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| Presenter: |
| AnneMarie Brescia |
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| Institute: |
Lab or Branch |
| NIAMS |
Autoimmunity Branch |
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| Title: |
| Proteomic confirmation of genes that define
human tonsillar B cell subsets using Immunoaffinity Capillary
Electrophoresis |
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| Authors: |
| A.C. Brescia, D.B. Thurber, T.M. Phillips,
P. Lipsky, A.C. Grammer |
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| Abstract: |
| Immunoaffinity capillary electrophoresis
(ICE) was used to confirm protein products of genes identified
by microarray that define human tonsillar B cell subsets.
Multiparameter flow cytometry was used to sort highly
purified populations of naïve, memory, germinal center
and Ig-secreting plasma cell B cell subsets from human
tonsil, an activated human secondary lymphoid tissue.
Following mRNA isolation from each subset, gene expression
was analyzed by microarray. Software analysis of the data
using TreeView and Cluster identified genes that were
threefold-higher in Ig-secreting plasma cells compared
to naïve, memory and germinal center populations.
Protein was isolated from each subset and tested by ICE
for the genes identified to be highly expressed in the
Ig-secreting plasma cell subset. ICE analysis is performed
with Fab fragments of antibodies to proteins of interest
that are attached inside a microcapillary. Lysates containing
protein from each subset are injected and proteins captured
specifically by Fabs are fluorescently labeled. Eluted
proteins are detected by an in-line laser-induced fluorescence
detector coupled to a fiber-optic mass spectrometer. ICE
analysis of human tonsillar B cell subsets confirmed unique
plasma cell expression of the transcription factors that
define Ig-secreting plasma cells, BLIMP1, XBP1 and IRF4,
as well as proteins that have been demonstrated to be
involved in Ig secretion such as GRP78, ERp72, HSP90,
and HSP70. Moreover, genes newly identified by microarray
to be markers of the tonsillar Ig-secreting plasma cell
subset such as the amino acid transporter CD98, the chemotactic
molecules HCC-4 and BMP6, along with signaling molecule
IkBE, were confirmed by ICE. Together, these results indicate
that ICE can be used as part of a multiomic approach to
identify the presence of B cell subsets, such as Ig-secreting
plasma cells that are elevated in the periphery of patients
with active systemic lupus erythematosus, in human samples.
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