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.