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Differentiation of cells by flow cytometry provides informative somatic cell counts (SCCs) that allow analyzing leukocyte population patterns in udder infections of different etiologies. Postulating that this approach also enhances the statistical power to detect genetic variants linked to cell levels in milk of healthy mammary glands, we used monoclonal antibodies anti-CD18, anti-CD4, anti--CD14, and anti-PMN to count cells presenting these surface antigens, and performed a genome-wide association study of these counts in 125 Israeli Holsteins genotyped using SNP BeadChips. We identified an informative haplotype of 15 SNPs in the centromeric end of BTA3 that was strongly associated with CD18 cells (p < 2.3 × 10-9). Within this region, examination of the network of genes interacting with ITGB2 (CD18) indicated an Fc-γ-receptor gene cluster, including FCGR2A (CD32). Sanger-sequence analysis of FCGR2s-linked exon 3 variation to CD18 counts. Meta-analysis of RNA-Seq data revealed a significant negative correlation (R = -0.51) between expression of CD32 and CD18 in milk. Assembly of DNA-Seq reads uncovered FCGR copy-number variation and a variant, designated V7, was abundant in dairy cattle, probably reflecting adaptation to selection pressure for low SCC in Holstein milk.
Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d)) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d) PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b) DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d)) are coinjected in the footpad of mice with autologous DC (H-2(b)). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.
In mammals, methylation of DNA within regulatory sites and histone deacetylase recruitment in transcriptional repressing domains are involved in the loss of the expression of retroviral DNA or repeat arrays transferred in cells for therapeutic purposes. Various investigation results suggest that methylation/deacetylation events are modulated by extracellular and cytoplasmic signal transduction pathways closely involved in regulating cell differentiation. To analyse gene silencing mechanisms and assess if potential pharmacological treatment affects gene silencing kinetics we transduced U937 myelomonocytic cells with a bicistronic retroviral construct carrying the herpes simplex virus thymidine kinase (HSV-TK) and beta-galactosidase (Lac-Z) genes. This vector can be employed in vivo and in vitro to render transduced cell populations susceptible to ganciclovir (GCV). We verified the effect of the histone deacetylase inhibitor Trichostatin A (TSA) alone or combined with 5'-azacytidine (5'aza-C) on transcription downmodulation. Our results indicate that in our in vitro model TSA is able to reactivate transgene expression, more efficiently and with quicker kinetics (12-24h) than 5'aza-C (36-48 h). The effect is dose dependent (between 1 and 50 nM), with no relevant toxicity. Treatment with both drugs is synergistic in gene reactivation in terms of extension and persistence, with low toxicity and no relevant differentiating effects. The cells in which transgene expression has been reactivated undergo progressive silencing, but once weekly drug treatment can maintain high transgene expression levels for more than 90 days with no evidence of selection. The results obtained by treating U937 transduced clones with TSA and/or 5'aza-C together with IL-3, G-CSF or GM-CSF cytokines suggest that transduced U937 differentiation levels do not affect basal expression, but render these cells more responsive to reactivation by TSA or TSA plus 5'aza-C, but not to 5'aza-C alone. In conclusion, the results suggest that in vitro inhibition of histone deacetylase by TSA can interfere with gene silencing mechanisms affecting 5' Moloney murine leukaemia virus long terminal repeat (MoMuLV-LTR) driven transgene expression thus providing the rationale for TSA and/or 5'aza-C administration in animal models for the translation on gene therapy applications.
Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid-associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3- cells by flow cytometry. Group 2/MYELO overexpressed myeloid-associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)- CD14+ and/or CD18 + CD4- cells. We proposed that >12.5% CD5 + CD3- cells in the blood was indicative of lymphoid lineage, and > 3.0% CD14 + MHCII- cells or > 18% CD18 + MHCII-CD4- cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes.
Monoclonal antibodies that recognize cell surface molecules have been used deliver antigenic cargo to dendritic cells (DC) for induction of immune responses. The encouraging anti-tumor immunity elicited using this immunization strategy suggests its suitability for clinical trials. This review discusses the complex network of DC, the functional specialization of DC subsets, the immunological outcomes of targeting different DC subsets and their cell surface receptors, and the requirements for the induction of effective anti-tumor CD4 and CD8 T cell responses that can recognize tumor-specific antigens. Finally, we review preclinical experiments and the progress toward targeting human DC in vivo.
Streptococcus pneumoniae causes infection-related mortality worldwide. Immunocompromised individuals, including young children, the elderly, and those with immunodeficiency, are especially vulnerable, yet little is known regarding S. pneumoniae-related pathogenesis and protection in immunocompromised hosts. Recently, strong interest has emerged in the gut microbiota's impact on lung diseases, or the "gut-lung axis." However, the mechanisms of gut microbiota protection against gut-distal lung diseases like pneumonia remain unclear. We investigated the role of the gut commensal, segmented filamentous bacteria (SFB), against pneumococcal pneumonia in immunocompetent and immunocompromised mouse models. For the latter, we chose the Rag-/- model, with adaptive immune deficiency. Immunocompetent adaptive protection against S. pneumoniae infection is based on antibodies against pneumococcal capsular polysaccharides, prototypical T cell independent-II (TI-II) antigens. Although SFB colonization enhanced TI-II antibodies in C57BL/6 mice, our data suggest that SFB did not further protect these immunocompetent animals. Indeed, basal B cell activity in hosts without SFB is sufficient for essential protection against S. pneumoniae. However, in immunocompromised Rag-/- mice, we demonstrate a gut-lung axis of communication, as SFB influenced lung protection by regulating innate immunity. Neutrophil resolution is crucial to recovery, since an unchecked neutrophil response causes severe tissue damage. We found no early neutrophil recruitment differences between hosts with or without SFB; however, we observed a significant drop in lung neutrophils in the resolution phase of S. pneumoniae infection, which corresponded with lower CD47 expression, a molecule that inhibits phagocytosis of apoptotic cells, in SFB-colonized Rag-/- mice. SFB promoted a shift in lung neutrophil phenotype from inflammatory neutrophils expressing high levels of CD18 and low levels of CD62L, to pro-resolution neutrophils with low CD18 and high CD62L. Blocking CD47 in SFB(-) mice increased pro-resolution neutrophils, suggesting CD47 down-regulation may be one neutrophil-modulating mechanism SFB utilizes. The SFB-induced lung neutrophil phenotype remained similar with heat-inactivated S. pneumoniae treatment, indicating these SFB-induced changes in neutrophil phenotype during the resolution phase are not simply secondary to better bacterial clearance in SFB(+) than SFB(-) mice. Together, these data demonstrate that the gut commensal SFB may provide much-needed protection in immunocompromised hosts in part by promoting neutrophil resolution post lung infection.
Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin-mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.
Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility to immune effector cells of MDR tumor cells. A cell line isolated from the malignant pleural effusion of a breast cancer patient was transfected with human and murine MDR1 genes, and four variants with different levels of MDR were obtained. Lymphokine-activated killer (LAK) activity was measured by a 51Chromium release, and conjugate formation assays. MDR1 transfectant P-gp+ breast carcinoma lines had increased LAK susceptibility compared to their parent line. Some part of the increased LAK susceptibility of drug-resistant cell lines was at the binding/recognition level as shown by conjugate formation assays. This suggests that differences may exist between paired cell lines with respect to the expression of cell adhesion molecules (CAMs). Monoclonal antibodies (mAbs) to CAMs and flow cytometry were used to quantitate these antigens. The CAMs studied were those previously found to be upregulated by stimulating NK cells with (interleukin-2) IL-2; ICAM-1 (CD54), LFA-3 (CD58), N-CAM (CD56), and the beta chain of LFA-1 (CD18). Although no differences in these CAMs were found between the breast carcinoma line and its MDR1-transfected variants, the target susceptibility results given above suggest that IL-2 treatment could be effective in combination with current protocols using chemotherapeutics, monoclonal antibodies (mAbs) and stem cell transplantation.
Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.
Cholesterol-laden, foam macrophages constitute the most characteristic component of human atherosclerotic plaques. Persistent uptake of oxLDLs results in accumulation of lipid bodies inside the cells and determines their phenotype and subsequent functions. In this work, we describe the phenotype of human monocyte-derived foam cells obtained by differentiation in the constant presence of oxLDLs for 30 days (prolonged-hMDFCs). Although neither the total cellular nor the cell surface expression of Toll-like receptors (TLR) was regulated by oxLDLs, the prolonged-hMDFCs changed dramatically their responsiveness to TLR ligands and inactivated bacteria. Using multiplex technology, we observed an acute decline in cytokine and chemokine production after surface and endosomal TLR stimulation with the exception of TLR2/6 triggering with agonists Pam2CSK4 and MALP-2. We also noted significant reduction of some surface receptors which can have accessory function in recognition of particulate antigens (CD47, CD81, and CD11b). In contrast, the prolonged-hMDFCs responded to inflammasome activation by LPS/nigericin with extensive, necrotic type cell death, which was partially independent of caspase-1. This pyroptosis-like cell death was aggravated by necrostatin-1 and rapamycin. These findings identify a potential contribution of mature foam cells to inflammatory status by increasing the immunogenic cell death burden. The observed cross-talk between foam cell death pathways may lead to recognition of a potential new marker for atherosclerosis disease severity. Overall, our study demonstrates that, in contrast to other cellular models of foam cells, the prolonged-hMDFCs acquire a functional phenotype which may help understanding the role of foam cells in the pathogenesis of atherosclerosis.
Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin-deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell-mediated immunity in response to a cutaneous antigen. These effects required platelet-expressed P-selectin that allows activated platelets to transiently form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent beta2 integrin-dependent firm adhesion. These profound effects of platelet-mediated cell-cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.
Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.
Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.
Peritoneal mesenchymal stromal cells (pMSCs) are isolated from peritoneal dialysis (PD) effluent, and treatment with the pMSCs reduces peritoneal membrane injury in rat model of PD. This study was designed to verify the identity of the pMSCs. pMSCs were grown in plastic dishes for 4-7 passages, and their cell surface phenotype was examined by staining with a panel of 242 antibodies. The positive stain of each target protein was determined by an increase in fluorescence intensity as compared with isotype controls in flow cytometrical analysis. Here, we showed that pMSCs predominantly expressed CD9, CD26, CD29, CD42a, CD44, CD46, CD47, CD49b, CD49c, CD49e, CD54, CD55, CD57, CD59, CD63, CD71, CD73, CD81, CD90, CD98, CD147, CD151, CD200, CD201, β2-micoglobulin, epithelial growth factor receptor, human leukocyte antigen (HLA) class 1, and, to a lesser extent, CD31, CD45RO, CD49a, CD49f, CD50, CD58, CD61, CD105, CD164, and CD166. These cells lacked expression of most hematopoietic markers such as CD11b, CD14, CD19, CD34, CD40, CD80, CD79, CD86, and HLA-DR. There was 38.55% difference in the expression of 83 surface proteins between bone marrow (BM)-derived MSCs and pMSCs, and 14.1% in the expression of 242 proteins between adipose tissue (AT)-derived MSCs and pMSCs. The BM-MSCs but not both AT-MSCs and pMSCs express cytokine receptors (IFNγR, TNFI/IIR, IL-1R, IL-4R, IL-6R, and IL-7R). In conclusion, pMSCs exhibited a typical cell surface phenotype of MSCs, which was not the same as on BM-MSCs or AT-MSCs, suggesting that the pMSCs may represent a different MSC lineage from peritoneal cavity.
The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis.
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