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Human leukocyte antigen (HLA) class I association is a well-established feature of common and uncommon inflammatory diseases, but it is unknown whether it impacts the pathogenesis of these disorders. The "arthritogenic peptide" hypothesis proposed initially for HLA-B27-associated ankylosing spondylitis (AS) seems the most intuitive to serve as a model for other HLA class I-associated diseases, but evidence supporting it has been scarce. Recent technological advances and the discovery of epistatic relationships between disease-associated HLA class I and endoplasmic reticulum aminopeptidase (ERAP) coding variants have led to the generation of new data and conceptual approaches to the problem requiring its re-examination. Continued success in these endeavors holds promise to resolve a Gordian Knot in human immunobiology. It may ultimately benefit patients by enabling the development of new therapies and precision tools for assessing disease risk and predicting treatment responses., Competing Interests: Declaration of competing interest We have no disclosures relevant to this article to report., (Copyright © 2024. Published by Elsevier Ltd.)

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., (Copyright © 2024 by The American Association of Immunologists, Inc.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manches, Um, Boudier, Maddouri, Lyon-Caen, Bayat, Slama, Philippat, Siroux and Chaperot.)

Virus-like particles (VLPs) are versatile protein-based platforms that can be used as a vaccine platform mainly in infectiology. In the present work, we compared a previously designed, non-infectious, adenovirus-inspired 60-mer dodecahedric VLP to display short epitopes or a large tumor model antigen. To validate these two kinds of platforms as a potential immuno-stimulating approach, we evaluated their ability to control melanoma B16-ovalbumin (OVA) growth in mice. A set of adjuvants was screened, showing that polyinosinic-polycytidylic acid (poly(I:C)) was well suited to generate a homogeneous cellular and humoral response against the desired epitopes. In a prophylactic setting, vaccination with the VLP displaying these epitopes resulted in total inhibition of tumor growth 1 month after vaccination. A therapeutic vaccination strategy showed a delay in grafted tumor growth or its total rejection. If the "simple" epitope display on the VLP is sufficient to prevent tumor growth, then an improved engineered platform enabling display of a large antigen is a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way for its potential utilization in humans as an off-the-shelf vaccine., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Virus-like particles constitute versatile vectors that can be used as vaccine platforms in many fields from infectiology and more recently to oncology. We previously designed non-infectious adenovirus-inspired 60-mer dodecahedric virus-like particles named ADDomers displaying on their surface either a short epitope or a large tumor/viral antigen. In this work, we explored for the first time the immunogenicity of ADDomers exhibiting melanoma-derived tumor antigen/epitope and their impact on the features of human dendritic cell (DC) subsets. We first demonstrated that ADDomers displaying tumor epitope/antigen elicit a strong immune-stimulating potential of human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific motifs to de-target epithelial cells and improve DCs' addressing. The improved engineered platform making it possible to display large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential utilization in humans as an off-the-shelf vaccine.

Objectives: The endoplasmic reticulum aminopeptidase ( ERAP1 ) haplotype Hap10 encodes for a variant allotype of the endoplasmic reticulum (ER)-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity. This haplotype recessively confers the highest risk for Behçet's diseases (BD) currently known, but only in carriers of HLA-B*51 , the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact., Methods: We genotyped and immune phenotyped a cohort of 26 untreated Turkish BD subjects and 22 healthy donors, generated CRISPR-Cas9 ERAP1 KOs from HLA-B*51 + LCL, analysed the HLA class I-bound peptidome for peptide length differences and assessed immunogenicity of genome-edited cells in CD8 T cell co-culture systems., Results: Allele frequencies of ERAP1-Hap10 were similar to previous studies. There were frequency shifts between antigen-experienced and naïve CD8 T cell populations of carriers and non-carriers of ERAP1-Hap10 in an HLA-B*51 background. ERAP1 KO cells showed peptidomes with longer peptides above 9mer and significant differences in their ability to stimulate alloreactive CD8 T cells compared with wild-type control cells., Conclusions: We demonstrate that hypoactive ERAP1 changes immunogenicity to CD8 T cells, mediated by an HLA class I peptidome with undertrimmed peptides. Naïve/effector CD8 T cell shifts in affected carriers provide evidence of the biological relevance of ERAP1-Hap10/HLA-B*51 at the cellular level and point to an HLA-B51-restricted process. Our findings suggest that variant ERAP1-Hap10 partakes in BD pathogenesis by generating HLA-B51-restricted peptides, causing a change in immunodominance of the ensuing CD8 T cell response., Competing Interests: Competing interests: GH, YO, and JN have received grant support as specified in the Funding section. GH has received research support from Celgene as well as speaker fees from Abbvie, Celgene, Novartis, and UCB Pharma, all paid to Istanbul University-Cerrahpasa. YO has received speaker fees from Pfizer, Novartis, and UCB Pharma, paid to Istanbul University-Cerrahpasa. JN has received lecture honoraria from Northwestern University, and Harvard University., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: L. Chaperot reports a patent to plasmacytoid dendritic cell line used in active or adoptive cell therapy issued and licensed. No disclosures were reported by the other authors., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Obstructive sleep apnea (OSA) syndrome is characterized by chronic intermittent hypoxia and is associated with an increased risk of all-cause mortality, including cancer mortality. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, characterized by increasing incidence and high mortality. However, the link between HCC and OSA-related chronic intermittent hypoxia remains unclear. Herein, we used a diethylnitrosamine (DEN)-induced HCC model to investigate whether OSA-related chronic intermittent hypoxia has an impact on HCC progression. To elucidate the associated mechanisms, we first evaluated the hypoxia status in the DEN-induced HCC model. Next, to simulate OSA-related intermittent hypoxia, we exposed cirrhotic rats with HCC to intermittent hypoxia during six weeks. We performed histopathological, immunohistochemical, RT-qPCR, and RNA-seq analysis. Chronic DEN injections strongly promoted cell proliferation, fibrosis, disorganized vasculature, and hypoxia in liver tissue, which mimics the usual events observed during human HCC development. Intermittent hypoxia further increased cell proliferation in DEN-induced HCC, which may contribute to an increased risk of HCC progression. In conclusion, our observations suggest that chronic intermittent hypoxia may be a factor worsening the prognosis of HCC.

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., Competing Interests: The authors report no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. The majority of HCC cases are associated with liver fibrosis or cirrhosis developing from chronic liver injuries. The immune system of the liver contributes to the severity of tissue damage, the establishment of fibrosis and the disease's progression towards HCC. Herein, we provide a detailed characterization of the DEN-induced HCC rat model during fibrosis progression and HCC development with a special focus on the liver's inflammatory microenvironment. Fischer 344 male rats were treated weekly for 14 weeks with intra-peritoneal injections of 50 mg/kg DEN. The rats were sacrificed before starting DEN-injections at 0 weeks, after 8 weeks, 14 weeks and 20 weeks after the start of DEN-injections. We performed histopathological, immunohistochemical, RT-qPCR, RNA-seq and flow cytometry analysis. Data were compared between tumor and non-tumor samples from the DEN-treated versus untreated rats, as well as versus human HCCs. Chronic DEN injections lead to liver damage, hepatocytes proliferation, liver fibrosis and cirrhosis, disorganized vasculature, and a modulated immune microenvironment that mimics the usual events observed during human HCC development. The RNA-seq results showed that DEN-induced liver tumors in the rat model shared remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.

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., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as "Treg" therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4 + CD25 + T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

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., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Despite major advances in rheumatoid arthritis outcome, not all patients achieve remission, and there is still an unmet need for new therapeutic approaches. This study aimed at evaluating in a pre-clinical murine model the efficacy of extracorporeal photopheresis (ECP) in the treatment of rheumatoid arthritis, and to provide a relevant study model for dissecting ECP mechanism of action in autoimmune diseases., Methods: DBA/1 mice were immunized by subcutaneous injection of bovine collagen type II, in order to initiate the development of collagen-induced arthritis (CIA). Arthritic mice received 3 ECP treatments every other day, with psoralen + UVA-treated (PUVA) spleen cells obtained from arthritic mice. Arthritis score was measured, and immune cell subsets were monitored., Results: ECP-treated mice recovered from arthritis as evidenced by a decreasing arthritic score over time. Significant decrease in the frequency of Th17 cells in the spleen of treated mice was observed. Interestingly, while PUVA-treated spleen cells from healthy mouse had no effect, PUVA-treated arthritic mouse derived-spleen cells were able to induce control of arthritis development., Conclusions: Our results demonstrate that ECP can control arthritis in CIA-mice, and clarifies ECP mechanisms of action, showing ECP efficacy and Th17 decrease only when arthritogenic T cells are contained within the treated sample. These data represent a pre-clinical proof of concept supporting the use of ECP in the treatment of RA in Human.

Introduction: The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training., Methods: We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets., Results: We developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health., Conclusions: We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets., (© 2019 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

γδT cells hold a pivotal role in tumor immunosurveillance through their prompt activation and cytokine secretion, their ability to kill tumor cells in an Human Leukocyte Antigen (HLA)-unrestricted manner, and their combination of features of both innate and adaptive immunity. These unique properties and functional plasticity render them very attractive both as targets and vectors for cancer immunotherapy. Yet, these potent and fascinating antitumor effectors have not been extensively explored in melanoma. We provided here a detailed investigation of the phenotypic and functional properties of circulating and tumor-infiltrating γδT cells in melanoma patients, and their impact on clinical evolution. High proportions of circulating- and tumor-infiltrating γδT and δ2+ subset were associated with better clinical outcome. We reported however that circulating and tumor-infiltrating γδT cells from melanoma patients displayed an altered expression of NCR, KIR, and immune checkpoints, and identified NKp44, PD1, 41BB/41BBL, TIM3, and LAG3 as crucial checkpoints allowing immune escape and tumor progression. Notably, melanoma drastically impaired the ability of γδT cells to exhibit activation molecules, secrete cytokines, and display cytotoxicity toward melanoma in response to stimulation with phosphoantigens. It drove them toward regulatory and Th17 profiles associated with poor clinical outcomes. Our study highlights that melanoma hijacked γδT cells to escape from immune control, and revealed that circulating and tumor-infiltrating γδT cell features are promising potential biomarkers of clinical evolution. Such understanding of the physiopathology of γδT cells may help designing new therapeutic approaches exploiting the antitumor potential of γδT cells while counteracting their skewing by tumors to improve patient outcomes.

Objective: Apoptotic cell receptors contribute to the induction of tolerance by modulating dendritic cell function following the uptake of apoptotic cells or microparticles. Dendritic cells that have bound or ingested apoptotic cells produce only low amounts of pro-inflammatory cytokines and fail to prime effector T cell responses. Specifically, ligation of the apoptotic cell receptor CR3 (CD11 b/CD18) on human monocyte-derived dendritic cells (moDC) down-modates proinflammatory cytokine secretion, but the consequences for human Th17 cell homeostasis and effector responses remain unknown. Here, we aimed to establish whether CD11b-ligated moDC modulate Th17 cell effector reponses to assess their potential for future use in moDC-based suppressive immunotherapy., Methods: We generated a bead-based surrogate system to target CD11b on monocyte-derived human dendritic cells and examined the effects of CD11b ligation on Th17-skewing cytokine secretion, priming, expansion and functional plasticity in DC/T cell co-culture systems at the poly- and monoclonal level., Results: We show that Th17 cell expansion within the human memory CD4 + T cell compartment was efficiently constricted by targeting the CD11b receptor on moDC. This tolerogenic capacity was primarily dependent on cytokine skewing. Furthermore, ligation of CD11b on healthy homozygous carriers of the rs11143679 (ITGAM) variant - a strong genetic susceptibility marker for human systemic lupus erythematosus - also down-modulated the secretion of Th17-skewing cytokines., Conclusion: Overall, our findings underline the potential of targeted CD11b ligation on human dendritic cells for the engineering of suppressive immunotherapy for Th17-related autoimmune disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Plasmacytoid dendritic cells (pDC) are innate immune cells that sense viral nucleic acids through endosomal Toll-like receptor (TLR) 7/9 to produce type I interferon (IFN) and to differentiate into potent antigen presenting cells (APC). Engagement of TLR7/9 in early endosomes appears to trigger the IRF7 pathway for IFN production whereas engagement in lysosomes seems to trigger the NF-κB pathway for maturation into APC. We showed previously that HIV-1 (HIV) localizes predominantly to early endosomes, not lysosomes, and mainly stimulate IRF7 rather than NF-κB signaling pathways in pDC. This divergent signaling may contribute to disease progression through production of pro-apoptotic and pro-inflammatory IFN and inadequate maturation of pDCs. We now demonstrate that HIV virions may be re-directed to lysosomes for NF-κB signaling by either pseudotyping HIV with influenza hemagglutinin envelope or modification of CD4 mediated-intracellular trafficking. These data suggest that HIV envelope-CD4 receptor interactions drive pDC activation toward an immature IFN producing phenotype rather than differentiation into a mature dendritic cell phenotype.

Although the major targets of HIV infection are CD4⁺ T cells, dendritic cells (DCs) represent a crucial subset in HIV infection because they influence viral transmission and target cell infection and presentation of HIV antigens. DCs are potent antigen-presenting cells that can modulate antiviral immune responses. Through secretion of inflammatory cytokines and interferons (IFNs), DCs also alter T cell proliferation and differentiation, participating in the immune dysregulation characteristic of chronic HIV infection. Their wide distribution in close proximity with the mucosal epithelia makes them one of the first cell types to encounter HIV during sexual transmission. We discuss here the multiple roles that DCs play at different stages of HIV infection, emphasizing their relevance to HIV pathology and progression., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF(V600) mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF(V600) mutant melanoma., Methods: We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib., Results: Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF(V600E) mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations., Conclusions: BRAF(V600E) mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.

Plasmacytoid dendritic cells (pDCs) are innate immune cells that are specialized to produce interferon-alpha (IFNα) and participate in activating adaptive immune responses. Although IFNα inhibits HIV-1 (HIV) replication in vitro, pDCs may act as inflammatory and immunosuppressive dendritic cells (DCs) rather than classical antigen-presenting cells during chronic HIV infection in vivo, contributing more to HIV pathogenesis than to protection. Improved understanding of HIV-pDC interactions may yield potential new avenues of discovery to prevent HIV transmission, to blunt chronic immune activation and exhaustion, and to enhance beneficial adaptive immune responses. In this chapter we discuss pDC biology, including pDC development from progenitors, trafficking and localization of pDCs in the body, and signaling pathways involved in pDC activation. We focus on the role of pDCs in HIV transmission, chronic disease progression and immune activation, and immunosuppression through regulatory T cell development. Lastly, we discuss potential future directions for the field which are needed to strengthen our current understanding of the role of pDCs in HIV transmission and pathogenesis.

Objective: Myeloid dendritic cell (mDC) dysfunction during HIV infection may hinder the formation of both innate and adaptive immune responses and contribute to pathogenesis. Our objective was to determine whether circulating factors during chronic HIV infection impair mDC function with respect to secretion of IL-12, a pro-Th1 cytokine, and T-cell stimulatory capacity. Particular focus was placed on the effect of combination antiretroviral therapy (cART) and the role of HIV itself on mDC function., Methods: Monocyte-derived DC (moDC) from uninfected donors were exposed to plasma from HIV-infected individuals before Toll-like receptor (TLR) stimulation. Cytokine secretion was measured via cytokine bead arrays, and T-cell proliferation and IFNγ secretion was evaluated after coculture with naive CD4 T cells. Expression of genes central to TLR-mediated signal transduction was analyzed via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) arrays and western blot., Results: Exposure of monocyte-derived DC to plasma from untreated HIV-infected donors suppressed secretion of IL-12, and impaired Th1-skewing of CD4 T cells. The suppressive effect was less by plasma donors receiving cART. Removal of virus from plasma did not relieve suppression nor was IL-12 secretion decreased on addition of HIV to control plasma. On a transcriptional level, decreased expression of IKKβ, a key regulator in the TLR/NF-kappaB signaling pathway, corresponded to suppressed cytokine secretion., Conclusions: Plasma factors during chronic HIV infection impair mDC function in a manner that likely impacts the formation of immune responses to HIV, opportunistic pathogens, and vaccines. Despite partial alleviation by cART, this suppression was not directly mediated by HIV.

HIV modulates plasmacytoid dendritic cell (pDC) activation via Toll-like receptor 7, inducing type I IFN and inflammatory cytokines. Simultaneously, pDCs up-regulate the expression of indoleamine 2,3 dioxygenase (IDO), which is essential for the induction of regulatory T cells (Tregs), which function to down-modulate immune activation. Here we demonstrate the crucial importance of the noncanonical NF-κB pathway in the establishment of this immunoregulatory phenotype in pDCs. In response to HIV, the noncanonical NF-κB pathway directly induces IDO and involves the recruitment of TNF receptor-associated factor-3 to the Toll-like receptor/MyD88 complex, NF-κB-inducing kinase-dependent IκB kinase-α activation, and p52/RelB nuclear translocation. We also show that pDC-induced Tregs can inhibit conventional DC (cDC) maturation partially through cytotoxic T-lymphocyte antigen (CTLA)-4 engagement. Furthermore, CTLA-4 induces IDO in cDCs in a NF-κB-inducing kinase-dependent way. These CTLA-4-conditioned cDCs can in turn induce Treg differentiation in an IDO-dependent manner. Thus, the noncanonical NF-κB pathway is integral in controlling immunoregulatory phenotypes of both pDCs and cDCs.

Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive.

CpG motifs in an A/U context have been preferentially eliminated from classical H1N1 influenza virus genomes during virus evolution in humans. The hypothesis of the current work is that CpG motifs in a uracil context represent sequence patterns with the capacity to induce an immune response, and the avoidance of this immunostimulatory signal is the reason for the observed preferential decline. To analyze the immunogenicity of these domains, we used plasmacytoid dendritic cells (pDCs). pDCs express pattern recognition receptors, including Toll-like receptor 7 (TLR7), which recognizes guanosine- and uridine-rich viral single-stranded RNA (ssRNA), including influenza virus ssRNA. The signaling through TLR7 results in the induction of inflammatory cytokines and type I interferon (IFN-I), an essential process for the induction of specific adaptive immune responses and for mounting a robust antiviral response mediated by IFN-α. Secretion of IFN-α is also linked to the activation of other immune cells, potentially amplifying the effect of an initial IFN-α secretion. We therefore also examined the role of IFN-α-driven activation of NK cells as another source of selective pressure on the viral genome. We found direct evidence that CpG RNA motifs in a U-rich context control pDC activation and IFN-α-driven activation of NK cells, likely through TLR7. These data provide a potential explanation for the loss of CpG motifs from avian influenza viruses as they adapt to mammalian hosts. The selective decrease of CpG motifs surrounded by U/A may be a viral strategy to avoid immune recognition, a strategy likely shared by highly expressed human immune genes.

Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme degrading the extracellular matrix and overexpressed by many tumors. Here, we documented the presence of MMP-2-specific CD4(+) T cells in tumor-infiltrating lymphocytes (TILs) from melanoma patients. Strikingly, MMP-2-specific CD4(+) T cells displayed an inflammatory T(H)2 profile, i.e., mainly secreting TNF-α, IL-4, and IL-13 and expressing GATA-3. Furthermore, MMP-2-conditioned dendritic cells (DCs) primed naïve CD4(+) T cells to differentiate into an inflammatory T(H)2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 degrades the type I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production. Active MMP-2, therefore, acts as an endogenous type 2 "conditioner" and may play a role in the observed prevalence of detrimental type 2 responses in melanoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Plasmacytoid DCs (pDCs) are innate immune cells that are specialized to produce IFN-α and to activate adaptive immune responses. Although IFN-α inhibits HIV-1 replication in vitro, the production of IFN-α by HIV-activated pDCs in vivo may contribute more to HIV pathogenesis than to protection. We have now shown that HIV-stimulated human pDCs allow for persistent IFN-α production upon repeated stimulation, express low levels of maturation molecules, and stimulate weak T cell responses. Persistent IFN-α production by HIV-stimulated pDCs correlated with increased levels of IRF7 and was dependent upon the autocrine IFN-α/β receptor feedback loop. Because it has been shown that early endosomal trafficking of TLR9 agonists causes strong activation of the IFN-α pathway but weak activation of the NF-κB pathway, we sought to investigate whether early endosomal trafficking of HIV, a TLR7 agonist, leads to the IFN-α-producing phenotype we observed. We demonstrated that HIV preferentially traffics to the early endosome in human pDCs and therefore skews pDCs toward a partially matured, persistently IFN-α-secreting phenotype.

Natural nonhuman primate hosts of SIV do not succumb to AIDS despite significant viral replication, a phenomenon attributed to reduced levels of chronic and deleterious "immune activation." Two studies in this issue of the JCI, by Bosinger et al. and Jacquelin et al., now show that SIV induces vigorous immune activation and upregulation of IFN-stimulated genes in both natural and susceptible hosts, but strikingly, the responses resolve only in the former (see the related articles, beginning on pages 3556 and 3544, respectively). Thus, natural hosts for SIV actively engage mechanisms to abort sustained immune activation and its associated harmful effects.

Among the different subsets of dendritic cells (DC), plasmacytoid dendritic cells (PDC) play a unique role in secreting large amounts of type I interferons upon viral stimulation, but their efficiency as antigen-presenting cells has not been completely characterized. We show here, by flow cytometry, with human primary blood PDC and with a PDC cell line, that PDC display poor endocytic capacity for soluble or cellular antigens when compared to monocyte-derived myeloid DC. However, immature PDC efficiently take up cellular material from live influenza-exposed cells, subsequently mature and cross-present viral antigens very efficiently to specific CD8+ T cells. Therefore, during viral infection PDC not only secrete immunomodulatory cytokines, but also recognize infected cells and function as antigen cross-presenting cells to trigger the anti-viral immune response.

Plasmacytoid DCs (pDCs) have been implicated as crucial cells in antiviral immune responses. On recognizing HIV, they become activated, secreting large amounts of IFN-alpha and inflammatory cytokines, thereby potentiating innate and adaptive antiviral immune responses. Here, we have shown that HIV-stimulated human pDCs can also induce the differentiation of naive CD4+ T cells into Tregs with suppressive function. This differentiation was independent of pDC production of IFN-alpha and primarily dependent on pDC expression of indoleamine 2,3-dioxygenase, which was induced through the TLR/MyD88 pathway, following binding of HIV to CD4 and triggering of TLR7 by HIV genomic RNA. Functionally, the Tregs induced by pDCs were shown to inhibit the maturation of bystander conventional DCs. This study therefore reveals what we believe to be a novel mechanism by which pDC may regulate and potentially limit anti-HIV immune responses.

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non-cytotoxic CD4(+) T cells are important for the induction and maintenance of anti-tumour immunity exerted by cytotoxic CD8(+) T cells. In contrast, the existence of direct anti-tumour, effector cytotoxic CD4(+) T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B-cell non-Hodgkin lymphomas. This study developed an appropriate, autologous follicular B-cell non-Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA-I) deficient B-cell line, and the generation of three autologous anti-tumour cytotoxic CD4(+) T-cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA-I-deficient B-cell line through a classical HLA-II restricted perforin-mediated pathway, but did not lyse the Epstein-Barr virus-infected autologous normal B lymphocytes. All three CD4(+)clones were T-cell receptor Vbeta17-Dbeta1-Jbeta1.2 and exhibited an identical complementarity-determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4(+) cytotoxic cells in immunotherapeutic strategies.

Among dendritic cells, plasmacytoid dendritic cells (PDC) represent a functionally distinct lineage. Regarding innate immunity, PDC secrete large amounts of type I IFN upon viral exposure or stimulation by microbial products such as unmethylated CpG-motif containing oligo-DNA due to their selective expression of TLR7 and TLR9. We asked whether they could acquire cytotoxic functions during the early phases of infection or after activation with TLR7 or TLR9 agonists. In the present study, we describe a human PDC cell line called GEN2.2, derived from leukemic PDC, that shares most of the phenotypic and functional features of normal PDC. We show that after contact with the influenza virus, GEN2.2, as well as normal PDC, acquires TRAIL and killer activity against TRAIL-sensitive target cells. Moreover, we show that activation of GEN2.2 cells by CpG-motif containing oligo-DNA or R848 also induces TRAIL and endows them with the ability to kill melanoma cells. Therefore, PDC may represent a major component of innate immunity that could participate to the clearance of infected cells and tumor cells. This phenomenon could be relevant for the efficacy of TLR7 or TLR9 agonists in the therapy of infectious disease and cancer.

HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS.

Human cytomegalovirus (HCMV) infection is associated with an increased susceptibility to opportunistic infections. Although the subversion of adaptive immune responses has been extensively studied, the consequences of HCMV infection on natural immune responses are not well documented. A striking selective downmodulation of CD11b/CD18 (CR3) or CD11c/CD18 (CR4) was found upon HCMV infection, on two models, the monocytic THP-1 cell line and monocyte- derived macrophages. HCMV-infected macrophages have an altered adhesion/phagocytic capacity to Candida albicans, a pathogen responsible for some opportunistic infections in immunocompromised patients. These results suggest a new mechanism implicated in the augmentation of opportunistic infections in HCMV patients., (Copyright (c) 2005 Wiley-Liss, Inc.)

Background and Objectives: The residual tumor cells remaining after completion of standard chemotherapy and radiation treatment in B lymphoma patients, may be eradicated by active immunotherapy that stimulates tumor-specific T lymphocytes. Irradiated autologous lymphoma cells expressing tumor-associated antigens (TAA) may serve as a potential tumor vaccine, provided that they are effectively targeted to the antigen-presenting cells (APC). We propose exploiting the natural anti-Gal antibody in order to target vaccinating tumor cells to APC. Anti-Gal constitutes 1% of IgG in human serum and interacts specifically with the alpha-gal epitope (Galalpha1-3Galphalbeta1-4GlcNAc-R)., Design and Methods: Alpha-gal epitopes were synthesized in vitro on the membrane of primary lymphoma cells by using the recombinant glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). Processed tumor cells were opsonized by purified anti-Gal antibodies and studied for uptake (phagocytosis) by APC including monocyte-derived macrophages and dendritic cells. Cross-presentation of tumor antigens after phagocytosis of processed MHC-I negative lymphoma cells was measured by activation of a tumor-specific CD8+ T-cell line., Results: We demonstrate synthesis of alpha-gal epitopes on freshly isolated B lymphoma cells of various types following the use of the recombinant enzyme alpha1,3GT. The subsequent binding of anti-Gal to the de novo synthesized alphagal epitopes opsonizes these tumor cells for effective uptake by macrophages and dendritic cells, through phagocytosis mediated by FcgammaR1 (CD64). Moreover, anti-Gal-mediated phagocytosis resulted in cross-presentation of TAA by dendritic cells., Interpretation and Conclusions: This study suggests that immunization with irradiated autologous lymphoma cells processed to express alpha-gal epitopes will result in anti-Gal-mediated, in vivo targeting of the autologous tumor vaccine to APC.

Protease inhibitor treatment strongly diminishes mortality in HIV-infected patients. This treatment has also been associated with lipodystrophy and has been shown to alter adipocyte differentiation. The protease inhibitor nelfinavir has been indirectly implicated in the appearance and development of lipodystrophic syndrome, as well as in adipocyte cell death. The aim of this study was to evaluate the effects of nelfinavir on the 3T3-F442A adipocyte cell line. Nelfinavir (30 microM) induced cell death of 3T3-F442A adipocytes by a necrotic process that was not mediated by TNF-alpha. Treatment of cells with this protease inhibitor led to a significant increase in expression of the heme oxygenase-1 gene that could be reduced by 100 microM of the antioxidant ascorbate. Moreover, ascorbate had a protective effect on nelfinavir-induced necrosis, decreasing the percentage of necrotic cells by 70%. Our results show that nelfinavir induces necrosis of adipocytes mediated by a cellular increase of reactive oxygen species. This deleterious effect could be counterbalanced by ascorbate.

In agreement with good practices for therapeutic use of human cells, quality control has to be performed to valid the process of extracorporeal photopheresis (ECP) with the Vilbert-Lourmat system. Since no protocol exists, we evaluated a technique based on the measurement of the inhibition of mitogen (PHA, Con-A, OKT3)-induced proliferation, in 164 procedures from 16 patients. Whatever the pathology, we observed a high proliferation rate in most samples, and we obtained over 90% ECP-induced inhibition in as many as 94% of the cases. Since this approach proved to be relevant regarding our objective, a protocol for the ECP process validation is proposed., (Copyright 2003 Elsevier Science Ltd.)