Vδ1 T cells are preferentially expanded by PHA and IL-7 with enhanced cytotoxicity against colon cancer

Since Vδ1 T cells show high cytotoxicity but is a minor subset of human PB γδT cells, protocols aim to achieve sufficient Vδ1 T cells number for cancer adoptive immunotherapy are needed. Upon stimulation with PHA and IL-7 for 14 d, we found that Vδ1 T cells in purified PB γδT cells (Fig. S2A) from 10 different donors were selectively enriched (average percentages from approximately 10% to 80%), but the percentages of Vδ2 T cells decreased significantly (Fig. 2A). Next, we noted that PHA and IL-7 induced Vδ1 T cell proliferation, but not Vδ2 T cell (Fig. 2B). In addition, we found that the expression levels of CD107a, GranzymeB, FasL, TRAIL, NKG2D, NKp30, NKp44, NKp46, CD57, LFA-1, HLA-DR, CD86, CCR3, CCR4, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR5, and CXCR7 were higher on expanded Vδ1 T cells than Vδ2 T cells (Figs. 2C and D). However, the expression levels of CD56, CCR1, CCR2, CCR6, CX3CR1, CXCR4, and CXCR6 were higher on Vδ2 T cells (Figs. 2C and D). There was no difference in the expression of Perforin, DNAM-1, CD16, and CD80 between Vδ1 and Vδ2 T cells (Fig. S2B). Interestingly, the expression levels of CTLA-4 and PD-1 were increased on PHA and IL-7 expanded Vδ2 T cells, but not Vδ1 T cells (Fig. 2C), suggesting expanded Vδ1 T cells may be resistant to suppressive regulation. The intracellular cytokine detection showed that PHA and IL-7 expanded Vδ1 T cells produced more IL-1α, IL-6, IL-8, IL-9, IL-10, IL-21, TNF-α, and GM-CSF, while Vδ2 T cells secreted more IL-4 and IFNγ (Fig. 2E). In contrast to our previous study on human colon cancer tissues,²⁰ neither expanded PB Vδ1 nor Vδ2 T cells produced IL-1β, IL-17, IL-22, and IL-12/23 (Fig. S2B). Noteworthily, the expression levels of GranzymeB, Perforin, FasL, TRAIL, NKG2D, LFA-1, NKp30, NKp44, and NKp46 were significantly increased on expanded Vδ1 T cells as compared to fresh Vδ1 T cells (Fig. 2F). Moreover, these ex vivo expanded Vδ1 T cells eradicated more adherent and sphere-forming colon cancer cells than Vδ2 T cells derived from the same sample in vitro (Fig. 2G). In addition, the cancer cell-killing capacity of expanded Vδ1 T cells against adherent and sphere-forming colon cancer cells were significantly enhanced compared with freshly isolated Vδ1 T cells (Fig. S2C). Similarly, PB Vδ1 T cells from colon cancer patients could also be induced proliferation by PHA and IL-7 efficiently (Fig. S2D). Moreover, expanded patient Vδ1 T cells also have higher cytolytic capacity against colon cancer cell line HT29 than Vδ2 T cells (Fig. S2E). These findings demonstrate that we have successfully developed an optimized protocol to preferentially promote Vδ1 T cell propagation with enhanced cytotoxicity against colon cancer in vitro.

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Figure 2 (See previous page) .

Expansion, characteristics, and cytotoxicity of Vδ1 T cells cultured with PHA plus IL-7. (A) Percentages of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells in CD45⁺ CD3⁺ TCRγδ⁺ T cells cultured with PHA and IL-7 on day 0 and day 14. Bars represent the median percentage; n = 10. (B) γδT cells were sorted by MACS, labeled with CFSE and cultured with PHA and IL-7. (Left) Proliferation of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells was evaluated on day 14 by FCM. (Right) Bar diagram summarizes the percentages of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ CFSE^- and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ CFSE^– cells; Data are shown as mean ± SEM; n = 6; ** p< 0.01. (C) Expression levels of CD107a, GranzymeB, FasL, TRAIL, NKG2D, NKp30, NKp44, NKp46, CD57, HLA-DR, CD56, CD86, LFA-1, CTLA-4 and PD-1 onCD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells cultured with PHA and IL-7 for 14 d were assessed by FCM. MFI of each molecular expression on CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ (red line) and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells (blue line) was represented in each histogram. Data are representative of six independent experiments with similar results. (D) Expression of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, and CX3CR1 on CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells cultured with PHA plus IL-7 for 14 d was assessed by FCM. MFI of each molecular expression on CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ (red line) and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells (blue line) was represented in each histogram. Data are representative of six independent experiments with similar results. (E) Intracellular expression of IL-1α, IL-6, IL-8, IL-9, IL-10, IL-21, TNF-α, GM-CSF, IL-4 and IFN-γ by CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells cultured with PHA plus IL-7 for 14 d was assessed by FCM. MFI of each molecular expression on CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ (red line) and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells (blue line) was represented in each histogram. Data are representative of six independent experiments with similar results. (F) MFI of GranzymeB, Perforin, FasL, TRAIL, NKG2D, LFA-1, NKp30, NKp44, and NKp46 expression on day 0 (fresh) and day 14 CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ cells cultured with PHA plus IL-7. Data are shown as mean ± SEM; n = 6; *p < 0.05; **p < 0.01; ***p < 0.001. (G) The susceptibility of three colon cancer cell lines, HT29, HCT116, Y and counterpart sphere-forming cells to CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ T cells cultured with PHA plus IL-7 for 14 d was tested. E:T ratio was 10:1. Data are shown as mean ± SEM; n = 6; *p < 0.05.

Expanded Vδ1 T cell-mediated colon cancer killing requires cell-to-cell contact via cytotoxicity-related receptors

To investigate the underlying mechanism of PHA and IL-7 expanded Vδ1 T cells mediated adherent and sphere-forming colon cancer cells killing, we performed cytotoxicity assay in a transwell co-culture system. As shown in Fig. 3A, tumor cell lysis was significantly decreased in transwell system, suggesting cell-to-cell contact is required for Vδ1 T cell-mediated tumor cell eradication. Consistent with previous reports,^21-23 we found Fas, Death Receptor (DR)4, DR5, MICA/B, and ICAM-1 were expressed on both adherent and sphere-forming colon cancer cells (Fig. 3B). Moreover, we demonstrated the eradication of adherent and sphere-forming colon cancer cells by Vδ1 T cells was significantly inhibited by blocking antibodies against FasL, TRAIL, NKG2D, NKp30, and LFA-1 (Fig. 3C), suggesting Vδ1 T cell-mediated cytotoxicity is partly dependent on these receptor-ligand interactions. These findings suggest that PHA and IL-7 expanded human PB Vδ1 T cells recognize adherent and sphere-forming colon cancer cells to eradicate them in cell-to-cell contact and cytotoxicity-related receptors dependent manner.

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Figure 3.

In vitro lysis of colon cancer by Vδ1 T cells requires cell-to-cell contact via cytotoxicity-related receptors. (A) The specific lysis of adherent and sphere-forming colon cancer cells by Vδ1 T cells stimulated with PHA and IL-7 in co-culture or transwell system, respectively. Data are shown as mean ± SEM; n = 6; *p< 0.05; **p < 0.01. (B) Representative flow cytometric analysis of expression levels of Fas, Death Receptor (DR)4, DR5, MICA/B, and ICAM-1 on both the adhered cells and sphere-forming cells of HT29, HCT116 and Y. Data are representative of six independent experiments with similar results. (C) In vitro tumor cytotoxicity assay performed in the presence or absence of blocking anti-FasL, TRAIL, NKG2D, NKp30, and LFA-1 antibodies with day 14 Vδ1 T cells stimulated with PHA and IL-7 as effectors and adherent and sphere-forming colon cancer cells as target cells. E:T ratio was 10:1. Data are shown as mean ± SEM; n = 3; *p < 0.05; **p < 0.01.

PHA and IL-7 expanded Vδ1 T cells exhibit greater cytotoxicity against colon cancer than Zol and IL-2 expanded Vδ2 T cells

Zol and IL-2 stimulation has been reported to efficiently expand γδT cells with cancer killing activity.²⁴ Indeed, combined Zol with IL-2 preferentially enriched Vδ2 T cells while PHA with IL-7 predominantly expanded Vδ1 T cells (Fig. 4A). We then found that total γδT cells expanded by PHA and IL-7 killed more adherent and sphere-forming colon cancer cells in vitro than that stimulated with Zol and IL-2 (Fig. 4B). A previous study reported that PHA and IL-2 also preferentially expanded Vδ1 T cells.²⁵ We found PHA and IL-2 expanded total γδT cells also showed higher cytotoxicity than that expanded by Zol and IL-2 (Fig. S3A). Interestingly, both total γδT and Vδ1 T cells expanded by PHA and IL-7 were more efficient in killing adherent and sphere-forming colon cancer cells than those expanded by PHA and IL-2 (Fig. S3B and D). Furthermore, we found that Vδ1 T cells derived from both PHA plus IL-7 and PHA plus IL-2 protocols significantly killed more adherent and sphere-forming colon cancer cells than Vδ2 T cells derived from Zol plus IL-2 system (Fig. 4C and Fig. S3C). These results indicate that human PB Vδ1 T cells expanded by our protocol are better killer cells of colon cancer than Vδ2 T cells.

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Figure 4.

Vδ1 T cells expanded with PHA and IL-7 exhibit stronger cytolytic effect than Vδ2 T cells stimulated with Zol and IL-2. (A) (Left) Representative flow cytometric analysis of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells in CD45⁺ CD3⁺ TCRγδ⁺ T cells cultured with Zol plus IL-2 or PHA plus IL-7 for 14 d, respectively. (Right) Bar diagram summarizes the percentages of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells in CD45⁺ CD3⁺ TCRγδ⁺ T cells in the above culture systems. Data are shown as mean ± SEM; n = 6；*** p< 0.001. (B) The specific lysis of adherent and sphere-forming colon cancer cells by day 14 γδT cells stimulated with Zol plus IL-2 and PHA plus IL-7, respectively. E:T ratio was 10:1. Data are shown as mean ± SEM; n = 6; *, p < 0.05; **, p < 0.01. (C) The specific lysis of adherent and sphere-forming colon cancer cells by sorted Zol plus IL-2 expaned CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ2⁺ cells and PHA plus IL-7 expanded CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ cells on day 14, respectively. E:T ratio was 10:1. Data are shown as mean ± SEM; n = 6; *, p < 0.05; **, p < 0.01.

Vδ1 T cells stimulated by PHA and IL-7 have higher expansion efficiency and survival advantage than those expanded by PHA and IL-2

To further decode the underlying mechanism of our protocol in human PB Vδ1 T cell proliferation and survival, we dissected the effect of each component in the expansion protocols and found that PHA alone, PHA plus IL-2, or PHA plus IL-2 and IL-7 were sufficient to expand Vδ1 T cells (Fig. S4A). However, PHA and IL-7 combination gave rise to more Vδ1 T cells with less dead cells (Fig. S4). After 21 d, PHA and IL-7 stimulation preferentially expanded Vδ1 T cells from 1 × 10⁵ initiating cells up to 1.2 × 10⁷ Vδ1 T cells, which represented over 100 fold increase and was superior to previous PHA and IL-2 expansion protocol (Fig. 5A). In addition, PHA and IL-7 were more efficient in inducing Vδ1 T cell proliferation with less cell apoptosis compared to PHA plus IL-2 (Figs. 5B and C). Moreover, PHA and IL-7 expanded Vδ1 T cells secreted more IL-2 than Vδ1 T cells expanded by PHA and IL-2 (Fig. 5D). Moreover, we found that PHA and IL-7 combination stimulated γδT cells to produce much more IL-2 than each component alone (Fig. 5E). Blocking experiments showed that the proliferation and survival of Vδ1 T cells were significantly inhibited by IL-2 neutralizing antibody (Fig. 5F and G). These results suggest that our protocol can induce γδT cells to produce IL-2 to promote the expansion and survival of Vδ1 T cells cooperated with exogenous IL-7.

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Figure 5.

PHA and IL-7 show more advantage in promoting Vδ1 T cell expansion and survival than PHA and IL-2. (A) The cell numbers and fold increase of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ cells stimulated with either PHA plus IL-2 or PHA plus IL-7 at day 0, 7, 14, and 21. Data are shown as mean ± SEM; n = 6; **, p < 0.01; ***, p < 0.001. (B) γδT cells were sorted by MACS, pre-labeled with CFSE and cultured with PHA plus IL-2 or PHA plus IL-7. (Left) Proliferation of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ cells expanded by PHA plus IL-2 or PHA plus IL-7 was evaluated on day 14 by FCM. (Right) Bar diagram summarizes the percentages of CD3⁺ TCRγδ⁺ TCRVδ1⁺ CFSE^- cells stimulated with PHA and IL-2 and PHA and IL-7. Data are shown as mean ± SEM; n = 6; *, p < 0.05. (C) (Left) CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ Annexin-V⁺ cells stimulated with PHA plus IL-2 or PHA plus IL-7 were detected on day 14 by FCM. (Right) Bar diagram summarizes the percentages of CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ Annexin V⁺ cells stimulated with PHA plus IL-2 and PHA plus IL-7. Data are shown as mean ± SEM; n = 6; ***, p < 0.001. (D) Expression of IL-2, IL-2R and IL-7R in fresh, and PHA plus IL-2 and PHA plus IL-7 expanded CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ cells was evaluated on day 14 by FCM. Data are representative of six independent experiments with similar results. (E) γδT cells were sorted by MACS and cultured in conditioned medium (Medium, PHA, IL-7 or PHA plus IL-7) for 14 d, and then IL-2 concentration in cultural supernatants was detected by ELISA. Data are shown as mean ± SEM; n = 6; ***, p < 0.001; ****, p < 0.0001. (F) γδT cells were sorted by MACS, labeled with CFSE and cultured in conditioned medium (Medium, PHA plus IL-7, PHA plus IL-7 and AbIL-2 or PHA plus IL-7 and AbIL-2 and AbIL-7) for 14 d, and CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ CFSE^– cells were detected by FCM. Data are representative of six independent experiments with similar results. (G) γδT cells were sorted by MACS and cultured in conditioned medium (Medium, PHA plus IL-7, PHA plus IL-7 and AbIL-2 or PHA plus IL-7 and AbIL-2 and AbIL-7) for 14 d, and then PI was add to the medium for another 15 min. CD45⁺ CD3⁺ TCRγδ⁺ TCRVδ1⁺ PI⁺ cells were detected by FCM. Data are shown as mean ± SEM; n = 6; ***, p < 0.001; ****, p < 0.0001.

Cell culture

Isolated γδT cells (1 × 10⁶ cells/mL) were cultured in RPMI 1640 medium (Invitrogen Gibco) containing 10% FBS, 50 mg/mL of penicillin and streptomycin (Invitrogen Gibco) at 37°C. The cells were cultured in the presence of rhIL-7 (20 ng/mL, Peprotech) and PHA (1 μg/mL, Sigma-Aldrich), rhIL-2 (100 IU/mL, R&D) and PHA (1 μg/mL), or rhIL-2 (100 IU/mL) and Zol (5 μM, Novartis Pharmaceutials) with or without IL-2 or IL-7 neutralizing antibody (2 μg/mL, Peprotech) for 14 d. Cells were washed and the conditioned medium was replaced every 2–3 d.

Colon cancer cell lines

Human colon cancer cell lines HT29 and HCT116 were purchased from the American Type Culture Collection (ATCC) and Y cell line, obtained from hepatic metastatic tissue of a colon cancer patient, was established in our laboratory as previously reported.⁴³ HT29 and HCT116 cells were cultured in McCoy's 5A medium (Invitrogen Gibco) and Y cells were cultured in RPMI 1640 medium containing 10% FBS, 50 mg/mL of penicillin and streptomycin at 37°C. All sphere-forming cells were obtained by culturing HT29, HCT116 and Y cells in DMEM/F12 medium (Invitrogen Gibco) containing 2% FBS, 50 mg/mL of penicillin and streptomycin, 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 4 μg/ml heparin, and 1.25 μg/mL amphotericin B (all from Life Technologies) in low adhesion culture plates (Corning Costar) at 37°C for 14 d.

Flow cytometry

For extracellular staining, γδT cells were pre-incubated in a mixture of PBS, 2% fetal calf serum, and 0.1% (w/v) sodium azide with FcgIII/IIR-specific antibody to block nonspecific binding and stained with different combinations of fluorochrome-coupled antibodies (Table S1). For intracellular staining, γδT cells were activated by Leukocyte Activation Cocktail (BD PharMingen) for 6 h following the manufacturer's protocol. Cells were collected on a FACSCanto II system (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

Fluorescence activated cell sorting

To sort Vδ1 and Vδ2 T cells, fresh PBMCs and expanded γδT cells by PHA plus IL-7, PHA plus IL-2, and Zol plus IL-2 were stained with anti-Vδ1 TCR and anti-Vδ2 TCR and sorted on a FACSAriaII cell sorter (BD Biosciences). The purity of sorted cells was above 90% assessed by flow cytometry (FCM).

In vitro cytotoxicity assay

The cytotoxicity of γδT cells against cancer cells was measured by using CellTrace Far Red DDAO-SE kit (1 mM, Invitrogen) following the manufacturer's instruction. γδT cells and DDAO-SE-labeled adherent or sphere-forming colon cancer cells were incubated at an effector : target (E:T) ratio of 10:1 at 37^oC and 5% CO₂ in a round-bottom 96 wells plate. On the 4th h, PI (1 mg/mL, BD Biosciences) was added to the medium for another 15 min and DDAO-SE⁺ PI⁺ cells were analyzed by FCM. All the expanded effector cells were cultured for 14 d in vitro and all the E:T ratio was 10:1 unless otherwise noted. The specific lysis of target cells was calculated as follows: % of specific lysis = (CT – TE) / CT × 100%, where CT indicates mean number of fluorescent target cells in control tubes without effector cells and TE indicates mean number of fluorescent cells in target plus effector cells.

RNA extraction and gene expression by qPCR

Total RNAs were extracted with the RNeasy Micro Kit (QIAGEN) and reverse transcribed into cDNA by oligo-dT primer (Invitrogen) and Superscript First Strand Synthesis System (Invitrogen). cDNA was analyzed by real-time PCR (RT-PCR) with SYBR Green I Master Mix (Invitrogen) on the StepOne Plus instrument (Applied Biosystems) for the target genes (Table S2). Data were presented as arbitrary units and calculated as 2^{(Ct(GAPDH–gene of interest))}.

γδT cell proliferation assay

Purified γδT cells were labeled with CFSE (0.5 mM, Invitrogen) and then stimulated with different stimuli for 14 d. γδT cells were collected and the CSFE^– cells were detected by FCM.

Apoptosis assay

The apoptosis of γδT cells was assessed by Annexin V-APC (BD PharMingen) and PI staining following the manufacturer's instruction. Annexin-V⁺ and PI⁺ cells were analyzed on a FACSCantoII system (BD Biosciences).

In vitro neutralizing antibody blocking experiment

To investigate the effects of FasL, TRAIL, NKG2D, NKp30, and LFA-1 in Vδ1 T cell-mediated cancer cell lysis, blocking antibodies (10 mg/mL each) against FasL, TRAIL, NKG2D, NKp30, and LFA-1 (all from BioLegend) were pre-incubated for 2 h and maintained in the co-culture medium during the killing assay. For Vδ1 T cell proliferation assay, CFSE-labeled cells were maintained in conditioned medium (Medium, PHA plus IL-7, PHA, IL-7, and anti-rhIL-2 neutralizing antibody (AbIL-2) or PHA, IL-7, AbIL-2, and AbIL-7) for 14 d, and TCRVδ1⁺ CFSE^– cells were detected by FCM. For apoptosis assay, unlabeled cells were maintained in the conditioned medium (Medium, PHA plus IL-7, PHA, IL-7 and AbIL-2 or PHA, IL-7, AbIL-2, and AbIL-7) for 14 d respectively, and then PI was added to the medium for another 15 min. The TCRVδ1⁺ PI⁺ cells were detected by FCM. The AbIL-2, AbIL-7 and control rabbit IgG (2 μg/mL, all from Peprotech) were used in neutralizing antibody blocking experiment.

ELISA assay

To detect IL-2 production by γδT cells, sorted γδT cells were stimulated with or without PHA, IL-7 or PHA plus IL-7 for 14 d. The concentration of IL-2 in the culture supernatants was measured by ELISA kit (R&D Systems) according to the manufacturer's protocol.

Immunofluorescent staining and H&E staining

Paraffin-embedded and formalin-fixed samples were cut into 5-μm sections, which were then processed for IF staining or H&E staining. After incubation with antibodies against human CD45 (BioLegend), followed by Dylight 633-conjugated goat anti-rabbit IgG (Invitrogen), images were acquired with a confocal microscopy (Zeiss LSM 710, Carl Zeiss).

In vivo human colon carcinoma xenograft tumor model

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice of male, 4- to 6-week old were used to determine the in vivo antitumor efficacy of the γδT cells expanded by PHA plus IL-7, and Zol plus IL-2, respectively. In brief, 5 × 10⁴ cells of human colon cancer cell line HT29 were subcutaneously injected. On the following day 3, 1 × 10⁶/100 μL γδT cells expanded with PHA plus IL-7, or Zol plus IL-2 were intravenously injected into mice (n = 5 for each group) for five times every third days. Mice injected with 100 μL PBS were used as controls (n = 5). Tumor progression was monitored by measuring tumor diameter with caliper. Tumor volume was calculated by length (mm) × width (mm) × height (mm). In some protocols, survival of tumor-bearing mice was also recorded.

The authors thank the technical assistance of Hao Wu.