The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway, and we propose one potential mechanism of action.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Human immunodeficiency virus-1 inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses.

Dendritic cells (DCs) in mucosal surfaces are early targets for human immunodeficiency virus-1 (HIV-1). DCs mount rapid and robust immune responses upon pathogen encounter. However, immune response in the early events of HIV-1 transmission appears limited, suggesting that HIV-1 evade early immune control by DCs. We report that HIV-1 induces a rapid shutdown of autophagy and immunoamphisomes in DCs. HIV-1 envelope activated the mammalian target of rapamycin pathway in DCs, leading to autophagy exhaustion. HIV-1-induced inhibition of autophagy in DC increased cell-associated HIV-1 and transfer of HIV-1 infection to CD4(+) T cells. HIV-1-mediated downregulation of autophagy in DCs impaired innate and adaptive immune responses. Immunoamphisomes in DCs engulf incoming pathogens and appear to amplify pathogen degradation as well as Toll-like receptor responses and antigen presentation. The findings that HIV-1 downregulates autophagy and impedes immune functions of DCs represent a pathogenesis mechanism that can be pharmacologically countered with therapeutic and prophylactic implications.

Triggering of TLR-3, -4, NOD2, and DC-SIGN reduces viral replication and increases T-cell activation capacity of HIV-infected human dendritic cells.

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T-cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C-type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV-specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand-driven triggering of TLR-3, -4, NOD2, and DC-SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen-presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.

HIV-Infected Dendritic Cells Present Endogenous MHC Class II-Restricted Antigens to HIV-Specific CD4+ T Cells.

It is widely assumed that CD4(+) T cells recognize antigenic peptides (epitopes) derived solely from incoming, exogenous, viral particles or proteins. However, alternative sources of MHC class II (MHC-II)-restricted Ags have been described, in particular epitopes derived from newly synthesized proteins (so-called endogenous). In this study, we show that HIV-infected dendritic cells (DC) present MHC-II-restricted endogenous viral Ags to HIV-specific (HS) CD4(+) T cells. This endogenous pathway functions independently of the exogenous route for HIV Ag presentation and offers a distinct possibility for the immune system to activate HS CD4(+) T cells. We examined the implication of autophagy, which plays a crucial role in endogenous viral Ag presentation and thymic selection of CD4(+) T cells, in HIV endogenous presentation. We show that infected DC do not use autophagy to process MHC-II-restricted HIV Ags. This is unlikely to correspond to a viral escape from autophagic degradation, as infecting DC with Nef- or Env-deficient HIV strains did not impact HS T cell activation. However, we demonstrate that, in DC, specific targeting of HIV Ags to autophagosomes using a microtubule-associated protein L chain 3 (LC3) fusion protein effectively enhances and broadens HS CD4(+) T cell responses, thus favoring an endogenous MHC-II-restricted presentation. In summary, in DC, multiple endogenous presentation pathways lead to the activation of HS CD4(+) T cell responses. These findings will help in designing novel strategies to activate HS CD4(+) T cells that are required for CTL activation/maintenance and B cell maturation.

Nonhuman TRIM5 Variants Enhance Recognition of HIV-1-Infected Cells by CD8+ T Cells.

UNLABELLED: Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8(+) T cells. We illustrate how TRIM5 restriction improves CD8(+) T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)-CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1ß expression in HIV-1-specific CD8(+) T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8(+) T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8(+) T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8(+) T-cell activation to increase species barriers against retrovirus infection. IMPORTANCE: New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), are attractive candidates owing to their potency in sensing HIV-1 and blocking its activity. Here, we show that expression of RhT5 and TCyp in HIV-1-infected cells improves CD8(+) T-cell-mediated inhibition through the direct activation of HIV-1-specific CD8(+) T-cell responses. We found that the potency in CD8(+) activation was stronger for RhT5 variants and capsid-specific CD8(+) T cells in a mechanism that relies on TRIM5-dependent particle recruitment to cellular proteasomes. This novel mechanism couples innate viral sensing with cellular immunity in a single protein and could be exploited to develop innovative therapeutics for control of HIV-1 infection.

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells.

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.

Langerhans Cell-Dendritic Cell Cross-Talk via Langerin and Hyaluronic Acid Mediates Antigen Transfer and Cross-Presentation of HIV-1.

Human epidermal and mucosal Langerhans cells (LCs) express the C-type lectin receptor langerin that functions as a pattern recognition receptor. LCs are among the first immune cells to interact with HIV-1 during sexual transmission. In this study, we demonstrate that langerin not only functions as a pattern recognition receptor but also as an adhesion receptor mediating clustering between LCs and dendritic cells (DCs). Langerin recognized hyaluronic acid on DCs and removal of these carbohydrate structures partially abrogated LC-DC clustering. Because LCs did not cross-present HIV-1-derived Ags to CD8(+) T cells in a cross-presentation model, we investigated whether LCs were able to transfer Ags to DCs. LC-DC clustering led to maturation of DCs and facilitated Ag transfer of HIV-1 to DCs, which subsequently induced activation of CD8(+) cells. The rapid transfer of Ags to DCs, in contrast to productive infection of LCs, suggests that this might be an important mechanism for induction of anti-HIV-1 CD8(+) T cells. Induction of the enzyme hyaluronidase-2 by DC maturation allowed degradation of hyaluronic acid and abrogated LC-DC interactions. Thus, we have identified an important function of langerin in mediating LC-DC clustering, which allows Ag transfer to induce CTL responses to HIV-1. Furthermore, we showed this interaction is mediated by hyaluronidase-2 upregulation after DC maturation. These data underscore the importance of LCs and DCs in orchestrating adaptive immunity to HIV-1. Novel strategies might be developed to harness this mechanism for vaccination.

B Cells Loaded with Synthetic Particulate Antigens: A Versatile Platform To Generate Antigen-Specific Helper T Cells for Cell Therapy.

Adoptive cell therapy represents a promising approach for several chronic diseases. This study describes an innovative strategy for biofunctionalization of nanoparticles, allowing the generation of synthetic particulate antigens (SPAg). SPAg activate polyclonal B cells and vectorize noncognate proteins into their endosomes, generating highly efficient stimulators for ex vivo expansion of antigen-specific CD4+ T cells. This method also allows harnessing the ability of B cells to polarize CD4+ T cells into effectors or regulators.

SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells.

UNLABELLED: Monocyte-derived dendritic cells (MDDC) stimulate CD8 cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses. IMPORTANCE: Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.

The HIV-1 antisense protein (ASP) induces CD8 T cell responses during chronic infection.

BACKGROUND: CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. RESULTS: A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient. CONCLUSION: Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines.

Long-term maintenance of skin immune system in a NOD-Scid IL2rγ(null) mouse model transplanted with human skin.

We developed a NOD-Scid IL2rγ(null) mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T-cell immunity. Indeed, T-cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long-term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation.

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1ß, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Escape from highly effective public CD8+ T-cell clonotypes by HIV.

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8(+) T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8(+) T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3 gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These "public" clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L(268)M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8(+) T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8(+) T-cell response against HIV.

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes.

BACKGROUND: Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8(+) T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8(+) T-cell responses. OBJECTIVE: We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)-mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. METHODS: Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8(+) T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4(+) T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. RESULTS: We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8(+) T-cell response compared with the earlier described efficient CD8(+) T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8(+) T-cell clones. CONCLUSION: Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs.

Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

Naive and memory CD4+ T cell subsets can contribute to the generation of human Tfh cells.

CD4+ T follicular helper cells (Tfh) promote B cell maturation and antibody production in secondary lymphoid organs. By using an innovative culture system based on splenocyte stimulation, we studied the dynamics of naive and memory CD4+ T cells during the generation of a Tfh cell response. We found that both naive and memory CD4+ T cells can acquire phenotypic and functional features of Tfh cells. Moreover, we show here that the transition of memory as well as naive CD4+ T cells into the Tfh cell profile is supported by the expression of pro-Tfh genes, including transcription factors known to orchestrate Tfh cell development. Using this culture system, we provide pieces of evidence that HIV infection differentially alters these newly identified pathways of Tfh cell generation. Such diversity in pathways of Tfh cell generation offers a new framework for the understanding of Tfh cell responses in physiological and pathological contexts.

Preclinical studies of a modified vaccinia virus Ankara-based HIV candidate vaccine: antigen presentation and antiviral effect.

Poxvirus-based human immunodeficiency virus (HIV) vaccine candidates are currently under evaluation in preclinical and clinical trials. Modified vaccinia virus Ankara (MVA) vectors have excellent safety and immunogenicity records, but their behavior in human cell cultures remains only partly characterized. We studied here various virological and immunological aspects of the interactions of MVA-HIV, a vaccine candidate developed by the French National Agency for AIDS Research (ANRS), with primary human cells. We report that MVA-HIV infects and drives Gag expression in primary macrophages, dendritic cells (DCs), and epithelial and muscle cells. MVA-HIV-infected DCs matured, efficiently presented Gag, Pol, and Nef antigens, and activated HIV-specific cytotoxic T lymphocytes (CTLs). As expected with this type of vector, infection was cytopathic and led to DC apoptosis. Coculture of MVA-HIV-infected epithelial cells or myotubes with DCs promoted efficient Gag antigen major histocompatibility complex class I (MHC-I) cross-presentation without inducing direct infection and death of DCs. Antigen-presenting cells (APCs) infected with MVA-HIV also activated HIV-specific CD4(+) T cells. Moreover, exposure of DCs to MVA-HIV or to MVA-HIV-infected myotubes induced type I interferon (IFN) production and inhibited subsequent HIV replication and transfer to lymphocytes. Altogether, these results show that MVA-HIV promotes efficient MHC-I and MHC-II presentation of HIV antigens by APCs without facilitating HIV replication. Deciphering the immune responses to MVA in culture experiments will help in the design of innovative vaccine strategies.

Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity.

CD8(+) T cells are major players in the immune response against HIV. However, recent failures in the development of T cell-based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell-mediated efficacy. CD8(+) T cells from HIV-1-infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8(+) T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8(+) T cells from infected donors. We report that attributes of CD8(+) T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8(+) T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8(+) T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.