Federica Bozzano

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM‐1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment‐naïve patients who underwent treatment with pegylated IFN‐α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56brightCD16+/− cell populations, increased CD56dullCD16+ NK cell proportions, and lower expression of NKp30, DNAM‐1, and CD85j. Similarly, reduced NK cell IFN‐γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56brightCD16+/− NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL‐2 and IFN‐α confirmed upregulation of NKp30 and also of NKp46 and DNAM‐1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV‐1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.

Natural killer cells in HIV controller patients express an activated effector phenotype and do not up-regulate NKp44 on IL-2 stimulation

Control of HIV replication in elite controller (EC) and long-term nonprogressor (LTNP) patients has been associated with efficient CD8+cytotoxic T-lymphocyte function. However, innate immunity may play a role in HIV control. We studied the expression of natural cytotoxicity receptors (NKp46, NKp30, and NKp44) and their induction over a short time frame (2–4 d) on activation of natural killer (NK) cells in 31 HIV controller patients (15 ECs, 16 LTNPs). In EC/LTNP, induction of NKp46 expression was normal but short (2 d), and NKp30 was induced to lower levels vs. healthy donors. Notably, in antiretroviral-treated aviremic progressor patients (TAPPs), no induction of NKp46 or NKp30 expression occurred. More importantly, EC/LTNP failed to induce expression of NKp44, a receptor efficiently induced in activated NK cells in TAPPs. The specific lack of NKp44 expression resulted in sharply decreased capability of killing target cells by NKp44, whereas TAPPs had conserved NKp44-mediated lysis. Importantly, conserved NK cell responses, accompanied by a selective defect in the NKp44-activating pathway, may result in lack of killing of uninfected CD4+NKp44Ligand+ cells when induced by HIVgp41 peptide-S3, representing a relevant mechanism of CD4+ depletion. In addition, peripheral NK cells from EC/LTNP had increased NKG2D expression, significant HLA-DR up-regulation, and a mature (NKG2A−CD57+killer cell Ig-like receptor+CD85j+) phenotype, with cytolytic function also against immature dendritic cells. Thus, NK cells in EC/LTNP can maintain substantially unchanged functional capabilities, whereas the lack of NKp44 induction may be related to CD4 maintenance, representing a hallmark of these patients.

Functionally relevant decreases in activatory receptor expression on NK cells are associated with pulmonary tuberculosis in vivo and persist after successful treatment.

Correlates for the initiation of Mycobacterium tuberculosis hominis (Mth) replication from latency are needed in order to improve Mth control. In order to analyze if perturbations of peripheral NK cells may be associated with exit from Mth latency, sequential patients with newly diagnosed lung tuberculosis (TB) were studied. Peripheral NK cells were analyzed by cytofluorometry, in vitro culture and functional assays. At the onset of lung TB, imbalances in NK cell subsets were evident. Decreased CD56(bright)CD16(+/-) subsets with significantly compromised NKp30 and NKp46 expression and with specifically decreased gamma-IFN production upon triggering were evident. These features were not completely restored when purified NK cells were cultured in vitro. Culture supplementation with alpha-IFN increased only NKp30 expression in TB and healthy donors. Extensive peripheral NK cell triggering was evident in these patients, as shown by the expression of NK cell activation markers and of the lymph node-homing chemokine receptor CCR7 on CD16(+) CD56(dull) cells. Significant persistence of decreased NKp30 and NKp46 after successful treatment with a standard four-drug regimen was detected after full recovery. NK cell function is deeply affected in patients at the onset of pulmonary TB. The involvement of multiple activatory receptors may provide a relevant contribution to the spread of mycobacteria exiting from latency.

Successfully treated HIV-infected patients have differential expression of NK cell receptors (NKp46 and NKp30) according to AIDS status at presentation.

Differences in innate immune responses may be associated with different capabilities of controlling HIV infection, not necessarily reflected by CD4(+) T-cell counts alone. We investigated by cytofluorometry the expression of NK cell receptors and ligands in 19 treated HIV-infected patients with CD4(+)<220 ml(-1) at presentation (11 AIDS, 8 non-AIDS) and 10 healthy donors. Expression of NKp46 and NKp30 was significantly higher in non-AIDS vs. AIDS patients. Overall, the level of NKp46 expression directly correlated with the degree of NK cell cytotoxicity. As compared to healthy donors, in both groups, there was a similar increase of CD69 and HLA-DR expression in NK cells that directly correlated with the presence of activation markers (HLA-DR) on CD4(+) and CD8(+) T cells. As compared to AIDS, in non-AIDS patients in vitro activated CD4(+) showed higher expression of MIC-A (NKG2D ligand), with significantly higher Nectin-2/DNAM-1 and MIC-A/NKG2D ratios. Thus, NK cell responses in AIDS and non-AIDS patients with similar CD4(+) counts significantly differ despite similar treatment. This suggests an involvement of innate mechanisms, in preventing AIDS-defining opportunistic infections in HIV infection and further suggests, that CD4(+) absolute counts alone, may be inadequate to explain differences in the clinical outcome.

Baseline and Dynamic Expression of Activating NK Cell Receptors in the Control of Chronic Viral Infections: The Paradigm of HIV-1 and HCV

Natural killer (NK) cell function is regulated by a balance between the triggering of activating and inhibitory receptors expressed on their surface. A relevant effort has been focused so far on the study of KIR carriage/expression setting the basis for NK cell education and self-tolerance. Focus on the evolution and regulation of activating NK receptors has lagged behind so far. Our understanding of activating receptor expression and regulation has recently improved by evidences derived from in vitro and in vivo studies. Virus infection – either acute or chronic – determines preferential expansion of NK cells with specific phenotype, activating receptors, and with recall-like functional activity. Studies on patients with viral infection (HIV and HCV) and specific diverging clinical courses confirm that inter-individual differences may exist in baseline expression of natural cytotoxicity receptors (NKp46 and NKp30). The findings that patients with divergent clinical courses have different kinetics of activating receptor density expression upon NK cell activation in vitro provide an additional, time-dependent, functional parameter. Kinetic changes in receptor expression thus represent an additional parameter to basal receptor density expression. Different expression and inducibilities of activating receptors on NK cells contribute to the high diversity of NK cell populations and may help our understanding of the inter-individual differences in innate responses that underlie divergent disease courses.

The ligurian human immunodeficiency virus clinical network: a web tool to manage patients with human immunodeficiency virus in primary care and multicenter clinical trials.

Background In recent years, Highly-Active Anti-Retroviral Therapies (HAARTs) have modified the Human Immunodeficiency Virus (HIV) life-cycle and the disease is now considered chronic. Consequently, a longitudinal and complex follow-up is now required for HIV positive patients during their lifetime. Moreover, patients often encounter various complications due to comorbidities, related to the immunodeficiency state and HAARTs’ side effects. Thus, HIV positive patients are involved in multicenter clinical trials (MCTs) to improve treatments and discover a preventive vaccine. Therefore, physicians require proper instruments to access comprehensive patient data for managing patients during follow-ups, and tools for data collection and analysis in MCTs. Objective The Ligurian HIV Clinical Network aims to provide physicians with a Web-tool to administrate HIV positive patients’ data within primary-care and to reuse the collected clinical information to perform MCTs in Northern Italy. Methods The key aspect of the system is a relational database which allows the storage of various types of clinical information (eg, related to HIV, cardiovascular, or hepatic diseases) in multiple formats. The modular design of the database permits a rapid insertion of new parameters without requiring any changes in the database structure. Furthermore, codes from biomedical ontologies controlled vocabularies (“Logical Observation Identifier Names and Codes”, and “International Classification of Diseases 9”) and ontologies (“Systematized Nomenclature of Medicine Clinical Terms”), units and normality ranges used by all partners participating in the project were collected to achieve a complete semantic interoperability. Accordingly, data can be automatically normalized through the z score formula and physicians can extract and correctly compare information with external statistical tools. Moreover, to respect patients’ privacy and legal issues, a local identifier, determined through an HASH cryptography algorithm, is assigned to each patient during the registration process. The database is managed by a user-friendly Web-platform which allows quick access to information during medical examinations and the reusing of the collected data for present and future MCTs. Furthermore, a bidirectional middleware was created in order to import/export information through HL7 messaging. Hence, data can be manually entered by physicians or automatically collected within HL7-compliant Hospital Information systems. Results Presently, the direct storage of patients’ information from the San Paolo Hospital (Savona, Italy), and San Martino and Galliera hospitals in Genoa is in a test phase. Currently, 8 centers of Infectious Diseases (located in Liguria and Piedmont) are participating in the project and almost 400 HIV positive patients have been recorded in the system. Patient data has been used for primary care and research purposes. Currently, there are 4 on-going MCTs and preliminary results have already been presented at International HIV congresses. Conclusions The Web-platform allows effective management, sharing and reuse of information within primary care and clinical research. In the future it is planned to share the clinical information from this network with other HL7-compliant workgroups and to extend the platform to other infective diseases (eg, hepatitis).

NK-cell phenotype at interruption underlies widely divergent duration of CD4+-guided antiretroviral treatment interruption.

Long-term side effects may represent a relevant burden of antiretroviral treatment (ART) in HIV-infected patients with good CD4 immune reconstitution over extended time spans. CD4-guided treatment interruption (TI) has been evaluated to address this point and may result in a wide spectrum of time off ART in different patient cohorts. We studied whether differences in innate immune responses, in particular NK cells, are associated to patterns of longer (LoTI) or a shorter (ShTI) TI. Clinical cohort parameters were analyzed on a group of patients widely diverging for TI duration (<9 versus >18 months) on samples before TI, including NK-cell analysis and function by natural cytotoxicity receptor (NCR)-triggered γ-IFN production. Although persistently reduced NCR expression (NKp30) and function were observed in both LoTI and ShTI patients on ART compared with healthy donors, relevant differences were observed at baseline TI in those patients who subsequently developed LoTI course. Lower expression of NKG2D and NKp46 on NK cells. This also translates in reduced γ-IFN production in redirected functional assays. Thus, differences in innate immune balance exist during ART, may be associated to differential control of HIV infection and their understanding could explain clinical differences in individual patients that are not reflected by CD4(+) cell counts alone.

HTLV-1/-2 and HIV-1 co-infections: retroviral interference on host immune status

The human retroviruses HIV-1 and HTLV-1/HTLV-2 share similar routes of transmission but cause significantly different diseases. In this review we have outlined the immune mediated mechanisms by which HTLVs affect HIV-1 disease in co-infected hosts. During co-infection with HIV-1, HTLV-2 modulates the cellular microenvironment favoring its own viability and inhibiting HIV-1 progression. This is achieved when the HTLV-2 proviral load is higher than that of HIV-1, and thanks to the ability of HTLV-2 to: (i) up-regulate viral suppressive CCL3L1 chemokine expression; (ii) overcome HIV-1 capacity to activate the JAK/STAT pathway; (iii) reduce the activation of T and NK cells; (iv) modulate the host miRNA profiles. These alterations of immune functions have been mainly attributed to the effects of the HTLV-2 regulatory protein Tax and suggest that HTLV-2 exerts a protective role against HIV-1 infection. Contrary to HIV-1/HTLV-2, the effect of HIV-1/HTLV-1 co-infection on immunological and pathological conditions is still controversial. There is evidence that indicates a worsening of HIV-1 infection, while other evidence does not show clinically relevant effects in HIV-positive people. Possible differences on innate immune mechanisms and a particularly impact on NK cells are becoming evident. The differences between the two HIV-1/HTLV-1 and HIV-1/HTLV-2 co-infections are highlighted and further discussed.

Natural killer cells in hepatitis C virus infection

Hepatitis C virus (HCV) infection induces the long-term risk of liver cirrhosis or hepatocellular carcinoma and in adults represents the most common cause of liver transplantation. Natural killer (NK) cells participate in innate immune responses with efficient direct antitumor and antiviral defense. Over the years, their complex interaction with downstream adaptive responses and with the regulation of immune responses has been increasingly recognized. Considerable advances have been made particularly in understanding the role of NK cells in the pathophysiology of HCV infection and their possible use as biological markers for clinical purposes. This review summarizes the available data on the role of NK cells in the natural history of HCV infection and their role in the outcome of treatment. The main objective of this review is to summarize recent advancements in the basic understanding of NK cell function highlighting their possible translational use in clinical practice. An integrated practical view on the possible use of currently available predictive immunogenetic and NK cell functional tests is provided, to support clinical management choices for optimal treatment of patients with both standard and new drug regimens.

IFN‐α‐mediated increase in cytolytic activity of maturing NK cell upon exposure to HSV‐infected myelomonocytes

Impaired control of chronic pathogen replication may be associated to alterations of NK‐cell function. Whether mechanisms underlying this dysfunction involve perturbations of differentiating NK cells is still unknown. We studied an “in vitro” model of differentiation from CD34+Lin− precursors growing only myelomonocytes and maturing NK cells and where myelomonocytes could be suitably infected with HSV, HIV, or vaccinia. Cultures were evaluated by cytofluorometry and cytotoxicity assays for perturbations in differentiating NK cells. Increased expression of natural cytotoxicity receptors on maturing NK cells with increased cytolytic activity was observed with HSV‐1 infection, and with vaccinia while no modulation of NK‐cell phenotype nor cytotoxic activity were evident with an ssRNA lentivirus (HIV‐1). In the presence of constant IL‐12 and IL‐15 concentrations, the observed effect did not require cell contact, involved IFN‐αand was not reproduced by the addition of TLR9 agonist, nor blocked by TLR9 antagonists. Virus replication at sites of NK‐cell precursor development may have different outcomes depending on the interaction between invading viruses and maturing NK cells. Thus, NK‐cell precursors may be involved in the immune response to dsDNA viruses and possibly contribute to efficient control of virus infection.