Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

Abstract

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM.

Keywords: HNSCC; HPV status; immune checkpoint molecules; immunotherapy; radiotherapy.

Figure A1

Gating strategy for cell death measurement with AnnexinV/Propidium iodide staining. Left: Forward-side-scatter to separate single cells from cell clusters. Middle: Separation of cells from cell debris. Right: Separation of the different cell death forms with AnnexinV-FITC and Propidium iodide. Primary necrotic cells: Ax+/Pi++, early apoptotic cells: Ax+/Pi−, late apoptotic cells: Ax+/Pi+. Shown is the gating strategy using an exemplary staining for HSC-4 cells that were evaluated on day 7 of the experiment.

Figure A2

Exemplary gating strategy for analyses of cell surface expression of immune checkpoint molecules via multicolor flow cytometry. Pre-gating: identification of single cells (“singlets”), differentiation of cells from debris (“cells”) and vital cells via Zombie live/dead stain (“vital”). X-Med of various antibodies for immune checkpoint molecules was taken and X-Med values of Zombie only stained samples was subtracted for ΔMFI calculation.

Figure 1

Cell death forms on day 7 for the two HPV-negative cell lines (HSC-4 and CAL33; grey dots) and the two HPV-positive cell lines (UD-Scc-2 and UM-Scc-47; white dots). The graphs show the percentage of early and late apoptotic (a) and necrotic cells (b) in the total cell count for all three treatment conditions, untreated (w/o), 5x3.0Gy and 1x19.3Gy n_hpv− = 5; n_hpv+ = 6 biological independent experiments. Graphs show median + min to max, and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05, ** p < 0.01.

Figure 2

Cell surface expression of immune suppressive checkpoint molecules PD-L1 (a), PD-L2 (b), and HVEM (c) on the cell surface of HPV-negative (grey dots) and HPV-positive (white dots) HNSCC cell lines with n_hpv− = 5; n_hpv+ = 6 biological independent experiments. Cells were subjected to either a hypofractionated irradiation regimen (5x3.0Gy) or a single high dose of 19.3Gy. ΔMFI was calculated by subtracting the respective stained samples from Zombie-only-treated samples. Graphs show median + min to max, and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05, ** p < 0.01.

Figure 3

Cell surface expression of immune stimulatory checkpoint molecules CD70 (a), CD137-L (b) and ICOS-L (c) on the cell surface of HPV-negative (grey dots) and HPV-positive (white dots) HNSCC cell lines with n_hpv− = 5; n_hpv+ = 6 biological independent experiments. Cells were subjected to either a hypofractionated irradiation regimen (5x3.0Gy) or a single high dose of 19.3Gy. ΔMFI was calculated by subtracting the respective stained samples from Zombie-only-stained samples. Graphs show Median + min to max and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05.

Figure 4

Normalized gene expression of immune suppressive (PD-L1 (a), PD-L2 (b), HVEM (c) and immune stimulatory (CD70 (d), CD137-L (e), ICOS-L (f)) checkpoint molecules of HPV-negative (grey dots) and HPV-positive (white dots) HNSCC cell lines. Cells were subjected to either a hypofractionated irradiation regimen (5x3.0Gy) or a single high dose of 19.3Gy. Normalized gene expression was calculated by normalizing samples to the following housekeeping genes: HPV−: ACTB, RPL27, RPL30, RPS18; HPV+: ACTB, RPL27, RPL30, RPS18, UBC. Graphs show Median + min to max for n_HSC-4 = 4, n_Cal33 = 5, n_UD-Scc-2 = 5, n_UM-Scc-47 = 3 biological independent experiments and a two-sided Mann–Whitney U test was performed for comparisons of treatments within one cell line: * p < 0.05.

Figure 5

Irradiation schedule of the HNSCC cell lines with 5x3.0Gy (hypofractionated) and 1x19.3Gy (high single dose). Seeding of the cells was carried out on day 1, irradiation with 5x3.0Gy on day 2–6 and 1x19.9Gy on day 6, respectively. On day 7, analysis of the cells via flow cytometry or collection of cell pellets in TriFast was performed. In the above scheme, each vertical line represents one day of the experiment.