Project description:Dendritic cells (DC) localize throughout the body, where they sense and capture invading pathogens to induce protective immune responses. Hence, harnessing the biology of tissue-resident DC is crucial for the rational design of vaccines against pathogens. Herein, we characterized the transcriptomes of four antigen presenting cell (APC) subsets from the human vagina (vLC, vCD14- DC, vCD14+ DC, vMM-NM-&) and compared them to those of three skin DC (sDC) subsets and blood myeloid DC. We find that APC genomic fingerprints are significantly influenced by the tissue of origin as well as by individual APC subsets. Nonetheless, CD14+ APC from both vagina and skin are geared towards innate immunity and pro-inflammatory responses, whereas CD14- DC, particularly sLC, vLC, and vCD14- DC, display both Th2-inducing and regulatory phenotypes. We also identified vAPC subset-specific cellular and functional biomarkers that will guide the design of mucosal vaccines against sexually transmitted pathogens. Vaginal and skin tissues were obtained from female patients who underwent pelvic or cosmetic surgeries under protocols approved by the Institutional Review Board (IRB) of Baylor Research Institute (BRI). Patients were not infected with HIV, HCV or TB and did not display inflammation in the tissues. No other diagnosis information was available. Blood from healthy female volunteers was obtained under a protocol approved by the IRB of BRI. 87 total samples. 6 Blood mDC; 16 Dermal CD1c+CD14-; 10 Epidermal LC; 12 Vaginal CD1c+CD14-; 13 Vaginal CD1c+CD14+; 7 Vaginal HLADR- w/ 2 replicates (Vaginal HLADR-_VM610 and Vaginal HLADR-_VM611); 9Vaginal LC; 14 Vaginal Macrophage.
Project description:Dendritic cells (DC) localize throughout the body, where they sense and capture invading pathogens to induce protective immune responses. Hence, harnessing the biology of tissue-resident DC is crucial for the rational design of vaccines against pathogens. Herein, we characterized the transcriptomes of four antigen presenting cell (APC) subsets from the human vagina (vLC, vCD14- DC, vCD14+ DC, vMΦ) and compared them to those of three skin DC (sDC) subsets and blood myeloid DC. We find that APC genomic fingerprints are significantly influenced by the tissue of origin as well as by individual APC subsets. Nonetheless, CD14+ APC from both vagina and skin are geared towards innate immunity and pro-inflammatory responses, whereas CD14- DC, particularly sLC, vLC, and vCD14- DC, display both Th2-inducing and regulatory phenotypes. We also identified vAPC subset-specific cellular and functional biomarkers that will guide the design of mucosal vaccines against sexually transmitted pathogens. Vaginal and skin tissues were obtained from female patients who underwent pelvic or cosmetic surgeries under protocols approved by the Institutional Review Board (IRB) of Baylor Research Institute (BRI). Patients were not infected with HIV, HCV or TB and did not display inflammation in the tissues. No other diagnosis information was available. Blood from healthy female volunteers was obtained under a protocol approved by the IRB of BRI.
Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins). Total RNA was extracted and hybridised to 24 bead arrays. Dendritic cells and monocytes from human blood and skin using magnetic bead and flow cytometry based cell sorting both before and after culture for 24 hours
Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins). Total RNA was extracted and hybridised to 62 cDNA arrays. Dendritic cells and monocytes from human blood and skin using magnetic bead and flow cytometry based cell sorting both before and after culture for 24 hours
Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins).
Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins).
Project description:Antigen presenting cells (APC) are a heterogenous population, comprised of macrophages/monocytes (CD14+ cells), classical dendritic cells (CD141+DC and CD1c+ DC) and pDC. Upon stimulation, APC migrate from peripheral organs to lymph nodes, where they drive T cell specific lineage fate, that is towards immune activation or suppression. APC in human tissues remain poorly defined. Through our previous published data we have charactised APC within adult skin and blood. Here we extend these findings, by increasing the sample for skin CD14+ DC and CD1c+ DC and performing gene array analysis of adult spleen CD14+ DC, CD141+DC and CD1c+ DC. Once, we were confident we could clearly distinguish our populations (CD14+ cell, CD141+ DC and CD1c+ DC) of interest from other cells, we sorted FACS purified the cells and prepared them for gene array analysis. Through generating subset specific gene signatures and comparing CMAP scores we confirmed we had identified equivalent APC subsets across human adult skin and spleen.
Project description:Inflammatory skin diseases including atopic dermatitis (AD) and psoriasis (PSO) are underpinned by dendritic cell- (DC) mediated T cell responses. Currently, the heterogeneous human cutaneous DC population is incompletely characterized, and its contribution to these diseases remains unclear. Here, we performed index-sorted single-cell flow cytometry and RNA-sequencing of lesional and non-lesional AD and PSO skin to identify macrophages and all DC subsets, including the newly-described mature LAMP3+BIRC3+ DC enriched in immunoregulatory molecules (mregDC) and DC3. By integrating our indexed data with published skin datasets, we generated a myeloid cell universe of DC and macrophage subsets in healthy and diseased skin. Importantly, we found that CD14+ DC3 were increased in PSO lesional skin and co-produced IL1B and IL23A, which are pathologic in PSO. Our study comprehensively describes the molecular characteristics of macrophages and DC subsets in AD and PSO at single-cell resolution, and identifies CD14+ DC3 as potential promotors of inflammation in PSO.