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Combination treatment immunotherapy (IO) and radiotherapy (RT), or immunoradiotherapy, is a promising therapeutic strategy in preclinical cancer models. However, the results of immunoradiotherapy clinical trials in locally-advanced head and neck squamous cell carcinoma (HNSCC) have been disappointing. This warrants re-evaluation of the translation between preclinical and clinical work. Standard-of-care treatment for unresected locally-advanced HNSCC is chemoradiation (CRT); however, there is a paucity of data on optimal scheduling of IO with respect to (C)RT and how combination therapy impacts on the anti-tumour T-cell response.

The preclinical model of HPV16-positive HNSCC used was subcutaneous mEER (a syngeneic murine cell line harbouring HPV16 E6 and E7) tumours established in the right flank of C57BL/6 mice. Treatment comprised RT 8 Gy × 3 on alternate days, Cisplatin 7 mg/kg intraperitoneally (i.p.) and anti-PD-1 antibody 200 µg delivered i.p. twice weekly for up to 6 doses. We used the Nr4a3-Tocky model, which allows analysis of transcriptional dynamics induced by T-cell receptor (TCR) stimulation, to demonstrate the temporal changes in effector T-cells following antigen recognition. Tumours were collected from Nr4a3-Tocky mice receiving RT alone versus control and analysed ex vivo, at day 7 or day 10 post-RT, using multi-parameter flow cytometry to characterise the dynamics of TCR signalling following engagement with cognate antigen.

Our preliminary data show improved tumour control and survival with combination treatment (CRT, aPD-1 and RT or triple combination of Cisplatin, RT and anti-PD-1) compared to controls. Scheduling of aPD-1 is important and adjuvant aPD-1, delivered 7 days after last fraction of (C)RT, provides superior tumour control compared to anti-PD-1 delivered concurrently with (C)RT. Multi-parameter flow cytometry on tumour samples from mEER-bearing Nr4a3-Tocky mice show dynamic changes between day 7 versus day 10 post-RT. Specifically, conventional CD4+ T-cells showed a significant increase in “new” TCR signalling at day 7 but not day 10 following RT. Tumour-infiltrating CD8+ T-cells expressed at high levels the activation markers, CD44 and CD69, and the immune checkpoints, PD-1 and TIGIT, with significantly increased “persistent-arrested” TCR antigen engagement between days 7 and 10 post-RT.

Our preclinical model supports starting anti-PD-1 therapy in the adjuvant setting with respect to radiotherapy. There may be a therapeutic window to initiate anti-PD-1 treatment, between day 7 and day 10 following RT, at which time there is active CD8+ and conventional CD4+ T-cell antigen engagement. Improved understanding of the fundamental biology underlying the anti-tumour T-cell response may help guide design of future immunoradiotherapy clinical trials.