The immunologically active site of prothymosin alpha is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Abstract

Prothymosin alpha (proTalpha) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTalpha is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTalpha presenting maximum immunomodulatory activity. Calf thymus proTalpha was trypsinised, and the five fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTalpha(89-102) and proTalpha(103-109) significantly fortified healthy donor-lymphocytes' immune responses to levels comparable to those induced by intact proTalpha. These effects were more pronounced in cancer patients, where peptides proTalpha(89-102) and proTalpha(103-109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTalpha. ProTalpha(1-14), proTalpha(21-30) and proTalpha(31-87) marginally upregulated lymphocyte activation. This is the first report showing that proTalpha's immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTalpha fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.

Fig. 1

ProTα and its C-terminal peptides increase lymphocyte proliferation of PBMC from healthy individuals (a) and cancer patients (b) during the autologous mixed lymphocyte reaction. Cultures were stimulated with autologous mitomycin C-inactivated PBMC at a responder-to-stimulator ratio of 2:1 in the presence of intact proTα and each individual peptide as described in ‘‘Materials and methods’’. Results are presented as mean stimulation index (SI) values ± SD from pooled data from 17 and 14 healthy donors and cancer patients, respectively—nonstimulated PBMC; *P<0.05 versus (−)

Fig. 2

Immunoenhancing synergistic effect of the C-terminal proTα fragments with IL-2 to healthy donors’ (a) and cancer patients’ (b) PBMC cytotoxicity. PBMC from both groups were incubated for 3 days with IL-2 (20 IU/ml) and proTα (160 ng/ml) or each of proTα’s peptides, at doses as indicated in ‘‘Materials and Methods’’, and tested as effectors for cytotoxicity against K562 and Daudi targets. In all experiments, the effector-to-target cell ratio was 40:1. Data are presented as mean % specific cytotoxicity ± SD from 17 and 14 healthy individuals and cancer patients tested, respectively. Other details as in Fig. 1

Fig. 3

Dot plot diagrams of 3-day cultured healthy donor (a) and cancer patient (b) PBMC with IL-2 (20 IU/ml) and proTα or its tryptic fragments. PBMC were labeled with anti-CD56-FITC (CD56)- and anti-perforin-PE (Per)-specific antibodies. Percentages of double positive cells refer to the lymphocyte population gated. Data are from one representative experiment out of 3 and 2, conducted for healthy donors and cancer patients, respectively— PBMC incubated with medium; IL-2, PBMC incubated with 20 IU/ml IL-2 alone