Adaptive immune responses to antigens released by dying cells play a critical role in the development of autoimmunity, allograft rejection, and spontaneous as well as therapy-induced tumor rejection. Although cell death in these situations is considered sterile, various reports have implicated type I IFNs as drivers of the ensuing adaptive immune response to cell-associated antigens. However, the mechanisms that underpin this type I IFN production are poorly defined. Here we show that dendritic cells (DCs) can uptake and sense nuclear DNA-associated entities released by dying cells to induce type I IFN. Remarkably this molecular pathway requires STING but not TLR or NLR function and results in the activation of IRF3 in a TBK1-dependent manner. DCs are shown to depend on STING function in vivo to efficiently prime IFN-dependent CD8+ T cell responses to tumor antigens. Furthermore, loss of STING activity in DCs impairs the generation of follicular helper T (Tfh) and plasma cells as well as anti-nuclear antibodies in an inducible model of systemic lupus erythematosus (SLE). These findings suggest that the STING pathway could be manipulated to enable the rational design of immunotherapies that enhance or diminish anti-tumor and autoimmune responses, respectively.

Impaired functionality of dendritic cells (DCs) significantly contributes to decreased adaptive immune responses in aged hosts. The expression of MHC-peptide on the DC surface is the critical first step in T cell priming, but few studies have addressed the effect of aging on antigen (Ag) acquisition, processing, and presentation by DCs. Here we show that aged murine DCs were less efficient in the cross-presentation of cell-associated Ag and subsequently in the cross-priming of CD8+ T cells than their young counterparts. The decreased cross-presentation was associated with a reduction in the frequency of CD8αDCs and merocytic (CD8α−CD11b−)DCs that could endocytose cell-associated Ag, as well as the number and the size of the endocytosed particles in the DC that did internalize cell-associated materials. Mechanistically, phagocytic capacity has been associated with mitochondrial activity and membrane potential (Δψm). Aged DCs exhibited profound signs of mitochondrial dysfunction, illustrated by lower Δψm, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation (OXPHOS), and greater proton leak and ROS production. Mimicking the aged metabolic phenotype in young DCs by pharmacologic manipulation indicated that the reductions in Δψm and ATP impeded the phagocytic capacity while ROS interfered with a later step in the cross-presentation process. Conversely, in vitro scavenging of ROS partially restored cross-presentation by aged DCs. Together, these data suggest that improvement of aged DC functionality might be feasible in the elderly, by targeting metabolic dysfunction, or its downstream sequelae, thereby opening new avenues for enhancing vaccine efficiency in this population.

Material

BackgroundThis study compared the level of advanced glycation end products (AGEs), N-(Carboxymethyl)lysine (CML) and N-(Carboxyethyl)lysine (CEL), in patients with multiple sclerosis (MS) and healthy controls (HCs), correlating these markers with clinical indicators of MS disease severity.MethodsCML and CEL plasma levels were analyzed in 99 MS patients and 43 HCs by tandem mass spectrometry (LC/MS/MS). Patients were stratified based on drug modifying therapies (DMTs) including interferon beta, glatiramer acetate and natalizumab.ResultsThe level of plasma CEL, but not CML, was significantly higher in DMT-naïve MS patients when compared to HCs (P < 0.001). Among MS patients, 91% had higher than mean plasma CEL observed in HCs. DMTs reduced CML and CEL plasma levels by approximately 13% and 40% respectively. CML and CEL plasma levels correlated with the rate of MS clinical relapse.ConclusionOur results suggest that AGEs in general and CEL in particular could be useful biomarkers in MS clinical practice. Longitudinal studies are warranted to determine any causal relationship between changes in plasma level of AGEs and MS disease pathology. These studies will pave the way for use of AGE inhibitors and AGE-breaking agents as new therapeutic modalities in MS.

SummaryVaccination with autologous cancer cells aims to enhance adaptive immune responses to tumour-associated antigens. The incorporation of Fms-like tyrosine kinase 3-ligand (FLT3L) treatment to the vaccination scheme has been shown previously to increase the immunogenicity of cancer vaccines, thereby enhancing their therapeutic potential. While evidence has been provided that FLT3L confers its effect through the increase of absolute dendritic cell (DC) numbers, it is currently unknown which DC populations are responsive to FLT3L and which effect FLT3L treatment has on DC functions. Here we show that the beneficial effects of FLT3L treatment resulted predominantly from a marked increase of two specific DC populations, the CD8 DCs and the recently identified merocytic DC (mcDC). These two DC populations (cross)-present cell-associated antigens to T cells in a natural killer (NK)-independent fashion. FLT3L treatment augmented the absolute numbers of these DCs, but did not change their activation status nor their capacity to prime antigen-specific T cells. While both DC populations effectively primed CD8 + T cell responses to cell-associated antigens, only mcDC were capable to prime CD4+ T cells to cell-associated antigens. Consequentially, the transfer of tumour vaccine-pulsed mcDC, but not of CD8 DCs, protected mice from subsequent tumour challenge in a vaccination model and resulted in eradication of established tumours in a therapeutic approach. These results show that the beneficial effect of FLT3L is associated with the induction of mcDC and suggests that selective targeting to mcDC or instilling mcDC 'characteristics' into conventional DC populations could significantly enhance the efficacy of tumour vaccines.

BackgroundDeveloping novel strategies to overcome the immunosuppressive tumor microenvironment is a critically important area of cancer therapy research. Here, we assess the therapeutic potential of CD244 (2B4/signaling lymphocyte activation molecule family 4), an immunoregulatory receptor found on a variety of immune cells, including exhausted CD8+T cells, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs).MethodsUsing de-identified human tumor and blood samples from patients with head and neck squamous cell carcinoma (HNSCC) and HNSCC models in WT and CD244-/-mice, we assessed the therapeutic potential of CD244 using flow cytometry, RT-PCR, Luminex immunoassays and histopathological analyses.ResultsCompared with healthy tissues, tumor infiltrating CD8+T cells from HNSCC patients and a HNSCC mouse model showed significant increased expression of CD244 expression that correlated with PD1 expression. Moreover, CD244 was increased on intratumoral DC and MDSC and high CD244 expression correlated with PD-L1 expression and increased spontaneous expression of immune-suppressive mediators. In addition, CD244 activation inhibited production of proinflammatory cytokines in human DC in vitro. Importantly, CD244-/-mice showed significantly impaired tumor growth of HNSCC and interventional treatment of WT mice with anti-CD244 monoclonal antibody significantly impaired the growth of established HNSCC tumors and increased tumor-infiltrating CD8+T cells.ConclusionsTogether these data suggest that CD244 contributes to the overall immune-suppressive environment and therefore has potential as a new immunotherapy target in the treatment of malignancies.

Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr−/− mice were transplanted with batf3−/− or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8α+ DC numbers in spleen and lymph nodes (>80%; P < 0,001). Concordantly, batf3−/− chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3−/− chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3−/− chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3−/− chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8α+ DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8α+ DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability.

Myriad studies have linked type I IFN to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). Although increased levels of type I IFN are found in patients with SLE, and IFN blockade ameliorates disease in many mouse models of lupus, its precise roles in driving SLE pathogenesis remain largely unknown. In this study, we dissected the effect of type I IFN sensing by CD4 T cells and B cells on the development of T follicular helper cells (T FH), germinal center (GC) B cells, plasmablasts, and antinuclear dsDNA IgG levels using the bm12 chronic graft-versus-host disease model of SLE-like disease. Type I IFN sensing by B cells decreased their threshold for BCR signaling and increased their expression of MHC class II, CD40, and Bcl-6, requirements for optimal GC B cell functions. In line with these data, ablation of type I IFN sensing in B cells significantly reduced the accumulation of GC B cells, plasmablasts, and autoantibodies. Ablation of type I IFN sensing in T cells significantly inhibited T FH expansion and subsequent B cell responses. In contrast to the effect in B cells, type I IFN did not promote proliferation in the T cells but protected them from NK cell-mediated killing. Consequently, ablation of either perforin or NK cells completely restored T FH expansion of IFNAR 2/2 T FH and, subsequently, restored the B cell responses. Together, our data provide evidence for novel roles of type I IFN and immunoregulatory NK cells in the context of sterile inflammation and SLE-like disease.