Cell-type specific promoters drive expression of the inhibitory receptor CD85j by hematopoietic lineages Público

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

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Immunology
Palavra-chave	Human Immunology Monocytes Lymphocytes Inhibitory Receptors CD85j Promoter Choice LILRB1
Committee Chair / Thesis Advisor	Lukacher, Aron E, Emory University
Committee Members	Boss, Jeremy, Emory University Cornelia M, Weyand, Stanford University Goronzy, Jorg J, Stanford University

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