Background: Immunotherapy (ITx) has become the standard of care in the treatment of recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, response rate is limited to 13-18% of patients, underlining the need to identify mechanisms implicated in response or resistance. This study aims to uncover novel biomarkers of ITx outcomes in R/M HNSCC using digital spatial profiling (DSP) technology. Methods: Pre-treatment biopsy samples of 50 ITx-treated R/M HNSCC patients, constructed in the form of tissue microarray (YTMA496), were included in our discovery cohort. Cases underwent DSP with the Human Immuno-Oncology protein panel (NanoString Technologies), comprising 71 photocleavable oligonucleotide-labeled primary antibodies, used for target-protein quantification in 4 distinct molecularly defined compartments: tumor (CK+), leukocyte (CD45+), macrophage (CD68+) and immune stroma (CD45+/CD68+). All markers were explored for associations with progression-free (PFS) and overall survival (OS) using a univariate Cox regression model. Significant markers were validated using an alternative quantitative immunofluorescence method as well as in an independent validation cohort of 29 ITx-treated R/M HNSCC cases (YTMA523). Results: Univariate DSP data analysis revealed high beta2-microglobulin (B2M), LAG-3, CD25 and 4-1BB in tumor, high B2M, CD45 and CD4 in stroma, and low fibronectin in the macrophage compartment, as markers associated with improved PFS. Increased levels of B2M and CD25 in tumor and CD11c in stroma were also correlated with prolonged OS. Focusing on B2M, cases at the top tertile of tumor B2M expression were associated with improved PFS and OS [HR, 0.43; 95% confidence interval (CI), 0.21-0.9; p = 0.034 and HR, 0.41; 95% CI, 0.18-0.90; p = 0.047, respectively], by an orthogonal QIF method. Findings were replicated in our validation cohort for PFS [HR, 0.41; 95% CI, 0.19-0.93; p = 0.034] and showed a similar trend for OS [HR, 0.44; 95% CI, 0.19-1.0; p = 0.074]. B2M-high tumors also had significant enrichment with immune-cell markers (CD3, CD4, CD8, CD11c, CD68 and CD163) and increased immune checkpoint expression (PD-L1, ICOS, TIM-3, LAG-3, IDO1, B7-H3), predominantly in the tumor compartment. Conclusions: Our study indicates that intact, highly functional antigen presentation, sustained by high B2M expression in tumor, confers survival benefit in ITx treated R/M HNSCC patients, an effect driven by increased intra-tumoral immunogenicity.