Fig 1: In the development of OSCC with periodontitis, IL-17+ ?d T cells are activated. (a) The mRNA expression of Il17, Il10, Il18, Ifn?, Il6, and Il1ß in the tumor tissues was detected via qRT-PCR, and expression was normalized to Gapdh. (b) IL-17A and IFN-? levels in serum were measured by ELISA. (c) IL-17 expression in ?d T cells from OSCC tissues was examined by flow cytometry. Representative plots are shown, and the frequencies of IL-17+ ?d T cells were quantified. Representative confocal immunofluorescence images of OSCC tissues are shown. Red, TCR ?d; green, IL-17. AO, 21 days after tumor initiation; AOP, the AO group treated with periodontitis; AOA, the AO group treated with 4Abx; EO, 7 days after tumor initiation; EOP, the EO group treated with periodontitis; EOA, the EO group treated with 4Abx; FSC, forward scatter; APC, allophycocyanin. The results are expressed as mean ± SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001, by ANOVA. The data represent the results of over 3 independent biological replicates. See also Fig. S3.
Fig 2: The activated ?d T cells are required for IL-17A expression and OSCC development. (a) SPF mice were ligated and infected with oral microbiota from periodontitis patients or healthy people. After treatment with the monoclonal antibody UC7-13D5, the OSCC tissues were collected at 21 days after SCC7 cell inoculation. The analysis of survival rate (number of mice in each group: AON, n = 6; AOP, n = 10; AOP-Anti, n = 6), the weight and volume of OSCC tissues, H&E staining, and immunohistochemistry analysis of Ki67 were performed, and representative pictures are shown. (b) IL-17 expression in ?d T cells from OSCC tissues was examined by flow cytometry and immunofluorescence staining. Red, TCR ?d; green, IL-17. (c) IL-17A and IFN-? levels in serum and OSCC tissues were measured by ELISA. AO, 21 days after tumor initiation; AON, the AO group treated with oral microbiota from healthy people and ligature; AOP, the AO group treated with periodontitis; AOP-Anti, the AOP group with UC7-13D5 injection to inhibit ?d T cells. *, P < 0.05; and **, P < 0.01, by ANOVA. The data represent the results of over 3 independent biological replicates. See also Fig. S5.
Fig 3: ?d T cells regulate the IL-17/STAT3 pathway in OSCC with periodontitis. (a) Correlation analysis of IL-17A or IL-17RA and STAT3 gene expression level in HNSC. TPM, transcripts per million; FPKM, fragments per kilobase per million. (b) With or without filters, SCC7 cells or Cal27 cells were cocultured with 200 pg/mL IL-17, the microbiota, and PBMCs (from mice or humans), respectively. The level of pSTAT3 was analyzed by immunofluorescence staining. Green, pSTAT3; the white arrows indicate pSTAT3+ cells. (c) Western blot analysis of pSTAT3 and STAT3 in OSCC tissues of different groups. Representative images and a summary of the normalized quantitative data are shown. The results are expressed as the mean ± SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001, by ANOVA. The data represent the results of over 3 independent biological replicates. (d) IL-17 expression in ?d T cells from clinical specimens was analyzed by immunofluorescence staining and quantitated. Red, TCR ?d; green, IL-17. The level of pSTAT3 from clinical specimens was analyzed by immunofluorescence staining and quantitated. Green, pSTAT3. Number of clinical samples: OSCC without periodontitis, n = 4; OSCC with periodontitis, n = 6. *, P < 0.05; and **, P < 0.01, by Student’s t test.
Fig 4: Proposed mechanistic diagram. The tumor-associated immune response could be shaped by the oral microbiota from periodontitis. The oral microbiota from periodontitis drive the activation of IL-17+ ?d T cells, which can promote tumor proliferation.
Fig 5: Oral microbiota from periodontitis activate ?d T cells to produce IL-17 and stimulate cell proliferation. (a) PBMCs were cocultured with live oral microbiota from periodontitis for 1 or 6 h. The frequencies of ?d T and IL-17+ ?d T cells were quantified. (b) With or without filters, PBMCs were cocultured with the microbiota treated in different ways. The frequencies of IL-17+ ?d T cells were quantified, and the representative histograms of flow cytometry are shown. (c) IL-17A levels of medium supernatant in panel b were measured. (d) SCC7 cells were cocultured with PBMCs and the live oral microbiota from periodontitis. IL-17A levels in the culture supernatant were measured. (e) CCK-8 assays were conducted to detect the cell viability of SCC7 cells. Results are expressed as the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001, by ANOVA; data represent at least 3 independent experiments. See also Fig. S4.
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