Francesca Urbani

IFN‐α‐conditioned dendritic cells are highly efficient in inducing cross‐priming CD8+ T cells against exogenous viral antigens

Dendritic cells (DC) generated after a short‐term exposure of monocytes to IFN‐α and GM‐CSF (IFN‐DC) are highly effective in inducing cross‐priming of CD8+ T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN‐DC and IL‐4‐derived DC. Both DC types efficiently cross‐presented soluble HCV NS3 protein to the specific CD8+ T cell clone, even though IFN‐DC were superior in cross‐presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV‐1 and injected into hu‐PBL‐SCID mice, the generation of virus‐specific CD8+ T cells was markedly higher in animals immunized with IFN‐DC than in mice immunized with CD40L‐matured IL‐4‐DC. Of interest, in experiments with purified CD8+ T cells, IFN‐DC were superior with respect to CD40L‐matured IL‐4‐DC in inducing in vitro cross‐priming of HIV‐specific CD8+ T cells. This property correlated with enhanced potential to express the specific subunits of the IL‐23 and IL‐27 cytokines. These results suggest that IFN‐DC are directly licensed for an efficient CD8+ T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL‐12 family cytokines.

Cell-mediated immunity and antibody responses to Bordetella pertussis antigens in children with a history of pertussis infection and in recipients of an acellular pertussis vaccine

Cell-mediated immunity (CMI) and antibody responses to Bordetella pertussis antigens were assessed 4-6 years after primary infant immunization with diphtheria-tetanus tricomponent acellular pertussis (DTaP) or diphtheria-tetanus (DT) vaccine in a country with high endemicity of B. pertussis infection. CMI to the B. pertussis antigens (especially to the pertussis toxin [PT]) was more frequent and/or intense in DTaP than in DT recipients. No lymphoproliferation differences were found between those with and without a history of pertussis although the DT recipients produced very little interferon-gamma after antigen (particularly PT and filamentous hemagglutinin [FHA]) stimulation. In contrast, seropositivity to PT, but not to pertactin or FHA, was more frequent in DT recipients with history of pertussis than in all other subjects. Thus, years after disease or vaccination, CMI response to PT or circulating PT antibodies appears to be the main distinctive feature of pertussis-protected DTaP recipients or pertussis-affected DT recipients.

Native and Genetically Inactivated Pertussis Toxins Induce Human Dendritic Cell Maturation and Synergize with Lipopolysaccharide in Promoting T Helper Type 1 Responses

The capacity of pertussis toxin (PT) to induce maturation and functional activities of human monocyte-derived dendritic cells (DCs) was investigated. Both native PT (nPT) and genetically detoxified PT (dPT) efficiently promoted expression on DCs of CD80, CD86, human leukocyte antigen-DR, and CD83 markers, alloreactive antigen presentation, and cytokine production, primarily interferon (IFN)-gamma. Although they did not affect interleukin (IL)-10 production by lipopolysaccharide (LPS)-stimulated DCs, both nPT and dPT strongly synergized with LPS for IL-12 production. PTs plus LPS-stimulated DCs secreted soluble factors fostering IFN-gamma but not IL-4 and IL-5 production by naive T cells. T helper type 1 (Th1) polarization was, as alloreactive antigen presentation, inhibited by anti-IL-12 monoclonal antibody. These findings support the notion that nPT, in addition to inducing specific immune response, is a potent Th1 adjuvant and that dPT fully preserves this adjuvanticity. The synergic interaction between PT and LPS in IL-12 production might be relevant for the mechanisms of vaccine-induced protection.

Cell-Mediated Immune Response of Healthy Adults to Bordetella pertussis Vaccine Antigens

Cell-mediated immunity (CMI) to Bordetella pertussis and acellular pertussis vaccine constituents (pertussis toxin, pertactin, and filamentous hemagglutinin) were studied in peripheral blood mononuclear cells (PBMC) and T cell cultures from healthy adults with no record of vaccination against, or history of, pertussis. Similarly to stimulation with common recall antigens, PBMC proliferation was induced in 80%-100% of the cultures, depending on the specific B. pertussis stimulant. Proliferation did not occur when antigen-presenting cells were ablated by chemical or physical methods or with naive cord blood lymphocytes. B. pertussis antigen stimulation resulted in a preferential induction of type 1 cytokine profile, as shown by interferon-gamma and interleukin-2 (but no interleukin-4 or interleukin-5) gene transcripts and actual cytokine production by T cells. The data suggest that most healthy adults are repeatedly exposed to B. pertussis, with natural acquisition of antigen-specific CMI and a putatively protective type 1 cytokine pattern.

Differential release of an immunodominant 65 kDa mannoprotein antigen from yeast and mycelial forms of Candida albicans

The release of mannoprotein (MP) antigen from Candida albicans grown at 28 degrees C (yeast form) or 37 degrees C (mycelial form), and the ability of each released material to stimulate a cell-mediated immune (CMI) response by human lymphocytes in vitro, were studied. Overall, the mycelial cells released more MP per unit of dry mass increase and the released material was relatively enriched with MP constituents of lower molecular mass with respect to the material released from yeast cells. Moreover, the mycelial MP contained a 65 kDa component (MP65) which was the largely predominant MP recognized by a rabbit anti-mycelium antiserum. When peripheral blood mononuclear cells from normal human subjects were stimulated in vitro with graded amounts of yeast or mycelial MP, the latter was about one order of magnitude more potent than the former in inducing lymphocyte proliferation. Following MP separation by gel permeation chromatography, an appreciable CMI response was stimulated only by the MP65-containing MP fractions, and to a degree apparently related to the amount of MP65 itself. Altogether, these data confirm our previous findings about the MP65 antigen as a major target of CMI response to C. albicans, and demonstrate that this antigen is released predominantly by the mycelial cells of the fungus in vitro.

Interleukin-6 production by U373 MG, a human astrocytoma cell line: different pathways involved in substance P and lipopolysaccharide activation

Substance P (SP) and lipopolysaccharide (LPS) stimulated interleukin-6 (IL-6) gene expression, as well as IL-6 protein secretion in the human astrocytoma cell line U373 MG. Staurosporine, an inhibitor of protein kinase C (PKC), entirely blocked SP- but not LPS-induced IL-6 release. In addition, the down regulation of PKC inhibited the SP response and only marginally altered LPS activation. Differently from SP, LPS-induced IL-6 release was markedly reduced by W7, a calmodulin antagonist. Moreover, SP interacted in a synergistic manner with LPS. Thus, neural (SP) and bacterial (LPS) mediators stimulate U373 MG IL-6 release via distinct, though not antagonistic, activation pathways.

Evaluation of the effects of human leukocyte IFN-α on the immune response to the HBV vaccine in healthy unvaccinated individuals

HBV vaccine needs 3 injections over 6 months to induce immunity. Thus, the use of adjuvants capable of inducing earlier immune protection would be highly desirable. Most adjuvants may act by inducing cytokines, and among them, type I interferons (IFNs), deserve a special attention in view of the potent immunomostimulatory activity observed in mouse models and on dendritic cell functions. The aim of the present trial was to evaluate the effects of IFN-alpha administered as an adjuvant of HBV vaccine in healthy unvaccinated individuals. No significant enhancing effect on the antibody response was observed, in spite of an early and transient upregulation of costimulatory molecule expression on peripheral blood mononuclear cells, which may be suggestive of an IFN-mediated activation of antigen presenting cells. We conclude that, under the conditions used in this trial, natural IFN-alpha does not act as an adjuvant of the HBV vaccine in healthy unvaccinated individuals.

Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

Concomitant detection of IFNα signature and activated monocyte/dendritic cell precursors in the peripheral blood of IFNα-treated subjects at early times after repeated local cytokine treatments

BackgroundInterferons alpha (IFNα) are the cytokines most widely used in clinical medicine for the treatment of cancer and viral infections. Among the immunomodulatory activities possibly involved in their therapeutic efficacy, the importance of IFNα effects on dendritic cells (DC) differentiation and activation has been considered. Despite several studies exploiting microarray technology to characterize IFNα mechanisms of action, there is currently no consensus on the core signature of these cytokines in the peripheral blood of IFNα-treated individuals, as well as on the existence of blood genomic and proteomic markers of low-dose IFNα administered as a vaccine adjuvant.MethodsGene profiling analysis with microarray was performed on PBMC isolated from melanoma patients and healthy individuals 24 hours after each repeated injection of low-dose IFNα, administered as vaccine adjuvant in two separate clinical trials. At the same time points, cytofluorimetric analysis was performed on CD14+ monocytes, to detect the phenotypic modifications exerted by IFNα on antigen presenting cells precursors.ResultsAn IFNα signature was consistently observed in both clinical settings 24 hours after each repeated administration of the cytokine. The observed modulation was transient, and did not reach a steady state level refractory to further stimulations. The molecular signature observed ex vivo largely matched the one detected in CD14+ monocytes exposed in vitro to IFNα, including the induction of CXCL10 at the transcriptional and protein level. Interestingly, IFNα ex vivo signature was paralleled by an increase in the percentage and expression of costimulatory molecules by circulating CD14+/CD16+ monocytes, indicated as natural precursors of DC in response to danger signals.ConclusionsOur results provide new insights into the identification of a well defined molecular signature as biomarker of IFNα administered as immune adjuvants, and for the characterization of new molecular and cellular players, such as CXCL10 and CD14+/CD16+ cells, mediating and possibly predicting patient response to these cytokines.

CD38 Ligation in Peripheral Blood Mononuclear Cells of Myeloma Patients Induces Release of Protumorigenic IL-6 and Impaired Secretion of IFNγ Cytokines and Proliferation

CD38, a surface receptor that controls signals in immunocompetent cells, is densely expressed by cells of multiple myeloma (MM). The immune system of MM patients appears as functionally impaired, with qualitative and quantitative defects in T cell immune responses. This work answers the issue whether CD38 plays a role in the impairment of T lymphocyte response. To this aim, we analyzed the signals implemented by monoclonal antibodies (mAb) ligation in peripheral blood mononuclear cells (PBMC) obtained from MM patients and compared to benign monoclonal gammopathy of undetermined significance (MGUS). PBMC from MM both failed to proliferate and secrete IFNγ induced by CD38 ligation while it retained the ability to respond to TCR/CD3. The impaired CD38-dependent proliferative response likely reflects an arrest in the progression of cell cycle, as indicated by the reduced expression of PCNA. CD38 signaling showed an enhanced ability to induce IL-6 secretion. PBMC from MM patients displays a deregulated response possibly due to defects of CD38 activation pathways and CD38 may be functionally involved in the progression of this pathology via the secretion of high levels of IL-6 that protects neoplastic cells from apoptosis.