Reto Schumacher

Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8 + T cells induced by TCR triggering or homeostatic cytokines

Production of indoleamine 2,3‐dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L‐kynurenine (L‐kyn) and 3‐hydroxyanthranilic acid (3‐HAA) on survival and antigen‐dependent and independent proliferation of CD8+ cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8+ T cells. Non toxic concentrations of L‐kyn or 3‐HAA did not significantly inhibit antigen‐specific CTL responses. However, 3‐HAA, but not L‐kyn markedly inhibited antigen‐independent proliferation of CD8+ T cells induced by common receptor γ‐chain cytokines IL‐2, ‐7 and ‐15. Our data suggest that CD8+ T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3‐HAA profoundly affects homeostatic proliferation of CD8+ T cells.

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Advanced Liposomal Vectors as Cancer Vaccines in Melanoma Immunotherapy

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.

Clinical applications of virosomes in cancer immunotherapy

Cancer immunotherapy is increasingly accepted as a treatment option for advanced stage disease. The identification of tumour-associated antigens in 1991 has prompted the development of antigen-specific immunotherapeutic strategies for a variety of cancers. Many of them result in some immunological responses in cancer patients; however, clinical results were not observed concomitantly with immunological responses; therefore, further improvements in the field of immunotherapy are urgently needed. Virosomes are lipidic envelopes devoid of genetic information, but which retain the antigenic profile and fusogenic properties from their viral origin. Virosomes are versatile antigen carriers and can be engineered to perform various tasks in cancer immunotherapy. Preclinical data have fostered the development of innovative clinical protocols. Hence, immunopotentiating reconstituted influenza virosomes will be assessed in breast and melanoma immunotherapy, and may contribute to the development of clinically effective cancer vaccines and ultimately improve patient outcomes. The objective of this review is to provide an overview of the potential clinical applications of virosomes as innovative and potentially effective reagents in active specific cancer immunotherapy.

Efficient stimulation of T cell responses by human IFN-alpha-induced dendritic cells does not require Toll-like receptor triggering.

Dendritic cells (DC) can be activated by proinflammatory cytokines or upon toll-like receptor (TLR) triggering. These stimuli induce specific patterns of phenotypic modulation and gene expression profiles. We investigated whether TLR triggering represents an indispensable requirement for the induction of T cell responses by human DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interferon-α (IFN-DC). As model stimulator we chose imidazoquinolone (3M-001), a synthetic TLR7 agonist used in the treatment of skin infections and tumors and as experimental adjuvant. At difference with DC generated upon culture of monocytes in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL-4) (IL-4–DC), IFN-DC display a semimature phenotype. Furthermore, IFN-DC, but not IL-4–DC are able to induce CD4+ and CD8+ T cell responses, in steady state, for example, in the absence of TLR triggering. 3M-001 treatment induces up-regulation of the surface expression of costimulatory molecules and “de novo” production of IL-12 and IL-6 in IFN-DC. However, TLR7 triggering fails to significantly enhance the capacity of IFN-DC to induce antigen-specific cytotoxic T lymphocytes and to stimulate allogeneic CD4+ T cells. These data indicate that TLR engagement and IL-12 production do not represent indispensable prerequisites for optimal antigen-presenting cell function in IFN-DC, qualifying these cells as powerful cellular reagents of potential use in active specific immunotherapy.

Selective responsiveness to common gamma chain cytokines in peripheral blood-derived cytotoxic T lymphocytes induced by Melan-A/MART-127–35targeted active specific immunotherapy

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan‐A/MART‐127–35 tumor associated antigen (TAA) to IL‐2, IL‐7 or IL‐15 cytokines, sharing a receptor common γ‐chain (cγ‐c cytokines). Twenty‐eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA‐A0201 and Melan‐A/MART‐1, and displayed phenotypic characteristics of effector/memory (CD45RA−/CCR7−) or CD45RA+/CCR7− effector cells in intermediate to late developmental stage (CD28−/CD276±) CTL. Proliferative responses could be elicited or enhanced by IL‐2 and IL‐15, but not IL‐7, in the absence or in the presence of T‐cell receptor (TCR) triggering, respectively. Accordingly, only IL‐2 and IL‐15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor cγ‐c (CD132), lower, but detectable, expression of IL‐7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan‐A/MART‐127–35 and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan‐A/MART‐127–35 tetramer positive cells was only observed in the presence of IL‐2 or IL‐15 but not IL‐7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL‐2 was able to promote the expansion of Melan‐A/MART‐127–35 tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA‐specific CTL to different cγ‐c cytokines.