Phenotypic and Functional Characterization of Tumor-reactive T Cells in Malignant Pleural Effusions

Pleural fluid drawn in an office procedure carries many of the same tumor-reactive T cell clones that are currently obtained by surgical tumor harvest, and those clones arrive in a less exhausted state. This work, published in Cancer Immunology Research comes from authors at the Yale School of Medicine and demonstrate malignant pleural effusion (MPE) as an accessible biological source for tumor-infiltrating lymphocyte (TIL) therapy.

TIL therapy is approved for advanced melanoma but depends on surgical tumor resection, which carries procedural risk, delays treatment, and fails to yield a usable product in up to 20% of cases. MPE can be collected by thoracentesis and is known to contain activated T cells, yet the frequency and proliferative potential of tumor antigen-specific cells there had not been compared against tumor. The investigators synchronously collected MPE, lung metastasis, and blood from a 51-year-old woman with BRAF wild-type, HLA-A*02:01-positive metastatic cutaneous melanoma previously treated with nivolumab and tebentafusp. Samples underwent 25-marker spectral flow cytometry, paired single-cell RNA and T cell receptor (TCR) sequencing, neoantigen-reactive signature scoring, TCR cloning into TCR-knockout Jurkat cells, and high-dose interleukin-2 (IL-2) expansion with cytotoxicity testing against an autologous cell line.

MPE held the highest fraction of CD45⁺ immune cells (94.8% vs 89.8% in blood, 14.7% in tumor) and CD3⁺ T cells (74.9% vs 58.6% in tumor, 48.7% in blood), while tumor held the most CD8⁺ T cells (55.8%) and CD103⁺CD69⁺ resident memory cells (24.6% vs 8.4% in MPE). Among 9,819 sequenced T cells (3,156 MPE, 3,553 tumor, 3,110 blood), 5,717 unique clonotypes were identified; 90 of 196 expanded tumor clonotypes (45.9%) were shared with MPE against 28 (14.3%) with blood. Of 37 expanded neoantigen-specific (NeoTCR) clonotypes, 23 (62.2%) were clonally related in MPE, where they carried higher stemness (p < 0.0001) and cytotoxicity (p = 0.02) and lower exhaustion (p < 0.0001) scores. Two of four cloned TCRs activated Jurkat reporters against beta-2 microglobulin-restored tumor cells, and MPE T cells outgrew tumor T cells under high-dose IL-2 with equivalent killing. These findings demonstrate that MPE supplied a polyclonal, tumor-reactive T cell pool that mirrored the tumor repertoire while remaining less terminally differentiated, supporting thoracentesis as a low-risk alternative starting material for TIL manufacturing.

Reference

Gaiger NS, Coburn J, Lee MN, et al. Phenotypic and functional characterization of tumor-reactive T cells in malignant pleural effusions. Cancer Immunol Res. 2026. https:doi.org/10.1158/2326-6066.CIR-26-0293