Tumor-infiltrating lymphocytes from human prostate tumors reveal anti-tumor reactivity and potential for adoptive cell therapy

Abstract

Advanced prostate cancer remains incurable and is the second leading cause of mortality in men. Immunotherapy based on the adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical results in patients with metastatic melanoma and lately also in other solid tumors. However, the ability to obtain TIL from patients with prostate cancer, considered poorly immunogenic, remains unknown. In this study, we investigate the feasibility of isolating and expanding TIL from primary prostate tumors. We collected tumor specimens from eight patients with diagnosed prostate adenocarcinoma undergoing radical prostatectomy and were able to successfully expand multiple autologous TIL cultures from all patients. Twenty-eight prostate-TIL cultures were further expanded using a standard rapid expansion procedure under Good Manufacturing Practice conditions. TIL cultures were phenotypically characterized for T cell subset composition, differentiation status and co-inhibitory/stimulatory markers such as PD-1, TIM-3, LAG-3, and CD28 and were found to have in general similarity to TIL obtained from patients with melanoma and lung carcinoma previously treated at our center. All analyzed TIL cultures were functional as determined by the capability to produce high level of IFNγ upon stimuli. Most importantly, co-culture assays of prostate-TIL with autologous tumors demonstrated anti-tumor reactivity. In conclusion, these findings demonstrate that functional and anti-tumor reactive TIL can be obtained, despite the immunosuppressive microenvironment of the cancer, thus this study supports the development of TIL therapy for prostate cancer patients.

Keywords: Tumor Infiltrating Lymphocyte (TIL); adoptive cell therapy (ACT); immunotherapy; primary prostate cancer cultures; prostate cancer (PCa).

Figure 1.

Expansion and phenotype analysis of TIL cultures from radical prostatectomy specimens. (A) Fold expansion during rapid expansion procedure (REP) of one or multiple TIL cultures obtained from patients PS-001-008. Frequency of CD3, CD4, and CD8 in 28 post-REP TIL cultures derived from PS-001-008 (B), and for each patient (C).

Figure 2.

Characterization of primary prostate cancer cultures (PCa). (A) Representative FACS graphs (PS-006) of epithelial markers cytokeratin 5 (CK5) and 8 (CK8), prostate-specific membrane antigen (PSMA), androgen receptor (AR) and the fibroblast surface marker CD90. Fibroblast cells and unstained sample of PS-006 served as controls. (B–D): Average expression of CK5, CK8, and CD90 (B), PSMA (C) and AR (D) in six patients (PS-002-006 and PS-008). (E) Representative FACS histogram plots (PS-008) of HLA-ABC, HLA-DP, DQ, DR, PD-L1, CD80 and CD86. Unstained samples served as control. (F,G): Average expression of HLA class 1 and class 2 (F) and PD-L1, CD80 and CD86 (G) in PCa cultures (n = 3) and melanoma cultures (n = 4).

Figure 3.

Prostate TIL functionality. (A) 12 TIL cultures from six PCa patients were stimulated overnight with the anti-CD3 antibody OKT-3 (10 µg/ml) and IFNγ secretion (pg/ml) was measured by ELISA and compared to non-stimulated TIL. (B–C) Frequency of IFNγ producing prostate-TIL reactivity measured by intracellular IFNγ flow cytometry after OKT-3 stimulation. (B) Representative FACS plots (PS-002 and PS-006), (C) Frequency of IFNγ producing cells within CD3, CD8 and CD4 T cells subsets (n = 11).

Figure 4.

Anti-tumor reactivity of post-REP prostate TIL. Anti-tumor reactivity measured after co-culture with PCa cultures as target. (A) Two TIL cultures of patient PS-004 were co-cultured with two different autologous PCa cultures (Target 1 and 2), HLA-mismatched PCa cultures from PS-006 (“HLA-mismatched target”) or without exposure to tumor cells (TIL only). IFNγ secretion (pg/ml) was measured by ELISA. Error bars represent the standard deviation of triplicate repetitions. (B) IFNγ secretion (pg/ml) after co-culture of pre-REP and post-REP TIL of PS-008 with autologous PCa with or without the addition of anti-MHC class 1 blocking antibody. (C) Representative FACS analysis of 4-1BB, OX-40 and CD107a for TIL only and after co-culture with autologous PCa target cells. (D) Average expression of 4-1BB and OX40 on CD8 or CD4 after co-culture (n = 7).

Figure 5.

Chemokine profiling of prostate-TIL. (A) Representative dot plots of PS-008. Average expression of chemokine receptors on CD3 positive post-REP TIL (B) and CD4 or CD8 T cells (n = 15) (C). Comparison of two post-REP and pre-REP TIL of PS-007 (D).