Development of a New Off-the-Shelf Plasmacytoid Dendritic Cell-Based Approach for the Expansion and Characterization of SARS-CoV-2-Specific T Cells.

Global vaccination against COVID-19 has been widely successful; however, there is a need for complementary immunotherapies in severe forms of the disease and in immunocompromised patients. Cytotoxic CD8+ T cells have a crucial role in disease control, but their function can be dysregulated in severe forms of the disease. We report here a cell-based approach using a plasmacytoid dendritic cell line (PDC*line) to expand in vitro specific CD8+ responses against COVID-19 Ags. We tested the immunogenicity of eight HLA-A*02:01 restricted peptides derived from diverse SARS-Cov-2 proteins, selected by bioinformatics analyses in unexposed and convalescent donors. Higher ex vivo frequencies of specific T cells against these peptides were found in convalescent donors compared with unexposed donors, suggesting in situ T cell expansion upon viral infection. The peptide-loaded PDC*line induced robust CD8+ responses with total amplification rates that led up to a 198-fold increase in peptide-specific CD8+ T cell frequencies for a single donor. Of note, six of eight selected peptides provided significant amplifications, all of which were conserved between SARS-CoV variants and derived from the membrane, the spike protein, the nucleoprotein, and the ORF1ab. Amplified and cloned antiviral CD8+ T cells secreted IFN-γ upon peptide-specific activation. Furthermore, specific TCR sequences were identified for two highly immunogenic Ags. Hence, PDC*line represents an efficient platform to identify immunogenic viral targets for future immunotherapies.

Melanoma tumour-derived glycans hijack dendritic cell subsets through C-type lectin receptor binding.

Dendritic cell (DC) subsets play a crucial role in shaping anti-tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C-type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant 'glyco-code,' and that circulating and tumour-infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s-Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)-signalling and pre-conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro-tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s-Tn/ C-type lectin-like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour-associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients.

Maternal imprinting and determinants of neonates' immune function in the SEPAGES mother-child cohort.

Introduction: Immune function in pregnancy is influenced by host-specific and environmental factors. This may impact fetal immune development, but the link between maternal and neonatal immune function is still poorly characterized. Here, we investigate the relationship between maternal and neonatal immune function, and identify factors affecting the association between maternal and child cytokine secretion. Methods: In the French prospective cohort SEPAGES, blood samples were obtained from pregnant women (n=322) at gestational week 20 ± 4 and from their child at birth (n=156). Maternal and cord blood cytokine and chemokine (CK) levels were measured at baseline in all subjects and after T cell or dendritic cell activation with phytohemagglutinin or R848 (in total 29 and 27 measures in maternal and cord blood samples, respectively). Associations between environmental, individual factors and CK level were estimated by linear regression modeling. The maternal-cord blood CK relations were assessed by Pearson correlation and regression models. Results: We observed that pregnant women and neonates displayed specific CK secretion profiles in the innate and adaptive compartments at baseline and upon activation. Activation of T cells in cord blood induced high levels of IL-2, but low levels of IFNγ, IL-13 or IL-10, in comparison to maternal blood samples. Elsewhere, neonatal innate immune responses were characterized by low production of IFNα, while productions of IL-1ß, IL-6, IL-8, IL-10 and TNFα were higher than maternal responses. Strong correlations were observed between most CK after activation in maternal and cord blood samples. Strikingly, a statistical association between global mother and child cytokine profiles was evidenced. Correlations were observed between some individual CK of pregnant women and their children, both at baseline (MCP1, RANTES) and after activation with R848 (IL-6, IL-8 and IL-10). We looked for factors which could influence cytokine secretion in maternal or cord blood, and found that leucocyte counts, maternal age, pre-conception BMI, smoking and season were associated with the levels of several CK in mothers or children. Discussion: Our study reveals in utero immune imprinting influencing immune responses in infants, opening the way to investigate the mechanisms responsible for this imprinting. Whether such influences have long lasting effects on children health warrants further investigation.

The melanoma tumor glyco-code impacts human dendritic cells' functionality and dictates clinical outcomes.

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs' functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor' hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.

A New Plasmacytoid Dendritic Cell-Based Vaccine in Combination with Anti-PD-1 Expands the Tumor-Specific CD8+ T Cells of Lung Cancer Patients.

The purpose of immune checkpoint inhibitor (ICI)-based therapies is to help the patient's immune system to combat tumors by restoring the immune response mediated by CD8+ cytotoxic T cells. Despite impressive clinical responses, most patients do not respond to ICIs. Therapeutic vaccines with autologous professional antigen-presenting cells, including dendritic cells, do not show yet significant clinical benefit. To improve these approaches, we have developed a new therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma. The goal of the study is to demonstrate the potential of this platform to activate circulating tumor-specific CD8+ T cells in patients with lung cancer, specifically non-small-cell lung cancer (NSCLC). PDC*line cells loaded with peptides derived from tumor antigens are used to stimulate the peripheral blood mononuclear cells of NSCLC patients. Very interestingly, we demonstrate an efficient activation of specific T cells for at least two tumor antigens in 69% of patients irrespective of tumor antigen mRNA overexpression and NSCLC subtype. We also show, for the first time, that the antitumor CD8+ T-cell expansion is considerably improved by clinical-grade anti-PD-1 antibodies. Using PDC*line cells as an antigen presentation platform, we show that circulating antitumor CD8+ T cells from lung cancer patients can be activated, and we demonstrate the synergistic effect of anti-PD-1 on this expansion. These results are encouraging for the development of a PDC*line-based vaccine in NSCLC patients, especially in combination with ICIs.

Leveraging a powerful allogeneic dendritic cell line towards neoantigen-based cancer vaccines.

In recent years, immunotherapy has finally found its place in the anti-cancer therapeutic arsenal, even becoming standard of care as first line treatment for metastatic forms. The clinical benefit provided by checkpoint blockers such as anti-PD-1/PD-L1 in many cancers revolutionized the field. However, too many patients remain refractory to these treatments due to weak baseline anti-cancer immunity. There is therefore a need to boost the frequency and function of patients' cytotoxic CD8+ cellular effectors by targeting immunogenic and tumor-restricted antigens, such as neoantigens using an efficient vaccination platform. Dendritic cells (DC) are the most powerful immune cell subset for triggering cellular immune response. However, autologous DC-based vaccines display several limitations, such as the lack of reproducibility and the limited number of cells that can be manufactured. Here we discuss the advantages of a new therapeutic vaccine based on an allogeneic Plasmacytoid DC cell line, which is easy to produce and represents a powerful platform for priming and expanding anti-neoantigen cytotoxic CD8+ T-cells.

Unique CLR expression patterns on circulating and tumor-infiltrating DC subsets correlated with clinical outcome in melanoma patients.

Subversion of immunity by tumors is a crucial step for their development. Dendritic cells (DCs) are strategic immune cells that orchestrate anti-tumor immune responses but display altered functions in cancer. The bases for such DCs' hijacking are not fully understood. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids. DCs express glycan-binding receptors, named C-type lectin receptors (CLR), allowing them to sense changes in glycan signature of their environment, and subsequently trigger a response. Recognition of tumor glycans by CLRs is crucial for DCs to shape antitumor immunity, and decisive in the orientation of the response. Yet the status of the CLR machinery on DCs in cancer, especially melanoma, remained largely unknown. We explored CLR expression patterns on circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs of melanoma patients, assessed their clinical relevance, and further depicted the correlations between CLR expression profiles and DCs' features. For the first time, we highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Furthermore, melanoma tumor cells directly altered CLR expression profiles of healthy DC subsets, and this was associated with specific glycan patterns (Man, Fuc, GlcNAc) that may interact with DCs through CLR molecules. Notably, specific CLR expression profiles on DC subsets correlated with unique DCs' activation status and functionality and were associated with clinical outcome of melanoma patients. Higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better survival, whereas elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Thus, melanoma tumor may shape DCs' features by exploiting the plasticity of the CLR machinery. Our study revealed that melanoma manipulates CLR pathways to hijack DC subsets and escape from immune control. It further paved the way to exploit glycan-lectin interactions for the design of innovative therapeutic strategies, which exploit DCs' potentialities while avoiding hijacking by tumor, to properly reshape anti-tumor immunity by manipulating the CLR machinery.

Diversification of circulating and tumor-infiltrating plasmacytoid DCs towards the P3 (CD80+ PDL1-)-pDC subset negatively correlated with clinical outcomes in melanoma patients.

Objectives: Plasmacytoid DCs (pDCs) play a critical yet enigmatic role in antitumor immunity through their pleiotropic immunomodulatory functions. Despite proof of pDC diversity in several physiological or pathological contexts, pDCs have been studied as a whole population so far in cancer. The assessment of individual pDC subsets is needed to fully grasp their involvement in cancer immunity, especially in melanoma where pDC subsets are largely unknown and remain to be uncovered. Methods: We explored for the first time the features of diverse circulating and tumor-infiltrating pDC subsets in melanoma patients using multi-parametric flow cytometry, and assessed their clinical relevance. Based on CD80, PDL1, CD2, LAG3 and Axl markers, we provided an integrated overview of the frequency, basal activation status and functional features of pDC subsets in melanoma patients together with their relationship to clinical outcome. Results: Strikingly, we demonstrated that P3-pDCs (CD80+PDL1-) accumulated within the tumor of melanoma patients and negatively correlated with clinical outcomes. The basal activation status, diversification towards P1-/P2-/P3-pDCs and functionality of several pDC subsets upon TLR7/TLR9 triggering were perturbed in melanoma patients, and were differentially linked to clinical outcome. Conclusion: Our study shed light for the first time on the phenotypic and functional heterogeneity of pDCs in the blood and tumor of melanoma patients and their potential involvement in shaping clinical outcomes. Such novelty brightens our understanding of pDC complexity, and prompts the further deciphering of pDCs' features to better apprehend and exploit these potent immune players. It highlights the importance of considering pDC diversity when developing pDC-based therapeutic strategies to ensure optimal clinical success.

Impaired Antitumor Immune Response in MYCN-amplified Neuroblastoma Is Associated with Lack of CCL2 Secretion and Poor Dendritic Cell Recruitment.

In neuroblastoma, MYCN amplification is associated with sparse immune infiltrate and poor prognosis. Dendritic cells (DC) are crucial immune sentinels but their involvement in neuroblastoma pathogenesis is poorly understood. We observed that the migration of monocytes, myeloid and plasmacytoid DC induced by MYCN-nonamplified neuroblastoma supernatants was abrogated by the addition of anti-CCL2 antibodies, demonstrating the involvement of the CCR2/CCL2 axis in their recruitment by these tumors. Using public RNA sequencing and microarray datasets, we describe lower level of expression of CCL2 in MYCN-amplified neuroblastoma tumors, and we propose a working model for T-cell recruitment in neuroblastoma tumors in which CCL2 produced by neuroblastoma cells initiates the recruitment of monocytes, myeloid and plasmacytoid DCs. Among these cells, the CD1c+ subset may recruit T cells by means of CCL19/CCL22 secretion. In vitro, supernatants from DCs cocultured with neuroblastoma cell lines and activated contain CCL22 and CCL19, and are chemotactic for both CD4+ and CD8+ T cells. We also looked at immunomodulation induced by neuroblastoma cell lines, and found MYCN-nonamplified neuroblastoma cell lines were able to create a microenvironment where DC activation is enhanced. Overall, our findings highlight a major role for CCL2/CCR2 axis in monocytes, myeloid and plasmacytoid cells recruitment toward MYCN-nonamplified neuroblastoma, allowing further immune cell recruitment, and show that these tumors present a microenvironment that can favor DC responses. Significance: In MYCN-nonamplified neuroblastoma, CCL2 produced by neuroblastoma cells induces the recruitment of antigen-presenting cells (DCs and monocytes/macrophages), allowing infiltration by T cells, in link with CCL19 and CCL22 production, hence favoring immune responses.

Dysfunctional BTN3A together with deregulated immune checkpoints and type I/II IFN dictate defective interplay between pDCs and γδ T cells in melanoma patients, which impacts clinical outcomes.

OBJECTIVES: pDCs and γδ T cells emerge as potent immune players participating in the pathophysiology of cancers, yet still remaining enigmatic while harbouring a promising potential for clinical translations. Despite strategic and closed missions, crosstalk between pDCs and γδ T cells has not been deciphered yet in cancers, especially in melanoma where the long-term control of the tumor still remains a challenge. METHODS: This prompted us to explore the interplay between pDCs and γδ T cells in the context of melanoma, investigating the reciprocal features of pDCs or γδ T cells, the underlying molecular mechanisms and its impact on clinical outcomes. RESULTS: TLRL-activated pDCs from the blood and tumor infiltrate of melanoma patients displayed an impaired ability to activate, to modulate immune checkpoints and trigger the functionality of γδ T cells. Conversely, γδ T cells from the blood or tumor infiltrate of melanoma patients activated by PAg were defective in triggering pDCs' activation and modulation of immune checkpoints, and failed to elicit the functionality of pDCs. Reversion of the dysfunctional cross-talks could be achieved by specific cytokine administration and immune checkpoint targeting. Strikingly, we revealed an increased expression of BTN3A on circulating and tumor-infiltrating pDCs and γδ T cells from melanoma patients, but stressed out the potential impairment of this molecule. CONCLUSION: Our study uncovered that melanoma hijacked the bidirectional interplay between pDCs and γδ T cells to escape from immune control, and revealed BTN3A dysfunction. Such understanding will help harness and synergise the power of these potent immune cells to design new therapeutic approaches exploiting their antitumor potential while counteracting their skewing by tumors to improve patient outcomes.

Identification of a predictive metabolic signature of response to immune checkpoint inhibitors in non-small cell lung cancer: METABO-ICI clinical study protocol.

BACKGROUND: Immune checkpoints inhibitors (ICI) are becoming new standards of care for the treatment of non-small cell lung cancer (NSCLC), both as first (alone or in association with chemotherapy) and second line. However, no powerful predictive biomarker of therapeutic response to ICI has been found to date. It has been recently shown that microbiota composition could influence the ability of patients to respond to ICI. Indeed, the microbiota produces circulating metabolites that will subsequently act on immune system, the investigators hypothesized that plasma metabolic signature, reflecting a global microbiota function, could represent a predictive biomarker of response to ICI. METHODS: Monocentric prospective study. Primary objective is to identify baseline metabolic signature (metabolomics analysis by mass spectrometry) associated to ICI response. Secondary objectives are to link metabolic signature with microbiota composition (metagenomics analysis RNA 16S) and immune profile, and altogether with clinic response to ICI. The study will include 60 NSCLC patients treated by ICI in 1st, 2nd or 3rd line of treatment at the Grenoble Alpes University hospital (CHUGA) in 18 months. Patients that have received antibiotic or steroid treatment, 2 or 4 weeks before ICI initiation, respectively, will be excluded. Blood and feces will be collected prior to, at 2 months after ICI treatment initiation, and at 6 months or at progression. EXPECTED RESULTS: We expect to highlight a metabolic profile predictive of response to ICI. By identifying factors associated with early progression, we could avoid to treat potential non-responding patients. Moreover, by restoring a favorable microbiota, patients' ability to respond to these treatments might be restored.

Engineering a Human Plasmacytoid Dendritic Cell-Based Vaccine to Prime and Expand Multispecific Viral and Tumor Antigen-Specific T-Cells.

Because dendritic cells are crucial to prime and expand antigen-specific CD8+ T-cells, several strategies are designed to use them in therapeutic vaccines against infectious diseases or cancer. In this context, off-the-shelf allogeneic dendritic cell-based platforms are more attractive than individualized autologous vaccines tailored to each patient. In the present study, a unique dendritic cell line (PDC*line) platform of plasmacytoid origin, already used to prime and expand antitumor immunity in melanoma patients, was improved thanks to retroviral engineering. We demonstrated that the clinical-grade PDC*line, transduced with genes encoding viral or tumoral whole proteins, efficiently processed and stably presented the transduced antigens in different human leukocyte antigen (HLA) class I contexts. Moreover, the use of polyepitope constructs allowed the presentation of immunogenic peptides and the expansion of specific cytotoxic effectors. We also demonstrated that the addition of the Lysosome-associated membrane protein-1 (LAMP-1) sequence greatly improved the presentation of some peptides. Lastly, thanks to transduction of new HLA molecules, the PDC platform can benefit many patients through the easy addition of matched HLA-I molecules. The demonstration of the effective retroviral transduction of PDC*line cells strengthens and broadens the scope of the PDC*line platform, which can be used in adoptive or active immunotherapy for the treatment of infectious diseases or cancer.

HIV-1 Nef Protein Affects Cytokine and Extracellular Vesicles Production in the GEN2.2 Plasmacytoid Dendritic Cell Line.

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1ß, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.

Hepatitis B virus exploits C-type lectin receptors to hijack cDC1s, cDC2s and pDCs.

OBJECTIVES: C-type lectin receptors (CLRs) are key receptors used by DCs to orchestrate responses to pathogens. During infections, the glycan-lectin interactions shape the virus-host interplay and viruses can subvert the function of CLRs to escape antiviral immunity. Recognition of virus/viral components and uptake by CLRs together with subsequent signalling cascades are crucial in initiating and shaping antiviral immunity, and decisive in the outcome of infection. Yet, the interaction of hepatitis B virus (HBV) with CLRs remains largely unknown. As HBV hijacks DC subsets and viral antigens harbour glycan motifs, we hypothesised that HBV may subvert DCs through CLR binding. METHODS: We investigated here the pattern of CLR expression on BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s from both blood and liver of HBV-infected patients and explored the ability of HBsAg to bind DC subsets through specific CLRs. RESULTS: We highlighted for the first time that the CLR repertoire of circulating and intrahepatic cDC2s, cDC1s and pDCs was perturbed in patients with chronic HBV infection and that some CLR expression levels correlated with plasma HBsAg and HBV DNA levels. We also identified candidate CLR responsible for HBsAg binding to cDCs (CD367/DCIR/CLEC4A, CD32/FcɣRIIA) and pDCs (CD369/DECTIN1/CLEC7A, CD336/NKp44) and demonstrated that HBsAg inhibited DC functions in a CLR- and glycosylation-dependent manner. CONCLUSION: HBV may exploit CLR pathways to hijack DC subsets and escape from immune control. Such advances bring insights into the mechanisms by which HBV subverts immunity and pave the way for developing innovative therapeutic strategies to restore an efficient immune control of the infection by manipulating the viral glycan-lectin axis.

BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s reveal distinct pathophysiologic features and impact on clinical outcomes in melanoma patients.

OBJECTIVES: Dendritic cells play a pivotal but still enigmatic role in the control of tumor development. Composed of specialised subsets (cDC1s, cDC2s, pDCs), DCs are critical in triggering and shaping antitumor immune responses. Yet, tumors exploit plasticity of DCs to subvert their functions and escape from immune control. This challenging controversy prompted us to explore the pathophysiological role of cDCs and pDCs in melanoma, where their precise and coordinated involvement remains to be deciphered. METHODS: We investigated in melanoma patients the phenotypic and functional features of circulating and tumor-infiltrating BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s and assessed their clinical impact. RESULTS: Principal component analyses (PCA) based on phenotypic or functional parameters of DC subsets revealed intra-group clustering, highlighting specific features of DCs in blood and tumor infiltrate of patients compared to healthy donors. DC subsets exhibited perturbed frequencies in the circulation and actively infiltrated the tumor site, while harbouring a higher activation status. Whereas cDC2s and pDCs displayed an altered functionality in response to TLR triggering, circulating and tumor-infiltrating cDC1s preserved potent competences associated with improved prognosis. Notably, the proportion of circulating cDC1s predicted the clinical outcome of melanoma patients. CONCLUSION: Such understanding uncovers critical and distinct impact of each DC subset on clinical outcomes and unveils fine-tuning of interconnections between DCs in melanoma. Elucidating the mechanisms of DC subversion by tumors could help designing new therapeutic strategies exploiting the potentialities of these powerful immune players and their cross-talks, while counteracting their skewing by tumors, to achieve immune control and clinical success.

Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes.

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

Potent Bidirectional Cross-Talk Between Plasmacytoid Dendritic Cells and γδT Cells Through BTN3A, Type I/II IFNs and Immune Checkpoints.

Plasmacytoid DCs (pDCs) and γδT cells are both critical players in immunosurveillance against pathogens and cancer due to their ability to sense microbes and cell stress through recognition of pathogen-associated molecular patterns or altered metabolism [phosphoantigens (PAgs)]. Their unique features, high functional plasticity and ability to interact with many immune cell types allow them to bridge innate and adaptive immunity, initiating and orientating widely immune responses, hence contributing to protective and pathogenic immune responses. Yet, despite strategic and closed missions, potential interactions between pDCs and γδT cells are still unknown. Here we investigated whether there is interplay between pDCs and γδT cells and the underlying molecular mechanisms. Purified human pDCs and γδT cells were cocultured in presence of TLR-L, PAg, and zoledronate (Zol) to mimic both infectious and tumor settings. We demonstrated that TLR7/9L- or Zol-stimulated pDCs drive potent γδT-cell activation, Th1 cytokine secretion and cytotoxic activity. Conversely PAg-activated γδT cells trigger pDC phenotypic changes and functional activities. We provided evidence that pDCs and γδT cells cross-regulate each other through soluble factors and cell-cell contacts, especially type I/II IFNs and BTN3A. Such interplay could be modulated by blocking selective immune checkpoints. Our study highlighted crucial bidirectional interactions between these key potent immune players. The exploitation of pDC-γδT cells interplay represents a promising opportunity to design novel immunotherapeutic strategies and restore appropriate immune responses in cancers, infections and autoimmune diseases.

An innovative plasmacytoid dendritic cell line-based cancer vaccine primes and expands antitumor T-cells in melanoma patients in a first-in-human trial.

The efficacy of immune checkpoint inhibitors has been shown to depend on preexisting antitumor immunity; thus, their combination with cancer vaccines is an attractive therapeutic approach. Plasmacytoid dendritic cells (PDC) are strong inducers of antitumor responses and represent promising vaccine candidates. We developed a cancer vaccine approach based on an allogeneic PDC line that functioned as a very potent antigen-presenting cell in pre-clinical studies. In this phase Ib clinical trial, nine patients with metastatic stage IV melanoma received up to 60 million irradiated PDC line cells loaded with 4 melanoma antigens, injected subcutaneously at weekly intervals. The primary endpoints were safety and tolerability. The vaccine was well tolerated and no serious vaccine-induced side effects were recorded. Strikingly, there was no allogeneic response toward the vaccine, but a significant increase in the frequency of circulating anti-tumor specific T lymphocytes was observed in two patients, accompanied by a switch from a naïve to memory phenotype, thus demonstrating priming of antigen-specific T-cells. Signs of clinical activity were observed, including four stable diseases according to IrRC and vitiligoïd lesions. Four patients were still alive at week 48. We also demonstrate the in vitro enhancement of specific T cell expansion induced by the synergistic combination of peptide-loaded PDC line with anti-PD-1, as compared to peptide-loaded PDC line alone. Taken together, these clinical observations demonstrate the ability of the PDC line based-vaccine to prime and expand antitumor CD8+ responses in cancer patients. Further trials should test the combination of this vaccine with immune checkpoint inhibitors.

T-cell receptor diversity as a prognostic biomarker in melanoma patients.

There is increasing evidence that T-cell receptor (TCR) repertoire diversity can be a predictive biomarker of immune responses in cancer patients. However, the characteristics of the T-cell repertoire together with its prognostic significance in melanoma patients and impact on disease progression remain unknown. We investigated the combinatorial TCR repertoire diversity by semi-quantitative multi-N-plex PCR in peripheral blood samples from 44 melanoma patients together with seven matched metastatic lymph nodes and explored its potential predictive value on clinical prognosis. The diversity was quantified by calculating both richness (number of different specificities) and evenness (relative abundance of the different specificities). Our results revealed that a higher TCR repertoire diversity in blood of patients was associated with a longer PFS, while divpenia (low repertoire diversity) was linked with poor prognosis. The diversity was significantly higher in patients undergoing late relapse and long survival compared to patients who progressed rapidly. Interestingly, the TCR repertoire diversity in tumor may have a potential prognostic value. Thus, our study highlights that the TCR repertoire diversity is a prognostic indicator of clinical outcome in patients with melanoma.

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display.

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.