Total Dendritic Cells Research Articles

Dendritic Cells in Subcutaneous and Epicardial Adipose Tissue of Subjects with Type 2 Diabetes, Obesity, and Coronary Artery Disease.

Dendritic cells (DCs) are professional antigen-presenting cells contributing to regulation of lymphocyte immune response. DCs are divided into two subtypes: CD11c-positive conventional or myeloid (cDCs) and CD123-positive plasmacytoid (pDCs) DCs. The aim of the study was to assess DCs (HLA-DR+ lineage-) and their subtypes by flow cytometry in peripheral blood and subcutaneous (SAT) and epicardial (EAT) adipose tissue in subjects with (T2DM, n = 12) and without (non-T2DM, n = 17) type 2 diabetes mellitus undergoing elective cardiac surgery. Subjects with T2DM had higher fasting glycemia (8.6 ± 0.7 vs. 5.8 ± 0.2 mmol/l, p < 0.001) and glycated hemoglobin (52.0 ± 3.4 vs. 36.9 ± 1.0 mmol/mol, p < 0.001) and tended to have more pronounced inflammation (hsCRP: 9.8 ± 3.1 vs. 5.1 ± 1.9 mg/ml, p = 0.177) compared with subjects without T2DM. T2DM was associated with reduced total DCs in SAT (1.57 ± 0.65 vs. 4.45 ± 1.56% for T2DM vs. non-T2DM, p = 0.041) with a similar, albeit insignificant, trend in EAT (0.996 ± 0.33 vs. 2.46 ± 0.78% for T2DM vs. non-T2DM, p = 0.171). When analyzing DC subsets, no difference in cDCs was seen between any of the studied groups or adipose tissue pools. In contrast, pDCs were increased in both SAT (13.5 ± 2.0 vs. 4.6 ± 1.9% of DC cells, p = 0.005) and EAT (29.1 ± 8.7 vs. 8.4 ± 2.4% of DC, p = 0.045) of T2DM relative to non-T2DM subjects as well as in EAT of the T2DM group compared with corresponding SAT (29.1 ± 8.7 vs. 13.5 ± 2.0% of DC, p = 0.020). Neither obesity nor coronary artery disease (CAD) significantly influenced the number of total, cDC, or pDC in SAT or EAT according to multiple regression analysis. In summary, T2DM decreased the amount of total dendritic cells in subcutaneous adipose tissue and increased plasmacytoid dendritic cells in subcutaneous and even more in epicardial adipose tissue. These findings suggest a potential role of pDCs in the development of T2DM-associated adipose tissue low-grade inflammation.

Negative impact of proteinuria on circulating myeloid dendritic cells

BackgroundA decrease in absolute numbers (abs.) of circulating dendritic cells (DCs) and recruitment into target organs has been reported, but whether the level of proteinuria associates with circulating DC abs. has not been clarified.MethodsWe conducted a cross-sectional study of 210 patients with kidney disease aged 21–96 years who were admitted to our hospital for kidney biopsy in 2007–2010. For accuracy, the level of proteinuria was thoroughly measured by 24-h urine collection from patients in their admitted condition. The abs. of total DCs (tDCs), myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) was measured by three-color fluorescence-activated cell sorting (FACS). Patients were divided into four groups based upon the quartile of each DC abs. and one-way ANOVA, and multivariable-adjusted regression analyses were performed.ResultsQuantile analysis showed that the level of daily proteinuria decreased with increasing blood mDC abs., with mean proteinuria levels (g/day) of 2.45, 1.68, 1.68, 1.10 for those in mDC abs. quartiles ≤ 445, < 686, < 907, ≥ 907 cells/102 µL (p = 0.0277), respectively. Multivariate-adjusted regression analysis revealed that the mDC abs. was negatively associated with proteinuria (95% CI − 57.0 to − 8.5) and positively associated with male gender (95% CI 66.2–250.5). Independent associations were also shown between pDCs abs. and estimated glomerular filtration rate (eGFR) (95% CI 0.14–2.67) and C-reactive protein (95% CI − 49.4 to − 9.9) and between tDCs abs. and male gender (95% CI 54.5–253.6) and C-reactive protein (95% CI − 80.5 to − 13.4).ConclusionWe first reported that circulating mDC abs. has a negative association with the level of proteinuria.

Changes of Immune Cells during the First Post-Transplant Year Guide the Way for Future Tolerance Induction Therapies

Purpose The development of efficacious tolerance induction therapies will progress when the immunological configuration after heart transplantation (HTx) is known. This study investigated the immunological changes of cell subsets involved in tolerance induction during the first year post-HTx and compared them with long-term HTx (LT-HTx) patients. Methods Blood samples were obtained from n=20 patients prior and at five time points during the first year post-HTx (month 1, 3, 6, 9 and 12) and from n=20 LT-HTx patients which were transplanted at least 5 years and never suffered from rejection. Percentages and mean fluorescence intensities (MFIs) of dendritic cell (DC) and regulatory T cell (Treg) subsets were quantified by flow cytometry. Results Total DCs and CD4+ T cells decreased in the first year post-HTx (Table 1). BDCA1+ (mo 1: 34.5±20.1%, mo 3: 43.4±12.9%, p 0.01) and BDCA3+ (mo 1: 44.2±31.7%, mo 3: 63.3±21.7%, p 0.01). MFIs of CD147, CD120b and CD39 expression in Tregs reduced from month 3 (p Conclusion Tolerance-induction therapies should aim at reinforcing the optimal configuration of DCs and Treg subsets, because these subsets underwent comprehensive changes in the first year post-HTx. Tolerance-inducing therapies should be initiated after regeneration of DC subsets took place in month 3-6 post-HTx.

Differences of Immune Reconstitution of Dendritic Cells in Pediatric GvHD Patients After Allogenic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is a life-saving procedure for children with a variety of (non) malignant conditions. GvHD is a severe complication with high morbidity and mortality. The pathogenesis remains unclear. We studied dendritic cell (DC) reconstitution to detect potential differences, which may improve our knowledge in the development of chronic GvHD (cGvHD). We examined immune reconstitution (T, B, and NK cells and dendritic cells) at defined time points in a pediatric cohort who underwent 61 allogeneic HSCTs. Regarding DC reconstitution we found a fast reconstitution of the DC compartment negatively correlated with age. After HSCT, both myeloid DC (mDC) and plasmacytoid DC (pDC) counts recover to pre-HSCT levels within 2 months. Higher CCR7 positive cell counts were found in patients receiving TBI during engraftment and during the whole posttransplant period we found a correlation with an improved outcome. In cGVHD patients decreased total DC counts and increased pDCs were found after day+100. No relevant correlation was achieved regarding to HLA-matching, stem cell manipulation of the graft as well as HSCT-indication compared with different DC counts. Pathogenesis of cGvHD remains complex. Our data suggest an influence of dendritic cells, which may contribute to the clinical picture and should be further investigated in future studies.

P054 CD103+SIRPα+ DC are specifically decreased in the inflamed colon from patients with ulcerative colitis but not with Crohn’s disease

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammation of the human gastrointestinal (GI) tract. Intestinal dendritic cells (DC) are essential to maintain the balance between immunity against pathogens and tolerance towards nutrients and commensals. However, there is not much information about DC composition in the human GI tract both in health and IBD. Human GI biopsies were obtained from healthy controls and IBD patients (including UC and CD; both active and quiescent). Tissue was disaggregated and lamina propria mononuclear cells (LPMC) characterised by flow cytometry. Human intestinal DC were identified within singlet viable leucocytes as CD14-CD64-HLA-DR+CD11c+. Type 1 DC were defined as CD103+SIRPα- while type 2 DC were identified as SIRPα+ and further divided into subsets based on the expression of CD103. The proportion of total DC displayed a gradient throughout the healthy human gut as it was higher in the colon (either distal or proximal) compared with the ileum. DC proportion was further decreased in the duodenum. Type 1 (minority) and type 2 (majority) conventional DC did not change their proportion throughout the healthy gut. However, CD103+SIRPα+ DC were the main subset in the duodenum as opposed to CD103-SIRPα+ DC which were predominant in the colon and the ileum. Compared with their CD103-SIRPα+ type 2 counterparts, CD103+SIRPα+ had higher levels of HLA-DR, CD40, CD86, CCR7, CD137L, ICOSL and PD-L1. CD103+SIRPα+ were also more phagocytic and had lower expression of blood-related markers like CLA and CCR2, suggesting that they are derived from CD103-SIRPα+ DC following mucosal conditioning. Indeed, CD103+SIRPα+ numbers were increased following LPMC culture, although this process was reverted in the presence of pro-inflammatory LPS. CD103+SIRPα+ DC displayed an enhanced production of IL-10, both in resting conditions and in the presence of LPS. In IBD, type 2 DC constitutively displayed lower expression of SIRPα irrespectively of IBD-type (CD or UC) or condition (active or quiescent). Nevertheless, the inflamed colon from UC patients, but not from CD, specifically displayed lower numbers of tolerogenic CD103+SIRPα+ DC. These results were in agreement with the colonic cytokine milieu, which was much more pro-inflammatory in UC patients compared with CD. Tolerogenic PD-L1 expression and IL-10 production was associated with CD103+SIRPα+ DC, confirming therefore their tolerogenic phenotype. Human colonic DC from IBD patients constitutively display lower levels of SIRPα. The specific reduction of CD103+SIRPα+ DC in the inflamed mucosa from UC, but not CD, suggests the presence of different pathogenic mechanisms occurring in IBD.

ICOSL+ Plasmacytoid Dendritic Cells As Biomarker and Inducer of Graft-Versus-Host Disease

Acute graft-versus-host disease (GVHD) remains one of the leading causes of death post allogeneic hematopoietic cell transplantation (HCT). Gastrointestinal GVHD (GI-GVHD), the most fatal type of GVHD, would benefit from additional biomarkers that are therapeutic targets. Using state-of-the-art quantitative proteomics we previously identified and validated an increased CD4+CD146+ T cell population in GI-GVHD patients. This population expressed a Th1 and Th17 phenotype and was induced by Inducible COStimulator (ICOS), a critical costimulatory molecule for the development of pathogenic Th17 (Li W. et al, J. Clin. Invest. Insights, 2016).ICOS binds its ligand, ICOSL, which is expressed on dendritic cells (DCs) that prime naïve T cells to initiate immune responses. This prompted us to examine ICOSL expression on the two blood DCs subsets that can be identified in human peripheral blood: Lineage-HLADR+CD11c+ myeloid DCs (mDCs) and Lineage-HLADR+CD123+ plasmacytoid DCs (pDCs). Using the same cohort of patients aforementioned, the frequency of ICOSL was significantly higher on pDCs in 64 GI-GVHD patients when compared to 22 non-GVHD enteritis patients, 35 skin GVHD patients, and 39 patients without GVHD (Figure 1). The numbers and frequencies of total DCs, mDCs and pDCs were similar between groups.The growth factor fms-related tyrosine kinase 3 ligand (Flt3l) is necessary for the development and differentiation of pDCs, and the transcription factor, Stat3, is required for Flt3l-dependent dendritic cell differentiation in mice. The role of pDCs in acute GVHD is still controversial (tolerogenic or initiator of GVHD depending on the murine model), and confirmatory studies about their functions are necessary before a therapeutic approach based on this mechanism can be contemplated. Based on the patients' data and previous knowledge, we hypothesized that absence of ICOSL signaling in donor DCs would protect against GVHD through Flt3l, Stat3, or both. We first found that knocking out (KO) ICOSL in the donor bone marrow (BM) extended survival compared to wild-type (WT) mice in the major mismatch (B6, H-2b à BALB/c, H-2d) experimental HCT model, while recipients of Stat3KO BM did not show any difference in GVHD mortality (Figure 2A). We also found a significant decrease of Flt3l levels in plasma collected at day 3 from ICOSLKO BM recipients compared to WT mice (Figure 2B). We then analyzed the recipients' infiltrating intestinal immune cells at day 10 post-HCT for the infiltration of pDCs and pathogenic Th17 cells. We found significantly lower frequencies of intestinal pDCs (CD11b-CD11c+B220+CD103+) (Figure 2C), and intestinal T cells coexpressing interferon (IFN)g and IL-17 (Figure 2D) in recipients of ICOSLKO BM compared to recipients of WT BM. Absolute counts of these two populations followed the same trend (data not shown). To confirm these data were not strain-specific, we performed similar analyses in the haplo-identical (B6, H-2b à B6D2F1, H-2d) experimental model showing similar outcomes for pDCs and IFNγ+IL-17+ T cells frequencies and counts in recipients of ICOSLKO BM compared to recipients of WT BM. Importantly, and in contrast to human T cells, CD146 is not expressed on naïve murine T cells and thus cannot be measured in vivo in acute GVHD models. Transcriptome analyses by Nanostring technology (Immunology panels) comparing 14 days post-HCT of sorted pDCs from ICOSLKO BM versus WT haplo-identical recipients showed increased expression of key molecules required for development (Itgax, Nos2, Socs1, Tcf4 and Bst2), costimulation (Cd80, Cd48, Cd74 and Cd86), and function (Tyrobp, Ikbkg, Nod2 and Irf7) of pDCs (Figure 3A). Lastly, we found increased T cell activation markers including Prf1, Il17a, eomes in sorted CD4+ T cells from ICOSLKO BM compared to WT recipients (Figure 3B).We conclude that early quantification of ICOSL+ pDCs frequency may allow identification of patients at risk of GI-GVHD development. Targeting ICOSL may represent a new avenue to treat acute GVHD. [Display omitted] DisclosuresPaczesny:Viracor IBT Laboratories: Patents & Royalties.

Acute aerobic exercise induces a preferential mobilisation of plasmacytoid dendritic cells into the peripheral blood in man.

Dendritic cells (DCs) are important sentinel cells of the immune system responsible for presenting antigen to T cells. Exercise is known to cause an acute and transient increase in the frequency of DCs in the bloodstream in humans, yet there are contradictory findings in the literature regarding the phenotypic composition of DCs mobilised during exercise, which may have implications for immune regulation and health. Accordingly, we sought to investigate the composition of DC sub-populations mobilised in response to acute aerobic exercise. Nine healthy males (age, 21.9 ± 3.6 years; height, 177.8 ± 5.4 cm; body mass, 78.9 ± 10.8 kg; body mass index, 24.9 ± 3.3 kg·m2; V̇O2 MAX, 41.5 ± 5.1 mL·kg·min-1) cycled for 20 min at 80% V̇O2 MAX. Blood was sampled at baseline, during the final minute of exercise and 30 min later. Using flow cytometry, total DCs were defined as Lineage- (CD3, CD19, CD20, CD14, CD56) HLA-DR+ and subsequently identified as plasmacytoid DCs (CD303+) and myeloid DCs (CD303-). Myeloid DCs were analysed for expression of CD1c and CD141 to yield four sub-populations; CD1c-CD141+; CD1c+CD141+; CD1c+CD141- and CD1c-CD141-. Expression of CD205 was also analysed on all DC sub-populations to identify DCs capable of recognising apoptotic and necrotic cells. Total DCs increased by 150% during exercise (F(1,10) = 60; p < 0.05, η2 = 0.9). Plasmacytoid DCs mobilised to a greater magnitude than myeloid DCs (195 ± 131% vs. 131 ± 100%; p < 0.05). Among myeloid DCs, CD1c-CD141- cells showed the largest exercise-induced mobilisation (167 ± 122%), with a stepwise pattern observed among the remaining sub-populations: CD1c+CD141- (79 ± 50%), followed by CD1c+CD141+ (44 ± 41%), with the smallest response shown by CD1c-CD141+ cells (23 ± 54%) (p < 0.05). Among myeloid DCs, CD205- cells were the most exercise responsive. All DC subsets returned to resting levels within 30 min of exercise cessation. These results show that there is a preferential mobilisation of plasmacytoid DCs during exercise. Given the functional repertoire of plasmacytoid DCs, which includes the production of interferons against viral and bacterial pathogens, these findings indicate that exercise may augment immune-surveillance by preferentially mobilising effector cells; these findings have general implications for the promotion of exercise for health, and specifically for the optimisation of DC harvest for cancer immunotherapy.

Vascular endothelial growth factor suppresses dendritic cells function of human prostate cancer.

PurposeProstate cancer (PCa) patients often have dendritic cell (DC) function defects, but the mechanism is not clear. The aim of this study was to detect the effect of vascular endothelial growth factor (VEGF) in mature DCs.Patients and methodsIn this study, we chose 30 PCa patients, 10 prostatic intraepithelial neoplasia (PIN) patients and 30 benign prostatic hyperplasia (BPH) patients, and compared the composition of peripheral blood T cells, the composition and function of local dendritic cells in prostate tissue, and the density of local VEGF.ResultsThe results showed that the numbers of total DCs, mature and functional DCs, and CD4+ T cells were inhibited in PCa, and the inhibitory effect was enhanced with increased malignancy. In addition, the infiltration density of VEGF-positive cells was increased in PCa, and this increase was associated with an increased malignant degree of PCa. The inhibition of tumor immunity in patients with PCa is achieved by inhibiting the function of dendritic cells.ConclusionVEGF plays an important role in the inhibition of the maturation and function of dendritic cells, and this inhibition is gradually increased with an increasing malignant degree of PCa.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Radiation- and Age-Associated Changes in Peripheral Blood Dendritic Cell Populations among Aging Atomic Bomb Survivors in Japan.

Previous immunological studies in atomic bomb survivors have suggested that radiation exposure leads to long-lasting changes, similar to immunological aging observed in T-cell-adaptive immunity. However, to our knowledge, late effects of radiation on dendritic cells (DCs), the key coordinators for activation and differentiation of T cells, have not yet been investigated in humans. In the current study, we hypothesized that numerical and functional decreases would be observed in relationship to radiation dose in circulating conventional DCs (cDCs) and plasmacytoid DCs (pDCs) among 229 Japanese A-bomb survivors. Overall, the evidence did not support this hypothesis, with no overall changes in DCs or functional changes observed with radiation dose. Multivariable regression analysis for radiation dose, age and gender effects revealed that total DC counts as well as subpopulation counts decreased in relationship to increasing age. Further analyses revealed that in women, absolute numbers of pDCs showed significant decreases with radiation dose. A hierarchical clustering analysis of gene expression profiles in DCs after Toll-like receptor stimulation in vitro identified two clusters of participants that differed in age-associated expression levels of genes involved in antigen presentation and cytokine/chemokine production in cDCs. These results suggest that DC counts decrease and expression levels of gene clusters change with age. More than 60 years after radiation exposure, we also observed changes in pDC counts associated with radiation, but only among women.

Profiling dendritic cell subsets in the patients with active pulmonary tuberculosis

Dendritic cell (DC) plays an important role in the immune response against pulmonary tuberculosis. However, the phenotypic profile of DC subsets in peripheral blood in individuals with active pulmonary tuberculosis (APT) is still inconclusive. Here, we demonstrated that the absolute numbers of total DC (tDC), myeloid DC (mDC) and plasmacytoid DC (pDC) in individuals with APT were decreased compared to healthy controls (HCs). The decreased number of DCs, especially of pDC, seems to be a useful diagnostic marker of APT. Meanwhile, the number of DCs was associated with the prolonged/complicated TB, ATD treatment effect and lymphocyte immune reactions, as manifested that relapsed APT patients with a higher number of tDC and lower number of pDC compared to newly diagnosed patients. Interestingly, mDC from APT patients displayed high expressions of CD83 and CCR7, but pDC displayed low expressions of CD83 and CCR7. Moreover, DCs from APT patients expressed lower levels of HLA-DR and CD80, but expressed a higher level of CD86 than those from HCs. However, the antigen uptake capacity of DC subsets was not different between APT and HCs, despite the antigen uptake capacity of pDC was much lower than that of mDC in both APT patients and HCs. Our data represent a systematic profile of DC subsets in the blood of APT patients, and would represent a useful biomarker for APT.

Graft-Infiltrating PD-L1hi Cross-Dressed Dendritic Cells Subvert Donor-Reactive T Cell Responses in Mouse Liver Transplant Tolerance

Introduction: The mechanisms underlying “spontaneous” liver allograft tolerance remain unclear. Given recent reports of the key role of host dendritic cells (DCs) in mouse spleens that express intact donor MHC (cross-dressing) in the instigation of graft rejection[1][2] we investigated the role of cross-dressed (CD) DCs in mouse liver transplantation tolerance. Methods: Liver allografts from C57BL/6 (B6; H-2b) or B6 SJL CD45.1 mice to C3H/HeJ (C3H; H-2 k) recipients were performed. In this combination, unlike heart, skin or kidney allografts, > 90% of liver grafts are accepted without immunosuppressive therapy[3]. Graft non-parenchymal cells (NPC) and splenocytes were examined by flow cytometry and imaging cytometry on post-operative days (POD) 1, 3, 7, 14, 30, and 300. Mixed leukocyte reactions (CFSE-MLR) were also performed to assess DC function. Results: Infiltration by recipient DCs (lineage (−), CD11c (+)) in liver allografts peaked on POD 7, while donor DCs in liver grafts gradually disappeared and could not be detected by POD 7. Interestingly, more than half of the graft-infiltrating recipient DCs at that time displayed donor MHC-I, indicating cross-dressing; these DCs persisted in the graft (approx. 20 % of recipient DC) at least until POD 300. In contrast, only a very minor fraction of cross-dressed DCs (CD-DCs) (0-2%) were detected in the spleen at any time point. Control staining for donor MHC-I using naïve C3H mouse liver NPC or splenocytes was negative. Moreover, especially on POD 7, and persisting until POD 300, CD-DCs isolated from liver grafts expressed higher levels of T cell inhibitory programed death ligand 1 (PD-L1) compared to non CD-DCs (nCD-DCs). Importantly, unlike nCD-DCs, CD-DCs from liver grafts did not stimulate proliferation of allo-reactive donor T cells and markedly suppressed donor-reactive host T cell proliferation in CFSE-MLR. Conclusions: A large proportion of CD recipient DCs with capacity to subvert donor-reactive host T cell responses are evident in liver allografts early post-transplant. Moreover, cross-dressing by graft-infiltrating DCs that also express high levels of PD-L1, persists indefinitely. This suggests that graft-infiltrating host CD-DC may play a key role in regulation of alloimmunity and the promotion of donor-specific liver transplant tolerance.Figure 1: Marked reduction in donor dendritic cells (DCs) and emergence of cross-dressed host DCs in the graft after liver allo-transplantaion. (A) (B) Graft and spleen DCs were analyzed using donor recipient MHC class 1 (A) or CD45.1/ CD 45.2 antibodies (B) after B6 WT graft to C3H recipient transplatation. Data shown are representative data from 3-5 mice.Figure 2: Infiltrating host DCs and CD-DCs peak on 7 POD and PD-L1high CD-DCs persist in the graft for at least 300 POD. (A) Chronological changes in (left) the incidence of total DC and (right) the incidence of CD-DC (as % total DC) in the graft. (B) Expression of PD-L1 by CD-DCs and non (n)CD-DCs in the graft. PD-L1 expression by CD-DCs was significally higher than of nCD-DC, especially on 7POD. 3-6 mice per time point. *: P<0.05, ***: P<0.001Figure 3: Liver CD-DCs markedly sppress donor-reactive host T cell proliferation in CFSE-MLR Spleen (Sp) or liver CD-DCs or nCD-DCs isolated from graft recipients on 7 POD were co- cultured with responder (C3H (host) spleen T cells) and stimulators (B6 (donor) spleen DC) in CFSE-MLR (T cells: DC (each) =10:1). Liver CD-DCs, markedly suppressed host T cell proliferation. % T cell proliferation in indicated. Data are representative of 3 experiments that gave similar results.

A pathogenic role for the integrin CD103 in experimental allergic airways disease.

The integrin CD103 is the α E chain of integrin α E β 7 that is important in the maintenance of intraepithelial lymphocytes and recruitment of T cells and dendritic cells (DC) to mucosal surfaces. The role of CD103 in intestinal immune homeostasis has been well described, however, its role in allergic airway inflammation is less well understood. In this study, we used an ovalbumin (OVA)‐induced, CD103‐knockout (KO) BALB/c mouse model of experimental allergic airways disease (EAAD) to investigate the role of CD103 in disease expression, CD4+ T‐cell activation and DC activation and function in airways and lymph nodes. We found reduced airways hyper‐responsiveness and eosinophil recruitment to airways after aerosol challenge of CD103 KO compared to wild‐type (WT) mice, although CD103 KO mice showed enhanced serum OVA‐specific IgE levels. Following aerosol challenge, total numbers of effector and regulatory CD4+ T‐cell subsets were significantly increased in the airways of WT but not CD103 KO mice, as well as a lack of DC recruitment into the airways in the absence of CD103. While total airway DC numbers, and their in vivo allergen capture activity, were essentially normal in steady‐state CD103 KO mice, migration of allergen‐laden airway DC to draining lymph nodes was disrupted in the absence of CD103 at 24 h after aerosol challenge. These data support a role for CD103 in the pathogenesis of EAAD in BALB/c mice through local control of CD4+ T cell and DC subset recruitment to, and migration from, the airway mucosa during induction of allergic inflammation.

Abstract # 1701 Anti-CD40 prevents the corticosterone-induced loss of splenic dendritic cells in C57BL/6 mice

Dendritic cells (DCs) are important immune cells linking the innate and adaptive immune response. DCs capture antigen to present to and activate naive T cells. Subsets of DCs vary based on protein expression. This research focused on total DCs (CD11c+), and the myeloid and cross-priming DC subsets. Corticosterone (CORT), a glucocorticoid hormone released in response to stress, greatly reduces DC numbers and alters DC function and subsequent T cell responses. The current study quantifies DC recovery after CORT exposure and demonstrates that the loss of DCs can be prevented. C57BL/6 mice were exposed to exogenous CORT in the drinking water for 24 h. The splenic DC populations were then quantified by flow cytometry. CORT exposure significantly decreased the numbers of all splenic DCs. Mice were then provided varying recovery times (3, 5, or 10 days) post CORT exposure. DCs quickly returned to normal levels, and even exceeded the percentages in control untreated mice. Injection of mice with anti-CD40 antibody triggers DC maturation. Thus, it was hypothesized that injection of anti-CD40 may prevent the CORT-induced loss of DCs. Mice received two injections of anti-CD40 antibody and were immediately supplied with CORT following the second injection. When provided anti-CD40 prior to CORT exposure, the loss of DCs was prevented. These studies contribute to the ongoing exploration of the mechanisms underlying the immunological effects of stress-induced glucocorticoids.

Association of peripheral blood dendritic cells with recurrent pregnancy loss: a case-controlled study.

Dendritic cells (DCs) have been reported to play an important role in pregnancy. However, the role of DCs in recurrent pregnancy loss (RPL) has not been investigated well. Forty-three women affected by RPL and 16 fertile controls were recruited from June 2013 to December 2014. The peripheral blood DCs subsets, including myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), the levels (%) of CD80(+) , CD86(+) , and CD200(+) DCs were analyzed using flow cytometry. The levels of total DCs, mDCs, and CD86(+) DCs were significantly higher (all P<.05); however, the level of CD200(+) DCs in the RPL group was significantly lower than that of the control group (P<.05). The logistical regression analyses showed that the elevated level of mDCs was significantly associated with RPL after adjustment for age (OR: 1.14, 95% CI, 1.01-1.29, P<.05). The elevated level of mDCs was significantly associated with RPL, which might lead to the intervention of targeted immunosuppression in women with RPL.

PWE-023 Vitamin D Enhances The Ability of Anti-TNF Therapy to Suppress Dendritic Cell Activity in Crohn’s Disease

Introduction Dendritic cells (DC) can determine whether the mucosal immune system mounts an inflammatory or regulatory response to antigen and may contribute to the pathogenesis of Crohn’s disease. Vitamin D down-regulates DC inflammatory responses and could prove beneficial as a treatment adjunct in Crohn’s. This study assessed the effect of high dose parenteral vitamin D treatment on circulating DC phenotype and function in patients with active luminal Crohn’s receiving anti-TNFα therapy. Methods Peripheral blood mononuclear cells were isolated from 13 patients with active luminal Crohn’s and suboptimal vitamin D levels prior to and 6 weeks after starting anti-TNFα (infliximab) therapy. Patients with low vitamin D ( 2 vitamin D3). Flow cytometry was used to identify total DC, (HLA-DR + cells negative for markers of other cell lineages (CD3, CD14, CD16, CD19 & CD34)). DC were further subtyped as myeloid (mDC, CD11c + CD123 − ). Expression of phenotypic markers (including maturation and homing markers and pattern recognition receptors) and on-going DC cytokine production during 4 hours’ culture were assessed. Results Production of TNFα by myeloid DC was significantly reduced (p = 0.016 Fig C) in those patients who received vitamin D alongside anti-TNFα therapy; without vitamin D treatment, TNFα production by myeloid DC did not decrease significantly after anti-TNFα therapy (p = 0.96 Fig B). There was a significant negative correlation between change in vitamin D level and change in TNFα production by myeloid DC (p = 0.025, correlation coefficient =0.68 Fig D). An increase of serum 25(OH)vitamin D greater than 20 nmol/m was associated with a decrease in myeloid DC TNFα production. Anti-TNFα therapy alone induced a significant upregulation of the skin homing marker cutaneous lymphocyte antigen (CLA) on myeloid DC (p = 0.0055), an effect which was not seen in patients receiving adjunctive vitamin D. Conclusion High doses of parenteral vitamin D in patients with Crohn’s promotes anti-TNFα down-regulation of circulating myeloid DC production of TNFα which may influence the subsequent interaction of DC and T cells. TNFα promotes a TH-1/ TH-17 response characteristic of Crohn’s inflammation; thus the ability of vitamin D to further block TNFα production may promote a more regulatory T cell response and improve outcomes when used as an adjunct to anti-TNFα therapy. The down-regulation of skin homing marker CLA by vitamin D may be clinically useful in those patients suffering cutaneous sequelae of anti-TNFα therapy. Disclosure of Interest None Declared

Tolerogenic effect of mouse fibroblasts on dendritic cells.

There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). To clarify this issue, in this study, we have evaluated different features of fibroblast-primed DCs including their ability to express co-inhibitory and co-stimulatory molecules, pro-inflammatory and anti-inflammatory cytokines and their ability to induce T-cell proliferation. We also examined migratory capacity of DCs to lymphatic tissues and present fibroblast-derived antigens after encountering fibroblasts. The results of our in vitro study showed that both co-inhibitory (programmed death ligand 1 and ligand 2 and B7H4) and co-stimulatory (CD86) molecules were up-regulated when DCs were co-cultured with fibroblasts. In an animal model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased both total DC count and expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4(+) and CD8(+) T cells. Even activation of fibroblast- primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4(+) T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and indoleamine 2, 3 dioxygenase. Fibroblast-primed DCs had a significantly reduced interleukin-12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin). In conclusion, after priming with fibroblasts, DCs gain tolerogenic features. This finding suggests the potential role of fibroblasts in the maintenance of immune tolerance.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

Dendritic cells inversely regulate airway inflammation in cigarette smoke-exposed mice.

The recruitment and activation of inflammatory cells into the respiratory system is considered a crucial feature in the pathophysiology of chronic obstructive pulmonary disease (COPD). Because dendritic cells (DCs) have a pivotal role in the onset and regulation of immune responses, we investigated the effect of modulating DC subsets on airway inflammation by acute cigarette smoke (CS) exposure. CS-exposed mice (5 days) were treated with fms-like tyrosine kinase 3 ligand (Flt3L) and 120g8 antibody to increase total DC numbers and deplete plasmacytoid DCs (pDCs), respectively. Flt3L treatment decreased the number of inflammatory cells in the bronchoalveolar lavage (BALF) of the smoke-exposed mice and increased these in lung tissue. DC modulation reduced IL-17 and increased IL-10 levels, which may be responsible for the suppression of the BALF cells. Furthermore, depletion of pDCs led to increased infiltration of alveolar macrophages while restricting the presence of CD103(+) DCs. This study suggests that DC subsets may differentially and compartment-dependent influence the inflammation induced by CS. pDC may play a role in preventing the pathogenesis of CS by inhibiting the alveolar macrophage migration to lung and increasing CD103(+) DCs at inflammatory sites to avoid extensive lung tissue damage.