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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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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%
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Fig4: 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%

Mentions: Interestingly, we also observed that these T lymphocytes exhibited cytotoxic activity, measured by CD107a mobilization and production of perforin, which correlated with that of IFN-γ production directed to the autologous melanoma line (Fig. 4).Fig. 4


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)

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%
© Copyright Policy
Related In: Results  -  Collection

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

Fig4: 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%
Mentions: Interestingly, we also observed that these T lymphocytes exhibited cytotoxic activity, measured by CD107a mobilization and production of perforin, which correlated with that of IFN-γ production directed to the autologous melanoma line (Fig. 4).Fig. 4

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