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Background The tumor immune microenvironment plays a critical role in the progression and therapeutic response of head and neck squamous cell carcinoma (HNSCC). TOX, a key regulator of T cell exhaustion, has gained attention as an indicator of immune disfunction within tumors. In HPV-associated HNSCC, which typically exhibits a distinct immune landscape and better prognosis, TOX expression may reflect unique patterns of immune exhaustion. Thus, this study evaluated TOX expression and compared to immune checkpoint markers in HNSCC stratified by smoking, alcohol use, and HPV status.Methods A total of 65 samples of human HNSCC were subjected to analysis and classification according to three groups: smoking/drinking (SD=31), non-smoking/non-drinking (NSND=25), and smoking/drinking + HPV (SD+HPV=9).Immunohistochemistry and multiplex immunofluorescence targeting TOX, FOXP3, PD-1, PD-L1, CD4, CD3, CD8, and CD103 were used.Results Patients were predominantly male, except for the NSND group. The NSND group encompassed the widest age range, including both the youngest and the oldest patients. Most tumors were in the oral cavity, with SD + HPV group having all oropharynx cases. TOX expression was significantly elevated in the SD + HPV group compared to both the SD (p = 0.0127) and NSND (p = 0.0237) groups. The percentage of CD3 +PD-1+ cells was significantly higher in the SD + HPV group compared to the SD group (p=0.0429). While co-localization of TOX with CD3, CD4, CD8, and FOXP3 showed no significant intergroup differences. Correlation analysis revealed a positive association between TOX and FOXP3 (p=0.011) and a negative association with CD3 (p=0.003) in the SD + HPV group. Additional moderate and weak positive correlations were found with PD-L1 and PD-1, respectively. Simple linear regression for CD3 and TOX expression indicated a weak negative correlation, where increased CD3 tended to correspond with decreased TOX in the SD + HPV group. Survival analysis indicated that high CD3 expression was associated with significantly improved overall survival (p=0.0271), while TOX expression did not correlate with survival outcomes (p=0.6299).Conclusions TOX expression varied significantly among the studied groups, with the highest percentage of TOX-positive cells observed in the SD + HPV group. These findings suggest that HPV-associated tumors exhibit increased TOX expression, reflecting enhanced T cell exhaustion in this subgroup, but it was not associated with significant differences in overall survival.The correlations between TOX, FOXP3, PD-1, and CD3 in HPV-associated cases suggest potential regulatory interactions, however larger sample sizes are needed to confirm these findings.Acknowledgements Biorepository and Molecular Pathology Core – Huntsman Cancer Institute, São Paulo Research Foundation - FAPESP, grant number: 2023/14770-4, and Doctoral Sandwich Program Abroad, CAPES, grant number: 88881.980919/2024-01.Ethics Approval The study was approved by the University of Campinas Ethics Board, approval number 6.498.683