Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy

Abstract

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

FIGURE 1.

General immune characteristics of GI and melanoma metastases. (A) Immunohistochemistry of MHC class I and HLA-DR expression on GI (upper panel) and melanoma (lower panel) visceral metastases, as well as intratumoral CD3⁺ T cell infiltrate. Semiquantification (<5%, 5–50%, >50%) for each tumor represents the percentage of cancer cells expressing Ag-presentation molecules and the estimated tumor area occupied by T cells; numbers on pie charts are percentage of tumors falling into each category. (B) Total number of live cells (trypan blue) recovered from GI and melanoma (Mel) metastases processed into cell suspensions (medians, 5.0 and 14.1). (C) Flow cytometry analysis of fresh tumor cell suspensions showing percentage of live cells expressing CD3 (medians, 24.0 and 49.7%) and percentage of CD8⁺ in CD3⁺ cells (medians, 38.1 and 30.8%).

FIGURE 2.

Tumor-infiltrating T cell phenotype in fresh tumors. (A) Flow cytometry characterization of the T cell phenotype in six liver and four lung metastases (T), associated peritumoral normal tissue (PT), and peripheral blood (B) from eight patients with colon adenocarcinoma, one with gastric adenocarcinoma, and one with biliary tract adenocarcinoma. Lines between dots represent samples from a given patient. *p < 0.05, **p < 0.01, ***p < 0.001 paired t test. (B) Flow cytometry characterization of tumor-infiltrating T cells in 10 GI and 10 melanoma (Mel) visceral metastases. Mean expression in GI compared with Mel was not statistically different for all markers (unpaired t test). Gating used forward and side light scatter lymphocytic gate, live singlet gate, CD3⁺ gate, and further subgating. CM, Central memory, CD62L⁺CD45RO⁺; EM, effector memory, CD62L⁻CD45RO⁺; EMRA, late effector memory, CD62L⁻CD45RO⁻; naive, CD62L⁺CD45RO⁻.

FIGURE 3.

Expansion of TILs from visceral metastases. (A) Expansion of TILs from tumor fragments. Bar height represents total number of fragments set up for TIL expansion in distinct culture wells; the white portion represents the number of fragments from which successful TIL outgrowth was obtained (percentage TIL-productive fragments), which was similar for GI or melanoma (Mel) lung and liver metastases (respective medians, 29 and 25%). Approximately 2 more days were needed for GI TILs to reach eight wells per fragment (p = 0.03). (B) TIL expansion from fresh tumor cell suspensions. TILs were counted at the end of the first outgrowth from tumors and compared with the total number of live cells initially plated. The mean absolute fold expansion of TILs from GI cancer is greater than from melanoma (27.5 versus 9.4, p = 0.02, bottom left panel), yielding more TILs (absolute counts, upper panels), but TILs from GI tumors were left in cultures longer (21.6 versus 17.3 d, p = 0.004). The difference between the maximal yield of cells and the initial number plated divided by the number of days in culture (Δ Expansion /days) is similar (mean, 22.0 versus 20.3 × 10⁶ cells/day, p = 0.9). (C) Percentage of CD3⁺CD8⁺ TILs out of live cells measured by flow cytometry (median days in culture, 18 and 19 for GI and Mel) after initiation with tumor fragments or cell suspension (GI versus Mel, p < 0.01). (D) Absolute (upper panel) and fold expansion (lower panel) of TILs derived from GI and melanoma metastases for 14 d in rapid expansion protocol. No significant differences between tumor types are seen for both bulk and CD8⁺ TIL–enriched samples. Dot plots are presented with medians (bars).

FIGURE 4.

Measurement of CD8⁺ TIL reactivity to autologous tumors by CD137 (4-1BB) upregulation. (A) Gating strategy: FACS plots (upper left) represent the effector TILs expanded with IL-2 from tumor fragments (average, 17 d) prior to tumor-recognition coculture assay. Effector TILs are typically bigger and more granular (red gate) than are the uncultured T cells (green gate) present in autologous cryopreserved tumor cell suspension (Tumor) used as stimulators in this assay. At rest, the percentage of 4-1BB expression in effector CD8⁺ TILs is low (2%). After coculture with tumor, 6% of the effector CD8⁺ TILs (expanded 17 d) upregulate 4-1BB expression (red gates), compared with 2% in the unstimulated condition. The CD8⁺ TILs found in the fresh tumor do not upregulate 4-1BB expression (green gates). (B) Representative examples of 4-1BB upregulation by CD8⁺ TILs grown from metastatic GI cancers in four patients after 24 h stimulation with autologous tumor in the presence or absence of a pan–MHC-I Ab. For each patient in at least two TIL cultures derived from distinct fragments (F), 4-1BB expression (gated on CD8⁺ cells, percentage in CD3⁺ cells stated below fragment number) is upregulated by at least 2-fold and can appear as a distinct cell population, whereas MHC-I blockade prevents this upregulation by at least 50%. Melanoma TILs with known reactivity to the cancer cell line Mel624 were used as a positive control in all assays.