Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Breast Cancer Res Treat. 2009 Sep 18;122(1):135–144. doi: 10.1007/s10549-009-0527-6

Adoptively-transferred ex vivo expanded γδ-T cells mediate in vivo antitumor activity in preclinical mouse models of breast cancer

Benjamin H Beck 1, Hyung-Gyoon Kim 1, Hyunki Kim 2, Sharon Samuel 2, Zhiyong Liu 3, Robin Shrestha 1, Hilary Haines 4, Kurt Zinn 2, Richard D Lopez 1,5,†
PMCID: PMC2883655  NIHMSID: NIHMS195640  PMID: 19763820

Abstract

In contrast to antigen-specific αβ-T cells (adaptive immune system), γδ-T cells can recognize and lyse malignantly transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immune system). Given the well-documented capacity of γδ-T cells to innately kill a variety of malignant cells, efforts are now actively underway to exploit the antitumor properties of γδ-T cells for clinical purposes. Here, we present for the first time preclinical in vivo mouse models of γδ-T cell-based immunotherapy directed against breast cancer. These studies were explicitly designed to approximate clinical situations in which adoptively-transferred γδ-T cells would be employed therapeutically against breast cancer. Using radioisotope-labeled γδ-T cells, we first show that adoptively-transferred γδ-T cells localize to breast tumors in a mouse model (4T1 mammary adenocarcinoma) of human breast cancer. Moreover, by using an antibody directed against the γδ-T cell receptor (TCR) we determined that localization of adoptively-transferred γδ-T cells to tumor is a TCR-dependant process. Additionally, biodistribution studies revealed that adoptively-transferred γδ-T cells traffic differently in tumor-bearing mice compared to healthy with fewer γδ-T cells localizing into the spleens of tumor-bearing mice. Finally, in both syngeneic (4T1) and xenogeneic (2Lmp) models of breast cancer, we demonstrate that adoptively-transferred γδ-T cells are both effective against breast cancer and are otherwise well-tolerated by treated animals. These findings provide a strong preclinical rationale for using ex vivo expanded adoptively-transferred γδ-T cells as a form of cell-based immunotherapy for the treatment of breast cancer. Additionally, these studies establish that clinically-applicable methods for radiolabeling γδ-T cells allows for the tracking of adoptively-transferred γδ-T cells in tumor-bearing hosts.

Keywords: γδ-T cells, Immunotherapy, Cell therapy, Innate Immunity

Introduction

Unlike αβ-T cells which require the recognition of specific processed peptide antigens presented by major histocompatibility complex (MHC) class-I or class-II molecules (adaptive immunity), γδ-T cells in contrast, appear to recognize and respond to a variety of stress-induced self antigens commonly displayed by cells having undergone malignant transformation [6, 12-15, 18]. Thus, while incapable of recognizing tumor-specific antigens per se, γδ-T cells can nonetheless recognize malignantly transformed cells – particularly malignant cells of epithelial origin – through less specific mechanisms that require no prior antigen exposure or priming (innate immunity). Consequently, γδ-T cells can recognize and lyse malignantly transformed cells almost immediately upon encounter – consistent with their role as a component of the innate immune system.

Given the well-documented capacity of γδ-T cells to innately kill a variety of malignant cells, efforts are now actively underway to develop and refine the means to exploit the antitumor properties of γδ-T cells for clinical purposes [4, 10, 11, 23, 42]. Over a decade ago, studies documented the presence of γδ-T cells among lymphocytic infiltrates within breast tumors and their principle draining lymph nodes [1, 3]. More recently, our group has reported that ex vivo expanded human γδ-T cells effectively killed a panel of human breast cancer cell lines in vitro, and notably, failed to lyse normal, control human fibroblasts [17]. These in vitro findings by our laboratory have since been confirmed and extended by other groups [30, 41]

While the previous findings by ourselves and others have been important in establishing the theoretical potential of γδ-T cell-based immunotherapy for the treatment of breast cancer, such studies have been until now, either observational or limited to in vitro studies. In this report, we present for the first time preclinical in vivo mouse models of γδ-T cell-based immunotherapy directed against breast cancer. These studies were explicitly designed to approximate clinical situations in which adoptively-transferred γδ-T cells would be employed therapeutically against breast cancer. Using radioisotope-labeled γδ-T cells, we show that adoptively-transferred γδ-T cells do indeed localize to breast tumors in a mouse model of human breast cancer. Subsequently, in both syngeneic and xenogeneic models of breast cancer, we demonstrate that adoptively-transferred γδ-T cells are both effective against breast cancer and are well-tolerated. Thus, these findings provide a strong biological rationale to justify the clinical use of adoptively-transferred γδ-T cells as a form of cancer immunotherapy for breast cancer.

Materials and Methods

Mice

Female BALB/c wild-type mice and BALB/c TCR αβ-deficient (TCRαβ−/−) mice were purchased from The Jackson Laboratory, and athymic mice (NCR-nu/nu) were purchased from Frederick Labs (National Cancer Institute). Mice were between 7 and 12 weeks of age and were maintained in pathogen-free facilities in accordance with the guidelines of the Animal Care and Use Committee at The University of Alabama at Birmingham (Birmingham, AL).

Cell lines

BALB/3T3 normal fibroblast (H-2d) and 4T1 mammary adenocarcinoma (H-2d) cell lines were purchased from the American Type Culture Collection. 4T1-luc2 was purchased from Caliper Life Sciences (http://www.caliperls.com). Cells were maintained as recommended by ATCC or the supplier. For xenograft studies, the human breast cancer cell line 2LMP/Luc was derived by transducing 2LMP cells to express firefly luciferase using the recombinant adeno-associated virus-2 transduction methods [7, 32-35].

Preparation of mouse and human γδ-T cells

γδ-T cells used in cytotoxicity assays and immunotherapy studies were obtained from spleen cells derived from BALB/c mice lacking αβ-T cells (TCRαβ−/−) as previously described [28]. Briefly, whole spleens were resected from TCRαβ−/− mice, homogenized, and then subjected to density gradient centrifugation. Cells were cultured as previously described [26] and were harvested after eight days in culture and were employed as effector cells at a 10:1 effector:target ratio against 4T1 and BALB/3T3 target cell lines in a standard four hour co-culture incubation period. Human γδ-T cells for xenograft studies were prepared as previously described [29].

Flow cytometry

To assess purity of γδ-T cells employed in in vitro cytolytic assays and adoptive transfer studies, flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The following antibodies were used for flow cytometry and were obtained from BDBiosciences: anti-CD3-APC (clone 145-2C11), anti-CD3-FITC (clone 145-2C11), anti-TCR-γδ-FITC (clone GL3), anti-TCR-γδ-PE (clone GL3), anti-CD16/CD32 (clone 2.4G2). Cell preparations were stained in FACS buffer (HBSS with 5% FBS). Living cells were distinguished from dead cells using propidium iodide (PI) uptake as previously described [16, 29].

In vitro cytotoxicity assay

The cytotoxicity of ex vivo expanded BALB/c-derived γδ-T cells against 4T1 and BALB/3T3 cell lines was measured using the standard 51Cr release assay as we have previously described [29] or using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, Madison Wisconsin), an assay which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. Both 4T1 and 4T1-luc2 cells were determined to be equivalently sensitive to γδ-T cell killing in vitro (data not shown).

Biodistribution studies using 111In-labeled γδ-T cells

Eight-week old female BALB/c mice were sham-injected with saline, or received 4×104 4T1 mammary adenocarcinoma cells in the mammary fat pad. After 20 days, mice were injected intravenously with 5×106 BALB/c-derived 111In-labeled γδ-T cells, labeled with the standard nuclear pharmacy 111In-oxine method at 5 pCi/cell (Birmingham Nuclear Pharmacy, Birmingham, AL) as previously described [8, 22]. For biodistribution experiments, animals were sacrificed at 48 hours and tissues were counted with a gamma scintillation counter with results expressed as % of injected dose (ID)/g of tissue. To inhibit γδ-T cell localization to tumor, γδ-T cells were first pretreated (prior to injection) with 10 μg/ml anti-γδ-TCR monoclonal antibody clone GL3 for 15 minutes on ice. The GL3 antibody has been shown to downregulate the γδ-TCR, which acts to functionally impair γδ-T cells [20, 24].

In Vivo SPECT/CT Imaging Study

Animals were maintained with isoflurane gas anesthesia for all studies, and monitored continuously to allow the lowest dose (typically 1.5-2.0%) to prevent movement. Imaging studies were conducted using X-SPECT, a SPECT/CT dual-modality imager (Gamma Medica-Ideas, Northridge, CA) as described previously [9]. The SPECT/CT images were acquired at 48 hours following injection of 111In-labeled γδ-T cells. Radiation decay of 111In was considered, and the same color scale was applied to all SPECT images.

Bioluminescence studies

To assess tumor response in the syngeneic mouse tumor model (i.e., BALB/c mice harboring 4T1 tumors treated with BALB/c γδ-T cells), tumors were first established in otherwise healthy mice. In these studies, 4×104 4T1-luc2 cells were introduced into the mammary fat pad. After two days, mice were imaged and matched into pairs based upon equivalent tumor burden and randomly assigned to a treated or a control group. Treated mice received three doses of syngeneic γδ-T cells (5×106 per dose) delivered by intravenous injection at 2, 5 and 9 days after establishment of tumors. For xenograft studies, human 2LMP/Luc cells (1×106 cells) were introduced subcutaneously into nude mice. By convention, the day of tumor implantation is designated day 0. On day 6, animals were imaged to determine the amount of detectable tumor, matched into pairs based upon equivalent tumor burden, then randomly assigned to a treated or a control group. Treated animals received 20 × 106 human γδ-T cells intravenously on days 6, 9, 13, 16, 20 and 23. Untreated animals received sham-injections. Bioluminescence images in both syngeneic and xenograft studies were obtained using the IVIS 100 imaging system (Xenogen) as has been described in detail elsewhere [21]. Briefly, each mouse was injected with luciferin (2.5 mg) and imaged after 10 min in a 37 °C chamber according to the following parameters: 25-cm axial field of view, 1-30 second exposure, photographic binning of 4 or 8, and an F-stop of 1. Identically sized regions of interest were manually drawn to surround all tumor cells, and the light emitted from the tumor cells within the region of interest was measured using Living Image 3.1 software.

Statistical analysis

In γδ-T cell biodistribution studies, the mean percentage of injected dose per gram in various tissues was compared in healthy and 4T1-bearing mice using the Student’s t test. In both the syngeneic and xenograft immunotherapy studies, the mean tumor burden was compared in treated and untreated mice using the Student’s t test.

Results

Syngeneic γδ-T cells are cytolytic in vitro against 4T1 mammary adenocarcinoma cells but not against non-malignant (control) fibroblasts

The 4T1 mammary adenocarcinoma cell line was derived from a spontaneously arising BALB/c mammary tumor and is commonly used in studies intended to approximate human breast cancer [2, 40]. As an initial step in the development of our syngeneic γδ-T cell immunotherapy model for breast cancer, we determined that syngeneic BALB/c cells could indeed kill 4T1 mammary adenocarcinoma cells in vitro. Importantly, we concurrently determined that normal syngeneic fibroblasts are not killed by these same γδ-T cells, indicating that γδ-T cells are capable of distinguishing malignant from non-malignant (i.e., normal) tissues. As shown in Figure 1, ex vivo expanded BALB/c-derived γδ-T cells (H-2d) kill syngeneic 4T1 (H-2d) cells but mediate reduced cytotoxicity against syngeneic non-malignant BALB/3T3 (H-2d) cells. This inability of activated (ex vivo expanded) γδ-T cells to kill non-malignant fibroblasts is important as this would suggest that in a clinical setting, ex vivo expanded, patient-derived (i.e., autologous) γδ-T cells will similarly not interact with normal tissues upon adoptive transfer. Together, these findings support our view that the 4T1 model is a feasible platform in which to continue the study of how ex vivo expanded γδ-T cells might be used in the treatment of breast cancer.

Fig. 1.

Fig. 1

Open in a new tab

Ex vivo expanded BALB/c-derived γδ-T cells kill syngeneic 4T1 mammary adenocarcinoma cells but not syngeneic normal fibroblasts. γδ-T cells were obtained and expanded from the spleens of BALB/c TCR αβ−/− mice. FACS-plot inset shows representative purity of ex vivo expanded γδ-T cells employed in the in vitro cytotoxicity assays (and adoptive transfer studies). BALB/c-derived effector γδ-T cells were co-cultured with syngeneic target cell lines 4T1 (BALB/c mammary adenocarcinoma) and BALB/3T3 (normal BALB/c fibroblast cell line) at a 10:1 effector:target ratio for 4 hours in a standard cytolytic assay. Cytotoxicity was determined and is expressed as percent specific lysis ± standard deviation. These results are from experiments performed three separate times

Adoptively-transferred γδ-T cells localize to tumors

We next designed a series of studies to determine the extent to which adoptively-transferred γδ-T cells could localize within primary mammary tumors. Previously, using a syngeneic mouse model of prostate cancer, we showed that γδ-T cells expressing green fluorescence protein (GFP) readily localized into established tumors [26]. However, in a clinical setting, it is unlikely that an approach employing GFP-expressing γδ-T cells would be feasible. Accordingly, in this present study, we employed radiolabeled (111In) γδ-T cells, noting that 111In-labeling is a clinically applicable method which can be used to assess the trafficking patterns of adoptively-transferred T cells in patients [31]. Using SPECT/CT imaging, Figure 2 shows that 111In-labeled γδ-T cells do indeed localize to 4T1 mammary fat pad tumors. Importantly, when γδ-T cells were first pretreated (prior to injection) with an anti-γδ-TCR monoclonal antibody which is known to downregulate the γδ-TCR [20, 24], a marked decrease in localization of γδ-T cells to tumor was observed (Figure 2). To support these SPECT/CT findings, biodistribution studies were also performed in which mice were treated with radiolabeled γδ-T cells, which were either pretreated (blocked) with the anti-γδ-TCR antibody GL3 antibody, or left untreated (unblocked). Tumors were then resected from these mice. As a quantitative measure of γδ-T cells localizing to tumors, radioactivity accumulating within tumor tissues was expressed (by convention) as the percent of injected radiation dose found per gram of tumor tissue. When comparing mice treated with either blocked or unblocked γδ-T cells, a 34.6% reduction in γδ-T cell localization to tumor was observed (2.6% of injected dose per gram of tissue when unblocked; 1.7% injected dose per gram of tissue when blocked; p = 0.004; data not shown). We interpret this to indicate that for adoptively-transferred γδ-T cells, either trafficking or the functional accumulation within tumor is to some degree dependent on the expression of a functional γδ-TCR.

Fig. 2.

Fig. 2

Open in a new tab

Adoptively-transferred γδ-T cells localize to 4T1 tumors. SPECT/CT fused images (axial view) showing distribution of 111In-labeled γδ-T cells in 4T1 mammary fat pad tumors at 48 hours post injection. To demonstrate that the γδ-T cell receptor (γδ-TCR) itself is involved in the localization of adoptively-transferred cells to tumors, imaging was performed using 111In-labeled γδ-T cells which were either (a) untreated or (b) pre-treated with an anti-γδ TCR monoclonal antibody (clone GL3) before injection into mice. Dotted circles in each image delineates tumor region. These results are representative of experiments performed on at least three separate mice in each group

γδ-T cell trafficking patterns differ between healthy and tumor bearing mice

The demonstration that adoptively-transferred γδ-T cells localize to mammary adenocarcinoma tumors is central to this present study. However, we were also able to assess the overall systemic tissue biodistribution of adoptively-transferred 111In-labeled γδ-T cells. In these studies, both healthy BALB/c mice and 4T1-bearing BALB/c mice were injected with 5 × 106 111In-labeled-γδ T cells and compared. After 48 h, mice were sacrificed and tissues were removed to assess the distribution of adoptively-transferred γδ-T cells within separate tissues — which by convention, is expressed as a percentage of injected radiation dose per gram of target tissue [5]. Figure 3 compares the tissue biodistribution of adoptively-transferred γδ-T cells in healthy BALB/c and tumor-bearing BALB/c mice. Curiously, when comparing healthy mice and 4T1-bearing mice, γδ-T cell biodistribution was similar in all tissues with the exception of the spleen where tumor-bearing mice had a significantly lower accumulation of γδ-T cells in the spleen compared to healthy mice (15.3% in 4T1-bearing mice; 37% in healthy mice; p = 0.003).

Fig. 3.

Fig. 3

Open in a new tab

Adoptively-transferred γδ-T cells distribute differently within tumor-bearing mice as compared to healthy mice. 111In-labeled γδ-T cells were injected into healthy mice or tumor-bearing mice. Biodistribution of administered γδ-T cells (expressed as a percent of injected radioactive dose per gram of target tissue) was determined by resecting tissues or organs isolated from healthy BALB/c mice (■; n = 10) or from BALB/c mice harboring 4T1 cells (□; n = 10). Tissues: HT, heart; LV, liver; ST, stomach; LI, large intestine; SI, small intestine; CE, cecum; SP, spleen; LU, lung; LK, left kidney; RK, right kidney; MU, muscle; BL, blood; RO, reproductive organs; BR, brain; FE, femur. These results are representative of experiments performed two separate times. Statistically significant differences between tissues taken from healthy mice or from tumor-bearing mice are indicated by asterisks (p = 0.003 for spleen)

Adoptively-transferred syngeneic mouse γδ-T cells moderate the growth of mammary adenocarcinoma tumors

Having established above that γδ-T cells do indeed localize to tumor, we next assessed to what extent adoptively-transferred γδ-T cells could moderate mammary tumor progression. For these studies, tumor was first established by injection of a luciferase-expressing 4T1 cell line (4T1-Luc2) into the mammary fat pads of otherwise healthy wild type female BALB/c mice. Figure 4 shows that tumor-bearing mice treated with γδ-T cells had a significant reduction in mammary tumor growth compared to untreated tumor-bearing mice. Importantly, during the course of these studies no untoward side effects were observed in mice treated with γδ-T cells.

Fig. 4.

Fig. 4

Open in a new tab

Bioluminescence studies: In vivo sensitivity of murine mammary adenocarcinoma cancer cell line 4T1-Luc2 to killing by adoptively-transferred syngeneic BALB/c γδ-T cells. 4T1-Luc2 cells were injected into the mammary fat pads of healthy female BALB/c mice. After 2 days, all animals were imaged and matched pairwise based on equivalence of tumor burden. Each animal from a given pair was then randomly assigned to a group receiving either treatment or no treatment. Treated animals (n = 9) received 5 × 106 BALB/c-derived γδ-T cells intravenously on days 2, 5 and 9. Untreated animals (n = 9) received sham-injections with saline. Whole animal images of tumor-bearing mice (19 days post-tumor injection) were obtained using an IVIS Imaging System Series 100 bioluminescence detector (Xenogen) 10 minutes after intraperitoneal injection of 2.5 mg luciferin (substrate for luciferase). Images of four representative untreated mice (panel a, top) and four representative treated mice (panel b, top) are shown. Light emission from tumor cells was measured using Living Image 3.1 software and is represented as a pseudo-color scaling of the bioluminescence data. Bioluminescence data are shown graphically where tumor burden is expressed in counts per second. Mean (+/− SD) and median tumor burdens of untreated and treated mice are shown. In these studies, diminished luciferase activity in tumor-bearing animals treated with syngeneic BALB/c-derived γδ-T cells is taken as evidence of in vivo γδ-T cell anti-tumor efficacy

Adoptive transfer of human γδ-T cells can moderate the growth of xenogeneic breast tumors

To further assess the therapeutic potential of administering γδ-T cells for the treatment of breast cancer, we used a human xenograft model of breast cancer. Here, we employed the 2Lmp/Luc cell line, a subclone of the human breast cancer cell line MDA-MB-231 that was engineered to express luciferase [7, 32-35]. As reported, human γδ-T cells expanded ex vivo have been shown to reduce tumor burden and improve survival when injected into SCID mice harboring melanoma or pancreatic cancer cells [19]. Consistent with this report, as shown in Figure 5, adoptively-transferred human γδ-T cells were clearly able to control the growth of human 2Lmp/Luc cells first xenografted into athymic (nude) mice. Similar to the above syngeneic studies, no untoward side effects were observed in mice treated with human γδ-T cells.

Fig. 5.

Fig. 5

Open in a new tab

Bioluminescence studies: In vivo sensitivity of human breast cancer cell line 2LMP/Luc to killing by human γδ-T cells. Human breast cancer cell line 2LMP/Luc was derived by transducing 2LMP cells to express firefly luciferase using the recombinant adeno-associated virus-2 transduction methods [7, 32-35]. 2LMP/Luc cells (1 × 106 cells) were introduced subcutaneously into 10 nude mice on day 0. After 6 days, all animals were imaged and matched pairwise based on equivalence of tumor burden. Each animal from a given pair was then randomly assigned to a group receiving either treatment or no treatment. Treated animals received 20 × 106 human γδ-T cells intravenously on days 6, 9, 13, 16, 20 and 23. Untreated animals received sham-injections. Serial images from a representative pair of mice are shown here (panel a, untreated mouse above; treated mouse below). A graphic representation of bioluminescence data expressed in counts per second is shown to the right of the images. Normalized data from all animals are shown in panel b. For each animal, all measurements of tumor size (in counts per second) were normalized to tumor size determined for that animal on day 6 after tumor implantation (panel b). Thus on day 6, each animal has a “measured tumor size to initial tumor size” ratio of 1. Data from subsequent days are expressed as a ratio of “measured tumor size to initial tumor size” (mean ± SD). Open bars, untreated animals (n=5). Solid bars, treated animals (n=5). In these studies, diminished luciferase activity in tumor-bearing animals treated with human γδ-T cells is taken as evidence of in vivo human γδ-T cell antitumor activity

Discussion

To date, most immune cell-based cancer immunotherapy strategies have focused on the stimulation of anti-tumor properties of the adaptive immune system, which are typically directed against tumor-specific or tumor-associated antigens. In contrast, γδ-T cells can recognize generic antigens commonly expressed by stressed cells such as malignantly-transformed cells. Indeed, cancerous cells can display a number of stress-induced antigens which while neither tumor-specific nor tumor-derived, can nonetheless serve as recognition determinants for human and mouse γδ-T cells [6, 12-15, 18]. Given the recognized capacity of γδ-T cells to directly recognize and kill malignant cells both in vitro and in vivo efforts are now actively underway to develop and refine the means to exploit the antitumor properties of γδ-T cells for clinical purposes [4, 10, 11, 17, 23, 28, 36-38, 42].

Although it remains to be determined how γδ-T cells might best be utilized clinically for the treatment of malignancies, two specific approaches are being developed in this regard. One approach relies upon the activation or expansion of the endogenous γδ-T cells within patients through the clinical administration of pharmacologic agents capable of stimulating human γδ-T cells. This includes the use of the aminobisphosphonate drugs, typically administered in conjunction with interleukin (IL)-2 [10, 11, 25, 39, 42].

Alternatively, the innate antitumor properties of γδ-T cells may also be exploited through the adoptive transfer of γδ-T cells first expanded ex vivo, then subsequently reinfused into tumor-bearing patients. This latter approach — approximated by these current animal studies — while technically more involved, is nevertheless now entirely feasible in the clinical setting as advances by our group as well as others have made possible the large-scale expansion of human γδ-T cells which retain antitumor activity against a variety of human tumor cell lines in vitro [4, 17, 28, 29]

With this in mind, our current report has several limited, but nevertheless important, objectives. Foremost, this work establishes for the first time that ex vivo expanded adoptively-transferred γδ-T cells can indeed limit the in vivo progression of disease in animal models of breast cancer. Although previously shown in other disease models [19] here for the first time, it is clearly demonstrated — using both a syngeneic mouse mammary tumor model (employing 4T1-Luc2 in immunocompetent BALB/c mice), as well as a complementary xenograft tumor model (employing human 2Lmp/Luc breast cancer cells) — that such approaches are directly relevant to breast cancer.

Second, and particularly important from a clinical perspective, we establish that ex vivo expanded γδ-T cells are not only effective against disease, but also do not cause untoward side effects upon adoptive transfer into tumor-bearing hosts. This is a key point to be made as clinical trials are developed to assess how γδ-T cells might best be administered therapeutically. Thus, we show that syngeneic BALB/c-derived γδ-T cells — which are capable of killing 4T1 cells in vitro and in vivo (Figures 1 and 4) — are nevertheless unreactive against normal BALB/c fibroblasts in vitro (Figure 1). Moreover, these ex vivo expanded γδ-T cells are well-tolerated by mice receiving treatments, even when delivered in multi-dose schedules. Similarly, from our studies shown in Figure 5, we infer that adoptively-transferred human γδ-T cells do not react with non-malignant (albeit, xenogeneic) tissues in vivo as these mice also tolerated multi-dose treatments well. This particular conclusion is indirectly supported by our previous work showing that in vitro, non-malignant human cell lines are not killed by ex vivo expanded human γδ-T cells which, nevertheless, readily recognized and killed human breast cancer cell lines [17].

The third objective of this current work is to highlight one of the potential advantages of adopting the approach whereby γδ-T cells are first expanded ex vivo, then reinfused. In contrast to the alternative approach whereby endogenous γδ-T cells are activated in vitro within patients, in using the approach taken here, it becomes possible for investigators to experimentally track γδ-T cells after administration — an important correlative tool for use in the design, interpretation and refinement of future clinical trials. Thus, in a manner analogous to our animal studies using 111In-labeled γδ-T cells (Figures 2 and 3), it will be possible to track therapeutically-administered human γδ-T cells to sites of disease employing the appropriate clinically-approved imaging techniques [31]. Moreover, in early human clinical trials which we are about to embark upon at our institution (UAB Breast Cancer SPORE Project 4, “Gamma-delta T cell Immunotherapy of Breast Cancer; Project co-leaders, R. Lopez, K. Zinn), the optimal γδ-T cell dose and schedule remain to be determined. Accordingly, in a manner similar to the studies presented in Figure 3, we will be able to perform critical clinical biodistribution studies, an important first step in the optimization of γδ-T cell-based immune therapies.

The observation that 111In-labeled γδ-T cells readily localize to mammary tumors is not surprising (Figure 2), especially given our previous report that adoptively-transferred γδ-T cells readily localize into tumors in a mouse model of prostate cancer [26]. Moreover, the finding that this localization to tumor appears to be only partially reduced by an anti-γδ-TCR antibody (mAb GL3) is consistent with our previous findings that in vitro, antibodies to the γδ-TCR only partially inhibit binding of γδ-T cells to sensitive tumor cell targets [27]. In any event, the altered homing displayed by antibody-treated γδ-T cells could reflect disrupted trafficking, or alternatively, antibody-treated γδ-T cells could display altered survival, be eliminated by virtue of being coated with antibody, or exhibit impaired proliferation within the local tumor site. This issue is currently under investigation.

Intriguingly, in the biodistribution studies (Figure 3), adoptively-transferred γδ-T cells were found to be more abundant in the spleens of healthy mice when compared to the spleens of tumor-bearing mice. Although we have no clear explanation for this finding, we surmise that γδ-T cells in tumor-bearing mice are trafficking differently — possibly as a result of altered homing receptors expressed on lymphoid tissues, including the spleen, within tumor-bearing mice. Conversely, intrinsic changes within the γδ-T cells may account for their observed altered biodistribution in tumor-bearing mice. We are currently undertaking studies to address this issue, as findings from such studies could have practical and clinical implications in the conduct of γδ-T cell-based therapeutic trials.

Given the biological, technological and pharmaceutical advances of the last several years, human clinical trials intended to exploit the innate antitumor properties of γδ-T cells are now a reality [10, 11, 23, 42]. Indeed as noted above, at our institution, we are preparing to embark upon our first generation of clinical trials — including a phase I trial in which patients with advanced breast cancer are to be treated with peripheral blood-derived autologous γδ-T cells first ex vivo expanded, then subsequently reinfused. Accordingly, in the performance of such early phase trials, a more thorough understanding of the in vivo activity and fate of adoptively-transferred γδ-T cells will be key to refining later generations of trials. In this context, the timeliness and relevance of our current findings are underscored.

Acknowledgments

This work was supported by Grants 5P50CA089019-07 from the National Cancer Institute, and the UAB Small Animal Imaging shared facility (P30CA013148). The authors thank Kyle Feeley for his thoughtful review of the manuscript.

References

  • 1.Alam SM, Clark JS, Leech V, Whitford P, George WD, Campbell AM. T cell receptor gamma/delta expression on lymphocyte populations of breast cancer patients. Immunol Lett. 1992;31:279–283. doi: 10.1016/0165-2478(92)90127-a. [DOI] [PubMed] [Google Scholar]
  • 2.Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52:1399–1405. [PubMed] [Google Scholar]
  • 3.Bank I, Book M, Huszar M, Baram Y, Schnirer I, Brenner H. V delta 2+ gamma delta T lymphocytes are cytotoxic to the MCF 7 breast carcinoma cell line and can be detected among the T cells that infiltrate breast tumors. Clin Immunol Immunopathol. 1993;67:17–24. doi: 10.1006/clin.1993.1040. [DOI] [PubMed] [Google Scholar]
  • 4.Bennouna J, Bompas E, Neidhardt EM, Rolland F, Philip I, Galea C, Salot S, Saiagh S, Audrain M, Rimbert M, Lafaye-de Micheaux S, Tiollier J, Negrier S. Phase-I study of Innacell gammadelta, an autologous cell-therapy product highly enriched in gamma9delta2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma. Cancer Immunol Immunother. 2008;57:1599–1609. doi: 10.1007/s00262-008-0491-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Buchsbaum DJ, Khazaeli MB, Axworthy DB, Schultz J, Chaudhuri TR, Zinn KR, Carpenter M, LoBuglio AF. Intraperitoneal pretarget radioimmunotherapy with CC49 fusion protein. Clin Cancer Res. 2005;11:8180–8185. doi: 10.1158/1078-0432.CCR-05-0607. [DOI] [PubMed] [Google Scholar]
  • 6.Cerwenka A, Bakker AB, McClanahan T, Wagner J, Wu J, Phillips JH, Lanier LL. Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity. 2000;12:721–727. doi: 10.1016/s1074-7613(00)80222-8. [DOI] [PubMed] [Google Scholar]
  • 7.Chaudhuri TR, Cao Z, Ponnazhagan S, Stargel A, Simhadri PL, Zhou T, Lobuglio AF, Buchsbaum DJ, Zinn KR. Bioluminescence imaging of non-palpable breast cancer xenografts during treatment with TRA-8, an anti-DR5 antibody and chemotherapy; AACR Meeting Abstracts; 2004; 2004. 481-a- [Google Scholar]
  • 8.Coito AJ, Binder J, Brown LF, de Sousa M, Van de Water L, Kupiec-Weglinski JW. Anti-TNF-alpha treatment down-regulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats. J Immunol. 1995;154:2949–2958. [PubMed] [Google Scholar]
  • 9.Cowey S, Szafran AA, Kappes J, Zinn KR, Siegal GP, Desmond RA, Kim H, Evans L, Hardy RW. Breast cancer metastasis to bone: evaluation of bioluminescent imaging and microSPECT/CT for detecting bone metastasis in immunodeficient mice. Clin Exp Metastasis. 2007;24:389–401. doi: 10.1007/s10585-007-9076-8. [DOI] [PubMed] [Google Scholar]
  • 10.Dieli F, Gebbia N, Poccia F, Caccamo N, Montesano C, Fulfaro F, Arcara C, Valerio MR, Meraviglia S, Di Sano C, Sireci G, Salerno A. Induction of gammadelta T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo. Blood. 2003;102:2310–2311. doi: 10.1182/blood-2003-05-1655. [DOI] [PubMed] [Google Scholar]
  • 11.Dieli F, Vermijlen D, Fulfaro F, Caccamo N, Meraviglia S, Cicero G, Roberts A, Buccheri S, D’Asaro M, Gebbia N, Salerno A, Eberl M, Hayday AC. Targeting human {gamma}delta} T cells with zoledronate and interleukin-2 for immunotherapy of hormone-refractory prostate cancer. Cancer Res. 2007;67:7450–7457. doi: 10.1158/0008-5472.CAN-07-0199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Girardi M. Immunosurveillance and immunoregulation by gammadelta T cells. J Invest Dermatol. 2006;126:25–31. doi: 10.1038/sj.jid.5700003. [DOI] [PubMed] [Google Scholar]
  • 13.Girardi M, Hayday AC. Immunosurveillance by gammadelta T cells: focus on the murine system. Chem Immunol Allergy. 2005;86:136–150. doi: 10.1159/000086658. [DOI] [PubMed] [Google Scholar]
  • 14.Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci U S A. 1999;96:6879–6884. doi: 10.1073/pnas.96.12.6879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gammadelta T cells. Science. 1998;279:1737–1740. doi: 10.1126/science.279.5357.1737. [DOI] [PubMed] [Google Scholar]
  • 16.Guo BL, Hollmig KA, Lopez RD. Down-regulation of IL-2 receptor alpha (CD25) characterizes human gammadelta-T cells rendered resistant to apoptosis after CD2 engagement in the presence of IL-12. Cancer Immunol Immunother. 2002;50:625–637. doi: 10.1007/s00262-001-0244-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Guo BL, Liu Z, Aldrich WA, Lopez RD. Innate anti-breast cancer immunity of apoptosis-resistant human gammadelta-T cells. Breast Cancer Res Treat. 2005;93:169–175. doi: 10.1007/s10549-005-4792-8. [DOI] [PubMed] [Google Scholar]
  • 18.Kabelitz D, Wesch D, He W. Perspectives of gammadelta T cells in tumor immunology. Cancer Res. 2007;67:5–8. doi: 10.1158/0008-5472.CAN-06-3069. [DOI] [PubMed] [Google Scholar]
  • 19.Kabelitz D, Wesch D, Pitters E, Zoller M. Characterization of tumor reactivity of human V gamma 9V delta 2 gamma delta T cells in vitro and in SCID mice in vivo. J Immunol. 2004;173:6767–6776. doi: 10.4049/jimmunol.173.11.6767. [DOI] [PubMed] [Google Scholar]
  • 20.Ke Y, Pearce K, Lake JP, Ziegler HK, Kapp JA. Gamma delta T lymphocytes regulate the induction and maintenance of oral tolerance. J Immunol. 1997;158:3610–3618. [PubMed] [Google Scholar]
  • 21.Kim H, Morgan DE, Buchsbaum DJ, Zeng H, Grizzle WE, Warram JM, Stockard CR, McNally LR, Long JW, Sellers JC, Forero A, Zinn KR. Early therapy evaluation of combined anti-death receptor 5 antibody and gemcitabine in orthotopic pancreatic tumor xenografts by diffusion-weighted magnetic resonance imaging. Cancer Res. 2008;68:8369–8376. doi: 10.1158/0008-5472.CAN-08-1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kircher MF, Grimm J, Swirski FK, Libby P, Gerszten RE, Allport JR, Weissleder R. Noninvasive in vivo imaging of monocyte trafficking to atherosclerotic lesions. Circulation. 2008;117:388–395. doi: 10.1161/CIRCULATIONAHA.107.719765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kobayashi H, Tanaka Y, Yagi J, Osaka Y, Nakazawa H, Uchiyama T, Minato N, Toma H. Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study. Cancer Immunol Immunother. 2007;56:469–476. doi: 10.1007/s00262-006-0199-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Koenecke C, Chennupati V, Schmitz S, Malissen B, Forster R, Prinz I. In vivo application of mAb directed against the gammadelta TCR does not deplete but generates “invisible” gammadelta T cells. Eur J Immunol. 2009;39:372–379. doi: 10.1002/eji.200838741. [DOI] [PubMed] [Google Scholar]
  • 25.Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP, Wilhelm M. Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood. 2000;96:384–392. [PubMed] [Google Scholar]
  • 26.Liu Z, Eltoum IE, Guo B, Beck BH, Cloud GA, Lopez RD. Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer. J Immunol. 2008;180:6044–6053. doi: 10.4049/jimmunol.180.9.6044. [DOI] [PubMed] [Google Scholar]
  • 27.Liu Z, Guo B, Lopez RD. Expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human gammadelta-T cells: Implications in the design of gammadelta-T-cell-based immunotherapies for pancreatic cancer. J Gastroenterol Hepatol. 2008 doi: 10.1111/j.1440-1746.2008.05668.x. [DOI] [PubMed] [Google Scholar]
  • 28.Liu Z, Guo BL, Gehrs BC, Nan L, Lopez RD. Ex vivo expanded human Vgamma9Vdelta2+ gammadelta-T cells mediate innate antitumor activity against human prostate cancer cells in vitro. J Urol. 2005;173:1552–1556. doi: 10.1097/01.ju.0000154355.45816.0b. [DOI] [PubMed] [Google Scholar]
  • 29.Lopez RD, Xu S, Guo B, Negrin RS, Waller EK. CD2-mediated IL-12-dependent signals render human gamma delta-T cells resistant to mitogen-induced apoptosis, permitting the large-scale ex vivo expansion of functionally distinct lymphocytes: implications for the development of adoptive immunotherapy strategies. Blood. 2000;96:3827–3837. [PubMed] [Google Scholar]
  • 30.Mattarollo SR, Kenna T, Nieda M, Nicol AJ. Chemotherapy and zoledronate sensitize solid tumour cells to Vgamma9Vdelta2 T cell cytotoxicity. Cancer Immunol Immunother. 2007;56:1285–1297. doi: 10.1007/s00262-007-0279-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Meidenbauer N, Marienhagen J, Laumer M, Vogl S, Heymann J, Andreesen R, Mackensen A. Survival and tumor localization of adoptively transferred Melan-A-specific T cells in melanoma patients. J Immunol. 2003;170:2161–2169. doi: 10.4049/jimmunol.170.4.2161. [DOI] [PubMed] [Google Scholar]
  • 32.Ponnazhagan S, Mahendra G, Curiel DT, Shaw DR. Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy. J Virol. 2001;75:9493–9501. doi: 10.1128/JVI.75.19.9493-9501.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ponnazhagan S, Mahendra G, Kumar S, Thompson JA, Castillas M., Jr. Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands. J Virol. 2002;76:12900–12907. doi: 10.1128/JVI.76.24.12900-12907.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ponnazhagan S, Mukherjee P, Wang XS, Qing K, Kube DM, Mah C, Kurpad C, Yoder MC, Srour EF, Srivastava A. Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation. J Virol. 1997;71:8262–8267. doi: 10.1128/jvi.71.11.8262-8267.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ponnazhagan S, Weigel KA, Raikwar SP, Mukherjee P, Yoder MC, Srivastava A. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes. J Virol. 1998;72:5224–5230. doi: 10.1128/jvi.72.6.5224-5230.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Salot S, Laplace C, Saiagh S, Bercegeay S, Tenaud I, Cassidanius A, Romagne F, Dreno B, Tiollier J. Large scale expansion of gamma 9 delta 2 T lymphocytes: Innacell gamma delta cell therapy product. J Immunol Methods. 2007;326:63–75. doi: 10.1016/j.jim.2007.07.010. [DOI] [PubMed] [Google Scholar]
  • 37.Sicard H, Al Saati T, Delsol G, Fournie JJ. Synthetic phosphoantigens enhance human Vgamma9Vdelta2 T lymphocytes killing of non-Hodgkin’s B lymphoma. Mol Med. 2001;7:711–722. [PMC free article] [PubMed] [Google Scholar]
  • 38.Sicard H, Ingoure S, Luciani B, Serraz C, Fournie JJ, Bonneville M, Tiollier J, Romagne F. In vivo immunomanipulation of V gamma 9V delta 2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. J Immunol. 2005;175:5471–5480. doi: 10.4049/jimmunol.175.8.5471. [DOI] [PubMed] [Google Scholar]
  • 39.Simoni D, Gebbia N, Invidiata FP, Eleopra M, Marchetti P, Rondanin R, Baruchello R, Provera S, Marchioro C, Tolomeo M, Marinelli L, Limongelli V, Novellino E, Kwaasi A, Dunford J, Buccheri S, Caccamo N, Dieli F. Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a gammadelta-T lymphocytes-mediated activation mechanism. J Med Chem. 2008;51:6800–6807. doi: 10.1021/jm801003y. [DOI] [PubMed] [Google Scholar]
  • 40.Tao K, Fang M, Alroy J, Sahagian GG. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer. 2008;8:228. doi: 10.1186/1471-2407-8-228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Tokuyama H, Hagi T, Mattarollo SR, Morley J, Wang Q, Fai-So H, Moriyasu F, Nieda M, Nicol AJ. V gamma 9 V delta 2 T cell cytotoxicity against tumor cells is enhanced by monoclonal antibody drugs--rituximab and trastuzumab. Int J Cancer. 2008;122:2526–2534. doi: 10.1002/ijc.23365. [DOI] [PubMed] [Google Scholar]
  • 42.Wilhelm M, Kunzmann V, Eckstein S, Reimer P, Weissinger F, Ruediger T, Tony HP. Gammadelta T cells for immune therapy of patients with lymphoid malignancies. Blood. 2003;102:200–206. doi: 10.1182/blood-2002-12-3665. [DOI] [PubMed] [Google Scholar]

RESOURCES