of February 13, 2018.This information is current as Compared with Inflammatory Lymph Nodes

Decreased in Tumor Lymph Nodes with Follicular Lymphoma, Whereas It IsExpandable in Peripheral Blood of Patients

T Lymphocytes Count Is Normal andδγ

Valérie Costes, Bernard Klein and Jean-François RossiSquiban,Sylvie Lafaye de Micheaux, Hélène Sicard, Patrick

Mounia Sabrina Braza, Anouk Caraux, Thérèse Rousset,

http://www.jimmunol.org/content/184/1/134doi: 10.4049/jimmunol.0901980November 2009;

2010; 184:134-140; Prepublished online 30J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

gd T Lymphocytes Count Is Normal and Expandable inPeripheral Blood of Patients with Follicular Lymphoma,Whereas It Is Decreased in Tumor Lymph Nodes Comparedwith Inflammatory Lymph Nodes

Mounia Sabrina Braza,* Anouk Caraux,* Therese Rousset,† Sylvie Lafaye de Micheaux,‡

Helene Sicard,‡ Patrick Squiban,‡ Valerie Costes,*,†,x Bernard Klein,*,x,{ and

Jean-Francois Rossi*,x,‖

gd 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 gd 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). gd and CD8 T lymphocytes

were less abundant in FL-LNs than in I-LNs (p £ 1027). These lymphocytes were localized in the perifollicular zone outside of the

tumor follicles. Perifollicular gd T lymphocytes expressed CCR7, in contrast to peripheral blood gd T lymphocytes and both

perifollicular and peripheral blood gd T lymphocytes expressed CXCR4. The very low number of perifollicular gd T lympho-

cytes 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 gd 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, gd T lymphocytes can be an

attractive source for adoptive immunotherapy in patients with FL, providing they may home in tumor LNs. The Journal of

Immunology, 2010, 184: 134–140.

Follicular lymphoma (FL) is characterized by the presenceof the t (14;18) (q32;q21) translocation in virtually allpatients. This leads to deregulation of the BCL-2 gene with

overproduction of the antiapoptotic BCL-2 protein (1). A sub-stantial body of evidence supports the hypothesis that the BCL-2/IgH translocation is necessary, but not sufficient, to cause FL. Geneexpression profiling of FL has revealed that the molecular char-acteristics of nonmalignant tumor–infiltrating immune cells havea major influence on survival. Indeed, two survival-associated genesignatures have been found. One, termed “immune response–2,” isan indicator of immune infiltration, mainly by macrophages and/ordendritic cells, and is associated with a bad prognosis and short

survival. The other one, defined as “immune response–1,” ischaracterized by a complex mixture of immune cells, such asT cells, and is associated with good prognosis and long survival (2–4). In the group of patients with the “immune response–1” signa-ture, two categories of CTLs—CD8 and gd T lymphocytes—couldbe of importance. CD8 T lymphocytes are able to kill efficientlytumor cells upon direct recognition of peptide Ags presented by thetumor cell MHC class I molecules (5). Cytotoxic gd T lymphocytesexhibit an innate reactivity to MHC-unrestricted microbial andtumor nonpeptidic phosphoantigens, which leads to proliferation,release of Th1 cytokines, and perforin-mediated killing (6–9). gd Tlymphocytes can be activated directly in vitro by py-rophosphomonoesters (10–12) like bromohydrin pyrophosphate(BrHPP/Phosphostim). Phosphostim is a synthetic g9d2 TCR ag-onist that mimics the biological properties of natural phos-phoantigens found in hydrosoluble mycobacterial extracts (13).Phosphostim and IL-2 allow the selective outgrowth of peripheralblood gd T lymphocytes from patients with renal colon carcinomaand multiple myeloma, which are highly cytotoxic toward autol-ogous primary tumor cells (10, 14, 15). Moreover, Phosphostimand IL-2 infusions in nonhuman primates trigger a transient butlarge expansion of circulating g9d2 T lymphocytes and a highproduction of Th1 cytokines (16).Traffic patterns and the inflammatory function of leukocytes, such

asgdT lymphocytes, are largely definedby their chemokine receptorexpression (17–19). Ag-driven T cell priming and differentiationinduce sequential changes in chemokine responsiveness (20–23).Chemokines are small proteins that play important roles in leukocytemigration, activation and degranulation (24). They are dividedinto two major subfamilies, “homeostatic” and “inflammatory”

*INSERM; †CHU Montpellier, Centre Hospitalier Universitaire de Montpellier, De-partment of Pathology; xUniversite Montpellier1, UFR Medecine, Unite de Forma-tion et de Recherche Medecine; {CHU Montpellier, Centre Hospitalier Universitairede Montpellier, Institute of Research in Biotherapy; ‖CHU Montpellier, Centre Hos-pitalier Universitaire de Montpellier, CIC BT, Centre d’Investigation Clinique Bio-therapie 509, Montpellier, Cedex 5; and ‡Innate Pharma, Marseille, 13009, France

Received for publication June 30, 2009. Accepted for publication October 21, 2009.

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

Address correspondence and reprint requests to Prof. Bernard Klein, INSERM, Unite847, Institute of Research in Biotherapy, 80 Avenue Augustin, Fliche 34295, Mont-pellier, Cedex 5, France. E-mail address: bernard.klein@inserm.fr; or Prof. JeanFrancois Rossi, Centre d’Investigation Clinique Biotherapie 509, Centre HospitalierUniversitaire, 375 Avenue du Doyen Giraud, Montpellier Cedex 5, France. E-mailaddress: jf-rossi@chu-montpellier.fr

Abreviations used in this paper: FL, follicular lymphoma; BrHPP, BromoHydrinPyroPhosphate; 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.

Copyright� 2009 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901980

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chemokines, and their distinctive roles are best exemplified inmatureT cells (20–23, 25). The homeostatic chemokines SLC/CCL21 andELC/CCL19 are produced by stromal cells in T cell zones of sec-ondary lymphoid tissues (26, 27) and selectively attract CCR7-bearing lymphocytes. These chemokines binds to the same receptorCCR7 (28). SDF1/CXCL12 is also a homeostatic chemokine ex-pressed in peripheral lymphoid tissues that enhances themigration ofnormal and malignant B lymphocytes and plays a role in the localdissemination of tumor cells (29). CCL21, CCL19, andCXCL12 areexpressed in high endothelial venules (HEVs) and lymphatic vessels(LVs) (30).In this study, we found that gd and CD8 T lymphocytes are lo-

calized in the perifollicular zone of FL lymph nodes (FL-LNs) andthat their count is lower than in inflammatory LNs (I-LNs). Becausemost LN gd Tlymphocytes expressed CCR7, unlike peripheralblood gd T lymphocytes, their decrease in FL-LNs may be ex-plained by the decreased expression of the CCR7-targeting CCL19chemokine in FL-LNs compared with I-LNs. In addition, the countof circulating gd T lymphocytes from FL patients was normal andthey could be easily expanded with Phosphostim and IL-2.

Materials and MethodsPatients

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

Patients’ samples and immunohistological analysis

Immediately after biopsy, a part of each FL-LNs or I-LNs was fixed informalin and Bouin’s fixative and paraffin embedded. The other part wasfrozen in liquid nitrogen and stored at 280˚C. Immunohistochemicalstaining with anti-gd T lymphocytes mAb (Immu510; Beckman Coulter,Paris, France) or anti-CD8 T lymphocytes mAb (clone C8/144 B; Dako-Cytomation, Paris, France) was done to determine the frequency, distri-bution, and topography of gd and CD8 T lymphocytes in 51 FL-LNs and28 I-LNs. CCL19, CCL21, and CXCL12 expression was determined inHEVs and LVs of 14 FL-LNs or I-LNs with anti-CCL19, -CCL21, or-CXCL12 mAbs (R&D Systems, Lille, France). Immunohistochemicalanalysis was performed on paraffin or frozen sections. Frozen sectionswere air-dried and fixed in cold acetone at room temperature for 10 min.After 30 min of incubation with blocking serum solution (DakoCytoma-tion) and Cas block (Zymed, Clinisciences, Paris, France), slides wereincubated with the primary mAb for 45 min. After washing, the bio-tinylated secondary Ab was added for 10 min and slides were rinsed.Three percent of H2O2 in methanol was used to inactivate the internalperoxidase 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 werecounterstained with blue hematoxylin. Labeled gd and CD8 T lympho-cytes were counted with an optical microscope (Laborluxe 12; Leica,Bannockburn, IL) in 14 random 2.25-mm2 fields. The median cell numberwas calculated in these 14 fields for every sample and for every stainingand was expressed as cell number per square millimeter. HEV and LVcells stained with anti-CCL19, -CCL21, and -CXCL12 mAbs werequantified similarly, and the level of expression was semiquantified usingthe following criteria (0 = negative; 1 = weak; 2 = moderate; and 3 =strong staining). Quantification of CCR7+ gd T in FL-LNs and I-LNs wasperformed by double immunostaining using anti-CCR7 (Clinisciences)and anti-gd T mAbs (Immu510; Beckman Coulter). CXCR4 im-munostaining in I-LNs could not be performed because cryopreserved I-LN samples were not available in our tissue bank.

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

PBMCs were isolated from fresh blood samples of 34 FL patients usingFicoll-Paque PLUS (Amersham Biosciences, Paris, France) and cultured ata density of 106 cells/ml in 24-well culture plates at 37˚C in 5% CO2 inRPMI 1640 medium and 10% FCS. Phosphostim (3 mM BrHPP; InnatePharma, Marseille, France) and 150 U/ml IL-2 (Proleukin; Chiron, Basel,Switzerland) were added for 8 and 14 d. Phosphostim was added once atthe onset of the culture, and then half of the culture medium volume wasreplaced every 3 d with fresh medium containing 150 U/ml IL-2. Theexpansion ability of gd T lymphocytes was evaluated at day 8 by de-termining the absolute counts (cell number in the culture 3 percentage ofgd T cells). According to this result, patients were classified in four groups:non responders (gd T lymphocyte fold expansion #2), weak responders(fold expansion .2 and ,8), intermediate responders (fold expansion $8and ,32), and high responders (fold expansion $32).

Immunophenotype analysis

The phenotype of T lymphocytes was evaluated at days 0 and 8 with thefollowing mAbs: PC5-conjugated anti-CD3, FITC-conjugated anti-Vd2TCR, 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 gdT lymphocytes of six healthy donors and five FL patients as well as inperifollicular gd T lymphocytes of five FL patients were determined usingthe following mAbs: PE-conjugated anti-CXCR4 (BD Pharmingen, LePont de Claix, France), PE-conjugated anti-CCR7 (clone 3D12; BDPharmingen), PE-conjugated anti-CCR7 (clone 150503; R&D Systems),

Table I. Patients’ characteristics

No. of Patients REAL Grade Sex (F/M) Mean Age (Range)

28 I 13/15 55 (25–79)8 II 6/2 58 (52–69)15 III 8/7 61 (38–92)8 IIIa 6/27 IIIb 2/5

FIGURE 1. In vitro expansion of peripheral blood

gd 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 gd T-lymphocytes were counted at the end

of the culture. A, Fold expansion of gd2 T lymphocytes

in 34 FL patients. B, Percentage of gd2 T lymphocytes

at the end of the expansion. The median is indicated by

a horizontal line.

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and FITC-conjugated anti-pan gd TCR (Beckman Coulter). Briefly, ap-propriate amounts of mAbs were added to 0.5 3 106 whole-blood cells,followed by 30 min of incubation at 4˚C. Cells were washed, red bloodcells lysed, and 50,000 total events in the lymphocyte gate were acquiredwith a FACScan4 cytometer (BD Pharmingen) and analyzed with theCellQuest software. CCR7 expression on gd T lymphocytes was evaluatedacquiring at least 3000 events in the gd T lymphocyte gate. CXCR4 im-munostaining 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 testand Mann–Whitney and Kruskal–Wallis nonparametric tests using theSPSS 10 software. The prognostic value of gd and CD8 T lymphocytecounts was evaluated with univariate Cox analysis.

ResultsNormal counts and in vitro expansion of peripheral blood gd Tlymphocytes from patients with FL

The mean count of circulating gd T lymphocytes in 34 patientswith 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). gd T lymphocytesfrom 80% of FL patients (27 of 34) could be highly expanded($32-fold at day 8, median expansion 174-fold, 84.7% gd Tlymphocytes) upon stimulation with BrHPP and IL-2 in vitro (Fig.1A, 1B). gd T lymphocytes from 18% of FL patients could beexpanded between 8- and 31-fold, and gd T lymphocytes fromonly 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 gd T lym-phocytes are present in FL patients, and they can be efficientlyexpanded using BrHPP and IL-2 in vitro.

gd and CD8 T lymphocytes are located in the perifollicularzone, and not inside follicles, in FL or I-LNs

Distribution of gd or CD8 T lymphocytes was examined in FL-LNs of 51 FL patients and in I-LNs of 28 patients with reactiveadenitis by immunohistochemistry. Patient clinical data are in-dicated in Table I. In most FL-LNs (47 of 51), gd T lymphocyteswere very rare and mostly (99.6%) localized in the perifollicularzone, which delineates the follicle contour (Fig. 2A, Table II) (theimage in Fig. 2A is from one of the four FL patients with thehighest gd T lymphocyte count to illustrate their perifollicularlocalization). Similarly, 98% of CD8 T lymphocytes were local-ized in the perifollicular zone (Fig. 2A, Table II). In I-LNs, gd andCD8 T lymphocytes were also mainly localized ($99%) in theperifollicular area (Fig. 2A, Table II).

Perifollicular gd and CD8 T lymphocyte counts are lower intumor LNs than in I-LNs

CD8 and gd T lymphocytes were then counted in 14 perifollicularfields of each FL-LNs (n = 51) and I-LNs (n = 28). Both gd andCD8 T lymphocytes were significantly reduced by 62% (p = 1.231027) and 18% (p = 0.004) in FL-LNs in comparison with I-LNs(Fig. 3A, 3B). Themedian gdT lymphocyte count was 18 cells/mm2

in FL-LNs and 47.5 cells/mm2 in I-LNs. The median CD8T lymphocyte countwas 1235cells/mm2 inFL-LNs and 1503.5 cells/mm2 in I-LNs. In FL-LNs, perifollicular gd and CD8 T lym-phocyte counts were not different between patients with newlydiagnosed FL (35 of 51) (48% of REAL grade I, 17% of gradeII, and 35% of grade III) and patients (16 of 51) with relapsingFL (p = 0.65) (63% of grade I, 19% of grade II, and 18% ofgrade III). The frequency of gd and CD8 T lymphocytes was notinfluenced by the FL grade (p = 0.5) either.

CCL19 is weakly expressed in FL-LNs

The lower T lymphocyte counts in FL-LNs compared with I-LNscould be due to changes in chemokines able to recruit these cells.A median of 86% of gd T lymphocytes in both FL-LNs and I-LNs expressed CCR7, the LN chemokine receptor (Fig. 4A).

FIGURE 2. Immunohistochemistry staining of FL- or I-LNs. A, Peri-

follicular distribution of gd and CD8 T lymphocytes in tumor LNs of FL pa-

tients (left panels, FL-LNs) or reactive adenitis (right panels, I-LNs) (for gd T

lymphocytes topography, we chose one of the rare patients with high gd T

lymphocyte count to show the perifollicular zone) (magnification 310). B,

Counting gd and CD8T lymphocytes. To quantify gd 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 gd T lymphocytes in FL-

LNs (left panels) and I-LNs (right panels) (magnification320).Bottompanels:

Immunostaining of CD8 T lymphocytes in FL-LNs (left panels) and I-LNs

(right panels) (magnification 340). The median counts of gd or CD8 T-lym-

phocytes 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) (magnification310). The median counts

of CCL19+ cells in the 14 FL-LN or I-LN samples are indicated in each panel.

136 LYMPH NODE gd T CELLS ARE RARE IN FL TUMORS

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Conversely, only 1.2 6 0.7% of circulating gd T lymphocytesfrom FL patients (n = 5) expressed CCR7 and 1.7 6 0.8% inhealthy individuals (n = 6). This low CCR7+ gd T lymphocyteswas evaluated acquiring at least 3000 events in the gd T lym-phocyte gate. The anti-CCR7 mAb used (clone 3D12; BDPharmingen) efficiently labeled some non-gd T cells present inthe peripheral blood (Fig. 4B), and the lack of CCR7 expressionin circulating gd T lymphocytes was confirmed with a secondPE-conjugated anti-CCR7 mAb (clone 150503; R&D Systems)(results not shown). The percentage of circulating CCR7+ gd Tlymphocytes did not increase after in vitro expansion withBrHPP and IL-2 (0.2 6 0.1% in FL patients, n= 5; and 1.26 0.6%in healthy donors, n = 6) (Fig. 4A), whereas some ab T lympho-cytes that were expanded together with gd T lymphocytes wereCCR7+ (Fig. 4B). Moreover, 84% of gd T lymphocytes in FL-LNsand 62% of circulating gd T lymphocytes from FL patients ex-pressed the chemokine receptor CXCR4. After expansion, CXCR4expression decreased ofz50% in circulating gd T lymphocytes atday 14 (p = 0.001) (Fig. 4C). CXCR4 expression could not beassessed in I-LNs, because CXCR4 immunolabeling can be car-ried out only on cryopreserved samples that were not available inour tissue bank.The CCL19 and CCL21 chemokines (CCR7 ligands) were

expressed mainly in T cell zones and CXCL12 (CXCR4 ligand) inboth T and B cell zones of 14 randomly chosen perifollicular fieldsfrom 14 FL-LNs and 14 I-LNs. CCL19 and CCL21 were expressedin HEVs and LVs. Specifically, the number of HEV/LVexpressingCCL19 was 3-fold lower in FL-LNs than in I-LNs (p = 2 3 1027)(Figs. 2C, 5A). Moreover, the intensity of CCL19 labeling wasreduced in FL-LNs (100% weak) in comparison with I-LNs (50%strong and 50% weak) (Figs. 2C, 5A). The number of cells ex-pressing CCL21 and the intensity staining for CCL21 wereslightly lower in FL-LNs versus I-LNs without reaching signifi-cance (p = 0.055) (Fig. 5B), whereas CXCL12 expression was notdifferent between FL-LNs and I-LNs (p = 0.4) (Fig. 5C). CXCL12was strongly expressed by stromal cells surrounding HEVs or LVsand diffuse in the perifollicular and tumor areas (data not shown).

Prognostic value of perifollicular CD8 T lymphocytes

A low count of perifollicular CD8 T lymphocytes per square mil-lilmeter was a poor prognostic factor for overall survival of patientswith FL (results not shown). gd T lymphocytes number and CCL19expression had no prognostic value (results not shown).

DiscussionThe aim of this study was to investigate the frequency and lo-calization of gd T lymphocytes in tumor LNs of patients with FL.We document here a lower count of gd T lymphocytes in FL-LNsthan I-LNs.Our finding is consistent with few other studies demonstrating

very low levels of intratumoral gd T lymphocytes in renal cellcarcinoma tumors. A possibility is that gd T lymphocytes dyesoon after interaction with tumor cells as previously suggested(33–35). If such intratumor death of gd T lymphocytes does oc-cur, injection of ex vivo expanded gd T lymphocytes could cor-rect the deficiency in intratumor gd T lymphocytes. Furthermore,ex vivo activation with BrHPP and IL-2 might confer to gd Tlymphocytes some resistance to apoptosis and increase their tu-moricidal effect.We report that the great majority of FL-LNs or I-LN gd T

lymphocytes are CCR7+, unlike peripheral blood gd T lympho-cytes, whereas expression of CCL19 chemokine was 3-fold lowerin FL-LNs than in I-LNs. CCL21 and CXCL12 immunostainingdid not reveal significant differences in their expression betweenthe two groups. These chemokines are likely to be synthesized bystromal cells, particularly fibroblastic reticular cells in the T cellzone as previously described and then to migrate to HEV/LV (36–38). Fibroblastic reticular cell networks are phenotypically andprobably functionally altered during FL development, eventuallycontributing to immune suppression (38). Therefore, the low gd Tlymphocyte count in FL-LNs could be explained in part by a de-fect in CCL19 and chemoattraction. Immunostaining of CXCL12revealed that CXCL12+ cells are stromal cells surrounding HEVand LV. CXCL12 could be a pivotal factor in the recruitment ofmalignant germinal center-derived B cells (29, 39). CXCL12 canalso promote tumor cell survival and proliferation.Given the low gd T lymphocyte count in FL-LNs, it should be

important to assess whether in vitro-expanded circulating gd Tlymphocytes injected into FL patients are able to home in tumorLNs. In this study, we report that peripheral blood gd T lym-phocytes from FL patients can be easily expanded using BrHPPand IL-2. However, only a very low percentage of circulating gd Tlymphocytes (1.7% in healthy donors) express CCR7, and thisvalue does not much change after expansion (1.2%). Becauseprevious reports have mentioned CCR7 expression in a subset ofperipheral blood gd T lymphocytes in association with a memoryfunction (18), we confirmed our findings with two different PE-

FIGURE 3. Significant decrease in gd and

CD8 T lymphocytes in FL-LNs compared

with I-LNs. For each LN, gd 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 gd (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 percen-

tiles and the centerline the median.

Table II. Distribution of gd and CD8 T lymphocytes in perifollicularand follicular zones

gd T Lymphocytes CD8 T Lymphocytes

n = 10Perifollicular

ZoneFollicularZone

PerifollicularZone

FollicularZone

FL-LN 99.6 6 0.3% 0.4 6 0.1% 98 6 1.0% 2 6 1.0%I-LN 99.5 6 0.5% 0.5 6 0.2% 99 6 0.4% 1 6 0.4%

To evaluate the percentage of gd and CD8 T lymphocytes in the perifollicular andfollicular zones, 14 fields (2.25 mm2) per LN were chosen randomly and the numbersof labeled gd or CD8 T lymphocytes per field were determined.

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conjugated anti-CCR7 mAb clones and acquiring at least 3000events in the gd T lymphocyte gate. In addition, anti-CCR7 la-beling was performed using whole blood to show that the mAbused efficiently labeled non-gd T lymphocytes together with thefew CCR7+ gd T lymphocytes. The efficacy of our anti-CCR7mAb was further checked using in vitro-generated mature den-dritic cells (40). Finally, a small number of ab T lymphocyteswere expanded concomitantly with gd T lymphocytes, and someof them showed a good labeling with the anti-CCR7 mAb, fur-ther indicating the efficacy of the anti-CCR7 mAb. Treatment ofin vitro expanded gd T lymphocytes with the PGE2, known toinduce CCR7 in dendritic cells (40), did not significantly in-crease CCR7 expression. This lack of CCR7 on expanded gd Tlymphocytes questions about the ability of ex vivo-expanded gdT lymphocytes, infused in vivo, to reach tumor sites. We showthat ex vivo-expanded gd T lymphocytes express CXCR4 andmay be thus recruited by CXCL12 as previously described inmultiple myeloma (10). Given the remarkable localization ofCXCL12+ stromal cells around HEV in FL-LNs, one could ex-pect that some ex vivo-expanded CXCR4+ gd T lymphocytes,infused in vivo, should be recruited into the tumor sites and killtumor cells (41). Ex vivo expanded gd T lymphocytes have beenshown to kill efficiently various tumors such as multiple mye-loma (10), renal carcinoma (15), and colon carcinoma (14). Inmonkeys, BrHPP associated with low IL-2 doses was shown toinduce a strong activation and amplification of g9d2 T lympho-

cytes accompanied by the production of considerable amounts ofcytokines, with no associated toxicity (16). A phase I study ofInnacell gd autologous-expanded g9d2 T lymphocytes in com-bination with IL-2 supports the therapeutic value of gd T lym-phocytes for patients with metastatic renal cell carcinomas (42).Our group carried out a phase I–II trial in which gd T lympho-cytes expanded in vivo with Phosphostim and IL-2 were ad-ministered to patients with relapsing FL to increase theircytotoxicity toward tumor cells. This trial has determined thefeasibility of this approach, and presently, we are assessing theclinical response (43).In conclusion, this study provides some rationale to further

develop gd T lymphocyte therapy, either by using ex vivo-ex-panded cells or by promoting gd T lymphocytes expansion in vivo.It should be now important to determine whether expanded gd Tlymphocytes can home at the tumor site and initiate tumor killing.As evidenced in tumor peptide vaccination trials, even a minorintratumor cell lysis can promote tumor Ag processing and pre-sentation to T lymphocytes. Recent imaging technique develop-ments that allow tracing activated immune cells in vivo (44, 45)could be of major use to investigate gd T lymphocytes homing inFL with noninvasive techniques.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 4. CCR7 and CXCR4 are strongly expressed in LN but not in circulating gd T lymphocytes. The percentage of CCR7+ or CXCR4+ gd 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 gd T lymphocytes from FL-LNs (s, n = 15), I-LNs (d, n = 15), and from peripheral blood of five FL

patients (4, N) and 6 HDs (:, n) at days 0 and 14 after expansion. CCR7+ gd T cells were determined by gating gd T lymphocytes within PBMCs. B,

Percentage of CCR7+ gd T lymphocytes in the peripheral blood of one FL patient at day 0 or that of gd T lymphocytes expanded in vitro with IL-2 and

BrHPP for 14 d. gd T lymphocytes were labeled with a FITC-conjugated pan-gd 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+ gd T lymphocytes were determined by analyzing at least 3,000 events in the gd 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 gd T lymphocytes from FL-LNs (s) and from peripheral

blood of FL patients and HDs (4, :) and in expanded gd T at day 14 (N, n).

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