BackgroundBlocking programmed cell death protein 1 (PD-1) has become a standard cancer immunotherapy, increasingly used in kidney, liver, or heart transplant recipients who develop skin cancer or hepatocellular carcinoma, despite the increased risk of graft failure or rejection. The mechanism of action of PD-1 blockade relies on stimulating CD8+ T cell activity, but its impact on humoral immunity in general and on alloimmunization in particular remains uncertain.ObjectiveThe aim of this study was to investigate the impact on anti-PD-1 treatment on alloimmunization.MethodsThe effect of anti-PD-1 treatment on the generation of anti-HLA (Human Leucocyte Antigen) antibodies was investigated in 72 patients with non-small cell lung cancer vaccinated with an allogeneic plasmacytoid dendritic cell line (PDC*line; six weekly injections), with or without pembrolizumab administered every three weeks. The kinetics and functionality of the anti-HLA generated were analyzed.ResultsThe results show that 51.4% of the patients developed anti-HLA antibodies, primarily dependent on the vaccine dose. In 60% of cases, the antibody response appeared after the sixth injection, peaked after one month, and then gradually declined over two years. Anti-HLA class II antibodies appeared earlier than class I antibodies. Functional assays demonstrated complement-dependent cytotoxicity against allogeneic B lymphocytes and PDC*line cells in the serum of some patients, with no difference related to treatment. PD-1 blockade did not alter the magnitude, kinetics, or cytotoxic potential of the vaccine-induced humoral response. ConclusionThese results indicate that, during allogeneic human vaccination, PD-1 signaling exerts a limited effect on antibody production and effector function, suggesting a more complex regulatory role in humoral immunity than previously thought.

Plasmacytoid dendritic cells (PDCs) represent a crucial bridge between innate and adaptive immunity as they can recognise viruses and viral-infected cells, secrete type I interferon and prime naive T-cell responses. Like naive T cells, PDCs express CD62L, an essential molecule involved in the trans-endothelial migration process, and are supposed to travel directly from blood to lymph nodes to prime naive T cells. We developed a therapeutic PDC line model (PDC*line) derived from a patient with blastic PDC neoplasm, and showing high antigen-presentation capacities. PDC*line-based vaccine was tested in clinical trials in melanoma and lung cancer. Efficient specific T lymphocyte priming was observed suggesting the efficient migration of injected PDC*line-based vaccine to lymph nodes. Here, we investigated the mechanisms involved in this migration and its regulation. In humanised mice, our results demonstrated the efficient migration of the PDC-based vaccine from blood to lymph nodes and its capacity to prime naive T cells. The expression of CD62L was evaluated following irradiation, which was mandatory in the vaccine manufacturing step and possibly important for its efficacy. CD62L was rapidly shed after irradiation of PDC*line cells and primary PDCs but not of NK-T cells, demonstrating the cell specificity of the mechanism. Our results show that p38 MAP kinase, known to be activated in response to irradiation and ADAM17 metalloprotease are involved in the rapid CD62L shedding. This new pathway could participate to the regulation PDC transmigration through High Endothelial Venules and homing to secondary lymphoid organs in cellular stress conditions.

BACKGROUND:HCC is the most common form of primary liver cancer, and despite recent advances in cancer treatment, it remains associated with poor prognosis and a lack of response to conventional therapies. Immunotherapies have emerged as a promising approach for cancer treatment, especially through the identification of tumor-specific immunogenic epitopes that can trigger a targeted immune response. This study aimed to identify immunogenic epitopes associated with HCC for the development of specific immunotherapies. METHODS:We used high-throughput data screening and bioinformatics tools for antigens and epitope selection. The immunogenicity of the selected epitopes was studied after coculture of peripheral blood mononuclear cells obtained from healthy donors or HCC patients with a plasmacytoid dendritic cell line loaded with the selected peptides. Specific CD8+ T cell amplification and functionality were determined by labeling with tetramers and by IFN-γ and CD107a expression (flow cytometry and ELISpot). RESULTS:We analyzed the transcriptional gene expression landscape of HCC to screen for a set of 16 ectopically expressed genes in a majority of HCC samples. Epitopes predicted to bind to HLA-A*02:01 with high affinity were further validated for their immunogenicity using the previously described plasmacytoid dendritic cell line in ex vivo CD8+ activation assays using patient immune cells. Three out of the 30 tested epitopes, namely FLWGPRALV (MAGE-A3), FMNKFIYEI (AFP), and KMFHTLDEL (LRRC46), elicited a strong T-cell response, in activation assays, degranulation assays, and IFN-γ secretion assays. CONCLUSIONS:These results highlight the potential of these peptides to be considered as targets for immunotherapies. The discovery of such immunogenic epitopes should improve immune-based treatments for liver cancer in combination with the current treatment approach.

The immune function is suspected to play an important role in the health effects of air pollution but it remains poorly investigated in pregnant women. One-week personal measurements of exposure to nitrogen dioxide (NO 2 ), particulate matter with an aerodynamic diameter of ≤ 2.5 µm mass concentration (PM 2.5 ) and PM 2.5 oxidative potential (OP) were assessed in 270 pregnant women from the French cohort SEPAGES. PM filters were analyzed for PM 2.5 OP using the dithiothreitol (DTT) and the ascorbic acid (AA) assays. From a blood sample withdrawn at the end of the exposure measurement week, levels of 29 cytokines and chemokines were measured at baseline and after T cell and dendritic cell activation with phytohemagglutinin (PHA) and resiquimod (R848), respectively. Associations between each air pollutant and each cytokine were assessed using adjusted linear regression models. An increase in NO 2 exposure was associated with higher interleukin 10 (IL-10) and lower PHA-activated tumor necrosis factor (TNF). No association with PM 2.5 concentration was observed, but increased exposure to PM $${\text{OP}}^{{{\text{AA}}}}$$ OP AA was associated with lower baseline and R848-activated IL-8 and increased exposure to PM $${\text{OP}}^{\text{DTT}}$$ OP DTT was associated with higher PHA-activated IL-17A. Our study provides insights into the relationships between air pollution exposure and immune function among pregnant women.

Introduction Les cellules dendritiques (DCs) jouent un rôle crucial dans l’immunité antitumorale. Les tumeurs échappent au contrôle du système immunitaire en détournant les fonctions des DCs. Les fondements d’une telle subversion ne sont que partiellement compris. Les cellules tumorales présentent des motifs glycans aberrants sur les glycoprotéines et les glycolipides de surface. Ces motifs carbohydrates peuvent être détectés par les DCs via les récepteurs lectine de type C (CLR) qui sont essentiels pour façonner et orienter les réponses immunitaires. Nous avons récemment démontré que les cellules tumorales du mélanome présentent un « glycocode » aberrant, et que les DCs circulantes et infiltrant les tumeurs des patients atteints de mélanome présentaient des perturbations majeures dans leurs profils d’expression des CLR. Matériel et méthodes Afin de déterminer si le mélanome, à travers des motifs glycans aberrants, peut exploiter les voies CLR pour détourner les DCs et échapper au contrôle immunitaire, nous avons exploré l’impact des motifs glycans présents de manière aberrante sur les cellules de mélanome avec des néoglycoprotéines (NeoGP) fonctionnalisées avec Gal, Man, GalNAc, s-Tn, Fucose [Fuc] et résidus GlcNAc sur les caractéristiques des sous-types de DCs (cDC2, cDC1, pDC). Nous avons examiné la capacité des glycans à se lier aux DCs purifiées et évalué leur impact sur les propriétés basales et les caractéristiques fonctionnelles des DCs par cytométrie en flux, microscopie confocale et analyse des protéines sécrétées. Résultats Les sous-types de DCs se lient et internalisent différentiellement les NeoGP en fonction de la nature du glycan. Fuc remodèle directement l’expression des marqueurs d’activation et des points de contrôle immunitaires, ainsi que le profil de sécrétion de cytokines/chimiokines des sous-types de DCs. De plus, les NeoGP interférent avec la signalisation des récepteurs TLR et induisent une réponse altérée des DCs à une stimulation TLR ultérieure, atténuant les médiateurs antitumoraux tout en favorisant la sécrétion de facteurs pro-tumoraux. Nous avons aussi démontré que les sous-types de DCs peuvent lier NeoGP via les CLR et identifié GalNAc/MGL et s-Tn/CLEC2 comme candidats potentiels. De plus, le dysfonctionnement des DCs induit par les motifs glycans associés à la tumeur peut être inversé en interférant avec l’axe glycan/CLR. Discussion Ces résultats révèlent que l’axe glycan/CLR est un point de contrôle prometteur à exploiter afin de remodeler l’immunité antitumorale tout en inhibant les voies immunosuppressives déclenchées par les motifs glycans aberrants. Conclusion Agir sur l’axe glycan/lectin permettrait d’épargner les DCs du détournement par les tumeurs et d’améliorer l’évolution clinique des patients atteints de cancer.

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 peptideloaded PDC*line induced robust CD8+ responses with total amplification rates that led up to a 198-fold increase in peptidespecific 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-g 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. The Journal of Immunology, 2024, 212: 825-833.

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-1β, 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.

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.

Supplementary figure 7 shows heat map and dot plots illustrating correlations between CD3E and chemokines levels, detailed migration of CD4 and CD8 lymphocytes driven by cell lines, correlations between CD3E and CD1c and between CCL19/CCL22 and APC specific markers.

Supplementary figure 9 shows IL-12 and IL-8 contents in supernatants after activation of PBMC in co-culture with NB cell lines.

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.

Supplementary figure 3 shows flow cytometry gating strategy and raw data in calcium mobilization experiments in all cell subsets

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.

Supplementary figure 5 shows analysis of MYCN level in NB tumors in GSE62564 according to MYCN amplification status

Supplementary figure 8 shows HLA class I level of expression in NB cell lines, and heat map illustrating relations between chemokines, immune cell subsets and HLA class I associated genes levels.

Supplementary figure 6 shows differential CCL2 level according to MYCN amplification status in GSE 3960, and correlations between CCL2 level and immune cell infiltrate

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.