Ex Vivo Staining of Metastatic Lymph Nodes by Class I Major Histocompatibility Complex Tetramers Reveals High Numbers of Antigen-experienced Tumor-specific Cytolytic T Lymphocytes

TLDR

Characterizing cytolytic T lymphocyte responses to tumor antigens has been limited by the lack of direct assays of CTL activity. We synthesized melanoma‑specific tetramers that stain CTLs and, using tetramer‑guided flow cytometry, isolated and confirmed their specificity and tumor‑killing ability. Tetramer staining of tumor‑infiltrated lymph nodes ex vivo revealed high frequencies of antigen‑experienced tumor‑specific CTLs, and in vitro cytokine culture expanded these cells in a tumor‑dependent manner, demonstrating their strong proliferative and tumoricidal potential and validating the reagents for monitoring responses and generating CTLs for adoptive immunotherapy.

Abstract

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents (“tetramers”) that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.