Javier Hernandez

A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCasE murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies.

Endogenous Cytotoxic T-Cell Response Contributes to the Long-Term Antiretroviral Protection Induced by a Short Period of Antibody-Based Immunotherapy of Neonatally Infected Mice

ABSTRACT Neutralizing monoclonal antibodies (MAbs) are increasingly being considered for blunting human viral infections. However, whether they can also exert indirect effects on endogenous antiviral immune responses has been essentially overlooked. We have recently shown that a short (several-day) period of immunotherapy with the neutralizing 667 MAb of mouse neonates shortly after infection with the lethal FrCasE retrovirus not only has an immediate effect on the viral load but also permits an endogenous antiviral immunity to emerge. Even though passive immunotherapy was administered during the particular period of immunocompetence acquisition, the endogenous response eventually arising was protective and persisted long (>1 year) after the MAb has disappeared. As very high levels of anti-FrCasE antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype and showing strong neutralization activity, were found in the sera of MAb-treated mice, it was necessary to address whether this humoral immunity was sufficient on its own to confer full protection against FrCasE or whether a cytotoxic T-lymphocyte (CTL) response was also necessary. Using a variety of in vivo assays in young and adult animals previously infected by FrCasE and treated by 667, we show here that transient 667 immunotherapy is associated with the emergence of a CTL response against virus-infected cells. This cytotoxic activity is indispensable for long-term antiviral protective immunity, as high neutralizing antibody titers, even enhanced in in vivo CD8+ cell depletion experiments, cannot prevent the FrCasE-induced death of infected/treated mice. Our work may have important therapeutic consequences, as it indicates that a short period of MAb-based immunotherapy conducted at a stage where the immune system is still developing can be associated with the mounting of a functional Th1-type immune response characterized by both CTL and IgG2a-type humoral contributions, the cooperation of which is known to be essential for the containment of chronic infections by a variety of viruses.

Gilz-Activin A as a Novel Signaling Axis Orchestrating Mesenchymal Stem Cell and Th17 Cell Interplay

Mesenchymal stem cells (MSC) are highly immunosuppressive cells able to reduce chronic inflammation through the active release of mediators. Recently, we showed that glucocorticoid-induced leucine zipper (Gilz) expression by MSC is involved in their therapeutic effect by promoting the generation of regulatory T cells. However, the mechanisms underlying this pivotal role of Gilz remain elusive. Methods and Results In this study, we have uncovered evidence that Gilz modulates the phenotype and function of Th1 and Th17 cells likely by upregulating the level of Activin A and NO2 secreted by MSC. Adoptive transfer experiments sustained this Gilz-dependent suppressive effect of MSC on Th1 and Th17 cell functions. In immunoregulatory MSC, obtained by priming with IFN-γ and TNF-α, Gilz was translocated to the nucleus and bound to the promoters of inos and Activin βA to induce their expression. The increased expression of Activin A directly impacted on Th17 cells fate by repressing their differentiation program through the activation of Smad3/2 and enhancing IL-10 production. Conclusion Our results reveal how Gilz controls inos and Activin βA gene expression to ultimately assign immunoregulatory status to MSC able to repress the pathogenic Th17 cell differentiation program and uncover Activin A as a novel mediator of MSC in this process.

CD4+ T Helper Cells Play a Key Role in Maintaining Diabetogenic CD8+ T Cell Function in the Pancreas

Autoreactive CD8+ and CD4+ T cells have been assigned independent key roles in the destruction of insulin-producing beta cells resulting in type 1 diabetes. Although CD4 help for the generation of efficient CD8+ T cell responses in lymphoid tissue has been extensively described, whether these two cell populations cooperate in islet destruction in situ remains unclear. By using intravital 2-photon microscopy in a mouse model of diabetes, we visualized both effector T cell populations in the pancreas during disease onset. CD4+ T helper cells displayed a much higher arrest in the exocrine tissue than islet-specific CD8+ T cells. This increased arrest was major histocompatibility complex (MHC) class II-dependent and locally correlated with antigen-presenting cell recruitment. CD8+ T cells deprived of continued CD4 help specifically in the pancreas, through blocking MHC class II recognition, failed to maintain optimal effector functions, which contributed to hamper diabetes progression. Thus, we provide novel insight in the cellular mechanisms regulating effector T cell functionality in peripheral tissues with important implications for immunotherapies.

Changes in metabolism affect expression of ABC transporters through ERK5 and depending on p53 status

Changes in metabolism require the efflux and influx of a diverse variety of metabolites. The ABC superfamily of transporters regulates the exchange of hundreds of substrates through the impermeable cell membrane. We show here that a metabolic switch to oxidative phosphorylation (OXPHOS), either by treating cells with dichloroacetate (DCA) or by changing the available substrates, reduced expression of ABCB1, ABCC1, ABCC5 and ABCG2 in wild-type p53-expressing cells. This metabolic change reduced histone changes associated to active promoters. Notably, DCA also inhibited expression of these genes in two animal models in vivo. In contrast, OXPHOS increased the expression of the same transporters in mutated (mut) or null p53-expressing cells. ABC transporters control the export of drugs from cancer cells and render tumors resistant to chemotherapy, playing an important role in multiple drug resistance (MDR). Wtp53 cells forced to perform OXPHOS showed impaired drug clearance. In contrast mutp53 cells increased drug clearance when performing OXPHOS. ABC transporter promoters contain binding sites for the transcription factors MEF2, NRF1 and NRF2 that are targets of the MAPK ERK5. OXPHOS induced expression of the MAPK ERK5. Decreasing ERK5 levels in wtp53 cells increased ABC expression whereas it inhibited expression in mutp53 cells. Our results showed that the ERK5/MEF2 pathway controlled ABC expression depending on p53 status.

The PDK1 Inhibitor Dichloroacetate Controls Cholesterol Homeostasis Through the ERK5/MEF2 Pathway.

Controlling cholesterol levels is a major challenge in human health, since hypercholesterolemia can lead to serious cardiovascular disease. Drugs that target carbohydrate metabolism can also modify lipid metabolism and hence cholesterol plasma levels. In this sense, dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, augments usage of the glycolysis-produced pyruvate in the mitochondria increasing oxidative phosphorylation (OXPHOS). In several animal models, DCA decreases plasma cholesterol and triglycerides. Thus, DCA was used in the 70u2009s to treat diabetes mellitus, hyperlipoproteinemia and hypercholesterolemia with satisfactory results. However, the mechanism of action remained unknown and we describe it here. DCA increases LDLR mRNA and protein levels as well as LDL intake in several cell lines, primary human hepatocytes and two different mouse models. This effect is mediated by transcriptional activation as evidenced by H3 acetylation on lysine 27 on the LDLR promoter. DCA induces expression of the MAPK ERK5 that turns on the transcription factor MEF2. Inhibition of this ERK5/MEF2 pathway by genetic or pharmacological means decreases LDLR expression and LDL intake. In summary, our results indicate that DCA, by inducing OXPHOS, promotes ERK5/MEF2 activation leading to LDLR expression. The ERK5/MEF2 pathway offers an interesting pharmacological target for drug development.

Systemic LPS Translocation Activates Cross-Presenting Dendritic Cells but Is Dispensable for the Breakdown of CD8 + T Cell Peripheral Tolerance in Irradiated Mice

Lymphodepletion is currently used to enhance the efficacy of cytotoxic T lymphocyte adoptive transfer immunotherapy against cancer. This beneficial effect of conditioning regimens is due, at least in part, to promoting the breakdown of peripheral CD8+ T cell tolerance. Lymphodepletion by total body irradiation induces systemic translocation of commensal bacteria LPS from the gastrointestinal tract. Since LPS is a potent activator of the innate immune system, including antigen presenting dendritic cells, we hypothesized that LPS translocation could be required for the breakdown of peripheral tolerance observed in irradiated mice. To address this issue, we have treated irradiated mice with antibiotics in order to prevent LPS translocation and utilized them in T cell adoptive transfer experiments. Surprisingly, we found that despite of completely blocking LPS translocation into the bloodstream, antibiotic treatment did not prevent the breakdown of peripheral tolerance. Although irradiation induced the activation of cross-presenting CD8+ dendritic cells in the lymphoid tissue, LPS could not solely account for this effect. Activation of dendritic cells by mechanisms other than LPS translocation is sufficient to promote the differentiation of potentially autoreactive CD8+ T cells into effectors in irradiated mice. Our data indicate that LPS translocation is dispensable for the breakdown of CD8+ T cell tolerance in irradiated mice.

Mitochondrial Complex I activity signals antioxidant response through ERK5

Oxidative phosphorylation (OXPHOS) generates ROS as a byproduct of mitochondrial complex I activity. ROS-detoxifying enzymes are made available through the activation of their antioxidant response elements (ARE) in their gene promoters. NRF2 binds to AREs and induces this anti-oxidant response. We show that cells from multiple origins performing OXPHOS induced NRF2 expression and its transcriptional activity. The NRF2 promoter contains MEF2 binding sites and the MAPK ERK5 induced MEF2-dependent NRF2 expression. Blocking OXPHOS in a mouse model decreased Erk5 and Nrf2 expression. Furthermore, fibroblasts derived from patients with mitochondrial disorders also showed low expression of ERK5 and NRF2 mRNAs. Notably, in cells lacking functional mitochondrial complex I activity OXPHOS did not induce ERK5 expression and failed to generate this anti-oxidant response. Complex I activity induces ERK5 expression through fumarate accumulation. Eukaryotic cells have evolved a genetic program to prevent oxidative stress directly linked to OXPHOS and not requiring ROS.

Integrin β1 Optimizes Diabetogenic T Cell Migration and Function in the Pancreas

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas (PA) during autoimmune diabetes remains unclear. Using intra-vital 2-photon microscopy in a mouse model of diabetes, we found that CXCR3 chemokine downregulated CD8+ T cell motility specifically within islets, promoting effector cell confinement to their target sites. By contrast, T cell velocity and directionality in the exocrine tissue were enhanced along blood vessels and extracellular matrix fibers. This guided migration implicated integrin-dependent interactions, since integrin blockade impaired exocrine T cell motility. In addition, integrin β1 blockade decreased CD4+ T cell effector phenotype specifically in the PA. Thus, we unveil an important role for integrins in the PA during autoimmune diabetes that may have important implications for the design of new therapies.