Abstract
Background
For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients’ immune system are not fully understood.
Methods
Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4+ and CD8+ T cells by multicolor flow cytometry, and polyfunctional cytokine expression profiles were determined on a single-cell basis.
Results
Expression levels of all measured cytokines in CD4+ T cells were higher in patients after chemoradiotherapy (CRT) as compared to untreated HNSCC patients or normal controls. After CRT, the frequency of polyfunctional CD4+ T cells, which simultaneously expressed multiple cytokines, was significantly increased as compared to untreated patients (p < 0.01).
Conclusion
CRT increases polyfunctionality of CD4+ T cells in HNSCC patients, suggesting that standard-of-care therapy can promote immune activity in immune cells. These polyfunctional CD4+ T cells in the blood of treated HNSCC patients are expected to be responsive to subsequent immunotherapeutic approaches.
Zusammenfassung
Hintergrund
Die Standardtherapie für Plattenepithelkarzinome im Kopf-Hals-Bereich (HNSCC) beinhaltet chirurgische Verfahren, Strahlentherapie und/oder Chemotherapie. Seit Kurzem ist eine Immuntherapie als zusätzliche palliative Therapieoption verfügbar und könnte auch in der adjuvanten Therapie nach primärer Chirurgie angewandt werden. Ein funktionierendes Immunsystem ist hierbei eine Grundvoraussetzung. Allerdings sind die Auswirkungen der herkömmlichen Therapien auf das Immunsystem noch nicht vollständig klar und bedürfen weiterer Untersuchungen.
Methoden
Es wurden periphere Monozyten („peripheral blood mononuclear cells“, PBMC) aus dem Blut von HNSCC-Patienten (n = 37) und gesunden Probanden (n = 10) zu unterschiedlichen Zeitpunkten isoliert. Zunächst wurden PBMCs mit Staphylokokken-Enterotoxin B (SEB) stimuliert. Anschließend wurde die Expression der Zytokine in CD4+ und CD8+ T‑Zellen mittels Mehrfarben-Durchflusszytometrie gemessen. Die Expressionsprofile und Polyfunktionalität wurden auf Einzelzellbasis ermittelt.
Ergebnisse
Die Expression aller gemessenen Zytokine in CD4+ T‑Zellen war für Patienten nach Radiochemotherapie höher, als für unbehandelte Patienten und gesunde Probanden. Die Polyfunktionalität der CD4+ T‑Zellen, also die Anzahl gleichzeitig exprimierter Zytokine, war nach Radiochemotherapie im Vergleich zu unbehandelten Patienten signifikant erhöht (p < 0,01).
Schlussfolgerung
Eine Steigerung der Polyfunktionalität von CD4+ T‑Lymphozyten nach Radiochemotherapie legt die Vermutung nahe, dass die Standardtherapie die Aktivität von Immunzellen fördert. Es ist daher gut möglich, dass diese polyfunktionalen CD4+ T‑Zellen auf eine nachfolgende Immuntherapie ansprechen könnten.
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Funding
The research was supported by the German Research Foundation (DFG) Grant # SCHU 2536/3 (PJS).
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J. Doescher, S. Jeske, S.E. Weissinger, C. Brunner, S. Laban, E. Bölke, T.K. Hoffmann, T.L. Whiteside, and P.J. Schuler declare that they have no competing interests.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.
Caption Electronic Supplementary Material
Fig. S1 Gating strategy flow cytometry analysis.
Examples of simultaneously expressed cytokines. IL‑2 and IL‑6 are mainly co-expressed with TNF-α. Fig. S2 Cytokine expression patterns. For each cohort and every treatment, median cytokine expression is shown. Exact percentages and p-values can be found in Table 2
Fig. S3 Polyfunctional cytokine expression in cohort 1. a
Pie charts show distribution of cytokine expression according to treatment. Expression of two or more cytokines at a time is higher after surgery and CRT compared to samples of untreated patients. b Boxplots display changes for CD8+ T cells expressing one cytokine or three cytokines simultaneously. After CRT significantly more CD8+ T cells express three cytokines (p = 0.04) compared to untreated patients whereas fewer CD8+ T cells express only one cytokine (p = 0.07)
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Doescher, J., Jeske, S., Weissinger, S.E. et al. Polyfunctionality of CD4+ T lymphocytes is increased after chemoradiotherapy of head and neck squamous cell carcinoma. Strahlenther Onkol 194, 392–402 (2018). https://doi.org/10.1007/s00066-018-1289-z
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DOI: https://doi.org/10.1007/s00066-018-1289-z