Peripheral blood samples were collected from healthy volunteers and MPM patients after approval of the Institutional Review Board of Nagasaki University Hospital and with written informed consent. All procedures were performed in accordance with the Guidelines and Regulations of Nagasaki University Hospital. The blood sample (10 ml) was treated with 0.1 ml of heparin sodium solution (Mochida Pharmaceutical., Co., Ltd., Shinjuku-ku, Tokyo, Japan) and diluted with 10 ml of phosphate-buffered saline. The diluted blood (20 ml) was loaded onto Ficoll-Paque™ PLUS (20 ml, Cytiva, Shinjuku-ku, Tokyo, Japan) in a 50 ml conical tube (Corning Inc., Corning, NY), which was centrifuged at 600 x g at room temperature for 30 min without acceleration and deceleration. The lymphocyte layer was collected into a 50 ml conical tube and diluted with 35 ml of PBS. The mixture of peripheral blood mononuclear cells (PBMC) and Ficoll was centrifuged at 900 x g and 4 °C for 10 min. After the supernatant was removed, the resulting cell pellets were dispersed by tapping and resuspended in 13 ml of PBS. Then, the cell suspension was transferred into a 15 ml conical tube, which was centrifuged at 600 x g and 4 °C for 5 min. The supernatant was removed and the resulting cell pellets were dispersed by tapping, to which was added Yssel’s medium to give a cell concentration of 2 x 10⁶ cells/ml (11).

The PBMC suspension (1.5 ml) was placed in a well of a 24-well plate (Corning Inc.), to which was added 1.5 μl of 1 mM PTA stock solution (Techno Suzuta Co., Ltd., Heiwa-machi, Nagasaki, Japan) in dimethyl sulfoxide (Nacalai Tesque Inc., Nakagyo-ku, Kyoto, Japan) to give a final concentration of 1 μM. The plate was incubated at 37 °C with 5% CO₂ overnight, to which was added interleukin-2 (IL-2, Shionogi Pharmaceutical Co., Ltd., Chuo-ku, Osaka, Japan) to give a concentration of 100 U/ml. After incubation at 37 °C with 5% CO₂ for 24 h, the medium was replaced with Yssel’s medium containing 100 U/ml IL-2. On day 2 through day 5, the culture medium was supplemented with 100 U/ml of IL-2 and γδ cells were expanded. On day 6, 1.5 ml of Yssel’s medium was added to the well. After being mixed with a pipet, 1.5 ml of the cell suspension was transferred to another well. On day 7, the medium was replaced with the complete RPMI1640 medium plus 100 U/ml IL-2, Then, γδ T cells were incubated by day 11, placed at -80 °C, and then stored in liquid nitrogen until used. Cells were observed under a microscope (Nikon Corp., Minato-ku, Tokyo, Japan) on days 2 through 8, and the proportion of Vδ2-positive cells was determined by flow cytometry before and after expansion.

Cells (2 x 10⁵ cells) were dispensed into a 96-well round bottom plate (Corning Inc.) and stained with monoclonal antibodies (mAbs), including fluorescein isothiocyanate (FITC)-conjugated anti-TCR Vδ2 mAb (BD Biosciences, San Diego, CA) and phycoerythrin (PE)-conjugated anti-CD3 mAb (Tombo Biosciences, Co., Ltd., Kobe, Hyogo, Japan), anti-NKG2D, DNAM-1, or CD16 mAbs (BioLegend Japan, Bunkyo-ku, Tokyo, Japan) in 50 μl of PBS containing 2% fetal calf serum (FCS, Merck, Darmstadt, Hessen, Germany). After the plate was incubated on ice for 15 min, 200 μl of PBS/2% FCS was added to each well of the plate. Then, the plate was centrifuged at 600 x g and 4 °C for 2 min. After the supernatants were removed, the cell pellets were dispersed by vortexing and resuspended in 200 μl of PBS/2% FCS. The cells were washed two more times with 200 μl of PBS/2% FCS and resuspended in 400 μl of PBS/2% FCS. The cell suspensions were passed through a mesh filter and analyzed using a FACS Lyric flow cytometer (Becton Dickenson, Franklin Lakes, NJ). The cell population was visualized using FlowJo ver. 10 (FlowJo LLC, Ashland, OR). For analysis of the expression of epidermal growth factor receptor (EGFR), MPM cell lines were stained with biotinylated anti-human EGFR mAb, followed by anti-biotin protein-labeled green fluorescence protein (Techno Suzuta Co., Ltd.)

Cytotoxic activity of γδ T cells against MPM cells was measured using a non-radioactive cellular cytotoxicity assay kit (Techno Suzuta Co., Ltd.). MESO-1 and MESO-4 human malignant pleural mesothelioma (MPM) cell lines were purchased from American Type Culture Collection (Manassas, VA). Daudi human Burkitt’s lymphoma, RPMI8226 human multiple myeloma, K562 human erythrocytoma, RAMOS-RAI human Burkitt’s lymphoma and U937 human monocyte-like cell lines were obtained from Health Science Research Resources Bank, Sennan, Osaka, Japan. The cells lines were maintained in complete RPMI1640 medium. Tumor cells (1 x 10⁶ cells in 1 ml) were pulsed with 25 μM bis(butyryloxymethyl) 4’-(hydroxymethyl)-2,2’:6’,2’’-terpyridine-6,6’’-dicarboxylate (BM-HT, Techno Suzuta Co., Ltd.) at 37 °C for 15 min. When BM-HT was internalized into tumor cells, the compound was hydrolyzed by intracellular esterases to yield 4’-(hydroxymethyl)-2,2’:6’,2’’-terpyridine-6,6’’-dicarboxylate (HT). After the cells were washed three times with 5 ml of complete RPMI140 medium, cell pellets were resuspended in 20 ml of the complete RPMI1640 medium. The tumor cell suspensions (5 x 10³ cells/100 μl) were dispensed into a 96-well round bottom plate, to which were added 100 μl of a serial dilution of γδ T cells at E/T ratios of 40:1, 20:1, 10:1 and 5:1 for hematopoietic tumor cells and those of 200:1, 100:1, 50:1 and 25:1 for MPM cells. The plate was centrifuged at 200 x g for 2 min and then incubated at 37 °C with 5% CO₂ for 40 min. Detergent (Techno Suzuta Co., Ltd.) was added to wells at a final concentration of 5 x 10^-5 M for maximum release and the plate was incubated at 37 °C for 20 more min. After the cell suspensions were mixed, the plate was centrifuged at 600 x g for 2 min and the supernatants (25 μl each) were transferred into a new 96-well round bottom plate containing 250 μl of europium (Eu³⁺) solution in 0.3 M sodium acetate buffer, pH 4 (Techno Suzuta Co., Ltd.). The culture supernatant/Eu mixtures (200 μl each) were transferred to a 96-well optical plate (Thermo Fisher Scientific Inc., Waltham, MA). Time-resolved fluorescence (TRF) was measured through an ARVO or NIVO multi-plate reader (PerkinElmer Inc., Waltham, MA). All measurements were performed in triplicate. Specific lysis (%) was calculated as 100 x [experimental release (counts) – spontaneous release (counts)]/[maximum release (counts) – spontaneous release (counts)]. To examine the effect of PTA on the cellular cytotoxicity, MESO-1 and MESO-4 were pulsed with 100 nM or 1 μM PTA at 37 °C for 1 h before being pulsed with BM-HT.

For determination of NK-like activity of γδ T cells, MESO-1 and MESO-4 cell suspensions (2 x 10⁴ cells/200 μl) were placed in a 96-well flat bottom plate and incubated at 37 °C with 5% CO₂ overnight. After the culture supernatants were aspirated, γδ T cells were added to the plate at effector-to-target (E/T) ratios of 0:1, 25:1, 50:1, 100:1 or 200:1 and incubated at 37 °C with 5% CO₂ for 72 h. Then, the culture supernatants were aspirated and the wells were gently washed three times with 200 μl of complete RPMI1640 medium. To each well was added 100 μl of CellTiterGlo^® Reagent (PerkinElmer Inc.), and the cell lysates were transferred into a 96-well Optiplate (PerkinElmer Inc.). Luminescence was measured through an ARVO or NIVO multi-plate reader (PerkinElmer Inc.). All measurements were performed in triplicate.

For determination of cellular cytotoxicity of γδ T cells in the presence of PTA, HMBPP, (1-hydroxy-2-imidazol-1-yl-1-phosphonoethyl)phosphonic acid (zoledronic acid, ZOL), or anti-EGFR mAb, MPM cell suspensions (2 x 10⁴ cells/200 μl) were placed in a 96-well flat bottom plate and incubated at 37°C with 5% CO₂ overnight. After the culture supernatants were aspirated, 100 μl of γδ T cell suspension (1.6 x 10⁶ cells/well) and 100 μl of a serially-diluted PTA, ZOL or HMBPP was added to each well in triplicate at concentrations of 0, 10⁰, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, or 10⁷ pM, or a serially-diluted anti-EGFR mAb at final concentrations of 0, 0.46, 1.37, 4.11, 12.3, 37, 111, 333 or 1000 ng/ml for HD06, HD07 and HDYT, or 78.125, 156.25, 312.5, 625, 1250, 2500, 5000 or 10000 ng/ml for HDYU. The plate was incubated at 37 °C with 5% CO₂ for 48 h. Then, the culture supernatants were aspirated and the wells were gently washed three times with 200 μl of complete RPMI1640 medium. To the well was added 100 μl each of CellTiterGlo Reagent^® (PerkinElmer Inc.), and the cell lysates were transferred into a 96-well optiplate (PerkinElmer Inc.). Luminescence was measured through an ARVO or NIVO multi-plate reader (PerkinElmer Inc.).

For measurement of IFN-γ production from γδ T cells in response to MPM cells in the absence or presence of PTA, ZOL, HMBPP or anti-EGFR mAb, γδ T cells (2 x 10⁴ cells in 100 μl of complete RPMI1640 medium) were placed in a 96-well flat-bottom plate (Corning Inc.), to which was added 100 μl each of PTA, ZOL or HMBPP at concentrations of 0, 0.1, 1 or 10 μM, or anti-EGFR mAb at concentrations of 0, 0.11, 0.33 or 1 μg/ml. The plate was incubated at 37°C with 5% CO₂. After 48 h incubation, the cell suspensions were mixed and the plate was centrifuged at 600 x g at 4 °C for 2 min. The supernatants were transferred into a 96-well round bottom plate and placed at -80 °C overnight. The samples were thawed and interferon-γ (IFN-γ) levels were determined by enzyme-linked immunosorbent assay (ELISA, Peprotech, Rocky Hill, NJ) according to the manufacturer’s instructions.

The statistical significance of differences was analyzed using GraphPad Prism ver. 9.0 with a p value less than 0.05 considered statistically significant.

In order to examine effector functions of human γδ T cells against malignant pleural mesothelioma (MPM) cells, we first obtained peripheral blood from healthy adult volunteers and purified peripheral blood mononuclear cells (PBMC). The proportions of Vδ2⁺ T cells in lymphocyte fractions were 2.34%, 1.61%, 15.2%, and 13.9% for HD06, HD07, HD11, and HD12, respectively ( Supplementary Figure 1A , upper panels). The majority of Vδ2⁺ T cells are known to express Vγ9 (also termed Vγ2) gene products and we hereafter use the term “γδ T cells” for Vγ9Vδ2-bearing T cells. When PBMC were stimulated with tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate (PTA), a nitrogen-containing bisphosphonate prodrug, and expanded with IL-2 for 11 days, the proportions of γδ T cells were increased to 99.2%, 98.9%, 99.0% and 99.5% for HD06, HD07, HD11, and HD12, respectively ( Supplementary Figure 1A , lower panels), consisting with previous studies. The numbers of γδ T cells before and after expansion were 1.07 x 10⁶ cells and 8.53 x 10⁸ cells for HD06 (797-fold expansion), 6.55 x 10⁵ cells and 1.50 x 10⁹ cells for HD07 (2290-fold expansion), 8.86 x 10⁶ cells and 3.73 x 10⁹ cells for HD11 (421-fold expansion), 2.05 x 10⁶ cells and 6.03 x 10⁸ cells for HD12 (294-fold expansion), respectively. It is worthy of note that a large number of highly purified γδ T cells could be obtained using the PTA/IL-2 stimulation/expansion system. Microscopic analysis revealed that cells started to form clusters in 3 to 5 days, depending on the initial proportion of γδ T cells in lymphocyte fractions ( Supplementary Figure 1B ).

On flow cytometric analysis, essentially all the expanded γδ T cells expressed NKG2D (CD314), an activating C-type lectin-like receptor and DNAX accessory molecule-1 (DNAM-1, CD226), a type I membrane protein containing 2 Ig-like C2-type domains as shown in Supplementary Figure 1C (upper and middle panels). In addition, γδ T cells expressed CD16 (also known as FcγRIII) to different degrees: weakly positive in HD06 and HD07 and highly positive in HD11 and HD12 ( Supplementary Figure 1C , lower panels). In order to examine the effect of the types of anti-CD16 mAb on the expression pattern of CD16, HD11 and HD12 γδ T cells were stained with 6 different clones of anti-CD16 mAbs, resulting in essentially the same patterns of CD16 expression ( Supplementary Figure 1D ).

Since NKG2D, DNAM-1 and CD16 are known to be expressed on natural killer (NK) cells, we examined cytotoxic activity of the PTA/IL-2-expanded γδ T cells using a time-resolved fluorescence (TRF)-based assay system (12). It has been shown that γδ T cells exhibited potent cytotoxicity against Daudi Burkitt’s lympnoma cells and RPMI8226 multiple myeloma cells in a T cell receptor (TCR)-dependent manner (13). As shown in Supplementary Figure 2A , HD13 γδ T cells efficiently killed Daudi and RPMI8226 cells in an effector-to-target (E/T) ratio-dependent manner in 60 min, indicating that healthy donor-derived γδ T cells had capacity to kill tumor cells of hematopoietic origin in a short period of time.

It has been known that K562 erythrocytoma cells are susceptible to NK cells. We thus examined NK cell-like cytotoxic activity of healthy donor-derived γδ T cells against K562. As shown in Supplementary Figure 2A , K562 was killed by HD13 γδ T cells in an E/T ratio-dependent manner in a short period of time. The specific lysis reached to around 40% in 60 min at an E/T ratio of 40:1, equivalent to that to TCR-dependent cytotoxicity against Daudi cells. In addition, RAMOS-RAI, another Burkitt’s lymphoma cell line, was also efficiently killed by HD14 γδ T cells. We then examined susceptibility of U937 histocytoma cells to γδ T cells derived from various healthy donors. As shown in Supplementary Figure 2B , U937 was killed by HD13, HD14, HD15 and HD16 γδ T cells, while the cytotoxic effect of γδ T cells against U937 was somewhat weaker than that against tumor panels in Supplementary Figure 1A . Whereas it is, strictly speaking, difficult to distinguish between NK cell-like activity and TCR-dependent activity, it is evident that tumor cells of hematopoietic origin were sensitive to γδ T cells and the cytotoxic effect could be observed within 1 h at an E/T ratio of 40:1.

We next examined NK-like cytotoxic activity of γδ T cells against MESO-1 and MESO-4, malignant pleural mesothelioma cell lines using the same TRF-based assay system. When MPM cell lines were challenged by HD05 and HD07 γδ T cell lines for 1 h, detectable cytotoxicity was not observed even at an E/T ratio of 200:1 ( Supplementary Figure 2C ). As controls, MPM cells were challenged by NK cells derived from healthy donors, showing that NK cells exhibited cellular cytotoxicity against MPM cells to different degrees ( Supplementary Figure 2D ). We next set out to examine the effect of healthy donor-derived γδ T cells using a luminescence-based cytotoxic assay system that allowed us to determine cytotoxic activity of γδ T cells over a long period of time. When MPM cells were incubated with a serial dilution of HD05 ( Figure 1A ) or HD07 ( Figure 1B ) γδ T cells for 72 h, about 60% of MESO-1 cells and 30% of MESO-4 cells were killed at an E/T ratio of 200:1. It is worthy of note that a relatively high E/T ratio and a long duration of time are required for γδ T cells to kill MPM cells, when NK-like effector functions of γδ T cells were determined.

We next examined TCR-dependent cytotoxic activity of healthy donor-derived γδ T cells against MPM cells. Since TCR-dependent cytotoxicity elicited by γδ T cells is expected to be more potent than NK-like activity, we again used TRF-based assay system to determine the specific lysis of PTA-pretreated MPM cells by γδ T cells in 1 h. When PTA is internalized into MPM cells, the prodrug is hydrolyzed by intracellular esterases to yield a biologically active N-BP, which increases intracellular concentrations of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by inhibiting farnesyl diphosphate synthase (FDPS). The resulting IPP and DMAPP bind to the B30.2 domain of butyrophilin (BTN) 3A1. Then, γδ T cells are expected to recognize the PTA-sensitized MPM cells in a TCR- and BTN3A1/2A1-dependent manner. As shown in Figure 1C , a significant level of cytotoxicity was observed in an E/T ratio- and PTA concentration-dependent manner when PTA-sensitized MPM cells were challenged by HD05 γδ T cells under conditions used for TRF-based assay. In case of HD07 γδ T cells, cytotoxic activity against MESO-1 and MESO-4 was marginal under the same conditions as shown in Figure 1D .

We thus set out to examine TCR-dependent cytotoxicity of healthy donor-derived γδ T cells against MPM cells in a long period of time using the luminescence-based assay system. When MPM cells were incubated with HD06 γδ T cells at an E/T ratio of 80:1 in the presence of a serially diluted PTA for 48 h, MESO-1 cells were killed by HD06 γδ T cells in a PTA concentration-dependent manner, in which 1 μM or greater concentration of PTA was required for efficient killing. By contrast, PTA-sensitized MESO-4 cells were much more sensitive to HD06 γδ T cells, in which only 1 pM PTA was required for HD06 γδ T cells to kill MESO-4 cells ( Figure 1E ). Essentially the same results were obtained for HD07 γδ T cells as shown in Figure 1F . Microscopic analysis revealed that cell clustering of HD07 γδ T cells was observed when MESO-1 cells were pretreated with 1 μM or 10 μM ( Figure 1G ). In case of MESO-4 cells, as low as 1 pM PTA induced a significant level of cell clustering in HD07 γδ T cells, indicating that MESO-4 cells were more efficiently sensitized by PTA for γδ T cell-mediated cytotoxicity ( Figure 1H ).

We next examined TCR-dependent cytotoxicity of healthy donor-derived γδ T cells against ZOL-sensitized MPM cells. When MESO-1 cells were challenged by HD04 γδ T cells at an E/T ratio of 80:1 with a serially diluted ZOL for 48 h, a marginal level of cytotoxicity was noted at a ZOL concentration of 10 μM for MESO-1 ( Supplementary Figure 3A , left). As for MESO-4 cells, a significant level of cytotoxicity was observed at 1 μM and 10 μM ZOL ( Supplementary Figure 3A , right), indicating that the efficiency of MPM sensitization by ZOL was much lower than that by PTA (1 μM for ZOL vs. 1 pM for PTA). Essentially the same results were observed for HD07 γδ T cells ( Supplementary Figure 3B ). As shown in Supplementary Figures 3C–F , both HD04 and HD07 γδ T cells formed a cluster at ZOL concentrations where a significant level of specific lysis was induced Supplementary Figures 3A, B .

We also examined the effect of HMBPP in the same assay system, As shown in Supplementary Figure 4A (left), a moderate level of cytotoxicity was observed at an HMBPP concentration of 10 pM or greater, when MESO-1 cells were challenged by HD07 γδ T cells in the presence of a serially diluted HMBPP. In case of MESO-4, an HMBPP concentration of 10 nM or greater induced a significant level of cytotoxicity in HD07 γδ T cells ( Supplementary Figure 4A , right). Similar results were observed for HD09 γδ T cells regarding cytotoxicity against HMBPP-sensitized MPM cells ( Supplementary Figure 4B ). Microscopic analysis demonstrated that HMBPP concentrations where a high level of specific lysis was induced triggered cell clustering in healthy donor-derived γδ T cells.

Since CD16 (also termed FcγRIII) expression was detected in γδ T cells derived from some healthy donors, we next analyzed antibody-dependent cellular cytotoxicity (ADCC) elicited by γδ T cells. Flow cytometric analysis revealed that both MESO-1 and MESO-4 expressed epidermal growth factor receptor (EGFR) ( Figure 2A ). Since HD06 and HD07 γδ T cells expressed only a marginal level of CD16, we first measured ADCC of these γδ T cell lines against MPM cells in the presence of serially diluted anti-EGFR mAb. As shown in Figure 2B (left panel), almost no cytotoxicity was observed when MESO-1 cells were incubated with HD06 γδ T cells in the presence of a serially diluted anti-EGFR mAb. As for MESO-4, only a marginal level of cytotoxicity was noted at mAb concentration of 1 μg/ml ( Figure 2B , right panel). HD07 γδ T cells exhibited a low level of cytotoxicity against both MESO-1 and MESO-4 cells in the presence of a serially diluted mAb ( Figure 2C ). When HD11 and HD12 γδ T cells with relatively high levels of CD16 expression were examined for ADCC activity, a significant level of cytotoxicity was induced in these γδ T cells when incubated with MPM cells in the presence of a serially diluted mAb as shown in Figures 2D, E . It is worthy of note that γδ T cell clustering was apparently not observed under the conditions used in this study, suggesting that CD16-mediated killing of MPM cells is intrinsically different from TCR-mediated cytotoxicity against MPM cells ( Figures 2F, G ).

We next determined IFN-γ production when MPM cells were challenged by healthy donor-derived γδ T cells in the presence of various stimulants. As shown in Figure 3A (right panel), HD06 γδ T cells produced a significant level of IFN-γ when incubated with MESO-4 cells in the presence of PTA at a concentration of 0.1 μM or greater, where the specific lysis was more than 90% ( Figure 1E ). By contrast, a detectable level of IFN-γ was not observed when MESO-1 cells were challenged by HD06 γδ T cells even in the presence of PTA at a concentration of 10 μM ( Figure 3A , left panel), where specific lysis was around 70% ( Figure 1E ), suggesting that the production of IFN-γ was induced in HD06 γδ T cells, when the specific lysis (%) was significantly high.

We then examined IFN-γ production when MPM cells were incubated with HD06 γδ T cells in the presence of anti-EGFR mAb. As shown in Figure 3B , no detectable IFN-γ was observed even at an mAb concentration of 1 μg/ml, consisting with the results of specific lysis ( Figure 2B ), where essentially no ADCC activity was observed in HD06 γδ T cells with low CD16 expression. As shown in Figure 3C , HD11 γδ T cells secreted a significant level of IFN-γ when incubated with MESO-1 or MESO-4 cells in the presence of PTA. By contrast, essentially no IFN-γ production was observed when HD11 γδ T cells were incubated with MPM cells in the presence of anti-EGFR mAb ( Figure 3D ). Since HD11 γδ T cells expressed a high level of CD16 ( Supplementary Figure 1C ) and showed an ADCC activity ( Figure 2D ) against MPM cells in the presence of anti-EGFR mAb, it is most likely that γδ T cells do not secrete IFN-γ by stimulation through CD16.

When HD07 γδ T cells were incubated with MPM cells in the presence of PTA, a significant level of IFN-γ was observed ( Figure 3E ). By contrast, almost no IFN-γ secretion was observed when incubated with MPM cells in the presence anti-EGFR mAb ( Figure 3F ), which was consistent with the findings that HD07 γδ T cells expressed only a marginal level of CD16 ( Supplementary Figure 1C ) and exhibited only a marginal level of ADCC activity ( Figure 2C ) when incubated with MPM cells and anti-EGFR mAb. In addition, HD07 γδ T cells secreted IFN-γ when treated with MESO-4 cells and ZOL, whereas MESO-1 cells with ZOL failed to induce IFN-γ production in HD07 γδ T cells, probably because the inhibition of FDPS was not sufficient in MESO-1 cells to yield a significant level of IFN-γ production ( Figure 3G ). HMBPP induced IFN-γ production in HD07 γδ T cells in the presence of MPM cells ( Figure 3H ). Taken together, γδ T cells derived from healthy donors produced a significant level of IFN-γ when γδ T cells recognized phosphoantigen-pulsed MPM cells through γδ TCR and when the cellular cytotoxicity against MPM cells was relatively high, whereas CD16-dependent cellular cytotoxicity by γδ T cells against MPM cells was not correlated with IFN-γ production, suggesting that there was an intrinsic difference between effector functions mediated by γδ TCR and CD16 in γδ T cells.

We next examined whether or not PTA induced expansion in γδ T cells derived from MPM patients. Peripheral blood samples were obtained from MPM patients, from which PBMC were purified and stimulated with PTA/IL-2 for 11 days. Supplementary Figure 5 illustrates flow cytometric analysis of γδ T cells before and after expansion. The proportions of γδ T cells in lymphocyte fractions were 0.13%, 0.68%, 0.20%, and 0.11% for MPM01, MPM02, MPM03, and MPM04, respectively ( Supplementary Figure 5 , upper panels). When PBMC were stimulated with PTA/IL-2 for 11 days, the proportions of γδ T cells were increased to 15.7%, 83.3%, 71.7% and 20.6% for MPM01, MPM02, MPM03, and MPM04, respectively ( Supplementary Figure 5 , lower panels). The numbers of γδ T cells before and after expansion were 4.64 x 10⁴ cells and 3.30 x 10⁷ cells for MPM01 (710-fold expansion), 1.92 x 10⁵ cells and 1.75 x 10⁸ cells for MPM02 (911-fold expansion), 7.48 x 10⁴ cells and 4.73 x 10⁷ cells for MPM03 (632-fold expansion), 4.28 x 10⁴ cells and 1.98 x 10⁷ cells for MPM04 (463-fold expansion), respectively. It is worthy of note that PTA/IL-2 combination induced the expansion of MPM patient-derived γδ T cells to different degrees. Whereas the proportions of γδ T cells both before and after expansion were low, the expansion rates of MPM patients-derived γδ T cells were equivalent to those of healthy donor-derived γδ T cells.

For example, the initial proportion of γδ T cells in MPM05 patients was 17.6% ( Figure 4A , right panel), which was relatively higher than that (2.34%, 1.61%, 15.2%, and 13.9%) in healthy donors ( Supplementary Figure 1A , upper panels) and the proportion after expansion with PTA/IL-2 was 97.6% ( Figure 4A , left panel), which was equivalent to that (99.2%, 98.9%, 99.0%, and 95.5%) in healthy donors ( Supplementary Figure 1A , lower panels). The expansion rate in MPM05 γδ T cells was 462-fold and cell clustering 5 to 8 days after stimulation was as vigorous as that of healthy donor-derived γδ T cells ( Figure 4B ), suggesting that proliferative responses of γδ T cells derived from MPM patients to phosphoantigens are intrinsically the same as those of healthy donor-derived γδ T cells. In addition, PTA/IL-2-expanded γδ T cells derived from MPM05 expressed a high level of NKG2D, DNAM-1 and CD16, suggesting that killing activity of MPM patient-derived γδ T cells might be equivalent to that of healthy donor-derived γδ T cells ( Figure 4C ).

We then examined TCR-mediated cytotoxicity of MPM05-derived γδ T cells. As shown in Figure 5A , MPM05 γδ T cells exhibited a high level of specific lysis against both MESO-1 and MESO-4 cells in the presence of a serial dilution of PTA. MPM01- and MPM04-derived γδ T cells also exhibited cellular cytotoxicity against MESO-1 and MESO-4 cells, confirming that MPM patient-derived γδ T cells have potential to kill MPM cells. Furthermore, a significant level of ADCC activity was observed when both MESO-1 and MESO-4 cells were challenged by MPM05 γδ T cells in the presence of anti-EGFR mAb ( Figure 5B ). Regarding IFN-γ production, MPM05 γδ T cells secreted a significant level of IFN-γ in response to MESO-1 and MESO-4 in the presence of PTA ( Figure 5C ), whereas essentially no IFN-γ was produced when MPM cells in the presence of anti-EGFR mAb were challenged by MPM05 γδ T cells ( Figure 5D ), consistent with the findings in healthy donor-derived γδ T cells, strongly suggesting that both healthy donor-derived and MPM patient-derived γδ T cells would be utilized for adoptive immunotherapy for MPM.