Characterisation of a novel dendritic-like cell in spleen

Abstract

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. Show more