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179 T-cells derived from malignant pleural effusions (MPE) are readily expandable, polyfunctional and cytotoxic to autologous tumor
  1. Vera Donnenberg1,
  2. Albert Donnenberg1,
  3. James Luketich1,
  4. Shannon Puhalla1,
  5. Yasa Mutlu1 and
  6. David Bartlett2
  1. 1University of Pittsburgh, Pittsburgh, PA, USA
  2. 2Ahn Research Institute, Pittsburgh, PA, USA


Background We have shown that malignant pleural effusions (MPE) are characterized by a distinct and complex pleural secretome1–3 dominated by IL-6, sIL-6Rα, CCL2, CXCL10, TGFβ, CCL22, IL-8. These cytokines favor tumor epithelial to mesenchymal transition, invasion and suppression of anti-tumor response among the abundant infiltrating T cells. The goal of this study was to determine whether brief ex vivo activation could induce potent effector activity.

Methods Pleural T cells were isolated from freshly drained pleural effusions from 6 breast cancer patients. Autologous pleural tumor was expanded in vitro using the Mammary Epithelial Growth Medium (Lonza). Pleural T cells were stimulated using antiCD3CD28 Dynal beads and low dose IL-2 (60 Cetus U/ml) for 2,4,7, 14 or 21 days. Expanded tumor targets (p0 and p1) were plated at 10,000 cells/96well cultured overnight before the addition of pleural effector T cells at effector to target ratios of 0.1, 3, 6, 12.5, 25 and 50. Cytotoxicity was assessed using a colorimetric assay for lactate dehydrogenase (LDH) release (PromegaCytox-96). Pleural T cells phenotype and activation/exhaustion markers. Cytokine secretion was measured by flow cytometry and (Luminex).

Results Ex vivo expanded pleural T cells were effector-memory phenotype (CD45RA-CD27-) and were highly cytotoxic against autologous tumor (89–100% Specific Lysis). Even a 2-day ex vivo activation generated highly cytotoxic T cells. The effectors were mostly CD4+ cells (62–90%). Majority of CD8+ T cells were central memory (CD45RA-/CD27-) or effector memory (CD45RA+/CD27-); a majority co-expressed intracellular granzyme B and perforin, 20–60% expressed PD-1. Most CD4+ co-expressed granzyme B and perforin, suggesting cytotoxic CD4+ T cells and were PD-1+. The in vitro cytotoxicity of expanded pleural T cells was highly reproducible among patients of significantly varied age (37 – 94) as well as patients with different molecular subtypes (triple negative, ER+PR+Her2-, ER+PR-Her2+, ER+PR+Her2+, ER+AR+PR-Her2-). Supernatants from cytotoxicity cultures evidenced secretion of G-CSF, IL-6, CXCL10, IFNgamma, MCP-3, MIP1alpha and beta, IL-13, IL-2 and TNFalpha in a dose dependent fashion. Polyfunctional T cells (single cells secreting IL-2, TNFalpha, IFNgamma, but not IL-10) were detectable among ex vivo expanded T cells.

Conclusions Pleural T cells are not exhausted and can be stimulated to become potent anti-tumor effectors that may be useful for adoptive cellular therapy.


  1. Donnenberg, A. D., Luketich, J. D., Dhupar, R., and Donnenberg, V. S. (2019) Treatment of malignant pleural effusions: the case for localized immunotherapy. Journal for ImmunoTherapy of Cancer 7, 110

  2. Donnenberg, A. D. D. V. S. (2019) The secretome of malignant pleural effusions: Clues to targets of therapy. Leukemia Research 85, S11-S12

  3. Donnenberg, A. D., Luketich, J. D., and Donnenberg, V. S. (2019) Secretome of pleural effusions associated with non-small cell lung cancer (NSCLC) and malignant mesothelioma: therapeutic implications. Oncotarget 10, 6456–6465

Ethics Approval Some samples were anonymized by an honest broker while some participants gave consent.

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