Cell-type specific promoters drive expression of the inhibitory receptor CD85j by hematopoietic lineages Public
Lamar, David Lewis (2010)
Abstract
CD85j (ILT2/LILRB1/LIR-1) is an inhibitory receptor that
recognizes MHC class Ia
and Ib alleles that are widely expressed on all cell types. Upon
ligand recognition,
CD85j diminishes kinase activity by recruiting phosphatases to
motifs within its
cytoplasmic domain. Within the hematopoietic system, CD85j is
expressed with
cell-specific patterns and cell surface densities which reflect the
different roles of
cell contact-mediated inhibition in these lineages. While monocytes
ubiquitously
have high cell surface expression, B lymphocytes start to express
CD85j at
intermediate levels during maturation on both early B cells in the
bone marrow
and, as we show here, on transitional B cells in the periphery. T
cells and NK
cells gain low expression only on a subset of cells. On NK cells,
CD85j is one
component of a repertoire of MHC class I-binding regulatory
receptors, each of
which may or may not be expressed on a given cell. CD85j expression
by T cells
is exclusive to memory cells and is more likely on CD8 T cells. We
show that
CD85j expression is predominant on CD45RA effector T cells and
that
expression increases with age in all memory subsets. The
cell-specific
expression pattern is accomplished by two complementing but not
independent
mechanisms. We show that lymphocytes and monocytes utilize
distinct
promoters to drive CD85j expression. The previously undescribed
lymphocyte
promoter maps 13 kb upstream of the monocyte promoter; its use
results in the
inclusion of a distant exon into the 5'UTR. A short sequence
stretch within this
exon has the unique function of repressing CD85j protein
translation and is
responsible for the subdued expression in lymphocytes. These
cell-specific
mechanisms allow tailoring of CD85j levels to the distinct roles it
plays in different
hematopoietic lineages.
Table of Contents
Table of Contents
Chapter 1: Introduction and Literature Review 1
Introduction 1
CD85j Literature Review 7
Original descriptions of CD85j 7
The defining of the LILR gene family 12
CD85j expression profile 14
Ligand-binding properties 18
Function of CD85j 22
Cell-type specific implications of CD85j expression and preview of our findings 35
Chapter 2: Distinct promoters drive expression of CD85j in hematopoietic lineages 39
CD85j expression differs among peripheral blood subsets 39
LILRB1 transcripts in lymphocytes contain 5'UTR sequences that are absent in monocytes 43
Lymphocytes initiate LILRB1 transcription from a site 13 kb upstream of the major site used by monocytes 45
Exon 1 sequences inhibit translation of CD85j 47
The sequence conferring translational repression of CD85j is mapped to 30 nt of exon 1 50
Chapter 3: Age-dependent acquisition of CD85j on CD8 T cells 54
CD85j is an age-dependent cell-surface marker on CD8 T cells 54
Increased LILRB1 transcription accounts CD85j surface expression on CD8 T cells 59
Chapter 4: Materials and Methods 68
Isolation of human mononuclear cells (Chapter 2) 68
Study population (Chapter 3) 68
Flow cytometry (Chapter 2) 69
Immunophenotyping and flow cytometry analysis (Chapter 3) 69
RNA isolation and cDNA synthesis 70
Quantitative real-time PCR 70
Western blotting 71
Plasmids 71
Transfection of human PBMCs 72
5'-rapid amplification of cDNA ends (5'RACE) 73
Luciferase reporter assay 73
In vitro cultures 74
Statistics (Chapter 2) 74
Statistical analysis (Chapter 3) 74
Primer Sequences 75
Chapter 5: Discussion 79
Acknowledgments 87
References 88
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