T Cells Derived From Human Melanoma Draining Lymph Nodes Mediate Melanoma-specific Antitumor Responses In Vitro and In Vivo in Human Melanoma Xenograft Model

Abstract

It has been established in murine models that lymph nodes draining a progressively growing tumor contain antigen-specific T cells capable of mediating protective immune responses upon adoptive transfer. However, naturally occurring human tumor-draining lymph nodes (TDLNs) have yet to be fully investigated. In this study, we analyzed TDLNs from patients with stage III melanoma who were undergoing routine lymph node dissection. Following short-term (14 d) culture activation with anti-CD3/anti-CD28 microbeads and expansion in low concentrations of IL-2, the melanoma-draining lymph node (MDLN) cells were ∼ 60% CD4-activated and ∼ 40% CD8-activated T cells. The activated MDLN cells demonstrated reactivity in response to overlapping peptides spanning the sequence of 4 different known melanoma antigens MAGEA1, Melan-A/MART-1, NY-ESO-1, and Prame/OIP4, suggesting the presence of melanoma-specific T cells. Coculture of activated MDLN T cells with cancer cells in vitro resulted in preferential apoptosis of human cancer cell lines that were cocultured with T cells with high degree of MHC matching. Adoptive transfer of MDLN T cells with high degree of MHC matching to A375 to mice-bearing human A375 melanoma xenografts resulted in dose-dependent improvement in survival. Although prior human studies have demonstrated the immune responses within melanoma vaccine-draining lymph nodes, this study presents evidence for the first time that naturally occurring human MDLN samples contain melanoma-experienced CD4 and CD8 T cells that can be readily cultured and expanded to mediate protective immune responses both in vitro and in vivo in a human melanoma xenograft model.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes

“…Our results have demonstrated that the MDLN samples contain antigen specific CD4 + and CD8 + T cells that could mediate melanoma cell apoptosis in vitro and improve mice survival in vivo (7, 8). MDLN is secondary lymphoid organ where both CD4 + and CD8 + T cells experience melanoma antigens in vivo in the presence of APCs, i.e., dendritic cells (DCs) and relevant cytokines (9).…”

“…Cells were washed twice with CM and counted using a cytometer and Trypan-blue exclusion to determine yield and viability. T cell activation and expansion were performed according to our previous publication (7, 8). Briefly, freshly harvested MDLN cells were incubated overnight with CM containing 30 I.U.…”

“…The re-stimulation of day-14 MDLN cells with melanoma antigen overlapping peptides has been conducted in our previous study and the results indicated that the MDLN CD4 + T cells could mediate diversified response to a broad range of antigens (7). Because of that, we determined to use whole tumor cells to re-stimulate MDLN cells as that whole tumor cells carry multiple and diversified antigens.…”

“…However, the MDLN samples investigated in our study were from patients with stage III melanoma, most of whom had the primary cutaneous melanoma previously resected and had no evidence clinically-evident melanoma in the regional lymph nodes. Because of this limitation, we had chosen to perform HLA typing (HLA-A, -DR and –DQ) for ten patients’ MDLN T cells and nine tumor cell lines in previous study (7). Our results of HLA-typing demonstrated that the patient 1–4 exhibited high degree of MHC-matching (50–87.5%) to A375 melanoma cells and low or no degree of MHC-matching to both U87 brain cancer cells (0–25%) and MDA-MB-231 breast cancer cells (12.5–25%) (Table 1).…”

“…As an IRB-approved protocol (CASE3610), we have recently investigated melanoma draining lymph nodes (MDLNs) from 25 patients with stage III malignant melanoma (7, 8). Our results have demonstrated that the MDLN samples contain antigen specific CD4 + and CD8 + T cells that could mediate melanoma cell apoptosis in vitro and improve mice survival in vivo (7, 8).…”