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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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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
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Fig8: 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

Mentions: The REP protocol was applied to both MLTC and TILs isolated from 10 cutaneous and ocular melanoma patients in order to determine whether comparable expansion in vitro of effector T cells could be obtained. The results shown in Fig. 8 indicate that efficient expansion (68–148 × 106 T cells) of TILs (#15765 and #4478D) and MLTCs (#4478D, #2710 and #0342) could be achieved with an increase value of 240–592 times (Fig. 8, insert). Notably, similar expansion of T lymphocytes was observed in TILs vs MLTCs of the same patient (#4478D) (240 and 272 times number, respectively) (insert of Fig. 8). Moreover, an efficient expansion of TILs from the ocular melanoma patient #15765 was achieved (8.4 × 107 cells starting from 1.5 × 105 cells with an increase of 560 times) as well, thus indicating that the possible anergic state of T cells isolated from ocular suppressive tumor milieu can be recovered ex vivo and such lymphocytes efficiently expanded.Fig. 8


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)

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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401505&req=5

Fig8: 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
Mentions: The REP protocol was applied to both MLTC and TILs isolated from 10 cutaneous and ocular melanoma patients in order to determine whether comparable expansion in vitro of effector T cells could be obtained. The results shown in Fig. 8 indicate that efficient expansion (68–148 × 106 T cells) of TILs (#15765 and #4478D) and MLTCs (#4478D, #2710 and #0342) could be achieved with an increase value of 240–592 times (Fig. 8, insert). Notably, similar expansion of T lymphocytes was observed in TILs vs MLTCs of the same patient (#4478D) (240 and 272 times number, respectively) (insert of Fig. 8). Moreover, an efficient expansion of TILs from the ocular melanoma patient #15765 was achieved (8.4 × 107 cells starting from 1.5 × 105 cells with an increase of 560 times) as well, thus indicating that the possible anergic state of T cells isolated from ocular suppressive tumor milieu can be recovered ex vivo and such lymphocytes efficiently expanded.Fig. 8

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