Uncovering the Expression Pattern of the Costimulatory Receptors ICOS, 4-1BB, and OX-40 in Exhausted Peripheral and Tumor-Infiltrating Natural Killer Cells from Patients with Cervical Cancer

Abstract

Cervical cancer (CC) poses a significant health burden, particularly in low- and middle-income countries. NK cells play a crucial role against CC; however, they can become exhausted and lose their cytotoxic capacity. This work explores the expression of costimulatory receptors (ICOS, 4-1BB, OX-40) in exhausted NK cells from CC patients. Peripheral blood and tumor biopsies were collected, and flow cytometry was used to evaluate the expression of costimulatory receptors in exhausted NK cells. There is an increase of peripheral exhausted NK cells (PD-1⁺TIGIT⁺) in CC patients; this subpopulation has a selectively increased expression of the costimulatory receptors ICOS and 4-1BB. An exhausted population is also highly increased in tumor-infiltrating NK cells, and it shows a dramatically increased expression of the costimulatory receptors ICOS (>15×) and 4-1BB (>10×) compared to peripheral NK cells. The exhausted cells, both in the periphery and in the tumor infiltrating lymphocytes (TILs), are also more likely than non-exhausted NK cell populations (PD-1^-TIGIT^-) to express these costimulatory receptors; increases ranging from 2.0× ICOS, 2.4× 4-1BB, and 2.6× OX-40 in CD56^dim PBMCs to 1.5× ICOS, 5× 4-1BB, and 10× OX-40 in TILs were found. Our study demonstrates for the first time the increased expression of the costimulatory receptors ICOS, 4-1BB, and OX-40 in peripheral CD56^dim, CD56^bright, and tumor-infiltrating NK cells in CC. Targeting these receptors for stimulation could reverse exhaustion and be a promising immunotherapy strategy.

Keywords: 4-1BB; ICOS; NK cells; OX-40; PD-1; TIGIT; cervical cancer; exhaustion.

Figure 1

Percentages of peripheral NK cells from HD and CC patients. (a) Gating strategy for the peripheral NK cell flow cytometry analysis in PBMCs from HD (n = 22) and CC patients (n = 22). Gated from the lymphocyte region in the singlet population, NK cells were determined as CD3^-CD56⁺ and classified as CD56^dim and CD56^bright NK cells. NK cells were further subdivided into four different subsets based on the expression of CD56 and CD16: CD56^dimCD16^bright, CD56^brightCD16^dim, CD56^dimCD16^dim, and CD56^negCD16^bright NK cells. (b) Percentages of total NK cells, CD56^dim, and CD56^bright NK cells in PBMCs from HD and CC patients. (c) Percentages of CD56^dimCD16^bright, CD56^brightCD16^dim, CD56^dimCD16^dim, and CD56^negCD16^bright NK cells in PBMCs from HD and CC patients. p-value: * p < 0.05; ns: non-significant.

Figure 2

Expression of costimulatory and inhibitory receptors in peripheral NK cells from HD and CC patients. (a) Representative dot plots of the costimulatory and inhibitory receptors in NK cells from HD and CC patients. (b) Receptor expression in peripheral CD56^dim NK cells from HD and CC patients. (c) Receptor expression in peripheral CD56^bright NK cells from HD and CC patients. p-value: * p < 0.05; ** p < 0.01; *** p < 0.005; ns: non-significant.

Figure 3

Expression of costimulatory receptors in peripheral exhausted CD56^dim NK cells from HD and CC patients. (a) Representative dot plot of the exhausted population (PD-1⁺TIGIT⁺) CD56^dim NK cells from CC patients. (b) Putatively exhausted CD56^dim NK cell population from HD and CC patients. (c) Coexpression of costimulatory receptors in the putatively exhausted CD56^dim NK cells. (d) Differential coexpression of costimulatory receptors in putatively exhausted (PD-1⁺TIGIT⁺) compared to non-exhausted (PD-1^-TIGIT^-) CD56^dim NK cells from the same patients. p-value: * p < 0.05; ** p < 0.01; *** p < 0.05; **** p < 0.01; ns: non-significant.

Figure 4

Expression of costimulatory receptors in peripheral exhausted CD56^bright NK cells from HD and CC patients. (a) Representative dot plot of the exhausted population (PD-1⁺TIGIT⁺) CD56^bright NK cells from CC patients. (b) Putatively exhausted CD56^bright NK cell population from HD and CC patients. (c) Coexpression of costimulatory receptors in the putatively exhausted CD56^bright NK cells. (d) Differential coexpression of costimulatory receptors in putatively exhausted (PD-1⁺TIGIT⁺) compared to non-exhausted (PD-1⁻TIGIT⁻) CD56^bright NK cells from the same patients. p-value: * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; ns: non-significant.

Figure 5

Expression of costimulatory and inhibitory receptors in tumor-infiltrating NK cells. (a) Gating strategy for the tumor-infiltrating NK cell flow cytometry analysis in biopsies from CC patients. Gated from the lymphocyte region in the singlet population, NK cells were determined as CD45⁺CD3⁻CD56⁺. (b) Receptor expression in peripheral NK cells from PBMCs (n = 22) compared to tumor-infiltrating NK cells (n = 8) from CC patients. (c) Putatively exhausted NK cell population in PBMCs and TILs from CC patients. (d) Coexpression of costimulatory receptors in the putatively exhausted NK cells from CC PBMCs compared to TILs. (e) Differential coexpression of costimulatory receptors in putatively exhausted (PD-1⁺TIGIT⁺) compared to non-exhausted (PD-1-TIGIT-) tumor-infiltrating NK cells from CC patients. p-value: * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; ns: non-significant.