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The Journal of Immunology, 2007, 179, 3332 -3341
Copyright © 2007 by The American Association of Immunologists, Inc.
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Tumor-Derived Chemokine MCP-1/CCL2 Is Sufficient for Mediating Tumor Tropism of Adoptively Transferred T Cells1

Christine E. Brown2,*, Reena P. Vishwanath*, Brenda Aguilar*, Renate Starr*, Joseph Najbauer{dagger}, Karen S. Aboody{dagger} and Michael C. Jensen*

* Division of Cancer Immunotherapeutics and Tumor Immunology and {dagger} Division of Hematology and Hematopoietic Cell Transplantation at the City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To exert a therapeutic effect, adoptively transferred tumor-specific CTLs must traffic to sites of tumor burden, exit the circulation, and infiltrate the tumor microenvironment. In this study, we examine the ability of adoptively transferred human CTL to traffic to tumors with disparate chemokine secretion profiles independent of tumor Ag recognition. Using a combination of in vivo tumor tropism studies and in vitro biophotonic chemotaxis assays, we observed that cell lines derived from glioma, medulloblastoma, and renal cell carcinoma efficiently chemoattracted ex vivo-expanded primary human T cells. We compared the chemokines secreted by tumor cell lines with high chemotactic activity with those that failed to elicit T cell chemotaxis (Daudi lymphoma, 10HTB neuroblastoma, and A2058 melanoma cells) and found a correlation between tumor-derived production of MCP-1/CCL2 (≥10 ng/ml) and T cell chemotaxis. Chemokine immunodepletion studies confirmed that tumor-derived MCP-1 elicits effector T cell chemotaxis. Moreover, MCP-1 is sufficient for in vivo T cell tumor tropism as evidenced by the selective accumulation of i.v. administered firefly luciferase-expressing T cells in intracerebral xenografts of tumor transfectants secreting MCP-1. These studies suggest that the capacity of adoptively transferred T cells to home to tumors may be, in part, dictated by the species and amounts of tumor-derived chemokines, in particular MCP-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The growing repertoire of molecularly identified Ags expressed by human tumors that are recognized by T cells, coupled with the development of methodologies to isolate and expand functional T cells, is creating new prospects for clinical adoptive therapy of cancer (1, 2, 3). Furthermore, understanding of the homeostatic milieu that facilitates the numerical expansion of transferred T cells has resulted in human adoptive therapy regimens using pretransfer iatrogenic lymphodepletion to facilitate engraftment and expansion of transferred T cells (4, 5). Significant challenges persist however, such as overcoming the mitigating effects of regulatory T cells, circumventing tumor escape mechanisms, and promoting efficient lymphocyte homing to all sites of metastatic disease (6, 7, 8).

The physiology of lymphocyte trafficking and tumor tropism in the context of malignant disease requires careful delineation. Chemotaxis, the directed movement of cells toward a chemotactic gradient, is a process by which lymphocytes traffic to sites of inflammation and infection (9). The extravasation of lymphocytes from the circulation into surrounding tissue is a multistep process involving attachment to the endothelium via selectin-mediated rolling and integrin-dependent adhesion, followed by transendothelial migration along established chemokine gradients (9, 10, 11). In addition to inducing cell migration, many chemokines play additional roles in tumor cell growth, tumor differentiation, angiogenesis, and tumor metastasis (12, 13, 14). Thus, the capacity of chemokines to contribute to cancer pathogenesis and their ability to promote leukocyte infiltration into tumor tissue may have opposing effects in tumor immunity.

Tumor tropism is a prerequisite for adoptively transferred T cells to exert a therapeutic effect. Blocking the ability of lymphocytes to respond to chemokines by pretreatment with pertussis toxin, an inhibitor of chemokine receptor signaling, abrogates the therapeutic efficacy of adoptive therapy with tumor-reactive T cells in mice (15). Genetic engineering of tumors to secrete chemokines specific for the recruitment of lymphocytes can inhibit the establishment of tumor xenografts and facilitate the regression of established tumors in mice (16, 17, 18). The efficiency of lymphocyte localization to tumors and clinical responses has been correlated in patients receiving tumor-infiltrating lymphocyte (TIL)3 infusions for metastatic melanoma (19). Patients in which adoptively transferred 111In-labeled TILs localized to tumor sites had a 38.5% clinical response, whereas no clinical responses were seen in patients in which transferred TILs did not localize to the tumor (19). In this study, we investigate the immunobiology of tumor tropism of ex vivo-expanded effector CD8+ human T cells. We show that the production of the MCP-1 chemokine by human tumors correlates with in vitro chemotactic responses of T cells and the accumulation in tumors of adoptively transferred T cells recruited from the systemic circulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
DNA constructs

The construction of ffLuc:Zeo and rLuc:Zeo mammalian expression vectors, consisting of engineered fusions between either firefly luciferase (ffLuc) or Renilla luciferase (rLuc) and zeocin resistance (Zeo) genes cloned into the pcDNA3.1(+) backbone (Invitrogen Life Technologies), has been described previously (20). The LRZ-pMG expression vector consists of the pMG vector backbone (InvivoGen) with an engineered fusion between rLuc and Zeo genes cloned downstream of the encephalomyocarditis virus internal ribosome entry sequence element. The rLuc gene was amplified by PCR from the phRL-CMV vector (Promega). To coexpress MCP-1 and rLuc, the human cDNA for MCP-1 was digested from the MCP1-pUC18 vector (American Type Culture Collection (ATCC)) with the EcoRI restriction enzyme, blunted, and then digested with BamHI. This product was ligated into the LRZ-pMG backbone that had been digested with NheI, blunted, and then digested with BglII to generate the MCP1-LRZ-pMG expression vector. Both the LRZ-pMG and MCP1-LRZ-pMG vectors also contain the glioma tumor-specific Ag IL-13R{alpha}2 in the second multiple cloning site (21).

Cell lines and growth conditions

Human glioma cell lines U87 and T98 were purchased from ATCC. The tumorigenic strain of U251, termed U251T, was a gift from Dr. W. Debinski (Wake Forest). The primary human glioma line UAB1074, explanted from a patient with glioblastoma multiforme (GBM) was a gift from Dr. G. Y. Gillespie (University of Alabama at Birmingham, Birmingham, AL). These glioma cell lines and the human medulloblastoma Daoy cell line (ATCC) were grown in DMEM (Irvine Scientific) supplemented with 10% heat-inactivated FCS (HyClone), 2 mM L-glutamine (Cambrex), and 25 mM HEPES buffer solution (Irvine Scientific). Human melanoma A2058, human kidney carcinoma Caki-1 and human colorectal adenocarcinoma SW480 cell lines were purchased by ATCC and cultured in medium/conditions recommended by ATCC. The Daudi lymphoma line and the 10HTB human neuroblastoma line were purchased from ATCC and grown in medium consisting of RPMI 1640 (Irvine Scientific), 2 mM L-glutamine (Irvine Scientific), and 10% heat-inactivated FCS (HyClone). The rLuc+ control and human MCP1-secreting Daudi lines (Daudi-LRZ and Daudi-LRZ-MCP1) were created by electroporating Daudi cells grown in log phase with linearized LRZ-pMG or MCP1-LRZ-pMG and selecting in 0.2 mg/ml Zeocin (InvivoGen).

The ffLuc+ bulk T cell line was created by electroporating PvuI-linearized ffLuc:Zeo into 3-day OKT3-stimulated PBMC from a healthy donor (HD004) and grown in 0.2 mg/ml zeocin (InvivoGen) for stable selection of a bulk population as previously described (22, 23). The protein fusion between ffLuc and the zeocin resistance gene dictates that all zeocin-resistant cells also express the ffLuc reporter gene. Firefly luciferase-positive T cells were propagated ex vivo on a 14-day stimulation cycle with 30 ng/ml OKT3, a 5:1 ratio of irradiated allogeneic PBMC and EBV-transformed lymphoblastoid (LCL) feeder cells, and 25 U/ml rhIL-2 (Chiron) as described previously (24). T cells were cultured in RPMI 1640 (Irvine Scientific) with 10% FCS (HyClone), 25 mM HEPES (Irvine Scientific), 2 mM glutamine (Cambrex), and fresh rhIL-2 was added to cultures every 48 h. All functional assays were performed with T cell effectors between days 10 and 14 from stimulation with OKT3.

For conditioned medium, adherent cell lines were grown to ~60–90% confluency and Daudi cells were grown to ~1–0.8 x 106/ml; cells were then transferred to serum-free medium and, after 72 h, the cell-free supernatants were collected. Patient cerebrospinal fluid (CSF) and resection cavity fluid were obtained from discarded samples under an institutional review board-approved protocol.

Chemotaxis assay

In vitro chemotaxis assays were conducted using 96-well ChemoTx plates with 5-µm pore diameter polycarbonate filters (no. 106-5; NeuroProbe) as described in detail by our laboratory (23). Diluted chemokines in serum-free medium, conditioned medium (serum-free) from cell lines, or cell-free cerebrospinal fluid were plated in the lower chamber, and ffLuc+ T cells (30 µl of 2.5 x 106/ml) were plated in the upper chamber. After 2 h at 37°C, the nonmigrated cells were scraped off the surface of the polycarbonate filter, ChemoTx plates were spun for 3 min at 150 x g, filters were removed, luciferin substrate was added at a final concentration of 0.14 mg/ml, and the luminescent photon flux in the lower chamber was measured using a Victor 3 luminometer (PerkinElmer). The numbers of migrated cells were quantified by comparing the luminescent flux of the experimental wells to an ffLuc+ responder cell standard curve plated in the same chemotaxis plate. Subtraction of background chemotaxis to medium alone was included for the analysis and quantitation of percent chemotaxis. For immunodepletion studies, conditioned medium was incubated overnight with 0.02 mg/ml anti-hMCP1 (AB-279-NA; R&D Systems) or anti-hIL-8 (AB-208-NA; R&D Systems) Ab, followed by depletion of cytokine-Ab complexes with 20 µl of protein G-agarose beads (Amersham Biosciences). Depletion of MCP-1 using this technique was confirmed by quantifying human MCP-1 levels before and after immunodepletion using the Chemokine I Cytometric Bead Array System (BD Biosciences). Recombinant human chemokines were purchased from R&D Systems.

Human chemokine profiling

Cytokine levels (picograms per milliliter) were determined using either the Chemokine I Cytometric Bead Array (BD Biosciences) or Bio-Plex Human Cytokine 9-Plex Panel (Bio-Rad) as per the manufacturers’ instructions. Cytokine profiles detected by the Cytokine Ab Array V protein chip technology were performed according to the manufacturer’s instructions (RayBiotech). Briefly, nitrocellulose blots were blocked for 1 h and then incubated for 4 h at room temperature with undiluted conditioned medium or human CSF. Blots were incubated overnight at 4°C with a 1/500 dilution of biotin-conjugated Ab, and the following day was incubated for 1 h at room temperature with a 1/10,000 dilution of HRP-conjugated streptavidin. Chemiluminescent detection of captured cytokines was quantified by densitometry using EPI Chemi II Darkroom and LabWorks software (Ultraviolet Laboratory Products). The binding of MCP-1 by T cells was demonstrated by FACS using biotinylated human MCP-1 and avidin-fluorescein according to the manufacturer’s instructions (Fluorokine Kit NFCP0; R&D Systems). CCR-2 expression on the T cells was examined by FACS using mouse anti-CCR2 primary mAb (Abcam) and FITC-conjugated goat anti-mouse Ab (Jackson ImmunoResearch Laboratories). CCR4 expression was examined using PE-conjugated anti-CCR4 mAb (BD Biosciences).

Tumor xenografts

Mice were maintained under specific pathogen-free conditions at the City of Hope Animal Resources Center and all procedures were reviewed and approved by the City of Hope Research Animal Care Committee. All procedures were performed with NOD-scid mice 6–8 wk of age. These mice lack T and B lymphocyte function and also have impaired NK and macrophage cell function, thus allowing for durable engraftment of human tumors. Intracerebral (i.c.) tumor xenografts required anesthetizing the mice with an i.p. injection of 132 mg/kg ketamine and 8.8 mg/kg xylazine. Mice received a stereotactically guided injection of tumor 2 mm lateral and 0.5 mm anterior to the bregma over 3–5 min. Tumor cells, suspended in 2 µl of phenol-free RPMI 1640 (Irvine Scientific), were injected at a depth of 2.5 mm from the dura. Animals received a s.c. injection of 0.1 mg/kg Buprenex for postsurgical recovery.

In vivo biophotonic imaging

Luminescent cells were imaged in mice using a charge-coupled device camera (Xenogen IVIS; Xenogen) coupled to the Living Image acquisition and analysis software (Xenogen). Mice were injected i.p. with D-luciferin substrate suspended in PBS at 4.29 mg/mouse. Images were captured while the mice were anesthetized by isoflurane (1.5 L/min oxygen + 4% isoflurane) and kept in an induction chamber. Light emission was measured over an integration time of 1 min at 14 min after injection of luciferin. The flux (photons/s) was quantified as total counts measured over time in the region of interest.

Immunohistochemistry

Histology was performed on 10-µm cryosections of paraformaldehyde-fixed mouse brain tissue. To detect T cells, tissue sections were incubated overnight at 4°C with 20 µg/ml anti-ffLuc goat polyclonal Ab (Promega) followed by biotinylated anti-goat IgG (Vector Laboratories) and FITC-conjugated streptavidin (Vector Laboratories). Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Calbiochem). In all staining experiments, negative controls were obtained by omission of the primary Ab. Sections were blindly examined using a fluorescence microscope (Nikon Eclipse TE2000-U). The number of positive cells was expressed as the mean ± SE in 20 different x30 fields per section for nine sections and brains from three mice per condition. The p value was obtained by the generalized estimating equations method under an unbalanced repeated measures design. The size of the tumor area (square millimeter) examined in each section was calculated using the NIH ImageJ software. To detect mouse monocytes/macrophages, formalin-fixed, paraffin-embedded tissue sections were stained for F4/80 (Abcam) following the manufacturer’s instructions using tissue sections of BALB/c spleens as a positive control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Adoptively transferred T cells traffic to glioma, but not Daudi lymphoma xenografts

While performing in vivo bioluminescent imaging experiments aimed at developing methods to follow the trafficking patterns of adoptively transferred ex vivo-expanded T cells, we unexpectedly discovered that T cells home to glioma, but not Daudi lymphoma xenografts independent of Ag recognition. Real-time bioluminescent imaging with firefly luciferase-positive (ffLuc+) cells and detection with low-light imaging cameras has been successfully used to quantitatively visualize tumor burden, bacterial and viral infection, reporter gene expression, and CD4+ T cell migration to sites of autoimmune disease in living mice (25, 26, 27, 28, 29, 30).

We assessed real-time in vivo homing patterns of i.v. administered T cells in tumor-bearing mice. For these studies, we used a primary human CD8high (79%), CD4low (17%), CD45RO+ (99%) T cell bulk line that has been stably transfected to express the ffLuc reporter (23). The cell surface receptor phenotype of the ffLuc+ T cell line remained stable upon ex vivo propagation with OKT3, irradiated feeder cells, and low-dose IL-2 (data not shown and Ref. 23). Palpable U251T and U87 glioma, as well as Daudi lymphoma tumors, engrafted into the flank of NOD-scid mice were challenged with i.v. administered ffLuc+ T cells. The TCR repertoire of this T cell line has not been selected for antiglioma or antilymphoma specificity, and these CTL do not exhibit tumor-specific cytolytic activity (data not shown), thus allowing us to limit our in vivo studies to tumor-dependent effects on tropism vs secondary T cell-dependent effects. The trafficking kinetics and tumor colocalization of i.v. injected ffLuc+ T cells was detected by i.p. administration of luciferin substrate, followed by imaging of the anesthetized mice with the Xenogen IVIS system. In Fig. 1A, the resultant T cell tracking patterns are presented as pseudocolor images of light intensity. During the first 24 h after i.v. T cell administration, the ffLuc+ CD8+ T cells primarily resided in the lung tissue (Fig. 1A). Between 24 and 48 h, a redistribution of the ffLuc+ T cells is observed to other organs, predominantly liver and spleen. By 48–72 h, a preferential colocalization of the ffLuc+ T cells to the U251T and U87 glioma flank xenografts was observed with an ~7-fold increase in luminescent intensity at the site of tumor flank vs the control, nontumor-bearing flank at 72 h (Fig. 1, A and B). The colocalization of T cells at the site of glioma flank tumors persisted for a time period greater than 14 days (data not shown). By comparison, i.v. administered ffLuc+ T cells localized to Daudi flank tumors less efficiently (Fig. 1A), and minimal ffLuc signal was detected above background due to the dispersion of surviving T cells throughout the body starting at 48 h. Furthermore, the modest 2-fold increase in T cell abundance at the Daudi tumor site at 72 h (Fig. 1B) was most likely due to the fact that the inflammatory environment inherent with tumor growth attracts T cells slightly more than a nontumor-bearing site in the opposite flank.


Figure 1
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  		FIGURE 1. Intravenously administered CD8+ T cells home to s.c. and i.c. glioma xenografts independent of Ag recognition. A, Mice were inoculated s.c. in the right flank with 5 x 106 of either U251T (top row), U87 (middle row), or Daudi (bottom row) cells. The location of the tumor xenograft is indicated with an arrow in the right-most panel. On day 13 after tumor injection, 5 x 106 ffLuc+ T cells were administered i.v. through the tail vein and Xenogen images at 2, 24, 48, and 72 h after T cell injection are shown. B, Plotted is the fold increase in the ffLuc+ T cell bioluminescent signal at U251T ({blacksquare}) and U87 (Figure 1) flank tumor sites vs contralateral non-tumor-bearing control flanks at 2, 24, 48, and 72 h after T cell injection. C, Three mice were inoculated s.c. with 107 U251T cells in the right flank and 107 Daudi cells in the left flank (flank sites indicated by ovals in top left panel). Twelve days after tumor injection, 5 x 106 ffLuc+ T cells were administered i.v. through the tail vein and Xenogen images at 2, 24, 48, and 72 h after T cell injection are shown. D, Plotted is the mean ± SE fold increase in the ffLuc+ T cell bioluminescent signal at U251T flank tumor sites vs contralateral Daudi tumor sites at 48 and 72 h post T cell injection. (E) Using mice bearing i.c. U87 tumor (top row) or no tumor (bottom row), 2 x 107 ffLuc+ T cells were administered i.v. through the tail vein at day 19 and Xenogen images at 24, 48, and 72 h after T cell injection are shown. F, Plotted is the mean ± SE of the photon flux at the i.c. site in nontumor-bearing (Control) or U87-bearing mice at 72 h after i.v. T cell injection (n = 3). *, p < 0.05 comparing the U251T values to controls using Student’s t test.

 
To directly compare the homing of T cells to glioma vs Daudi xenografts, mice were engrafted with U251T tumors in the right flank and Daudi tumors in the left flank, and challenged with i.v. administered ffLuc+ T cells. Consistent with our previous observations, i.v. administered T cells preferentially traffic to sites of glioma (Fig. 1C). Quantifying the photon flux from the tumor region of interest for the three experimental mice at 48 and 72 h after T cell administration revealed an ~3-fold increase in ffLuc+ T cell homing to the U251T vs Daudi xenografts (Fig. 1D). Subsequent to the 72-h imaging time point, mice were sacrificed and tumors measured by caliper to ensure that tumors were of similar size (mouse 578: 12 x 15 mm U251T, 8 x 18 mm Daudi; mouse 579: 15 x 15 mm U251T, 15 x 15 mm Daudi; mouse 580: 10 x 10 mm U251T, 15 x 15 mm Daudi). In a few animals (for instance, mouse 580), T cells were observed to colocalize with the Daudi tumors; however, for these animals the Daudi tumors were always larger than the glioma tumors, and T cell homing was still preferentially associated with the glioma xenografts (Fig. 1D). The observed in vivo tropic response of T cells to glioma tumors is therefore unlikely solely a consequence of tumor-induced tissue trauma, because T cells do not efficiently colocalize to Daudi tumors, which would be expected to induce a similar injury response.

We also investigated whether T cells could colocalize to i.c. glioma xenografts as an orthotopic model for the trafficking of T cells to human brain tumors. For these studies, U87 cell line was engrafted i.c. for 19 days, followed by i.v. administration of ffLuc+ T cells, and tumor engraftment was confirmed by H&E staining at the end of the experiment (day 25; data not shown). As shown in Fig. 1E, ffLuc+ T cells were capable of homing from the systemic circulation to i.c. engrafted glioma tumor, whereas T cells did not efficiently home to the brain of control animals in which no tumor was engrafted. Comparison of the photon flux from nontumor bearing cranial sites showed a significant enhancement of ffLuc+ T cells localizing to sites bearing U87 tumors (Fig. 1F). Intravenously administered T cells also homed to i.c. engrafted U251T (data not shown). Taken together, these in vivo studies demonstrate that adoptively transferred T cells, independent of Ag recognition, can home to both s.c. and i.c. sites of glioma tumor engraftment.

Primary human CD8+ effector T cells chemotax in response to glioma-derived conditioned medium

Based on our in vivo trafficking studies, we hypothesized that gliomas may secrete a chemokine to which T cells respond, and that other tumor types, such as Daudi lymphoma, do not secrete this chemokine. To directly test the ability of tumor-derived chemokines to chemoattract ex vivo-expanded T cells, we compared the chemotactic response of ex vivo-expanded CD8+ T cells to conditioned medium from various tumor types. For these studies, a luminescent-based in vitro chemotaxis assay was used (23), whereby the percentage of ffLuc+ T cells that chemotax in response to a chemokine gradient to the lower well of a chemotaxis chamber is quantified based on the biophotonic flux generated by the enzymatic reaction between luciferase and its substrate luciferin.

Using the luminescent-based chemotaxis assay, conditioned media from glioma cell lines were shown to efficiently chemoattract the polyclonal ffLuc+ T cell line used in our in vivo studies. For a typical experiment, the chemotactic response of the ffLuc+ T cells to glioma cell lines U251T, U87, T98, and UAB1074 was between 10 and 20% (Fig. 2A). T cell locomotion was determined to be directional toward glioma supernatants because dilutions of the glioma-conditioned medium correspondingly reduced the chemotactic response (Fig. 2A). Also, a dramatic reduction in the migrational response was observed in chemokinesis control wells, where glioma-conditioned medium was present in both the upper and lower chambers. Consistent with these findings, CSF from a patient with GBM-CSF more efficiently chemoattracted T cells than CSF from a control patient who had recently recovered from bacterial meningitis (control CSF (c-CSF); Fig. 2B). Taken together, these studies suggest that many high-grade gliomas secrete a chemokine(s) that chemoattracts ex vivo-expanded T cells.


Figure 2
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  		FIGURE 2. In vitro chemotaxis of ex vivo-expanded T cells in response to tumor-conditioned medium. Plotted is the percentage of ffLuc+ T cells that have chemotaxed in response to (A) glioma U251T-, U87-, T98-, and UAB1074-conditioned medium; (B) human CSF fluid from a patient with GBM-CSF as compared to the c-CSF from a patient with bacterial meningitis; (C) Daoy-, Caki-1-, and SW480-conditioned medium; and (D) Daudi-, 10HTB-, and A2058-conditioned medium. Conditioned media were tested neat (undiluted) and diluted (1/2 and 1/10) with DMEM. Chemokinesis was calculated as the percentage of cells that migrated to the lower chamber when both top and bottom chambers contained undiluted conditioned medium. Data represent mean ± SD from triplicate wells of a representative experiment.

 
The T cell migrational response to tumor types other than glioma was also assessed. Interestingly, effector T cells were found to chemotax in response to Daoy medulloblastoma-, Caki-1 renal cell carcinoma-, and SW480 colon tumor-conditioned medium (Fig. 2C). However, in contrast to that seen with the glioma cell lines, high levels of chemokinesis was observed when effector T cells were incubated in Daoy, Caki-1, or SW480-conditioned medium. These chemokinesis results indicate that the induced cell movement does not specifically reflect the potential for directional migration of T cells toward these tumors. By contrast, conditioned medium from Daudi lymphoma, 10HTB neuroblastoma, and A2058 melanoma cell lines did not chemoattract ex vivo-expanded T cells (Fig. 2D). The extent of chemotaxis in response to supernatants derived from these tumor cell lines was typically <5% and dilutions of the conditioned medium did not correspondingly reduce the percent migration. Furthermore, conditioned medium from the Be2 neuroblastoma, Raji lymphoma, and a primary acute lymphoblastic leukemia line also did not chemoattract ex vivo-expanded T cells (data not shown). It remains to be resolved whether this lack of tropism is a general feature of these tumor types or a characteristic of the individual cell lines analyzed in this study. Overall, these in vitro observations expand upon our in vivo tumor tropism findings, demonstrating that tumors differ in their intrinsic ability to chemoattract ex vivo-expanded T cells.

Chemokine profiles of tumor cell lines

To evaluate differences in the cytokine secretion profiles between various tumor types and identify putative chemokine candidates responsible for the observed differential trafficking of T cells to tumor targets, we simultaneously compared the relative expression profiles of 79 cytokines using the Cytokine Ab Array V protein chip technology (RayBiotech). The list of cytokines detected by the Cytokine Ab Array System and a map of their location on the nitrocellulose chip is depicted in Fig. 3A. For these experiments, cytokine array blots were incubated with conditioned medium from glioma, lymphoma, and neuroblastoma cell lines. Comparison of the cytokine array blots identified both the CXC chemokine IL-8/CXCL8 and the CC chemokine MCP-1/CCL2 as being highly expressed in glioma-conditioned medium compared with Daudi and 10HTB (Fig. 3B). Other nonchemotactic cytokines detected by this method that appeared to be specifically expressed in the glioma-conditioned medium include IL-6, insulin-like growth factor binding protein, tissue inhibitor of metalloproteinase 1, tissue inhibitor of metalloproteinase 2, and vascular endothelial growth factor.


Figure 3
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  		FIGURE 3. Chemokine expression profiles of glioma-, Daudi lymphoma-, and 10HTB neuroblastoma-conditioned medium. A, Map of the capture cytokine-specific Abs spotted onto RayBio Cytokine Ab Array V (RayBiotech) protein chips. B, Cytokine protein profiles of conditioned medium from glioma, Daudi, and 10HTB cell lines detected using the Cytokine Ab Array V. Detected levels of MCP-1 and IL-8 are indicated with an oval and a square, respectively.

 
We next quantitatively compared MCP-1 and IL-8 levels produced by each of the tumor types previously examined in the chemotaxis assays to assess whether the level of either of these chemokines correlated to the observed chemotaxis (Table I). In agreement with the cytokine array blots, MCP-1 and IL-8 were secreted at high levels by glioma cell lines U251T, U87, T98, and UAB1074. A 6- to 7-fold increase in MCP-1 and IL-8 levels was also detected in the GBM-CSF vs the c-CSF (Table I). MCP-1 and IL-8 were also detected at high levels in Daoy and Caki-1 supernatants that exhibited high chemokinetic activity, thus suggesting that the migrational response observed in Fig. 2C may be a consequence of both chemokine-dependent directional migration as well as a yet to be identified chemokinesis-inducing factor. By contrast, SW480, Daudi, and 10HTB secreted MCP-1 and IL-8 at low levels (<1 ng/ml). Interestingly, the non-chemoattracting A2058 melanoma line secreted low levels of MCP-1 (<1 ng/ml), but moderate levels of IL-8 (>6 ng/ml), suggesting that IL-8 may not be responsible for driving T cell migration in this system. Other chemokines were also examined (i.e., IFN-{gamma}-inducible protein 10 (IP-10/CXCL10), RANTES/CCL5, MIP-1{alpha}/β{alpha}β/CCL3, and (MIG/CXCL9) monokine induced by IFN-{gamma}; data not shown), but were either 1) not detected by the majority of cell lines that elicited a CTL chemotactic response or 2) present at similar levels in both the chemotactic and nonchemotactic supernatants. Together, these results identify MCP-1 and, to a lesser extent, IL-8 as chemokine candidates capable of mediating the observed tropism of ex vivo-expanded T cells.


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  		Table I. Levels of chemokines in conditioned media from tumor cell lines and in CSF samples

 
MCP-1 chemoattracts ex vivo-expanded primary human T cells

To evaluate the chemotactic response of ex vivo-expanded effector T cells to MCP-1 and IL-8, the two chemokines identified as being most highly expressed by glioma tumor lines, a chemotactic dose response was compared. We found that the ex vivo-expanded T cells are efficiently chemoattracted by recombinant human MCP-1 (rhMCP-1) with an ED50 of 10 ng/ml (Fig. 4A), and that the rhMCP-1-dependent migrational response is chemotactic, not chemokinetic (Fig. 4B). By comparison, the ex vivo-expanded T cells did not migrate in response to rhIL-8, even at concentrations as high as 100 ng/ml (Fig. 4A). Consistent with the observed MCP-1-dependent chemotactic response, ex vivo-expanded T cells were shown to bind biotinylated rhMCP-1 and to express the MCP-1 receptor CCR2 on the cell surface (Fig. 4C). Interestingly, T cell expression of another MCP-1 receptor, CCR4, was not significant (data not shown). This suggests that CCR2 is the functional receptor on these T cells that is responsible for the MCP-1-mediated tumor tropism.


Figure 4
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  		FIGURE 4. Primary human T cells are chemoattracted by rhMCP-1, but not rhIL-8. A, Plotted is the percentage of ffLuc+ T cells that have migrated through to the lower chamber in response to various concentrations of rhMCP-1 and rhIL-8 after 2 h at 37°C. B, The percentage of ffLuc+ T cells that have migrated through to the lower chamber in response to 100 ng/ml rhMCP-1 and rhIL-8 is compared for chemotaxis (directional migration) vs chemokinesis (random migration). Chemokinesis was calculated as the percentage of cells that migrated to the lower chamber when both top and bottom chambers contained 100 ng/ml chemokine. Data represent mean ± SD from triplicate wells from a representative experiment. C, Flow cytometry analysis of ffLuc+ T cells incubated with biotin-rhMCP-1 (top, filled histogram) or the biotinylated soybean trypsin inhibitor control protein (top, open histogram) followed by avidin-fluorescein; and mouse anti-CCR2 followed by FITC-conjugated goat anti-mouse (bottom, filled histogram) or FITC-conjugated goat anti-mouse alone (bottom, open histogram).

 
To further determine whether MCP-1 is required for the T cell chemotactic response in our system, immunodepletion studies were performed. Conditioned supernatants from U251T, T98, and U87 were incubated with either anti-MCP-1 or anti-IL-8 polyclonal Abs; Ab-cytokine complexes were depleted using protein G-agarose beads; and immunodepleted supernatants were tested for their chemotactic capacity (Fig. 5). The efficient immunodepletion of rhMCP-1 (150 ng/ml) chemotactic activity validates the removal of MCP-1 by this procedure. Immunodepletion of MCP-1 abolished the chemoattractant ability of the glioma-conditioned medium (Fig. 5A). The chemotactic response of T cells was unaffected by immunodepletion with anti-IL-8 and restored by readdition of rhMCP-1, supporting the specificity of the response to MCP-1 removal. Neutralization of the chemokines IP-10/CXCL10, IFN-inducible T cell {alpha} chemoattractant (ITAC/CXCL11), stromal cell-derived factor 1 (SDF-1/CXCL12), macrophage-derived chemokine (MDC/CCL22), and thymus and activation-regulated chemokine (TARC/CCL17) in glioma-conditioned supernatants did not reduce levels of chemotaxis (data not shown). Moreover, blocking Abs to chemokine receptors CXCR2, CXCR3, and CXCR4 also showed no decrease in glioma-mediated chemotaxis (data not shown). Taken together, these studies identify MCP-1 as the primary chemokine responsible for glioma-specific recruitment of ex vivo-expanded T cells in vitro.


Figure 5
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  		FIGURE 5. Immunodepletion of MCP-1 nullifies the in vitro chemotactic response of ex vivo-expanded T cells to glioma-conditioned medium. Plotted is the percentage of ffLuc+ T cells that have chemotaxed in response to medium containing rhMCP-1, or conditioned medium (CM) from glioma cell lines (A) or nonglioma cell lines (B) that had been immunodepleted using either anti-MCP-1 or anti-IL-8 Abs.

 
The chemotactic capacities of conditioned medium from nonglioma Daoy, Caki-1, and SW480 cells were also examined for their dependence on MCP-1 (Fig. 5B). Immunodepletion of MCP-1 from Daoy and Caki-1 samples reduced the chemotactic activity, compared with IL-8 depletion, by ~68 ± 21% and 33 ± 7%, respectively, consistent to the observation that Daoy supernatants contain an average of 46,788 pg/ml MCP-1 and Caki-1 supernatants contain an average of 11,822 pg/ml MCP-1 (Table I). However, the incomplete depletion of chemotactic activity and the observed chemokinesis (Fig. 2C) suggests that T cell-attracting chemokines other than MCP-1 may be present in these supernatants. It is of interest to note that immunodepletion of MCP-1, a well-established chemotactic chemokine, reduces the migrational response of the highly chemokinetic Daoy and Caki-1 extracts. This observation was unexpected but highly reproducible, and may possibly suggest a physical or functional interaction between MCP-1 and a yet to be identified chemokinetic factor. In contrast, the chemotactic activity of SW480-conditioned medium is not affected by immunodepletion of MCP-1, consistent with the observation that this tumor line does not secrete significant levels of MCP-1 (Table I). Thus, the in vitro chemotaxis of ex vivo-expanded T cells directed by these representative medulloblastoma, renal cell carcinoma, and colon tumor cell lines does not appear to be as strongly dependent on MCP-1 as that seen with the glioma tumor cell lines.

MCP-1 is sufficient for T cell recruitment both in vitro and in vivo

Our in vitro studies have identified an important role for tumor-derived MCP-1 in directing the migration of effector T cells. Therefore, to address whether MCP-1 is sufficient to mediate T cell recruitment to sites of tumor burden in vivo, Daudi cells were engineered to secrete rhMCP-1. As shown in Fig. 6A, conditioned medium prepared from vector control-transfected Daudi-LRZ and the MCP-1-engineered Daudi-LRZ-MCP1 cell lines produce similar levels of IP-10 and MIG, but only the Daudi-LRZ-MCP1 line secretes MCP-1. The MCP-1 chemokine level produced by the Daudi-LRZ-MCP1 line was sufficient to chemoattract T cells in an in vitro chemotaxis assay (Fig. 6B).


Figure 6
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  		FIGURE 6. The i.c. tumor infiltration of adoptively transferred i.v. T cells is dependent on MCP-1. A, Secreted MCP-1, MIG, and IP-10 cytokine levels for Daudi-LRZ-MCP1- and Daudi-LRZ- conditioned medium as quantified by the Chemokine I Cytometric Bead Array System (BD Biosciences). B, In vitro chemotaxis of ffLuc+ T cells in response to human Daudi-LRZ-MCP1-conditioned medium, Daudi-LRZ-conditioned medium, and 100 ng/ml rhMCP-1. C, Mice were inoculated i.c. with 5 x 105 Daudi-LRZ or Daudi-LRZ-MCP1, and on day 19 after tumor injection, 10 x 107 ffLuc+ T cells were administered i.v. through the tail vein and Xenogen images (maximum (MAX) 8000; minimum (MIN) 3000) at 72 h after T cell injection are shown. D, Upper panels, Horizontal tissue sections of mouse brains with established i.c. xenografts of Daudi-LRZ and Daudi-LRZ-MCP1 tumor lines (scale bar, 1 mm). Representative sections were stained with H&E. Inset highlights magnified tumor image (scale bar, 100 µm). Lower panels, Corresponding anti-ffLuc-stained brain sections by immunohistochemistry indicating T cell infiltration into tumor mass and counterstained with DAPI (scale bar, 100 µm). Inset is a magnified image of ffLuc+ T cells. E, Average number of infiltrated ffLuc+ T cells within i.c. tumor mass (+SE) as quantified by immunohistochemistry using 20 different x30 fields from one to three sections per mouse (total of seven sections per experimental group). *, p = 0.03 when compared with the Daudi-LRZ group.

 
We next evaluated whether tumor-derived MCP-1 is sufficient for mediating T cell tumor tropism in vivo. Daudi-LRZ and Daudi-LRZ-MCP1 tumor cell lines were engrafted i.c., and animals were challenged with i.v. administered ffLuc+ T cells. In vivo biophotonic imaging in mice demonstrate a preferential homing of the ffLuc+ T cells to intracranial MCP-1-secreting Daudi tumors (Fig. 6C). The ffLuc+ T cell biophotonic flux for one mouse was significantly stronger than either of the other two mice. Upon tumor dissection, we observed that the i.c. Daudi tumors for one mouse in each experimental group (Daudi-LRZ-MCP1 and Daudi-LRZ) extended into the subarachnoid space. Although this extraparenchymal tumor growth did not alter the incidence of T cell infiltration, it dramatically increased the observed luminescent flux. To quantitatively compare the preferential homing of adoptively transferred T cells to MCP-1-secreting tumors, brain specimens were processed for histological analysis. Engrafted Daudi-LRZ and Daudi-LRZ-MCP1 tumors were confirmed to be of comparable size (1–2 mm in diameter) by H&E staining of the frozen brain sections (Fig. 6D). The similar tumor sizes suggest that tumor-derived cytokines other than MCP-1 and tumor-dependent brain injury responses are similar between the two experimental groups. The ffLuc+ T cells were detected by immunohistochemical staining of the brain sections with an anti-ffLuc Ab. Only the Daudi-LRZ-MCP1 brain sections exhibited significant numbers of ffLuc+ T cells that extravasated out of the blood stream to infiltrate the tumor site (Fig. 6D). This increase in the number of ffLuc+ T cells that had infiltrated the MCP-1-producing tumor mass was found to be statistically significant (Fig. 6E). Because MCP-1 is a potent monocyte chemoattractant, we also investigated the possibility that the difference in observed T cell homing could be mediated by differences in monocyte infiltration of the Daudi tumors. However, upon examination of the levels of infiltrating mouse monocytes by immunohistochemistry, no significant mouse monocyte population was found to have infiltrated either Daudi-LRZ or Daudi-LRZ-MCP1 tumors (data not shown). Furthermore, it must be noted that monocytes in NOD-scid mice are functionally deficient in their ability to secrete cytokines in response to stimuli (31). Thus, together these findings demonstrate that tumor-derived MCP-1 is sufficient to promote tumor tropism of adoptively transferred T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
One prerequisite for effective adoptive immunotherapy of malignancy is the localization of systemically administered effector cells to anatomically disparate sites of tumor metastases. Although T cells can, upon activation, produce chemokines that recruit more effector cells, a purely stochastic mechanism of T cell tumor tropism would predict suboptimal therapeutic effects in tissues that are not efficiently accessed by T cells following adoptive transfer. In this study, we show that tumors can direct the tropism of adoptively transferred ex vivo-expanded T cells propagated using culture systems currently used for human clinical studies. Our studies demonstrate that adoptively transferred T cells can efficiently transmigrate across endothelial barriers into both s.c. and i.c. tumor xenografts in response to the tumor-derived chemokine MCP-1. Although our data suggest that this chemotaxis may be mediated through CCR2, future studies will better define the receptors involved in the observed MCP-1-mediated homing of T cells to tumors, since others have found that MCP-1 directed migration can be dependent on CCR4 (32, 33).

We have shown that MCP-1 is secreted by a variety of glioma cell lines and is present in glioma resection cavity CSF, and therefore MCP-1 production may be a general feature of high-grade gliomas. Our data are consistent with other reports that have shown MCP-1 to be expressed in glioblastomas (34, 35). We have also found that the medulloblastoma cell line Daoy and the renal cell carcinoma line Caki-1 produce sufficient levels of MCP-1 to chemoattract ex vivo-expanded T cells in vitro. Other tumor types are also known to constitutively produce high levels of MCP-1, including melanoma (36), ovarian cancer (37), breast carcinoma (38), Hodgkin’s lymphoma (39), and lung cancer (38). However, as exemplified by the SW480 colon tumor, Daudi lymphoma, 10HTB neuroblastoma and A2058 melanoma, MCP-1 does not appear to be produced by all tumors. Recently, Metelitsa et al. (40) have shown that 53% of primary neuroblastomas from patients with metastatic disease (stage 4) secrete MCP-1, suggesting that there is heterogeneity of MCP-1 expression even within a certain type of tumor. This may help explain the fact that our studies with the human melanoma A2058 cells are in direct contrast to the recent report by Zhang et al. (33), where it was found that human T cell migration toward autologous melanoma cells in culture was dependent on MCP-1/CCL2. Furthermore, our studies do not preclude the necessity of future studies aimed at identifying chemokines other than MCP-1 that mediate T cell tumor tropism, especially because the chemotactic capacity of SW480 cells appeared to be independent of MCP-1 (and IL-8). Additionally, several tumor cell lines evaluated in this study, including Daoy, Caki-1, and SW480, appear to secrete a yet to be identified chemokine(s) that elicits random T cell movement (chemokinesis), and the significance of this chemokine for tumor tropism remains to be resolved. Overall, these studies emphasize the differences in chemokine secretion profiles between tumors and raise the possibility that these tumor-specific chemokine signatures may be important in tumor progression, metastasis, and biology.

We envision that human cancers that produce MCP-1 or other analogous chemokines capable of promoting the efficient extravasation of adoptively transferred T cells across endothelial cell barriers and into tumor metastases will likely be more amenable targets for adoptive immunotherapy. Tumors engineered to overexpress chemokines for augmented lymphocyte recruitment have increased levels of endogenous infiltrated T cells resulting in delayed tumor growth (41) and enhanced antitumoral responses to adoptive T cell therapy (16, 42).

The MCP-1 chemokine has multifunctional activities that appear to have opposing effects on tumor biology. MCP-1 can directly promote angiogenesis in vitro (43) and may also increase the levels of angiogenic factors in vivo through the recruitment of tumor-associated macrophages (44). MCP-1 has been shown to be critical for glioma tumor cell proliferation (45), cancer cell metastasis (46, 47), and is an important determinant of tumor aggressiveness (48, 49, 50, 51). Conversely, MCP-1 is a potent leukocyte chemoattractant and may play an important role in the host antitumor immune response. MCP-1 has been shown to promote in vitro chemotaxis of both CD8+ and CD4+ cells (52, 53, 54). Levels of tumor-derived MCP-1 often correlate with macrophage infiltration of malignant tissue. V{alpha}24-J{alpha}18-invariant NK T cells have also been shown to traffic to and infiltrate neuroblastoma tumors in a MCP-1-dependent manner (40). We demonstrate in this study that MCP-1 increases the efficiency by which adoptively transferred ex vivo-expanded T cells home to sites of tumor burden.

Although CD8+ and CD4+ T cells chemotactically respond to MCP-1 in vitro, patient specimens of malignant gliomas are not routinely permeated with large numbers of lymphocytes. Possible explanations of this apparent discrepancy include the possibility that tumor-induced myeloid suppressor cells and/or regulatory T cells negatively regulate immune surveillance against established cancers. Alternatively, it has been observed that immune cells are rapidly desensitized to MCP-1 chemokine gradients, and high systemic concentrations of tumor-derived MCP-1 may render cells in the circulation refractory to MCP-1 gradients created by tumors. In support of these hypotheses, a reduction in T cell and monocyte chemotactic responses have been observed in patients with malignant melanomas that secrete MCP-1 (55, 56), and MCP-1 binding to its receptor CCR2 has been found to promote rapid desensitization of chemotactic calcium flux responses (57). Furthermore, transgenic mice engineered to constitutively express MCP-1 protein in various organs resulted in monocyte and T cell nonresponsiveness to the locally produced MCP-1 (58).

This is one of the first studies to follow in real time the trafficking patterns of i.v. administered T cells to sites of tumor burden in animals. Our findings highlight a critical role for the MCP-1 chemokine in mediating immune surveillance by ex vivo-expanded T cells. Our demonstration that tumor infiltration does not solely depend on Ag recognition, but is also regulated by tumor-derived chemokines, identifies polyclonal ex vivo-expanded T cells as a readily accessible cellular delivery vehicle for tumor-toxic molecules. We are currently investigating the implications of the observed MCP-1-dependent tumor tropism for Ag-specific T cells to determine whether tumors capable of chemoattracting adoptively transferred T cells may exhibit a greater clinical response to T cell adoptive therapy. Alternatively, improved clinical outcome in patients with tumors that do not produce MCP-1 may be achieved by the expression of chemokine receptor transgenes in therapeutic T cells that respond to alternate tumor-derived chemokines such as IL-8, thus endowing tropism to tissues that harbor tumor metastasis. A more thorough understanding of the factors that influence treatment efficacy, such as tumor tropism of adoptively transferred T cells, will hopefully improve future clinical trial designs and cure rates.


   Acknowledgments
 
We thank W. Chang, E. Lin, C. Wright, A. Naranjo, B. Meechoovet, and J. Wagner for technical assistance; J. R. Ostberg for preparation of this manuscript; and L. J. Cooper, M. Kalos, and S. Riddell for their comments and critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health (R01-CA103959); Michael Hoefflin Foundation, Ronald Flax Memorial Pediatric Cancer Research Fund; The Estelle, Abe, and Marjorie Sanders California Foundation; and Cancer Center Support Grant (5P30-CA33572-21). M.C.J. is a recipient of the Stop Cancer Foundation Career Development Award. C.E.B. is a Leukemia and Lymphoma Society of America Research Fellow. Back

2 Address correspondence and reprint requests to Dr. Christine Brown, Division of Cancer Immunotherapeutics and Tumor Immunology, City of Hope National Medical Center, KCRB 3009, 1500 East Duarte Road, Duarte, CA 91010. E-mail address: cbrown{at}coh.org Back

3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; c-CSF, control cerebrospinal fluid; i.c., intracerebral; IP-10, IFN-{gamma}-inducible protein 10; rh, recombinant human; GBM, glioblastoma multiforme. Back

Received for publication January 20, 2006. Accepted for publication June 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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