Eleonora Gallerani

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.

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.

The HIV-1 Tat Protein Induces the Activation of CD8+ T Cells and Affects In Vivo the Magnitude and Kinetics of Antiviral Responses

T cells are functionally compromised during HIV infection despite their increased activation and proliferation. Although T cell hyperactivation is one of the best predictive markers for disease progression, its causes are poorly understood. Anti-tat natural immunity as well as anti-tat antibodies induced by Tat immunization protect from progression to AIDS and reverse signs of immune activation in HIV-infected patients suggesting a role of Tat in T cell dysfunctionality. The Tat protein of HIV-1 is known to induce, in vitro, the activation of CD4+ T lymphocytes, but its role on CD8+ T cells and how these effects modulate, in vivo, the immune response to pathogens are not known. To characterize the role of Tat in T cell hyperactivation and dysfunction, we examined the effect of Tat on CD8+ T cell responses and antiviral immunity in different ex vivo and in vivo models of antigenic stimulation, including HSV infection. We demonstrate for the first time that the presence of Tat during priming of CD8+ T cells favors the activation of antigen-specific CTLs. Effector CD8+ T cells generated in the presence of Tat undergo an enhanced and prolonged expansion that turns to a partial dysfunctionality at the peak of the response, and worsens HSV acute infection. Moreover, Tat favors the development of effector memory CD8+ T cells and a transient loss of B cells, two hallmarks of the chronic immune activation observed in HIV-infected patients. Our data provide evidence that Tat affects CD8+ T cell responses to co-pathogens and suggest that Tat may contribute to the CD8+ T cell hyperactivation observed in HIV-infected individuals.

HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors

Objective:HIV infection is characterized by several immune dysfunctions of both CD8+ and CD4+ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8+ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4+ T cells; however, its effects on CD8+ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8+ T-cell functionality and programming. Methods:CD8+ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Results:Tat favors the secretion of interleukin-2, interferon-&ggr; and granzyme B in CD8+ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8+ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Conclusion:Tat deeply alters the programming and functionality of CD8+ T lymphocytes.

Proteasome inhibitors induce the presentation of an Epstein–Barr virus nuclear antigen 1‐derived cytotoxic T lymphocyte epitope in Burkitt’s lymphoma cells

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV‐associated tumours and is therefore an interesting target for immunotherapy. However, evidence for the recognition and elimination of EBV‐transformed and Burkitt’s lymphoma (BL) cells by cytotoxic T lymphocytes (CTLs) specific for endogenously presented EBNA1‐derived epitopes remains elusive. We confirm here that CTLs specific for the HLA‐B35/B53‐presented EBNA1‐derived HPVGEADYFEY (HPV) epitope are detectable in the majority of HLA‐B35 individuals, and recognize EBV‐transformed B lymphocytes, thereby demonstrating that the GAr domain does not fully inhibit the class I presentation of the HPV epitope. In contrast, BL cells are not recognized by HPV‐specific CTLs, suggesting that other mechanisms contribute to providing a full protection from EBNA1‐specific CTL‐mediated lysis. One of the major differences between BL cells and lymphoplastoid cell lines (LCLs) is the proteasome; indeed, proteasomes from BL cells demonstrate far lower chymotryptic and tryptic‐like activities compared with proteasomes from LCLs. Hence, inefficient proteasomal processing is likely to be the main reason for the poor presentation of this epitope in BL cells. Interestingly, we show that treatments with proteasome inhibitors partially restore the capacity of BL cells to present the HPV epitope. This indicates that proteasomes from BL cells, although less efficient in degrading reference substrates than proteasomes from LCLs, are able to destroy the HPV epitope, which can, however, be generated and presented after partial inhibition of the proteasome. These findings suggest the use of proteasome inhibitors, alone or in combination with other drugs, as a strategy for the treatment of EBNA1‐carrying tumours.

Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice

BackgroundAs HIV-specific cytotoxic T cells play a key role during acute and chronic HIV-1 infection in humans, the ability of potential anti-HIV vaccines to elicit strong, broad T cell responses is likely to be crucial. The HIV-1 Gag antigen is widely considered a relevant antigen for the development of an anti-HIV vaccine since it is one of the most conserved viral proteins and is also known to induce T cell responses. In the majority of studies reporting Gag-specific cellular immune responses induced by Gag-based vaccines, only a small number of Gag T cell epitopes were tested in preclinical mouse models, thus giving an incomplete picture of the numerous possible cellular immune responses against this antigen. As is, this partial knowledge of epitope-specific T cell responses directed to Gag will unavoidably result in a limited preclinical evaluation of Gag-based vaccines.ResultsIn this study we identified new Gag CD8+ T cell epitopes in BALB/c mice vaccinated with the HIV-1 Gag antigen alone or in combination with the HIV-1 Tat protein, which was recently shown to broaden T cell responses directed to Gag. Specifically, we found that CTL responses to Gag may be directed to nine different CTL epitopes, and four of these were mapped as minimal CTL epitopes.ConclusionThese newly identified CTL epitopes should be considered in the preclinical evaluation of T cell responses induced by Gag-based vaccines in mice.

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.

The HIV-1 Tat protein affects human CD4+ T cell programming and activation, and favors the differentiation of naïve CD4+ T cells

Objective: HIV infection is characterized by several immune dysfunctions, such as chronic activation of the immune system, premature aging and loss of CD4+ T cells, in particular within the naïve compartment. The Tat protein of HIV is released extracellularly and enters neighboring cells affecting their functionality, for instance impacting on CD8+ T-cell programs and activity. As the presence and/or induction of anti-Tat immune responses is associated with reduced T-cell dysfunction and CD4+ T-cell loss, we investigated whether Tat impacts human resting or activated CD4+ T cells. Methods: Purified CD4+ T cells were activated by T cell receptor engagement in the presence or absence of Tat. Cytokine production, surface phenotype and expression of transcription factors important for T-cell programing were measured. Purified naïve CD4+ T cells were cultured in nonpolarizing conditions in the presence or absence of Tat and their proliferation and differentiation was evaluated. Results: Tat favors the secretion of IL2, IFN&ggr; and TNF&agr; in CD4+ T cells, as well as the upregulation of T-bet and Eomes expression. Naïve CD4+ T cells cultured in the presence of Tat showed enhanced expansion and differentiation toward memory phenotype, showing in particular recruitment into the effector memory T-cell pool. Conclusion: Tat affects the programing and functionality of CD4+ T lymphocytes favoring the differentiation of naïve CD4+ T cells.

Studies of C-terminal naphthoquinone dipeptides as 20S proteasome inhibitors

Abstract The ubiquitin proteasome pathway is crucial in regulating many processes in the cell. Modulation of proteasome activities has emerged as a powerful strategy for potential therapies against much important pathologies. In particular, specific inhibitors may represent a useful tool for the treatment of tumors. Here, we report studies of a new series of peptide-based analogues bearing a naphthoquinone pharmacophoric unit at the C-terminal position. Some derivatives showed inhibition in the µM range of the post-acidic-like and chymotrypsin-like active sites of the proteasome.

Systemic immunodominant CD8 responses with an effector-like phenotype are induced by intravaginal immunization with attenuated HSV vectors expressing HIV Tat and mediate protection against HSV infection

Mucosal HSV infection remains a public health issue in developing and developed world. However, an effective vaccine is still missing, partly because of the incomplete knowledge of correlates of protection. In this study we have investigated the kinetics and quality of immunity elicited by an attenuated HSV1 vector expressing the immunomodulatory Tat protein of HIV-1 (HSV1-Tat). Animals were immunized by intravaginal (IVag) or intradermal (ID) route with HSV1-Tat or with a control HSV1 vector expressing the LacZ gene (HSV1-LacZ) and immune responses were characterized in different anatomical districts. IVag immunization with HSV1-Tat enhanced both expansion and memory phases of HSV-specific immunodominant CD8 responses at systemic, but not local, level and induced short- and long-term protection against mucosal challenge. Conversely, ID immunization with HSV1-Tat favored HSV-subdominant CD8 responses, which protected mice only at early time points after immunization. IVag immunization, in particular with HSV1-Tat, compared to ID immunization, induced the differentiation of CD8(+) T lymphocytes into short-lived effector (SLEC) and effector memory (Tem) cells, generating more robust recall responses associated with increased control of virus replication. Notably, systemic SLEC and Tem contributed to generate protective local secondary responses, demonstrating their importance for mucosal control of HSV. Finally, IgG responses were observed mostly in IVag HSV1-Tat immunized animals, although seemed dispensable for protection, which occurred even in few IgG negative mice. Thus, HSV1 vectors expressing Tat induce protective anti-HSV1 immune responses.