CCR7, with resulting responsiveness to CCL19/CCL21 (24)
. However, whether DC chemoattraction to LNs and DC maturation are concomitant processes remains controversial (25
, 26)
. As reported in psoriasis (27)
, rheumatoid arthritis (28)
, and in some reports, cancer (9
, 29)
, our findings of dominant CCR6/CCL20 expression suggest that the tumor-invaded LN should be considered as an inflammatory site rather than a typical lymphoid organ. Therefore, in this metastatic LN, maturing DCs might indeed display significant heterogeneity regarding expression of CCR receptors and could even transiently coexpress CCR6 and CCR7 in an intermediate stage of maturation, as has been reported recently (30)
. Altogether, these data point to a potential role of MIP3/CCL20 in the local recruitment of DCs in metastatic LNs.
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. In the SLN/DC-Lamplow group, DC-Lamp+ cells displayed a predominant dot-like lysosomal staining, whereas in the SLN/DC-Lamphigh group, DC-lamp+ cells displayed a predominant dendritic staining (P = 0.019) and an increased frequency of tumor infiltration and DC clustering, as mentioned in the case report. Furthermore, there was a striking inverse correlation between the density of DC-Lamp infiltrates and the occurrence of at least one melanoma-positive non-SLN in the subsequent regional completion lymphadenectomy performed a few weeks after the SLN resection. Seven of 11 patients in the SLN/DC-Lamplow group had at least one metastatic non-SLN, in contrast to none of 8 patients in the SLN/DC-Lamphigh group (P = 0.013). We will attempt to validate this correlation between the presence of mature DCs in the initial metastatic lesion and the absence of secondary metastatic spread in a comprehensive multicenter study aimed at evaluating the prognostic value of DC-Lamp and other dendritic cell markers in disease-free and overall survival of a large cohort of patients with primary cutaneous melanoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: M. Movassagh and A. Spatz contributed equally to this work. J. Davoust is currently at UMR-CNRS 8115, Genethon, 91002 Evry, France.
Requests for reprints: Eric Angevin, M.D. Ph.D, Unité d’Immunologie/INSERM ERM0208, Département de Biologie Clinique, Institut Gustave Roussy, 94805 Villejuif Cedex, France. Phone: (33) 1 42 11 50 35; Fax: (33) 1 42 11 60 94; E-mail: angevin{at}igr.fr
Received 9/24/03. Revised 1/14/04. Accepted 1/19/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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/CCL20 and CC chemokine receptor 6 in psoriasis. J Immunol, 164: 6621-32, 2000.This article has been cited by other articles:
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  R. J. Prestwich, F. Errington, L. P. Steele, E. J. Ilett, R. S. M. Morgan, K. J. Harrington, H. S. Pandha, P. J. Selby, R. G. Vile, and A. A. Melcher Reciprocal Human Dendritic Cell-Natural Killer Cell Interactions Induce Antitumor Activity Following Tumor Cell Infection by Oncolytic Reovirus J. Immunol., October 1, 2009; 183(7): 4312 - 4321. [Abstract] [Full Text] [PDF]
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F. Errington, L. Steele, R. Prestwich, K. J. Harrington, H. S. Pandha, L. Vidal, J. de Bono, P. Selby, M. Coffey, R. Vile, et al. Reovirus Activates Human Dendritic Cells to Promote Innate Antitumor Immunity J. Immunol., May 1, 2008; 180(9): 6018 - 6026. [Abstract] [Full Text] [PDF]
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 B. Elliott, R. A. Scolyer, S. Suciu, S. Lebecque, D. Rimoldi, O. Gugerli, E. Musat, R. N. Sharma, D. Lienard, U. Keilholz, et al. Long-Term Protective Effect of Mature DC-LAMP+ Dendritic Cell Accumulation in Sentinel Lymph Nodes Containing Micrometastatic Melanoma Clin. Cancer Res., July 1, 2007; 13(13): 3825 - 3830. [Abstract] [Full Text] [PDF]
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 A. Faith, E. Peek, J. McDonald, Z. Urry, D. F. Richards, C. Tan, G. Santis, and C. Hawrylowicz Plasmacytoid Dendritic Cells from Human Lung Cancer Draining Lymph Nodes Induce Tc1 Responses Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 360 - 367. [Abstract] [Full Text] [PDF]
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 A. Bergeron, F. El Hage, M. Kambouchner, D. Lecossier, and A. Tazi Characterisation of dendritic cell subsets in lung cancer micro-environments Eur. Respir. J., December 1, 2006; 28(6): 1170 - 1177. [Abstract] [Full Text] [PDF]
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 K. Bennaceur, I. Popa, J. Portoukalian, O. Berthier-Vergnes, and J. Peguet-Navarro Melanoma-derived gangliosides impair migratory and antigen-presenting function of human epidermal Langerhans cells and induce their apoptosis Int. Immunol., June 1, 2006; 18(6): 879 - 886. [Abstract] [Full Text] [PDF]
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J. H. Lee, H. Torisu-Itakara, A. J. Cochran, A. Kadison, Y. Huynh, D. L. Morton, and R. Essner Quantitative Analysis of Melanoma-Induced Cytokine-Mediated Immunosuppression in Melanoma Sentinel Nodes Clin. Cancer Res., January 1, 2005; 11(1): 107 - 112. [Abstract] [Full Text] [PDF]
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 R. J. C. L. M. Vuylsteke, B. G. Molenkamp, H. A. Gietema, P. A. M. van Leeuwen, P. G. J. T. B. Wijnands, W. Vos, P. J. van Diest, R. J. Scheper, S. Meijer, and T. D. de Gruijl Local Administration of Granulocyte/Macrophage Colony-stimulating Factor Increases the Number and Activation State of Dendritic Cells in the Sentinel Lymph Node of Early-Stage Melanoma Cancer Res., November 15, 2004; 64(22): 8456 - 8460. [Abstract] [Full Text] [PDF]
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) inhibited by the anti-MHC class I W6.32 monoclonal antibody (
) and T2 cells pulsed with Mart-126–35 or irrelevant peptides (
and 
, respectively). C, analysis of Melan-A/Mart-1-specific T cells in immunopurified CD8+ peripheral T cells according to CD45RA and CCR7 phenotyping. Although a majority of HLA-A*0201/Mart-126–35+ T cells in the CD8+ cells, as well as in the Vß3+ T-cell subset, displayed a naive CD45RA+CCR7+ (red dots) phenotype, CD45RA-/dim/CCR7- (green dots) memory cells were preferentially found in the Vß2+ T-cell subpopulation. PE, phycoerythrin; ECD, phycoerythrin-Texas Red; APC, allophycocyanin.
