Eur J Cancer. 2013 May;49(7):1706-14.

Ovarian malignant ascites-derived lymphocytes stimulated with prothymosin α or its immunoactive decapeptide lyse autologous tumour cells in vitro and retard tumour growth in SCID mice.

Voutsas IF a, Pistamaltzian N b, Tsiatas ML a,b, Skopeliti M a, Katsila T a, Mavrothalassiti I a, Spyrou S c, Dimopoulos MA b, Tsitsilonis OE a, Bamias A b

a Department of Animal and Human Physiology, Faculty of Biology and b Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens; c Department of Sports Medicine, Olympic Village Polyclinic, Athens, Greece

 

ABSTRACT

BACKGROUND: Tumour-associated lymphocytes (TALs) present in effusions of ovarian cancer patients exhibit impaired activities, due to the immunosuppressive environment of the ascites. Means to enhance their cytotoxicity against autologous tumour cells are of clinical importance. The immunomodulator prothymosin alpha (proTα) increases the specific lysis of tumour cells by activated CD8+ T lymphocytes and its immunoreactivity is exerted by the carboxy-terminal decapeptide, proTα(100-109). These two molecules were studied on TALs in vitro, and in SCID mice bearing human ovarian tumours.

METHODS: TALs and tumour cells were isolated from 41 ovarian cancer patients and co-cultured in the presence of proTα or proTα(100-109). The cytotoxicity of peptide-stimulated TALs was tested against autologous tumour cells and K562. Ex vivo proTα – or proTα (100-109)-stimulated TALs from three patients were adoptively transferred intraperitoneally in SCID mice, inoculated with each patient’s autologous tumour cells.

RESULTS: ProTα and its immunoreactive peptide proTα(100-109), enhanced the cytotoxic activity of TALs against autologous tumour cells in vitro, but marginally affected the lysis of K562. The effect of proTα and proTα(100-109) was higher after 7-14 days of stimulation, whereas TAL cytotoxicity was significantly decreased after 21 days. Mice administered TALs, ex vivo activated with proTα or proTα(100-109) for 7 days, showed a relatively lower tumour increase rate and prolongation of their survival.

CONCLUSION: Our data demonstrate that, in the presence of tumour antigens, proTα and proTα (100-109) enhance the depressed cytotoxicity of TALs against autologous tumour cells in vitro and retard tumour growth in vivo.

 

SUPPLEMENT

Prothymosin alpha (proTα) is a 109 residue long acidic polypeptide with immunomodulatory activity. ProTα acts in synergy with cytokines (eg. IL-2, IFN-γ) and enhances tumour-reactive cytotoxicity of cancer patient-derived PBMC, shown to be predominantly mediated by activated CD8+ T lymphocytes [1]. The carboxy-terminal fragment of proTα, the decapeptide proTα(100-109), exerts similar immunoreactivity to the parental molecule [2]. We initially evaluated the in vitro effect of proTα and proTα(100-109) in MLTCs set up for 7-21 days, comprising TALs and autologous tumour cells from ovarian cancer patients. TALs and tumour cells were isolated from the ascites of patients with histologically confirmed epithelial ovarian adenocarcinomas and co-cultured, at a TAL:tumour cell ratio of 10:1, in the presence of proTα or proTα(100-109) in synergy with low dose IL-2. The cytotoxic activity of MLTC-derived TALs was determined using as targets autologous tumour cells and the NK-sensitive cells K562. Both proTα and its immunoreactive peptide proTα(100-109) enhanced TAL cytotoxicity in vitro. To further verify whether a similar effect can be exerted in vivo, we inoculated sc SCID mice with human ovarian tumour cells isolated from selected patients. Upon palpable tumour formation, mice were thrice adoptively transferred autologous TALs, ex vivo activated with proTα or proTα(100-109). This immunotherapeutic intervention resulted in delaying tumour increase rate and prolonging the survival of the animals.

Our results show that proTα and proTα(100-109) reverse the immunosuppression of TALs isolated from malignant ascites and induce the in vitro expansion of tumour-reactive T lymphocytes, which efficiently lyse autologous tumour cells, both in vitro and in vivo. These data, in conjunction with previous reports from us [3] and others [4] suggest that proTα and proTα(100-109) act as adjuvants and stimulate anticancer immune responses. Specifically, proTα and proTα(100-109) ligate TLRs (TLR-4 and possibly others) and induce the maturation of ascites-derived antigen-presenting cells (Fig. 1). Now immunocompetent, these cells effectively uptake cancer antigens provided by irradiated autologous tumor cells and present them to T cells. Within 7 days, in the presence of low dose IL-2, activated tumor-reactive CD8+ effectors proliferate, secrete perforin and lyse tumor cells. Adoptive transfer of proTα- and proTα(100-109)-stimulated T effectors in tumor-bearing mice prolongs their survival, suggesting that the enhanced cytotoxic activity of these effectors is also retained in vivo. We propose that incorporation of proTα or preferably, of its immunoreactive decapeptide proTα(100-109), in peptide-based immunobiotherapeutic vaccination strategies may further assist anticancer-reactive T cell expansion and activation.

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REFERENCES

[1] Voutsas IF, Baxevanis CN, Gritzapis AD, et al. Synergy between interleukin-2 and prothymosin alpha for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas. Cancer Immunol. Immunother. 2000, 49(8): 449–58.

[2] Skopeliti M, Iconomidou VA, Derhovanessian E, et al. Prothymosin alpha immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a beta-sheet conformation in a sequence-specific manner. Mol. Immunol. 2009, 46(5): 784–92.

[3] Ioannou K, Derhovannesian E, Tsakiri E, et al. Prothymosin alpha and a prothymosin alpha-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes. BMC Immunol. 2013, 14:43-56.

[4] Mosoian A, Teixeira A, Burns CS, et al. Prothymosin alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction. Proc. Natl. Acad. Sci. USA 2010, 107(22): 10178-83.

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