Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients’ plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumor- or T cell-derived exosomes in patient’s plasma are immunosuppressive and impact on disease activity, we separated CD3(-) from CD3(+) exosomes by immunocapture using anti-CD3 Abs. The exosome protein cargo was evaluated for immunoregulatory proteins using on-bead flow cytometry. Tumor protein-enriched CD3 (-) exosomes were CD44v3(+). Surprisingly, mean levels of PD-L1, CTLA-4 and COX-2 were similar in CD3(+) and CD3(-) exosomes, although the latter induced higher (p<0.0025) ex vivo apoptosis of CD8(+) T cells and greater (p<0.005) conversion of CD4+ T cells to CD4(+)CD39(+) Treg. CD3(+) and CD3(-) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with UICC stage III/IV disease had higher levels of PD-L1 and COX-2 than stage I/II patients (p<0.005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (p<0.03). CD3(+) and CD3(-) exosomes of HNSCC patients had higher PD-L1, COX-2 and CD15s levels than healthy donors' exosomes (p<0.009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3(-)/CD44v3-enriched and CD3(+) exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers. This article is protected by copyright. All rights reserved.