Purpose: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. Experimental Design: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)–induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). Results: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8þCD103þCD161þ effector T cells and less CD4þCD161þ effector memory T cells than OPSCC. CD161þ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4þ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4þ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. Conclusions: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.@en