A comprehensive evaluation of human plasmacytoid dendritic cells using small volumes of human blood.
Elucidation of the role played by cells of the innate immune system, particularly the dendritic cell (DC) populations, has led to a precipitous growth in our understanding of immunity to pathogens and foreign antigens. Much of this information has been derived from studies using mouse model systems. However, mice and human DCs differ drastically in the relative distribution of the toll-like receptors (TLRs) critical for immune activation. This is particularly true for the plasmacytoid DCs (pDCs), which are activated almost exclusively through TLR signaling. Variation in this DC subpopulation has been implicated in a number of pathological syndromes; therefore, a thorough understanding of their steady state and activation profiles in human patients is essential. A number of factors, including the relatively low numbers of these cells in blood, have precluded careful analysis in clinical trials. To overcome these limitations, we have developed a technique for studying the steady state and activation profile of pDCs collected from small amounts of human blood. This technique can be performed with 10,000 cells to obtain the immune transcriptome of the pDCs analyzed by quantitative PCR using amplified RNA. In addition, we have used multiplex enzyme-linked immunosorbent assays to measure secreted proteins. We demonstrate the validity of this technique and document its potential for use with blood from human study populations.
Phipps-Yonas
Hannah
H
Department of Microbiology, Mount Sinai School of Medicine, New York, New York, USA.
Jiang
Ke
K
Hartmann
Boris Michael
BM
Ge
Yongchao
Y
Sealfon
Stuart C
SC
Moran
Thomas M
TM
D000818:Animals; D000941:Antigens / Q000276:immunology; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D051379:Mice; D010950:Plasma Cells / Q000166:cytology / Q000276:immunology*; D012313:RNA / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000276:immunology; D051193:Toll-Like Receptors / Q000276:immunology; D014158:Transcription, Genetic / Q000276:immunology*
D000941:Antigens; D051193:Toll-Like Receptors; D012313:RNA