Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα). ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100-109). In conjunction with data from others, we also revealed that proTα and proTα(100-109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100-109) are viewed as candidate adjuvants for cancer immunotherapy. Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα's mode of action, which could eventually contribute to its clinical application.

Infection risk dictates immunological divergence among populations in a Mediterranean lizard.

The ability of vertebrates to evolve different defence strategies in response to varying parasitism regimes remains poorly understood. Hosts may adopt two different strategies to defend themselves against parasites: tolerance (hosts alleviate the negative fitness consequences of parasite infection) and resistance (hosts strengthen their immune response as parasite burden increases). Both strategies are effective, but fitness has been reported to decline faster in less-tolerant individuals. Here, we assessed the number of splenocytes and the cell-mediated response (proxies for resistance) and body condition (a proxy for tolerance) in four populations of a Greek endemic lizard (Podarcis gaigeae), each exposed to different infection risks (defined as the cumulative effect of parasite burden and duration of exposure). We anticipated that populations with heavy parasite burden would enhance the efficacy of their immune response (resistance) compared to lizards deriving from parasite-poor habitats. We also predicted that populations with longer exposure to parasites would be adopted and be more tolerant. Each factor (duration of exposure and parasite burden) had a distinct effect on the immune response, and thus, our results were rather complicated. Lizards with heavy parasite burden and aperiodic exposure demonstrated resistance, whereas lizards with heavy parasite burden and chronic exposure were more tolerant. Populations with low parasite burden and minimal exposure were more resistant. Our results suggest that the development of some immunological strategies may be differentiated under different infection risks, even within the same species.

Immune changes in patients with colorectal cancer treated by adjuvant therapy with monoclonal antibody 17-1A: a pilot study.

We investigated the efficacy of Mo17-1A to improve important immunological parameters in vivo and in vitro in colorectal cancer (CRC) patients receiving no second-line treatment. Eighteen CRC patients stage Dukes' C were treated postoperatively with 5 doses of Mo17-1A. Peripheral blood mononuclear cells (PBMC) were analyzed for proliferative responses and cytolytic activity. Serum levels of cytokines were determined during treatment. Enhancement in the proliferative activity of patients' T cells, improvement in their lymphocytes' killing ability of natural killer (NK) and lymphokine-activated killer (LAK) sensitive tumor targets, and an in vivo increase in serum levels of interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) were noted. We conclude that treatment of CRC patients with Mo17-1A partially restores deficient cellular immune responses and the secretion of cytokines with immuno-enhancing properties. The development of novel immunotherapeutic protocols using combinations of Mo17-1A with stimuli that enhance the lytic capacity of effector cells (e.g. cytokines), may improve clinical responses in these types of patients.

Compromised anti-tumor responses in tumor necrosis factor-alpha knockout mice.

The response of lymphokine-activated killer (LAK) and natural killer (NK) cells from mice lacking tumor necrosis factor-alpha (TNF-alpha-/- mice) was impaired in cytotoxicity assays against various tumor cell targets. Furthermore, allogeneic cytotoxic T lymphocyte (CTL) responses were also impaired as compared to TNF-alpha+/+ littermates (control mice). Cytotoxicity was restored both upon in vitro incubation of TNF-alpha-/- lymphocytes with recombinant TNF-alpha (rTNF-alpha) or upon in vivo treatment of TNF-alpha-/- mice with rTNF-alpha. Using combinations of monoclonal antibodies we were able to show that TNF-alpha-/- effector lymphocytes exhibit both perforin- and Fas ligand-based cytotoxicity. Furthermore, upon in vivo administration of rTNF-alpha these effectors, in addition to perforin and Fas ligand, are also armed with TNF-alpha cytotoxic molecules, thus resembling to the cytotoxic effectors from control mice. In a tumor model, immunized TNF-alpha-/- mice failed to reject the syngeneic fibrosarcoma MC57X, but did so when injected with rTNF-alpha. In vivo administration of anti-TNF-alpha mAb neutralized the effect of rTNF-alpha supporting the growth of MC57X cells. Our data provide novel evidence for TNF-alpha as an essential factor in (i) controlling cytotoxicity in vitro and in vivo and (ii) promoting tumor rejection in vivo.

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor.

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.

Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin alpha.

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin alpha (ProTalpha) is a biological response modifier that exerts its effects primarily on mononuclear cells, especially when these cells' effector functions are impaired. In this study, we report that ProTalpha enhances the AAK cytotoxicity in PBMC from healthy donors. This effect was more profound with cancer patients' PBMC, which were deficient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTalpha, exhibited increased AAK activity and efficiently lysed both autologous tumor and Daudi targets. The ProTalpha effect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Moreover, effector CD8+ and CD56+ cells pretreated with anti-CD3 and ProTalpha contained high cytoplasmic perforin levels and increased expression of IL-1beta- and TNFalpha-specific receptors. The induction of autologous-tumor-reactive CD8+ and CD56+ lymphocytes in anti-CD3-activated PBMC by ProTalpha provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity.

This report demonstrates that in vitro activation of human cells with the beta-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK- and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and IL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.

The prognostic value of alpha-thymosins in breast cancer.

The prognosis of breast cancer is of major clinical importance and several histopathological, biochemical and immunological variables have been reported to be useful prognostic factors. In the present study, we investigated the clinical significance of the levels of alpha-thymosins in relation to established prognostic factors, both in breast cancer and non-malignant breast lesions, alpha-thymosin levels were measured in breast tissue extracts by specific radioimmunoassays (RIAs) developed for human prothymosin alpha (ProT alpha) and parathymosin alpha (ParaT alpha) and were found to be significantly higher (up to 17.2-fold) in malignant but not in benign breast lesions, as compared to the values of the neighbouring tissues. When alpha-thymosin levels of the tumor samples were correlated with various known prognostic parameters a statistically significant correlation (p < 0.05) was observed between the levels of ProT alpha in malignant tissues to the grade of cancer and the lymph node status of the patient. An association between ProT alpha levels with increase in risk of death from breast cancer was also noticed. These results suggest that the expression of alpha-thymosins in human breast cancer a) depends on the proliferation status of the tumor, b) associates with established prognostic factors describing the metastatic potential of the tumor and c) is related to the overall survival of the patient. The fact that such relationships hold only for cancer tissues encourages the future use of alpha-thymosins as potent prognostic factors in breast cancer.

Prothymosin alpha: efficient sequence determination by experimental and theoretical capillary electrophoretic support.

A rapid and efficient primary structure elucidation of prothymosin (ProT alpha) and its enzymatic fragments was performed by a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI), automatic N-terminal Edman degradation, and the available theoretical predictions of electrophoretic mobility in capillary electrophoresis (CE) as a basis suggesting active sites of ProT alpha in the different bioassays.

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin alpha.

We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.