Article Figures & Data
Figures
- Figure 1.
Anti–FSHR-IR construction and expression in primary human T cells. A, schematic of lentiviral anti–FSHR-IR vectors containing an anti-FSHR peptide linked to intracellular signaling domains from CD3-z (IR-z) alone and in tandem with CD28 (IR-28z). Anti-FSHR constructs also encode GFP separated by a viral T2A (2A) ribosomal skip peptide. B, transgene expression in primary human T cells. Expression of anti–FSHR-IR constructs in primary human T cells was measured by GFP. TM, transmembrane domain; UNT, untransduced T cells. C, FSHR expression detected by flow cytometry using rabbit anti–FSHR-APC IgG (open histogram) and isotype control (gray). Specific mean fluorescence intensity (MFI) is shown on each plot. D, expression of human FSHR in ovarian cancer cell lines and control 293T cells determined by RT-PCR using FSHR-specific PCR primers. Controls included 293T cells and no input (H2O). RT-PCR using FSHR primers based on human sequence (NM_0) amplified the predicted 234-bp product from cDNA templates. Five-fold less PCR-amplified cDNA from CaOV434 cells was loaded relative to other lanes to avoid spillover.
- Figure 2.
Anti-FSHR T cells recognize human and mouse ovarian cancer cells expressing FSHR but not cells without the FSHR expression. A, levels of IFNγ in supernatants following overnight coculture of anti–FSHR-IR T cells with FSHR+ and FSHR− CaOV3 or FSHR− 293T target cells detected by ELISA. Cocultures were established at 1:1 E:T ratio. B, IFNγ in overnight cocultures of anti–FSHR-IR T cells with mouse FSHR+ ID8 cells or FSHR− AE17 mesothelioma cell lines. C, lack of immune recognition of human recombinant FSHR protein by anti–FSHR-IR T cells. Results are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student t test).
- Figure 3.
Anti–FSHR-IR T cells react against and lyse FSHR-expressing human and mouse cancer cells in vitro. A, cytokine secretion by anti–FSHR-redirected T cells and control GFP–transduced primary human T cells. IFNγ, IL2, MIP1α, TNFα, IL4, and IL10 secretion was detected by CBA 16 hours after tumor stimulation (data represent three independent experiments in triplicates). Results are presented as mean ± SD. Values of P < 0.01 and P < 0.05 were considered statistically significant. We observed a specific and statistically significant production of cytokines; IFNγ, IL2, MIP1α, and TNFα by anti-FSHR 33–53b-28z and anti-FSHR agonist A-28z against FSHR+ targets human CaOV3 and mouse ID8 cell lines when compared with cytokine levels in cocultures with FSHR− targets in human 293T and mouse AE17 cell lines. B, anti-FSHR+ T cells upregulate surface CD69 expression upon 24-hour exposure to FSHR-expressing targets. The FSHR+ IR T cells are identified by GFP expression. Graph, percentage of CD69-positive cells gated on the viable CD3+/GFP-positive T-cell population. C, cytotoxicity of anti-FSHR T cells. Anti-FSHR 33–53β-28z or agonist A-28z IR T-cell killing of FSHR-expressing human CaOV3 and mouse ID8, or control 293T or AE17 cells, was assessed in a 16-hour luciferase-based killing assay. T cells were cocultured with target cells expressing firefly luciferase at E:T ratios of 0:1, 1:1, or 3:1. Residual luciferase signal was determined after 16 hours. The percentage of lysis was determined by luminescence in comparison with untreated target wells. Results, mean ± SD. *, P < 0.05 was considered statistically significant.
- Figure 4.
Suppressed ovarian cancer growth in mice receiving anti–FSHR-IR T-cell treatment. A, 5 × 106 luciferase-expressing CaOV3 cells were injected s.c. into NSG mice on day 0. A total of 5 × 106 T cells were injected i.v. on days 20 and 25. Tumor growth was monitored by caliper measurement. Graphs, mean ± SEM of 5 mice per experiment. P values were calculated compared with GFP T-cell– and PBS-treated control mice. *, P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.01. B, preferential expansion and survival of human T cells in peripheral blood of anti–FSHR-IR T-cell–treated mice, compared with control GFP T-cell and PBS-treated groups. Peripheral blood was collected 20 and 35 days after T-cell injection and absolute number of human CD3+ T cells was quantified by flow cytometry using TruCount beads and reported as total cells per microliter of blood. Bar graphs, mean ± SD for 5 mice per group. P values were determined compared with the control GFP T-cell–treated group. *, P < 0.05.
Tables
- Table 1.
FSH peptides used for anti–FSHR-IR construction
Construct namea FSH-derived peptides Sequence Predicted affinity Predicted cross-reactivity Anti-FSHR 33–53β-IR β-Chain 33–53aa YTRDLVYKDPARPKIQKTCTF 10−5 mol/L Human/mouse Anti-FSHR 51–65β-IR β-Chain 51–65aa CTFKELVYETVRVPGC 10−4 mol/L Human Anti-FSHR 81–95β-IR β-Chain 81–95aa QCHCGKCDSDSTDCT 10−5 mol/L Human/mouse Anti-FSHR antagonist A-IR β-Chain (87–94aa) + α-chain (25–42aa) CDSDSTDCILQCMGCCFSRAYPTPLR 10−8 mol/L Human/mouse Anti-FSHR agonist A-IR β-Chain (87–94aa) + α-chain (25–42aa) CDSDSTDCILQCMGCCFSRAYPTPLRWCAGYCYCYTRD 10−7 mol/L Human/mouse +b (27–45aa) VKDPARP Anti-FSHR a+b-IR FSH (α-chain + β-chain) GenBank Gene IDs: 1081 (FSH, α subunit) + 2488 (FSH, β subunit) NA Human/mouse ↵Abbreviation: NA, not available.
aFinal chimeric IR constructs were engineered to encode for either an intracellular CD3z (-z) or a tandem CD3z and CD28 (-28z) domain.
Supplementary Data
- Supplementary Figure 1 - The expression of 18S used as a internal control (housekeeper). cDNA was constructed from total RNA isolated from 10e5 cells (cultured at 60-70% confluence). PCR performed using intron-spanning primers. Validation criteria included water blanks, and NO RT samples. For human 18SrRNA 5’CAGCCACCCGAGATTGAGCA3’ and Rev: 5’TAGTAGCGACGGGCGGTGTG3’ (amplicon 253bp) (Genbank:: NR_003286.2). For mouse ID8 cell line Ribosomal RNA for mouse Eukaryotic small ribosomal subunit [Genbank: NR_003278] primers Fw: 5’AGGGGAGAGCGGGTAAGAGA-3’ and Rev: 5’GGACAGGACTAGGCGGAACA3’ were used (amplicon size of 249bp).
- Supplementary Figure Legend
Volume 3, Issue 10
