Showing papers by "Chiara Castelli published in 2002"

Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles.

Abstract: The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going?

Abstract: Many human tumor-associated antigens (TAAs) have recently been identified and molecularly characterized. When bound to major histocompatibility complex molecules, TAA peptides are recognized by T cells. Clinical studies have therefore been initiated to assess the therapeutic potential of active immunization or vaccination with TAA peptides in patients with metastatic cancer. So far, only a limited number of TAA peptides, mostly those recognized by CD8 + T cells in melanoma patients, have been clinically tested. In some clinical trials, partial or complete tumor regression was observed in approximately 10%–30% of patients. No serious side effects have been reported. The clinical responses, however, were often not associated with a detectable T-cellspecific antitumor immune response when patients’ T cells were evaluated in ex vivo assays. In this review, we analyze the available human TAA peptides, the potential immunogenicity (i.e., the ability to trigger a tumor-specific T-cell response) of TAA peptides in vitro and ex vivo, and the potential to construct slightly modified forms of TAA peptides that have increased T-cell stimulatory activity. We discuss the available data from clinical trials of TAA peptide-based vaccination (including those that used dendritic cells to present TAA peptides), identify possible reasons for the limited clinical efficacy of these vaccines, and suggest ways to improve the clinical outcome of TAA peptide-based vaccination for cancer patients. [J Natl Cancer Inst 2002;94:805–18] Tumor cells express antigens that can be recognized by the host’s immune system. These tumor-associated antigens (TAAs) can be injected into cancer patients in an attempt to induce a systemic immune response that may result in the destruction of the cancer growing in different body tissues. This procedure is defined as active immunotherapy or vaccination inasmuch as the host’s immune system is either activated de novo or restimulated to mount an effective, tumor-specific immune reaction that may ultimately lead to tumor regression. However, until now, the vaccination approach for cancer has been carried out in the presence of the disease (i.e., in immunocompromised subjects) and not, as it occurs in prophylactic vaccination against infectious diseases, in healthy individuals. Moreover, although in infectious disease vaccination, the antibody response is of major importance, in anticancer vaccination, the focus is on the induction of T-lymphocyte responses. In fact, a considerable body of data from animal models and with human cells in vitro indicates that T cells are the major factor for the immunologic control of

Immunity to cancer: attack and escape in T lymphocyte–tumor cell interaction

Abstract: Tumor cells may express antigens which are recognized in a form of HLA/peptide complexes by T cells. The frequency at which different antigens are seen by T cells of melanoma patients and healthy donors was evaluated by human leukocyte antigen (HLA)/peptide tetramer technology which stains T cells bearing the specific receptor for a given epitope. By this technique, it was found that the majority of metastatic melanoma patients can recognize differentiation antigens (particularly Melan-A/MART-1), whereas such a recognition is scanty in the early phase of the disease and in healthy subjects. Despite the presence of melanoma-specific T cells infiltrating tumor lesions, tumor rejection rarely occurs. Among the different mechanisms of such inefficient antitumor response, this review discusses the possible anti-T-cell counterattack mediated by FasL-positive tumor cells, and shows that FasL is located in the cytoplasm of melanoma cells and is transported in the tumor microenvironment through the release of melanosomes. Additionally, mechanisms of suboptimal T cell activation through tumor cell expression of peptide analogs with antagonist activity are described, together with the possibility of overcoming such anergy induction by the usage of optimized tumor epitopes. Down-modulation of HLA expression by target tumor cells and its multiple mechanisms is also considered. Finally, we discuss the role of inducible nitric oxide synthases in determining the inhibition of apoptosis in melanoma cells, which can make such tumor cells resistant to the T-cell attack.

Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1.

Abstract: Monitoring of CD8+ T-cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T-cell responses in 3 melanoma patients who were immunized with peptides derived from Melan-A/MART-1. Recombinant HLA-A2 tetramers loaded with the naturally presented Melan-A/MART-1 nonamer peptide (AAGIGILTV) and the Melan-A/MART-1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T-cell subsets including naive T cells, effector T cells, "true memory" T cells and "memory effector" T cells, based on CD45RA/RO and CCR7-expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7-) T-cell subset. Molecular and functional analysis of tetramer-binding T cells revealed that the T-cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV-reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV-reactive T cells prior to vaccination. The TCR repertoire reactive with Melan-A/MART-1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer-binding T cells from 2/3 patients secreted granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma but not interleukin-2 (IL-2) in response to Melan-A/MART-1 peptides. In the third patient, tetramer-binding T cells secreted IL-2 exclusively. Our results show that T-cell responses to peptide vaccination consist of different T-cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T-cell population induced by peptide vaccination.

T-cell response to unique and shared antigens and vaccination of cancer patients

Abstract: Most vaccination studies of cancer patients find no clear association between clinical and immunological responses to the vaccine. We discuss the possible kinetics of the T cell response in melanoma patients against unique or shared tumor antigens. We hypothesize that a response against unique antigens prevails during primary melanoma growth, causing the selection of tumor cells lacking most of these antigens unless these are necessary to maintain the neoplastic state. After a subset of tumor cells metastasize to the lymph nodes, T cells are activated against previously ignored shared, differentiation-like antigens, owing to a new environment where pro-inflammatory cytokines can be present. The development of a T cell response to such normal epitopes then associates with tumor growth, but remains clinically inefficient. We predict that two immunologically different subsets of melanoma patients may exist, one that mounted an early immune response against melanoma antigens and one that did not. A paradox may emerge when vaccination is attempted in these two groups of subjects, with the second group being more prone to develop an effective immune response if the vaccine is potent enough to activate naive T cells, while the first has probably already eliminated most of the tumor antigens potentially recognizable by the host T cells owing to the previous selection made by the immune response developed early during tumor growth. Thus, it is likely that the subgroup of metastatic patients with a high frequency of anti-melanoma memory T cells may not show a clinical response to vaccination.

Improved detection of melanoma antigen-specific T cells expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide analogue.

Abstract: MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen-specific CD8(+) T-cell responses We demonstrate here that binding of HLA-A2 tetramers to CD8(+) T cells specific for the melanoma-associated antigen Melan-A/MART-1 can be fine-tuned by altering either the bound peptide epitope or residues in the alpha 3 domain of HLA-A2, which is important for CD8 binding Antigen-specific T cells expressing high levels of CD8 could be detected using HLA-A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan-A/MART-1 In contrast, low CD8-expressing, antigen-specific T cells could be detected efficiently only by using a mutated HLA-A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild-type HLA-A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen-specific T-cell responses Our results suggest ways to optimize the identification and expansion of antigen-specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T-cell receptor binding to peptide variants

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes.

Abstract: Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/ζ-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8 + T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanine→valine aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR ζ-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/ζ-chain regulation and cytokine release, including IL-2, IFN-γ, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.

The T-cell response in patients with cancer

Abstract: Publisher Summary The chapter examines several methods to measure human T-cell responses, including ELISPOT analysis, intracellular cytokine staining of immune cells after antigenic stimulation, limiting dilution analysis, conventional cloning, and molecular definition of the T-cell response either in the peripheral circulation or in situ in patients with cancer The chapter presents the cellular immune response in patients with cancer. The chapter explores the recent studies that suggest humoral immunity and T-cell-mediated immunity are closely linked. In addition, most of the data concerning antitumor immune responses have been generated using MHC class I tetramer reagents. The ultimate goal of immunomonitoring in patients with cancer is the association of the biomarkers with the clinical outcome or with response to therapy. The enumeration of MHC-restricted and antigen-specific T cells in patients with cancer allows to address the question of whether the number of antigen-experienced T cells are associated with the disease, or alternatively, with antigen-specific vaccination. The ultimate goal would be that either the number of antigen-specific T cells or the quality of T cells that is, defined by cytokine release, or by homing markers be associated with a good or bad prognosis in the course of the natural disease or predict response to vaccination.