Monitoring Circulating γδ T Cells in Cancer Patients to Optimize γδ T Cell-Based Immunotherapy

The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients’ γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.


INTRODUCTION
Human γδ T cells (γδTc) represent a small subset (1-10%) of CD3 + T lymphocytes with several unconventional features. Similar to antigen presenting cells (APC), γδTc can phagocytose and present soluble antigens to CD3 + αβ T cells (1,2). Additionally, γδTc can induce the maturation of dendritic cells (DCs), and kill various tumor cells in a HLA-independent manner (3,4). Thus, there is a substantial interest in γδTc in the context of T cellbased immunotherapeutic strategies (5,6). Several pilot studies have described a partial success of γδ T cell-based immunotherapy in different types of cancer after the application of aminobisphosphonates (n-BP) or phosphorylated antigens (PAg) plus IL-2 in vivo or after repetitive transfer of in vitro expanded Vδ2expressing γδTc (7)(8)(9)(10). Although γδ T cell-based immunotherapy has delivered promising results, sustained stimulation of Vδ2 γδTc by n-BP or PAg often leads to Vδ2 T cell exhaustion (8,11,12). Additionally, a low number of functionally unresponsive γδTc has been described in patients with chronic lymphocytic leukemia or multiple myeloma (13)(14)(15). Novel bispecific antibodies (with concomitant specificity for epitopes on both γδTc and tumor cells) provide a tool to enhance cytotoxic activity of γδTc against cancer cells by selectively targeting γδTc to antigens expressed by tumor Abbreviations: BrHPP, bromohydrin-pyrophosphate; γδTc, γδ T cells; mAb, monoclonal antibody; n-BP, nitrogen-containing bisphosphonate; PAg, phosphorylated antigens; PDAC, pancreatic ductal adenocarcinoma; RTCA, real-time cell analyzer; TCR, T cell-antigen receptor. cells (16). Additionally, independent of previous immunotherapeutic strategies and prior to the application of a γδ T cell-based immunotherapy, it is mandatory to analyze the number and functional capacity of patients' γδTc in a simple manner. This article demonstrates that the analysis of absolute cell numbers of circulating γδTc from patients as well as the determination of the cytotoxic capacity against tumor cells of interest can give a better assessment of subsequent personalized tumor treatment.

MONITORING OF ABSOLUTE CELL NUMBERS
The monitoring system that uses the BD Multitest 6-color TBNK (M6T) Reagent with BD Trucount™ Beads (www.bd.com/ resource.aspx?IDX=17743, BD Biosciences, San Jose, CA, US) allows determination of absolute cell numbers of αβ T and B lymphocytes and NK cells as well as CD4 + and CD8 + T cell subsets (17,18). Since γδ T lymphocytes and their subpopulations are not detected by the M6T, we adapted γδTc staining from the BD Tru-count™ Tube technical data sheet (version 8/2010) as follows: 50 µl whole blood from cancer patients were stained with anti-CD45-PE/Cy7 (clone HI30), CD3-PE (clone SK7) pan-TCRγδ-APC (clone 11F2, customized) (all from BD Biosciences, Heidelberg, Germany), and Vδ2-PerCP (clone B6, Biolegend, Fell, Germany) mAbs and occasionally with Vδ1-FITC mAb (clone TS8.2, Thermo Fisher Scientific, Germany) in BD Trucount™ Tubes as described (16). After staining, red blood cells were lysed with 200 µl BD Lysing buffer and analyzed using the FACS Canto flow cytometer and FACS Diva software (both from BD Biosciences). For two www.frontiersin.org FIGURE 1 | Determination of the absolute cell number of circulating γδ T cells and their subsets in blood of PDAC patients. Fifty microliters whole blood samples from PDAC patients were stained with the indicated mAb in BD Trucount™ Tubes. These mAbs were previously titrated and a final concentration of 2-5 µg/ml was used. The mAb cocktail can be prepared in advance in bulk. The BD Trucount™ tubes contain lyophilized pellets that dissolve after adding liquid, thereby releasing a known number of fluorescent beads. Two hundred microliters of BD Lysing buffer was added to lyse red blood cells. To distinguish lymphocytes and beads from granulocytes and monocytes, an appropriate gate was set on CD45 + cells or beads using side scatter and CD45 or CD3 expression, respectively (upper panel). The ratio of the event number in the bead gate was compared to the total number of beads originally in the tube. The absolute cell number (Abs. Counts) of CD3 + (CD3), CD3 + TCRγδ + (γδ), TCRγδ + TCRnon-Vδ2 + (non-Vδ2), and TCRγδ + TCRVδ2 + (Vδ2) within CD45 + lymphocytes was calculated as follows: (cells/microliter of whole blood) = [(events of cells of interest)/(ratio of acquired bead events to total beads in pellet)]/50 µl. Two representative determinations (PDAC-Donor 7 and 2) of 21 are shown, as are the percentages of the different cell populations.
representative donors, the absolute numbers of total γδTc as well as Vδ2 and non-Vδ2 subsets are shown (Figure 1). Moreover, cells can be stained with anti-Vδ1 mAb labeled with an additional fluorochrome (data not shown).
Certainly, other bead-based detection systems could be used alternatively to determine absolute cell numbers. Importantly, however, these strategies must allow this determination from a small volume of patient's blood.
In addition, a possible influence of radio-or chemotherapy on circulating immune cell numbers can be easily determined by this monitoring system. For instance, our own data reveal that the absolute number of Vδ2 γδTc in a cohort of 10 breast cancer patients receiving chemotherapy did not differ from age-matched breast cancer patients without treatment (Adam-Klages et al., unpublished data). Moreover, in a cohort of 41 patients with pancreatic ductal adenocarcinoma (PDAC, stage pT3-4, pN0-1, L0-1 and V0-1), we recently observed that the decrease in absolute numbers of Vδ2 γδTc did not correlate with cancer stage/progression, but rather with patient age (16).
While determination of the absolute γδ T cell numbers and that of their subsets provides no information about their cytotoxic capacity, this can be addressed in an additional functional assay.

DETERMINATION OF CYTOTOXIC CAPACITY
We recently examined the functional capacity of γδTc from patients with PDAC (16). PDAC is a highly aggressive gastrointestinal malignancy characterized by the presence of Frontiers in Immunology | T Cell Biology desmoplastic stromal microenvironment where conventional treatment approaches including surgery, chemotherapy, and/or radiation are often not effective (19). The observed decrease in absolute Vδ2 T cell numbers in untreated patients with advanced PDAC is attributable to age, not disease status, as similar numbers were found in age-matched healthy controls (16). In an attempt to avoid Vδ2 T cell exhaustion through repetitive n-BP stimulation and overcome the immunosuppressive activity of PDAC stromal cells on cytotoxic γδ T cells, novel bispecific antibodies such as [Her2xCD3] and [(Her2) 2 xVγ9] were designed. [(Her2) 2 xVγ9] is specific for Vγ9 on γδTc (associated with Vδ2) and for human epidermal growth factor receptor HER2/neu overexpressed on PDAC, breast, and prostate cancer cells. The [(Her2) 2 xVγ9] tribody design allows monovalent binding to γδTc and bivalent HER2targeting, which enhances avidity to the tumor cell and thereby increases cytolytic activity. Both bispecific antibodies selectively target γδTc to tumor antigens, thereby enhancing the cytotoxic activity of γδTc in vitro as well as in vivo in a PDAC grafted SCID-Beige mouse model (16).
In previous studies, we usually examined the functional capacity of γδ T cell lines or freshly isolated γδ Tc. Aiming to simplify handling of cells from patients with a low γδ T cell number in the following experiments, we investigated the functional capacity of cytotoxic γδTc within PBMC. We observed that the functional cytotoxic activity of circulating γδTc from patients can be determined in as few as of 1-2 × 10 6 PBMC, readily obtainable from 2 to 4 ml of patients' blood. We analyzed blood from 21 patients with PDAC after obtaining their informed consent and relevant institutional review board approvals (code number: D401/14). As a read out system for cytotoxic activity of γδTc within freshly isolated PBMC, the real-time cell analyzer (RTCA) single-plate system (ACEA, San Diego, CA, USA) was used. RTCA measures the impedance of adherent tumor cell monolayers, but not of suspended cells such PBMCs with electronic sensors. The measurement of impedance in arbitrary cell index units reflects changes in cellular parameters of tumor cells, which allows monitoring of cellular events in real time without the incorporation of labels over time periods of several days. The loss of impedance correlates with the γδ T cell-mediated lysis of tumor cells (16). A further advantage of measuring impedance over an extended time is that it enables us to observe whether tumor cells can regenerate when lysis is incomplete.
To ensure adherence of tumor cells, PDAC cells were cultured for 24-27 h in RTCA plates before the addition of γδTc alone with or without additional substances. Thereafter, PDAC cells were still cultured alone or together with PDAC patient-derived PBMC in (i) medium, (ii) PAg such as bromohydrin-pyrophosphate (BrHPP), or (iii) [(Her2) 2 xVγ9]. During the extended time course, we observed that γδTc within PBMC required almost 24-36 h after initial stimulation to exert their cytotoxic capacity (Figure 2A, red arrow with a star). Moreover, we observed that [(Her2) 2 xVγ9] triggered tumor cell lysis more efficiently than PAg in 30% of PDAC patient samples (Figure 2A, responder), while neither substance was effective in 70% of patient samples (Figure 2A, nonresponder). The unexpected cytotoxicity against PDAC cells in the absence of a stimulus (medium, orange line) is likely due to the reactivity of NK cells in the presence of IL-2 (Figure 2A), because additional experiments with untouched, freshly isolated γδTc demonstrated that cytotoxic activity of γδTc is not induced by IL-2 alone (16).
Regarding the absolute Vδ2 T cell numbers presented in Figure 2B (table), we correlated the unresponsiveness of the majority of the tested patient samples [negative [(Her2) 2 xVγ9] reactivity] with their low initial Vδ2 T cell number. PBMC from patients with more than 30 Vδ2 + γδTc/µl blood were responsive (responder in Figure 2A and "positive" in Figure 2B), whereas in samples with <30 Vδ2 + γδTc/µl blood, no induction of cytotoxic activity to PAg or [(Her2) 2 xVγ9] stimulation was observed (non-responder in Figure 2A and "negative" in Figure 2B).
The weak capacity of bispecific antibodies to induce γδ T cell proliferation could explain the observed unresponsiveness to [(Her2) 2 xVγ9]. Therefore, PBMC from the same patients were stimulated with the PAg BrHPP or, as presented in Figure 2C, with n-BP zoledronic acid for 7-14 days. Although the responder cells expanded to 80% γδTc in culture, while non-responders comprised only 7% after n-BP stimulation, this small population of non-responders exhibited nearly the same degree of cytotoxicity as responders after re-stimulation with [(Her2) 2 xVγ9], despite the lower effector/target ratio ( Figure 2C).
Taken together, our results demonstrate that prior analysis of absolute circulating cell numbers of immune cell subsets as well as determination of their cytotoxic capacity against tumor cells of interest may provide a better assessment of whether a particular personalized tumor treatment will be effective.

WHAT CAN WE LEARN FROM THIS MONITORING SYSTEM?
γδ T cell monitoring can provide an estimate for a potential treatment of cancer patients. Although knowledge of the functional capacity of γδTc within PBMC does not provide information about their migration and infiltration into the tumor, characterization of these circulating γδTc is useful since they are activated by intravenous n-BP or PAg administration (8,10). In clinical trials where γδTc were repetitively activated with n-BP or PAg together with low-dose IL-2, effects on tumor growth were observed; however, this was associated with exhaustion, anergy, or depletion of γδTc due to repetitive stimulation (8,11,12). In light of these observations, it is necessary to optimize cytotoxic activity, which can be achieved with bispecific antibodies such as the tribody [(Her2) 2 xVγ9]. Adoptive transfer of γδTc with [(Her2) 2 xVγ9] and IL-2 significantly reduced growth of pancreatic tumors grafted into SCID-Beige mice in comparison to adoptively transferred γδTc together with n-BP and IL-2 (16).
Vδ2 γδTc used for adoptive transfer are cells within PBMC that are initially activated with n-BP or PAg plus IL-2 (7,20). Such initial activation with n-BP or PAg plus IL-2 causes selective Vδ2 T cell-expansion, while [(Her2) 2 xVγ9] does not induce strong proliferation of γδTc (unpublished data). Independently of the proliferative response of γδ Tc, the cytotoxic activity of PAg or n-BP expanded Vδ2 T cell lines can be significantly enhanced after re-stimulation with [(Her2) 2 xVγ9]. Moreover, the addition of [(Her2) 2 xVγ9] did not induce cell death of Vδ2 T cells, in contrast to restimulation of Vδ2 T cell lines with PAg (unpublished data). Thus, [(Her2) 2 xVγ9] provides a tool to further enhance cytotoxic activity of adoptively transferred γδTc, whereas PAg or www.frontiersin.org

FIGURE 2 | Correlation between absolute cell number and functional capacity of Vδ2 T cells. (Continued)
Frontiers in Immunology | T Cell Biology n-BP failed because they induce cell death in almost half of the activated cells (unpublished data). The observation that the majority of elderly people has a low frequency of γδTc hampers the expansion of autologous γδTc required for adoptive transfer. Considering these challenges, one might suggest adoptively transferring allogeneic or haploidentical γδTc from (younger) healthy donors or activating γδTc within PBMC in vivo with bispecific antibodies (21)(22)(23). To investigate the effect of bispecific antibodies on unstimulated γδTc, we monitored whether [(Her2) 2 xVγ9] can induce cytotoxic activity in γδTc within PBMC. As described above, no or weak responses to [(Her2) 2 xVγ9] were obtained with PBMC from PDAC donors with a lower frequency of Vδ2 γδTc (non-responder), whereas PBMC with a higher Vδ2 γδ T cell frequency responded to [(Her2) 2 xVγ9] resulting in enhanced cytotoxicity (responder) (Figure 2A). Interestingly, n-BP-or PAg-mediated enrichment of non-responder γδTc within PBMC for 7-14 days led to enhanced cytotoxic activity after restimulating the cells with [(Her2) 2 xVγ9] ( Figure 2C).
The validity of this monitoring system to determine γδ T cellreactivity within PBMC needs to be confirmed in patients undergoing γδ T cell-targeting therapy. Based on our experience, one might suggest initially administration of n-BP together with IL-2 in cancer patients to induce proliferation of Vδ2 γδTc followed by treatment with bispecific antibodies engaging γδTc plus IL-2 in order to avoid the Vδ2 T cell exhaustion observed in patients mediated by repetitive application of n-BP plus IL-2.

WHAT ARE THE BENEFITS OF COMBINING γδ T CELL-BASED IMMUNOTHERAPY WITH BISPECIFIC ANTIBODIES?
Therapeutic antibodies such as rituximab (anti-CD20 mAb) and trastuzumab or pertuzumab (both anti-HER2 mAb) as well as different combined therapies have clearly improved the treatment outcome of patients with B-cell lymphoma or breast cancer, respectively (24,25). Furthermore, combining these therapeutic antibodies with γδ T cell-based immunotherapy seems very promising. Rituximab enhanced cytotoxic activity of ex vivo expanded CD16 + (FcRγIII) γδTc against CD20 + chronic lymphocytic leukemia, while Trastuzumab increased γδ T cell cytotoxicity against HER2 + breast cancer cells (26).
The success of such therapeutic antibodies has inspired antibody engineers to improve the antibody efficacy. One promising approach to enhance cytotoxicity and selectively target T cells to tumor-associated antigens is based on the usage of single-chain bispecific antibody constructs. One such construct is Blinatumomab with specificity for CD19 on lymphoma or leukemia and CD3 on T cells, which has proved efficient for the treatment of patients with hematological malignancies (27). The short half-life of only a few hours in serum requires continuous intravenous infusion of Blinatumomab, which induces an almost complete molecular response and prolonged leukemia-free survival in patients with minimal residual B-lineage acute lymphoblastic leukemia (28). The favorable characteristics of bispecific antibodies such as high specificity, high cytotoxic potential, and low immunogenicity, led us to design a bispecific antibody targeted to Vγ9 instead of CD3 and to HER2 expressed on several PDAC as well as on breast and prostate cancer, which could be easily replaced by another tumor target antigen of interest.
Of course, the question arises as to what differentiates bispecific antibodies with specificity for γδTc and those with specificity for CD3 T cells. For instance, a target group could be patients with advanced hematological malignancies (e.g., AML) who require allogeneic stem cell transplantation. A major advantage of γδ T cell-based immunotherapy is the HLA-independent killing of tumor cells, thereby reducing the risk of graft-versus-host disease often caused by alloreactive CD3 + αβ T cells (21,22,29,30). A successful anti-tumor activity was described for patients with refractory hematological malignancies after adoptive transfer of haploidentical γδTc (23). Labeling ex vivo expanded haploidentical γδTc with bispecific antibodies could perhaps further enhance the cytotoxic capacity of these cells. A further advantage could be envisioned with respect to the innate lymphocyte capacity of γδTc to phagocytose and present antigens to αβ T cells, an activity that may be enhanced in the presence of a bispecific antibody. In the treatment of solid tumors, the initial administration of n-BP/IL-2 followed by infusion of bispecific antibody together with IL-2 could probably enhance cytotoxic activity of γδTc, which infiltrate several different tumor types at low frequency.

CONCLUDING REMARKS
Bispecific antibodies have been designed in different formats. Clinical trials with bispecific antibodies such as Catumaxomab (TriomAb [EpCAMxCD3]), Ertumaxomab (Triomab [HER2xCD3]), and Blinatumomab (Bispecific T Cell Engager (BiTE) [CD19xCD3]) have delivered impressive therapeutic results. Additional clinical studies are certainly required to deeper www.frontiersin.org evaluate and improve their therapeutic potential. Bispecific antibodies with specificity for CD3 enhance the cytotoxic potential of αβ as well γδ T cells. However, under certain circumstances, it would be desirable to activate only γδTc rather than a polyclonal population of T cells. For instance, CD8 + γδTc were presented at low frequency but at higher number than CD8 + αβ T cells in ductal epithelium and nearby stroma in PDAC tissues. This γδTc accumulation suggests an important role of γδTc in the immune response against PDAC, which is apparently suppressed by the pronounced immunosuppressive PDAC-microenvironment.
Together with the monitoring system described in this article, the tribody [(Her2) 2 xVγ9], which selectively targets γδTc and enhances their cytotoxic activity, provides a tool to determine the functional capacity of γδTc within the blood or within tumorinfiltrating T lymphocytes isolated from fresh tumor tissue of tumor patients. Whether bispecific antibodies targeting γδTc have the capacity to overcome the immunosuppressive stroma in PDAC patients, has yet to be investigated in further in vivo studies.