Daniele Accapezzato

Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo

The presentation of exogenous protein antigens in a major histocompatibility complex class I–restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination.

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.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus‐specific effector CD8+ T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV‐specific CCR7+/CD8+ T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7– cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi‐effectors). However, we report strong evidence in support of IL‐2 being capable of pushing semi‐effector CTL to complete their effector cell program. Therefore, IL‐2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL‐2 to rebuild the effector T cell pool in these patients.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: the role of the virus

In a companion study, we showed a dichotomy between the expansion of central memory (CCR7+) hepatitis C virus (HCV)‐specific CTL and the incomplete memory effector differentiation in patients with acute HCV infection. Indeed, effector cells were unable to perform immediate functions, despite expressing the tissue‐homing phenotype of effector memory cells (CCR7–; semi‐effectors). However, since they promptly differentiated into full‐effectors upon IL‐2 contact, we suggested that the inhibitory effect by environmental (possibly viral) factors on IL‐2 production may have a pivotal role in generating the large population of semi‐effector CCR7–/IFN‐γ– CTL. In accord with this view, we report here strong evidence in support of circulating HCVcore protein (HCVcore) playing a central role in inhibiting effector CTL differentiation, but not memory CTL expansion. The regulatory HCVcore effect is related to inhibition of the signal transduction pathway instrumental for IL‐2 production, supporting the evidence that IL‐2 was capable both of pushing semi‐effector CTL to complete their effector cell program and of restoring the HCVcore‐dependent inhibitory effect. Therefore, the strength of CTL activation is dependent on the balance between the threshold of stimulatory signals and the viral interference capacities provided during priming.

Integrin β2-chain (CD18) over-expression on CD4+ T cells and monocytes after ischemia/reperfusion in patients undergoing primary percutaneous revascularization

β2-integrin subunit (CD18) plays an essential role in leukocyte recruitment and adhesion in sites of endothelial injury. We analyzed the surface expression of CD18 on T lymphocytes and monocytes in a series of patients presenting acute coronary syndrome (ACS) who underwent primary percutaneous intervention (PCI) for coronary artery revascularization. We found that basal CD18 expression on peripheral blood-derived CD4+ (but not CD8+) T lymphocytes was significantly increased in ACS patients as compared with age-matched healthy volunteers. During primary PCI, a significant increase in CD18 molecule density was detected immediately after balloon deflation (reperfusion) on both CD4+ T cells and monocytes obtained from the right atrium (RT) as compared with basal values. These data suggest that upregulation of CD18 molecules plays an important role in local recruitment of CD4+ T cells and monocytes to the site of endothelial damage after ischemia/reperfusion, therefore being responsible, at least in part, for the inflammatory-mediated complications associated with primary PCI.

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8+ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8+ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8+ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8+ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection.

Hepatitis C virus infection and autoimmune diseases

Hepatitis C virus (HCV) infection is associated with a number of extrahepatic disorders. The most studied conditions associated with HCV are type II mixed cryoglobulinemia and B cell lymphoma. However, many reports suggest that HCV might also be associated with a number of autoimmune disorders, both organ-specific and not organ-specific. Although concomitant treatment of HCV infection is a confounding factor when ascertaining the actual role of HCV in inducing autoimmune disease, a considerable amount of experimental data indicates that HCV is able to subvert the immune system and consequently induce autoimmunity. In the present review, we report a series of observations which associate chronic HCV infection with the onset of autoimmune disorders.

Discovery of chemotherapy-associated ovarian cancer antigens by interrogating memory T cells

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8+ and CD4+ T‐cell responses against tumor cells. To discover chemotherapy‐associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two‐dimensional electrophoresis gel‐eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass‐spectrometry‐based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy‐induced apoptosis as an adjuvant of anti‐tumor immunity. The strength of both memory CD4+ and CD8+ T cells producing either IFN‐γ or IL‐17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T‐cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.

Mechanisms Inducing or Controlling CD8+ T Cell Responses against Self- or Non-Self-Antigens

Abstract: Cytotoxic T lymphocytes (CTLs) generally recognize antigens en‐dogenously synthesized within the cells and presented in the form of peptides on class I molecules. However, a large body of evidence suggests that dendritic cells (DCs) have the capacity to capture and deliver exogenous antigens into the major histocompatibility complex (MHC) class I processing pathway. In this paper, we discuss this function, defined as cross‐presentation, and how it is directed, particularly in inducing T cell tolerance, and how it requires special activating signals (such as CD40 ligand) to transform into a mechanism that provides either protective immunity or autoimmunity.

Hepatitis C flare due to superinfection by genotype 4 in an HCV genotype 1b chronic carrier

We report here on a patient affected by chronic hepatitis C who developed acute hepatitis C virus (HCV) superinfection with replacement of genotype 1b by genotype 4. The history revealed no risk factors for a new exposure to HCV, with the exception of colonoscopy with mucosal biopsy performed about 3 months before. This report underlines the absence of an effective immune-mediated cross-protection against different HCV genotypes. Moreover, the possible relationship between HCV infection and colonoscopy points out the importance of strict adherence to international guidelines for disinfection and cleaning of invasive diagnostic tools for all subjects examined, including HCV chronic carriers.