Marino Paroli

PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV

CD4+CD25+Foxp3+ Tregs suppress autoimmune responses. In addition, they limit T cell responses during chronic infection, thereby minimizing T cell-dependent immunopathology. We sought to investigate how Tregs are regulated in the livers of patients chronically infected with HCV, where they control the balance between an adequate protective immune response and suppression of immunopathology. We found that, despite accumulating and proliferating at sites of infection in the livers of patients chronically infected with HCV, Tregs were relatively less expanded than CD4+CD25+Foxp3- effector T cells. The relative lower expansion of intrahepatic Tregs coincided with their upregulation of programmed death-1 (PD-1). PD-1 expression inversely correlated with both Treg proliferation and clinical markers of immune suppression in vivo. Consistent with the possibility that PD-1 controls Tregs, blockade of the interaction between PD-1 and programmed death-1 ligand 1 (PD-L1) enhanced the in vitro expansion and function of Tregs isolated from the livers of patients chronically infected with HCV. Blockade of the interaction between PD-L1 and B7.1 also improved the proliferation of these cells. Interestingly, both PD-1 and phosphorylated STAT-5 were overexpressed in intrahepatic Tregs in a parallel fashion in steady disease conditions, and in an alternate-fluctuating fashion during the course of severe hepatitis reactivation. Notably, PD-L1 blockade upregulated STAT-5 phosphorylation in Tregs ex vivo. These data suggest that PD-L1 negatively regulates Tregs at sites of chronic inflammation by controlling STAT-5 phosphorylation.

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.

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1–infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Primary biliary cirrhosis induced by interferon-α therapy for hepatitis C virus infection

SummaryInterferon-α is known to exacerbate and in some cases induce a variety of autoimmune disorders. In this report we describe the onset of primary biliary cirrhosis in a 55-year-old woman without evidence of preexisting autoimmune diseases receiving recombinant interferon-α2a for chronic active hepatitis C. Shortly after discontinuating interferon therapy, alkaline phosphatase levels started to rise up to three times the normal range. Anti-mithocondrial antibodies were found to be positive at a high titer, and liver biopsy showed a picture of chronic active hepatitis along with primary biliary cirrhosis features (overlap syndrome). Primary biliary cirrhosis should be considered in the differential diagnosis in any patient treated with interferon-α with unexplained elevation of serum alkaline phosphatase.

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.

Immunoglobulin a nephropathy complicating pulmonary tuberculosis

A 31-year-old man who presented with smear- and culture-negative pulmonary tuberculosis had associated macroscopic hematuria, elevation of serum creatinine and immunoglobulin A (IgA) levels, overt proteinuria, and peripheral edema. Renal biopsy revealed focal mesangial proliferation with IgA deposits, and a diagnosis of IgA nephropathy was made. The patient received treatment with isoniazide and rifampin. After 4 months, pulmonary lesions were almost completely healed, and a significant improvement of creatinine clearance with normalization of serum creatinine and IgA levels and disappearance of proteinuria were observed. Treatment with isoniazide and rifampin was discontinued after 6 months, without reappearance of either pulmonary or renal symptoms. Two years after the diagnosis of IgA nephropathy, the patient is in good general condition. Serum creatinine and IgA levels are normal, proteinuria is absent, and there is neither macrohematuria nor microhematuria. These findings suggest that IgA nephropathy may be a consequence of tuberculosis, possibly due to an abnormal IgA-mediated immune response against Mycobacterium tuberculosis with formation of nephrotoxic immune complexes.

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.