Follicular B Lymphomas Generate Regulatory T Cells Via the ICOS

Published OnlineFirst May 31, 2016; DOI: 10.1158/0008-5472.CAN-15-0589

Cancer Microenvironment and Immunology Research

Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy Kieu-Suong Le1,2, Marie-Laure Thibult1,3, Sylvain Just-Landi3, Sonia Pastor1, Francoise¸ Gondois-Rey1, Samuel Granjeaud1, Florence Broussais3, Reda Bouabdallah3, Renaud Colisson4, Christophe Caux4, Christine Men etrier-Caux 4, Dominique Leroux5, Luc Xerri1,2,3, and Daniel Olive1,2,3

Abstract

The prognosis of follicular lymphoma (FL) patients is sus- not only conventional T cells, but also FL B cells. These FL B pected to be influenced by tumor-infiltrating regulatory T cells cells were able to express ICOSL in vitro andtogenerate (Treg). The mechanism of Treg enrichment in FL and their impact CD25highFoxP3high Tregs expressing ICOS. Treg generation was on malignant FL B cells remains to be elucidated. We analyzed associated with ICOS/ICOSL engagement and was abrogated by 46 fresh lymph node biopsy samples, including FL (n ¼ 20), antagonist anti-ICOS and anti-ICOSL antibodies. Interactions diffuse large B-cell lymphoma (n ¼ 10), classical Hodgkin lym- between Tregs and FL B cells resulted in ICOSL downregulation phoma (n ¼ 9), and reactive lymphadenitis (n ¼ 7). Using on FL B cells. Our results highlight a key role for Tregs in FL multicolor flow cytometry and cell sorting, we observed an pathogenesis and suggest that targeting the ICOS/ICOSL path- accumulation of CD25highCD127low/neg Tregs in FL tissues. These way may be a promising immunotherapy for FL treatment. þ Tregs comprised activated ICOS Tregs that were able to suppress Cancer Res; 76(16); 4648–60. 2016 AACR.

appears paradoxical, because Tregs from FL tissues were shown Introduction þ þ to inhibit the function of antitumor CD4 and CD8 T cells Follicular lymphoma (FL) is usually considered as an indolent (14–16). Of note, a similar favorable influence of Tregs has been disease, but some patients present resistance to treatment and/or also reported in classical Hodgkin lymphoma a lymphoma sub- transformation into aggressive lymphoma (1). The clinical out- type in which the microenvironment is known to play a key come of FL patients was shown to be influenced by tumor- þ role (17). Nonetheless, the favorable prognostic value of Tregs in infiltrating macrophages and T cells (2–5), including CD4 FL remains controversial because it was not clearly confirmed in regulatory T cells (Treg). recent studies (18–20). Tregs comprise natural and inducible Tregs, that can modulate Tregs lack CD127 (IL7 receptor a chain) and usually express immune responses by suppressive mechanisms (6). Accumula- CD25 (IL2 receptor a chain), GITR (glucocorticoid-induced tion of Tregs was found in various hematologic and nonhema- TNFR-related protein), CTLA-4 (cytotoxic T lymphocyte associ- tologic malignancies (6). In contrast with solid tumors, in which ated antigen 4), and CD45RO (21). Expression of the transcrip- Tregs suppress antitumor immunity and promote tumor progres- þ tion factor FoxP3 (forkhead box P3) is considered as a crucial sion (7–10), high amounts of intratumoral FoxP3 Tregs were feature for the development and function of Tregs (21). Lympho- associated with a favorable outcome in FL (11–13). This result ma B cells produce the CCL22 chemokine inducing Treg recruitment (15) and converting conventional T cells (Tconv) to Tregs (22–24). Cell contacts were shown to be required for this con- 1Centre de recherche en Cancerologie de Marseille, Inserm U1068/ CNRS U7258, Marseille, France. 2Aix Marseille Universite, Marseille, version, which involves CD70, CD80, and CD86 expression on B France. 3Institut Paoli – Calmettes, Marseille, France. 4Centre de cells (22, 24). Recherche en Cancerologie de Lyon, Inserm U1052/CNRS 5286, Lyon, – 5 ICOS (inducible T cell costimulator, CD278)/ICOSL pathway France. Institut Albert Bonniot, Grenoble, France. driven generation of Tregs has been recently demonstrated in the Note: Supplementary data for this article are available at Cancer Research periphery (25) and microenvironment of solid tumors (26–28). Online (http://cancerres.aacrjournals.org/). ICOS, a costimulatory molecule of the CD28 family, is expressed Corresponding Author: Daniel Olive, Centre de Recherche en Cancerologie de on activated T cells. ICOS is involved in T-cell responses upon Marseille, Inserm U1068/CNRS U7258, Aix Marseille Universite, Institut Paoli - engagement with its ligand, ICOSL (ICOSLG, B7-H2, CD275), Calmettes, 27 Bd Le€ Roure, 13009 Marseille, France. Phone: 33-486-977-271; which is normally expressed on B cells, dendritic cells, and Fax: 33-486-977-499/498; E-mail: [email protected] monocytes (29). ICOS expression confers an activated phenotype doi: 10.1158/0008-5472.CAN-15-0589 and a strong suppressive capacity to intratumoral Tregs (30). 2016 American Association for Cancer Research. Particular Tregs displaying strong expression of ICOS and CXCR5

4648 Cancer Res; 76(16) August 15, 2016

ICOS Costimulation of Tregs in Follicular Lymphoma

þ and inhibiting CD4 effector T cells were recently identified in FL evaluation committee (CECCAP ethical committee agreement tissues, and called follicular regulatory T cells (TFR; ref. 31). This no. CECCAPP-2013-010 and registered at the MESR, #02312.01). subset is reminiscent of TFR observed in normal follicles in mice Mice were injected with either anti-mICOS mAb (17G9, rat (32, 33). Nonetheless, the hypothesis of a direct suppressive IgG2) or isotype control (BioXcell; 100 mg/mL) by i.p. route and function of Tregs on FL B cells has not been fully investigated. sacrificed after 24 hours or 48 hours. Blood was collected by These observations prompted us to clarify the role of Tregs in FL, intracardiac function, and lymph node and spleen were subjected using a combination of flow cytometry, cell sorting, and IHC on to mechanical disaggregation to obtain homogenous single-cell tissue samples. As a control group, we analyzed tissue samples suspension. Membrane multiparametric flow cytometry stainings from not only benign lymphadenitis, but also lymphoma sub- (CD45-AF-700, CD3-APC-H7, CD19-BV605, ICOS-L-PE, and types in which the microenvironment is suspected to play a live/dead Aqua) were performed to evaluate ICOS expression on þ significant role, especially cHL, and to a lesser extent diffuse large CD19 B cells after gating on viable cells (live/dead Aquaneg). A B-cell lymphoma (DLBCL). multistaining including an IgG2a-PE in place of anti-ICOSL Mab (FMO) was used as a negative control. Flow cytometry analyses Materials and Methods were performed on an LSR-II (Becton Dickinson), and results were analyzed using FlowJo software. Patients Fresh biopsy samples from 46 patients were collected at diagnosis. Samples were classified as FL (n ¼ 20), DLBCL (n ¼ 10), cHL T-cell proliferation and suppression assays (n ¼ 9), and reactive lymphadenitis (rL; n ¼ 7) according to the To study proliferative capacity, CellTrace violet (Life Technol- – World Health Organization classification (IARC, Lyon, 2008). ogies) labeled Tregs and Tconv were activated by CD3/CD28 DLBCL cases were considered as de novo cases, due to the lack of dynabeads (Life Technologies; 1 bead for 1 cell) in the presence of recombinant human IL2 (rIL2, 100 IU/mL; Chiron) for 5 days. To previous history of FL and absence of FL histologic features. All þ patients gave informed consent and the study was approved by the evaluate the suppressive effects of Tregs on CD4 Tconv, CellTrace – ethical board of the Paoli-Calmettes and Albert Bonniot institute. violet labeled Tconv were activated by CD3/CD28 dynabeads in the presence of rIL2 (100 IU/mL) and Tregs for 4 days. Cells were Cell lines harvested and stained with anti-CD2-PECF-594 and live/dead Burkitt lymphoma cell line Raji and COS-7 cells were obtained near-IR. Proliferation was evaluated by Division index (DI) using from American Type Culture Collection (ATCC). Raji and COS-7 the proliferation tool of FlowJo software. Supernatants of cocul- cells were respectively cultured in RPMI 1640 Medium (Life ture were collected and IFNg was further measured by ELISA (BD Technologies) supplemented with 10% fetal calf serum (FCS; Biosciences). Lonza) and in DMEM (Life Technologies) supplemented with 10% FCS. These cell lines were not authenticated at the time we B-cell suppression assay performed the study. FL B cells purified from tissues were stained with CellTrace violet and were cultured with autologous or allogeneic Tregs Cell isolation and purification (1/1 ratio) in the presence of rIL2 (100 IU/mL) for 5 days. Sample tissues were mechanically disrupted and passed Anti-IgA/G/M (10 mg/mL; Jackson ImmunoReseach) and CpG- through a nylon filter (BD Bioscience) to obtain mononuclear B(2mg/mL; Invivogen) were added to stimulate FL B cells as – cells (MC), which were frozen or used immediately for functional previously described (34). To study T-cell dependent suppres- assay. sion of Tregs, an equal number of Tconv and/or Tregs were added þ B cells and CD4 T cells were obtained from MCs by negative to stimulated B cells. Supernatant of coculture was collected to selection (Stemcell Technologies). To isolate Tregs and Tconv, measure IL6 by ELISA (R&D Systems). þ purified intratumoral CD4 T cells were labeled with anti-CD4- PC7, anti-CD25-APC, anti-CD127-FITC, and a viability marker Kinetics of ICOSL expression on lymphoma B cells (live/dead aqua) and sorted by FACs (BD Biosciences). To sep- Purified FL B cells or total MCs were cultured in culture þ þ þ arate ICOS and ICOSneg CD4 T cells, intratumoral CD4 T cells medium. Cells were harvested and stained with live/dead aqua, were labeled with anti-ICOS-PE and live/dead aqua. anti-CD19-FITC, anti-ICOSL-PE, and anti-CD86-APC before and during culture. FL B cells were cultured overnight until ICOSL was þ Antibodies and flow cytometry expressed. Then, different ratios of intratumoral ICOS or neg þ Antibodies are detailed in Supplementary Table S1. To analyze ICOS CD4 T cells were added on the B cell culture. In some intracellular markers like FoxP3, Helios, Bcl-2, and Ki67, cells experiments, anti-ICOS–blocking antibody (314.8 clone, pro- were fixed/permeabilized after surface staining and then incubat- duced in our laboratory) or isotype control (Ctrl) IgG1 (Sigma) ed with specific antibodies using Foxp3 staining buffer set were added at 10 mg/mL. (eBioscience). Data were acquired on LSRII (BD Biosciences) and analyzed using FACS Diva and FlowJo softwares. Treg induction assay þ CellTrace violet-labeled intratumoral CD4 T cells were cul- Mice model tured alone or with autologous or allogeneic FL B cells expressing Na€ve wild-type FVB/N (Charles River Laboratory) were used at ICOSL at 1.5/1 ratio in the presence of rIL2 (100 IU/mL). Anti- 6 weeks of age. Mice were maintained in the pathogen-free animal ICOS (314.8 clone) or anti-ICOSL (MIH.12 clone, eBioscience) facility "AniCan" at the Cancer Research Center of Lyon. Experi- blocking antibodies or isotype Ctrl IgG1 were added at 10 mg/mL ments were conducted in accordance with the European and for each. After 5 to 6 days, cells were harvested and surface stained French laws and were validated by the local animal ethical with anti-CD4-PC7, anti-CD19-PECF-594, anti-CD25-APC, anti-

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Le et al.

ICOS-PE, and live/dead near-IR before fixation/permeabilization Results and intracellular staining with FoxP3-FITC. þ – Intratumoral T cells with CD4 CD25highCD127low/ profile Cells harvested after 5 to 6 days of coculture were restimulated have characteristics of effector Tregs with PMA (10 ng/mL; Sigma) and Ionomycin (1 mg/mL, Sigma) Coexpression of CD25high and CD127low/neg was used to iden- for 4 hours. After stimulation, cells were stained with anti-CD4- tify and isolate Tregs in FL tissue samples (Fig. 1A). CD25high V450, anti-CD19-PECF-594, anti-CD25-PC7, anti-FoxP3-PE, CD127low/neg cells expressed high levels most Treg markers such as anti-IFNg-FITC, anti-IL2-APC and live/dead near-IR. FoxP3 (86% 9.1%, mean SD), Helios (84% 7.6%; Fig. 1B) and strongly expressed other Tregs markers such as GITR (72 IHC 13.4), surface CTLA-4 (32.8% 11%), and CD39 (80.2% IHC was performed on whole sections of paraffin blocks used 16.1%; Fig. 1B). Most CD25highCD127low/neg Tregs expressed for diagnosis. After dewaxing and pressure-cooker antigen retriev- CD27 (99.2% 1.1%) and were CD45RA-negative (3.2% al, mAbs directed against ICOS (clone sp98; Abcam), and FoxP3 1.6%; Fig. 1B), suggesting a memory T-cell phenotype. Tconv (clone 236A/E7; eBioscience) were incubated in an automated þ were identified as CD25neg/low among viable CD4 T cells (Fig. immunostainer (Dako) using a standard avidin biotin peroxidase 1A). When compared to Tconv, purified CD25highCD127low/neg technique. Double staining experiments were performed using Tregs had a low proliferation rate in response to CD3/CD28 the K5361 Envision G/2 Doublestain System (Dako) according to stimulation (87% and 10% for Tconv and Tregs, respectively; the supplier's instructions. ICOSL IHC was performed on frozen Fig. 1C). Tconv proliferation and IFNg production was strongly tissue sections using the Envision-Flex K8000 kit (Dako) and the inhibited when CD25highCD127low/neg cells were added in culture Autostainer Plus device (Dako). The anti-ICOSL mAb HIL-270 (Fig. 1D). Taken together, these results demonstrate that CD25high (provided by Pr. Richard Kroczek) was diluted at 10 mg/mL and CD127low/neg T cells have the phenotypic and functional profile of incubated for 45 minutes at room temperature. As a control for the effector Tregs. specificity of the HIL-270 ICOSL mAb, paraffin-embedded cell ICOSL blocks were constructed using -transfected COS-7 cells and þ parental COS-7 cells as negative control. Briefly, COS-7 cells Intratumoral accumulation of ICOS Tregs is a specific feature of FL transfected for 48 hours with bDNA4 vector containing full-length þ human ICOSL (produced in our laboratory) using X-tremGENE 9 The mean percentage of Tregs within CD4 T cells was signif- DNA Transfection reagent (Roche) according to the manufac- icantly increased in FL tissues as compared to rL (7.5-fold, P ¼ 0.0005), cHL (2.6-fold, P ¼ 0.003) and DLBCL (2.3-fold, P ¼ turer's instructions. After 48 hours, transfected and parental COS- þ 7 cells were detached and prefixed in 4% formaldehyde solution 0.01; Fig. 2A). Both Tregs/Tconv and Tregs/TCD8 ratios were significantly higher in FL than in other samples (Fig. 2B). ICOS and then suspended in agarose 1% LMT to form a cell-agarose þ gel, which was fixed in 4% formaldehyde solution for 24 hours. was mostly expressed in CD4 T cells, especially in Tregs (65.3% fi fi 14.4%), as compared with other tumor infiltrating lymphocytes After xation, the cell-agarose gel was xed, dehydrated, par- þ fi (TIL) like CD8 T cells, gd T cells, and natural killer cells (Fig. 2C). af n impregnated on a Leica ASP300S processor (Leica Biosys- þ þ tems), and finally embedded in paraffin using a Leica Histo- Accordingly, the percentage of ICOS Tregs among CD4 T cells fi was significantly increased in FL tumors (Fig. 2D). Like the Embedded processor. The method for ICOSL IHC on paraf n þ embedded cell blocks was similar to that described for ICOS majority of Tregs in FL, ICOS Tregs expressed CXCR5 (mean SD: 83% 7.5%; Fig. 2E). When compared to ICOSneg Tregs, and FoxP3. As expected, positive HIL-270 immunostaining was þ present only in ICOSL-transfected cells. For all antibodies, ICOS Tregs in FL tissues displayed significant upregulation of including HIL-270, a control was also done by omitting the surface CTLA-4 (40.1% 10.1%), GITR (83.6% 10.2%), CD39 primary mAb on serial sections of frozen tissues analyzed, (85% 15.9%), PD1 (90.4% 7.9%) as well as higher Ki67 expression (19.1% 7.2%; Fig. 2E). These data indicate a specific which resulted in a lack of positive signal. þ accumulation of activated and expanding ICOS Tregs in the FL Quantitative RT-PCR microenvironment. Total RNA was extracted from purified FL B cells and Burkitt lymphoma cell line Raji with TRIzol reagent (Life Technolo- ICOSL is occasionally expressed in neoplastic B cells from gies) according to the manufacturer's protocol and was then DLBCL, but is downregulated in FL B cells þ reverse transcribed into cDNA using M-MLV Reverse Transcrip- To identify a putative cell population responsible for ICOS tase (Life Technologies). Quantitative PCR was performed Tregs accumulation in fresh FL tissues, we assessed the expression using Taqman Universal PCR Master Mix and primers as of ICOSL and CD86 on FL B cells, reactive T cells, myeloid dendritic follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cells (mDC) and plasmacytoid DC (pDC). FL B cells were iden- þ Unigene reference: Hs.544577) and human ICOSL (Hs. 14155; tified as CD3neg/CD16neg/CD56neg/CD20 , because most of these Life Technologies). Gene expression was measured using the cells in FL samples were shown to be neoplastic B cells due to 7900HT Fast Real-Time PCR System (Life Technologies). For coexpression of CD10 and Bcl-2 (Supplementary Fig. S1A). pDCs þ þ þ each sample, mRNA expression was normalized to GAPDH as a were identified as lineageneg/HLA-DR /CD33neg/ /CD123 and þ þþ reference gene. mDCs as lineageneg/HLA-DR /CD33 /CD123neg (Fig. 3A). CD86 was strongly expressed on mDCs (90.6% 10.1%), pDCs Statistical analysis (21.8% 15.9%), and FL B cells (52.6% 27.3%), but ICOSL was Quantitative variables were expressed as mean SEM. Statis- weak on all cell populations (Fig. 3B) including reactive T cells tical analysis was performed with GraphPad Prism 5 software (Supplementary Fig. S1A). Surprisingly, only ICOSL expression (GraphPad software) using the Mann–Whitney and Wilcoxon was rapidly upregulated on FL B cells after a few hours of culture in nonparametric t test (, P < 0.05; , P < 0.01). vitro of purified FL B cells (Fig. 3C). ICOSL expression was weak on

4650 Cancer Res; 76(16) August 15, 2016 Cancer Research

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Le et al.

AB*** 4 40 *** 0.5 ** ** * ** * * 0.4 3 30

0.3 20 2 0.2

% Tregs/TCD4 10 1 Tregs/TCD8 Ratio

Tregs/Tconv Ratio 0.1

0 0.0 0

rL FL rL FL rL FL cHL cHL cHL DLBCL DLBCL DLBCL

C Tregs Tconv TCD8 Tγδ NK

65.3 29.5 1.7 1 0.3

ICOS

D E Gated on Tregs ** 15 ** * 12.4 68.6 5.49 52.7 5.93 51.7

10 ICOS 9.52 9.52 28 14.3 32.7 9.61 Tregs/TCD4 + 5 CXCR5 PD1 GITR

% ICOS 45.8 17.8 6.03 50.6 54.7 5.31

rL FL cHL DLBCL 34.7 1.71 19.9 23.4 38.8 1.24

CTLA-4 CD39 Ki67

Figure 2. Frequency and phenotype of Tregs in FL. MCs extracted from lymphoma and rL samples were stained with antibodies and analyzed by ﬂow cytometry. A, percentage of Tregs among CD4þ T cells in FL compared with controls. B, ratio of Tregs to Tconv (left) and to TCD8 (right) in FL compared with controls. þ C, representative histogram for ICOS expression (white) and isotype control (gray) on different lymphocytes, including Tregs, Tconv, TCD8 ,Tgd, and NK. Mean of percentage of 14 FL samples was indicated. D, percentage of Tregs expressing ICOS among CD4þ T cells in FL compared with controls. E, representative dot plots for expression of various molecules on ICOSþ Tregs and ICOSneg Tregs from FL samples (n ¼ 9–10). Mann–Whitney nonparametric t tests were performed (, P 0.05; , P < 0.01; , P < 0.001).

FL B cells after puriﬁcation (1.7% 1.6%), but it was induced by culture of total MCs (Supplementary Fig. S1B). We then examined short in vitro culture (15.1% 18.0% after 2 hours of culture) and the expression of ICOSL transcripts on puriﬁed FL B cells by increased during culture (45.1% 34.6% at 48 hours). Further- quantitative RT-PCR. The Burkitt lymphoma cell line Raji was more, ICOSL upregulation on FL B cells was also observed in vitro used as a positive control due to its high ICOSL expression detected

4652 Cancer Res; 76(16) August 15, 2016 Cancer Research

ICOS Costimulation of Tregs in Follicular Lymphoma

B cells/ A Viable cells CD3/16/56negCD20neg DCs mDC/pDC CD20 SSC-A CD33 HLA DR

Live/dead CD3 CD16 CD14 CD123 CD56

B C B cells mDC pDC 64

0.6 1.9 1.9 32 16 ICOSL 8 CD86 4 % FL B Cells 2 ICOSL 1 0.25 0.5 1 2 4 8 16 32 64 52.6 90.6 21.8 Time (h)

D ICOSL 3

CD86 2

1 mRNA (fold change)

Raji

FL B Cells

Figure 3. ICOSL and CD86 expression on immune cells in FL. MCs extracted from lymphoma samples were stained with antibodies and analyzed by flow cytometry. A, gating strategy used to identify lymphoma B cells and DCs: lymphoma B cells were identified as CD3negCD16negCD56negCD20þ. Within DCs defined as lineage (CD3/CD16/CD56/CD20/CD14)neg HLA DRþ, pDCs were CD33þ/negCD123þ, whereas mDCs were CD33þþCD123neg. B, representative histogram for ICOSL and CD86 expression (white) as compared with isotype control (gray) on mDCs, pDCs, and lymphoma B cells from 14 samples of FL. Mean of percentage was indicated. C, purified FL B cells were cultured in vitro and percentage of expression of ICOSL and CD86 were analyzed at different times (n ¼ 5). D, mRNA from purified FL B cells (n ¼ 5) and Raji cell line were extracted and expression of ICOSL transcripts was detected by quantitative RT-PCR. The amount of ICOSL transcripts on purified FL B cells was compared with value of the positive control Raji used as a calibrator.

by ﬂow cytometry (Supplementary Fig. S1C). ICOSL transcripts immunostaining was absent or faint in neoplastic follicles in the were detectable on puriﬁed FL B cells and Raji cell line but its 10 FL cases analyzed (Fig. 4). Internal positive controls were mRNA level was 2-fold higher on FL B cells than on positive control mainly located outside the B-cell follicles and often displayed Raji (Fig. 3D). In contrast to FL B cells, ICOSL was expressed on morphologic features of macrophages with large cytoplasm and lymphoma B cells from some DLBCL samples, whereas ICOS was oval nucleus (Fig. 4). Capillary venules also displayed positive absent on TILs (Supplementary Fig. S1D). signals of variable intensity (Fig. 4) in accordance with a previous þ ICOSL IHC was performed on 16 frozen samples, including report (35). A few ICOSL cells are also present in the interfolli- human tonsils (n ¼ 2), reactive lymph node (n ¼ 1), low-grade cular areas of benign tonsils and lymphadenitis, whereas normal follicular lymphoma (n ¼ 10), and DLBCL (n ¼ 3). ICOSL B cells in reactive follicles appeared mainly negative. In contrast,

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Le et al.

A B

FOL FOL

Figure 4. ICOS IHC features of ICOS, Foxp3, and ICOSL expression. Serial sections from ICOS F D a representative formalin-ﬁxed parafﬁn-embedded FL case show C the predominant intrafollicular localization of ICOSþ cells (A and B) and the presence of FoxP3þ cells within neoplastic follicles (FOL) of the same tumor area (C). C, FoxP3- positive cells were also observed FOL within interfollicular areas (star). Flow cytometry indicated that this FL tissue sample contained 35% of ICOSþ CD4þ T cells and 15% of CD25highCD127low/neg Tregs among CD4þ T cells. D, double staining FoxP3 ICOS/FoxP3 experiments highlight FoxP3/ICOS coexpression in TILs localized within E F follicles (black arrows) and intermingled with ICOSþ TILs (red arrow) and FoxP3þ TILs (brown arrow). E, ICOSL immunodetection on a frozen FL sample shows no FOL FOL detectable positivity in FL cells (FOL), whereas positive signals are seen outside neoplastic follicles, especially on venules (arrow). The negative control of the same FL case is shown in F. G and H, high-power view shows ICOSL that some ICOSL-positive cells display morphologic features of reactive G H macrophages with large cytoplasm and oval or reniform nucleus (arrows).

ICOSL

DLBCL cells displayed heterogeneous ICOSL positivity in two of experiments and were located within the follicles (Fig. 4), which three analyzed cases (data not shown). implies vicinity and possible interaction with neoplastic B cells.

þ þ Intrafollicular localization of FoxP3 and ICOS cells in FL ICOS/ICOSL interaction induced downregulation of ICOSL on cases FL B cells FoxP3 and ICOS IHC were performed on 6 FL and DLBCL cases ICOSL downregulation through ICOS binding was previously that were also analyzed by flow cytometry. IHC results confirmed described in normal murine B cells (36) and in human pDC (27). þ flow cytometry data, because numerous ICOS were present in FL We evaluated the impact of FL T cells on ICOSL expression by cases, with a predominant intrafollicular localization (Fig. 4). cocultures of conditioned FL B cells with either autologous þ þ þ Variable amounts of FoxP3 cells were also observed within intratumoral ICOS or ICOSneg CD4 T cells at different ratios þ neoplastic follicles of FL cases (Fig. 4). In addition, ICOS during 24 hours. An important decrease of ICOSL expression on þ /FoxP3 TILs could be detected in FL cases using double staining FL B cells (about 67% at 1:1 ratio after 24 hours of coculture) was

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ICOS Costimulation of Tregs in Follicular Lymphoma

ICOS+ CD4 T Cells A 4096 ICOSneg CD4 T Cells 2048

1024

512 FL B Cells (MFI) + 256 ICOSL Figure 5. 128 0 1 2 3 4 5 Kinetic for expression of ICOSL and CD86 on FL B cells in vitro culture. Purified FL B cells TCD4:B Ratio were cocultured in vitro with intratumoral T cells, and their expression of ICOSL and CD86 was evaluated by flow cytometry. B B + T B + T A, ICOSL expression on FL B cells at 24 hours B Alone B + T Ctrl IgG1 Anti-ICOS in the presence of different ratios of autologous intratumoral ICOSþ CD4þ T cells 1238 379 373 1398 or ICOSneg CD4þ T cells (n ¼ 3). B, representative dot plots for ICOSL and CD86 expression on purified FL B cells at 24 hours

in coculture with autologous intratumoral CD19 ICOSþ CD4þ T cells (CD4þ T- vs. B-cell ratio 1:1) in the presence of anti-ICOS antagonist (clone 314.8) or isotype Ctrl IgG1. Median of MFI of three independent ICOSL experiments was indicated.

93 111 123 100 CD19

CD86

þ þ observed in the presence of ICOS CD4 T cells (Fig. 5A). A high cells. The number of CD25highFoxP3high Tregs was virtually unde- level of ICOSL expression could be restored on FL B cells using an tectable without FL B cells, but clearly increased during coculture. þ antagonist anti-ICOS antibody (314.8; Fig. 5B). Similar results Their median percentage was 3.0% and 9.4% per total CD4 T were also observed in both in vitro culture of total MCs and in vivo cells and per total Tregs, respectively (P ¼ 0.03). Proliferation of using normal murine model (Supplementary Fig. S2). In contrast, Tregs in culture with FL B cells was significant, reaching 47.9% and þ þ no alteration of CD86 expression on FL B cells was observed (Fig. 20.5% of CD25highFoxP3high Tregs and CD25 FoxP3 Tregs, 5B). These data suggest that ICOS/ICOSL interaction is involved respectively (Fig. 6B, top). ICOS expression was markedly higher þ þ in downregulating ICOSL expression on FL B cells. on CD25highFoxP3high Tregs than on CD25 FoxP3 Tregs [mean fluorescence intensity (MFI): 1451 vs. 573, respectively; Fig. 6B, ICOS/ICOSL interaction induced enrichment of CD25high bottom]. After restimulation of cell suspension using PMA/iono- FoxP3high Tregs in FL mycin, intracellular cytokines were detected (Fig. 6C). FoxP3neg þ We next explored the possible causes of Treg accumulation in FL CD4 T cells strongly secreted IL2 (median 23%) and IFNg tissues. We cocultured purified FL B cells conditioned to express (median: 13.4%). In contrast, both putative Treg subsets showed þ ICOSL by in vitro culture, with intratumoral autologous or allo- weak IL2 and IFNg secretion. Median percentages among CD25 þ þ geneic CD4 T cells in the presence of rIL2 for 5 to 6 days. FoxP3 Tregs were 5.3% and 1.9% for IL2 and IFNg, respectively. þ Intratumoral CD4 T cells were labeled with CellTrace violet Median percentages among CD25highFoxP3high Tregs were 4.6% prior coculture to evaluate the division rate. We first observed and 1.1% for IL2 and IFNg, respectively. These data indicate that þ an enhancement of CD25 and FoxP3 expression on CD4 T cells both cell populations were indeed Tregs rather than activated T (data not shown). Consequently, the number of Tregs, defined by cells. Thus, FL B cells expressing ICOSL could favor intratumoral þ CD25 and FoxP3 coexpression, was significantly increased in the enrichment of ICOS Tregs. presence of FL B cells (median, 33.5% versus 20.0%; n ¼ 6; P ¼ When neutralizing anti-ICOS and anti-ICOSL mAbs were 0.03; Fig. 6A, left). These Tregs comprised two distinct subsets of added in coculture, there were not significant change in the þ þ þ þ þ CD25 FoxP3 and CD25highFoxP3high Tregs (Fig. 6A, right). percentage of CD25 FoxP3 Tregs among CD4 T cells (Fig. þ þ Median percentages of CD25 FoxP3 Tregs were 29.2% follow- 6D, left), whereas both mAbs induced a significant reduction in ing coculture with FL B cells, versus 19.9% (P ¼ 0.03) without FL B the percentage of CD25highFoxP3high Tregs (Fig. 6D, right). No

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Le et al.

impact was observed after the addition of an isotype control IgG1. 40 * A These results indicate that generation of CD25highFoxP3high Tregs expressing ICOS is strongly dependent on ICOSL expression by FL TCD4 30 TCD4 + B B cells. 0.05 2.2 30.7 35.3 20 Intratumoral Tregs directly inhibit FL B cells for both T-cell–dependent and T-cell–independent responses 10 CD25 % Tregs/TCD4% Our observation of Tregs overrepresentation in FL tumors prompted us to test their capacity to inhibit FL B cells. We selected 0 FoxP3 IL6 as one of the main B cell–produced cytokine (37). We first tested whether Tregs could act on B cells activated in a T-cell– TCD4 independent way. FL B cells were activated by CpG-B and anti-IgA/ TCD4 + B G/M prior to culture with Tregs at 1:1 ratio. We observed a significant attenuation of CD80 and CD86 expression on FL B B TCD4 TCD4 + B cells. This was evidenced by the percentage for CD80 (approxi- mately 30% of inhibition, P ¼ 0.0078, n ¼ 9) and by MFI for Tregs CD25+ FoxP3+ CD25high FoxP3high CD80 and CD86 (about 35% and 31% inhibition for CD80 and CD86, respectively (Fig. 7A). Similarly, Tregs abrogated IL6 secre- 1.7 20.2 58.7 tion in the supernatant of FL B cells (median percentage of 26% inhibition, P ¼ 0.0078, n ¼ 8; Fig. 7B). Tconv in FL samples could also be suppressed by Tregs (Fig. 1D). About a quarter (25.2% 16.1%, n ¼ 12) of these Tconv CellTrace were TFH cells characterized by ICOS and CXCR5 coexpression MFI: MFI: (data not shown). We next searched for a putative suppression 287 738 of Tconv-driven B-cell responses by Tregs. For this purpose, stimulated B cells were cocultured with Tconv, with or without Tregs at 1:1 ratio. Tconv induced a significant increase of both CD80 and CD86 expression on stimulated B cells, and of IL6 ICOS secretion (median increase of 67%, 121%, and 49% for CD80 MFI, CD86 MFI, and IL6, respectively). B-cell responses were neg + + C FoxP3 CD25 FoxP3 CD25high FoxP3high inhibited after addition of Tregs (inhibition of 44%, 53%, and 43.5 6.1 5.6 0.4 2.2 0.2 39%forCD80MFI,CD86MFI,andIL6,respectively;Fig.7C and D). Of note, there was also a tendency for Tregs to suppress B-cell IL2 proliferation, although it was not statistically significant for all 1.2 1.7 0.6 samples (Supplementary Fig. S3). These data suggest that intra- IFNγ tumoral Tregs can directly or indirectly suppress FL B-cell responses upon activation.

D 40 6 * * Discussion 30 /TCD4 Clarifying the impact of Tregs on FL pathogenesis is a critical high /TCD4 4 + issue for clinical implications. However, the lack of strictly a ﬁ 20 speci c Treg marker may result in discrepancies between studies FoxP3

FoxP3 focusing on Tregs in human. FoxP3, which is considered as the + 2 high most speciﬁc Treg marker, was reported to be transiently expressed 10 þ þ in activated CD4 and CD8 T cells (38, 39), which, in contrast to % CD25 g

% CD25 Tregs, produce effector cytokines like IL2 and IFN (23, 39, 40). 0 0 Using the CD25high/CD127low/neg combination, we could isolate T cells displaying Treg phenotypic markers and strong suppressive high low/neg high Medium MediumCtrl IgG1 Ctrl IgG1Anti-ICOS activity. Most CD25 CD127 T cells were FoxP3 and Anti-ICOSL Anti-ICOS Anti-ICOSL hardly secreted IL2 and IFNg, further supporting their Tregs' nature. Thus, our results corroborate previous investigations Figure 6. high low/neg Treg enrichment driven by FL B cells dependent on ICOS/ICOSL interaction. (41, 42) suggesting that the CD25 CD127 combination þ ﬁ CellTrace violet-labeled intratumoral CD4 T cells were cultured alone or with FL is useful for Tregs' identi cation. B cells expressing ICOSL, and cell suspension was stained for CD25, FoxP3, ICOS after 5 to 6 days of culture. A, frequency among CD4þ T cells of total Tregs þ (n ¼ 6; left) and each Treg subset from a representative case (right). B, stimulation after 5 to 6 days of coculture (n ¼ 2–4). D, percentage of CD25 þ high high þ representative histogram for proliferation (top) and ICOS expression (bottom) FoxP3 Tregs (left) and CD25 FoxP3 Tregs (right) among CD4 T cells in of CD25þFoxP3þ Tregs and CD25highFoxP3high Tregs in coculture with FL B cells. coculture with FL B cells in the presence of neutralizing anti-ICOS or C, production of intracellular IL2 and IFNg on FoxP3neg CD4þ T cells, CD25þ anti-ICOSL antibodies or isotype Ctrl IgG1. Statistical analyses were done FoxP3þ Tregs, and CD25highFoxP3highTregs in response to PMA/ionomycin using Wilcoxon nonparametric t tests ( , P < 0.05).

4656 Cancer Res; 76(16) August 15, 2016 Cancer Research

ICOS Costimulation of Tregs in Follicular Lymphoma

150 A B Alone B/Tregs B B Alone 39.4 14.1 B/Tregs ** 100

50 CD80 % Inhibition (IL6)

150 * B Alone 0 ** B/Tregs

100

50 % Inhibition (MFI)

0 CD80 CD86

CDB/Tconv/ B Alone B/Tconv 250 Tregs **** B Alone 39.4 49.6 31.7 200 B/Tconv B/Tconv/Tregs 150

100 CD80 % Inhibition (IL6) 50

500 * ** B Alone 0

400 B/Tconv * ** B/Tconv/Tregs 300

200

% Inhibition (MFI) 100

0 CD80 CD86

Figure 7. Inhibition of FL B-cell responses by intratumoral Tregs. CellTrace violet-labeled FL B cells activated by anti-IgA/G/M and CpG-B were cocultured with Tregs for direct suppression assay (A and B) or Tconv with or without Tregs for indirect suppression assay (C and D) during 5 days. A and C, representative histogram for the percentage of CD80 (white) versus isotype Ctrl (gray; top) and MFI of CD80 and CD86 expression (bottom) on FL B cells were evaluated by ﬂow cytometry (n ¼ 9). B and D, IL6 secretion in supernatant of coculture measured by ELISA (n ¼ 8). All data were normalized to "B-alone" condition that is considered as 100%. Wilcoxon nonparametric t tests were used: , P < 0.05; , P < 0.01.

In this study, we confirm using multiparameter flow cytometry CXCR5, PD1, GITR, and CTLA-4, and thus fit the profile of TFR that þ that not only Tregs, but also ICOS Tregs, are overrepresented in were described in mice and humans (32, 33) and in FL tissues as þ FL tumors. We observed that ICOS Tregs expressed high levels of well (31). Of note, we demonstrate herein that FL B cells are able

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Le et al.

þ to promote generation of these intratumoral ICOS /CD25high- well as migration of Tregs induced by CCL17- and CCL22- FoxP3high Tregs via ICOS/ICOSL interaction, which induced secreting FL B cells (55). ICOSL downregulation on FL B cells. Furthermore, we provide Survival, expansion, and suppressive functions of human evidence of a strong suppressive effect of intratumoral Tregs on Tregs are known to be strongly dependent on ICOS costimula- malignant B cells. tion in physiologic settings (25). Accordingly, we show that þ Several studies have shown a favorable outcome for FL patients coculture of ICOSL FL B cells with T cells induced generation þ with high amounts of intratumoral Tregs, although this remains of ICOS /CD25highFoxP3high Tregs. Using neutralizing antibo- controversial (18, 43). These observations support the hypothesis dies, we show that ICOS/ICOSL interaction is critical for this that Tregs could control malignant FL cells, but no demonstration induction. ICOSL expression on FL B cells was downregulated þ of such functional interaction was reported so far. In nontumoral following interaction with ICOS T cells, which may explain human tonsils, B-cell responses were shown to be inhibited by why ICOSL was undetectable in vivo. It remains, however, Tregs (44, 45). We report herein that Tregs are able to suppress the difficult to decipher what could be the primum movens of the activation process and cytokine secretion of FL B cells, either cascade of ICOS/ICOSL interactions in vivo. Whatever the directly or indirectly. triggering event could be in FL, such a mechanism is globally Tregs are known to use various mechanisms to suppress reminiscent to that described in other human cancers, in which þ immune responses, including cell-to-cell contact (45). Addi- ICOS Tregs' enrichment is triggered by ICOSL produced by tionally, Tregs are known to kill B cells through Fas–FasL pDCs or tumor cells (26–28). Thus, our findings provide a basis interaction or through the release of cytolytic molecules to target the ICOS/ICOSL pathway for the development of (46, 47). So, further study is needed to identify mechanisms future immunotherapies in FL patients. involved in direct FL B cell inhibition. The indirect suppressive effect of Tregs upon FL B cells is in accordance with a report Disclosure of Potential Conflicts of Interest showing that T cells support FL B-cell activity via IL4 and FH No potential conflicts of interest were disclosed. CD40L (31). Our data suggest that Tregs are able to inhibit IL4/ CD40L-producing T cells, probably including TFH, which could hamper FL growth. Authors' Contributions Nonetheless, the favorable prognostic value of high numbers Conception and design: K.-S. Le, S. Pastor, C. Caux, C. Menetrier-Caux, L. Xerri, of Tregs in FL tumors remains debated (11–13). In mouse D. Olive models, depletion of Tregs induced decreased lymphoma bur- Development of methodology: K.-S. Le, M.-L. Thibult, S. Just-Landi, S. Pastor, F. Gondois-Rey, C. Caux, D. Olive den (48). Unfavorable outcome was reported for FL patients Acquisition of data (provided animals, acquired and managed patients, with high levels of peripheral blood Tregs (49). A possible provided facilities, etc.): M.-L. Thibult, F. Broussais, R. Bouabdallah, R. Colis- explanation that may reconcile the latter studies with our son, D. Leroux, L. Xerri, D. Olive results may reside in the polarization of Tregs into distinct Analysis and interpretation of data (e.g., statistical analysis, biostatistics, functional subsets. Tregs expressingTbet,IRF4,STAT3,andBcl6 computational analysis): K.-S. Le, F. Gondois-Rey, S. Granjeaud, R. Colisson, appear to control Th1-, Th2-, Th17-, and T -mediated immune C. Menetrier-Caux, L. Xerri, D. Olive FH Writing, review, and/or revision of the manuscript: K.-S. Le, R. Bouabdallah, responses, respectively (32, 50–52). Therefore, it is possible þ C. Caux, C. Menetrier-Caux, L. Xerri, D. Olive that intratumoral ICOS Tregs include multiple subsets, with Administrative, technical, or material support (i.e., reporting or organizing opposite effects on tumor growth. The balance between these data, constructing databases): K.-S. Le, M.-L. Thibult, S. Just-Landi, R. Bouab- antagonist Tregs subsets may vary according to complex cues dallah, R. Colisson, D. Olive from the microenvironment. In this extent, it is noteworthy that Study supervision: L. Xerri, D. Olive we also observed a significant Tregs' increase in tissue samples Other (performed experiments): K.-S. Le from cHL, a lymphoma subtype that is known to undergo, like FL, crucial influences from the microenvironment, including a Acknowledgments suspected favorable effect of Treg accumulation. This raises the The authors thank Pr. Thierry Fest (CHU de Rennes, Rennes, France), Pr. question as to whether similar signaling networks could regu- Richard Kroczek (Molecular Immunology, Robert Koch-Institute, Berlin, Ger- late Tregs in FL and cHL. The dependence of Tregs' status on the many), Cytometry platform, Experimental Histo-Pathology (ICEP) platform, IBiSA Cancer Immunomonitoring platform, and Department of Biopathology microenvironment of particular lymphoma subtypes is also for their help. supported by the previous observation that impoverishment of the Treg pool in angio-immunoblastic T-cell lymphoma (AITL) resulted from a proinflammatory microenvironment Grant Support involving Th17 cells (53, 54). This work was supported by grants from Institute National du cancer (INCa), Agence Nationale de la Recherche (ANR-11-EMMA-0045), Canceropole PACA, We observed an increase in the amount of Tregs, including þ high high þ the pharmaceutical company GlaxoSmithKline (GSK) and by fellowships (K.-S. ICOS /CD25 Foxp3 Tregs, among CD4 T cells in cocul- Le) from Ministere de l'Enseignement Superieur et de la Recherche and the Ligue ture with FL B cells. This result is consistent with recent reports Nationale contre le Cancer (LIGUE). D. Olive team was labeled "Equipe FRM of Treg differentiation induced by malignant B cells (22–24). DEQ 201 40329534." D. Olive is a senior scholar of the Institut Universitaire de Tregs' increase could be due to either Tconv conversion or Tregs France. expansion. Our observation of significant Ki67 expression in The costs of publication of this article were defrayed in part by the þ payment of page charges. This article must therefore be hereby marked ICOS Tregs favors the view of Tregs' expansion. On the other advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate hand, it is also possible that lymphoma cells could convert this fact. Tconv to Tregs, as previously reported (22, 23). Tregs enrichment in FL tumors probably result from the combination of Received March 4, 2015; revised April 3, 2016; accepted May 9, 2016; diverse mechanisms, including conversion and expansion, as published OnlineFirst May 31, 2016.

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ICOS Costimulation of Tregs in Follicular Lymphoma

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Follicular B Lymphomas Generate Regulatory T Cells via the ICOS/ICOSL Pathway and Are Susceptible to Treatment by Anti-ICOS/ICOSL Therapy

Kieu-Suong Le, Marie-Laure Thibult, Sylvain Just-Landi, et al.

Cancer Res 2016;76:4648-4660. Published OnlineFirst May 31, 2016.

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