Co-Stimulatory and Immune Checkpoint Molecule Expression on Peripheral Immune Cells Differs Age Dependently Between Healthy Donors and Patients with Head and Neck Squamous Cell Carcinoma

Abstract

Introduction: The analysis of exhaustion marker expression, like immune checkpoint molecules (ICMs) on tumor-infiltrating lymphocytes as well as peripheral immune cells of patients with head and neck squamous cell carcinoma (HNSCC), is of high interest since it reveals information about the patient's immune status and the effectiveness of immune modulation. However, data regarding age-dependent expression are lacking. Here, we demonstrate an increase in most ICMs associated with age and disease, suggesting immune checkpoint modulation as an evidence-based option for the treatment of aged HNSCC patients.

Material and methods: Peripheral blood mononuclear cells (PBMCs) (healthy: n = 30 with an age range from 21 to 84 years/HNSCC: n = 37 with an age range from 37 to 94 years) were analyzed via flow cytometry following (density gradient separation) standard protocol. Flow cytometry was performed using CD4 and CD8 as backbone markers as well as co-stimulatory molecules like CD137, OX40, GITR, and CD27 or ICM like PD-1, CTLA4, BTLA, LAG3, or TIM3 using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

Results: We found a statistically significant decreased ICM expression on the PBMCs of healthy donors with increasing age. However, the expression of ICMs in HNSCC was significantly increased (PD1 on CD4⁺ T cells: p = 0.001), while we found a decreased co-stimulatory molecule expression (e.g., CD27 on CD4⁺ T cells and CD39⁺ T cells: p = 0.003 and p = 0.009, respectively). Immune cells of HPV^neg HNSCC have a significant age-dependent decrease in CD27 expression on CD8⁺, CD4⁺, and CD39⁺ T cells (p = 0.0426, p = 0.0078, and p = 0.0078, respectively).

Conclusions: This study sheds light on the changing co-stimulatory molecule/ICM expression regarding the patient's age and reveals an increase in most immune checkpoint molecules for HNSCC patients according to their age. This new evidence is valuable in ensuring individualized therapeutic approaches in the increasingly relevant field of checkpoint inhibition, even in old age.

Keywords: HNSCC; ageing; immune checkpoints; immune senescence; targeted immunotherapy.

Figure 1

Dot plots depict the expression of immune checkpoint molecules and co-stimulatory molecules on CD8⁺ T cells for healthy donors (black) and HNSCC samples (red). The straight line represents Pearson’s correlation analysis along with the corresponding p-values. (A): Selected immune checkpoint molecules; (B): selected co-stimulatory molecules. ns = not significant.

Figure 2

Dot plots depict the expression of immune checkpoint molecules and co-stimulatory molecules on CD4⁺ T cells for healthy donors (black) and HNSCC samples (red). The straight line represents Pearson’s correlation analysis along with the corresponding p-values. (A): Selected immune checkpoint molecules; (B): selected co-stimulatory molecules. ns = not significant.

Figure 3

Dot plots depict the expression of immune checkpoint molecules and co-stimulatory molecules on CD39⁺ T cells for healthy donors (black) and HNSCC samples (red). The straight line represents Pearson’s correlation analysis along with the corresponding p-values. (A): Selected immune checkpoint molecules; (B): selected co-stimulatory molecules. ns = not significant.

Figure 4

Dot plots depict the expression of selected immune checkpoint molecules and co-stimulatory molecules on different T cells for HNSCC samples grouped by HPV status: HPV^pos: red, HPV^neg: green. The straight line represents Pearson’s correlation analysis along with the corresponding p-values. (A): Selected immune checkpoint molecules; (B): selected co-stimulatory molecules. ns = not significant.

Figure 5

Dot plots depict the expression of the costimulatory molecule CD27 on CD8+ T cells for healthy donors (black) and HNSCC samples (red). The straight line represents Pearson’s correlation analysis along with the corresponding p-values. Female and male patients are compared. ns = not significant.