Riccardo Gavioli

Altered miRNA expression in T regulatory cells in course of multiple sclerosis.

OBJECTIVES Multiple sclerosis (MS) is a chronic inflammatory response against constituents of the central nervous system. It is known that regulatory T cells (Tregs) play a key role in the autoimmune balance and their improper function may facilitate the expansion of autoaggressive T cell clones. Recently, microRNAs (miRNAs) have been involved in autoimmune disorders and their loss-of-function in immune cells was shown to facilitate systemic autoimmune disorders. Here, we analyzed the miRNA expression profile in Tregs from MS-RR. METHODS We assessed miRNA genome-wide expression profile by microarray analysis on CD4(+)CD25(+high) T cells from 12 MS relapsing-remitting patients in stable condition and 14 healthy controls. Since CD4(+)CD25(+high) T cells comprise both T regulatory cells (CD4(+)CD25(+high)CD127(dim/-)) and T effector cells (CD4(+)CD25(+high)CD127(+)), we performed a quantitative RT-PCR on CD4(+)CD25(+high)CD127(dim/-) and CD4(+)CD25(+high)CD127(+) cells isolated from the same blood sample. RESULTS We found 23 human miRNAs differentially expressed between CD4(+)CD25(high)bona fide Treg cells from MS patients vs. healthy donors, but, conversely, among the deregulated miRNAs, members of the miR-106b-25 were found down-regulated in MS patients when compared to healthy donors in CD4(+)CD25(high)CD127(dim/-) T regulatory cells. More interesting, the ratio between Treg/Teff showed an enrichment of these microRNA in T regulatory cells derived from patients if compared to healthy controls. CONCLUSION miR-106b and miR-25 were previously shown to modulate the TGF-β signaling pathway through their action on CDKN1A/p21 and BCL2L11/Bim. TGF-β is involved in T regulatory cells differentiation and maturation. Therefore, the deregulation of this miRNA cluster may alter Treg cells activity in course of MS, by altering TGF-β biological functions.

c- myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells

Burkitts lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein–Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

HIV-1 Tat Protein Modulates the Generation of Cytotoxic T Cell Epitopes by Modifying Proteasome Composition and Enzymatic Activity

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

Effect of interferon-α therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals

The majority of hepatitis C virus (HCV)‐infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV‐specific CTL responses directed to different HCV‐derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA‐A2‐presented, HCV‐derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon‐α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4‐derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV‐specific CTL responses and therapy‐induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3‐derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN‐α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3‐derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV‐derived epitopes with a dominant response to the NS3‐derived peptide antigen. This suggests that CTL responses directed to this NS3‐derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

The Toll-like receptor ligand MALP-2 stimulates dendritic cell maturation and modulates proteasome composition and activity

A 2‐kDa synthetic derivative of the macrophage‐activating lipopeptide (MALP‐2) from Mycoplasma fermentans is a potent inducer of monocytes/macrophages and improves the immunogenicity of antigens co‐administered by systemic and mucosal routes. Dendritic cells (DC) are the most potent antigen‐presenting cells, which are able to prime naive T cells in vivo. To elucidate the underlying mechanisms of MALP‐2 adjuvanticity, we analyzed its activity on bone marrow‐derived murine DC. In vitro stimulation of immature murine DC with MALP‐2 resulted in the induction of maturation with up‐regulated expression of MHC class II, costimulatory (CD80, CD86) and adhesion (CD40, CD54) molecules. MALP‐2 also enhances the secretion of cytokines (IL‐1α, IL‐6 and IL‐12), and increases DC stimulatory activity on naive and antigen‐specific T cells. Further studies demonstrated that MALP‐2 treatment of DC results in a dose‐dependent shift from the protein pattern of proteasomes to immunoproteasomes (up‐regulation of LMP2, LMP7 and MECL1), which correlates with an increased proteolytic activity. Thus, the adjuvanticity of MALP‐2 can be mediated, at least in part, by the stimulation of DC maturation, which in turn leads to an improved antigen presentation. Therefore, MALP‐2 is a promising molecule for the development of immune therapeutic or prophylactic interventions.

α,β-Unsaturated Carbonyl System of Chalcone-Based Derivatives is Responsible for Broad Inhibition of Proteasomal Activity and Preferential Killing of Human Papilloma Virus (HPV)-Positive Cervical Cancer Cells

Proteasome inhibitors have potential for the treatment of cervical cancer. We describe the synthesis and biological characterization of a new series of 1,3-diphenylpropen-1-one (chalcone) based derivatives lacking the boronic acid moieties of the previously reported chalcone-based proteasome inhibitor 3,5-bis(4-boronic acid benzylidene)-1-methylpiperidin-4-one and bearing a variety of amino acid substitutions on the amino group of the 4-piperidone. Our lead compound 2 (RA-1) inhibits proteasomal activity and has improved dose-dependent antiproliferative and proapoptotic properties in cervical cancer cells containing human papillomavirus. Further, it induces synergistic killing of cervical cancer cell lines when tested in combination with an FDA approved proteasome inhibitor. Exploration of the potential mechanism of proteasomal inhibition by our lead compound using in silico docking studies suggests that the carbonyl group of its oxopiperidine moiety is susceptible to nucleophilic attack by the γ-hydroxythreonine side chain within the catalytic sites of the proteasome.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV‐8) is a human γ2‐herpesvirus that is implicated in the development of Kaposis sarcoma (KS), primary effusion lymphoma and Castelmans disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV‐8 viral antigens. In this study, using peptide‐binding motifs, we selected potential human leucocyte antigen (HLA)‐A2‐binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV‐8 antigens, respectively. HLA‐A2‐binding peptides were tested for their capacity to induce CTL responses in HHV‐8‐negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV‐8‐derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease‐free individuals infected by HHV‐8 demonstrating that the two epitopes are relevant targets of CTL‐mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV‐8‐associated malignancies.

Recent advances in the development of HIV-1 Tat-based vaccines.

Over the last two decades most of the efforts in HIV vaccine development have been based on the use of the HIV Env with the goal to induce sterilizing immunity. However, as a result of Env variability disappointing results have been obtained in preclinical and phase III clinical trials. Although the objective of a preventive immunity still remains a priority, secondary endpoints (e.g. block of virus replication and disease onset) are being considered at the present as more achievable end-points in HIV vaccine development. This is based on accumulating evidence indicating that low viral load correlates with maintenance of immune functions and slow progression to disease, and that cell-mediated immunity plays a major protective role in the absence of sterilizing immunity. The promising results obtained in non-human primates with a vaccine based on a native Tat protein (B-clade), which is an early regulatory protein key for HIV replication and AIDS pathogenesis, highlights the importance of targeting the virus very early after infection. In particular, the immune response against Tat appears to modify the virus-host interactions at the very beginning of infection, thus containing the depletion of critical immune cells and the progression of infection. Moreover, since Tat targets and induces maturation of dendritic cells, has immunomodulatory activities and drives Th-1 and CTL responses, immunization with Tat may drive or increase these immune responses also against other HIV antigens to support an effective, long-lasting and hopefully even sterilizing antiviral immunity. Finally, Tat B-clade is similarly recognized by sera from individuals infected by different virus clades (A, B, C, D) supporting the concept of a cross-clade vaccine. Therefore, the Tat-vaccine should contain virus replication protecting from disease progression (non-sterilizing immunity) or even favoring an abortive infection. Although only a phase III clinical trial will establish the efficacy of this vaccine strategy, the Tat-vaccine has recently entered preventive and therapeutic phase I clinical testing in Italy to establish safety (primary-end-point) and immunogenicity (secondary end-point) and phase II studies are being prepared.

Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1

The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)-GAS (gamma-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1-IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.

Induction of humoral and enhanced cellular immune responses by novel core-shell nanosphere- and microsphere-based vaccine formulations following systemic and mucosal administration

Anionic surfactant-free polymeric core-shell nanospheres and microspheres were previously described with an inner core constituted by poly(methylmethacrylate) (PMMA) and a highly hydrophilic outer shell composed of a hydrosoluble co-polymer (Eudragit L100-55). The outer shell is tightly linked to the core and bears carboxylic groups capable of adsorbing high amounts (antigen loading ability of up to 20%, w/w) of native basic proteins, mainly by electrostatic interactions, while preserving their activity. In the present study we have evaluated in mice the safety and immunogenicity of new vaccine formulations composed of these nano- and microspheres and the HIV-1 Tat protein. Vaccines were administered by different routes, including intramuscular, subcutaneous or intranasal and the results were compared to immunization with Tat alone or with Tat delivered with the alum adjuvant. The data demonstrate that the nano- and microspheres/Tat formulations are safe and induce robust and long-lasting cellular and humoral responses in mice after systemic and/or mucosal immunization. These delivery systems may have great potential for novel Tat protein-based vaccines against HIV-1 and hold promise for other protein-based vaccines.