Induction of Anti-Tumour Immune Responses by Dendritic Cells Generated with Flt3-Ligand

<p>Dendritic cells (DCs) are potent antigen presenting cells that are crucial for the initiation of an immune response. Due to this property, DCs have been used as the basis of cancer vaccines in immunotherapy. In clinical trials, DCs used for vaccination are commonly generated by culturing monocytes from each patients' blood with the growth factors GM-CSF and IL-4 (GMCSF/IL-4 DCs). The DCs generated are reportedly similar to those that arise in vivo during inflammation and trials using these DCs have been met with some success. A recently developed method of generating mouse or human DCs in vitro, involves the culture of bone marrow (BM) precursors with the cytokine Flt3-Ligand (Flt3L-DCs). Flt3L-DCs differ substantially in phenotype from GMCSF/IL-4 DCs and more closely resemble steady-state DCs in vivo. This thesis investigated the suitability of Flt3L-DCs for cancer immunotherapy. Murine BM cells cultured in Flt3L generated three DC subsets. These consisted of plasmacytoid DCs (pDCs) that were CD11c⁺B220⁺, and conventional DCs (cDCs) that were CD11c⁺B220⁻ and could be further subdivided into CD11bhigh and CD24high populations. We observed that cDCs responded to stimulation with a variety of Tolllike receptor (TLR) agonists, as evaluated by the up-regulation of activation markers. However pDCs responded to the agonist CpG at a higher extent compared to all other agonists used. In addition, combining TLR agonists could further enhance the activation of Flt3L-DCs. Among all combinations tested, Pam3Cys/Poly I:C was the most optimal at inducing the secretion of inflammatory cytokines IL-12p70 and TNF-α. Furthermore, Pam3Cys/Poly I:C stimulated Flt3L-cDCs exhibited a greater ability at inducing CD4⁺ T cell proliferation and cross-presentation of soluble antigen to CD8⁺ T cells, compared to Flt3L-cDCs activated with the respective individual agonists. Studies have shown that GM-CSF DCs are highly reliant on glycolytic metabolism during activation in order to up-regulate activation markers. Therefore, we also characterised Flt3L-cDCs for their ability to up-regulate activation markers following stimulation with the agonist LPS and treatment with the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). In line with previous reports, DCs generated in culture with GMCSF/IL-4 were unable to up-regulate activation markers at all the 2-DG concentrations used. In contrast, Flt3L-cDCs appeared to have a threshold level where only high concentrations of 2-DG inhibited their ability to up-regulate activation markers. This result indicates that steady-state and inflammatory DCs preferentially use different metabolic pathways upon activation. The ability of optimally activated Flt3L-cDCs and GMCSF/IL-4 DCs to confer tumour protection was also examined. While unstimulated Flt3L-cDCs or GMCSF/IL-4 DCs could protect mice from tumour growth, vaccination with activated DCs from either population was required for complete tumour protection. Furthermore, we found that even in optimal conditions of activation, 1x10⁵ Flt3LcDCs were required for maximal tumour protection, whereas 1x10⁴ GMCSF/IL-4 DCs provided sufficient protection. These findings indicate that Flt3L-cDCs can be used as the basis of a therapeutic cancer vaccine, but are not superior to GMCSF/IL- 4 DCs. Further studies are required to establish conditions that can enhance the efficacy of Flt3L-cDCs.</p>

Induction of Anti-Tumour Immune Responses by Dendritic Cells Generated with Flt3-Ligand

<p>Dendritic cells (DCs) are potent antigen presenting cells that are crucial for the initiation of an immune response. Due to this property, DCs have been used as the basis of cancer vaccines in immunotherapy. In clinical trials, DCs used for vaccination are commonly generated by culturing monocytes from each patients' blood with the growth factors GM-CSF and IL-4 (GMCSF/IL-4 DCs). The DCs generated are reportedly similar to those that arise in vivo during inflammation and trials using these DCs have been met with some success. A recently developed method of generating mouse or human DCs in vitro, involves the culture of bone marrow (BM) precursors with the cytokine Flt3-Ligand (Flt3L-DCs). Flt3L-DCs differ substantially in phenotype from GMCSF/IL-4 DCs and more closely resemble steady-state DCs in vivo. This thesis investigated the suitability of Flt3L-DCs for cancer immunotherapy. Murine BM cells cultured in Flt3L generated three DC subsets. These consisted of plasmacytoid DCs (pDCs) that were CD11c⁺B220⁺, and conventional DCs (cDCs) that were CD11c⁺B220⁻ and could be further subdivided into CD11bhigh and CD24high populations. We observed that cDCs responded to stimulation with a variety of Tolllike receptor (TLR) agonists, as evaluated by the up-regulation of activation markers. However pDCs responded to the agonist CpG at a higher extent compared to all other agonists used. In addition, combining TLR agonists could further enhance the activation of Flt3L-DCs. Among all combinations tested, Pam3Cys/Poly I:C was the most optimal at inducing the secretion of inflammatory cytokines IL-12p70 and TNF-α. Furthermore, Pam3Cys/Poly I:C stimulated Flt3L-cDCs exhibited a greater ability at inducing CD4⁺ T cell proliferation and cross-presentation of soluble antigen to CD8⁺ T cells, compared to Flt3L-cDCs activated with the respective individual agonists. Studies have shown that GM-CSF DCs are highly reliant on glycolytic metabolism during activation in order to up-regulate activation markers. Therefore, we also characterised Flt3L-cDCs for their ability to up-regulate activation markers following stimulation with the agonist LPS and treatment with the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). In line with previous reports, DCs generated in culture with GMCSF/IL-4 were unable to up-regulate activation markers at all the 2-DG concentrations used. In contrast, Flt3L-cDCs appeared to have a threshold level where only high concentrations of 2-DG inhibited their ability to up-regulate activation markers. This result indicates that steady-state and inflammatory DCs preferentially use different metabolic pathways upon activation. The ability of optimally activated Flt3L-cDCs and GMCSF/IL-4 DCs to confer tumour protection was also examined. While unstimulated Flt3L-cDCs or GMCSF/IL-4 DCs could protect mice from tumour growth, vaccination with activated DCs from either population was required for complete tumour protection. Furthermore, we found that even in optimal conditions of activation, 1x10⁵ Flt3LcDCs were required for maximal tumour protection, whereas 1x10⁴ GMCSF/IL-4 DCs provided sufficient protection. These findings indicate that Flt3L-cDCs can be used as the basis of a therapeutic cancer vaccine, but are not superior to GMCSF/IL- 4 DCs. Further studies are required to establish conditions that can enhance the efficacy of Flt3L-cDCs.</p>

Human Endogenous Retrovirus Type W Envelope from Multiple Sclerosis Demyelinating Lesions Shows Unique Solubility and Antigenic Characteristics

In multiple sclerosis (MS), human endogenous retrovirus W family (HERV-W) envelope protein, pHERV-W ENV, limits remyelination and induces microglia-mediated neurodegeneration. To better understand its role, we examined the soluble pHERV-W antigen from MS brain lesions detected by specific antibodies. Physico-chemical and antigenic characteristics confirmed differences between pHERV-W ENV and syncytin-1. pHERV-W ENV monomers and trimers remained associated with membranes, while hexamers self-assembled from monomers into a soluble macrostructure involving sulfatides in MS brain. Extracellular hexamers are stabilized by internal hydrophobic bonds and external hydrophilic moieties. HERV-W studies in MS also suggest that this diffusible antigen may correspond to a previously described high-molecular-weight neurotoxic factor secreted by MS B-cells and thus represents a major agonist in MS pathogenesis. Adapted methods are now needed to identify encoding HERV provirus(es) in affected cells DNA. The properties and origin of MS brain pHERV-W ENV soluble antigen will allow a better understanding of the role of HERVs in MS pathogenesis. The present results anyhow pave the way to an accurate detection of the different forms of pHERV-W ENV antigen with appropriate conditions that remained unseen until now.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address their limited clinical efficacy. Soluble Antigen Arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using hydrolysable linkers (hSAgA) or stable “click” chemistry linkers (cSAgA). They were evaluated for the ability to block the spontaneous development of disease in the non-obese diabetic mouse model of Type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgA) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers such as CD73, IL-10, PD-1, KLRG-1. Anaphylaxis caused by the free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely non-overlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to soluble peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address their limited clinical efficacy. Soluble Antigen Arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using hydrolysable linkers (hSAgA) or stable “click” chemistry linkers (cSAgA). They were evaluated for the ability to block the spontaneous development of disease in the non-obese diabetic mouse model of Type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgA) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers such as CD73, IL-10, PD-1, KLRG-1. Anaphylaxis caused by the free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely non-overlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to soluble peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2–RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD–immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2–RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

Immunoprophylactic Potential of a New Recombinant Leishmania infantum Antigen for Canine Visceral Leishmaniasis: An In Vitro Finding

The development and application of safe and effective immunoprophylactic/immunotherapeutic agents against canine visceral leishmaniasis (CanL) have been pointed out as the only means for the real control of the disease. Thus, this study aimed to evaluate the in vitro cellular immune response of dogs, elicited by the new recombinant proteins of Leishmania infantum, Lci10 and Lci13, in order to investigate their potential for vaccinology. Twenty-four dogs were submitted to clinical, parasitological, serological and molecular tests, and then separated into two study groups: 12 infected (InD) and 12 non-infected dogs (NInD), and six of each group were directed for Lci10 and Lci13 evaluation. Peripheral blood mononuclear cells (PBMC) were cultured and stimulated with Lci10 (10 μg/ml) or Lci13 (5 μg/ml), and with L. infantum soluble antigen (LSA) (25 μg/ml) or no stimulus (NS) as controls. Afterwards, the mRNA levels of different cytokines were quantified through qPCR, and Nitric Oxide (NO) production was assessed in the culture supernatants. Significant differences were considered when p ≤ 0.05. The comparative analysis revealed that, in the NInD group, Lci13 promoted a significant increase in the expression of IFN-γ in relation to LSA (p = 0.0362), and the expression of this cytokine in NInD was significantly higher than that presented in the InD (p = 0.0028). A negative expression for TGF-β was obtained in both groups. Lci13 also induced a greater production of NO in relation to the NS sample in the NInD group. No significant differences were observed after stimulation with Lci10. In conclusion, the results suggest a protective role of Lci13 for uninfected animals, thus with a potential for immunoprophylaxis. The results will help to direct the antigen Lci13 for further studies (pre-clinical trials), in order to determine its immunogenicity and reactogenicity effects, as a way to consolidate its real applicability for vaccinology against CanL.

Mast Cells-derived Exosomes Promote the Development of Experimental Cerebral Malaria

Background: Cerebral malaria (CM) is a severe neurological manifestation caused by Plasmodium infection, with high morbidity and mortality rate, and long-term cognitive impairments in survivors. Exosomes are cell-derived nano-vesicles secreted by virtually all types of cells and serve as mediators of intercellular communication. Studies have demonstrated that mast cells (MCs) play a critical role in mediating malaria severity, however, the potential functions and pathological mechanisms of MCs-derived exosome (MCs-Exo) impacting on CM pathogenesis remain largely unknown. Methods: Herein, we utilized an experimental CM (ECM) murine model (C57BL/6 mice infected with P. berghei ANKA), and then intravenously (i.v.) injected MCs-Exo into ECM mice to investigate the effect of MCs-Exo on ECM pathogenies. We also used an in vitro model by investigating the pathogenesis development of brain microvascular endothelial cells line (bEnd.3 cells) upon MCs-Exo treatment after P. berghei ANKA blood-stage soluble antigen (PbAg) stimulation. Results: MCs-Exo were successfully isolated from culture supernatants of mouse MCs line (P815 cells) stimulated with PbAg, characterized by spherical vesicles with the diameter of 30–150 nm, expressing of typical exosomal markers, including CD9, CD81, and CD63. In vivo and ex vivo tracking showed that DiR-labeled MCs-Exo were taken up by liver and brain tissues after 6 h of i.v. injection. Compared with naive mice, ECM mice exhibited higher numbers of MCs and higher levels of MCs degranulation in various tissues (e.g., brain, cervical lymph node, and skin). The present of MCs-Exo dramatically shortened survival time, elevated incident of ECM, exacerbated liver and brain histopathological damage, promoted Th1 cytokine response, and aggravated brain vascular endothelial activation and blood brain barrier breakdown in ECM mice. Interestingly, compared with bEnd.3 cells stimulated with PbAg, the treatment of MCs-Exo led to decrease of cells viability, increase the mRNA levels of Ang-2, CCL2, CXCL1, and CXCL9, and decrease the mRNA levels of Ang-1, ZO-1, and Claudin-5. Conclusions: Thus, our data suggest that MCs-Exo could promote pathogenesis of ECM in mice.