CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment After Allogeneic Transplantation of Hematopoietic Cells

Cancer Microenvironment and Immunology Research

CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment after Allogeneic Transplantation of Hematopoietic Cells

Christoph Durr€ 1,2, Dietmar Pfeifer1, Rainer Claus3, Annette Schmitt-Graeff4, Ulrike V. Gerlach4, Ralph Graeser5, Sophie Kruger€ 1, Armin Gerbitz6, Robert S. Negrin7,Jurgen€ Finke1, and Robert Zeiser1

Abstract Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen- presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)- based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12- dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT. Cancer Res; 70(24); 10170–81. 2010 AACR.

Introduction immunosuppressive effect of Treg in the tumor microenvironment was shown not to occur randomly but to be due to the Allogeneic hematopoietic cell transplantation (alloHCT) specific recruitment of Tregs (4) or the conversion of con- was shown to be an effective treatment option in patients ventional T cells into Tregs (5). Increased numbers of Tregs with refractory or recurrent non-Hodgkin lymphoma (1–3). within the tumor tissue, in the tumor draining lymphatic þ þ CD4 FoxP3 regulatory T cells (Treg) interfere with antitu- tissue, or in the peripheral blood were found in human mor immunity in the presence of autologous tumor (4). The samples from solid tumors (4, 6, 7), non-Hodgkin B-cell lymphoma (BCL; ref. 8), Hodgkin lymphoma (9), late stage myelodysplastic syndrome (10), and acute myeloid leukemia Authors' Affiliations: 1Division of Hematology and Oncology, Department (11). Within the tumor microenvironment, Tregs were shown of Medicine, Freiburg University Medical Center, and 2Department of to suppress natural killer (NK) cell–mediated cytotoxicity by Biology, Albert-Ludwigs-University, Freiburg, Germany; 3Department of Epigenomics and Cancer Risk Factors, German Cancer Research Center affecting NKG2D receptor expression (12), interfering with (DKFZ), Heidelberg, Germany; 4Department of Pathology, Freiburg Uni- dendritic cell (DC)-mediated cytotoxicity (13) and T-cell 5 versity Medical Center, Albert-Ludwigs-University; Tumor Biology Cen- maturation (14). ter, Freiburg; 6Universitatsklinikum€ Erlangen, Medizinische Klinik 5, Erlangen, Germany; and 7Division of Blood and Marrow Transplantation, Following reduced intensity conditioning, residual active Department of Medicine, Stanford University School of Medicine, Stan- BCL tissue is still present in the recipient and could attract ford, California functionally immunosuppressive Tregs to the BCL microen- Corresponding Author: Robert Zeiser, Division of Hematology and vironment, resulting in local immunosuppression (15, 16). Oncology, Department of Medicine, Freiburg University Medical Center, Albert Ludwigs University, Hugstetterstr. 55, 79106 Freiburg, Germany. Therefore, the goal of this study was to delineate factors Phone: (49)-0761-270-3401; Fax: (49)-0761-270-3658. E-mail: robert. that impact Treg migration and accumulation in the presence [email protected] of active BCL tissue. We employed in vivo bioluminescence doi: 10.1158/0008-5472.CAN-10-1943 imaging (BLI)–based Treg tracking methodologies (17) in 2010 American Association for Cancer Research. combination with neutralization or blockade of different

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chemokines that were identified by microarray analysis. Our 13-3 (pTRE Luc) and the neomycin resistance gene from data clearly indicate that the CXCR4/CXCL12 axis is critical pcDNA 3.1 (Invitrogen), using L1_eGFP_IRES as a backbone for Treg accumulation within the tumor microenvironment (23). The transduction of the 291PC cells using a VSV-G (BD and has a major impact on antitumor effects after alloHCT Clontech) pseudotyped retrovirus was performed according to that could be exploited in the clinical situation. the instructions from the manufacturer. After selecting suc- cessfully transduced cells using 1 to 3 mg/mL of neomycin, Methods their luciferase activity was tested with a standard luciferase assay (Promega E4550) in a Luminometer (BMG Lumistar; Mice BMG Labtech GmbH). C57Bl/6 (H-2Kb, Thy-1.2), FVB/N (H-2Kq, Thy-1.2), and Balb/c (H-2Kd, Thy-1.2) mice were purchased from the local In vivo chemokine neutralization experiments stock of the animal facility at Freiburg University. Only gender- AMD3100 (Sigma) was administered at a previously matched combinations (6 and 12 weeks old) were used for þ reported dosage of 5 mg/kg for 7 days after Treg infusion transplant experiments. Luciferase (luc ) transgenic C57Bl/6 (24). Anti-CXCL3 (clone:CXCR3-173; Biolegend) was injected mice have been previously described (18). All animal protocols intraperitoneally at a dosage of 200 mg per mouse, which was (G07-19, G08-8) were approved by the University Committee on previously shown to have in vivo activity (25). Control groups the Use and Care of Laboratory Animals at Albert-Ludwigs received matched isotype antibody (Ab) at the same dosage University. CD11c.DTR-Tg mice (19) were kindly provided by (clone:HTK888; Biolegend). A. Diefenbach. In vivo BLI Human subjects In vivo BLI was performed as previously described (17). Mice All samples were collected after approval of the study were imaged using an IVIS100 charge-coupled device imaging protocol by the Ethics Committee of the Albert-Ludwigs system (Xenogen) for 5 minutes or Berthold Nightowl (Bert- University and after written informed consent. Diffuse, large hold Technologies). Cell expansion was quantified in photons/ B-cell lymphoma (DLBCL) samples were collected in a pro- s/cm2 with Living Image 3.0 Software (Calipers). spective manner.

Bone marrow transplantation model and In vitro proliferation assays þ histopathologic scoring CD4 T cells from C57Bl/6 mice and irradiated splenocytes Bone marrow transplantation (BMT) experiments were from Balb/c mice were purified by MACS-positive selection performed as previously described (20). Briefly, recipients (Miltenyi). CFSE labeling of cells was done with Vybrant CFDA were injected intravenously with 5 106 wild-type (wt) bone SE (Molecular Probes) as previously described (26). To test the þ marrow cells after lethal irradiation with 900 cGy. The follow- suppressor activity of CD4 T cells, these cells were isolated ing transplant models were employed: C57Bl/6!Balb/c and from either spleen or lymphoma tissues as indicated in the FVB/N!C57Bl/6. Slides of liver, small bowel, and large bowel respective experiments. samples collected on day 7 were stained with hematoxylin and eosin (H&E) and scored by an experienced pathologist (U.V. Coculture experiments Gerlach) according to a previously published histopathologic Bone marrow DCs were prepared basically as described scoring system (21). (27), except that interleukin 4 (IL-4) was not used. A total of 5 104 Balb/c DCs per well, together with 2 105 A20 B-cell lymphoma models lymphoma cells, were cultivated using supernatant from A20 BCL cells were injected subcutaneously into the shaved right lymphoma cells in 96-well plates in a total volume of 200 mLat flank on the day of alloHCT after irradiation. When indicated, þ 37 C, 5% CO . Supernatants were collected after 24 hours for wt or luc transgenic A20 BCL (Balb/c background, 5 106), or 2 chemokine measurement. the 291PC Burkitt lymphoma (C57Bl/6, 2 105 and 5 104), was employed. The Burkitt lymphoma cell line 291PC was established from the human l-c-myc transgenic mouse (22). Migration assay Primary lymphoma cells from 1 C57BL/6 l-hu-c.myc trans- For Treg migration studies, 24-well, flat-bottomed plates genic mouse were cultured on irradiated MRC5 feeder cells in with a pore size of 5 mm (Costar; Corning) were employed. Iscove's MDM medium supplemented with 20% fetal calf RPMI medium with 2% FCS was used in the transwells. CXCL12 serum (FCS). The animals were sacrificed when the tumor (R&D Systems) was added to the medium at a concentration reached a diameter above 15 mm or showed ulceration. of 100 ng/mL. After incubation for 2 hours at 37 C, the number of cells migrated across the transwell was quantified by Generation of luciferase-expressing 291PC Burkitt fluorescence-activated cell sorting (FACS) analysis. lymphoma cells The retrovirus encoding the luciferase–aminoglycoside Preparation of tumor-infiltrating lymphocytes phosphotransferase (neomycin resistance) fusion gene Tumors were removed, homogenized, and digested in (Luci–Neo) was constructed from the luciferase gene of pUHC RPMI 1640, 20% FCS, and 400 units/mL of collagenase

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(Worthington) at 37C for 4 hours. The cell suspension was script Sense Target Labeling Assay as described by the man- then filtered through a 40-mm nylon mesh (BD Bioscience). ufacturer. We used the Genedata Expressionist software The cells for intracellular chemokine staining were treated (version 5.1.2) for further data analysis. CEL files were with BD GolgiPlug at 37C for 5 hours and stained according imported into the Refiner module of Expressionist where to manufacturer's instruction (BD Cytofix/Cytoperm; BD quantile normalization and probe summarization were per- Bioscience). formed using the Refiner RMA condensing algorithm. To identify differentially expressed genes between the A20 cul- Flow cytometry tured cell and the A20 grown in vivo, the unpaired Bayes t test All Abs were purchased from BD Pharmingen, Biolegend, (CyberT; ref. 29), with the Bayes confidence estimate value set eBiosciences, and R&D and used as FITC, PE, Alexa647, or to 10 and a window size of 101 genes, was performed with the eFluor450 conjugates. The following Abs were used: CD3 Analyst module of Expressionist. To control the false discovery (17A3), CD4 (GK 1.5/RM4-5), CD8 (53-6.7), CD25 (PC61), rate, the Benjamini–Hochberg q-value was calculated. We CD11c (HL3/N418), CD19 (6D5), Thy-1.1 (H1S51), Foxp3 then used the "N-fold regulation" activity of Analyst to calcu- (FJK-16s), CXCR3 (CXCR3-173), CXCR4 (TG12), H-Kb (AF6- late the median ratio between the A20 without stroma (w/o 88.5), H-Kd (GF1 1.1), H-Kq (KH114), NK1.1 (PK136), CD11b stroma) group and the A20 with stroma in vivo group. (M1/70), PDGFR (APB5), CD34 (MEC 14.7), and CXCL12 (79018). Data were acquired with a CyanADP flow cytometer Statistical analysis (Beckman Coulter) and then analyzed with FlowJo 7/8 soft- Differences in animal survival (Kaplan–Meier survival ware (Tristar). curves) were analyzed by log-rank test. For comparison of proliferation of conventional luc transgenic T cells, cytokines, Conventional histology and immunohistchemistry mean fluorescence, and graft-versus-host disease (GvHD) Fresh frozen sections of 5-mm thickness were mounted on histopathology scores between experimental groups, the microscope slides (Superfrost/Plus; R. Langenbrink). Evalua- Student’s t test was used. P < 0.05 was considered to be tion of tissue sections was performed on a Zeiss Axioplan 2 statistically significant. microscope. The standard objectives used were 20/numerical aperture 0.45 and 40/numerical aperture 0.60. Digital Results photographs from the microscope were obtained using a Spot digital camera. For immunoenzymatic staining, the tissue was Donor-type Tregs accumulate within malignant BCLs fixed for 10 minutes in acetone (Sigma) and the primary after alloHCT and are functionally suppressive biotinylated Ab was applied (anti-CXCL12; R&D systems To study their migratory pattern, Tregs were isolated from clone: 79014). For visualization, streptavidin alkaline phos- luciferase transgenic donors and 106 cells were administered phatase–coupled Ab and corresponding substrate (Vector to the recipients on day 7 after alloHCT. We observed that Labs) or the DAB-system (Dako Cytomation) was used. Tregs migrated to and accumulated in lymphoma tissue (Fig. 1A, left), which was not the case when benign B cells ELISA-based cytokine analysis for CXCL-12 were injected into the same anatomic area (data not shown). Cell culture supernatants were obtained at the indicated As the purity of adoptively transferred Treg was greater than þ time points. ELISA was performed according to the man- 90% CD4 Foxp3 cells (data not shown), at most 10% of the þ ufacturer's instruction (R&D Systems). Briefly, samples were transferred luc cells could be conventional T cells (Tconv), diluted 1:10 to 1:50, and the cytokine was captured by the which would be equivalent to 1 105 T cells. To study whether specific primary monoclonal antibody (mAb) precoated on the this amount of Tconv would display the same trafficking þ microplate and then detected by horseradish peroxidase– pattern, 1 105 CD4 CD25 cells were adoptively transferred labeled secondary mAbs. Plates were read at 450/570 nm on day 7 after alloHCT and followed by BLI. In contrast to þ using a microplate reader (Tecan model: Sunrise). Recombi- Tregs, CD4 CD25 cells accumulated in secondary lymphoid nant cytokines were used as standards. Samples and stan- organs, GvHD target organs including the abdominal area, dards were run in duplicate, and the sensitivity of the assay skin (ears), and also the A20 BCL region (Fig. 1A, right). On the was 47 pg/mL. basis of this obvious difference, we hypothesized that the Treg trafficking pattern mirrored specific recruitment and was not Luminometric analysis of different tissues simply due to random accumulation. A comparable Treg Organs were collected from Balb/c recipients on day 21 recruitment phenomenon was observed in the murine after transplantation, homogenized, and their protein content 291PC Burkitt lymphoma model as shown in later studies. determined by Bradford assay. Luminometric analysis was To further determine the amount of Treg infiltration in performed as previously described (28). different tissues independent of their depth within the body, which could in turn influence signal intensity, we isolated Microarray analysis individual organs and evaluated their luminescence intensity RNA was isolated from A20 tumor cells grown in cell culture normalized to the protein content of the tissue (Fig. 1B). When þ or from A20 tumor grown in vivo following alloHCT. RNA luc Tregs were transferred, the highest luciferase activity was samples with an RNA integrity number greater than 7 were found within the A20 BCL tissue (Fig. 1B), corresponding to further processed with the Affymetrix GeneChip Whole Tran- the BLI data. To determine whether or not the observed BLI

10172 Cancer Res; 70(24) December 15, 2010 Cancer Research

A Day A20 + Tregluc A20 + CD4 + luc after Lateral Ventral Lateral Ventral BMT: Image Min = -9.29e3 d13 Figure 1. Adoptively transferred Max = 3.20e7 lucþ Tregs are recruited toward 5.0 A20 BCL. A, A20 (5 106) cells were given subcutaneously in the 4.0 right flank after irradiation of Balb/c alloHCT recipients (H2-Kd). Luciferase transgenic Treg or 3.0 þ d17 CD4 CD25 cells (H2-Kb) were ×105 given 7 days after alloHCT. 2.0 Experiments were repeated 3 times with a total of at least 10 mice per group. B, organs from 1.0 the indicated groups were harvested and homogenized, and d21 p/sec/cm^2/sr luciferase activity relative to the protein content was measured for Color bar the indicated organs. A Min = 2.00e4 Max = 5.00e5 representative experiment with 2 mice per group is displayed. C, proliferation of CD4 T cells (C57Bl/ B 4,000 C 6) stimulated with irradiated Subcutis right flank Balb/c splenocytes is displayed 75 with the addition of the indicated 3,000 Mesenterial LN cell populations. Pooled data from Liver 50 2 independent experiments is 2,000 Lung shown (*, P < 0.05). D, amount of þ þ þ 25

Thy1.1 CD4 Foxp3 cells within (photons) Spleen tumor tissue for the indicated Luminescence 1,000 groups is shown. Thy1.1 is used to % Proliferating CD4 0 identify adoptively transferred Suppressors: Treg/CD4 cells in contrast to bone 0 CD4 from spleen marrow–derived cells (Thy1.2). A20 CD4 from tumor luc Gating is on CD4þ cells. A Treg w/o suppressor representative FACS plot for the indicated group is shown. luc + luc Experiments were repeated 3 D A20 + Treg A20 + CD4 times (n ¼ 6) with comparable 22.56 66.11 45.65 2.79 results. Thy1.1 Thy1.1 10.97 0.35 50.22 1.34 Foxp3 Foxp3

signal at the A20 BCL sites was derived from functionally Chemokine production within the lymphoma at the suppressive Tregs, CD4 T cells from the A20 BCL or the spleen time point of Treg transfer were used as suppressors in vitro. We observed the strongest To screen for factors that may be causative for the observed suppressor activity when CD4 T cells isolated from A20 BCL Treg accumulation within the A20 BCL in vivo, we performed tissue were used (Fig. 1C), which was compatible with FACS comparative microarray analysis of A20 BCL cells. We com- þ analysis, indicating that more than 66% of the CD4 popula- pared A20 BCL grown in vitro (w/o stroma) to A20 BCL grown tion isolated from the A20 BCL after alloHCT were donor in Balb/c mice following alloHCT and containing a tumor derived (Thy1.1) and Foxp3 positive when Tregs were trans- stroma or benign B cells (Fig. 2A). On using a fold-change ferred (Fig. 1D). threshold value of greater than 3 and a q-value of less than

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0.05, 652 genes fulfilled these criteria (619 upregulated and 43 (Fig. 4B, bottom). On the basis of the observation that CXCL12 downregulated in A20 BCL þ stroma vs. A20 BCL w/o stroma). was not produced by the A20 BCL cells themselves, and that in As we were mainly interested in the chemokine profile, we vitro APCs were the major source of the chemokines, we aimed selected all genes that had an annotation as "chemokine" or to assess the relevance of the BCL infiltrating, host-derived "chemokine receptor." This resulted in 58 genes, of which 11 APCs for Treg migration in a functional in vivo study. There- fulfilled these criteria. We selected the 2 receptor/ligand(s) fore, CD11c.DTR Tg mice (19) were used as alloHCT recipi- combinations CXCR3/CXCL9/10 and CXCR4/CXCL12 for later ents, and CD11c depletion by diphtheria toxin (DT) yielded functional in vivo analysis, based on their significantly higher potent APC reduction when given 2 days prior and 2 days after expression in A20 BCL þ stroma (Fig. 2B) and the high alloHCT (data not shown). In these experiments, the Burkitt- þ þ expression of CXCR4 on CD4 Foxp3 cells prior to their type 291PC BCL was used, as it was on the same genetic adoptive transfer (Fig. 2C). In contrast to CXCR4, CXCR3 background as the CD11c.DTR Tg mice (both C57Bl/6). The þ þ was sparsely expressed on sorted CD4 Foxp3 cells absence of host APCs caused a reduction in Treg recruitment (Fig. 2C). Therefore, we included CXCR3 blocking in the signal toward the established 291PC BCL, as shown for an following functional studies as a negative control. individual time point (Fig. 4C, left) and in the quantification of the luc signal over the 291PC BCL region (Fig. 4C, right). In Interference with CXCR4 but not CXCR3 blocks Treg keeping with the functional studies, CXCL12 was not detect- migration toward A20 BCL able in the 291PC BCL tissue of CD11c-depleted CD11c.DTR To systematically investigate the relevance of the receptor/ Tg mice (Fig. 4D). These data are indicative of host APCs ligand(s) pairs for Treg migration, we blocked their function playing a major role after alloHCT in the process of CXCL12- either with an Ab against CXCR3 or with the CXCR4 inhibitor mediated chemoattraction of Treg toward the 291PC BCL AMD3100, which has been shown to prevent CXCR4 engage- tissue. These data confirm the presence and biological activity ment (30). Consistent with the increased expression of of host APCs following alloHCT (32). CXCL12 RNA in A20 BCL tissue, CXCL12 protein could be detected by immunohistochemistry in different murine BCLs Low level of CXCR4 expression allows effector cells to (A20, 291PC) at significant levels (Fig. 3A, left). Within the infiltrate BCL tissue independent of CXCR4 tumor, we could identify antigen-presenting cells (APC), neutralization fibroblast cells, and endothelial cells as CXCL12-producing Because the usage of AMD3100 affected Treg migration to cells (Fig. 3A, middle). AMD3100 but not anti-CXCR3 or PBS BCL tissue, we next studied the impact of AMD3100 on the þ was found to reduce the accumulation of luc Treg toward immune compartment within the 291PC BCL. The numbers of established A20 BCL after alloHCT, as shown for individual CD4, CD8 T cells, and NK cells in the BLC tissue were not time points (Fig. 3B, top), or when plotted as photons per significantly different whether or not AMD3100 was adminis- second over time for the lymphoma area (Fig. 3B, bottom). tered (Fig. 5A). This obvious difference in comparison with This observation was consistent with enhanced Treg migra- Tregs was based on the low to absent CXCR4 expression on tion upon CXCL12 stimulation in vitro (Fig. 3C). Because the surface of conventional CD4 T cells, CD8 T cells, and NK1.1 recent evidence suggests a therapeutic role for AMD3100 in cells among the lymphocytes isolated from the spleen non-Hodgkin lymphoma (31), the drug could reduce A20 BCL (Fig. 5B). These data indicate that cytotoxic T cells and NK viability and thereby have an indirect impact on Treg traffick- cells were not prevented from reaching the 291PC tissue when ing. However, we found that AMD3100 did not interfere with CXCR4 was blocked. the viability of the murine A20 BCL during the observation period (data not shown). To study whether or not AMD3100 Impact of CXCR4 neutralization on antitumor effects affected Treg-mediated GvHD protection, the inhibitor was and survival given in comparison with PBS. Importantly, neither the pro- To study the effects of CXCR4 blocking by AMD3100 on tective effect of Treg on survival (Fig. 3D, top) nor that on the rejection of the BCL by allogeneic T cells, growth of GvHD severity (Fig. 3D, bottom) was affected when AMD3100 luciferase transgenic 291PC lymphoma after alloHCT was was given. Interestingly, CXCL12 expression was also found in monitored by BLI in a longitudinal fashion. The addition of 9 of 15 human DLBCL samples of which 1 representative donor type T cells decelerated the growth of the lymphoma, example is shown (Fig. 3A, right). although in all groups several mice developed strong tumor growth (Fig. 6A). The strength of the antitumor effect was Host-derived APCs residing within the BCL tissue are reduced when Tregs were adoptively transferred and only critically involved in CXCL12 production, and their PBS treatment was given (Treg/PBS). The group that targeted deletion abrogates Treg recruitment received Tregs and AMD3100 displayed significantly less In vitro CXCL12 production by A20 cells alone was either tumor growth than the group receiving Treg/PBS low or absent. However, it could be significantly increased (P ¼ 0.026; Fig. 6A). Reduced tumor growth translated into when APCs were included in the culture (Fig. 4A). Immuno- improved survival of the Treg/AMD3100 group as compared histochemical staining of A20 BCL tissue isolated after with the Treg/PBS group (P ¼ 0.04;Fig.6B).Thesedata þ alloHCT showed the presence of CD11c APCs within A20 indicate that AMD3100 enhances antilymphoma effects BCL tissue (Fig. 4B, top). The majority of APCs residing in the postallografting most likely by blocking Treg recruitment tumor microenvironment were of recipient (H2-Kd) origin toward the lymphoma tissue.

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ABCXCL9 CXCL10 4 P = 1.03E-08 P = 1.57E-07 10 400 BCL B- 400 cells 300 300 Stroma – + 200 200 Cc122 Xcr1 Cxc19 100 100 Ccrl1 Ccr6 Cxcl114 0 0 Cmklr1 Ccr10 Ccl11 103 Cxcl5 CXCL12 w/o stroma Cxcl11 + storma Cc128 Cxcr6 150 P = 8.82E-07 Ccr4 Cxcl3 Ccl25 Ccr7 100 Ccr5 Cxcl12 signal values (arbitrary units) RMA Darc Ccl3 50 Ccl5 Ccr3 Ccrl2 Cxcr3 C 0 100 Cxcr7 Ccl25 0.13 1.44 3.78 96.21 Ccl8 Ccl1 Cxcl10 Cx3cr1 Ccbp2 Ccr1l1 Cxcl1 Ccr2

Cxcl17 CXCR3 CXCR4 Cxcr4 2.46 95.98 0.00 0.01 Ccl12 Xcl1 Foxp3 Cklf Cxcl16 10 Cxcl13 Ccl17 Ccl4 Ccl9 100 Cxcl15 Ccr8 Cx3cl1

3 80 Ccr9 Ccl6

Ccl2 Foxp 60 Ccl26 + Ccl24 Ccr5 40 Ccr1 Ccl7 Cxcl2 20 % of CD4 1 Ccl20 0

CXCR4 CXCR3

Figure 2. Differential chemokine expression within the A20 BCL and corresponding receptors on Tregs. A, RNA was isolated from A20 BCL tissue grown for 7 days in vivo (þ stroma) or from A20 BCL grown in vitro cultures (w/o stroma). Experiment names are aligned as columns and genes as rows. The gene expression values are color-coded, and the color bar on the left displays the coding for the expression values in a logarithmic scale. B, absolute gene expression for the indicated chemokines is displayed as RMA signal values. The P values are indicated for the individual comparisons on top of each bar diagram. C, surface expression of the corresponding receptors, CXCR3 and CXCR4, after gating on CD4þ cells prior to adoptive transfer is displayed. Top, representative FACS plots; bottom, percentages of CD4þFoxP3þCXCR3þ and CD4þFoxP3þCXCR4þ are displayed for sorted CD4þCD25þ cells. Each data point represents an individual donor mouse (n ¼ 9; *, P < 0.0001).

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Ag-presenting cells A 25 P = .0005 CXCL12 human CD11c+CD11b+ 20 P = .0059 CD11c+CD11b– cells/

+ 15 CD11c–CD11b+ (MΦ) 10 Fibroblast cells 5 CXCL12 high-power field Endothelial cells 0

SB LB 0.0 iLN liver A20 0.5 1.0 spleen 291 PC CXCL12 producing cells in % of total tumor B Tregluc + PBS Tregluc + AMD 5.0 5.0 C 2 × 10 04 4.0 4.0

d10 3.0 3.0 1 × 10 04 ×107 No Stim ×107

2.0 2.0 CXCL12 Cell count 5 × 10 03

1.0 1.0 0 d14 p/s/cm^2/sr

Color scale Color scale Min = 2.00e6 Min = 2.00e4 Max = 5.00e7 Max = 5.00e7 D TC + PBS (n = 11) TC + AMD (n = 12)

d17 TC + Treg + PBS (n = 10) TC + Treg + AMD (n = 11) 100

60 d21 40

Survival % 20

0 Lymphoma area 0102050607080 1 × 1010 Days after BMT

1 × 1008 Bone marrow Bone marrow +Tregluc PBS × 06 2 +TC+PBS 1 10 luc Photons/s +Treg AMD luc +TC+AMD 1 × 1004 +Treg antiCXCR3 8121620 1 +TC+Treg+PBS Hist GvHD Days after BMT +TC+Treg+AMD 0

Figure 3. Impact of in vivo CXCR4 blocking on Treg recruitment to A20 BCL tissue. A, indicated tissues (SB, small bowel; LB, large bowel; iLN, inguinal lymph nodes; and spleen, liver, 291PC lymphoma, A20 lymphoma) were isolated on day 7 after alloHCT and analyzed by immunohistochemistry for the expression of CXCL12. Left, number of CXCL12þ cells/high-power field (12 high-power fields per organ analyzed). The P values are indicated for the individual comparison in the figure. Middle, A20 lymphoma tissue was isolated on day 15 after alloHCT performed as described for the C57Bl/6 ¼ >BALB/c combination. Pregating was on CXCL12þ cells. Displayed are APCs (CD11cþCD11b, CD11cCD11bþ, CD11cþCD11bþ), CD11cþCD11bþ, CD11cþCD11b, CD11cCD11bþ, fibroblast cells (PDGFRþCD19), and endothelial cells (CD34þCD19). A total of 7 or more individual mice were analyzed, and the experiment was performed twice. Right, representative human DLBCL tissue stained for CXCL12 (red) as described earlier (original magnification 200). A total of 15 individual lymphoma patients were analyzed (9/15 positive). B, Treg in vivo migration was assessed as described for Fig. 1. AMD3100 (AMD, n ¼ 9, 5 mg/kg of bodyweight), or PBS (n ¼ 6) was injected daily from day 7 to 13. Top, representative time points of BLI. Bottom, signal intensity derived from lymphoma area (red circle) region (y-axis) and time (x-axis) is plotted for the indicated groups. Experiments were repeated 3 times with total number of mice indicated earlier (SB, n ¼ 15/LB, n ¼ 12). C, in vitro migration of Tregs was assessed in a transwell chamber with or w/o CXCL12 as a stimulus (*, P < 0.05). The experiment was repeated 3 times. A representative experiment is shown. D, top, survival of C57Bl6 recipients that received lethal irradiation (9 Gy) and 5 106 bone marrow cells (H-2Kq) and where indicated T cells (day 2, intravenously 1 106) and Tregs (day 0, intravenously 5 105). AMD3100 (5 mg/kg of bodyweight) or PBS were injected intraperitoneally day 0 to 6. Bottom, 7 days after transplantation, mice from the indicated groups (each n ¼ 3) were sacrificed and analyzed for evidence of GvHD (AMD ¼ AMD3100).

10176 Cancer Res; 70(24) December 15, 2010 Cancer Research

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CD11c.DTR Tg d Research. H2-K n 51 0.25 15.14 79.69 ¼ p/s/cm^2/sr Min =-9.55e3 Max =9.05e5 Max =1.00e6 Min =1.00e4 Color Bar CD11c )teteta niae;luc indicated; as treatment 3) Image 0.2 0.4 0.6 0.8 1.0 n ¼ ×10 ) ecnae frcpet(-K)dnrtp (H22-K (H-2Kd)/donor-type recipient of percentages 5), 7 photons/s 1 ×10 2 ×10 2 ×10 4 ×10 4.92 ¼ 06 06 05 05 1 *, 11; P

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0.05). within CD11c population acrRs 02)Dcme 5 2010 15, December 70(24) Res; Cancer rg eegvndy7atralHT et 2 Left, alloHCT. after 7 day given were Tregs XL2Mdae Immunosuppression CXCL12-Mediated Day 10 100 50 P wt CD11c.DTR Tg 0

¼ + CXCL12 cells/ high-power field Tumor .6;woD s DT, vs. DT w/o 0.066; 10 15 20 25 0 5 Spleen ,0) otm representative Bottom, 1,000). P onesann with counterstaining , < .5 ,CD11c B, 0.05. Day 13 Donor Host b P CD11c ) ¼ þ .8.The 0.08). Psare APCs þ APCs b ). 10177 Dürr et al.

Tumor A Spleen PBS (n = 7) PBS (n = 7) AMD (n = 6) AMD (n = 6) ns 15,000 10,000 total

total Figure 5. Effector cell expression 5 5 5,000 of CXCR4 and accumulation 10,000 3,000 independent of CXCR4 blocking. A, 291PC lymphoma cells (2 2,000 105) were given subcutaneously in 5,000 the right flank after irradiation and 1,000 BMT of C57Bl6 alloHCT recipients 0 (H2-Kb). Tconv (1 106, H2-Kq) 0 Counts from 1x10 Counts from 1x10 + + – + + – were given when the tumor was established following alloHCT. CD4 CD8 CD3 CD4 CD8 CD3 + + Tumor-infiltrating cell populations were isolated 5 days after T-cell NK1.1 NK1.1 transfer from lymphoma tissue. Each data point represents an individual mouse (PBS: n ¼ 7; AMD: n ¼ 6). No significant difference was noted between the B PBS and the AMD3100 group ¼ 8.05 0.88 6.76 0.85 (AMD AMD3100). B, CXCR4 100 expression in different cell populations isolated from spleen of untreated mice was measured. CD4 CD8 Left, representative FACS plots for 50 þ þ þ NK1.1 , CD4 , CD4/FoxP3 , and 89.80 1.27 91.29 1.10 CD8 T cells with respect to CXCR4 2.15 0.65 31.21 68.79 expression. Right, percentage of 0 CXCR4þ cells within the NK1.1þ, Amount of cells in % +– +– +– +– CXCR4 CD4þ, CD4/FoxP3þ, and CD8þ populations. NK1.1 CD4/FoxP3 95.89 1.30 0.00 0.00 CD4 CD8 Treg NK1.1 CXCR4

þ Discussion Under physiologic conditions, CXCR4 Tregs are recruited to the bone marrow where significant levels of CXCL12 are The failure of immune responses to eliminate tumors has produced (35). Our observations that Tregs accumulate in been convincingly attributed to active suppression mediated BCL tissue after alloHCT are compatible with studies indi- by Tregs recruited toward the tumor (8) or its draining lymph cating their recruitment toward the bone marrow compart- nodes (33). In contrast, the link between donor-derived Treg ment in patients with bone marrow infiltration by multiple accumulation in BCL and the local effector immune response myeloma (36). In solid tumors, it has been previously shown after alloHCT is poorly understood. that the chemokine is involved in Treg recruitment toward By tracking Tregs in vivo, we observed their migration to pleural mesothelioma (37). different BCL tissues after alloHCT, suggesting their immu- Furthermore, it was reported that the CXCR4/CXCL12 nomodulatory effect at this site. Mechanistically, we could axis is crucial for tumor trafficking of plasmacytoid DCs in show that A20 BCL tissue displayed increased expression of patients with ovarian cancer (38). Apart from cell recruit- CXCL12 and that this chemokine was functionally relevant for ment, CXCL12 induced a regulatory phenotype in conven- Treg recruitment whereas CXCL9–11, which binds to CXCR3, tional CD4 T cells involved in experimental autoimmune was not. CXCL12 was dependent on the presence of tumor- encephalomyelitis (39), suggesting its immunomodulatory infiltrating host APCs as shown by immunohistologic analysis. role. Besides their endogenous CXCL12 production, APCs þ The in vivo depletion of host-type CD11c cells in CD11c- residing within the lymphoma microenvironment may indir- DTR-Tg recipients was sufficient to reduce Treg accumula- ectly enhance the production of the chemokine by stroma tion in 291PC BCL tissue. These findings indicate that host cells including fibroblasts and endothelial cells. It was pre- APCs that survive irradiation (34) infiltrate BCL tissue and viously shown that different cell populations, including are essential for Treg recruitment. Selective recruitment of tumor cells (40, 41), stromal fibroblast cells (42), and vas- CXCR4-expressing Tregs to the tumor may be the underlying cular endothelial cells (43, 44), can contribute to the pro- causeforthehighexpressionofCXCR4ontumorTregs. duction of CXCL12.

10178 Cancer Res; 70(24) December 15, 2010 Cancer Research

alloHCT. This is in line with previous reports on the impaired BMT + PC291-luc (n = 11) rejection of P815 mastocytoma cells when Tregs were given A + Tc + PBS (n = 11) + Tc + AMD (n = 12) following alloHCT (46). One could speculate that this impair- + Tc Treg + PBS (n = 11) ment of tumor control due to transfer of Tregs after allograft- 1 × 1011 + Tc Treg + AMD (n = 11) P = 0.026 ing is caused by rapid tumor growth as well as the 1 × 1010 subcutaneous location, which may support immune escape, 1 × 1009 as has been described for immune-privileged regions such as the eye (47) or the skin (48). In such settings, Tregs may tip the × 08 1 10 balance toward the failure of T cell–mediated tumor rejection 1 × 1007

Photons/s despite the strength of alloreactivity. In light of the current use 1 × 1006 of AMD3100 in patients undergoing hematopoietic cell transplantation (49) and our finding that CXCL12 is detectable in 1 × 1005 human DLBCLs, AMD3100 treatment may be a justified 1 × 1004 addition to adoptively transferred Tregs. 0102030 In conclusion, we observed that Tregs are recruited toward Days after BMT BCL after alloHCT and that this immunosuppressive process B is dependent on BCL-infiltrating host-type APCs. Affecting 100 Treg recruitment toward BCL tissue through the blockade of the CXCR4/CXCL12 axis by the use of AMD3100 enhances antilymphoma effects, which may have important clinical 50 implications.

Survvival (%) P = 0.02 Disclosure of Potential Conflicts of Interest

0 No potential conflicts of interest were disclosed. 0 20406080 Days after BMT Authors’ Contributions

C. Durr€ designed the experiments, performed experiments, analyzed data, Figure 6. Impact of AMD3100 on antilymphoma effects; luc 291PC and helped to write the manuscript; D. Pfeifer supervised and analyzed micro- lymphoma cells (5 104) were given subcutaneously in the right flank array experiments; R. Claus performed and analyzed experiments; A. Schmitt- after irradiation of C57Bl6 alloHCT recipients (H2-Kb). Tconv (1 106, H2- Graeff supervised immunohistology of human lymphoma samples; U.V. Gerlach Kq) were given on day 2. Treg (6 105, H2-Kq) were given on day 3 performed histophathologic scoring; R. Graeser helped to generate expression of following alloHCT. AMD3100 or PBS was given for 7 consecutive days luc 291PC Burkitt lymphoma cells; A. Gerbitz provided Burkitt lymphoma cells, helped to design tumor experiments, and to write the manuscript; R.S. Negrin starting on day 3 (AMD ¼ AMD3100). The number of animals in each group and J. Finke helped to design experiments and to write the manuscript; and R. is indicated in the top of the figure. A, growth of luc 291PC lymphoma cells Zeiser designed the studies, analyzed data, and wrote the manuscript. All is analyzed by BLI. Tumor growth is significantly reduced in the Treg/ authors have read and agreed to the final version of the manuscript. AMD3100 (filled circle) group as compared with the Treg/PBS (filled P ¼ triangle) group; *, 0.026. B, survival of the groups indicated earlier is Acknowledgments shown. Survival is significantly prolonged in the Treg/AMD3100 (filled circle) group as compared with the Treg/PBS (filled triangle) group; *, P ¼ 0.02. The authors are grateful to Klaus Geiger and Astrid Petersen for excellent technical assistance and to Dr. Marie Follo and Prof. Evelyn Ullrich for critically reading the manuscript. CXCR4 blocking did not affect A20 BCL rejection by conventional T cells and also allowed for protection from GvHD. This would suggest that blocking the CXCR4/CXCL12 axis Grant Support may have clinical potential in modifying the immune response after alloHCT. Such modification could be particularly impor- This study was supported by the Deutsche Krebshilfe, Germany, grant 108034, to R. Zeiser and J. Finke and in part by the DFG (SFB850) tant in the setting of residual tumor burden after alloHCT, and to R. Zeiser. in light of the observation that high Treg counts were asso- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in ciated with increased relapse rates in patients with chronic accordance with 18 U.S.C. Section 1734 solely to indicate this fact. myeloid leukemia after alloHCT (45). This hypothesis is supported by our finding that Tregs Received 06/01/2010; revised 08/11/2010; accepted 08/25/2010; published influenced 291PC lymphoma rejection when transferred after Online 12/15/2010.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment after Allogeneic Transplantation of Hematopoietic Cells

Christoph Dürr, Dietmar Pfeifer, Rainer Claus, et al.

Cancer Res 2010;70:10170-10181.

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