Year: 2009
Type: dissertation
Abstract: Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response.Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8 + and CD8 -subtypes.Each subtype appears to have a specialised role in the various arms of immunity and tolerance.Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes.The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation.Particular was given to DC of the spleen.Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steadystate DC subtypes.From the spleen of mice I isolated a novel DC precursor population that generated only CD8 + and CD8 -steady-state cDC upon transfer.These precursors had the phenotype CD11c int CD43 + SIRP-α int MHC II -and, as they were strictly cDCcommitted in their development, were termed pre-cDC.In contrast to many reports that monocytes are the precursors of DC, I found that pre-cDC were not classical monocytes.Pre-cDC were a relatively late-stage precursor population as they only transiently gave DC progeny within a few divisions.Importantly, there was already evidence of pre-commitment to either CD8 + or CD8 -cDC when separated by CD24, eatablishing that these subtypes represent different terminal lineages.The DC subtypes and their precursors are rare in a mouse and so their extensive study has been hampered.I investigated an in vitro system in which bone marrow (BM) is cultured with flt3 ligand (FL) to generate DC.I discovered that the DC products included, in addition to pDC, two cDC subtypes that were equivalent DC subtypes to in vivo steady-state CD8 + and CD8 -cDC.Thus this culture system reflected in vivo steady-state DC development.These FL BM cultures were probed at earlier time points during culture for the precursor stages that gave rise to the DC subtypes.In this way, I identified two novel precursors that each generated pDC, CD8 + cDC and CD8 -cDC.These two precursors were related in a linear developmental pathway such that the upstream "pro-DC" divided and gave rise to "pre-DC", en route to DC generation.As monocytes had been assumed to be precursors of steady-state DC, it was surprising that the precursors identified above were not monocytes.Therefore, I tested whether either of the characterised Ly6C hi and Ly6C lo monocytes were efficient precursors of spleen DC in the steady-state state.They were not.However, when mice were subject to inflammation, Ly6C hi monocytes were then able to generate DC in spleen.This was a novel DC subtype that was CD11 int CD11b hi Mac-3 + , distinct from steady-state CD11c hi CD11b lo Mac-3 -spleen cDC.These induced "inflammatory" DC appeared to actually be the in vivo 'myeloid' DC equivalent to those generated when monocytes or BM are cultured with granulocyte macrophage colony-stimulating factor (GM-CSF).Thus, I have identified some of the key steps and branch points in the development of DC downstream of the early haematopoietic precursors.I have also dissected the contribution of different precursors to the DC subtypes in both the steady-state and during inflammation.The culture system I have developed will allow clonal analysis of DC development.It may also help guide the identification of the human equivalents of the various mouse DC precursors and DC subtypes, and hasten clinical applications.Cell suspensions ........
Author: Shalin H. Naik
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2736805708