The Inhibitory Effect of Kell Blood Group Antibodies on Erythroid Progenitor Cell Growth

98 Pages·2008·3.86 MB·English

by  Eva Seto

The Inhibitory Effect of Kell Blood Group Antibodies on Erythroid
Progenitor Cell Growth

By

Eva Seto

A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Laboratory Medicine & Pathobiology
University of Toronto
© Copyright by Eva Seto 2008
The Inhibitory Effect of Kell Blood Group Antibodies on Erythroid
Progenitor Cell Growth

Eva Seto
Master of Science
Department of Laboratory Medicine & Pathobiology
University of Toronto
2008

Abstract
The clinical manifestations of hemolytic disease of the fetus and newborn mediated by anti-K, an
antibody of the Kell blood group system, have features that are distinguishable from the classical
form of the disease. Affected fetuses may have low numbers of circulating reticulocytes. As
well, antibody titers and amniotic fluid bilirubin levels are not reliable predictors of anemia.
These observations suggest that antibodies to Kell glycoprotein lead to anemia through
suppression of erythropoiesis. This study established a liquid in vitro erythroid progenitor cell
culture model in which to perform biochemical analyses on the mechanism of the suppressive
growth effect of anti-Kell glycoprotein. Using this culture model, this study demonstrated the
requirement for co-ligation of Kell glycoprotein by a bivalent antibody for growth suppression.
The absence of markers of apoptosis in cell cultures treated with anti-Kell glycoprotein suggests
that the mechanism of growth suppression is distinct from programmed cell death and necrosis.
Furthermore, this growth suppression cannot be rescued by direct addition of erythropoietin.
ii
Acknowledgements

I would like to thank my supervisor, Dr. Greg Denomme, for his mentorship and continued
support. His efforts to ensure my graduate training was a fulfilling and rewarding experience are
greatly appreciated.

I would also like to thank my committee members, Dr. B. Fernandes, Dr. G. Seaward, Dr. F.
Tsui, and Dr. C. Wang, for their guidance and valuable input throughout this project.

To all members of the Denomme lab, thank you for your friendships, great conversation and
many wonderful lunches. I would particularly like to thank Dr. D. Wang for his technical
expertise and support.

I would like to extend my sincerest gratitude to my parents, Jim and Bernice, and Gary. They
have always believed in me and provided me with unwavering support.

Finally, I would like to acknowledge funding support during my Master’s program, awarded
through the Ontario Graduate Scholarship.

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Table of Contents

Abstract ii
Acknowledgements iii
Table of Contents iv
List of Tables vii
List of Figures viii
List of Appendices ix
Abbreviations x
1.0 Introduction 1
1.1 History of hemolytic disease of the fetus and newborn (HDFN) 2
1.2 Blood group antigens that can cause HDFN 4
1.3 Laboratory and clinical management of HDFN 5
1.4 Clinical features of anti-K HDFN 6
1.5 The Kell blood group system 7
1.6 The KEL and XK genes 8
1.7 Kell glycoprotein polymorphisms 8
1.8 Structure of Kell glycoprotein 9
1.9 Kell glycoprotein as an enzyme 11
1.10 Structure of XK 11
1.11 Function of XK 12
1.12 The Kell-Kx complex 13
1.13 Kell glycoprotein and XK are connected to the cell cytoskeleton 14
1.14 Kell and erythropoiesis 16
1.15 In vitro studies of antibodies to the Kell blood group system 18
1.16 Objectives and Hypothesis 20
2.0 Retrospective Review 21
2.1 Predictive fetal blood group genotyping 22
2.1.1 Laboratory review of fetal blood group genotyping tests 22
2.2 Clinical management of HDFN 23
2.2.1 Retrospective review of health care resource utilization 24
iv
3.0 Materials and Methods 28
3.1 In vitro suppression of erythroid progenitor cells 29
3.1.1 Preparation of CD34+ cells 29
3.1.2 Preparation of antibodies 29
3.1.3 Liquid cell culture system 30
3.1.4 Immunophenotype analysis 30
3.2 Effect of anti-Kell glycoprotein Fab on erythroid progenitor proliferation 31
3.2.1 Preparation of anti-Kell glycoprotein Fab and anti-GYPC Fab 31
3.2.2 Characterization of anti-Kell glycoprotein Fab and anti-GYPC Fab 32
product
3.2.3 Effect of anti-Kell glycoprotein Fab on erythroid progenitor liquid 32
cultures
3.3 Role of apoptosis in the growth inhibitory effect of anti-Kell glycoprotein 33
3.3.1 Phosphatidylserine asymmetry in anti-Kell glycoprotein induced 33
erythroid cells
3.3.2 Effect of a general caspase inhibitor on the growth inhibitory effect of 33
anti-Kell glycoprotein
3.3.3 Caspase activation in anti-Kell glycoprotein induced erythroid cell 34
cultures
3.3.4 Mitochondrial membrane potential in anti-Kell glycoprotein treated cells 34
3.4 The ability of erythropoietin to rescue the growth inhibitory effect of anti-Kell 34
glycoprotein
3.5 Statistical analysis 35
4.0 Results 36
4.1 Erythroid progenitor proliferation in liquid culture system 37
4.2 Immunophenotype of erythroid progenitors in liquid culture 38
4.3 Effect of antibodies to Kell glycoprotein and blood group specific antibodies 39
on erythroid progenitor cells in liquid culture
4.4 Characterization of anti-Kell glycoprotein Fab product 40
4.5 Effect of anti-Kell glycoprotein Fab on erythroid progenitor proliferation 41
4.6 Phosphatidylserine asymmetry in anti-Kell glycoprotein induced erythroid 43
progenitors
4.7 Effect of a general caspase inhibitor on the growth inhibitory effect of anti- 44
Kell glycoprotein
v
4.8 Caspase activation in anti-Kell glycoprotein induced erythroid cell cultures 47
4.9 Mitochondrial membrane potential of anti-Kell glycoprotein treated cells 49
4.10 Ability of serial additions of erythropoietin to rescue growth suppression 51
caused by anti-Kell glycoprotein
5.0 Discussion 53
5.1 Clinical and laboratory impact of HDFN 54
5.1.1 Laboratory review of fetal blood group genotyping tests 54
5.1.2 Retrospective review of health care resource utilization 55
5.2 In vitro suppression of erythroid progenitor cells 57
5.2.1 Progression through erythropoiesis in liquid cell culture 57
5.2.2 Suppression by antibodies to Kell glycoprotein and blood group specific 58
antibodies
5.3 Requirement for antigen co-ligation for growth inhibitory effect 59
5.3.1 Characterization of Fab products 59
5.3.2 Effect of Fab products on erythroid progenitor proliferation 60
5.4 Role of apoptosis in the growth inhibitory effect of anti-Kell glycoprotein 61
5.4.1 Phosphatidylserine asymmetry in anti-Kell glycoprotein induced 61
erythroid progenitors
5.4.2 Effect of a general caspase inhibitor on the growth inhibitory effect of 62
anti-Kell glycoprotein
5.4.3 Caspase activation in anti-Kell glycoprotein induced erythroid cell 62
cultures
5.4.4 Mitochondrial membrane potential of anti-Kell glycoprotein treated cells 63
5.5 Ability of serial additions of erythropoietin to rescue growth suppression 63
caused by anti-Kell glycoprotein
5.6 Summary 64
5.7 Future Directions 65
6.0 References 68
7.0 Appendices 80

vi
List of Tables

Table 1. Order of expression of cell-surface markers on differentiating erythroid cells. 18
Table 2. Summary of cost analysis for fetal blood group genotyping. 27
Table 3. Immunophenotype of CD34+ cells seeded in liquid culture. 38
Table 4. Percent of events with loss of JC-1 red fluorescence. 50

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List of Figures

Figure 1. Diagram of the Kell-XK complex. 10

Figure 2. Schematic representation of Kell glycoprotein and XK within the 4.1R 15
multiprotein complex in the red cell membrane.

Figure 3. Distribution of antigens for which fetal blood group was tested. 23

Figure 4. Comparison of the mean number of clinic visits or procedures per woman 26
with Kell-positive, antigen-positive, or antigen-negative fetus.

Figure 5. Representative cell proliferation in two-stage culture protocol in the absence 37
of suppressive antibody.

Figure 6. Percent growth suppression of treated erythroid progenitors. 39

Figure 7. Suppression of progenitor proliferation by anti-Kell glycoprotein Fab and 42
anti-(heavy and light chain).

Figure 8. Annexin V-FITC and PI labeling of erythroid progenitors treated with anti- 43
Kell glycoprotein.

Figure 9. Suppression of progenitor proliferation by anti-Kell glycoprotein in the 45
presence of Z-VAD-FMK.

Figure 10. Suppression of progenitor proliferation by etoposide and partial rescue by Z- 46
VAD.

Figure 11. Mean fluorescence intensity of ApoStat of erythroid progenitors treated with 48
anti-Kell glycoprotein.

Figure 12. JC-1 assay of anti-Kell glycoprotein treated erythroid progenitors. 49

Figure 13. Proliferation of erythroid progenitors in the presence of serially added excess 52
erythropoietin and anti-Kell glycoprotein.

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List of Appendices

Appendix A. Agglutination grading of indirect agglutination test. 81
Appendix B. Cell surface marker expression of CD34+ cells seeded in liquid culture. 82
Appendix C. Non-reducing SDS-PAGE analysis of Kell glycoprotein Fab product. 83
Appendix D. Characterization of anti-GYPC Fab product. 84
Appendix E. Non-reducing SDS-PAGE analysis of anti-GYPC Fab product. 85
Appendix F. Effect of anti-GYPC Fab on erythroid progenitor proliferation. 86
Appendix G. Caspase activation of erythroid progenitors treated with anti-Kell 87
glycoprotein

ix
Abbreviations

~(H+L) anti-(heavy and light chain)
4.1R protein 4.1
BCA bicinchoninic acid
BFU-E burst-forming unit-erythroid
BrdU 5-bromo-2-deoxyuridine
CCCP carbonyl cyanide 3-chlorophenylhydrazone
cdk cyclin-dependent kinase
CFU-E colony-forming unit-erythroid
CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte
DIM detergent-insoluble material
DMSO dimethyl sulfoxide
Epo erythropoietin
ET-3 endothelin-3
FACS fluorescence activated cell sorting
FITC fluorescein isothiocyanate
Ge Gerbich
GM-CSF granulocyte-macrophage colony stimulating factor
GP glycoprotein
GYPA glycophorin A
GYPC glycophorin C
HDFN hemolytic disease of the fetus and newborn
IgG immunoglobulin G
IL interleukin
IMDM Iscove’s Modified Dulbecco’s Medium
IVIG intravenous immune globulin
JAK2 Janus kinase 2
JC-1 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazol-carbocyanine iodide
K Kell, KEL1
K Kell-null
0
k Cellano, KEL2
MAP mitogen-activated protein
MFI mean fluorescence intensity
MWCO molecular weight cut off
OHIP Ontario Health Insurance Plan
PBS phosphate buffered saline
PI propidium iodide
pRb retinoblastoma protein
PS phosphatidylserine
RBC red blood cell
Rh Rhesus
RhAG Rh-associated glycoprotein
SCF stem cell factor
SDS-PAGE sodium dodecyl sulfate – polyacrylamide gel electrophoresis
x