Stefania Mazza

Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients.

Hepatitis C virus (HCV) readily establishes high‐level lifelong persistent infection in the majority of immunocompetent adults with failure of HCV‐specific CD8+ CTL to clear viral replication. Virus‐induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus‐specific CD8+ CTL responses. Here, we analyzed whether triggering of NK cell receptor expression and function is affected during chronic viremic HCV infection. Flow cytometric analysis of purified resting peripheral NK cells showed no evidence of NK cell activation, while analysis of natural cytotoxicity receptors (NCR) showed that NK cells from HCV‐infected patients had selective increased expression of NKp30 and NKp46. NK cells had corresponding conserved cytotoxic activity against all targets with the exception of HepG2 hepatoma cells. Freshly separated NK cells from HCV patients showed significant production of IL‐10 and normal concentrations of IFN‐γ upon cell‐mediated triggering. Thus, increased expression of NKp30 during HCV infection with increased IL‐10 production could contribute, once NK cells localize in the liver, to a NK‐DC crosstalk leading to skewing of subsequent adaptive immune responses and lack of virus control.

Highly stable oligomerization forms of HIV‐1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV‐1 LTR

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV‐1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N‐terminal, C‐terminal and conformation‐dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV‐LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV‐1 LTR promoter.

The MHC class II transactivator: prey and hunter in infectious diseases

The MHC class II transcriptional activator (CIITA) is the major regulator of expression of MHC class II genes. Thus, CIITA plays a fundamental role in the regulation of the immune response. Here, we discuss our findings on the dual role of CIITA during infections, as the target (prey) for certain pathogens but the host effector (hunter) against other pathogens, including HIV-1. This dual role is placed in an evolutionary context as a rather peculiar example of a strategy used by pathogens to evade host defenses and a counteraction of the host to minimize the survival and spread of the pathogen.

The HLA class II transcriptional activator blocks the function of HIV-1 Tat and inhibits viral replication

The expression of HLA class II genes is under the control of a transcriptional activator, CIITA, encoded by the AIR‐1 locus. Here we show that CIITA inhibits HIV‐1 LTR transactivationmediated by Tat. The inhibition occurred when CIITA and Tat were transiently expressed in cells after transfection and, most importantly, when tat cDNA was transfected in cells expressing CIITA in a constitutive fashion and at physiological levels. Furthermore, CIITA inhibited the HIV‐1 LTR transactivation mediated by extracellular Tat protein. CIITA inhibition of Tat function could be reversed by overexpression of Cyclin T1, the cellular cofactor used by Tat to facilitate elongation of viral transcripts. CIITA inhibition of Tat function had a dramatic effect on HIV‐1 productive infection of human T cells because CIITA+ T cells supported very poorly, if any, viral replication. These results indicate that sustained expression of CIITA in HIV‐1‐susceptible targets may down‐regulate viral expression both in cells actively replicating the virus and in silently infected cells requiring exogenous Tat to reactivate virus from latency.

Differential NKp30 inducibility in chimpanzee NK cells and conserved NK cell phenotype and function in long-term HIV-1-infected animals

HIV-1 infection in chimpanzees, the closest human relative, rarely leads to disease progression. NK cells contribute to the shaping of adaptive immune responses in humans and show perturbed phenotype and function during HIV-1 infection. In this study, we provide full phenotypic, molecular, and functional characterization for triggering molecules (NKp46, NKp30 NKp80, and NKG2D) on Pan troglodytes NK cells. We demonstrate that, in this AIDS-resistant species, relevant differences to human NK cells involve NKp80 and particularly NKp30, which is primarily involved in NK-dendritic cell interactions. Resting peripheral chimpanzee NK cells have low or absent NKp30 molecule expression due to posttranscriptional regulation and increase its levels upon in vitro activation. Following long-standing HIV-1 infection, peripheral NK cells in chimpanzees have conserved triggering receptor expression and display moderate phenotypic and functional decreases only once activated and cultured in vitro. These data suggest that one of the keys to successful lentivirus control may reside in part in a different regulation of NK cell-triggering receptor expression.

CD8+ NK cells are predominant in chimpanzees, characterized by high NCR expression and cytokine production, and preserved in chronic HIV-1 infection

HIV‐1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an “anergic” CD56− CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56bright and CD56dim subset but, as shown here, could be subdivided into functionally different CD8+ and CD8− subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN‐γ, TNF‐α and CD107 than their CD8− counterparts. In addition, chimpanzee CD8− NK cells had relatively high levels of HLA‐DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up‐regulated in CD8+ but not in CD8− NK cells and were functionally capable of inhibiting NKp30‐triggered killing. In contrast to HIV‐1‐infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.

Identification of immunodominant epitopes in inactivated Tat-vaccinated healthy and HIV-1-infected volunteers.

We analyzed the epitopes and the molecular forms of Tat recognized by the antibodies raised by Tat-toxoid vaccination in both healthy and HIV-infected volunteers. Tat-toxoid-vaccinated healthy volunteer sera reacted predominantly with peptides covering amino acids 1 through 24 and 46 through 60, corresponding to the N-terminus and basic domains of Tat. In contrast, whereas all sera from vaccinated HIV-1-positive patients reacted with the N-terminus and (with a single exception) with the basic domain, most of these sera also recognized peptides encompassing distinct domains of Tat, particularly the C-terminus (79-86). The sera of vaccinated individuals recognized both monomeric and oligomeric forms of Tat 1 through 86 or of Tat 1 through 101 and also blocked the ability of cell-released extracellular Tat to transactivate the HIV-1 LTR promoter. Synthetic Tat preincubated with sera from vaccinated individuals lost its functional activity as well. This is probably because of its inability to enter the cells as a result of immune complex formation with anti-Tat IgG. These data demonstrate that Tat-toxoid vaccination induces an efficient antibody response blocking the functional activity of Tat.