Ourania E. Tsitsilonis

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-γ, TNF-α, 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.

Compromised anti‐tumor responses in tumor necrosis factor‐α knockout mice

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

VEGF directly suppresses activation of T cells from ovarian cancer patients and healthy individuals via VEGF receptor Type 2.

The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis is well characterized; nevertheless, it is also a key element in promoting tumor evasion of the immune system by downregulating dendritic cell maturation and thus T cell activation. We sought to investigate the possible direct effect of VEGF on T cell activation and through which type of VEGF receptor (VEGFR) it exerts this effect. Circulating T cells from healthy donors and ovarian cancer patients were expanded in cultures with anti‐CD3 and IL‐2 with or without VEGF for 14 days, and the number of T cells was assessed. Cultured T cells were also tested for their cytotoxic activity in a standard 4‐hr 51Cr‐release assay, and the expression of VEGFRs 1, 2 and 3 was assayed by flow cytometry, immunocytochemistry and Western blotting. To assess the ability of activated T cells to secrete VEGF, levels in culture supernatants were measured by enzyme linked immunosorbent assay. The addition of VEGF in cultures significantly reduced T cell proliferation in a dose‐dependent manner. Protein expression studies demonstrated that CD3+ T cells express VEGFR‐2 on their surface upon activation. Experiments with anti‐VEGFR‐2 antibodies showed that the direct suppressive effect of VEGF on T cell proliferation is mediated by VEGFR‐2. We also showed that VEGF significantly reduced the cytotoxic activity of T cells and that activated T cells secrete VEGF in the culture environment. Overall, our study shows that T cells secret VEGF and expresses VEGFR‐2 upon activation. VEGF directly suppresses T cell activation via VEGF receptor type 2.

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy.

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as “thymic hormones,” are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting.

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

Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα 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. ProTα(89–102) and proTα(103–109) significantly fortified healthy donor-lymphocytes’ immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89–102) and proTα(103–109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1–14), proTα(21–30) and proTα(31–87) marginally upregulated lymphocyte activation. This is the first report showing that proTα’s immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα 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.

Toll‑like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies

Toll-like receptor (TLR) agonists possess remarkable properties, particularly with regard to dendritic cell activation, promoting Th1-type cytokine production and optimizing cytotoxic T-cell responses. Preclinical and clinical studies conducted to date show that TLR agonists can improve currently applied anticancer vaccination protocols. Although these have resulted in the US FDA approval of three TLR agonists for use in humans, their abundant application encounters limitations, principally due to dose-limiting toxicity evoking from systemic cytokine production. Here, using selected examples of clinical studies, we provide a concise review regarding the knowledge acquired thus far on the adjuvant use of TLR agonists as cancer vaccine components. We also provide evidence on the exploitation of a novel TLR agonist, prothymosin-α, which enhances the efficacy of tumor-reactive effectors without causing severe adverse effects.

Prothymosin α immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a β-sheet conformation in a sequence-specific manner

Prothymosin alpha (ProTalpha) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTalphas C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTalphas immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTalpha stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTalpha forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel beta-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTalpha during apoptosis, strengthen our hypothesis that immune response stimulation by ProTalpha is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific beta-sheet conformation and induce DC maturation.

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

We have recently reported that administration of Pro Tα 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 α [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T α, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8+ cytotoxic T lymphocytes (CTL). The Pro T α-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 Pro T α developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T α and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T α-induced CD8+ CTL activity was regulated by Pro T α-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 Pro T α 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 Pro T α is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T α may prove to be useful in clinical protocols aimed at cancer immunotherapy.

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTalpha), i.e. against either non-conjugated ProTalpha (isolated from bovine thymus) or ProTalpha conjugated to keyhole limpet hemocyanin (ProTalpha/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTalpha, while antibodies Y against ProTalpha/KLH had a better apparent titer, could better discriminate between ProTalpha and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.

Chronic antidepressant treatment exerts sexually dimorphic immunomodulatory effects in an experimental model of major depression: do females lack an advantage?

Major depression is a stress-related disorder that affects about 20% of the population, with women outnumbering men by 2:1. However, research focusing on stress/antidepressant-related immunomodulation overlooks sex differences, although an established sexual dimorphism also characterizes the immune system. We report for the first time that both chronic clomipramine treatment (10 mg/kg, twice daily) and chronic mild stress (CMS) application in rats, exert sexually dimorphic effects on cellular immunoreactivity (natural killer and lymphokine-activated killer cell cytotoxicity and interleukin-2-induced T-cell proliferation), with females presenting a relatively immunosuppressed phenotype compared to males. Moreover, following chronic antidepressant treatment, thymic monoamines presented sex-related alterations, as well as intriguing associations with peripheral T-cell responses. This study highlights the sex-related effects of chronic clomipramine treatment and CMS application on the cellular arm of immunity, and represents a preliminary exposé of a thymus-dependent route pertaining to the interactions between antidepressants and the immune system.