Number Of Plasmacytoid Dendritic Cells Research Articles

Bioinformatic analysis reveals an association between Metadherin with breast cancer prognosis and tumor immune infiltration

Breast cancer metastasis and invasion are both promoted by the oncoprotein Metadherin (MTDH). However, the the role of Metadherin in breast cancer progression and its role in the immune microenvironment. Are not clear. A bioinformatic analysis was performed to demonstrate the prognostic value of Metadherin in BC. In the present study, we found that Metadherin is overexpressed in BC and is significantly correlated with individual cancer stage, age, subclasses, menopause and nodal metastasis status. Metadherin overexpression was associated with a significant decrease in OS and DSS. Cox multivariate analysis indicated that Metadherin was an independent negative prognostic indicator for OS and DSS. Moreover, Metadherin hypomethylation status was associated with poor prognosis. A negative correlation was also noted between Metadherin overexpression and the number of plasmacytoid dendritic cells, cluster of differentiation 8+ T cells, and natural killer cells. Association patterns varied with different subtypes. Various associations between Metadherin levels and immune cell surface markers were revealed. A total of 40 groups of BC and adjacent normal breast tissue samples were collected. Metadherin mRNA was detected by PCR, and its expression levels in BC tissues were significantly increased compared with those noted in normal tissues. The expression levels of Metadherin were also measured in normal and BC cell lines, respectively, and similar conclusions were obtained. The Metadherin mRNA levels were knocked down in SK-BR3 and MDA-MB-231 cell lines and the cell proliferative and migratory activities were determined using Cell Counting Kit-8 and scratch assays, respectively. The results indicated that the cell proliferative and migratory abilities were reduced following knockdown of Metadherin expression. Therefore, Metadherin may be considered as a novel prognostic biomarker in BC.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

The role of mast cells in cellular modifications evoked by Exendin-4 in treated wounds: a preclinical study.

To assess the response of cellular infiltration in wounds treated with Exendin-4. In this study, 16 mice were used. On each mouse, two wounds were produced, one above the other, in order to study the effects of the various treatments carried out. The wounds then received an intradermal injection of either saline (20μl; Group 1) or Exendin-4 (Exe4, 62ng; Group 2) in the upper and lower wounds, respectively. The mice were euthanised in order to collect the wounds at time of abrasion (T0), at 48 hours (T1), 96 hours (T2) and 144 hours (T3). The expression of the glucagon-like peptide-1 receptor (GLP-1R) was evaluated by Western blot in wound lysates. Histological and histochemistry methods were applied in cryosections. In T2 and T3 treated wounds, the mast cells degranulation index increased while GLP-1R expression, tumour necrosis factor (TNF)-α, or heat shock protein (HSP)47 antigens were detected in their cytoplasm. These cells interacted with dendritic cells, vessels or granulocytes. The density of dendritic cells increased progressively, and intercellular connections were found between these cells and vessels. Among the dendritic cells at T2, only M2 macrophages increased. However, M1 cells expressed transforming growth factor (TGF)-β and both interacted with either fibroblasts or with vessels. The number of plasmacytoid dendritic cells increased and established close contacts with regulatory T cells. We propose that after treatment with Exe4, early activation of mast cells is critical in wound healing acceleration. This is crucial in understanding the potential effect of this drug for viable clinical therapies. No potential conflict of interest was reported by the authors.

PB2206: FULL SPECTRAL FLOW CYTOMETRY ANALYSIS OF THE BONE MARROW IMMUNE MICROENVIRONMENT IN PATIENTS WITH MYELODYSPLASTIC SYNDROME

Background: The bone marrow (BM) microenvironment is actively studied in patients with myelodysplastic syndrome (MDS). Full-spectral flow cytometry (FSFCM) is a novel technique that makes it possible to simultaneously study the immune system and malignant cells. Aims: to study the microenvironment and immune checkpoint of MDS by FSFCM. Methods: Retrospective analysis of BM samples from 22 MDS patients and 22 healthy donors who received treatment at the Hebei Yanda Lu Daopei Hospital between January 2020 and September 2020 using 26 parameter FSFCM in a single tube. Multiple immune subsets, myeloblasts and their expression of CD96 and CD200 were also analyzed. Results: The percentage of multiple lymphocytic subsets and number of plasmacytoid dendritic cells (PDCs) were significantly significantly lower in the samples from MDS patients compared to the samples from non-malignant controls.Additionally, expression of the CD96 surface marker were significantly lower on T-cell subsets and CD56bri NK cells,and was significantly higher levels on PDCs in the MDS patient samples compared to the healthy control samples. in the MDS samples CD200 expression on immune cells and myeloblasts were significantly higher in the MDS samples compared to those from healthy controls. The overexpression of CD200 on CD56dim natural killer (NK) cells was an independent risk factor for MDS. Image:Summary/Conclusion: The percentage of immune cells and expression of immune checkpoint proteins on immune cells from BMs of MDS were distinct from that of healthy control samples.

Altered innate immune profile in blood of systemic mastocytosis patients.

BackgroundMast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM.MethodsOverall, we studied 115 SM patients ‐ 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)‐ and 32 healthy donors (HD). Spontaneous and in vitro‐stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated.ResultsSM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1β, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ‐stimulated blood monocytes to produce IL1β and TGFβ. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non‐classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL+ DC was specifically encountered in BMM cases.ConclusionThese results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.

Do reduced numbers of plasmacytoid dendritic cells contribute to the aggressive clinical course of COVID-19 in chronic lymphocytic leukaemia?

Infections with SARS‐CoV‐2 have been unduly severe in patients with haematological malignancies, in particular in those with chronic lymphocytic leukaemia (CLL). Based on a series of observations, we propose that an underlying mechanism for the aggressive clinical course of COVID‐19 in CLL is a paucity of plasmacytoid dendritic cells (pDCs) in these patients. Indeed, pDCs express Toll‐like receptor 7 (TLR7), which together with interferon‐regulatory factor 7 (IRF7), enables pDCs to produce large amounts of type I interferons, essential for combating COVID‐19. Treatment of CLL with Bruton's tyrosine kinase (BTK) inhibitors increased the number of pDCs, likely secondarily to the reduction in the tumour burden.

Machine learning identification of specific changes in myeloid cell phenotype during bloodstream infections

The early identification of bacteremia is critical for ensuring appropriate treatment of nosocomial infections in intensive care unit (ICU) patients. The aim of this study was to use flow cytometric data of myeloid cells as a biomarker of bloodstream infection (BSI). An eight-color antibody panel was used to identify seven monocyte and two dendritic cell subsets. In the learning cohort, immunophenotyping was applied to (1) control subjects, (2) postoperative heart surgery patients, as a model of noninfectious inflammatory responses, and (3) blood culture-positive patients. Of the complex changes in the myeloid cell phenotype, a decrease in myeloid and plasmacytoid dendritic cell numbers, increase in CD14+CD16+ inflammatory monocyte numbers, and upregulation of neutrophils CD64 and CD123 expression were prominent in BSI patients. An extreme gradient boosting (XGBoost) algorithm called the “infection detection and ranging score” (iDAR), ranging from 0 to 100, was developed to identify infection-specific changes in 101 phenotypic variables related to neutrophils, monocytes and dendritic cells. The tenfold cross-validation achieved an area under the receiver operating characteristic (AUROC) of 0.988 (95% CI 0.985–1) for the detection of bacteremic patients. In an out-of-sample, in-house validation, iDAR achieved an AUROC of 0.85 (95% CI 0.71–0.98) in differentiating localized from bloodstream infection and 0.95 (95% CI 0.89–1) in discriminating infected from noninfected ICU patients. In conclusion, a machine learning approach was used to translate the changes in myeloid cell phenotype in response to infection into a score that could identify bacteremia with high specificity in ICU patients.

Environmental Air Pollutants Inhaled during Pregnancy Are Associated with Altered Cord Blood Immune Cell Profiles.

Air pollution exposure during pregnancy may be a risk factor for altered immune maturation in the offspring. We investigated the association between ambient air pollutants during pregnancy and cell populations in cord blood from babies born to mothers with asthma enrolled in the Breathing for Life Trial. For each patient (n = 91), daily mean ambient air pollutant levels were extracted during their entire pregnancy for sulfur dioxide (SO2), nitric oxide, nitrogen dioxide, carbon monoxide, ozone, particulate matter <10 μm (PM10) or <2.5 μm (PM2.5), humidity, and temperature. Ninety-one cord blood samples were collected, stained, and assessed using fluorescence-activated cell sorting (FACS). Principal Component (PC) analyses of both air pollutants and cell types with linear regression were employed to define associations. Considering risk factors and correlations between PCs, only one PC from air pollutants and two from cell types were statistically significant. PCs from air pollutants were characterized by higher PM2.5 and lower SO2 levels. PCs from cell types were characterized by high numbers of CD8 T cells, low numbers of CD4 T cells, and by high numbers of plasmacytoid dendritic cells (pDC) and low numbers of myeloid DCs (mDCs). PM2.5 levels during pregnancy were significantly associated with high numbers of pDCs (p = 0.006), and SO2 with high numbers of CD8 T cells (p = 0.002) and low numbers of CD4 T cells (p = 0.011) and mDCs (p = 4.43 × 10−6) in cord blood. These data suggest that ambient SO2 and PM2.5 exposure are associated with shifts in cord blood cell types that are known to play significant roles in inflammatory respiratory disease in childhood.

Graft Versus Host Disease Activity of Donor T Cells Is Regulated By Vasoactive Intestinal Polypeptide Synthesized By Donor Plasmacytoid Dendritic Cells

Introduction: Increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft improve clinical outcomes with decreased graft-versus-host disease (GvHD) and improved survival in previous pre-clinical murine experiments (Lu Y, Blood. 2012;119:1075-1085) and in clinic data from BMT CTN 201 (Waller JCO 2014; 32:2365-72). The mechanism by which donor pDC limit severe GvHD and improve survival after allo-BMT is unknown. We have previously described higher levels of donor T cell activation and expansion (measured as bio-luminescent radiance) among recipients of VIP-KO pDC (Li, J.X Blood 2016 128:2154). We hypothesize that blocking VIP signaling augment the magnitude of T cell activation and expansion, and that modulating the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-HSCT.Methods:We used a MHC mismatched C57BL/6 àB10BR allo-BMT model, transplanting purified populations of FACS sorted Lin-Sca-1+C-kit+ (LSK) stem cells combined with FACS purified BM pDC and MACS purified splenic T cells. Lethally irradiated B10BR recipients received 5,000 HSC, 50,000 pDC from wild type (WT) or VIP-KO donor and 1×106 T cells from WT C57BL6 donor mice. Survival and GvHD were measured based on fur texture, posture, activity, skin integrity and weight body loss. To test VIP production by pDC cells, we purified donor pDC from VIP-GFP C57 BL/6 mice, in which GFP transcription is under the control of the VIP gene promotor, and transplanted them into lethally irradiated MHC mis-matched B10.BR recipients. VIP expression in mice receiving pDC cells from a VIP-GFP transgenic donor was detected by fluorescence confocal microscopy. Immune polarization of donor T cells was accessed by intracellular cytokine staining and flow cytometry. Mice transplanted with VIP-GFP pDC cells were sacrificed on D10 and spleens were collected in OCT.Results: Survival of recipients receiving HSC, T cells and WT pDC was 71% on day100 post-transplant while survival of those receiving VIP-KO pDC was 31% (Fig 1A). Splenocytes were obtained from recipients on day 3 post-transplant. IFN-γexpression was higher and IL-10 epression was significantly lower in donor CD8+ T cells in mice receiving VIP-KO pDC compared with WT pDC (Fig 1B). Confocal microscopy showed VIP expression in donor pDC among recipients of VIP-GFP pDC (Fig 2)Conclusion:These data indicated VIP signaling occurs via interactions between donor T cells and donor pDC. Local production of VIP inhibits activation and Th1 immune polarization of donor T cells. In murine allo-BMT, absence of VIP expression in pDC leads to increased T cell activation, expansion, allo-reactivity and Th1 polarization resulting in worse survival. Thus VIP is a critical pathway in regulating GvHD and GvL action of donor T cells and an attractive pharmacological target for GvHD prevention. [Display omitted] DisclosuresWaller:AMGEN: Consultancy; PRA: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Celldex: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Helocyte: Consultancy; Cerus: Equity Ownership; National Institutes of Health: Research Funding; Chimerix: Equity Ownership; Coulter Foundation: Research Funding; Katz Foundation: Research Funding.

Plasmacytoid Dendritic Cells Depletion and Elevation of IFN-γ Dependent Chemokines CXCL9 and CXCL10 in Children With Multisystem Inflammatory Syndrome.

BackgroundSARS-CoV-2 occurs in the majority of children as COVID-19, without symptoms or with a paucisymptomatic respiratory syndrome, but a small proportion of children develop the systemic Multi Inflammatory Syndrome (MIS-C), characterized by persistent fever and systemic hyperinflammation, with some clinical features resembling Kawasaki Disease (KD).ObjectiveWith this study we aimed to shed new light on the pathogenesis of these two SARS-CoV-2-related clinical manifestations.MethodsWe investigated lymphocyte and dendritic cells subsets, chemokine/cytokine profiles and evaluated the neutrophil activity mediators, myeloperoxidase (MPO), and reactive oxygen species (ROS), in 10 children with COVID-19 and 9 with MIS-C at the time of hospital admission.ResultsPatients with MIS-C showed higher plasma levels of C reactive protein (CRP), MPO, IL-6, and of the pro-inflammatory chemokines CXCL8 and CCL2 than COVID-19 children. In addition, they displayed higher levels of the chemokines CXCL9 and CXCL10, mainly induced by IFN-γ. By contrast, we detected IFN-α in plasma of children with COVID-19, but not in patients with MIS-C. This observation was consistent with the increase of ISG15 and IFIT1 mRNAs in cells of COVID-19 patients, while ISG15 and IFIT1 mRNA were detected in MIS-C at levels comparable to healthy controls. Moreover, quantification of the number of plasmacytoid dendritic cells (pDCs), which constitute the main source of IFN-α, showed profound depletion of this subset in MIS-C, but not in COVID-19.ConclusionsOur results show a pattern of immune response which is suggestive of type I interferon activation in COVID-19 children, probably related to a recent interaction with the virus, while in MIS-C the immune response is characterized by elevation of the inflammatory cytokines/chemokines IL-6, CCL2, and CXCL8 and of the chemokines CXCL9 and CXL10, which are markers of an active Th1 type immune response. We believe that these immunological events, together with neutrophil activation, might be crucial in inducing the multisystem and cardiovascular damage observed in MIS-C.

CXCR3 chemokine receptor contributes to specific CD8+T cell activation by pDC during infection with intracellular pathogens.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.

Immune reconstitution following umbilical cord blood transplantation: IRES, a study of UK paediatric patients.

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1‐12 months post‐transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti‐thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

Phenotypical Characterization of Spleen Remodeling in Murine Experimental Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum or L. donovani infection. One of the main problems related to this disease is the emergence of severe clinical forms with a lethality of 5–20%, even while under specific treatment. In humans and other species susceptible to fatal VL, such as dogs and hamsters, the disruption of splenic white pulp (WP) is accompanied by disease progression. Control of VL progression is seen in BALB/c mice, as evidenced by a mild clinical presentation and controlled parasite replication in the liver and spleen. In this study, we investigated the features involved in the morphological remodeling of splenic compartments associated with the control of VL progression to death.Methods: We evaluated cohorts of BALB/c mice after 30, 60, and 90 days of infection by L. infantum. Spleen morphology, cell population subsets and cytokine production were studied in the spleen using flow- and histo-cytometry.Results: Intraperitoneal infection with 108 promastigotes of L. infantum led to progressive increases in spleen size at 60 and 90 days after infection. Splenomegaly was the only clinical sign of disease observed. At 30 days after infection, hyperplasia in the WP and decreased numbers of plasmacytoid dendritic cells were observed. The WP hyperplasia subsided at 60 days post-infection. However, the splenomegaly remained in association with increased numbers of macrophages, B and T lymphocytes and plasma cells. An increased number of lymphoid tissue inducer (LTi) cells was observed; these were distributed around the periarteriolar lymphoid sheath in control mice and scattered throughout the red pulp in the Leishmania-infected mice. After 90 days of infection, increased IL-6 and IFN-γ production was seen in the spleen, as well as higher frequencies of follicular and plasmacytoid dendritic cells.Conclusion: The data presented herein emphasizes the potential role of spleen remodeling in the control of severe forms of VL and highlights features potentially involved in this process.

Different distribution patterns of plasmacytoid dendritic cells in discoid lupus erythematosus and lichen planopilaris demonstrated by CD123 immunostaining

BackgroundClinical and histological features may overlap between lichen planopilaris-associated and discoid lupus erythematosus-associated scarring alopecia. ObjectivesThe aim of this study was to demonstrate the cutaneous infiltration of plasmacytoid dendritic cells and to compare their distribution pattern in discoid lupus erythematosus and lichen planopilaris. MethodsTwenty-four cases of discoid lupus erythematosus and 30 cases of lichen planopilaris were examined for immunostaining of the CD123 marker. The percentage and distribution pattern of plasmacytoid dendritic cells and the presence of the plasmacytoid dendritic cells clusters were evaluted in the samples. ResultsThe number of plasmacytoid dendritic cells was higher in the discoid lupus erythematosus specimens. Aggregations of 10 cells or more (large cluster) were observed in half of the discoid lupus erythematosus specimens and only 2 lichen planopilaris, with 50% sensitivity and 93% specificity for differentiating discoid lupus erythematosus from lichen planopilaris. Study limitationsIncidence and prevalence of discoid lupus erythematosus-associated scarring alopecia in the scalp are low, so the samples size of our study was small. ConclusionsWe suggest that a plasmacytoid dendritic cells cluster of 10 cells or more is highly specific for distinguishing discoid lupus erythematosus from lichen planopilaris. It also appears that CD123 immunolabeling is valuable in both active and late stages of the disease.

Novel Hemizygous IL2RG p.(Pro58Ser) Mutation Impairs IL-2 Receptor Complex Expression on Lymphocytes Causing X-Linked Combined Immunodeficiency

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient’s clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.

Interaction of Plasmacytoid Dendritic Cells and Myeloma Cells Trigger Tumor Promoting Transcriptional Changes in Multiple Myeloma Cells

Introduction We have previously shown that increased numbers of plasmacytoid dendritic cells (pDCs) in bone marrow (BM) of multiple myeloma (MM) patients promote tumor cell growth, survival, and drug resistance; as well as suppress T and NK cell mediated anti-MM immunity (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444). Here, we analyzed genetic changes in MM cells triggered by coculture with pDCs using next generation sequencing (NGS). Functional validation of NGS data was performed in our coculture models of pDC-T-MM cells to assess impact on tumor cell surface phenotype, growth, survival, and drug resistance; as well as cytotoxic T lymphocyte (CTL) activity against MM. We identified and validated the metabolic ectoenzyme CD73/NT5E, implicated in cancer metabolism and immunosuppression via nucleotide degradation pathway, as a novel therapeutic target in MM. Methods Purified MM patient pDCs were cocultured with autologous MM cells or allogeneic MM cell lines (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using flow cytometry. Total RNA from MM cells was subjected to RNAseq analysis using Illumina Next Generation Sequencing (NGS). Raw sequence data were analyzed using VIPER workflow generating differential expression (DEseq2) and KEGG pathway. Statistical significance: log2FC (fold change) values in coculture vs control, with an FDR (False Discovery Rate) value of &amp;lt;0.05, was considered significant (CI &amp;gt; 95). Linear model for RNAseq analysis (Limma) and its GUI (Glimma) were also utilized for the visualization of data. Results RNA-seq data was analyzed using negative binomial distribution (DEseq2) and linear (Limma) models. Results showed 9200 and 9250 genes were differentially expressed (p &amp;lt; 0.05). MM cells cultured with or without pDCs clustered into two distinct groups, based upon pDC-MM contact-dependent transcriptional changes. Pathway enrichment analysis showed that pDCs interaction with MM cells regulates multiple physiological processes in MM cells including DNA replication/repair, purine/pyrimidine metabolism, and cell cycle. Hierarchical clustering showed increased expression of genes in MM cells after coculture with pDCs (log2FC range: ± 6.0): TLR7/9 (0.5; p=0.02), HDAC6 (0.65; p=0.00002), CD274 (0.6; p=0.02), or IL3Rα/CD123(0.1; p&amp;lt;0.05). On the other hand, pDCs reduces expression of CASP3 (-1.049; p= 1.1e-7), BAK1 (-0.5; p=0.000043), ADAM33 (-1.36; p=0.004), and BAD (-0.14; p = 0.0048) in MM cells. We validated the functional significance of pDC-induced gene alterations in MM cells using coculture model of patient MM-pDCs and autologous tumor cells. For example CD73 levels further increases in MM cells after coculture with pDCs (MFI: 1.2-fold vs MM; p = 0.008; CD73+ cells: 1.15-fold vs MM; n = 5; p = 0.005); and anti-CD73 Ab (1.0 µg/ml) treatment of autologous pDC-T cell cocultures (1pDC/10T cells) induces MM-specific CD8+ CTL activity against both autologous and allogeneic tumor cells. Furthermore, combining anti-CD73 Ab and TLR7 agonist triggers more robust MM-specific CD8+ CTL activity than either agent alone (% MM lysis: anti-CD73 Ab plus TLR7 agonist: 60-70%; TLR7 agonist: 40%; and anti-CD73 Ab: 30%; p = 0.009; n = 5). Conclusions Our RNA-seq and NGS analysis of pDCs-triggered transcriptome changes in MM cells identifies genes and pathways mediating tumor growth and immunosuppression, which therefore represent targets for novel therapeutics to improve patient outcome. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Helicobacter pylori: An Underrated Cause of Immune Thrombocytopenic Purpura. A Comprehensive Review.

Idiopathic thrombocytopenic purpura (ITP) is the autoimmune-mediated destruction of platelets. ITP is a diagnosis of exclusion after other identifiable etiologies have been ruled out. After the first report by Gasbarrini et al. (1998) showing rising platelet counts in ITP patients following Helicobacter pylori (HP) eradication therapy, there is growing evidence that highlights the role of HP in triggering ITP. However, the exact pathophysiology of HP-associated ITP is still unclear, but many theories have been implicated in this regard. According to various reports, the postulated mechanisms for the role of HP in cITP include molecular mimicry, increased plasmacytoid dendritic cell numbers, phagocytic perturbation, and variable host immune response to HP virulence factors. One famous theory suggested molecular mimicry between platelet surface antigen and bacterial virulence factor, i.e. cytotoxin-associated gene A (CagA). It is thought that a chronic inflammatory response following an HP infection induces the host autoantibodies' response against CagA, which cross-reacts with platelet surface glycoproteins; therefore, it may accelerate platelet destruction in the host reticuloendothelial system. However, further studies are mandated to better understand the causal link between ITP and HP and study the role of biogeography. Nowadays, it is recommended that every patient with ITP should undergo HP diagnostic testing and triple therapy should be administered in all those candidates who test positive for HP infection. In our review, there were a few pregnant female ITP patients who took HP eradication therapy mainly after 20 weeks of gestation without maternal or fetal worst outcomes. However, large-scale studies are advisable to study the adverse fetal outcomes following triple therapy use.

Type I Interferon Predicts an Alternate Immune System Phenotype in Systemic Lupus Erythematosus.

ObjectiveType I interferon (IFN) is important to systemic lupus erythematosus (SLE) pathogenesis, but it is not clear how chronic elevations in IFN alter immune function. We compared cytokine responses after whole blood stimulation with Toll‐like receptor (TLR) agonists in high‐ and low‐IFN SLE patient subgroups.MethodsSLE patients and nonautoimmune controls were recruited, and SLE patients were categorized as either high or low IFN. Whole blood was dispensed into tubes coated with lipopolysaccharide (LPS), oligonucleotides with cytosine‐guanine repeats, Resiquimod, IFN‐α, and IFN‐α + LPS. Cytokine production in patient sera and after whole blood TLR stimulation was measured by multiplex assay, and type I IFN was assessed using a functional assay.ResultsCirculating plasmacytoid dendritic cell numbers were specifically reduced in high‐IFN SLE patients and not in low‐IFN SLE patients. In serum, we observed that the correlations between cytokines in serum differed to a much greater degree between the high‐ and low‐IFN groups (P < 0.0001) than the absolute cytokine levels differed between these same groups. In stimulated conditions, the high‐IFN patients had less cytokine production in response to TLR ligation than the low‐IFN SLE patients. LPS produced the most diverse response, and a number of interactions between type I IFN and LPS were observed.ConclusionWe find striking differences in resting and stimulated cytokine patterns in high‐ vs. low‐IFN SLE patients, which supports the biological importance of these patient subsets. These data could inform personalized treatment approaches and the pathogenesis of SLE flare following infection.

Different doses of vitamin C supplementation enhances the Th1 immune response to early Plasmodium yoelii 17XL infection in BALB/c mice

Vitamin C (ascorbate) is maintained at high levels in most immune cells and can affect many aspects of the immune response. Here, we evaluated the effect of vitamin C supplementation on the immune response to Plasmodium yoelii 17XL (P. yoelii 17XL) infection in BALB/c mice. Two orally administered doses (25 mg/kg/day and 250 mg/kg/day) of vitamin C significantly reduced levels of parasitemia during the early stages of P. yoelii 17XL infection. The numbers of activated Th1 cells and macrophages in the groups receiving vitamin C supplementation were both higher than those in the untreated group. Meanwhile, vitamin C administration reduced the levels of tumor necrosis factor α secreted by splenocytes. Vitamin C also regulated the protective anti-malarial immune response by increasing the number of plasmacytoid dendritic cells, as well as the expression of dendritic cell maturation markers, such as major histocompatibility complex class II and cluster of differentiation 86. In conclusion, the doses of vitamin C (25 mg/kg/day, 250 mg/kg/day) during the early stages of malaria infection may better enhance host protective immunity, but have no dose dependence.

Innate immune cell infiltration in melanoma metastases affects survival and is associated with BRAFV600E mutation status.

Little is known about the infiltrative pattern of innate immune cells in primary melanoma compared with their paired metastases and in BRAF-mutated tumors. Therefore, our aim was to characterize the inflammatory microenvironment in primary ulcerated and nonulcerated melanomas and paired metastases, to investigate the relation between inflammation and BRAF mutation in primary melanoma and paired metastases, and to evaluate the effect of the analyzed biomarkers on melanoma-specific survival. A total of 385 primary tumors and 96 paired metastases were stained with immunohistochemistry for BRAF, CD163+ macrophages, CD123+ plasmacytoid dendritic cells, CD66b+ neutrophils, and E-cadherin and estimated using objective computer-assisted image analysis. BRAF was semiquantitatively scored as either present or absent. In metastases of nonulcerated melanomas, we observed higher neutrophil (P=0.02) and macrophage (P=0.01) numbers. In the metastases of ulcerated melanomas, we found a higher number of macrophages (P<0.0001). Increase in the neutrophil numbers in the metastases was associated with poor patient survival after first relapse (hazard ratio=1.19, 95% confidence interval: 1.03-1.38, P=0.02). BRAF-positive primary tumors (P=0.02) and metastases (P=0.01) exhibited increased plasmacytoid dendritic cell numbers compared with BRAF-negative tumors. Lastly, primary melanomas in men had higher neutrophil numbers than women (P≤0.0001), and men had worse melanoma-specific survival (hazard ratio=1.52, 95% confidence interval: 1.04-2.21, P=0.03). Our data show that melanoma metastases are densely infiltrated with neutrophils, which affects survival. Our results also highlight the importance of recognizing the presence of inflammatory cells in the metastases as a prognostic marker, and that they may potentially be used to improve the precision of immunotherapy and BRAF targeted therapy.