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

Mazzarella T, Cambiaghi V, Rizzo N, Pilla L, Parolini D, Orsenigo E, Colucci A, Modorati G, Doglioni C, Parmiani G, Maccalli C - Cancer Immunol. Immunother. (2011)

Bottom Line: 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.The functional activity of these T lymphocytes was characterized and compared with that of TILs.Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients.

View Article: PubMed Central - PubMed

Affiliation: Unit of Immuno-Biotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy.

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.

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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)
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Fig3: 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)

Mentions: Independent MLTC cultures were set up from PBMCs isolated from 6 metastatic cutaneous melanoma patients (#2710, 4478D, JOFR-IA, DAJU, 0342 and 7). T cells were stimulated weekly with irradiated autologous tumor cells in the presence of IL-2 and IL-15. Following the two rounds of in vitro stimulation (day 15), the tumor specificity of these MLTCs was assessed by IFN-γ release (ELISPOT assay). Representative data of patients #2710 are shown in Fig. 3a. In addition, results of patient #4478D are shown in Fig. 1S Panel A of the supplementary online data. These T lymphocytes exhibit specific recognition of the autologous tumor lines (285 spots/5,000 cells for #2710 Fig. 3a) with significant inhibition (53%; P ≤ 0.001) of cytokine release after pre-treatment of target cells with the W6/32 mAb. Instead, low inhibition (26% for #2710) of IFN-γ release was found in the presence of the anti-MHC class II (L243) mAb (Fig. 3a).Fig. 3


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

Mazzarella T, Cambiaghi V, Rizzo N, Pilla L, Parolini D, Orsenigo E, Colucci A, Modorati G, Doglioni C, Parmiani G, Maccalli C - Cancer Immunol. Immunother. (2011)

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)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401505&req=5

Fig3: 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)
Mentions: Independent MLTC cultures were set up from PBMCs isolated from 6 metastatic cutaneous melanoma patients (#2710, 4478D, JOFR-IA, DAJU, 0342 and 7). T cells were stimulated weekly with irradiated autologous tumor cells in the presence of IL-2 and IL-15. Following the two rounds of in vitro stimulation (day 15), the tumor specificity of these MLTCs was assessed by IFN-γ release (ELISPOT assay). Representative data of patients #2710 are shown in Fig. 3a. In addition, results of patient #4478D are shown in Fig. 1S Panel A of the supplementary online data. These T lymphocytes exhibit specific recognition of the autologous tumor lines (285 spots/5,000 cells for #2710 Fig. 3a) with significant inhibition (53%; P ≤ 0.001) of cytokine release after pre-treatment of target cells with the W6/32 mAb. Instead, low inhibition (26% for #2710) of IFN-γ release was found in the presence of the anti-MHC class II (L243) mAb (Fig. 3a).Fig. 3

Bottom Line: 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.The functional activity of these T lymphocytes was characterized and compared with that of TILs.Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients.

View Article: PubMed Central - PubMed

Affiliation: Unit of Immuno-Biotherapy of Melanoma and Solid Tumors, Division of Molecular Oncology, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy.

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.

Show MeSH
Related in: MedlinePlus