γδ T Lymphocytes Count Is Normal and Expandable in Peripheral Blood of Patients with Follicular Lymphoma, Whereas It Is Decreased in Tumor Lymph Nodes Compared with Inflammatory Lymph Nodes

Mounia Sabrina Braza, Anouk Caraux, Thérèse Rousset, Sylvie Lafaye de Micheaux, Hélène Sicard, Patrick Squiban, Valérie Costes, Bernard Klein and Jean-François Rossi
J Immunol January 1, 2010, 184 (1) 134-140; DOI: https://doi.org/10.4049/jimmunol.0901980

Abstract

γδ T lymphocytes are attractive effector cells for immunotherapy. In vitro, they can be expanded and kill efficiently a variety of tumor cells. The frequency and distribution of γδ T lymphocytes were compared in tumor lymph nodes of 51 patients with follicular lymphoma lymph nodes (FL-LNs) and 28 patients with inflammatory lymph nodes (I-LNs). γδ and CD8 T lymphocytes were less abundant in FL-LNs than in I-LNs (p ≤ 10−7). These lymphocytes were localized in the perifollicular zone outside of the tumor follicles. Perifollicular γδ T lymphocytes expressed CCR7, in contrast to peripheral blood γδ T lymphocytes and both perifollicular and peripheral blood γδ T lymphocytes expressed CXCR4. The very low number of perifollicular γδ T lymphocytes in FL-LNs could be explained in part by migratory problems because of absence of CCL19 expression in FL-LNs compared with I-LNs. Conversely, CCL21 and CXCL12 were similarly expressed in both FL-LNs and I-LNs. CCL19 and CCL21 were expressed in high endothelial venules and lymphatic vessels, whereas CXCL12 was expressed by stromal cells surrounding high endothelial venules and lymphatic vessels. Peripheral γδ T lymphocytes from 34 patients with FL, expanded with Phosphostim and IL-2 in vitro, had the same expansion capacity as those from healthy individuals. Thus, γδ T lymphocytes can be an attractive source for adoptive immunotherapy in patients with FL, providing they may home in tumor LNs.

Follicular lymphoma (FL) is characterized by the presence of the t (14;18) (q32;q21) translocation in virtually all patients. This leads to deregulation of the BCL-2 gene with overproduction of the antiapoptotic BCL-2 protein (1). A substantial body of evidence supports the hypothesis that the BCL-2/IgH translocation is necessary, but not sufficient, to cause FL. Gene expression profiling of FL has revealed that the molecular characteristics of nonmalignant tumor–infiltrating immune cells have a major influence on survival. Indeed, two survival-associated gene signatures have been found. One, termed “immune response–2,” is an indicator of immune infiltration, mainly by macrophages and/or dendritic cells, and is associated with a bad prognosis and short survival. The other one, defined as “immune response–1,” is characterized by a complex mixture of immune cells, such as T cells, and is associated with good prognosis and long survival (2–4). In the group of patients with the “immune response–1” signature, two categories of CTLs—CD8 and γδ T lymphocytes—could be of importance. CD8 T lymphocytes are able to kill efficiently tumor cells upon direct recognition of peptide Ags presented by the tumor cell MHC class I molecules (5). Cytotoxic γδ T lymphocytes exhibit an innate reactivity to MHC-unrestricted microbial and tumor nonpeptidic phosphoantigens, which leads to proliferation, release of Th1 cytokines, and perforin-mediated killing (6–9). γδ T lymphocytes can be activated directly in vitro by pyrophosphomonoesters (10–12) like bromohydrin pyrophosphate (BrHPP/Phosphostim). Phosphostim is a synthetic γ9δ2 TCR agonist that mimics the biological properties of natural phosphoantigens found in hydrosoluble mycobacterial extracts (13). Phosphostim and IL-2 allow the selective outgrowth of peripheral blood γδ T lymphocytes from patients with renal colon carcinoma and multiple myeloma, which are highly cytotoxic toward autologous primary tumor cells (10, 14, 15). Moreover, Phosphostim and IL-2 infusions in nonhuman primates trigger a transient but large expansion of circulating γ9δ2 T lymphocytes and a high production of Th1 cytokines (16).

Traffic patterns and the inflammatory function of leukocytes, such as γδ T lymphocytes, are largely defined by their chemokine receptor expression (17–19). Ag-driven T cell priming and differentiation induce sequential changes in chemokine responsiveness (20–23). Chemokines are small proteins that play important roles in leukocyte migration, activation and degranulation (24). They are divided into two major subfamilies, “homeostatic” and “inflammatory” chemokines, and their distinctive roles are best exemplified in mature T cells (20–23, 25). The homeostatic chemokines SLC/CCL21 and ELC/CCL19 are produced by stromal cells in T cell zones of secondary lymphoid tissues (26, 27) and selectively attract CCR7-bearing lymphocytes. These chemokines binds to the same receptor CCR7 (28). SDF1/CXCL12 is also a homeostatic chemokine expressed in peripheral lymphoid tissues that enhances the migration of normal and malignant B lymphocytes and plays a role in the local dissemination of tumor cells (29). CCL21, CCL19, and CXCL12 are expressed in high endothelial venules (HEVs) and lymphatic vessels (LVs) (30).

In this study, we found that γδ and CD8 T lymphocytes are localized in the perifollicular zone of FL lymph nodes (FL-LNs) and that their count is lower than in inflammatory LNs (I-LNs). Because most LN γδ Tlymphocytes expressed CCR7, unlike peripheral blood γδ T lymphocytes, their decrease in FL-LNs may be explained by the decreased expression of the CCR7-targeting CCL19 chemokine in FL-LNs compared with I-LNs. In addition, the count of circulating γδ T lymphocytes from FL patients was normal and they could be easily expanded with Phosphostim and IL-2.

Materials and Methods

Patients

Fifty-one patients consecutively diagnosed with FL have been followed at the Hemato-Oncology Unit of the University Hospital of Montpellier between 1988 and 2005. Patients’ characteristics and Revised European-American Classification of Lymphoid Neoplasms (REAL) grade are indicated in Table I (31). Twenty-eight patients with inflammatory adenitis were also included. Their median age was 49 y (range, 2–83 y), and the male/female ratio was 57/43%.

Patients’ samples and immunohistological analysis

Immediately after biopsy, a part of each FL-LNs or I-LNs was fixed in formalin and Bouin’s fixative and paraffin embedded. The other part was frozen in liquid nitrogen and stored at −80°C. Immunohistochemical staining with anti-γδ T lymphocytes mAb (Immu510; Beckman Coulter, Paris, France) or anti-CD8 T lymphocytes mAb (clone C8/144 B; DakoCytomation, Paris, France) was done to determine the frequency, distribution, and topography of γδ and CD8 T lymphocytes in 51 FL-LNs and 28 I-LNs. CCL19, CCL21, and CXCL12 expression was determined in HEVs and LVs of 14 FL-LNs or I-LNs with anti-CCL19, -CCL21, or -CXCL12 mAbs (R&D Systems, Lille, France). Immunohistochemical analysis was performed on paraffin or frozen sections. Frozen sections were air-dried and fixed in cold acetone at room temperature for 10 min. After 30 min of incubation with blocking serum solution (DakoCytomation) and Cas block (Zymed, Clinisciences, Paris, France), slides were incubated with the primary mAb for 45 min. After washing, the biotinylated secondary Ab was added for 10 min and slides were rinsed. Three percent of H2O2 in methanol was used to inactivate the internal peroxidase for 5 min. Slides were then covered with a streptavidin-peroxidase complex (HRP) for 10 min, rinsed, and finally incubated with a peroxidase substrate (diaminobenzidine) for 10 min. Slides were counterstained with blue hematoxylin. Labeled γδ and CD8 T lymphocytes were counted with an optical microscope (Laborluxe 12; Leica, Bannockburn, IL) in 14 random 2.25-mm2 fields. The median cell number was calculated in these 14 fields for every sample and for every staining and was expressed as cell number per square millimeter. HEV and LV cells stained with anti-CCL19, -CCL21, and -CXCL12 mAbs were quantified similarly, and the level of expression was semiquantified using the following criteria (0 = negative; 1 = weak; 2 = moderate; and 3 = strong staining). Quantification of CCR7+ γδ T in FL-LNs and I-LNs was performed by double immunostaining using anti-CCR7 (Clinisciences) and anti-γδ T mAbs (Immu510; Beckman Coulter). CXCR4 immunostaining in I-LNs could not be performed because cryopreserved I-LN samples were not available in our tissue bank.

Expansion of γδ T lymphocytes with BrHPP and IL-2 in vitro

PBMCs were isolated from fresh blood samples of 34 FL patients using Ficoll-Paque PLUS (Amersham Biosciences, Paris, France) and cultured at a density of 106 cells/ml in 24-well culture plates at 37°C in 5% CO2 in RPMI 1640 medium and 10% FCS. Phosphostim (3 μM BrHPP; Innate Pharma, Marseille, France) and 150 U/ml IL-2 (Proleukin; Chiron, Basel, Switzerland) were added for 8 and 14 d. Phosphostim was added once at the onset of the culture, and then half of the culture medium volume was replaced every 3 d with fresh medium containing 150 U/ml IL-2. The expansion ability of γδ T lymphocytes was evaluated at day 8 by determining the absolute counts (cell number in the culture × percentage of γδ T cells). According to this result, patients were classified in four groups: non responders (γδ T lymphocyte fold expansion ≤2), weak responders (fold expansion >2 and <8), intermediate responders (fold expansion ≥8 and <32), and high responders (fold expansion ≥32).

Immunophenotype analysis

The phenotype of T lymphocytes was evaluated at days 0 and 8 with the following mAbs: PC5-conjugated anti-CD3, FITC-conjugated anti-Vδ2 TCR, and FITC-conjugated anti-CD8. Fluorochrome-conjugated isotype-matched mAbs, recognizing no human Ag, were used as negative controls: FITC-conjugated IgG1, PE-conjugated IgG1, and PC5-conjugated IgG1 (Beckman Coulter). CCR7 and CXCR4 expression in peripheral blood γδ T lymphocytes of six healthy donors and five FL patients as well as in perifollicular γδ T lymphocytes of five FL patients were determined using the following mAbs: PE-conjugated anti-CXCR4 (BD Pharmingen, Le Pont de Claix, France), PE-conjugated anti-CCR7 (clone 3D12; BD Pharmingen), PE-conjugated anti-CCR7 (clone 150503; R&D Systems), and FITC-conjugated anti-pan γδ TCR (Beckman Coulter). Briefly, appropriate amounts of mAbs were added to 0.5 × 106 whole-blood cells, followed by 30 min of incubation at 4°C. Cells were washed, red blood cells lysed, and 50,000 total events in the lymphocyte gate were acquired with a FACScan4 cytometer (BD Pharmingen) and analyzed with the CellQuest software. CCR7 expression on γδ T lymphocytes was evaluated acquiring at least 3000 events in the γδ T lymphocyte gate. CXCR4 immunostaining in I-LNs could not be performed because cryopreserved I-LN samples were not available in our tissue bank.

Statistical analysis

The significance of the data was evaluated with the Student parametric test and Mann–Whitney and Kruskal–Wallis nonparametric tests using the SPSS 10 software. The prognostic value of γδ and CD8 T lymphocyte counts was evaluated with univariate Cox analysis.

Results

Normal counts and in vitro expansion of peripheral blood γδ T lymphocytes from patients with FL

The mean count of circulating γδ T lymphocytes in 34 patients with FL was 20 cells/mm3 (range, 1–104). They represented 2.2% (0.1–11.4%) of the circulating CD3 cells (results not shown). These findings were similar to those previously described in age-related healthy donors (HDs) by our group (10). γδ T lymphocytes from 80% of FL patients (27 of 34) could be highly expanded (≥32-fold at day 8, median expansion 174-fold, 84.7% γδ T lymphocytes) upon stimulation with BrHPP and IL-2 in vitro (Fig. 1A, 1B). γδ T lymphocytes from 18% of FL patients could be expanded between 8- and 31-fold, and γδ T lymphocytes from only 1 patient could not be expanded (<2-fold) (Fig. 1A, 1B). Their expansion was in the same range as that reported for HDs (10, 11, 15, 32). Thus, normal levels of circulating γδ T lymphocytes are present in FL patients, and they can be efficiently expanded using BrHPP and IL-2 in vitro.

FIGURE 1.
FIGURE 1.

In vitro expansion of peripheral blood γδ T lymphocytes from patients with FL using BrHPP and IL-2. Peripheral blood mononuclear cells from 34 patients with FL were cultured with BrHPP and IL-2 for 8 d, and γδ T-lymphocytes were counted at the end of the culture. A, Fold expansion of γδ2 T lymphocytes in 34 FL patients. B, Percentage of γδ2 T lymphocytes at the end of the expansion. The median is indicated by a horizontal line.

γδ and CD8 T lymphocytes are located in the perifollicular zone, and not inside follicles, in FL or I-LNs

Distribution of γδ or CD8 T lymphocytes was examined in FL-LNs of 51 FL patients and in I-LNs of 28 patients with reactive adenitis by immunohistochemistry. Patient clinical data are indicated in Table I. In most FL-LNs (47 of 51), γδ T lymphocytes were very rare and mostly (99.6%) localized in the perifollicular zone, which delineates the follicle contour (Fig. 2A, Table II) (the image in Fig. 2A is from one of the four FL patients with the highest γδ T lymphocyte count to illustrate their perifollicular localization). Similarly, 98% of CD8 T lymphocytes were localized in the perifollicular zone (Fig. 2A, Table II). In I-LNs, γδ and CD8 T lymphocytes were also mainly localized (≥99%) in the perifollicular area (Fig. 2A, Table II).

View this table:
Table I. Patients’ characteristics
FIGURE 2.
FIGURE 2.

Immunohistochemistry staining of FL- or I-LNs. A, Perifollicular distribution of γδ and CD8 T lymphocytes in tumor LNs of FL patients (left panels, FL-LNs) or reactive adenitis (right panels, I-LNs) (for γδ T lymphocytes topography, we chose one of the rare patients with high γδ T lymphocyte count to show the perifollicular zone) (magnification ×10). B, Counting γδ and CD8 T lymphocytes. To quantify γδ and CD8 T lymphocytes in perifollicular zones, cells were counted in 14 randomly chosen 2.25-mm2 fields for each LN. Top panels: Immunostaining of γδ T lymphocytes in FL-LNs (left panels) and I-LNs (right panels) (magnification ×20). Bottom panels: Immunostaining of CD8 T lymphocytes in FL-LNs (left panels) and I-LNs (right panels) (magnification ×40). The median counts of γδ or CD8 T-lymphocytes in the 14 FL-LN or I-LN samples are indicated in each panel. C, Expression of CCL19 in FL-LNs and I-LNs. Immunohistochemical analysis of paraffin sections of FL-LNs (left panels) and I-LN (right panels) shows a weak expression of CCL19 in the HEVs (necklace-shaped structures, black arrows) and LVs (similar to a ball of wool, white arrows) of FL-LNs (left panels), compared with I-LNs (right panels) (magnification ×10). The median counts of CCL19+ cells in the 14 FL-LN or I-LN samples are indicated in each panel.

View this table:
Table II. Distribution of γδ and CD8 T lymphocytes in perifollicular and follicular zones

Perifollicular γδ and CD8 T lymphocyte counts are lower in tumor LNs than in I-LNs

CD8 and γδ T lymphocytes were then counted in 14 perifollicular fields of each FL-LNs (n = 51) and I-LNs (n = 28). Both γδ and CD8 T lymphocytes were significantly reduced by 62% (p = 1.2 × 10−7) and 18% (p = 0.004) in FL-LNs in comparison with I-LNs (Fig. 3A, 3B). The median γδ T lymphocyte count was 18 cells/mm2 in FL-LNs and 47.5 cells/mm2 in I-LNs. The median CD8 T lymphocyte count was 1235 cells/mm2 in FL-LNs and 1503.5 cells/mm2 in I-LNs. In FL-LNs, perifollicular γδ and CD8 T lymphocyte counts were not different between patients with newly diagnosed FL (35 of 51) (48% of REAL grade I, 17% of grade II, and 35% of grade III) and patients (16 of 51) with relapsing FL (p = 0.65) (63% of grade I, 19% of grade II, and 18% of grade III). The frequency of γδ and CD8 T lymphocytes was not influenced by the FL grade (p = 0.5) either.

FIGURE 3.
FIGURE 3.

Significant decrease in γδ and CD8 T lymphocytes in FL-LNs compared with I-LNs. For each LN, γδ and CD8 T lymphocytes were counted in 14 randomly chosen fields (2.25 mm2) in perifollicular zones, and the mean counts were determined. Shown are the box plot distributions of γδ (A) and CD8 (B) T lymphocyte counts in the LNs of 51 patients with FL and 28 patients with reactive adenitis. The bottom and top edges of the boxes indicate the 25th and 75th percentiles and the centerline the median.

CCL19 is weakly expressed in FL-LNs

The lower T lymphocyte counts in FL-LNs compared with I-LNs could be due to changes in chemokines able to recruit these cells. A median of 86% of γδ T lymphocytes in both FL-LNs and I-LNs expressed CCR7, the LN chemokine receptor (Fig. 4A). Conversely, only 1.2 ± 0.7% of circulating γδ T lymphocytes from FL patients (n = 5) expressed CCR7 and 1.7 ± 0.8% in healthy individuals (n = 6). This low CCR7+ γδ T lymphocytes was evaluated acquiring at least 3000 events in the γδ T lymphocyte gate. The anti-CCR7 mAb used (clone 3D12; BD Pharmingen) efficiently labeled some non-γδ T cells present in the peripheral blood (Fig. 4B), and the lack of CCR7 expression in circulating γδ T lymphocytes was confirmed with a second PE-conjugated anti-CCR7 mAb (clone 150503; R&D Systems) (results not shown). The percentage of circulating CCR7+ γδ T lymphocytes did not increase after in vitro expansion with BrHPP and IL-2 (0.2 ± 0.1% in FL patients, n = 5; and 1.2 ± 0.6% in healthy donors, n = 6) (Fig. 4A), whereas some αβ T lymphocytes that were expanded together with γδ T lymphocytes were CCR7+ (Fig. 4B). Moreover, 84% of γδ T lymphocytes in FL-LNs and 62% of circulating γδ T lymphocytes from FL patients expressed the chemokine receptor CXCR4. After expansion, CXCR4 expression decreased of ~50% in circulating γδ T lymphocytes at day 14 (p = 0.001) (Fig. 4C). CXCR4 expression could not be assessed in I-LNs, because CXCR4 immunolabeling can be carried out only on cryopreserved samples that were not available in our tissue bank.

FIGURE 4.
FIGURE 4.

CCR7 and CXCR4 are strongly expressed in LN but not in circulating γδ T lymphocytes. The percentage of CCR7+ or CXCR4+ γδ T lymphocytes were determined by immunostaining (CCR7 expression in 15 FL-LNs and 15 I-LNs) or flow cytometry. For each group, the median value is indicated by a horizontal line. CXCR4 immunostaining in I-LNs could not be performed because no cryopreserved I-LN samples were available in our tissue bank. A, Percentage of CCR7+ cells within the γδ T lymphocytes from FL-LNs (○, n = 15), I-LNs (●, n = 15), and from peripheral blood of five FL patients (△, □) and 6 HDs (▴, ▪) at days 0 and 14 after expansion. CCR7+ γδ T cells were determined by gating γδ T lymphocytes within PBMCs. B, Percentage of CCR7+ γδ T lymphocytes in the peripheral blood of one FL patient at day 0 or that of γδ T lymphocytes expanded in vitro with IL-2 and BrHPP for 14 d. γδ T lymphocytes were labeled with a FITC-conjugated pan-γδ T lymphocyte mAb and CCR7 cells using the PE-conjugated anti-CCR7 mAb (clone 3D12; BD Pharmingen). Lymphocytes were first gated according to their forward-side scatter (FSC/SSC) distribution, including activated lymphocytes, and CCR7+ γδ T lymphocytes were determined by analyzing at least 3,000 events in the γδ T lymphocyte gate (8,300 events at day 0 and 10,900 events at day 14, in the two samples shown in the figure). C, Percentage of CXCR4+ within γδ T lymphocytes from FL-LNs (○) and from peripheral blood of FL patients and HDs (△, ▴) and in expanded γδ T at day 14 (□, ▪).

The CCL19 and CCL21 chemokines (CCR7 ligands) were expressed mainly in T cell zones and CXCL12 (CXCR4 ligand) in both T and B cell zones of 14 randomly chosen perifollicular fields from 14 FL-LNs and 14 I-LNs. CCL19 and CCL21 were expressed in HEVs and LVs. Specifically, the number of HEV/LV expressing CCL19 was 3-fold lower in FL-LNs than in I-LNs (p = 2 × 10−7) (Figs. 2C, 5A). Moreover, the intensity of CCL19 labeling was reduced in FL-LNs (100% weak) in comparison with I-LNs (50% strong and 50% weak) (Figs. 2C, 5A). The number of cells expressing CCL21 and the intensity staining for CCL21 were slightly lower in FL-LNs versus I-LNs without reaching significance (p = 0.055) (Fig. 5B), whereas CXCL12 expression was not different between FL-LNs and I-LNs (p = 0.4) (Fig. 5C). CXCL12 was strongly expressed by stromal cells surrounding HEVs or LVs and diffuse in the perifollicular and tumor areas (data not shown).

FIGURE 5.
FIGURE 5.

Low expression of CCL19 in FL-LNs. Immunohistochemical analysis of paraffin sections of 14 FL-LNs and 14 I-LNs were performed. The number of HEV and LV expressing CCL19, CCL21, and CXCL12 were counted with an optical microscope (Laborluxe 12; Leica) in 14 random 2.25-mm2 fields. The level of expression was semiquantified using the following criteria (0 = negative; 1 = weak; 2 = moderate; and 3 = strong staining). A, A significant decrease in CCL19 expression in FL-LNs compared with I-LNs. All FL-LNs examined expressed a weak level of CCL19 in HEV and LV, compared with I-LNs. B, There was trend for weaker CCL21 intensity in FL-LNs compared with I-LNs, P = 0.055. Expression of CCL21 in FL-LNs was classified as strong (50%) and weak (50%) and as strong (36%) and weak (64%) in I-LNs. C, No significant difference was observed in the expression of CXCL12 in FL-LNs compared with I-LNs. The level of expression was classified as strong (64%), moderate (7%), and weak (29%) in FL-LNs and strong (36%), moderate (14%), and weak (50%) in I-LNs.

Prognostic value of perifollicular CD8 T lymphocytes

A low count of perifollicular CD8 T lymphocytes per square millilmeter was a poor prognostic factor for overall survival of patients with FL (results not shown). γδ T lymphocytes number and CCL19 expression had no prognostic value (results not shown).

Discussion

The aim of this study was to investigate the frequency and localization of γδ T lymphocytes in tumor LNs of patients with FL. We document here a lower count of γδ T lymphocytes in FL-LNs than I-LNs.

Our finding is consistent with few other studies demonstrating very low levels of intratumoral γδ T lymphocytes in renal cell carcinoma tumors. A possibility is that γδ T lymphocytes dye soon after interaction with tumor cells as previously suggested (33–35). If such intratumor death of γδ T lymphocytes does occur, injection of ex vivo expanded γδ T lymphocytes could correct the deficiency in intratumor γδ T lymphocytes. Furthermore, ex vivo activation with BrHPP and IL-2 might confer to γδ T lymphocytes some resistance to apoptosis and increase their tumoricidal effect.

We report that the great majority of FL-LNs or I-LN γδ T lymphocytes are CCR7+, unlike peripheral blood γδ T lymphocytes, whereas expression of CCL19 chemokine was 3-fold lower in FL-LNs than in I-LNs. CCL21 and CXCL12 immunostaining did not reveal significant differences in their expression between the two groups. These chemokines are likely to be synthesized by stromal cells, particularly fibroblastic reticular cells in the T cell zone as previously described and then to migrate to HEV/LV (36–38). Fibroblastic reticular cell networks are phenotypically and probably functionally altered during FL development, eventually contributing to immune suppression (38). Therefore, the low γδ T lymphocyte count in FL-LNs could be explained in part by a defect in CCL19 and chemoattraction. Immunostaining of CXCL12 revealed that CXCL12+ cells are stromal cells surrounding HEV and LV. CXCL12 could be a pivotal factor in the recruitment of malignant germinal center-derived B cells (29, 39). CXCL12 can also promote tumor cell survival and proliferation.

Given the low γδ T lymphocyte count in FL-LNs, it should be important to assess whether in vitro-expanded circulating γδ T lymphocytes injected into FL patients are able to home in tumor LNs. In this study, we report that peripheral blood γδ T lymphocytes from FL patients can be easily expanded using BrHPP and IL-2. However, only a very low percentage of circulating γδ T lymphocytes (1.7% in healthy donors) express CCR7, and this value does not much change after expansion (1.2%). Because previous reports have mentioned CCR7 expression in a subset of peripheral blood γδ T lymphocytes in association with a memory function (18), we confirmed our findings with two different PE-conjugated anti-CCR7 mAb clones and acquiring at least 3000 events in the γδ T lymphocyte gate. In addition, anti-CCR7 labeling was performed using whole blood to show that the mAb used efficiently labeled non-γδ T lymphocytes together with the few CCR7+ γδ T lymphocytes. The efficacy of our anti-CCR7 mAb was further checked using in vitro-generated mature dendritic cells (40). Finally, a small number of αβ T lymphocytes were expanded concomitantly with γδ T lymphocytes, and some of them showed a good labeling with the anti-CCR7 mAb, further indicating the efficacy of the anti-CCR7 mAb. Treatment of in vitro expanded γδ T lymphocytes with the PGE2, known to induce CCR7 in dendritic cells (40), did not significantly increase CCR7 expression. This lack of CCR7 on expanded γδ T lymphocytes questions about the ability of ex vivo-expanded γδ T lymphocytes, infused in vivo, to reach tumor sites. We show that ex vivo-expanded γδ T lymphocytes express CXCR4 and may be thus recruited by CXCL12 as previously described in multiple myeloma (10). Given the remarkable localization of CXCL12+ stromal cells around HEV in FL-LNs, one could expect that some ex vivo-expanded CXCR4+ γδ T lymphocytes, infused in vivo, should be recruited into the tumor sites and kill tumor cells (41). Ex vivo expanded γδ T lymphocytes have been shown to kill efficiently various tumors such as multiple myeloma (10), renal carcinoma (15), and colon carcinoma (14). In monkeys, BrHPP associated with low IL-2 doses was shown to induce a strong activation and amplification of γ9δ2 T lymphocytes accompanied by the production of considerable amounts of cytokines, with no associated toxicity (16). A phase I study of Innacell γδ autologous-expanded γ9δ2 T lymphocytes in combination with IL-2 supports the therapeutic value of γδ T lymphocytes for patients with metastatic renal cell carcinomas (42). Our group carried out a phase I–II trial in which γδ T lymphocytes expanded in vivo with Phosphostim and IL-2 were administered to patients with relapsing FL to increase their cytotoxicity toward tumor cells. This trial has determined the feasibility of this approach, and presently, we are assessing the clinical response (43).

In conclusion, this study provides some rationale to further develop γδ T lymphocyte therapy, either by using ex vivo-expanded cells or by promoting γδ T lymphocytes expansion in vivo. It should be now important to determine whether expanded γδ T lymphocytes can home at the tumor site and initiate tumor killing. As evidenced in tumor peptide vaccination trials, even a minor intratumor cell lysis can promote tumor Ag processing and presentation to T lymphocytes. Recent imaging technique developments that allow tracing activated immune cells in vivo (44, 45) could be of major use to investigate γδ T lymphocytes homing in FL with noninvasive techniques.

Acknowledgments

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported in part by grants from the Goelams Group.

  • Abreviations used in this paper:

    FL
    follicular lymphoma
    BrHPP
    BromoHydrin PyroPhosphate
    FL-LN
    follicular lymphoma lymph node, HD, healthy donor
    HEV
    high endothelial venule
    I-LN
    inflammatory lymph node
    LV
    lymphatic vessel
    REAL
    Revised European-American Classification of Lymphoid Neoplasms.

  • Received June 30, 2009.
  • Accepted October 21, 2009.

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