Quantitation and visualization of tumor-specific T cells in the secondary lymphoid organs during and after tumor elimination by PET

Nuclear Medicine and Biology 31 (2004) 1021–1031

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Quantitation and visualization of tumor-specific T cells in the secondary lymphoid organs during and after tumor elimination by PET Ken Matsuia, Zheng Wangb, Timothy J. McCarthyc, Paul M. Allena, and David E. Reichertd,* a

Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, USA b University of Texas Health Science Center, Department of Radiology, 7703 Floyd Curl Dr., San Antonio TX 78229-3900, USA c World Wide Clinical Technology, Pfizer Global R&D, Eastern Point Rd., MS8260-2605, Groton, CT 06340, USA d Department of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., Campus Box 8225, St Louis, MO 63110, USA Received 20 January 2004; received in revised form 4 June 2004; accepted 8 June 2004

Abstract Increased understanding in the area of trafficking behavior of adoptively transferred tumor-specific T cells could help develop better therapeutic protocols. We utilized the DUC18/CMS5 tumor model system in conjunction with a microPET scanner to study the DUC18 T cell distribution pattern in spleens and lymph nodes in live mice. Anti-Thy1.2 antibodies conjugated to 1,4,7,10-tetraazacyclododecaneN,N⬘,N⬘⬘,N⬘⬘-tetraacetic acid (DOTA) and radiolabeled with 64Cu were administered to three groups of BALB-Thy1.1 mice on days 4, 7, or 14 post-DUC18 T cell transfer. We were able to detect the transferred cells in all the major lymph nodes, spleens, and in tumors. Our findings suggest that tumor-specific T cells do not all preferentially localize to the tumors but they also home to all the major lymphoid organs; additionally the number of DUC18 T cells remains relatively constant during and after tumor elimination within each lymphoid organ. © 2004 Elsevier Inc. All rights reserved. Keywords: DUC18; CMS5; PET; DOTA;

64

Cu; Immunotherapy

1. Introduction Antigen-specific CD8⫹ T cells have been utilized in the treatment of cancer [1–10]. Although these tumor-specific T cells have been shown to infiltrate into the tumors [1,6,9 – 11], surprisingly little is known about the in vivo distribution pattern of the infused T cells, and it is not clearly known whether tumor-specific T cells preferentially accumulate in certain organs during the tumor ablation process. It is of a great interest to study T cell trafficking in live hosts because the information gained from such study could potentially lead to the development of more effective protocols. Positron emission tomography (PET) can be utilized to detect cells, proteins, and gene expressions in vivo [12–17]. It is a non-invasive imaging modality that allows the acquisition of whole body static or dynamic images. In the past, PET, utilizing clinical scanners, has been used to follow the

* Corresponding author. Tel.: ⫹1-314-362-8461; fax: ⫹1-314-3629940. E-mail address: [email protected] (D. E. Reichert). 0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nucmedbio.2004.06.002

in vivo distribution of natural killer cells and metastatic tumor cells [18 –24]. These studies relied on labeling the cells of interest with short-lived positron emitting labels such as 2- [18F] fluorodeoxyglucose (18F t1/2⫽109.71 min) or [11C]methyl iodide (11C t1/2⫽20.4 min). The short halflives of these isotopes limited the time frames in which the transferred cells can be imaged. The development of dedicated small animal positron emission tomography (microPET) has been applied to several biological systems [25–28]. In a proof of concept study, Adonai et al. utilized copper-64-pyruvaldehyde-bis(N4methylthiosemicarbazone) (64Cu-PTSM) to label C6 rat glioma cell line and splenocytes to examine if these cells can be tracked in vivoin live mice [25]. The study showed that the experiment could be performed with a little adverse effect on cell viability and function. Although 64Cu has a longer half-life than more common PET isotopes, it still imposes time constraints on the imaging studies. The 64CuPTSM labeled cells allowed the investigators to perform the scanning only up to 36 hours post-cell labeling. Copper-64 is a positron emitting isotope with a longer half-life than that of 18F or 11C, t1/2⫽12.7 hours, and is readily available

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through the 64Ni(p,n)64Cu nuclear reaction on a biomedical cyclotron [29]. Copper-64 is a particularly useful PET isotope for imaging biological processes of moderate duration such as antibody localization; due to its relatively long half-life and mild labeling conditions to previously synthesized bioconjugates. Subsequently, Witte and colleagues retrovirally tranduced the HSV1-sr39TK PET reporter gene into T cells [26]. These cells were transferred into tumor-bearing mice and in vivo visualization of these cells were achieved by administering 9-[4-[18F]fluoro-3 (hydroxymethyl) butyl] guanine ([18F]FHBG), a substrate for sr39TK. In this system, the same mice were monitored over a period of days, and the localization of T cells to the tumors was successfully visualized. Though well designed, the study did not address how T cell distribution might change in different secondary lymphoid organs, and whether the system was sensitive enough to detect T cells in small organs such as lymph nodes. Previously, we applied microPET imaging to a murine arthritis model to study the kinetics and trafficking pattern of the infused arthritogenic antibodies [28]. Antibody molecules were conjugated and labeled with 1, 4, 7, 10-tetraazacyclododecane-N,N⬘,N⬘⬘,N⬘⬘-tetraacetic acid (DOTA) and with 64Cu, respectively. The method allowed us to capture the dynamic images of antibody molecule trafficking within the same mice in a real-time fashion. In the current study, we have applied the use of 64Cu-labeled antibodies to our DUC18/CMS5 murine tumor model system [30,31]. Our primary goal was to examine the homing pattern of in vitro stimulated DUC18 T cells. Specifically, we were interested in examining how the infused T cells’ overall distribution pattern might change during and after tumor elimination at the level of secondary lymphoid organs. Using BALB/c-Thy1.1 congenic mouse strain as tumor hosts, we specifically detected the transferred DUC18 T cells (Thy1.2) by administering anti-Thy1.2 antibody molecules that have been conjugated and labeled with DOTA and 64Cu. The microPET scanner provided us with whole body images of mice that displayed the locations of CMS5specific DUC18 CD8⫹ TCR transgenic T cells in live hosts. We found that DUC18 T cells localize to all the major secondary lymphoid organs, including small structures such as iliac, brachial, and axillary lymph nodes. In addition, we were able to discern the presence of tumor-infiltrating DUC18 T cells. The results demonstrated the power and sensitivity of the microPET scanner when used in combination with 64Cu-labeled antibody, as it detected tumorspecific T cells that localized to small lymph nodes. More importantly, we found that the infused DUC18 T cells home to all the major secondary lymphoid organs and we did not observe major changes in the T cell number within individual lymphoid compartment during and after tumor elimination.

2. Materials and methods 2.1. Cell lines and reagents The maintenance of the fibrosarcoma cell lines CMS5 was carried out as described in [30,31]. The tERK-I (QYIHSANVL) epitope was synthesized, purified, and characterized as described [30,32]. Antibodies used in this study were purchased from BD Biosciences (San Diego, CA, USA) and Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). 2.2. Mice All animal studies were performed in accordance with procedures approved by the Washington University Animal Studies Committee. All mice were maintained in a specific pathogen-free barrier facility at Washington University School of Medicine. BALB/c-Thy1.1 congenic mice were a kind gift from Dr. Hyam Levitsky (Johns Hopkins School of Medicine). The generation and characterization of the DUC18 CD8⫹ TCR transgenic mouse is described elsewhere [30]. 2.3. Preparation of in vitro activated DUC18 T cells In vitro stimulation of DUC18 splenocytes were performed as described [31]. Briefly, a single cell suspension of DUC18 splenocytes (5⫻106/mL) were cultured in R102-ME in 10 cm tissue culture dishes with 0.5 ␮M of tERK-I peptide. After 3 days of culture, the cells were split 1:1 with R10-2ME medium [30,31] alone and incubated for an additional 24 hours. On the day of adoptive transfer, the cells were purified using ficoll-paque, (Pharmacia Biotech, Uppsala, Sweden), washed three times in HBSS, and resuspended in PBS (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 0.9% NaCl) for injection. The determination of the percentage of DUC18 T cells was carried out as described [30,31]. The activated T cells consisted of 90-95 % DUC18 T cells as determined by the flow cytometry analysis. 2.4. Tumor transplantation, adoptive T cell transfer, and measurement of tumor growth Cultured CMS5 cells were harvested by trypsinization, washed three times in HBSS, and resuspended in PBS at 15⫻106 cells/mL. Three million CMS5 cells were inoculated into BALB/c or BALB/c-Thy1.1 mice subcutaneously on the right hind flank. Eight days later, tERK-I peptidestimulated DUC18 T cells were adoptively transferred i.v. After 2 days, the tumor size was measured with calipers, and the measurements are expressed as the product of two orthogonal diameters [30,31].

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2.5. DOTA linker conjugation to antibody The anti-CD90.2 (Thy1.2) antibody molecules used in this work were conjugated to 1,4,7,10-tetraazacyclododecane-N,N⬘,N⬘⬘,N⬘⬘-tetraacetic acid (DOTA) following the method of Lewis [33]. The following molar ratios of mAb to reagents were utilized, mAb (1):DOTA (1000):sulfoNHS (1,000):EDC (100) (sulfo-NHS ⫽Nhydroxysulfosuccinimide, EDC ⫽1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide). After conjugation, the reaction mixture was repeatedly centrifuged through a Centricon-30 spin column (Millipore, Bedford, MA, USA) with 30 mM ammonium citrate buffer pH 6.5. The purified conjugates were concentrated to 2 mg/mL in PBS and stored at ⫺80°C for further use. The concentrations of antibody-conjugates were determined by UV spectroscopy. The effective number of chelates conjugated to anti-CD90.2 was determined to be approximately eight, using the method of Meares and coworkers [34]. 2.6. Flow cytometry Three groups of day-8 established CMS5 tumor-bearing BALB/c- Thy1.1 mice were treated with in vitro-stimulated DUC18 T cells. These mice were sacrificed for flow cytometric analysis for the presence of transferred DUC18 T cells. The mice were analyzed on days 4, 7, and 14 post-T cell transfers. On the appropriate day, spleen, inguinal and axillary/brachial lymph nodes were dissected. A single cell suspension was prepared, cell number was counted, and the cells were stained with antibodies against Thy1.2 and CD8. Flow cytometry was performed using a FACS Calibur (Becton Dickinson), and 0.1– 0.5⫻106 events were collected. Results were analyzed using Cell Quest 3.3 software. The percentages of the Thy1.2/CD8 double positive were used to enumerate the absolute number of DUC18 T cells in each organ. Biotin-conjugated anti-Thy1.2, DOTA-conjugated anti-Thy1.2, and cold copper-labeled DOTA-antiThy1.2 antibodies were also used to stain DUC18 (Thy1.2) and BALB/c-Thy1.1 splenocytes. BALB/c-Thy1.1 and DUC18 spleens were dissected and a single cell suspension was prepared. One million cells were incubated with 250 ng of the antibodies on ice for 30 minutes. The samples were washed in PBS 0.5% BSA, followed by 30 minutes incubation with fluorescein isothiocyanate-conjugated goat antirat antibody on ice. The samples were washed with PBS 0.5% BSA and analyzed with a FACS Calibur. 2.7. 64Cupreparation and labeling of antibody 64

Cu (64CuCl2 in 0.1 M HCl; radionuclide purity⬎99%) was produced with an in-house cyclotron from enriched 64 Ni targets by a previously published method [29]. On the day of the experiment, typically 100 ␮g of DOTAconjugated anti-Thy1.2 antibody molecules and 1 mCi of

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CuCl2 were incubated in 0.03 M ammonium citrate, pH 6.5, at 43°C for 45 minutes. The reaction was terminated by the addition of 57 ␮l 10 mM diethylenetrinitrilopentaacetic acid (DTPA) solution. The labeled antibody molecules were then separated from unincorporated 64Cu by utilizing size exclusion BioSpin6 columns (BioRad, Richmond, CA, USA). The radiolabeling efficiency was determined by integrating areas on the fast protein liquid chromatography (FPLC) joined to a flow-through radiodetector and calculating the percentage of radioactivity associated with the corresponding protein peaks for both the relevant and irrelevant mAbs. The isotype-matched anti-TNP rat antibody was also prepared the same way (data not shown). Natural CuDOTA-antiThy1.2 antibody was prepared in the same fashion as the radioactive, using natCuCl2 in ammonium citrate pH 6.5 (1 ␮g/␮L) and incubating at RT for 1 hour. The reaction was terminated by the addition of diethylenetrinitrilopentaacetic acid (DTPA) solution, followed by isolation on a BioSpin6 column. 2.8. microPET scanning and ROI analysis Description and the procedure of microPET scanning have been described in our previous study [28]. Briefly, the microPET-R4 rodent scanner (Concorde Microsystems, Knoxville, TN, USA) provides a 10 cm by 8 cm field of view with a reconstructed resolution of 2.25 mm in the central 40 mm of the field of view. Images are reconstructed using Fourier re-binning followed by two-dimensional filtered backprojection. A total of 14 CMS5 tumor bearing mice were imaged on the microPET for the work reported in this study, the details of the model used follows. On the appropriate days, three groups of BALB/c-Thy1.1 mice were inoculated with 3⫻106 CMS5 cells into the hind flank. On days ⫺14, ⫺7, and ⫺4 prior to the 64Cu-labeled DOTA-anti-Thy1.2 antibody administration, these groups of mice were given forty million activated DUC18 T cells. On day 0, the antibody was infused into all mice, and they were scanned with the microPET scanner 12 hours later. Approximately 25 ␮g of 64 Cu-labeled antibody molecules (300 ␮Ci) were drawn and administered into mice via tail vein using 27 gauge needles. The activity of the delivered doses was determined by counting each sample syringe prior to and following the infusion using a dose calibration instrument (Radioisotope Calibrator CRC-12, Capintec Inc., NJ, USA). Twelve hours later, the mice were anesthetized with 1–2% vaporized isofluorane for microPET scanning. A single mouse or a pair of mice was immobilized in a supine position upon custom-built support beds with attached anesthetic gas nose cones for data collection in the microPET-R4 scanner. The mice are warmed via a heat lamp while on the scanner resulting in a average temperature of 32–34°C. Data were collected continuously for 15–20 minutes per mouse. The images were then reconstructed and quantitation of the regions of interest was performed on the selected tissues,

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corrected for the decay of 64Cu activity. Mandibular, brachial/axillary, mesenteric, inguinal, popliteal, and inguinal lymph nodes were detected in all DUC18 T cell recipient mice. The ROI analysis was performed with ASIPro software developed by Concorde Microsystems in IDL (International Data Language, Research Scientific Inc., Boulder, CO, USA). The data were expressed as percent of input dose (%ID) for each organ we analyzed (%ID ⫽total amount of activity per organ/the amount of activity administered). Two-dimensional images of the experiment was extracted from different frames of three-dimensional images and imported into Adobe Photoshop. 2.9. Immunofluorescent staining of DUC18 T cells in tumors CMS5 tumor-bearing BALB/c- Thy1.1 mice that received activated DUC18 T cells were sacrificed four days after the T cell administration. Tumors were placed in Tek Optimal Cutting Temperature (O.C.T) compound (VWR, West Chester, PA, USA) and frozen in liquid nitrogen. The frozen blocks were stored at ⫺70°C. The tissues were sectioned by using a cyrostat, model JUNG CM1800 (Leica, Plymouth, MN, USA), to generate 6 ␮m thick tissue sections. The sections were air-dried, fixed in 95% ethanol, and dried. The tissue sections were blocked with 0.5% BSA in PBS for 30 minutes at room temperature, washed once in plain PBS for 5 minutes, followed by 15 minute incubations with avidin then biotin (Vector Laboratories, Inc., Burlingame, CA, USA). The samples were then incubated with biotin-conjugated anti-Thy1.2 antibody or with an isotype matched control (data not shown) for 1 hour at room temperature, washed in PBS, and stained with streptavidinphycoerythrin (Caltag Laboratories, Burlingame, CA, USA) for 1.5 hours at room temperature. The sections were washed twice in PBS and cover slips were gently placed with fluoromount-G (Southern Biotechnology Associations Inc., Birmingham, AL, USA). Pictures were taken by using Optronics digital camera Magna Fire Model-S99802 (Optronics, Goleta, CA, USA) with Nikon Eclipse microscope with a 10⫻ objective lens and 10⫻ eyepieces. Images were captured by using a software analySIS version 3.2 (Soft Imaging System, Lakewood, CO, USA) and imported into Adobe Photoshop.

Fig. 1. Activated DUC18 T cells successfully ablate day-8 established tumors. Day-8 established tumor-bearing BALB/c mice were treated either with PBS vehicle control or with forty million in vitro-stimulated DUC18 splenocytes. Tumor size was measured as described in Materials and Methods. The means from three independent experiments are shown (n⫽10 for each group). Bars indicate standard error of the mean.

We transferred antigen-specifically stimulated DUC18 T cells into day-8 established CMS5 tumor-bearing BALB/c mice. We observed that the tumors were visibly smaller by day 4 post-transfer and were completely ablated within 1 week (Fig. 1). Also, the DUC18 T cell recipient mice remained tumor-free for the duration of the experiment and no outgrowth of escape variants were observed (Fig. 1, data not shown). In contrast, a vehicle control with PBS failed to retard the tumor growth (Fig. 1). Of interest, on day 2 post-transfer, the tumor size was the same in the PBS and the DUC18 T cell recipient groups, indicating that DUC18 T cells have not shown enough anti-tumor activity at this time point (Fig. 1). Therefore, we reasoned that it would be pertinent to define day 4 post-transfer as a point in which the tumor is actively getting eliminated. Overall, the data show the reproducibility and reliability of the DUC18/CMS5 system, and it provides an ideal model to study the in vivo distribution pattern of tumor-specific T cells. 3.2. DOTA conjugated anti-Thy1.2 and copper-labeled DOTA-anti-Thy1.2 antibodies retain antigen-specificity

3. Results 3.1. Anti-tumor activity of DUC18 CD8⫹ T cells against day-8 established CMS5 tumors We previously reported that activated DUC18 T cells can potently and specifically eliminate subcutaneously transplanted fibrosarcoma cell line CMS5 but not an irrelevant fibrosarcoma cell line MethA [31].

To ensure that DOTA-conjugated anti-Thy1.2 antibody retained its specificity with or without the presence of copper we first performed flow cytometry on splenocytes. We stained spleen cells from DUC18 (Thy1.2) and BALB/cThy1.1 mice with either DOTA-conjugated rat anti-Thy1.2 antibody or with natural copper-labeled DOTAconjugated rat anti-Thy1.2 antibody, followed by FITC-conjugated anti-rat antibody. As a control, we utilized the biotin-conju-

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Fig. 2. DOTA-conjugated anti-Thy1.2 antibody retains its specificity. Antibodies were utilized to stain BALB/c-Thy1.1 and DUC18 (Thy1.2) spleen cells. DUC18 splenocytes were also stained with anti-rat-FITC antibody alone. A representative histogram from the three separate flow cytometry analyses is shown. Horizontal bars indicate the region of Thy1.2 positive cell population. Arrows point to the cell population that was stained with anti-rat-FITC antibody alone and Thy1.1 splenocytes that were stained with the Thy1.2 antibodies.

gated rat-anti-Thy1.2 antibody to stain DUC18 and BALB/c- Thy1.1 splenocytes. All three antibodies detected the Thy1.2 expressing cells in DUC18 splenocytes; however, they did not recognize BALB/c-Thy1.1 cells (Fig. 2). We stained three different DUC18 splenocytes with the three antibodies. We found that the average percentages of Thy1.2 expressing cells detected by the antibodies were 30.02% for biotin-Thy1.2, 31.45% for DOTA-Thy1.2, and 30.86% for cold copper labeled-DOTA-Thy1.2. Similar results were obtained with other preparations of DOTA-conjugated anti-Thy1.2 antibodies (data not shown). Further, all three antibodies showed similar dose responses when we performed a dose titration experiment (data not shown). The findings show that the conjugation of DOTA to the antibody and the presence of copper do not alter the antigen specificity and the antibodies can detect all the Thy1.2 positive cell population. 3.3. DOTA-conjugated 64Cu-labeled anti-Thy1.2 antibody specifically detects DUC18 T cells in spleens, lymph nodes, and tumors Next, we utilized a microPET scanner to examine the DUC18 T cell distribution. We were particularly interested in studying how the T cell distribution in various secondary lymphoid organs is affected during and after the tumor elimination. In addition, we examined for the presence of tumor-infiltrating T cells. Because the experiment takes days, direct labeling of DUC18 T cells with 64Cu-PTSM (t1/2⫽12.7 hours) was not feasible. Therefore, we decided to

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administer 64Cu-labeled DOTA-anti-Thy1.2 antibody on different days: (1) 4 days post-DUC18 T cell transfer; (2) 1 week post-transfer; and (3) 2 weeks post-transfer. As mentioned before, the 4 days post-transfer date was selected because the established CMS5 tumors are actively being eliminated at this time point (Fig. 1). The later time points were chosen to determine whether DUC18 T cells might preferentially home to certain lymphoid compartments after tumors have been ablated and how this distribution might differ from the 4 days post-transfer time point. We utilized the BALB/c mouse strain that is congenic for the Thy1 gene. BALB/c- Thy1.1 mice express only the Thy1.1 allele, whereas DUC18 T cells express the Thy1.2 allele. Hence, DUC18 cells can be distinguished from the endogenous host cells. We inoculated all the BALB/cThy1.1 mice with CMS5 cells 8 days prior to the DUC18 T cell infusion. And on days 4, 7, and 14 post-T cell infusions, we administered 64Cu-labeled anti-Thy1.2 antibodies to these mice. The groups were setup in a staggered fashion that allowed us to administer the antibody and perform microPET imaging on all the mice on the same day [Fig. 3(A)]. Twelve hours post-anti-Thy1.2 antibody administrations, we collected the static images of the mice. This time point was chosen because we have empirically determined that it will provide the strongest signal/noise ratio (data not shown). The ventral, dorsal and sagittal views of representative mice from three separate experiments (n⫽14) are shown in Figs. 3(B–M). We captured the images of nearly all the major lymph nodes and spleens from the experimental mice. In the control mice, however, we did not detect any of the secondary lymphoid organs except for the very faint images of spleen [Figs. 3(B–D)]. In the 4 days post-transfer group, CMS5 tumors were clearly detected [Figs. 3(E–G)]. And, in the 1-week post-transfer group, we detected an activity that is clearly distinct from the draining lymph node [Figs. 3(H and J)]. This spot is the location where the CMS5 tumors were located before the DUC18 T cell administration [Figs. 3(H–J)]. This piece of data suggests that there was a very small residual tumor mass with the T cells. Interestingly, upon dissection, we could not find the tumors in these mice (data not shown). More impressively, iliac and popliteal lymph nodes, as well as a string of mesenteric lymph nodes that are located in the center of the torso were detected [Figs. 3(K–M), supplemental data]. Subsequent to the scanning, we have sacrificed the mice and dissected several of the secondary lymphoid organs and tumors to detect the presence of radioactivity. We detected radioactivity in all the organs examined, indicating that the labeled antibody did localized to these lymphoid organs. The data collectively demonstrate the exquisite sensitivity of the system, and that we were able to discern the presence of the transferred DUC18 T cells in small lymph nodes that are located in obscure anatomical positions. Interestingly, in the control mice, we also detected CMS5 tumors [Figs. 3(B–D)]. This was also true when we administered control anti-TNP antibody into day-8 tumor-

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Fig. 3. 64Cu-labeled DOTA-Thy1.2 antibody specifically detects the major lymph nodes and spleens. (A) The flow chart of the experimental protocol is shown. From this experiment, 6 mice were imaged on the same day. (B-M). Two-dimensional images of representative experimental and control mice are shown. For the control mice (B-D), the PBS vehicle control (n⫽4) was given to day-8 tumor-bearing mice 4 days prior to the antibody administration. In the 4 days (EG) (n⫽4), 1 week (H-J) (n⫽2), and 2 weeks (K–M) (n⫽4) post-transfer groups, all the major lymph nodes were detected. Upon visual examination, it appears that the lymph nodes are better observed in the 2 weeks post-transfer group. However, this is likely due to difference in clearance rate of the infused antibodies by different mice. Black and white (K) arrowheads and arrows were used to indicate some organs only in certain pictures for the purpose of presentation.

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Fig. 4. Flow cytometry corroborates the ROI analysis: T cell distribution in individual lymphoid compartment does not change. (A–E) The PBS control group (n⫽4, data not shown), the 4 days post-transfer group (n⫽6), the 1 week post-transfer group (n⫽4), and the 2 weeks post-transfer group (n⫽4) were analyzed as described in Sec. 2. As expected, in the PBS control group, we did not detect any Thy1.2/CD8 double positive cells. Averages⫾SD of each organ from each group are shown. There are no statistically significant mean differences between the 4 days post-transfer group and the 1 week, or the 2 weeks post-transfer group (p values⬎0.05). A representative experiment from two independent experiments is shown (total n⫽8 –10 for each group).

bearing BALB/c-Thy1.1 mice (Fig. 4). Because TNP does not naturally exist in mice, perhaps the vasculature in the progressively growing tumors is responsible for these results. Consistent with this explanation, we also detected heart and liver, which are well-vascularized organs, in all mice. Also, as mentioned earlier, we detected images of spleen. Further, we do not think the residual tumors we detected in the 1-week post-transfer group are due to the circulation. We believe this is the residual T cells that are still harboring at the tumor site. However, because it is not visible by naked eye, we could not confirm this by biodistribution or by flow cytometry in separate experiments.

3.4. Quantitative analysis of the reconstructed microPET images show that the T cell distribution pattern remains unchanged during and after tumor elimination One of the advantages of PET imaging is that the reconstructed images can be used to perform quantitative analyses. Region of interest analysis (ROI) was performed on spleens, brachial/axillary, and inguinal lymph nodes, and on tumors to determine the amount of radioactivity in each tissue. We calculated for the proportion of radioactivity that exists in each organ by dividing the total activity found in organ by the total amount of 64Cu administered. The results were expressed as percent of total input dose (% ID) (Table

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Table 1 DUC18 T cell distribution does not change during and after tumor eliminationa Organ

Spleen Draining inguinal LN Contralateral inguinal LN Lt. brachial/axillary LN Rt. brachial/axillary LN CMS5 tumor

% ID 4 days post-transfer

1 week post-transfer

2 weeks post-transfer

Control

0.81% 0.19% 0.06% 0.09% 0.23% 1.97%

1.76% 0.24% 0.10% 0.18% 0.18% 0.71%c

1.43% 0.16% 0.13% 0.14% 0.27% NDb

0.40% NDb NDb NDb NDb 0.53%

Average % IDs of organs from each group are shown (n ⫽ 2– 4). ND, not determined. Lymph nodes in the control mice were not detected, and there were no tumors in the 2 weeks post-transfer group. c For the 1-week post-transfer group, even though we did not see the tumors in these mice upon dissection (data not shown), the activity we detected in the CMS5 inoculation site was considered to be the residual tumors for the purpose of analysis. a

b

1). Interestingly, the analyses showed that the activities found in various secondary lymphoid organs remained relatively unchanged during and after tumor elimination, indicating that the number of DUC18 T cells in each lymphoid compartment did not drastically fluctuate before and after the tumor elimination. The data also showed that in the control spleens (PBS recipients), there was approximately 2-4 times less activity (Table 1). A similar result was found for the tumors (Table 1). This is consistent with the notion that the observed radioactivity we detected in the control mice is due to the still circulating unbound antibodies, as they do not bind to Thy1.1 molecules expressed on the endogenous cell (Fig. 2).

tumors from BALB/c-Thy1.1 mice that received either DUC18 T cells or PBS were dissected and stained with anti-Thy1.2 antibody. The tumors were dissected 4 days after the T cell infusion. Although the tumors from the DUC18 T cell recipient mice showed the presence of DUC18 T cells, we did not find any T cells in the PBS control tumors (Fig. 6). Isotype-matched control antibody did not show any staining (data not shown). The data suggest that the radioactivity we detected in the tumors from

3.5. FACS analysis also shows that the T cell distribution remains unchanged To corroborate the ROI analysis, we performed flow cytometry. Again, BALB/c-Thy1.1 mice were utilized as tumor hosts and activated DUC18 T cells were infused into these mice. We analyzed spleens, inguinal, and axillary/ brachial lymph nodes that were dissected at various times following the DUC18 T cell transfer. Axillary and brachial lymph nodes were pooled together because of low cell numbers. The cells were stained with anti-Thy1.2 and -CD8 antibodies, and the percentage of the double positive cells was used to enumerate the DUC18 T cells in each lymphoid organ. We found that there was no major difference in the number of DUC18 T cells in each lymphoid compartment amongst different groups of mice (Fig. 5). Within each secondary lymphoid compartment, the number of DUC18 T cells we detected remained the same during and after the tumor elimination. The results corroborated the ROI analysis of the PET studies, with the T cell distribution in the secondary lymphoid organs remaining unchanged. 3.6. Adoptively transferred DUC18 T cells have infiltrated into CMS5 tumors Next, we utilized the immunofluorescent technology to detect tumor-infiltrated DUC18 T cells. Day-8 established

Fig. 5. Activated DUC18 T cells infiltrate into day-8 established CMS5 tumors. BALB/c- Thy1.1 mice were inoculated with CMS5 cells and treated with either activated DUC18 T cells (n⫽3) or PBS (n⫽3) as before. Four days later, the mice were sacrificed and the tumors were dissected, frozen, processed, and stained as described in Sec. 2. Representative staining samples for each group from three independent experiments are shown (n⫽9 for each group). Red indicates the specific presence of DUC18 T cells. The scale bar shown in the top panel is equal to 100 ␮m.

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the control mice [Fig. 3(B–D) and Table 1] is likely due to the unbound antibody molecules that are circulating in the blood. And, the increased activity we observed in the 4-day transfer group was due to the antibodies that specifically bound to DUC18 T cells.

4. Discussion We have utilized 64Cu-labeled DOTA-conjugated antiThy1.2 antibodies in conjunction with a microPET scanner to visualize CMS5-specific DUC18 T cells in BALB/cThy1.1 mice. The primary aim of our study was to examine how the DUC18 T cell distribution changes in secondary lymphoid organs by comparing the mice that were actively eliminating the tumors to those that have eliminated the tumors. We successfully visualized the presence of DUC18 T cells in all the major lymph nodes and spleens in the DUC18 T cell recipient mice but not in the control animals. The level of detection was extremely sensitive in that we detected iliac, popliteal, and mandibular lymph nodes, which are difficult to locate. In addition, the tumors that were actively being eliminated were clearly visualized in the 4 days post-transfer group. Interestingly, we also obtained tumor images in the control PBS recipient group. This is likely due to the free 64 Cu-labeled DOTA-conjugated anti-Thy1.2 antibodies that were circulating through the extensive network of vasculature that forms in the progressively growing tumors. The ROI analyses, flow cytometry, and histology data are consistent with this notion. The most intriguing and important finding from our study was that there was no major change in the T cell distribution pattern within each secondary lymphoid organs. Our results showed that, during and after tumor elimination, the number of DUC18 T cells remained relatively constant within the organs. From the immunological point of view, microPET technology is attractive because it allows us to conduct in vivo studies in living animals in a real-time fashion whereby obtaining high-resolution two- and three-dimensional images that can provide quantitative data. The important immunological findings were that the infused DUC18 T cells localized to all the major secondary lymphoid organs, and the distribution pattern of the T cells did not change in the lymphoid compartments after the tumor ablation when compared to the mice whose tumors were being eliminated. We hypothesized that DUC18 T cells would concentrate more in the vicinity of the tumors. However, to our surprise, activated DUC18 T cells localized throughout the body in all the groups. Compared to the 1and 2-weeks post-transfer groups, we were expecting to find more T cells in the tumor-draining inguinal lymph node in the 4 days post-transfer group. However, the data showed that the number remained relatively constant in the draining inguinal lymph nodes in all the groups. The observations suggest that once the activated T cells are transferred into

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tumor-bearing host, many of the cells may not be maintaining the physiological phenotypes that are required to directly migrate into the tumors. Our preliminary data show that after the administrations, the surface CD62 ligand expression on DUC18 T cells becomes heterogeneous whereas at the time of transfer all the cells are CD62 ligand low (data not shown). Down regulation of CD62 ligand has been shown to correlate with the T cell tumor infiltration and effective anti-tumor activity [35]. Our findings may be indicating that we need to somehow manipulate these infused T cells to maintain the necessary phenotypes to be more effective effectors. Compared to the other reported methods [25,26], our approach provides several distinct advantages. The level of detection and the sensitivity was higher, and our system added a level of flexibility in conducting studies that require days to perform. While directly labeling the cells in vitro with 64Cu-PTSM is a simple and efficient process, two major limitations exist [25]. First, 64Cu-PTSM leaks out of cells, and this poses a possible complication in imaging due to increased background signal. Second, the half-life of 64 Cu is only 12.7 hours, which limits the duration of the experiment. Although utilizing larger amounts of 64Cu would extend the feasible imaging time period, preliminary studies suggested that the level of radiation toxicity mediated by 64Cu seems to vary among different types or different conditioned cells, and lymphocytes are known to be sensitive to radiation. Moreover, we found that the rate of 64 Cu-PTSM leakages in activated DUC18 T cells was rather high (data not shown). Our system also enables one to perform studies on experiments that require days by introducing the radiolabel at the appropriate time point, thereby overcoming the timeconstraint imposed by 64Cu’s half-life. Although Witte and colleagues performed their experiment on the same mice for days [26], their method required the transduction of T cells with a retroviral construct for each experiment. In this setup, the quality of the experiment heavily depends on the efficiency of the transduction. Although this may be a useful system, the level of transduction efficiency can vary from experiment to experiment, and the efficiency is generally low. The consequences are such that one would have to further purify the transduced T cells since the usage of a heterogeneous polyclonal T cell population may lead to the underestimation of the results. In our system, 90 –95 % of the T cells transferred are CMS5-specific and all the DUC18 cells express surface Thy1.2 marker [31]. Conversely, there are several issues associated with our system. Unlike the aforementioned system, we can only monitor the mice for a limited time period determined by the 64Cu half-life. Also, one requires a system in which the adoptively transferred cells must express a unique surface marker in order to distinguish them from the endogenous host cells. As was previously mentioned, there are unbound 64 Cu-labeled antibody molecules in circulation that contribute to an increased background signal. To overcome this

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problem, one may need to label the antibody molecules at a higher specific-activity, or to develop a pre-targeting system capable of clearing the unbound antibodies from the circulation. Our ROI analysis of the CMS5 tumors in the DUC18 T cell recipients may be an underestimated result. As tumors progressively grow, it would be reasonable to assume that the vasculature has increased. Therefore, the larger the tumor, the more activity from unbound antibody would be detected in the tissue. In contrast, in the tumors that are being eliminated, there would be less vasculature. Hence, the activity from unbound antibody would be reduced and the specific signal would increase. Therefore, comparing the activity found in the tumors of the PBS recipients with that of the DUC18 T cell recipients may well under-represent the specific signals detected in the experimental mice. Further, the tumor host BALB/c-Thy1.1 mice we utilized only express Thy1.1 molecule, and the antibody is very specific against Thy1.2 molecule. Therefore, it is highly unlikely that these circulating anti-Thy1.2 antibody molecules are actually binding to the tumor vasculature. In addition, endothelial cells that are lining the blood vessels are not known to express Thy1 molecule. The data we obtained with anti-TNP control antibody would be consistent with this explanation. However, at this point we cannot definitively rule out a possibility that the captured tumor images from the control mice are not due to a general uptake of protein by the tumors. Another potential issue associated with our system is that the binding of antibodies to the T cell surface might lead to loss of cells. The preliminary data show that at least 12 hours post-antibody-administration, there is no detectable level of cell loss (data not shown). In addition, depending on the cell surface marker targeted by the antibody, it may also lead to cell activation or inactivation, which in turn might alter the T cell behavior. For this, we are currently developing a system that expresses a unique surface protein that does not have the capability of generating T cell activation signals upon antibody binding. We know from our flow cytometry analyses that in different lymph nodes, the number of DUC18 T cells is different. Yet, in the ROI analysis, the % ID for each lymph node is the same. One explanation for this is that the rate of antibody trafficking into the lymph nodes is less efficient since they are not as vascularized as spleens. Hence, the amount of antibody molecules that enter the lymph node becomes limiting. Overall, we believe our approach in detecting transferred T cells by antibody provides a simple way of obtaining high-quality images. The method is extremely sensitive, and it could be suitable for many different systems. Our method represents a powerful way for tracking adoptively transferred cells and studying their in vivo distribution. Information obtained from the in vivo T cell trafficking studies could aid in designing protocols that would enhance the efficacy of tumor-specific T cells. We are only beginning to understand the complexity of the biological system that is involved in the immunotherapy. The advance-

ment in the basic knowledge of T cell behaviors and their fate, and the advancement in the area of imaging technology should help us further our understanding.

Acknowledgments The authors wish to acknowledge the following individuals for technical support of this work; Darren Kreamalmeyer, Donna Thompson, Steve Horvath, Joon Young Kim, Jerrel Rutlin, John Engelbach, Nicole Mercer, and Lynne Jones. They would also like to thank Jerri Smith for her secretarial support. Supported by funds from National Institutes of Health, (P50 CA94056). The production of 64Cu at Washington University is supported by the NCI (R24 CA86307). MicroPET imaging is supported by an NIH/NCI SAIRP grant (1 R24 CA83060). They would also like to thank the Small Animal Imaging Core of the Alvin J. Siteman Cancer Center at Washington University and BarnesJewish Hospital in St. Louis, MO, USA, for additional support of the microPET imaging.

Supplemental material Three-dimensional movies of the mice from Fig. 3 clearly show the lymph nodes. (A–D) Four three-dimensional images of the mice shown in Fig. 3 are presented here in the rotational movie format. (A) Control group. Two day-8 tumor-bearing mice had received PBS 4 days prior to the antibody administration and they were scanned with the mPET_ scanner as described in Sec. 2. Only in this group two mice were scanned simultaneously. One mouse received 64Cu-labeled DOTA-anti-Thy1.2 antibody and the second mouse received 64Cu-labeled DOTA-anti-TNP antibody. (B-D) As described in Fig. 3, the mice from the experimental groups all received DUC18 T cells and 64Culabeled DOTA-anti- Thy1.2 antibody. (B), 4 days posttransfer group; (C), 1 week post-transfer group; and (D), 2 weeks post-transfer group.

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