Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Abstract

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC's samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated T(EM) cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.

Fig. 1

Expansion in vitro of T lymphocytes cultured with different cytokines or their combinations. PBMCs (1 × 10⁶ cells) isolated from the cutaneous melanoma patient 2710 were stimulated in vitro with irradiated autologous tumor cells at 1:5 tumor cell lymphocyte ratio in the presence of cytokines indicated in the figure. Fresh medium was replaced every 3 days. T-cell growth was assessed at days 9, 14 and 21. Results represent averages of triplicates of the fold increase number with SD ≤ 10%; statistical analysis of differences between means of fold of increase of T-cell numbers was performed by two-tailed t test (P ≤ 0.01)

Fig. 2

Tumor-specific recognition of MLTCs from patient #2710 cultured in vitro in the presence of different cytokines or of their combinations. The specific tumor recognition of lymphocytes isolated from PBMCs of the cutaneous melanoma patient 2710 and cultured for 2 weeks in the presence of different cytokines was analyzed for IFN-γ release (ELISPOT assay). IFN-γ secretion by T cells was assessed following the incubation with the autologous melanoma line (#2710 mel) pre-treated or not with anti-HLA class I (W6/32) or anti-HLA class II (L243) mAbs. The recognition of the allogeneic HLA-matched (HLA-A*0201⁺ 501 mel) or HLA-mismatched (15392 mel) melanoma lines and of the NK target cell line K562 was also determined. PHA/Con-A was used as a positive control for IFN-γ release. Statistical analysis of differences between means of IFN-γ released by T cells was done by two-tailed t test (P ≤ 0.01)

Fig. 3

Isolation of highly tumor reactive T lymphocytes by the MLTC protocol from cutaneous and ocular melanoma patients. PBMC from #2710 cutaneous and #15765 ocular melanoma patients were stimulated in vitro with irradiated autologous tumor cells at 1:5 tumor cell lymphocyte ratio in the presence of IL-2 + IL-15. Following 2 weekly stimulations, the tumor specificity of T cells was assessed by measuring IFN-γ secretion (ELISPOT assay) after the incubation with the autologous melanoma cells (#2710 and #15765, a, b, respectively) pre-treated or not with anti-HLA class I (W6/32) or anti-HLA class II (L243) mAbs. Moreover, T cells were also pre-incubated or not with the anti-NKG2D mAb. The recognition of the allogeneic HLA-matched (HLA-A*0201⁺ 501 mel, a) or HLA-mismatched (#49318 mel, a) melanoma line and of the NK target cell line K562 was also determined. For patient #2710, the recognition of HLA-A2-restricted TAA-isolated epitopes (Melan-A/MART-1, Panel A and Gp100, SVV-1, COA-1, MAGE-A3, Tyr, data not shown) loaded onto T2 cells was assessed. PHA/Con-A was used as a positive control for IFN-γ release. Statistical analysis of differences between means of IFN-γ released by T cells was done by two-tailed t test (P ≤ 0.01)

Fig. 4

Cytotoxic activity of the MLTC isolated from the ocular melanoma patient 15765. The cytotoxic activity against the autologous tumor by the MLTCs from patient 15765 was assessed as CD107a mobilization and the production of perforin and IFN-γ (evaluated by immunofluorescence and cytofluorimetric analysis) by CD8⁺ T cells (a logical gate was used to identify CD8⁺ T cells) following the incubation with the autologous melanoma line (b, c) or allogeneic HLA-mismatched melanoma lines (501 and 2710 mel; data not shown). OKT3 stimulation of T cells was used as a positive control (data not shown). Data are expressed as % of positive cells and represent averages of duplicates with SD ≤ 10%

Fig. 5

Phenotype analysis of MLTCs. The phenotype analysis of T lymphocytes isolated in vitro by MLTC before and after REP was assessed by multiparametric IF and cytofluorimetric analysis (see “Materials and methods”). mAbs directed to the following molecules were used: CD3, CD4, CD8, CD45RO, CD45RA, CCR7, CD62L, NKG2D, CD27, CD28, OX40, 41BB, CD25, CD127, CD16, CD56 and CD57. Representative data of T cells (pre-REP MLTC) analysis of patient #JOFR-IA are represented in the Figure (CD4 vs. CD8 a; CD8 vs. NKG2D, b; CD8 vs. CD27, c; CD8 vs. CD28, Panel d; CD8 vs CD137, e; CD8 vs. CD134, f). Data are expressed as percentage of positive cells

Fig. 6

Characterization TILs isolated from both cutaneous and ocular melanoma patients. TILs isolated by the mechanical processing of surgically resected tumor lesions (N = 4 metastatic cutaneous melanoma and N = 3 primary ocular melanoma patients) were cultured for 5–7 days in X-Vivo-15 plus 5% HS and 600 IU/ml of rh-IL-2 in order to enrich T cells and to remove tumor cell contaminations. IF and cytofluorimetric analysis was carried out according the procedures indicated in “Materials and methods”. mAb directed to the following molecules were used: CD3, CD4, CD8, CD45RO, CD45RA, CCR7, CD62L, NKG2D, CD27, CD28, OX40, 41BB, CD25, CD127, CD16, CD56, CD57. Representative results of CD4, CD8, CD27, CD28, CD134 and CD137 are shown. Data are expressed as percentage of positive cells (a–f). IHC analysis of the immune infiltration in ocular melanoma patients was carried out (g–n). CD4 (brown staining)/CD8 (red staining) (h, m) and FOXP3 (brown)/CD25 (red) (i, n) in two ocular melanoma tissues (# 050306324–0503161250). Hematoxylin/eosin staining is shown in g, l). The scale bars represent 100 μm. Magnification ×10

Fig. 7

Functional activity of TILs isolated from cutaneous and ocular melanoma patients. TILs isolated from the mechanical processing of surgically resected tumor lesions were cultured for 5–7 days in X-Vivo-15 plus 5% HS and 600 IU/ml of rh-IL-2 in order to enrich T cells and to remove tumor cell contaminations. The tumor recognition of these TILs was assessed by measuring IFN-γ release (ELISPOT assay) after the incubation with the autologous melanoma cells (the cutaneous melanoma #4478D mel and the ocular melanoma #15765 mel, a, b, respectively) pre-treated or not with anti-HLA class I (W6/32) or anti-HLA class II (L243) mAbs. The recognition of the allogeneic HLA-mismatched (1067 mel, a) or of the allogeneic HLA-matched (HLA-A3⁺ 15392 mel, b) melanoma lines and of the NK target cell line K562 was also determined. PHA/Con-A was used as a positive control for IFN-γ release. Statistical analysis of differences between means of IFN-γ released by T cells was done by two-tailed t test (P ≤ 0.01)

Fig. 8

Efficiency of expansion in vitro of anti-tumor T cells from both MLTCs and TILs. T cells from MLTCs or TILs of cutaneous and ocular melanoma patients were stimulated in vitro by REP in the presence of irradiated (50 Gy) allogeneic PBMCs from 3 healthy donors plus OKT3 (30 ng/ml), and at day 4, 6,000 IU/ml of rh-IL-2 was added. Fresh medium with 6,000 IU/ml of rh-IL-2 was replaced every 3 days. After 15 days of in vitro culture, the growth of T cells was evaluated. Data are represented as cell number and are the mean of three independent experiments with SD ≤ 10%. In the insert, the fold increase number of these T-cell cultures is indicated