Elisabetta Padovan

IFN-α2a induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived dendritic cells and enhances their capacity to attract and stimulate CD8+ effector T cells

Type I IFNs are immunomodulatory factors that possibly influence the properties of tissue‐resident dendritic cells. Here, we have investigated the capacity of IFN‐α2a to enhance DC chemoattractive and stimulatory capacity toward CD8+ T lymphocytes. Phenotypically, IFN‐α2a‐treated DC (IFN‐DC) showed an increased expression of costimulatory and antigen‐presenting molecules, maintained even after withdrawal of the cytokine. IFN‐α2a enhanced DC stimulatory capacity toward CD8+ T cells, as assessed by increased MLR responses and induction of MART‐126–35‐specific CTLs in vitro. No functional CCR7 chemokine receptor could be induced. Instead, high amounts of IP‐10/CXCL10 and MIG/CXCL9 chemokines were produced. Freshly isolated CD8+RO+ cells and PHA‐activated CD8+ T cells migrated efficiently in response to IFN‐DC‐conditioned medium, and the migration could be inhibited by neutralizing the CXCR3 receptor on responder cells. These results suggest that type I IFNs could enhance the elicitation of class I‐restricted effector functions in vivo in the periphery by modulating DC chemoattractive properties.

Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

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.

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG‐1 and RAG‐2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re‐express RAG‐1 and RAG‐2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Dual receptor T-cells. Implications for alloreactivity and autoimmunity.

Using monoclonal antibodies to human V alpha, we have estimated that up to one-third of mature T-cells express two V alpha chains as part of two functional and independent T-cell receptors. Cells belonging to this dual TCR subset may be specific for a broader range of antigens than cells with a single receptor. We discuss the possibility that dual receptor T-cells may be involved in alloreactivity and autoimmunity.

The ester-bonded palmitoyl side chains of Pam3CysSerLys4 lipopeptide account for its powerful adjuvanticity to HLA class I-restricted CD8+ T lymphocytes

Molecularly defined adjuvants are urgently required to implement immunization protocols by which CD8+ T cells induction is envisaged. We show here that the synthetic lipopeptide Pam3CysSerLys4 (P3CSK4) strongly enhances the expansion of antigen‐specific IFN‐γ+CD8+ cells in vitro. These effects critically depend on the presence of two ester‐bonded palmitoylated side chains. In fact, T cell expansion is impaired in the presence of derivatives bearing two non‐palmitoylated fatty acid chains, while derivatives with only one amide‐bonded palmitoylated residue are completely inactive and behave like the non‐lipidated peptide backbone. P3CSK4 is not mitogenic for T lymphocytes and can modulte DC immune biological properties. Indeed, doses as low as 100 ng/ml increase CD86, CD83 and CD40 surface expression on DC, fail to induce CCR7, and trigger a defined pattern of soluble factors associated to immune effector functions. In particular, substantial amounts of TNF‐α, IL‐6, CCL2 and CXCL10, in the absence of IFN‐α, IFN‐γ, IL‐15, IL‐12p70 and CX3CL1, can be measured. Accordingly, antigen‐specific CD8+ T cells expanded in vitro express CCR2 and CXCR3 chemokine receptors. Altogether our data suggest that human DC are able to respond to chemically different synthetic lipopeptide analogs and that optimal adjuvanticity to CD8+ T cell induction is achieved by the palmitoylated structures.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer

Background The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. Objective To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. Methods IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. Results IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. Conclusions Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)–targeting signal and the HLA-A201 binding 27–35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27–35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus–encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-γ release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201–restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation. Cancer Gene Therapy (2001) 8, 655–661.

Anti-Inflammatory/Tissue Repair Macrophages Enhance the Cartilage-Forming Capacity of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Macrophages are key players in healing processes. However, little is known on their capacity to modulate the differentiation potential of mesenchymal stem/stromal cells (MSC). Here we investigated whether macrophages (Mf) with, respectively, pro‐inflammatory and tissue‐remodeling traits differentially modulate chondrogenesis of bone marrow derived‐MSC (BM‐MSC). We demonstrated that coculture in collagen scaffolds of BM‐MSC with Mf derived from monocytes polarized with M‐CSF (M‐Mf), but not with GM‐CSF (GM‐Mf) resulted in significantly higher glycosaminoglycan (GAG) content than what would be expected from an equal number of BM‐MSC alone (defined as chondro‐induction). Moreover, type II collagen was expressed at significantly higher levels in BM‐MSC/M‐Mf as compared to BM‐MSC/GM‐Mf constructs, while type X collagen expression was unaffected. In order to understand the possible cellular mechanism accounting for chondro‐induction, developing monoculture and coculture tissues were digested and the properties of the isolated BM‐MSC analysed. We observed that as compared to monocultures, in coculture with M‐Mf, BM‐MSC decreased less markedly in number and exhibited higher clonogenic and chondrogenic capacity. Despite their chondro‐inductive effect in vitro, M‐Mf did not modulate the cartilage tissue maturation in subcutaneous pockets of nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. Our results demonstrate that coculture of BM‐MSC with M‐Mf results in synergistic cartilage tissue formation in vitro. Such effect seems to result from the survival of BM‐MSC with high chondrogenic capacity. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of cartilage repair. J. Cell. Physiol. 230: 1258–1269, 2015.