The TLR agonist prothymosin α (proTα) pleiotropically stimulates immune cells via generating the appropriate cytokine milieu for their activation. The C-terminal decapeptide proTα(100-109) is considered the immunologically active moiety of proTα, as it restores in vitro the deficient immune responses of cancer patients equally well to proTα. ProTα and proTα(100-109) ligate TLR-4, signal through the TRIF/MyD88-dependent pathways, and promote the maturation of dendritic cells. The latter further stimulate TH1-type immune responses and prime tumor antigen-reactive T cell functions. We evaluated whether proTα and proTα(100-109) function correspondingly in vivo.
C57BL/6 mice were subcutaneously inoculated with the syngeneic melanoma B16.F1 cells. Upon palpable tumor formation, mice were intraperitonealy treated with 2 cycles of GM-CSF, proTα(100-109) or proTα, in conjunction with a B16.F1-specific peptide vaccine. Tumor growth and animal survival were monitored. Splenocytes from selected animals were tested for ex vivo cytotoxicity by 51Cr-release assay and CD107 expression. Excised tumors were analyzed by immunohistochemistry, while serum cytokines were quantified by flow cytometry.
Both peptides therapeutically administered in melanoma-bearing mice in the presence of cancer antigens, retarded tumor growth and prolonged the survival of treated animals by 25 days. Ex vivo analysis of spleen cell cytotoxicity confirmed the in vivo induction of B16.F1-specific and non-specific anti-tumor responses. Tumors of mice treated with proTα/proΤα(100-109) did not show infiltration of smooth muscle fibers and vessels, produced less melanin, presented limited necrotic areas and were characterized by sparse T cell infiltration. Sera from the same animals contained more IFN-γ, whereas the concentration of IL-4 was marginally increased.
Our results show that proTα and proTα(100-109) induce TH1-biased immune responses in vivo. As both peptides are non-toxic to normal cells, their ability to orchestrate and modulate the desired anti-tumor immune responses in mice, suggests their eventual exploitation as adjuvants in anti-cancer peptide vaccines in humans.
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All authors have declared no conflicts of interest.