Vittorio Francavilla

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.

Virus-specific CD8+ T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection

The present study demonstrates that the quality of the virus‐specific CD8+ T cell responses, as detected by both enzyme‐linked immunospot assay and specific MHC‐peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus‐infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8+ T cells producing IFN‐γ (Tc1), but inversely to the frequencies of those producing both IL‐4 and IL‐10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer‐positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver‐infiltrating lymphocytes indicated that they produced both IFN‐γ and IL‐4 with an evident bias towards the Tc1‐like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long‐lasting low‐level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40–CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L−, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

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.

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.

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.

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.

Effect of Coronary Percutaneous Revascularization on Interferon-γ and Interleukin-10 Producing CD4+ T Cells during Acute Myocardial Infarction:

T lymphocytes play an important role in the induction and progression of acute coronary syndromes (ACS). To gain insight into how different T cell subsets can influence ACS, we analyzed the frequencies of circulating CD4+T cells producing either pro-inflammatory interferon(IFN)-γ or anti-inflammatory interleukin (IL)-10 in subjects presenting with ST-elevation myocardial infarction (STEMI). The effect of coronary bare metal (BS) and paclitaxel-eluting stent (PES) on the balance between CD4+IFN-γ+ and CD4+IL-10+ lymphocytes was also investigated. Peripheral blood mononuclear cells (PBMC) were isolated from 38 consecutive patients with STEMI before and 48 hrs or 6 days after implantation of either BS or PES. Twenty patients with no history of coronary artery disease were included as basal controls. PBMC were stimulated in vitro with anti-CD3/anti-CD28 monoclonal antibodies, and CD4+IFN-γ+ or CD4+IL-10+ T cells were detected by flow cytometry intracellular staining. The frequency of peripheral CD4+IL-10+ T cells was significantly higher in STEMI patients as compared with controls. Conversely, the frequency of CD4+IFN-γ+ T lymphocytes did not differ between STEMI and subjects without history of coronary artery disease. Six days after the revascularization procedure, the percentage of CD4+IL-10+ T cells was significantly decreased in BS but not in the PES group, whereas the relative percentage of CD4+IFN-γ+ T lymphocytes were diminished in both groups as compared with baseline levels. Our data indicate that STEMI is associated with a peripheral expansion of CD4+IL-10+T lymphocytes, and that primary coronary revascularization with implantation of either BS or PES is followed by a reduction in circulating CD4+IFN-γ+ T lymphocytes. PES implantation, however, appears to inhibit the relative decrease of the IL-10 producing lymphocyte as observed in BS implanted patients, shifting the balance between pro-inflammatory and anti-inflammatory T cell populations in favor of the latter.