Multiple Dendritic Cells Research Articles

Role of Thymic Stromal Lymphopoietin in the Pathophysiology of Asthma and Clinical and Biological Effects of Blockade With Tezepelumab.

The airway epithelium is the first line of defense of the respiratory system against the external environment. It plays an active role in the initiation of immune and allergic responses against potential hazards. Among the various specialized cells and cytokines that participate in epithelium-induced responses, alarmins are particularly interesting, given their ample role in mediating T2 and non-T2 inflammatory mechanisms involved in the pathogenesis of asthma. Thymic stromal lymphopoietin (TSLP) is an alarmin with broad effects in asthma that result from its widespread action on multiple cell types, including eosinophils, mast cells, dendritic cells, and group-2 innate lymphoid cells. Its role in allergy-mediated responses, eosinophilic inflammation, airway hyperresponsiveness, mucus hyperproduction, viral tolerance, and airway remodeling is of the utmost importance, as more comprehensive asthma assessments have been developed to explore these pathogenic features. Therefore, blockade with targeting molecules, such as monoclonal antibodies, has emerged as a promising therapeutic option, particularly in patients with multiple pathogenic pathways. In this review, we examine the roles of alarmins (mainly TSLP) in the pathogenesis of asthma and clinical expression and discuss the effects of inhibiting TSLP on several inflammatory and clinical outcomes. We also review the literature supporting treatment with anti-TSLP biologics and the unanswered questions and unmet needs associated with targeting alarmins in asthma.

CD91 and its ligand gp96 confer cross-priming capabilities to multiple APCs during immune responses to nascent, emerging tumors.

During cancer immunosurveillance, dendritic cells (DCs) play a central role in orchestrating T-cell responses against emerging tumors. Capture of miniscule amounts of antigen along with tumor-initiated costimulatory signals can drive maturation of DCs. Expression of CD91 on DCs is essential in cross-priming of T-cell responses in the context of nascent tumors. Multiple DC and macrophage subsets express CD91 and engage tumor-derived gp96 to initiate antitumor immune responses, yet the specific CD91+ antigen-presenting cells (APCs) that are required for T-cell cross-priming during cancer immunosurveillance are unknown. In this study, we determined that CD91 expression on type 1 conventional DCs (cDC1) is necessary for cancer immunosurveillance. Specifically, CD91-expressing cDC1 were found to capture the CD91 ligand gp96 from tumors and, upon migration to the lymph nodes, distribute gp96 among lymph-node resident APCs. However, cDC1 that captured tumor-derived gp96 only provided early tumor control, while sustained and long-term tumor rejection was bestowed to the host by other gp96+ cross-priming DCs. We further found that the CD91-induced transcriptome in APCs promoted cross-priming of T-cell responses while downregulating immune regulatory pathways. Our results show an elaborate and synchronized division of labor of APCs in the successful elimination of cancer cells via CD91. We predict that the specialized functions of APC subsets can be harnessed for immunotherapy of disease.

Mannosylated STING Agonist Drugamers for Dendritic Cell-Mediated Cancer Immunotherapy.

The Stimulator of Interferon Genes (STING) pathway is a promising target for cancer immunotherapy. Despite recent advances, therapies targeting the STING pathway are often limited by routes of administration, suboptimal STING activation, or off-target toxicity. Here, we report a dendritic cell (DC)-targeted polymeric prodrug platform (polySTING) that is designed to optimize intracellular delivery of a diamidobenzimidazole (diABZI) small-molecule STING agonist while minimizing off-target toxicity after parenteral administration. PolySTING incorporates mannose targeting ligands as a comonomer, which facilitates its uptake in CD206+/mannose receptor+ professional antigen-presenting cells (APCs) in the tumor microenvironment (TME). The STING agonist is conjugated through a cathepsin B-cleavable valine-alanine (VA) linker for selective intracellular drug release after receptor-mediated endocytosis. When administered intravenously in tumor-bearing mice, polySTING selectively targeted CD206+/mannose receptor+ APCs in the TME, resulting in increased cross-presenting CD8+ DCs, infiltrating CD8+ T cells in the TME as well as maturation across multiple DC subtypes in the tumor-draining lymph node (TDLN). Systemic administration of polySTING slowed tumor growth in a B16-F10 murine melanoma model as well as a 4T1 murine breast cancer model with an acceptable safety profile. Thus, we demonstrate that polySTING delivers STING agonists to professional APCs after systemic administration, generating efficacious DC-driven antitumor immunity with minimal side effects. This new polymeric prodrug platform may offer new opportunities for combining efficient targeted STING agonist delivery with other selective tumor therapeutic strategies.

Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy.

Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3+ DC (cDC1) and CD1c/BDCA1+ DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy.

A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas

BackgroundCell-based immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells.MethodsIn this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18–27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily.ResultsFrom October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months.ConclusionsOverall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended.Trial registrationClinicalTrials.gov, NCT04074564.

The safety and anti-tumor effect of multiple peptides-pulsed dendritic cells combined with induced specific cytotoxic T lymphocytes for patients with solid tumors

ObjectiveThe aim of this study was to explore the safety and efficacy of multiple peptide-pulsed autologous dendritic cells (DCs) combined with cytotoxic T lymphocytes (CTLs) in patients with cancer.MethodsFive patients diagnosed with cancer between November 2020 and June 2021 were enrolled and received DC-CTLs therapy. Peripheral blood was collected and antigenic peptides were analyzed. The phenotype and function of DC-CTLs and the immune status of patients were detected using flow cytometry or IFN-γ ELISPOT analysis.ResultsDCs acquired a mature phenotype and expressed high levels of CD80, CD86, CD83, and HLA-DR after co-culture with peptides, and the DC-CTLs also exhibited high levels of IFN-γ. Peripheral blood mononuclear cells from post-treatment patients showed a stronger immune response to peptides than those prior to treatment. Importantly, four of five patients maintained a favorable immune status, of which one patient’s disease-free survival lasted up to 28.2 months. No severe treatment-related adverse events were observed.ConclusionOur results show that multiple peptide-pulsed DCs combined with CTLs therapy has manageable safety and promising efficacy for cancer patients, which might provide a precise immunotherapeutic strategy for cancer.

Genome-wide open reading frame profiling identifies fibroblast growth factor signaling as a driver of PD-L1 expression in head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCC) are associated with significant treatment-related morbidity and poor disease-free and disease-specific survival, especially in the recurrent and metastatic (R/M HNSCC) setting. Inhibition of the programmed death-1/ligand-1 (PD-1/PD-L1) immune checkpoint is accepted as a first-line treatment strategy for R/M HNSCC and has expanded into the neoadjuvant, definitive, and adjuvant settings. To understand cellular signals modulating the PD-L1 in HNSCC, we profiled a HNSCC cell-line with a genome-wide open reading frame (ORF) library of 17,000 individual constructs (14,000 unique genes). We identified 335 ORFs enriched in PD-L1high cells and independently validated five of these ORFs (FGF6, IL17A, CD300C, KLR1C and NFKBIA) as drivers of PD-L1 upregulation. We showed that exogenous FGF ligand is sufficient to induce PD-L1 expression in multiple HNSCC cell lines and human immature dendritic cells. Accordingly, overexpression of FGFR1, FGFR3 or the FGFR3 S249C and D786N mutants common to HNSCC tumors also induced PD-L1 overexpression on tumor cells. Small molecule inhibition of FGF signaling abrogated PD-L1 upregulation in these models and also blocked “classical” IFNγ-regulated PD-L1 expression in a STAT1-independent manner. Finally, we found that FGF specifically upregulated a glycosylated form of PD-L1 in our study, and exogenous FGF led to concomitant upregulation of glycosyltransferases that may stabilize PD-L1 on the surface of HNSCC cells. Taken together, our study supports a potential role for FGF/FGFR pathway signaling as a mechanism driving immune escape and rationalizes further exploration of novel combination therapies to improve clinical responses to PD-1/PD-L1 axis inhibition in HNSCC.

OS08.4.A Analysis of melanoma brain metastasis immune microenvironment through multiplex gene expression profiling

Abstract Background Novel immunotherapies based on targeting of specific immune checkpoints enabled a significant improvement of melanoma outcome, but melanoma brain metastases (BM) remain an unmet oncological need with an overall 2-year survival rate lower than 10%. Tumour immune microenvironment has been demonstrated to play a key role in BM establishment and development, but data regarding the specific milieu of melanoma BM is limited. Material and Methods Gene expression profiles of 55 samples of primary melanoma and BM were evaluated using the nCounter PanCancer IO 360 Panel (NanoString Technologies) targeting 770 mRNA involved in tumor immune microenvironment modulation. The case series consisted of 10 primary melanomas and their 10 matched BM, 25 unmatched BM, and 10 locally advanced control melanomas without evidence of BM after >5 year follow up. Results Among BM samples, most patients (25/45) were males and median age at BM diagnosis was 61,2 years with a median time to BM development of 2,1 years. Median OS from BM diagnosis was 1,3 years. Several genes resulted significantly downregulated in BM compared to primary melanomas, including SERPINB5 (p<0.001), ARG1 (p=0.0067), S100A8 (p<0.001), S100A9 (p<0.001), S100A12 (p=0.0037), IL1RN (p=0.0012), CCL21 (p=0.0012), CCL22 (p=0.0012) and CCL13 (p=0.037) and SELE (p=0.026); conversely, C7 was upregulated (p<0.001). Downregulated signatures in BM involved those associated with multiple immune cell populations, including neutrophils, dendritic cells, mast cells and Treg, as well as inflammatory chemokines, the CTLA4 immune checkpoint and ARG1 enzyme function; conversely, MAGEs-related signature was upregulated. Comparison between primary melanomas which developed BM and those which did not showed a significant overexpression of RRM2 (p=0.0247) and TNFRSF1A (p=0.032) genes in the latter group and an upregulation of the PD-1 pathway. Analysis according to tumour mutational status showed an upregulation of signatures associated with inflammatory chemokines, dendritic and myeloid cells and neutrophils. No differences were observed according to time to BM development and survival from BM diagnosis. Conclusion Our findings show that melanoma BM harbor distinct immunosuppressive mechanisms compared to primary tumors: this data elicits the importance of investigating the heterogeneity of BM microenvironment. Genes and pathways selectively overexpressed or downregulated in melanoma BM should be validated to be possibly considered as novel therapeutic targets.

Expansion of Phenotypically Altered Dendritic Cell Populations in the Small Airways and Alveolar Parenchyma in Patients with Chronic Obstructive Pulmonary Disease

Contrasting the antigen-presenting dendritic cells (DCs) in the conducting airways, the alveolar DC populations in human lungs have remained poorly investigated. Consequently, little is known about how alveolar DCs are altered in diseases such as chronic obstructive pulmonary disease (COPD). This study maps multiple tissue DC categories in the distal lung across COPD severities. Specifically, single-multiplex immunohistochemistry was applied to quantify langerin/CD207+, CD1a+, BDCA2+, and CD11c+ subsets in distal lung compartments from patients with COPD (GOLD stage I–IV) and never-smoking and smoking controls. In the alveolar parenchyma, increased numbers of CD1a+langerin− (p < 0.05) and BDCA-2+ DCs (p < 0.001) were observed in advanced COPD compared with controls. Alveolar CD11c+ DCs also increased in advanced COPD (p < 0.01). In small airways, langerin+ and BDCA-2+ DCs were also significantly increased. Contrasting the small airway DCs, most alveolar DC subsets frequently extended luminal protrusions. Importantly, alveolar and small airway langerin+ DCs in COPD lungs displayed site-specific marker profiles. Further, multiplex immunohistochemistry with single-cell quantification was used to specifically profile langerin DCs and reveal site-specific expression patterns of the maturation and activation markers S100, fascin, MHC2, and B7. Taken together, our results show that clinically advanced COPD is associated with increased levels of multiple alveolar DC populations exhibiting features of both adaptive and innate immunity phenotypes. This expansion is likely to contribute to the distal lung immunopathology in COPD patients.

Deconstructing cold-induced brown adipocyte neogenesis in mice.

Cold exposure triggers neogenesis in classic interscapular brown adipose tissue (iBAT) that involves activation of β1-adrenergic receptors, proliferation of PDGFRA+ adipose tissue stromal cells (ASCs), and recruitment of immune cells whose phenotypes are presently unknown. Single-cell RNA-sequencing (scRNA-seq) in mice identified three ASC subpopulations that occupied distinct tissue locations. Of these, interstitial ASC1 were found to be direct precursors of new brown adipocytes (BAs). Surprisingly, knockout of β1-adrenergic receptors in ASCs did not prevent cold-induced neogenesis, whereas pharmacological activation of the β3-adrenergic receptor on BAs was sufficient, suggesting that signals derived from mature BAs indirectly trigger ASC proliferation and differentiation. In this regard, cold exposure induced the delayed appearance of multiple macrophage and dendritic cell populations whose recruitment strongly correlated with the onset and magnitude of neogenesis across diverse experimental conditions. High-resolution immunofluorescence and single-molecule fluorescence in situ hybridization demonstrated that cold-induced neogenesis involves dynamic interactions between ASC1 and recruited immune cells that occur on the micrometer scale in distinct tissue regions. Our results indicate that neogenesis is not a reflexive response of progenitors to β-adrenergic signaling, but rather is a complex adaptive response to elevated metabolic demand within brown adipocytes.

Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Optimal CD8+ T-cell memory formation following subcutaneous cytomegalovirus infection requires virus replication but not early dendritic cell responses.

SummaryCytomegalovirus (CMV) induction of large frequencies of highly functional memory T cells has attracted much interest in the utility of CMV‐based vaccine vectors, with exciting preclinical data obtained in models of infectious diseases and cancer. However, pathogenesis of human CMV (HCMV) remains a concern. Attenuated CMV‐based vectors, such as replication‐ or spread‐deficient viruses, potentially offer an alternative to fully replicating vectors. However, it is not well understood how CMV attenuation impacts vector immunogenicity, particularly when administered via relevant routes of immunization such as the skin. Herein, we used the murine cytomegalovirus (MCMV) model to investigate the impact of vector attenuation on T‐cell memory formation following subcutaneous administration. We found that the spread‐deficient virus (ΔgL‐MCMV) was impaired in its ability to induce memory CD8+ T cells reactive to some (M38, IE1) but not all (IE3) viral antigens. Impaired‐memory T‐cell development was associated with a preferential and pronounced loss of polyfunctional (IFN‐γ+ TNF‐α+) T cells and also reduced accumulation of TCF1+ T cells, and was not rescued by increasing the dose of replication‐defective MCMV. Finally, whilst vector attenuation reduced dendritic cell (DC) recruitment to skin‐draining lymph nodes, systematic depletion of multiple DC subsets during acute subcutaneous MCMV infection had a negligible impact on T‐cell memory formation, implying that attenuated responses induced by replication‐deficient vectors were likely not a consequence of impaired initial DC activation. Thus, overall, these data imply that the choice of antigen and/or cloning strategy of exogenous antigen in combination with the route of immunization may influence the ability of attenuated CMV vectors to induce robust functional T‐cell memory.

Multiple Myeloma and Dendritic Cell Vaccines

Despite advances in the treatment of multiple myeloma, most of patients after its completion retain minimal residual disease (MRD-positive status), which increases the risk of relapse. Antigen-specific immunotherapy of tumors contributes to improving the clinical outcomes in such patients by the killing of cancer drug resistant clone of tumor cells without any damage to normal tissues. Dendritic cells (DC) are antigen-presenting elements with the main function of antigen-capturing, processing, and presenting them to naive T-lymphocytes for the activation of immune response against the captured antigen. The unique ability of DC to activate T-helpers and cytotoxic T-lymphocytes as well as to target thereby the immune reactions was used in developing DC-based tumor immunotherapy. This approach suggests the implementation of the so-called ‘DC-vaccines’. The clinical trials performed by now also showed the results of using DC-vaccines in various tumors including hematological ones. On the whole, according to the studies DC-vaccines are characterized by satisfactory safety profile, moderate immunological activity, and moderate clinical efficacy. The present review provides the results of clinical trials dealing with the use of DC-based vaccines in multiple myeloma patients. Besides, the potentials of improving the clinical efficacy of this therapy are discussed.

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses.

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126−35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors.

Computational Models Accurately Predict Multi-Cell Biomarker Profiles in Inflammation and Cancer

Individual computational models of single myeloid, lymphoid, epithelial, and cancer cells were created and combined into multi-cell computational models and used to predict the collective chemokine, cytokine, and cellular biomarker profiles often seen in inflamed or cancerous tissues. Predicted chemokine and cytokine output profiles from multi-cell computational models of gingival epithelial keratinocytes (GE KER), dendritic cells (DC), and helper T lymphocytes (HTL) exposed to lipopolysaccharide (LPS) or synthetic triacylated lipopeptide (Pam3CSK4) as well as multi-cell computational models of multiple myeloma (MM) and DC were validated using the observed chemokine and cytokine responses from the same cell type combinations grown in laboratory multi-cell cultures with accuracy. Predicted and observed chemokine and cytokine responses of GE KER + DC + HTL exposed to LPS and Pam3CSK4 matched 75% (15/20, p = 0.02069) and 80% (16/20, P = 0.005909), respectively. Multi-cell computational models became ‘personalized’ when cell line-specific genomic data were included into simulations, again validated with the same cell lines grown in laboratory multi-cell cultures. Here, predicted and observed chemokine and cytokine responses of MM cells lines MM.1S and U266B1 matched 75% (3/4) and MM.1S and U266B1 inhibition of DC marker expression in co-culture matched 100% (6/6). Multi-cell computational models have the potential to identify approaches altering the predicted disease-associated output profiles, particularly as high throughput screening tools for anti-inflammatory or immuno-oncology treatments of inflamed multi-cellular tissues and the tumor microenvironment.

Widespread alterations in the peripheral blood innate immune cell profile in cystic fibrosis reflect lung pathology.

Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTR-targeted drug efficacy.

Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs.

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+ T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

Dendritic Cell Subsets and Effector Function in Idiopathic and Connective Tissue Disease-Associated Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease characterized by an incurable condition of the pulmonary vasculature, leading to increased pulmonary vascular resistance, elevated pulmonary arterial pressure resulting in progressive right ventricular failure and ultimately death. PAH has different underlying causes. In approximately 30–40% of the patients no underlying risk factor or cause can be found, so-called idiopathic PAH (IPAH). Patients with an autoimmune connective tissue disease (CTD) can develop PAH [CTD-associated PAH (CTD-PAH)], suggesting a prominent role of immune cell activation in PAH pathophysiology. This is further supported by the presence of tertiary lymphoid organs (TLOs) near pulmonary blood vessels in IPAH and CTD-PAH. TLOs consist of myeloid cells, like monocytes and dendritic cells (DCs), T-cells, and B-cells. Next to their T-cell activating function, DCs are crucial for the preservation of TLOs. Multiple DC subsets can be found in steady state, such as conventional DCs (cDCs), including type 1 cDCs (cDC1s), and type 2 cDCs (cDC2s), AXL+Siglec6+ DCs (AS-DCs), and plasmacytoid DCs (pDCs). Under inflammatory conditions monocytes can differentiate into monocyte-derived-DCs (mo-DCs). DC subset distribution and activation status play an important role in the pathobiology of autoimmune diseases and most likely in the development of IPAH and CTD-PAH. DCs can contribute to pathology by activating T-cells (production of pro-inflammatory cytokines) and B-cells (pathogenic antibody secretion). In this review we therefore describe the latest knowledge about DC subset distribution, activation status, and effector functions, and polymorphisms involved in DC function in IPAH and CTD-PAH to gain a better understanding of PAH pathology.

Glyco-Dendrimers as Intradermal Anti-Tumor Vaccine Targeting Multiple Skin DC Subsets.

The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4+ and CD8+ T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes.Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (LeY), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN+ monocyte derived DC (moDC) and Langerin+ primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8+ T cell activation in vitro. In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated.Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a+ and CD14+ dermal DC in situ. Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8+ T cell activation.Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8+ T cell responses.

Determination of T Follicular Helper Cell Fate by Dendritic Cells.

T follicular helper (Tfh) cells are a specialized subset of CD4+ T cells that collaborate with B cells to promote and regulate humoral responses. Unlike other CD4+ effector lineages, Tfh cells require interactions with both dendritic cells (DCs) and B cells to complete their differentiation. While numerous studies have assessed the potential of different DC subsets to support Tfh priming, the conclusions of these studies depend heavily on the model and method of immunization used. We propose that the location of different DC subsets within the lymph node (LN) and their access to antigen determine their potency in Tfh priming. Finally, we provide a three-step model that accounts for the ability of multiple DC subsets and related lineages to support the Tfh differentiation program.