Supplementary Figure 1 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

Cytotoxic activity of iNKT cells toward K562 cells gradually decreases daily.</p

FIGURE 1 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

Soluble factors in iNKT cell cultures mediate degranulation of iNKT cells toward K562 cells. A, Jurkat and K562 cells were stained with an anti-CD1d Ab and analyzed by flow cytometry. B–E, PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. iNKT cells were then treated with IL2 until analysis. B, iNKT cells maintained for 3 days after sorting were cocultured with K562, Jurkat, or α-GalCer–pulsed Jurkat cells at an E/T ratio of 2:1 and then a CD107a assay was performed. C and D, iNKT cells maintained overnight after sorting were washed and cultured in fresh medium or maintained in the same medium (control) for 2 days. C, iNKT cells were cocultured with K562 cells at an E/T ratio of 2:1 and then a CD107a assay was performed. D, iNKT cells were cocultured with K562 cells at 10:1, 5:1, and 2.5:1 E/T ratios. An LDH release assay was performed and the percentage cytotoxicity was calculated. E, Sorted iNKT cells were maintained overnight and then the culture supernatant was collected. iNKT cells maintained overnight after sorting were washed and then maintained for 4 days. The culture supernatant was added to an iNKT cell culture overnight prior to coculture with K562 cells for the CD107a assay. Data are representative of two independent experiments. Data represent the mean ± SEM. ***, P P < 0.0001. HD, healthy donor.</p

FIGURE 6 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

Primary tumor cells express CD32. A, Expression levels of FCGR2A across TCGA tumors were assessed by GEPIA. TCGA study abbreviations: LAML, acute myeloid leukemia; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; KIRP, kidney renal papillary carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; STAD, stomach adenocarcinoma; UCS, uterine carcinosarcoma. B, Lung tumor tissue arrays were subjected to IHC to detect CD32 expression in LUAD and LUSC (×400). C, LAML cells were stained with anti-CD32 or isotype control Abs and analyzed by flow cytometry. D, iNKT cells sorted using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, washed twice, and then cultured in fresh medium with IL2 for 4 or 5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with LAML or K562 cells (control) at an E/T ratio of 2:1. A CD107a assay was then performed. PT, patient.</p

FIGURE 4 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb treatment induces degranulation of iNKT cells toward K562 cells in a CD32-dependent manner. A, K562 cells were stained with anti-FC receptor (CD16, CD32, and CD64) mAbs and assessed by flow cytometry. B, E, G, iNKT cells sorted by using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, and then iNKT cells were washed twice and cultured in fresh medium with IL2 for 4–5 days. B, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with K562 cells at an E/T ratio of 2:1 in the presence of an anti-CD32 mAb or isotype (mIgG1, 10 µg/mL). An CD107a assay was then performed. C, The indicated cell lines were stained with an anti-CD32 mAb and assessed by flow cytometry. D, CD32 was constitutively overexpressed via lentiviral transduction in A549 cells. A549 cells, which express CD32 at various levels, were stained with an anti-CD32 mAb and then assessed by flow cytometry. E, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with A549 cells overexpressing CD32 at an E/T ratio of 2:1. A CD107a assay was then performed. F, U937 cells were stained with anti-CD16 and anti-CD64 mAbs and then assessed by flow cytometry. G, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with U937 cells at an E/T ratio of 2:1 in the presence of anti-CD32, anti-CD64, or isotype control Abs. A CD107a assay was then performed. Data are representative of two independent experiments. Data represent the mean ± SEM. ***, P < 0.001. HD, healthy donor.</p

FIGURE 2 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb induces degranulation of iNKT cells toward K562 cells. PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. Sorted iNKT cells were maintained in the presence of IL2 overnight and then washed. iNKT cells were then maintained in the presence of IL2 until analysis. A, Sorted iNKT cells were maintained overnight and then the culture supernatant was collected. The culture supernatant depleted with anti-mouse Ig beads or the control was added to iNKT cell cultures overnight prior to coculture with K562 cells for the CD107a assay. B, Sorted iNKT cells treated with the 6B11 mAb (10, 50, and 100 ng/ mL) or isotype mAb (mIgG1, 50 ng/mL) were cocultured with K562 cells for CD107a assays. C, Sorted iNKT cells treated with the 6B11 mAb (50 ng/mL) or isotype mAb (50 ng/mL) were cocultured with K562 cells at E/T ratios of 10:1, 5:1, and 2.5:1. An LDH release assay was then performed. D, Sorted iNKT cells treated with the 6B11 mAb (50 ng/ mL) or isotype (mIgG1, 50 ng/mL) were cocultured with K562 cells. Culture supernatants were collected for Cytometric Bead Array to assess IFNγ and TNFα production. E, iNKT cells were treated with the 6B11 mAb and anti-CD3e (UCHT1), anti-CD28, anti-CD2, or isotype control Abs overnight. iNKT cells were then washed and cocultured with K562 cells. Data are representative of two independent experiments. Data represent the mean ± SEM. *, P P P P < 0.0001. HD, healthy donor.</p

FIGURE 8 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb-treated iNKT cells suppress tumor growth in vivo. A,E,G, Schematic of iNKT cell therapy in vivo. B–D, Human IL7/IL15 knockin NSG mice were subcutaneously injected with K562 cells on day 0. iNKT cells treated with the 6B11 mAb (50 ng/mL) or mIgG1(50 ng/mL) overnight were washed and resuspended in PBS. B, iNKT cells were intratumorally injected on days 3, 5, 7, and 9 (n = 6 or 7 mice/group). B and C, Tumor size was measured every 2 days. D, Tumors were excised at day 13 and their weight was measured. F, Human IL7/IL15 knock-in NSG mice were subcutaneously injected with K562 cells on day 0. iNKT cells treated with the 6B11 mAb as described in B were intratumorally or intravenously injected on days 3, 5, 7, and 9 (n = 5 or 6 mice/group). H, Human IL7/IL15 knock-in NSG mice were subcutaneously injected with K562 cells on day 0. iNKT cells treated with the 6B11 mAb as described in B or untreated iNKT cells were intratumorally injected on days 3, 5, 7, and 9 (n = 5 or 6 mice/group). The 6B11 mAb was further intraperitoneally administrated to mice injected with untreated NKT cells. Data are representative of two independent experiments. Data represent the mean ± SEM. *, P P P P < 0.0001.</p

Supplementary Figure 2 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

Plate-bound 6B11 or anti-CD3 mAb stimulation induces degranulation of iNKT cells.</p

FIGURE 7 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

CD32-expressing normal cells barely induce degranulation of 6B11 mAb- bound NKT cells. A, PBMCs isolated from healthy donors were stained with Abs against FC receptors (CD16, CD32, and CD64), a monocyte marker (CD14), B-cell marker (CD19), and neutrophil marker (CD66b). Stained cells were analyzed by flow cytometry. Data are representative of three independent experiments. B, iNKT cells sorted using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, washed twice, and then cultured in fresh medium with IL2 for 4 or 5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with monocytes, B cells, neutrophils, or K562 cells (control) at an E/T ratio of 2:1. A CD107a assay was then performed. HD, healthy donor.</p

TABLE 1 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

IHC scoring of CD32 in the patients with lung cancer</p

FIGURE 3 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb treatment induces degranulation of iNKT cells toward some cell lines. A, The indicated cell lines were stained with an anti-CD1d mAb and analyzed by flow cytometry. B, PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. iNKT cells sorted by MACS were maintained in the presence of IL2 overnight, and then iNKT cells were washed twice and cultured in fresh medium with IL2 for 4–5 days. iNKT cells treated with the 6B11 mAb (50 ng/mL) or isotype (mIgG1) were cocultured with the indicated cell lines at an E/T ratio of 2:1. A CD107a assay was then performed. Data are representative of two independent experiments.</p