Ioannis F. Voutsas

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.

HER-2/neu as a target for cancer vaccines

A novel modality toward the treatment of HER-2/neu-positive malignancies, mostly including breast and, more recently prostate carcinomas, has been the use of vaccines targeting HER-2/neu extracellular and intracellular domains. HER-2/neu-specific vaccines have been demonstrated to generate durable T-cell anti-HER-2/neu immunity when tested in Phase I and II clinical trials with no significant toxicity or autoimmunity directed against normal tissues. Targeting of HER-2/neu in active immunotherapy may involve peptide and DNA vaccines. Moreover, active anti-HER-2/neu immunization could facilitate the ex vivo expansion of HER-2/neu-specific T cells for use in adoptive immunotherapy for the treatment of established metastatic disease. In addition, early data from trials examining the potential use of HER-2/neu-based vaccines in the adjuvant setting to prevent the relapse of breast cancer in high-risk patients have shown promising results. Future approaches include multiepitope preventive vaccines and combinatorial treatments for generating the most efficient protective anti-tumor immunity.

Induction of potent CD4+ T cell-mediated antitumor responses by a helper HER-2/neu peptide linked to the Ii-Key moiety of the invariant chain.

The Ii‐Key fragment from the MHC class II‐associated invariant chain (or Ii protein) has been shown to facilitate direct charging of MHC class II epitopes to the peptide binding groove. The purpose of the present study was to test the potential of a series of Ii‐Key/HER‐2/neu(776–790) hybrid peptides to generate increased frequencies of peptide‐specific CD4+ T cells over the native peptide in mice transgenic (Tg) for a chimeric human mouse class II molecule (DR4‐IE) (H‐2b) as well as their antitumor potency. Following in vivo priming, such hybrid peptides induced increased proliferation and frequencies of IFN‐γ producing CD4+ T cells in response to either syngeneic dendritic cells pulsed with native peptide, or HLA‐DR4+ human tumor cell lines expressing HER‐2/neu. Hybrid peptides were more stable in an off‐rate kinetics assay compared to the native peptide. In addition, antigen‐specific CD4+ T cells from hybrid peptide immunized DR4‐IE Tg mice synergized with HER‐2/neu(435–443)‐specific CD8+ T cells from HLA‐A2.1 Tg HHD (H‐2b) mice in producing antitumor immunity into SCID mice xenografted with the HER‐2/neu+, HLA‐A2.1+ and HLA‐DR4+ FM3 human melanoma cell line. High proportions of these adoptively transferred HER‐2/neu peptide‐specific CD4+ and CD8+ T cells infiltrated FM3‐induced tumors (tumor infiltrating lymphocytes; TIL) in SCID mice. CD8+ TIL exhibited long‐lasting antitumor activity when cotransferred with CD4+ TIL, inducing regression of FM3 tumors in a group of untreated, tumor‐bearing SCID mice, following adoptive transfer. Our data show that Ii‐Key modified HER‐2/neu(776–790) hybrid peptides are sufficiently potent to provide antigen‐specific CD4+ TH cells with therapeutic antitumor activity.

Ii-Key/HER-2/neu(776-90) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ tumors

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-90) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-γ-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-43) CTL epitope, as illustrated by either IFN-γ ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-43) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-90) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-90) hybrid peptides.

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 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.

Peptide Vaccination Breaks Tolerance to HER-2/neu by Generating Vaccine-Specific FasL+ CD4+ T Cells: First Evidence for Intratumor Apoptotic Regulatory T Cells

BALB/c mice transgenic (Tg) for the transforming rat neu oncogene (BALB-neuT) are genetically predestined to develop mammary carcinogenesis in a process similar to that in humans. We crossed HLA-A2.1/HLA-DR1 (A2.1/DR1) Tg mice with BALB-neuT mice to generate A2.1/DR1 x BALB-neuT triple Tg (A2.1/DR1 x neuT(+)) mice, which represent an improvement over BALB-neuT mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. A vaccine formulation strategy, consisting of synthetic peptides from the rat HER-2/neu oncogene combined with granulocyte macrophage colony-stimulating factor, was highly effective in preventing the growth of established transplantable tumors in male A2.1/DR1 x neuT(+) mice. Vaccination with HER-2(435-443) (p435) CTL peptide alone induced weak antitumor responses, which were characterized by increased numbers of regulatory T cells (Treg) and low numbers of vaccine-specific CD8(+) CTL and helper T cells (Th). The administration of p435 plus HER-2(776-790) (p776; helper peptide) reversed this situation, inducing functionally active, peptide-specific CTL and Th. There was a striking change in the intratumoral balance of Tregs (decrease) and vaccine-specific Th (increase) that directly correlated with tumor rejection. Intratumoral administration of anti-FasL antibody promoted tumor growth. The decrease in Tregs (Fas(+)) was due to apoptosis induced by cell contact with Fas ligand(+) (L)(+) Th. Mice vaccinated with p435 plus p776 exhibited long-lasting antitumor immunity. Our vaccine regimen also significantly delayed the outgrowth of mammary carcinomas in female A2.1/DR1 x neuT(+) animals. We provide a mechanism to overcome tolerance against HER-2/neu, which proposes a combined vaccination with two (Th and CTL) HER-2 peptides against HER-2/neu-expressing tumors.

New use for old drugs? Prospective targets of chloroquines in cancer therapy.

During the last decade research is gradually repositioning the antimalarial drug chloroquine, and certain related quinoline derivatives, as anticancer agents. Chloroquine and hydroxychloroquine, in particular, have relatively well-characterized toxicity profiles due to several decades of use for treatment of malaria. Previously published review articles provide an excellent overview of the diversity of chloroquine effects on cancer cells, both in the cell culture as well as on human tumors grafted into mice; and an account of the increasing pace of incorporation of hydroxychloroquine in combination treatment schemes for clinical studies. In this review we present some features that are common between cancers that are sensitive to quinoline derivatives, in particular features that are amenable to pharmaceutical intervention.