(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

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.