The Activatory Receptor 2B4 Is Expressed In Vivo by Human CD8+ Effector αβ T Cells

Daniel E. Speiser, Marco Colonna, Maha Ayyoub, Marina Cella, Mikaël J. Pittet, Pascal Batard, Danila Valmori, Philippe Guillaume, Danielle Liénard, Jean-Charles Cerottini and Pedro Romero
J Immunol December 1, 2001, 167 (11) 6165-6170; DOI: https://doi.org/10.4049/jimmunol.167.11.6165

Abstract

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ αβ T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-γ production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.

T cell activity and differentiation are regulated by TCR binding to Ag/MHC complexes and a combination of multiple additional receptor-ligand interactions. Adhesion molecules such as LFA-1 and ICAM-1 promote the communication between T cells and APCs. Signals mediated by the costimulatory molecule CD28 enhance T cell activation (1, 2, 3). A further group of receptors belongs to the CD2 family, which is comprised of a number of cell-surface molecules with two Ig superfamily domains. Members of the CD2 family are CD48, CD58 (the human ligand for CD2), CD84, the signaling lymphocytic activation molecule (SLAM),3 Ly-9, and 2B4 (4, 5, 6). These proteins are expressed by various leukocyte populations and contribute to the activation of T cells and NK cells, as shown for CD2 that enhances T cell Ag recognition by setting quantitative thresholds (7, 8).

The 2B4 Ag was identified on all NK cells, all γδ T cells, a subset of CD8+ αβ T cells, and all CD14+ monocytes and basophils. 2B4 shares a signaling pathway with SLAM, in that they both bind to the SLAM-associated protein, which is mutated in immunodeficiency patients with X-linked lymphoproliferative syndrome (9, 10, 11, 12). The function of 2B4 has primarily been analyzed in NK cells, where 2B4 triggering was found to enhance cytotoxicity (5, 13, 14, 15) and IFN-γ production (16). There is evidence that 2B4 also promotes T cell cytotoxicity, because it can mediate non-MHC-restricted killing, and experiments with CD3 and 2B4 mAbs suggest that 2B4 can enhance signals through the TCR (6).

In this study, we analyzed CD8+ αβTCR+ cells ex vivo. We show that 2B4 was predominantly expressed by differentiated T cells with high levels of granzyme B and perforin and rapid production of IFN-γ. Naive resting tumor Ag-specific CD8+ T cells were 2B4 negative, whereas effector cells were 2B4 positive. Thus, in contrast to NK cells and γδ T cells, which express 2B4 constitutively (5, 6), the expression of this molecule is regulated in CD8+ αβ T cells and correlates with distinct cellular differentiation stages.

Materials and Methods

Healthy donors and melanoma patients

Peripheral blood was obtained from healthy donors and patients with advanced stage malignant melanoma. Tumor-infiltrated lymph nodes (TILNs) were obtained from patients by surgical dissection. All patients had no irradiation, chemotherapy, or immunotherapy during several weeks before blood or lymph node withdrawal, except the melanoma patient (LAU 337) shown in Fig. 6⇓, who received immunotherapy consisting of peptides (Melan-A/MART-126–35 EAAGIGILTV and influenza matrix protein58–66 GILGFVFTL, each 100 μg) plus adjuvant SB-AS2 (600 μl) containing the saponin QS21 and monophosphoryl lipid A in an oil-in-water emulsion. Immunotherapy was given i.m. at weeks 0, 4, 8, and 12 (17). SB-AS2 was provided by SmithKline Beecham (Rixensart, Belgium).

Blood and lymph node samples

PBLs were separated from heparinized blood by centrifugation over Ficoll-Paque (Pharmacia, Peapack, NJ), washed three times, and cryopreserved in RPMI 1640, 40% FCS, and 10% DMSO. Vials containing 5–10 × 106 cells were stored in liquid nitrogen. Lymph nodes were dissociated to single-cell suspensions in sterile RPMI 1640 supplemented with 10% FCS, washed, and cryopreserved as indicated above for PBLs. For tetramer analysis, the donors were selected on the basis of HLA-A2 Ag expression. PBLs from patient LAU 337 were obtained before (week −1) and 14 days after two vaccines (week 6) and after four vaccines (week 14).

mAbs and tetramers

mAbs were obtained from BD Biosciences (Mountain View, CA), except anti-CD28FITC and anti-αβTCRPE (Immunotech, Marseille, France), anti-IFN-γFITC and anti-IL-2FITC (BD PharMingen, San Diego, CA), anti-perforinFITC (Ancell, Bayport, MN), anti-granzyme BFITC (Hölzel Diagnostica, Köln, Germany), and goat anti-ratAPC (Caltag Laboratories, Burlingame, CA). The mouse Ab 1C7 (anti-2B4) was coupled with the fluorescent dye PE-cyanin 5 (18). The rat IgG2a mAb 3D12 (anti-CCR7) was obtained from R. Förster and M. Lipp (Max Delbrueck Center for Molecular Medicine, Berlin, Germany) (19). Tetramer complexes were synthesized as described (20, 21, 22). The peptides used for the tetramers were GILGFVFTL (influenza matrix protein58–66), NLVPMVATV (CMV pp65495–503), and ELAGIGILTV (Melan-A/MART-126–35, with A to L substitution at position 2).

Flow cytometry

PBLs were thawed and, where indicated, CD8+ T cells were purified in two rounds of positive sorting using a Minimacs device (Miltenyi Biotec, Auburn, CA). The resulting cells were >98% CD3+CD8+. Cells (5–10 × 105) were stained with tetramers, FITC, PE, PerCP, PE-Texas Red, PE-cyanin 5, or APC-labeled mAb conjugates in 50 μl of PBS, 2% BSA and 0.2% azide, during 30 min at 4°C. Samples with tetramers were stained by first incubating them with tetramers (50 μg/ml) for 30 min at room temperature and then adding the fluorescent Abs for 30 min at 4°C. Cells were washed once in the same buffer and analyzed immediately in a FACSCalibur (four color) or in a FACSVantage (five color) machine (BD Biosciences). The “lymphocyte forward/side scatter gate” was set for a cell population expressing >98% CD45 and <1% CD14 (as determined by control samples labeled for CD45 and CD14). The staining for IFN-γ and IL-2 was done before and after stimulation with 1 μg/ml PMA and 0.25 μg/ml ionomycin, followed by 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) 1 h later. For all intracellular stainings, cells were first stained with cell-surface mAbs, washed, and then fixed and permeabilized in 1 ml of Orthopermeafix (Ortho Diagnostics Systems, Raritan, NJ) for 30 min at room temperature, and then washed twice and incubated with mAb conjugates for 30 min at 4°C. Data analysis was performed using CellQuest software (BD Biosciences, San Jose, CA). The values obtained with isotype-matched fluorescent control Abs were <1% in all cases. Statistical comparisons were done using the t test for two samples with equal variance.

FACS sorting and in vitro T cell stimulation

PBLs from healthy donors were thawed and kept in “CTL medium” overnight. CD8+ T cells were purified by positive sorting using a Minimacs device (Miltenyi Biotec). Using 2B4- and CD8-specific mAbs, FACS sorting with the FACSVantage machine was performed to obtain 2B4-positive and -negative CD8+ T cells. The sorted cells were stimulated with either CD3 (10 μg/ml) and CD28 (1 μg/ml) specific mAbs or PHA (1 μg/ml) and were incubated in the presence of rhIL-2 (150 U/ml), rhIL-7 (10 ng/ml), and irradiated allogeneic feeder cells. After 6 days, the percentages of 2B4 expression were determined through staining with anti-CD8 and anti-2B4 mAbs and flow cytometry.

Results

Activated and effector CD8+ αβ T cells express 2B4

Many human CD8+ T cells up-regulate HLA-DR (MHC class II) upon activation, and such cells can be found in PBLs from healthy donors and patients. In CD8+ αβTCR+ PBLs from a healthy donor, most HLA-DR-positive T cells expressed 2B4, whereas the 2B4-negative cells were largely HLA-DR negative (Fig. 1⇓a). A marker for effector cells is neural cell adhesion molecule (CD56), which is expressed by human CD8+ T cells that are cytolytic ex vivo (23). We found that most CD56+ cells were 2B4 positive (Fig. 1⇓b). Recently, the chemokine receptor CCR7 was identified to be expressed by naive and “central memory” T cells residing in secondary lymphoid organs. In contrast, CD8+ effector T cells were CCR7 negative (19). Interestingly, most CCR7-negative cells expressed 2B4, in contrast to the CCR7-positive cells that were primarily 2B4 negative (Fig. 1⇓c). Results representative for a group of 12 individuals confirmed that HLA-DR+, CD56+, and CCR7− cells were significantly increased in 2B4+ as compared with 2B4− cells (Fig. 1⇓, histograms).

FIGURE 1.
FIGURE 1.

HLA-DR+, CD56+, and CCR7− αβ T cells express 2B4. The dot plots show CD8+ αβTCR+ cells from a representative healthy donor, analyzed for expression of 2B4 and HLA-DR (a), CD56 (b), or CCR7 (c). The histograms below symbolize the corresponding mean percentages (and SDs) from 12 individuals. PBLs were analyzed ex vivo by four-color flow cytometry and gating for αβTCR+/CD8+ cells.

Granzyme B and perforin expression is confined to 2B4-positive cells

Effector CD8+ T cells express the cytotoxic molecules granzyme B and perforin, which are released during cytolytic effector activity leading to target cell lysis (24, 25). Intracellular staining in fixed CD8+ PBLs revealed that high levels of granzyme B and perforin expression were essentially restricted to 2B4-positive cells (Fig. 2⇓a). This correlation was statistically significant when analyzed in six individuals (Fig. 2⇓b). However, lower levels of positive perforin staining were also found in the 2B4-negative cells (Fig. 2⇓a).

FIGURE 2.
FIGURE 2.

Effector molecules expressed by 2B4-positive cells. a, Intracellular staining revealed that high level expression of the cytotoxic molecules granzyme B and perforin was confined to 2B4-positive cells. b, Mean percentages (and SDs) from six healthy individuals demonstrating a statistically significant association between 2B4 and granzyme B/perforin expression. c, After stimulation with PMA and ionomycin, 2B4+ cells produced IFN-γ more rapidly than 2B4− cells, whereas IL-2 production was largely confined to 2B4− cells (mean percentages from six healthy donors). For all experiments, CD8+ PBLs were prepared ex vivo by Minimacs sorting, and flow cytometry included gating for αβTCR+ cells. Negative control stains with irrelevant, isotype-matched FITC-labeled Abs revealed >99.9% negative αβTCR+ cells with an FL1 fluorescence intensity of >30 (data not shown).

Rapid onset of IFN-γ expression in 2B4-positive cells

In contrast to naive cells, effector CD8+ T cells synthesize IFN-γ in <3 h after stimulation, whereas IL-2 production is less efficient (19, 26). We stimulated CD8+ T cells with PMA and ionomycin and stained intracellularly with FITC-labeled cytokine-specific Abs. Whereas 2B4+ cells rapidly became IFN-γ positive, it took longer until the 2B4-negative cells expressed IFN-γ. In contrast, IL-2 expression was preferentially found in 2B4-negative cells (Fig. 2⇑c).

CD8+ T cell differentiation and 2B4 expression

Upon Ag encounter, CD8+ T cells differentiate from naive to “memory” and effector cells. During this process, they down-regulate CD45RA (27) and CCR7 (19), and these two markers are useful to separate naive cells (RA+CCR7+) from “memory” cells (RA−CCR7+) and effector cells (CCR7−). Fig. 3⇓a shows that these three populations differed with respect to 2B4 expression, whereby naive cells were mostly negative, effector cells were positive, and “memory” cells were intermediate. Analysis of 2B4 expression in 11 individuals revealed a statistically significant difference between the three populations (Fig. 3⇓b). Because effector cells may re-express the CD45RA isoform (28), we also compared 2B4 expression in CCR7−RA+ vs CCR7−RA− cells and found comparable mean fluorescent intensities (54.8 vs 64.1; data not shown).

FIGURE 3.
FIGURE 3.

Distinct 2B4 expression by naive, “memory,” and effector CD8+ T cells. a, PBLs from a healthy donor showed that 2B4 was mostly absent on naive (CCR7+CD45RA+) cells, partially up-regulated on “memory” (CCR7+CD45RA−) cells, and positive on effector (CCR7−) cells. b, Individual (and mean) fluorescence intensities of 2B4 expression in the three CD8+ T cell subsets in PBLs from 11 individuals. The differences were statistically significant, including the comparison of naive (left) with effector (right) cells (p < 0.00001). CD8+ PBLs were prepared ex vivo by Minimacs sorting, and flow cytometry gates were set for αβTCR+ cells and for expression of CD45RA and/or CCR7 corresponding to the compartments shown.

In vitro 2B4 up-regulation by CD8+ T cells

To test for changes in 2B4 expression upon in vitro stimulation, we sorted CD8+ T cells from PBLs of healthy donors in 2B4-negative and -positive populations (Fig. 4⇓). Subsequently, the cells were stimulated in vitro with anti-CD3 and anti-CD28 mAbs or with PHA. After 6 days, 2B4 was up-regulated in cells from all cultures. The mean values from four healthy donors showed that 2B4 up-regulation was significant (Fig. 4⇓d). Thus, in vitro stimulation of CD8+ PBLs induced surface expression of 2B4.

FIGURE 4.
FIGURE 4.

In vitro up-regulation of 2B4 by CD8+ T cells. CD8+ PBLs from a representative healthy donor were FACS sorted resulting in 2B4 negative and positive cells (a). Staining with 2B4-specific fluorescent mAb was done 6 days after stimulation with Abs specific for CD3 and CD28 (b) or PHA (c), both in the presence of IL-2 and IL-7 and irradiated allogeneic feeder cells. d, Mean values and SDs from PBLs of four healthy donors demonstrated that 2B4 was significantly up-regulated in 2B4-negative sorted cells (p values marked with an asterisk) and that the 2B4 positively sorted population expressed significantly higher levels of 2B4 in all cases (p values marked with #).

Ag-specific T cells show characteristic 2B4 expression depending on the differentiation stage

To investigate Ag-specific T cells, we prepared fluorescent HLA-A*0201 tetramers for ex vivo analysis of T cells specific for the tumor Ag Melan-A/MART-1 and the viral Ags influenza matrix protein and CMV protein pp65. In healthy donors, Melan-A-specific T cells are found at relatively high frequencies, even though they are not activated (i.e., they are CD45RA+, CCR7+, CD28+, and HLA-DR−) (17, 29). These cells are generally considered naive and resting, and our analysis showed that most were 2B4 negative (Fig. 5⇓a). In the majority of healthy adults, influenza-specific cells are prototype “memory” cells, and we found that they expressed 2B4 at low but measurable levels. This was in contrast to CMV-specific cells, which were mostly 2B4 positive. CMV-specific CD8+ T cells are preferentially found as effector cells with high cytolytic activity and strong IFN-γ expression upon short-term stimulation (30, 31). The results from six healthy donors confirmed that 2B4 expression differed significantly between the T cells with the three different Ag specificities (Fig. 5⇓b).

FIGURE 5.
FIGURE 5.

2B4 expression by Ag-specific CD8+ T cells. PBLs from healthy donors were analyzed ex vivo by flow cytometry. a, The dot plots show sorted CD8+ cells stained with 2B4-specific Abs and HLA-A*0201 tetramers containing peptides derived from Melan-A/MART-1 (left), influenza matrix protein (center), and CMV pp65 (right). b, Individual (and mean) percentages of 2B4-positive cells (within tetramer+CD8+ cells) from six healthy donors for the three Ag specificities. The differences were statistically significant, including the comparison of Melan-A-specific (left) with CMV-specific (right) cells (p < 0.00001).

In melanoma patients, tumor Ag-specific T cells may express 2B4

We have recently described that TILNs in melanoma patients often contain Melan-A-specific T cells in large numbers as a result of tumor Ag-driven T cell expansion (22, 29). Here, we analyzed TILNs ex vivo from nine patients by flow cytometry and found measurable numbers of Melan-A-specific cells in five of these nine patients. These tumor Ag-specific cells were preferentially 2B4 positive (Fig. 6⇓a). The summarized data from Melan-A-specific cells in TILNs revealed that 2B4 was expressed at high frequencies, in contrast to low expression of CCR7 (Fig. 6⇓a). In contrast, Melan-A-specific T cells were largely CCR7+ and 2B4− in disease-free lymph nodes (data not shown). Recent evidence suggests that increased tumor cell infiltration may correlate with increased percentages of CCR7-negative T cells in TILNs (32). The fact that tumor infiltration leads to progressive destruction of the lymph node tissue architecture and progressive T cell activation and differentiation to effector cells (22) fits well with the finding that the CCR7 expression by Melan-A-specific T cells was low, whereas 2B4 was up-regulated.

FIGURE 6.
FIGURE 6.

Tumor Ag-specific T cells expressing 2B4 in melanoma patients. a, Ex vivo staining with HLA-A*0201/Melan-A tetramers and 2B4-specific Abs shows that the majority of Melan-A-specific T cells from TILNs (patient LAU 362) expressed 2B4 in vivo. The histogram shows the summarized data (mean and SD) from six TILNs from five different patients (LAU 306, 343, 359, 362, and 392) for 2B4 and CCR7 expression in Melan-A-specific T cells. b, In PBLs from patient LAU 337, increasing numbers of Melan-A-specific T cells were found: 0.1% of CD8+ T cells before peptide vaccination (week −1, left), 0.8% after two vaccine injections (week 6, center), and 2.3% after four vaccine injections (week 14, right). 2B4 expression increased from 50 to 80 and to 93%, and HLA-DR expression increased from 23 to 46% and decreased back to 22%. Immunotherapy was given four times (symbolized by arrows), as described in Materials and Methods. The dot plots show the Melan-A-specific cells (gated for positive tetramer staining), and the numbers in the quadrants indicate the percentages of them negative or positive for 2B4 and HLA-DR expression, respectively.

Expanded tumor Ag-specific CD8+ T cells are 2B4 positive

In PBLs from patient LAU 337, the percentages of Melan-A-specific cells increased during immunotherapy. They were 0.1% of CD8+ T cells 1 wk before peptide vaccination, 0.8% after two vaccine injections, and 2.3% after four vaccine injections. On the surface of these Melan-A-specific cells, 2B4 was partially expressed before immunotherapy and increased considerably upon in vivo peptide immunization (Fig. 6⇑b). Comparable observations were made in three other melanoma patients with in vivo CTL differentiation upon peptide immunization (data not shown). Thus, in Melan-A-specific cells, 2B4 was not expressed in healthy donors (Fig. 5⇑) but progressively up-regulated with in vivo T cell activation and differentiation in melanoma patients. Activated cells also up-regulated HLA-DR (Fig. 6⇑b), especially during the period of strong cellular proliferation and expansion (17), but this was only transient, despite subsequent differentiation to effector cells and persistence at high cell numbers during the following 6 mo (data not shown).

Discussion

To investigate 2B4 expression in various stages of CD8+ T cell differentiation, we used the previously applied markers CD45RA and CCR7. In a large number of Ag-experienced cells, the long CD45 isoform RA may be replaced by the alternatively spliced short isoform CD45RO (27). Some differentiated cells are also found in the CD45RA+ compartment, indicating that at least one additional marker must be used to distinguish naive from non-naive cells (28). The lymph node homing chemokine receptor CCR7 has recently been shown to be down-regulated in effector T cells, an observation allowing to phenotypically differentiate so called “central memory” cells (CCR7+RA−) from effector cells (CCR7−) (19). In the present study, we show that the CCR7+RA+ naive cells are mostly 2B4−, whereas the CCR7− effector cells are 2B4+. The CCR7+RA− “central memory” cells express 2B4 at intermediate frequencies and at lower surface levels than CCR7− effector cells.

Effector T cells were described to have a CD45RA+ CD27− phenotype (28). Our findings are compatible with this observation, because CD45RA+ CD27− cells are primarily 2B4+ (18). CD27− T cells are generally 2B4+ when analyzed in a larger number of individuals, and the same is true for CD28− T cells, which are largely overlapping with the CD27− subset (data not shown). CD28−/CD27− CD8+ T cells produce large amounts of IFN-γ and are cytolytic. These cells appear to be terminally differentiated effector cells (33, 34), and their capacity to proliferate and differentiate (back) to “memory” cells seems low. However, CD28−/CD27− CD8+ T cells are only partly overlapping with CCR7− cells (data not shown), and further studies are necessary to characterize the subpopulations defined by these markers.

To study 2B4 expression in Ag-specific T cells, we applied tetramers and focused on three different Ags. 1) In CMV seropositive individuals, CMV-specific T cells are considerably expanded and persist at high numbers in circulation. They express cytokines and cytolytic function and are thus considered to be effector cells (30, 35). In contrast to CMV, 2) influenza virus infection is cleared after a few weeks, and influenza-specific cells persist in a not activated or only weakly activated state thereafter (20, 36). These so-called “memory” cells remain at increased numbers in circulation and are relatively rapidly reactivated upon Ag encounter, thus protecting from severe re-infections. 3) In healthy donors, Melan-A/MART-1-specific cells present all features of naive cells that have not been activated in vivo, despite the fact that they are present at relatively high numbers in the circulation (29). Although the distinction of naive, “memory,” and effector cells is not clear cut and many issues remain unresolved (37, 38), the three populations specific for Melan-A, influenza, and CMV epitopes are among the best representatives to cover the spectrum of CD8+ T cell differentiation stages in humans. Using these three Ag-specific cell populations, our data show significant differences in 2B4 expression, suggesting that differentiation to effector cells is associated with 2B4 up-regulation.

In contrast with healthy individuals, melanoma patients may have Melan-A-specific T cells that are activated in vivo through Melan-A expressed by tumor cells (39, 40) and/or through peptide immunization (17). Such cells show a progressive shift in phenotype consisting of CCR7 down-regulation (32) and 2B4 up-regulation, which persisted even many weeks after immunotherapy, suggesting that this phenotype remains relatively stable. In contrast, HLA-DR expression, which was also up-regulated upon cellular activation, decreased afterward in parallel to reduced proliferative and telomerase activity (17). Thus, the data indicate that HLA-DR expression correlated with ongoing proliferative activity, whereas CCR7 down-regulation and 2B4 up-regulation correlated with differentiation to effector T cells, at least in part independently of the cellular activation status.

The primary ligand of 2B4 is CD48, which is ubiquitously expressed in humans (14, 41). NK cells constitutively express 2B4, and it has been demonstrated that signaling through 2B4 enhances NK cell-mediated cytotoxicity when triggered with specific Abs or with the ligand CD48. The function of 2B4 in NK cells is regulated, at least in part, by the signaling molecule SLAM-associated protein, which is up-regulated after 24 h and rapidly degraded during the second day after activation. Thus, despite the fact that NK cells express 2B4 constitutively, it may only be functional shortly after activation.

The finding that the CD2 family member 2B4 is expressed by effector T cells raises the hypothesis that 2B4 may promote effector cell function. Only little is known on 2B4 function in Ag-specific T cells. A major reason for this is that human Ag-specific T cells are difficult to assess ex vivo, due to the low numbers of available cells. We are currently generating target cells that express the appropriate HLA molecule with or without CD48 to investigate the influence of 2B4 triggering on MHC-restricted killing. As mentioned, many effector T cells down-regulate the costimulatory molecules CD28 (33, 34) and CD27 (28) and can thus no longer receive costimulatory signals known to enhance T cell activation (1, 2, 3). Instead, these cells up-regulate 2B4, which raises the possibility that effector function is promoted by 2B4-dependent signals that may allow efficient T cell activity in the absence of costimulation via CD28 and CD27.

In conclusion, the data establish 2B4 as a useful marker for CD8+ αβ T cell differentiation, which can be applied to monitor and study human T cells in disease and vaccination. The data suggest that 2B4-mediated co-activatory signals may be involved in the control of cellular activation, expansion, and/or effector function.

Acknowledgments

We thank the patients and the healthy donors for blood donation. We also thank Reinhold Förster and Martin Lipp for the CCR7-specific mAb and Ferdy Lejeune, Guiseppe Pantaleo, H. Robson MacDonald, Immanuel Lüscher, and Anne Wilson for support. We acknowledge the excellent technical and secretarial help of Andrée Porret, Danielle Minaïdis, Christine Geldhof, Nicole Montandon, and Martine van Overloop.

Footnotes

  • ↵1 This work was supported in part by a grant from the Leenaards Foundation. M.J.P. was supported by Swiss Cancer League Grant KFS 633-2-1998.

  • ↵2 Address correspondence and reprint requests to Dr. Daniel E. Speiser, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Niveau 5, Aile Est, Av. Pierre-Decker 4, CH-1005 Lausanne, Switzerland. E-mail address: daniel.speiser{at}hospvd.ch

  • ↵3 Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; TILN, tumor-infiltrated lymph node.

  • Received April 11, 2001.
  • Accepted September 21, 2001.

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