Figure legends
Figure 1. Percentages of peripheral blood NK and T cells in healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group), and cervical cancer patients (CC group). (A) Frequency of CD56dim NK cells, (B) CD56bright NK cells, and (C) CD56- CD3+T cells were evaluated in PBMCs from HD (n=17), LG (n=19), HG (n=8) and CC (n=19) groups by flow cytometry. (D-F) A correlation analysis between CD56dim NK cells and CD3+ T cells was also performed for LG, HG, and CC groups. Frequency data are shown as individual percentages of expression and their mean. Comparison between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. A Pearson correlation analysis was performed to verify the linear associations between CD56dim NK cells and CD3+ T cells. The correlation coefficient, r, andp values are shown on the figures. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 2. Expression of PD-1, TIGIT and Tim-3 on peripheral blood CD56dim NK cells from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). (A) t-SNE analysis was used to visualized expression distribution within the CD3-CD56dim NK cell population of DNAM-1, CD16, NKG2D, TIGIT, PD-1 and Tim-3 in HD (n= 17), LG (n= 19), HG (n= 8) and CC (n= 19) groups. PD-1 expression patterns for all 4 groups are highlighted in red and common staining regions in the CC group for PD-1, TIGIT, Tim-3, and NKG2D are outlined in black. Flow cytometry gating of the data was performed to analyze the single expression of the immune checkpoints: (B) PD-1, (C) TIGIT, (D) Tim-3; double expression (E) PD-1+TIGIT+, (F) PD-1+Tim-3+; and triple expression (G) of PD-1+TIGIT+Tim-3+on CD56dim NK cells. Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 3. Expression of PD-1, TIGIT and Tim-3 on peripheral blood CD56bright NK cell from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). (A) t-SNE analysis was used to visualized expression distribution within CD3-CD56bright NK cell population of DNAM-1, CD16, NKG2D, TIGIT, PD-1 and Tim-3 in HD (n=13), LG (n=20), HG (n=7) and CC (n=19) groups. PD-1 expression patterns for all 4 groups are highlighted in red and common staining regions in the CC group for PD-1, TIGIT, Tim-3, and NKG2D are outlined in black; the red box in the HD group indicates the TIGIT high region in those samples. Flow cytometry gating of the data was performed to analyze the single expression of the immune checkpoints: (B) PD-1, (C) TIGIT, (D) Tim-3; double expression (E) PD-1+TIGIT+, (F) PD-1+Tim-3+; and triple expression (G) of PD-1+TIGIT+Tim-3+on CD56bright NK cell. Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 4. Expression of PD-1, TIGIT and Tim-3 on peripheral blood T cells from from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). (A) t-SNE analysis was used to visualized expression distribution within CD56-CD3+ cells of DNAM-1, CD16, NKG2D, TIGIT, PD-1 and Tim-3 in HD (n=13), LG (n=19), HG (n=8) and CC (n=19) groups. PD-1 and TIGIT expression patterns for all 4 groups are highlighted in red and common staining regions in the CC group for PD-1, TIGIT, Tim-3, DNAM-1 and NKG2D are outlined in black. Flow cytometry gating of the data was performed to analyze the single expression of the immune checkpoints: (B) PD-1, (C) TIGIT, (D) Tim-3; double expression of (E) PD-1+TIGIT+, (F) PD-1+Tim-3+; and triple expression (G) of PD-1+TIGIT+Tim-3+on CD3+ T cells. Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 5. NKG2D and DNAM-1 expression on peripheral blood CD3+ cells from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). (A) NKG2D expression alone and (B) with PD-1 co-expression was assessed. (C) PD-1 was also evaluated on NKG2Dneg CD3+ cells. (D) DNAM-1 expression and (E) co-expression with its antagonist receptor, TIGIT, was evaluated in the same groups: HD (n=13), LG (n=19), HG (n=8) and CC (n=19). Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 6. PD-1 intermediate (PD-1int) and PD-1 high (PD-1hi) expression on peripheral blood T cells from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). PD-1 expression was measured by flow cytometry and staining was characterized as high or intermediate depending on MFI expression. (A) Examples of high and intermediate PD-1 expression in HD versus CC. In (B-E) PD-1int and (F-I) PD-1hi, double positive and triple positive checkpoint marker populations were evaluated. Data are shown as individual percentages of expression and their mean from HD (n=13), LG (n=19), HG (n=8) and CC (n=19) groups. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure 7. Expression of PD-1, TIGIT and Tim-3 on peripheral blood CD4 and CD8 T cells from low grade lesions (LG group). (A) Expression of PD-1, TIGIT and Tim-3 in CD4 versus CD8 T cells from the low grade lesion patient group. (B) double and triple co-expression: PD-1+TIGIT+, PD-1+Tim-3+ and PD-1+TIGIT+Tim-3+in CD4 versus CD8 T cells from the low grade patient group. PD-1 expression was divided according to the MFI in two populations, PD-1 intermediate (PD-1int) and PD-1 high (PD-1hi). The co-expression of PD-1 TIGIT, Tim-3 and both receptors in CD4 versus CD8 cells was also evaluated on (C-E) PD-1int and (F-H) PD-1hiCD3+ cells. Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure 8. Soluble PD-1L (sPD-1L) in serum from healthy donors (HD group), low grade lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). The concentration of soluble PD-1L in the serum of the different groups was measured by ELISA. Data are shown as pg/ml of sPD-1L and their mean. Comparisons between HD (n=24), LG (n=24), HG (n=9) and CC (n=21) groups was performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Supplemental Figure 1. Gating strategy for the flow cytometric analysis of PBMCs from patients and controls. (A) representative workflow showing identification of NK and T cell populations. (B) representative CD56dim NK cells single antibody staining and FMO controls; left healthy donor, right cervical cancer. (C) representative CD56dim NK cells double and triple staining examples, the fifth plot in the row shows PD-1+TIGIT+Tim-3+cells derived from the double positive populations; left healthy donor, right cervical cancer. (D) representative CD56bright NK cells single antibody staining and FMO controls; left healthy donor, right cervical cancer (E) representative CD56bright NK cells double and triple staining examples, the fifth plot in the row shows PD-1+TIGIT+Tim-3+cells derived from the double positive populations; left healthy donor, right cervical cancer. (F) representative CD3+ T cells single antibody staining and FMO controls; left healthy donor, right cervical cancer. (G) representative CD3+ T cells double and triple staining examples, the fifth plot in the row shows PD-1+TIGIT+Tim-3+cells derived from the double positive populations; left healthy donor, right cervical cancer.
Supplemental Figure 2. NKG2D and DNAM-1 expression on peripheral blood CD56dim NK cells from healthy donors (HD group), low grade Lesions (LG group), high grade lesions (HG group) and cervical cancer patients (CC group). (A) NKG2D expression alone and (B) with PD-1 co-expression was assessed. (C) PD-1 was also evaluated on NKG2Dneg CD3+ cells. (D) DNAM-1 expression and (E) co-expression with its antagonist receptor, TIGIT, was evaluated in the same groups: HD (n=17), LG (n=20), HG (n=7) and CC (n=19). Data are shown as individual percentages of expression and their mean. Comparisons between the groups were performed using ANOVA with Dunnett’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.