Characterisation of a novel dendritic-like cell in spleen

Dendritic cells (DC) are specialised antigen presenting cells, which induce and control the adaptive immune response. DC mediate a wide range of immune responses including generation of T helper cells, development of cytotoxic T cell responses and induction of tolerance. In this lab, a splenic longterm culture (LTC) was established that produces distinct dendritic-like cells (LTC-DC) continuously in the absence of added growth factors or cytokines. Previous studies have identified an in vivo equivalent of LTC-DC in murine spleen. This thesis further characterises this novel dendritic-like subset (L-DC). The relationship between L-DC and other known dendritic and myeloid subsets has also been investigated through phenotypic, morphological and functional studies, by comparing gene expression, and by analysis of cell development in mutant mouse models. In order to identify L-DC, it was necessary to redefine splenic dendritic and myeloid subsets. L-DC have now been characterised with the CD11bhiCD11cloMHCII-Ly6C-Ly6G-CD43+CX3CR1+Siglec-F- phenotype. CD43 expression, lack of MHCII expression, and a low level of CD11c expression best differentiate L-DC from conventional DC (cDC). Like CD8+ cDC, L-DC have high capacity for receptor-medicated endocytosis and induce activation and proliferation of CD8+ T cells, although not CD4+ T cells. L-DC also show capacity to induce an in vivo cytotoxic T lymphocyte response equivalent to that induced by CD8+ cDC. L-DC are distinguishable from neutrophils by the absence of Ly6G and 7/4 expression, and from eosinophils by lack of Siglec-F expression. L-DC can be distinguished from monocytes by their lack Ly6C expression. Morphological studies revealed L-DC to be mononuclear cells with vacuoles in the cytoplasm, and distinct from other myeloid cell types. All of these findings have identified L-DC as a novel antigen presenting cell subset, distinct from other known dendritic and myeloid subsets in murine spleen. Attempts have been made to understand the lineage relationship between L-DC and the other splenic dendritic and myeloid subsets, and to identify new markers for easier delineation of L-DC. Transcriptome analysis (Affymetrix) was performed on L-DC, dendritic and myeloid subsets. The L-DC gene expression profile was found to be distinct from that of cDC. However, the L-DC gene expression profile was also found to be distinct from that of monocytes subsets. L-DC specifically expressed genes like Siglec-e, Igsf2, CD300ld, CD300e and CD9. Overall, gene expression analysis showed that L-DC resembled a myeloid lineage cell having dendritic-like characteristics and function in antigen presentation. Mouse strains carrying mutations which affect the development of dendritic and myeloid cell types were investigated for changes in L-DC development in spleen. However L-DC numbers were not altered in any of the mutant mice studied, suggesting that L-DC is a distinct lineage of cells that arise from endogenous splenic progenitors, separate from the dendritic and monocyte. This study has revealed a novel dendritic-like cell type distinct from known dendritic and myeloid subsets in spleen, and having specialised function in cross-priming CD8+ T cells. The findings presented here predict an important role for L-DC in the immune response against blood-borne antigens which enter the splenic microenvironment

Antigen presenting capacity of murine splenic myeloid cells

BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named ‘L-DC’ was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8(+) T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII(−) cells and unable to activate CD4(+) T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4(+) T cells or CD8(+) T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8(+) T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4(+) T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell

Murine spleen contains a diversity of myeloid and dendritic cells distinct in antigen presenting function

The spleen contains multiple subsets of myeloid and dendritic cells (DC). DC are important antigen presenting cells (APC) which induce and control the adaptive immune response. They are cells specialized for antigen capture, processing and presentation to naıve T cells. However, DC are a heterogeneous population and each subset differs subtly in phenotype, function and location. Similarly, myeloid cell subsets can be distinguished which can also play an important role in the regulation of immunity. This review aims to characterize splenic subsets of DC and myeloid cells to better understand their individual roles in the immune response.NHMRC (National Health and Medical Research Council of Australia

Spleen as a site for hematopoiesis of a distinct antigen presenting cell type

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen

Spleen as a Site for Hematopoiesis of a Distinct Antigen Presenting Cell Type

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.This work was supported by a project grant #585443 to HO from the National Health and Medical Research Council (NHMRC) of Australia. PP was supported by an NHMRC CJ Martin Fellowship. YH was supported by a postgraduate scholarship from the Australian National Universit

A mixed methods comparative evaluation of a low cost otoscope (Arclight) with a traditional device in twenty-one clinicians

According to the World Health Organisation estimate, the global burden of illness from chronic ear infection affects about 4% of the world population (up to 330 million) with ear discharge and 60% of whom (up to 200 million) suffer from significant hearing impairment.1 Alarmingly, over 90% of the burden of chronic ear infections is borne by low‐ and middle‐income countries (LMICs).PostprintPeer reviewe