Introduction: MPE are uniformly fatal. It is estimated that the incidence of MPE in the United States is more than 150,000 cases per year, making this a common terminal pathway for a variety of cancers and a dire problem without a solution. Currently available cellular therapeutics are costly and often lack polyclonality, polyfunctionality, and the ability to persist as central memory. The treatment of this deadly complication is potentially at a turning point if the rich immune infiltrates that characterize the majority of effusions can be redirected to an efficacious anti-tumor response. Despite this promise, pleural immune infiltrates have not been used to generate effector cells for adoptive cellular therapy.
Objectives: We have exploited the heterogeneous cellular composition of MPE by piloting the generation of therapeutic T-cell products, using conventional methods used for expanding tumor-infiltrating lymphocytes (TIL). The advantages of plural T cells are: (1) Fewer cycles of expansion owing to several orders of magnitude greater starting number of T cells; (2) Greater initial clonal and functional heterogeneity; (3) Likelihood of preserving polyclonality, polyfunctionality and central memory.
Results: MPE have abundant tumor infiltrating CD3+ T-cells, CD19+ B-cells, CD14+ macrophages, and EpCAM-/Cytokeratin+ mesothelial cells. Regulatory T-cells, which may be abundant in TIL, are low or absent in MPE. Our laboratory's average recovery of viable nucleated cells per MPE is 7.8±4.0×108 cells, with viability exceeding 95%. The cellular composition (tumor, lymphocytes, macrophages, neutrophils, mesothelial cells) varies from patient to patient, but T-cell recovery averages 2.0±1.6×108 (mean, SD). In pilot experiments we cultured whole breast cancer MPE in the presence of anti-CD3/anti-CD28 Dynal beads, IL-2 and IL-7 for 96h. CD3+ T cells were FACS-sorted and added to autologous tumor monolayer cultures and expanded for an additional passage (2 weeks). Expanded passage 2 T cells were compared to freshly isolated T cells (2nd MPE drainage) for ability to kill autologous tumor and non-tumor targets (live cell imaging). Expanded T cells were potently cytotoxic, whereas freshly isolated MPE had no activity against autologous tumor. Expanded T cells did not kill the autologous non-tumor target (adherent cells isolated from peripheral blood). Additionally, we tested freshly isolated breast cancer MPE T cells for the ability to secrete cytokines associated with expansion and effector generation (IL-2, IFNγ and TNFα). We also measured the immunosuppressive cytokine IL-10. Freshly isolated plastic nonadherent cells from a breast cancer MPE were incubated with TPA+ ionomycin for 1h, followed by brefeldin for 2h. CD4+ T cells (85%) and CD8+ T cells (9%) were gated on cells co-expressing intracellular IL-2 and IFNγ. Polyfunctional T cells, defined as IL-2+/IFNγ+/TNFα+/IL-10-, comprised 0.38%, and 0.82% of CD4+ and CD8+ T cells. Unstimulated control cultures constitutively secreted IL-10 and IFNγ but not IL-2 or TNFα.
Conclusions: Pleural infiltrating T-cells represent an attractive source of T cells for immunotherapy. They are numerous, readily expandable without protracted passage and can be induced to secrete immunostimulatory and effector cytokines and specifically kill autologous tumor.
