Specificity and Properties of Anti-HLA Antibodies ... · Specificity and Properties of Anti-HLA Antibodies Associated with Renal Allograft Rejection . Hooi Sian Eng . Bachelor of

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Specificity and Properties of Anti-HLA Antibodies Associated with Renal Allograft Rejection

Hooi Sian Eng Bachelor of Biomedical Science (Hon)

Master of Medical Science

A thesis submitted for the degree of PhD

School of Medicine Faculty of Health Science

University of Adelaide, South Australia

February 2009

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i. Contents i. Contents ................................................................................................................................................ 2 ii. List of Tables ......................................................................................................................................... 6 iii. List of Figures ........................................................................................................................................ 8 iv. Summary ............................................................................................................................................... 9 v. Dedication ............................................................................................................................................ 11 vi. Acknowledgements ............................................................................................................................. 12 vii. Declaration........................................................................................................................................... 13 viii. Thesis Structure .................................................................................................................................. 14 ix. Abbreviations ....................................................................................................................................... 15 x. Publications ......................................................................................................................................... 17 xi. Presentations ....................................................................................................................................... 19 xii. Awards ................................................................................................................................................. 22 1 Chapter 1 Literature Review ................................................................................................................ 23

1.1 Overview of Human Major Histocompatibility Complex (MHC) – Gene Discovery ......................... 24 1.1.1 Chronology of HLA Discovery ................................................................................................ 25

1.2 Genetics of MHC ............................................................................................................................. 25 1.2.1 Evolution of MHC Genes ........................................................................................................ 28 1.2.2 MHC Gene Polymorphism ...................................................................................................... 29

1.3 Human Leukocyte Antigen (HLA) System ...................................................................................... 31 1.3.1 HLA Gene Inheritance and Frequency ................................................................................... 33 1.3.2 HLA Class I, II and III Regions ............................................................................................... 33

1.4 Nomenclature .................................................................................................................................. 35 1.5 Structure of HLA Molecules ............................................................................................................ 37

1.5.1.1 HLA Peptide-Binding Grooves ....................................................................................... 40 1.5.2 HLA Epitopes .......................................................................................................................... 42 1.5.3 HLA Expression ...................................................................................................................... 43

1.6 Roles of HLA Molecules .................................................................................................................. 43 1.6.1 Antigen Presentation .............................................................................................................. 43

1.6.1.1 Endogenous Presentation Pathway............................................................................... 43 1.6.1.2 Exogenous Presentation Pathway ................................................................................. 45

1.6.2 T-cell Receptor Repertoire Selection ..................................................................................... 47 1.6.3 Regulation of NK Cell Activity ................................................................................................. 50

1.7 Significance of HLA in Renal Transplantation ................................................................................. 52 1.7.1 Mechanism of Renal Allograft Rejection ................................................................................ 52

1.7.1.1 Isolated MHC Class I Disparity ...................................................................................... 53 1.7.1.2 Isolated MHC class II Disparity ...................................................................................... 56 1.7.1.3 MHC Class I and II Disparity .......................................................................................... 58

1.8 Pre-transplantation HLA Antibody Detection and Crossmatching .................................................. 60 1.8.1 Negative T-cells / Positive B-cells Crossmatches (TN/BP) .................................................. 60 1.8.2 Crossmatch: Current Serum Negative / Historic Serum Positive (CN/HP) ............................ 61 1.8.3 Crossmatch: DTT-Reducible Positivity ................................................................................... 63

1.9 Post-transplantation Monitoring – Donor-Specific Antibodies ......................................................... 64 1.10 MHC Class I Chain Related (MIC) Genes: MICA and MICB .......................................................... 65 1.11 HLAMATCHMAKER – Amino Acid Triplet Program ....................................................................... 66 1.12 Antibody Detection Techniques ...................................................................................................... 67

1.12.1 Complement-dependent Lymphocytotoxicity Assay .............................................................. 67 1.12.2 Flow Cytometry ....................................................................................................................... 68 1.12.3 Solid Phase ELISA ................................................................................................................. 69 1.12.4 Solid Phase Luminex® ............................................................................................................ 69 1.12.5 Method Comparison ............................................................................................................... 70

2 Chapter 2 Materials & Methods ........................................................................................................... 72

2.1 HLA Antigen Typing ........................................................................................................................ 73 2.1.1 Dynabead® T-cell Isolation ..................................................................................................... 73 2.1.2 Dynabead® B-cell Isolation ..................................................................................................... 73 2.1.3 Serology HLA Antigen Typing ................................................................................................ 74

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2.1.4 DNA Quantitation .................................................................................................................... 74 2.1.5 Gel Electropheresis ................................................................................................................ 74 2.1.6 Visualization of PCR Product ................................................................................................. 75 2.1.7 Sequence Base Typing .......................................................................................................... 75

2.1.7.1 Prepare PCR Mixture ..................................................................................................... 75 2.1.7.2 PCR Product Visualization ............................................................................................. 77 2.1.7.3 AMPure PCR Purification .............................................................................................. 77 2.1.7.4 ABI Big Dye Terminator Ready Reaction Kit ................................................................. 78 2.1.7.5 CleanSEQ Dye Terminator Removal ............................................................................. 79

2.1.8 Luminex® Single Stranded Oligonucleotide (SSO) HLA Typing ............................................. 80 2.1.8.1 Prepare PCR Mixture ..................................................................................................... 80 2.1.8.2 Visualization of PCR Product ......................................................................................... 81 2.1.8.3 Hybridisation .................................................................................................................. 81

2.2 Crossmatch ..................................................................................................................................... 82 2.2.1 Direct Crossmatch .................................................................................................................. 82 2.2.2 DTT Crossmatch .................................................................................................................... 83

2.3 Anti-HLA Antibodies Screening ....................................................................................................... 84 2.3.1 SeraClean ............................................................................................................................... 84 2.3.2 Luminex® Screen and PRA .................................................................................................... 84 2.3.3 Luminex® Single Antigen Bead ............................................................................................... 85 2.3.4 Luminex® Single Antigen Beads – Anti-HLA antibodies IgG subclass ................................... 85

2.4 Database ......................................................................................................................................... 86 2.4.1 National Organ Matching System (NOMS) ............................................................................ 86

2.5 Statistics .......................................................................................................................................... 88 2.6 Reagent Preparation ....................................................................................................................... 89 2.7 Equipments ..................................................................................................................................... 94 2.8 Kits .................................................................................................................................................. 95 2.9 Chemicals ........................................................................................................................................ 95 2.10 Consumables .................................................................................................................................. 96

3 Chapter 3 Clinical Relevance of A positive B-cell Crossmatch in Renal transplantation .................... 97

3.1 Introduction ...................................................................................................................................... 98 3.2 Objectives ........................................................................................................................................ 99 3.3 Materials and Methods .................................................................................................................. 100

3.3.1 Study Cohort ......................................................................................................................... 100 3.3.2 HLA Typing ........................................................................................................................... 100 3.3.3 Screening for Autoantibodies ............................................................................................... 100 3.3.4 Luminex® .............................................................................................................................. 101 3.3.5 Antibody Specificity .............................................................................................................. 101 3.3.6 Clinical Data ......................................................................................................................... 101 3.3.7 Statistics ............................................................................................................................... 102

3.4 Results .......................................................................................................................................... 103 3.4.1 Characteristics of Patients with T–B+ crossmatch ............................................................... 103 3.4.2 Luminex® Specificity of Anti B-cell Antibodies ...................................................................... 106 3.4.3 Early Graft Rejection ............................................................................................................ 106

3.4.3.1 Correlation of T–B+ and Early Graft Rejection ............................................................ 107 3.4.3.2 Correlation of Early Graft Rejection and Antibody Specificity in T–B+ ........................ 109

3.4.4 Graft Function at 6, 12 and 36 month Post-transplantation ................................................. 114 3.4.5 Graft Survival ........................................................................................................................ 116

3.4.5.1 Significance of T–B+ in Graft Survival ......................................................................... 116 3.4.5.2 Correlation of Antibody Specificity in T–B+ with Graft Survival ................................... 116

3.4.6 Historic Positive B-cell Crossmatch ...................................................................................... 121 3.4.7 BXM as A Predictor of Graft Failure in 5 years Post-transplantation ................................... 127

3.5 Discussion ..................................................................................................................................... 128 4 Chapter 4 Anti-HLA Antibodies and Risk Factors Associated with Transplant Glomerulopathy ...... 132

4.1 Introduction .................................................................................................................................... 133 4.2 Objectives ...................................................................................................................................... 134 4.3 Materials and Methods .................................................................................................................. 134

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4.3.1 Study Cohorts ....................................................................................................................... 134 4.3.2 HLA Typing ........................................................................................................................... 135 4.3.3 Detection of Anti-HLA Antibodies ......................................................................................... 135 4.3.4 Clinical Data – ANZDATA Analysis ...................................................................................... 135 4.3.5 Statistics ............................................................................................................................... 136

4.4 Results .......................................................................................................................................... 137 4.4.1 Univariate Risk Factors ........................................................................................................ 137

4.4.1.1 Demographic Comparison ........................................................................................... 137 4.4.1.2 Immunosuppression..................................................................................................... 140

4.4.2 Early Graft Rejection ............................................................................................................ 142 4.4.3 Significance of Anti-HLA Antibodies in Transplant Glomerulopathy .................................... 144

4.4.3.1 CDC Class I PRA defined Presensitization ................................................................. 144 4.4.3.2 Luminex® defined Presensitization .............................................................................. 144 4.4.3.3 Pre-transplant DSA as A Risk Factor of TG ................................................................ 146 4.4.3.4 De novo DSA Predict TG ............................................................................................. 146

4.4.4 Multivariate Analysis ............................................................................................................. 148 4.4.5 DSA as A Predictor of TG ..................................................................................................... 150

4.4.5.1 Time to Biopsy ............................................................................................................. 150 4.4.5.2 Association of TG and Graft Survival........................................................................... 151 4.4.5.3 Pre-transplant DSA Associated with Early TG Development ...................................... 156 4.4.5.4 Significance of DSA in TG Progression ....................................................................... 157

4.4.6 DSA are Predictive for Graft Loss in TG .............................................................................. 159 4.5 Discussion ..................................................................................................................................... 161

5. Chapter 5 A Retrospective Study of B-cell Crossmatch with Luminex® Technology in Well-Matched

Highly Sensitized Patients from The Australian National Renal Exchange Programme (ANREP) ... 165 5.1 Introduction .................................................................................................................................... 166 5.2 Objectives ...................................................................................................................................... 167 5.3 Materials and Methods .................................................................................................................. 168

5.3.1 Study Cohort ......................................................................................................................... 168 5.3.2 Crossmatch .......................................................................................................................... 169 5.3.3 HLA Typing ........................................................................................................................... 170 5.3.4 Luminex® Antibody Analysis ................................................................................................. 170 5.3.5 Clinical Data ......................................................................................................................... 171 5.3.6 Statistics ............................................................................................................................... 171

5.4 Results .......................................................................................................................................... 171 5.4.1 Characteristics of Study Cohort ............................................................................................ 171 5.4.2 Graft Outcomes of Highly Sensitized Patients ..................................................................... 176

5.4.2.1 Graft Rejection ............................................................................................................. 176 5.4.2.2 Graft Function .............................................................................................................. 178 5.4.2.3 Graft Survival ............................................................................................................... 179

5.4.3 Predictive Value of B-cell Crossmatch in Highly Sensitized Patients .................................. 181 5.4.3.1 Prevalence of Positive B-cell Crossmatch ................................................................... 181 5.4.3.2 Characteristics of Patients with Positive B-cell Crossmatch ....................................... 181 5.4.3.3 Correlation of B-cell Crossmatch and Graft Rejection ................................................. 185 5.4.3.4 Graft function B-cell Crossmatch and Graft Function .................................................. 187 5.4.3.5 Correlation of B-cell Crossmatch and Graft Survival ................................................... 188

5.4.4 Predictive Value of Luminex® defined Donor-specific Antibodies ........................................ 190 5.4.4.1 Presence of Donor-specific Antibodies ........................................................................ 190 5.4.4.2 Specificity of DSA defined by Luminex® ...................................................................... 194 5.4.4.3 Correlation of DSA and Graft Rejection ....................................................................... 196 5.4.4.4 Correlation of DSA and Graft Function ........................................................................ 198 5.4.4.5 Correlation of Luminex® defined DSA and Graft Survival ............................................ 199

5.4.5 Luminex® defined DSA in B-cell Crossmatches ................................................................... 201 5.4.5.1 Correlation of Luminex® defined DSA and BXM with Graft Rejection ......................... 202 5.4.5.2 Multivariate analysis of BXM and Luminex® defined DSA in predicting rejection........ 202

5.5 Discussion ..................................................................................................................................... 204

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6 Chapter 6 Discussion ........................................................................................................................ 209 6.1 Significance of Preformed Anti-HLA Antibodies Detected by B-cell Crossmatch ......................... 210

6.1.1 Antibodies Causing Positive B-cell Crossmatches and Correlation with Graft Outcomes ... 214 6.2 Luminex® Defined Donor-specific Antibodies as A Predictor of Graft Outcomes ......................... 214

6.1.2 Solid Phase Antibody Analysis: Virtual Crossmatch ............................................................ 215 6.2.2.1 Prediction of Flow Crossmatch Outcomes by Luminex® Assays................................. 216 6.2.2.2 Improved Transplant Rate by Defining Acceptable Mismatches ................................. 219 6.2.2.3 Cost-Effectiveness of Solid Phase Assays Virtual Crossmatch .................................. 219 6.2.2.4 Possible Weaknesses of Solid Phase Assays Virtual Crossmatch ............................. 220

6.1.3 Luminex® Antibody Analysis and Luminex® Lysate Crossmatch ......................................... 221 6.1.4 DSA in Transplant Glomerulopathy – Significance of Post-transplant Monitoring ............... 222

6.3 Summary of Conclusions .............................................................................................................. 223 References ................................................................................................................................................. 225 Appendix .................................................................................................................................................... 243

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ii. List of Tables Chapter 2 Table 2.1.1: Preparation of PCR Mixture for Sequence Base Typing ......................................................... 75Table 2.1.2: PCR conditions for Sequence Base Typing ............................................................................. 76Table 2.1.3: Conditions of sequencing amplification .................................................................................... 79Table 2.1.5: Preparation of PCR mixtures for Luminex® SSO ..................................................................... 80Table 2.1.6: Condition of PCR amplification for Luminex® SSO .................................................................. 81Table 2.1.7: Hybridisation steps for Luminex® SSO .................................................................................... 82 Chapter 3 Table 3.4.1: Descriptive statistics of cohorts by the result of B-cell crossmatching .................................. 104Table 3.4.2: Immunosuppresion in T–B– and T–B+ groups ...................................................................... 105Table 3.4.3: Prevalence of cellular, vascular and glomerular rejection in All grafts, T–B– and T–B+ groups

.................................................................................................................................................................... 108Table 3.4.4: Graft rejection at 6 months according to the antibody specificity in T–B+ ............................. 110Table 3.4.5: Risk of cellular rejection according to the antibody specificity in T–B+ ................................. 111Table 3.4.6: Risk of vascular rejection according to the antibody specificity in T–B+ ............................... 112Table 3.4.7: Risk of glomerular rejection according to the antibody specificity in T–B+ ............................ 113Table 3.4.8: eGFR at 6, 12 and 36 months by B-cell crossmatch and antibody specificity, ................. 115Table 3.4.9: Graft survival rates and hazard ratios for graft loss by antibody specificity ........................... 118Table 3.4.10: Prevalence of cellular, vascular and glomerular rejection in T–B+ subgroups .................... 122Table 3.4.11: Odds ratios of graft rejection at 6 months in T–B+ subgroups ............................................ 123Table 3.4.12: eGFR at 6, 12 and 36 months post-transplant in T–B+ subgroups ..................................... 124Table 3.4.13: Graft survival rates and hazard ratios for graft loss in T–B+ subgroups .............................. 125Table 3.4.14: Graft outcomes at 5 years post-transplant ........................................................................... 127 Chapter 4 Table 4.4.1: Descriptive statistics of cohort and univariate analyses of risk factor of TG .......................... 138Table 4.4.2: Immunosuppressants at induction and prior to diagnosis of TG ............................................ 141Table 4.4.3: Cellular, vascular and glomerular rejection as a risk factor of TG ......................................... 143Table 4.4.4: Univariate effects of HLA presensitization, defined by CDC or Luminex® techniques, in the development of TG ..................................................................................................................................... 145Table 4.4.5: Univariate effects of anti-HLA antibodies in the development of TG ..................................... 147Table 4.4.6: In multiple regression analysis, DSA was the only independent predictor of TG. ................. 149Table 4.4.7: Graft survival rates in patients with TG, according to antibody specificity ............................. 152Table 4.4.8: Graft survival and risk of graft loss in TG patients, according to antibody specificity, in 20 years of follow up ....................................................................................................................................... 153 Chapter 5 Table 5.3.1: Patient recruitment ................................................................................................................. 168Table 5.4.1: Descriptive statistics of study cohort, compared to all other grafts transplanted within the same period in participating centres .......................................................................................................... 173Table 5.4.2: Immunosuppressants at induction ......................................................................................... 175Table 5.4.3: Risk of cellular, vascular and glomerular rejection in highly sensitized patients ................... 177Table 5.4.4: Graft function, eGFR, of highly sensitized patients ................................................................ 178Table 5.4.5: Total graft survival rates in highly sensitized patients, compared to all grafts ....................... 180Table 5.4.6: Demographics of T–B– and T–B+ groups ............................................................................. 182Table 5.4.7: Immunosuppressants at induction for T–B– and T–B+ groups ............................................. 184Table 5.4.8: Correlations of T–B+ with cellular, vascular and glomerular rejection ................................... 186Table 5.4.9: Comparison of graft function (MDRD eGFR) between T–B– and T–B+ groups. ................... 187Table 5.4.10: Correlation of T–B+ and total graft survival ......................................................................... 189Table 5.4.11: Demographics of DSA and Non-DSA groups ...................................................................... 191Table 5.4.12: Immunosuppressants at induction for DSA and Non-DSA groups ...................................... 193Table 5.4.13: Correlations of DSA with cellular, vascular and glomerular rejection .................................. 197Table 5.4.14: Comparison of graft function (MDRD eGFR) between Non-DSA and DSA groups ............ 198Table 5.4.15: Correlation of DSA and total graft survival ........................................................................... 200Table 5.4.16: BXM results and specificity of DSA in highly sensitized patients ........................................ 201Table 5.4.17: Independent predictors of graft rejection ............................................................................. 203

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Chapter 6 Table 6.1.1: A summary of studies investigated clinical significance of positive BXM in renal transplantation ............................................................................................................................................ 212Table 6.2.1: A review of clinical significance of low titer DSA detected by Luminex® or Flow crossmatch in sera with negative CDC / AHG-CDC .......................................................................................................... 218

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iii. List of Figures Chapter 1 Figure 1.2.1: Human MHC gene map .......................................................................................................... 27Figure 1.2.2: Timescale of MHC evolution ................................................................................................... 29Figure 1.3.1: The number of antigens and alleles from 1968 to September 2008. ..................................... 32Figure 1.3.2: HLA gene map illustrates clustering of immune system ......................................................... 34Figure 1.5.1: Structure of HLA class I molecules ......................................................................................... 38Figure 1.5.2: Ribbon structure of class I peptide binding groove ................................................................. 38Figure 1.5.3: Structure of HLA class II molecule .......................................................................................... 39Figure 1.5.4: Peptide in HLA peptide-binding groove .................................................................................. 40Figure 1.5.5: Amino acid pockets in the floor of binding groove determine the binding of peptide ............. 41Figure 1.5.6: HLA molecule epitopes ........................................................................................................... 42Figure 1.6.1: Roles of peptide loading complex in MHC class I assembly and peptide loading. ................ 44Figure 1.6.2: Endogenous peptide presentation pathway ............................................................................ 45Figure 1.6.3: Exogenous antigen presentation pathway .............................................................................. 46Figure 1.6.4: Positive and negative selection of T-cell receptor repertoire .................................................. 49Figure 1.6.5: Regulation of NK activity by HLA class I molecules ............................................................... 51Figure 1.7.1: Effector mechanisms of rejection in graft carrying mismatched HLA class I antigen. ............ 55Figure 1.7.2: Effector mechanism of rejection in graft carrying mismatched HLA class II antigens. ........... 57Figure 1.7.3: Effector mechanisms of rejection in graft carrying mismatched HLA class I and class II antigens ........................................................................................................................................................ 59 Chapter 3 Figure 3.4.1: T–B+ as a group was not associated with inferior graft survival .......................................... 119Figure 3.4.2: Graft survival according to antibody specificity in T–B+ ....................................................... 120Figure 3.4.3: Significance of DSA class in graft survival ............................................................................ 121Figure 3.4.4: 5 years graft survival in T–B+ subgroups stratified by peak/current status .......................... 126 Chapter 4 Figure 4.4.1: Cumulative graph for biopsy events in relation to time post-transplantation ........................ 150Figure 4.4.2: Graft survival in the controls and patients. ............................................................................ 154Figure 4.4.3: Graft survival before and after diagnosis of TG .................................................................... 155Figure 4.4.4: Cumulative TG diagnosis events in relation to time post-transplant, according to presence of DSA ............................................................................................................................................................ 156Figure 4.4.5: Graft survival in patients with TG according to presence of DSA ......................................... 158Figure 4.4.6: Graft survival in TG subgroups, compared to the control group ........................................... 160 Chapter 5 Figure 5.4.1: Graft function of highly sensitized patients, compared to all grafts ...................................... 178Figure 5.4.2: Total graft survival of highly sensitized patients, compared to all grafts .............................. 179Figure 5.4.3: Correlation of graft function and BXM ................................................................................... 187Figure 5.4.4: Significance of positive BXM in relation to graft survival ...................................................... 188Figure 5.4.5: Specificity of HLA class I donor-specific antibodies in highly sensitized patients ................ 195Figure 5.4.6: Specificity of HLA class II donor-specific antibodies in highly sensitized patients ............. 195Figure 5.4.7: A trend of poor graft function in DSA group within 1 year post-transplant ........................... 198Figure 5.4.8: Graft survival in the presence of DSA ................................................................................... 199

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iv. Summary

Identification of the complement C4d fragment in peritubular capillaries as a

specific marker for antibody mediated rejection in renal transplantation revealed the

critical role of antibodies in graft survival. In this thesis, I document the design and

findings of studies performed to investigate the clinical impact of anti-HLA antibodies

present before and/or after transplantation.

Over time, the detection techniques for anti-HLA antibodies has evolved from

the less sensitive complement-dependent lymphocytotoxicity (CDC) crossmatching (XM)

to more sensitive solid phase assays such as Luminex®. Studies have been conducted

to compare the predictive value of different antibody detection techniques.

The first result chapter presents antibody specificity in positive CDC B-cell

crossmatch (BXM), analysed with highly specific Luminex® assays. The study also

investigates the predictive value of BXM in the general transplant population. I found

that donor-specific anti-HLA antibodies (DSA) are only present in one third of positive

BXM and are associated with poor outcomes. The novel finding is that >80% of the DSA

detected by BXM are complement-fixing IgG1 and IgG3 subclasses.

Transplant glomerulopathy (TG) is type of chronic renal graft rejection. The

pathogenesis of TG is unclear. In the second result chapter, I report risk factors and

involvement of anti-HLA antibodies in the development of TG. This study shows that

glomerular rejection, delayed graft function, HLA presensitization and DSA have a

univariate effect on TG development. Multivariate analysis revealed that DSA are an

independent predictor of TG, after adjustment for other risk factors.

I have further investigated the role of BXM in a unique, well-matched, highly

sensitized patient group transplanted under the national renal exchange programme. I

compared Luminex® antibody analysis with BXM in predicting transplant outcomes. In

highly sensitized patients, DSA are found in two thirds of positive BXM. In univariate

analyses, BXM is associated with humoral rejection whereas DSA defined by Luminex®

are associated with total and all rejection types. The major finding is that, by

multivariate analysis, DSA defined by Luminex® are an independent predictor of total

and humoral rejection, but BXM are not. These interesting findings are reported in the

third result chapter.

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Studies reported in this thesis define the clinical significance of anti-HLA

antibodies in renal transplant outcomes. Method comparison studies provide useful

information on antibody specificity and their impact on graft survival. Collectively, a

better understanding of alloantibodies associated with graft rejection and limitation of

antibody detection methods may facilitate donor selection and choice of

immunosuppressants, and consequently improve transplant outcomes.

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v. Dedication

For my father Kheng Choo, mother Sew Kee, siblings Hooi Ling, Inn Soo, Hooi Ping,

Soo Tchuen, Chuen Sin, Sin Zheng and husband Lee Khuan.

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vi. Acknowledgements

A thesis, with multiple research studies and high quality collaboration, such as

this can only be completed with a brilliant and exceptional supportive team of

supervisors, collaborators, and team assistance. I have had the privilege to be

supervised by Professor Graeme Russ, Dr. Toby Coates and, and Dr. Peter Bardy,

without whom, my study would not be possible. I am so honoured and most grateful to

have the opportunity to work in such an amazing team environment. My most heartfelt

thanks for all your professional advice, your guidance, and unwavering support.

To my wonderful collaborators, of whom I am most appreciative and

honoured to have work with. Firstly Dr. Brian Tait (Royal Melbourne Hospital) and his

team for their involvement in the National B-cell Cross-match Study, through data and

serum collection, and presentation and further learning and networking opportunities

within the local and international research and clinical community. Many thanks, it is

such an honour to work with such a gracious and encouraging individual.

My thanks to all the participating hospitals, and tissue-typing laboratories,

who have collected serum and data for contribution to this research. Your professional

accurate and timely support has made this research possible.

To the ANZDATA team who has worked tirelessly, sometimes with

unimaginable deadlines, and overtime hours, for such high quality data analysis.

Special thanks to Ms. Hannah Dent, Mr. Brian Livingstone, and Dr. Sean Chang.

Thank you to my colleagues at the South Australian Tissue Typing Laboratory

(Pirie Street, Adelaide) of National Transplantation Immunology Services, Australian

Red Cross Blood Service, and The Queen Elizabeth Hospital’s Transplantation

Immunology Team. The support, assistance and encouragement, and positive learning

and working environments they have provided have been enjoyable and exceptional,

and fundamental to my completion of this work.

Finally I would like to thank the organisations who have made many

educational, financial and facility contributions, to this thesis possible. Thank you

especially to the National Transplantation Services of Australian Red Cross Blood

Service, the Australian Department of Education, Science and Technology, and

University of Adelaide, and Department of Nephrology and Transplantation services

Queen Elizabeth Hospital, Woodville, South Australia for their support.

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vii. Declaration This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue, the Australasian Digital Theses Program (ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time. ----------------------- Hooi Sian Eng

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viii. Thesis Structure

Chapter 1 is a review of Human Leukocyte Antigen (HLA) system consists of

the genetics, structures and functions of HLA molecules. It also describes role of HLA

antigen in renal graft rejection, and current tissue typing and antibody detection

techniques.

Chapter 2 contains the materials and methods used to perform the studies

described in Chapter 3 to 5. The steps of tissue typing and Luminex® assays and

reagent preparation are documented. Statistical tests for patient demographics and

transplant outcomes comparisons are recorded in this chapter.

This thesis consists of three studies performed on patients with different

transplant characteristics. The first study investigated the clinical significance of positive

B-cell crossmatch in an Australian renal transplant population using Luminex® assays.

This study also examined the specificity of antibody causing positive B-cell crossmatch

and correlation with renal transplant outcomes. The design and results of the study are

described in Chapter 3.

The pathogenesis of transplant glomerulopathy remains unknown. The

second study described in Chapter 4 reported the involvement of anti-HLA antibodies in

development of transplant glomerulopathy. This study also indentified the influence of

anti-HLA antibodies on the progression of transplant glomerulopathy.

The Australia National Renal Exchange Programme commenced in October

2004 to improve the HLA antigen matching and transplant rate for highly sensitized

patients. Chapter 5 reported the overall performance of patients transplanted under this

programme in comparison to general transplant population. Clinical predictive value of

B-cell crossmatch and Luminex® antibody analysis in highly sensitized patients were

also compared.

Chapter 6 brings together the findings of the studies reported in Chapter 3 to

5, and examined the uses of BXM and Luminex® assays in current tissue typing

protocol. The results are compared to previous studies and appropriate comments are

included.

A list of references and then an appendix are attached after Chapter 6. The

appendix consists of the published journal article that has arisen from the findings

presented in this thesis.

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ix. Abbreviations

AAT amini acid triplets ADCC antibody–mediated cell cytotoxicity ELISA enzyme-linked immunoabsorbent assay AHR acure humoral rejection APC antigen- presenting cells BXM B-Cell crossmatch CAN chronic allograft nephropathy CDC complement-dependent lymphocytotoxicity CN/HP current serum negative/ historic serum positive CNX calnexin CREGs cross-reactive groups CRT chaperone calreticulin CTLRs C-type lectin receptors cTLs cytotoxic T-lymphocytes DISC death-inducing signal complex DS/SAPE dilution Solution/PE Streptavidin DSA donor specific antibodies DTH delayed hypersensitivity reaction DTT dithiothreitol EPO eosinophil persoxide ER endoplasmic reticulum ERAAP or ERAP 1 ER associated aminopeptidase ERp57 thiol oxidoreductase FasL Fas-ligand Fc-receptors fragment , crystallisable H histocompatibility HLA human leukocyte antigen IFN interferon IL interleukin IMGT ImMunoGeneTics IR Immune Response ITIMs immunonoreceptor tyrosine-based inhibitory motifs KIR killer like receptor LD linkage disequilibrium Li Invariant chain MESF Molecules of Equivalent Soluble Fluorochrome MFI Median Fluorescence Intensity MHC Major Histocompatalibity complex MIC MHC class 1 chain-related genes MMF mycophenolate mofetil Mya million years ago NK natural killer

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NO nitric oxide PBS phosphate buffered saline PBS/citrate trisodium citrate PFD probability of finding a donor RSA recurrent spontaneous abortions TAP transporter associated with antigen processing TCR T-cell receptor TG transplant glomerulopathy TH1 T helper TN/BP negative T-cells/positive B-cells crossmatches TNF tumour necrosis factor TPSN Tapasin TXM T-cell crossmatch UNOS United Network for Organ Sharing Registry XM crossmatching

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x. Publications

1. HS Eng, G Bennett, I Humphreys, E. Tsiopelas, M Lake, SH Chang, P Bardy, PTH

Coates and G. Russ. Luminex® Investigation: Low titer HLA donor-specific

antibodies in positive B-cell crossmatches predict late graft loss. Nephrology

2007:Supp 1;A19.


Coates and G. Russ. Luminex® Investigation of Positive B-cell Crossmatches: low

titler HLA donor-specific antibodies associated with inferior outcomes. Human

Immunology 2007:68 Supp 1:S31.


Coates and G. Russ. Anti-HLA donor-specific antibodies detected in positive B-cell

crossmatches by Luminex® predict late graft loss. American Journal of

Transplantation 2008 8(11):2335-42.

4. Hooi Sian Eng, Scott Campbell, Graeme Russ, P. Toby H. Coates, Peter Bardy,

Greg Bennett, Sean Chang, Brian Tait, On behalf of the Renal Transplant Advisory

Committee (RTAC). A retrospective study of B-cell crossmatch with Luminex®

technology in well-matched highly sensitized patients from the AUSTRALIAN

interstate exchange. Nephrology 2008:13;A112.

5. Hooi Sian Eng, Greg Bennett, Eleni Tsiopelas, Sean Chang, Peter Bardy, Graeme

Russ, P. Toby H. Coates. Alloantibody involvement and risk factors in transplant

glomerulopathy. Nephrology 2008:13;A99.


Greg Bennett, Sean Chang, Brian Tait, On behalf of the Renal Transplant Advisory

Committee (RTAC). Predictive value of B-cell crossmatch and Luminex® antibody

analysis in well-matched highly sensitized patients from the Australian National

Interstate Exchange (ANIE). Human Immunology 2008:69;S13.

18

7. Hooi Sian Eng, Greg Bennett, Eleni Tsiopelas, Sean Chang, Peter Bardy, Graeme

Russ, P. Toby H. Coates. Donor specific anti-HLA antibodies in transplant

glomerulopathy. Human Immunology 2008:69;S109.

8. Hooi Sian Eng, Greg Bennett, Peter Bardy, Patrick Coghlan, Graeme R. Russ, P.

Toby H. Coates. Clinical significance of anti-HLA antibodies detected by Luminex®:

Enhancing the interpretation of CDC-BXM and important post-transplant monitoring

tools. Human Immunology 2009 In press.

19

xi. Presentations

1. HS Eng, G Bennett, I Humphreys, E. Tsiopelas, M Lake, SH Chang, P Bardy,

PTH Coates and G. Russ. Clinical relevance of a positive B-cell crossmatch on

renal transplantation: A single centre study. The Queen Elizabeth Hospital

Research Day 20 October 2006, Adelaide, Australia.


PTH Coates and G. Russ. Analysis of the specificity and properties of

alloantibodies associated with rejection of renal transplant. The 3rd International

Summer School on Immunogenetics 17-21 November 2006, Bangkok, Thailand.


PTH Coates and G. Russ. B-cell crossmatching and kidney allograft outcomes in

cadaveric transplant recipients. The 30th Annual Scientific Meeting of The

Australasia and South East Asia Tissue Typing Association 22-25 November

2006, Chiang Mai, Thailand.


PTH Coates and G. Russ. Luminex® investigation of positive B-cell crossmatches

in renal transplantation. The 31st Annual Scientific Meeting of The

Transplantation Society of Australia and New Zealand 28-30 March 2007,

Canberra, Australia. TSANZ Young Investigator Award 2007


PTH Coates and G. Russ. Luminex® Investigation: Low Titer HLA Donor-specific

Antibodies in Positive B-cell Crossmatches Predict Late Graft Loss. The 43rd

Annual Scientific Meeting of The Australia and New Zealand Society of

Nephrology (ANZSN) 8-12 September 2007, Queensland, Australia. Kidney Health Australia (KHA) Award for The Best Clinical Science Presentation

20


PTH Coates and G. Russ. Luminex® Investigation of Positive B-cell

Crossmatches: Low Titer HLA Donor-specific Antibodies Associated with Inferior

Outcomes. The 33rd Annual Meeting of American Society For Histocompatibility

and Immunogenetics (ASHI) 8-12 October 2007, Minnesota, USA.


PTH Coates and G. Russ. Low Titer Donor-specific Antibodies in Positive B-cell

Crossmatches Predict Late Graft Loss. The 31st Annual Scientific Meeting of

Australasia and South East Asia Tissue Typing Association 15-18 November

2008, Perth, Australia.


Greg Bennett, Sean Chang, Brian Tait, On behalf of the Renal Transplant

Advisory Committee (RTAC). Clinical significance of B-cell positive crossmatches

and Luminex® defined donor-specific antibodies in well-matched highly sensitized

patients from the AUSTRALIAN interstate exchange.

The XXII International Congress of The Transplantation Society 10-14 August

2008, Sydney, Australia.

• Top 10 Abstracts Young Investigation Award of The XXII International Congress of The

Transplantation Society 10-14 August 2008, Sydney, Australia.

• Nominee for The President’s Award of The Transplantation Society of Australia and New Zealand

(TSANZ).

• TSANZ Young Investigator Award

• Amgen Young Investigator Award

21

9. Hooi Sian Eng, Greg Bennett, Eleni Tsiopelas, Sean Chang, Peter Bardy,

Graeme Russ, P. Toby H. Coates. Risk factors and associations with

alloantibodies in transplant glomerulopathy. The XXII International Congress of

The Transplantation Society 10-14 August 2008, Sydney, Australia.



Advisory Committee (RTAC). A retrospective study of B-cell crossmatch with

Luminex® technology in well-matched highly sensitized patients from the

AUSTRALIAN interstate exchange. The 44th Annual Scientific Meeting of

Australia and New Zealand Society of Nephrology (ANZSN) 6-10 September

2008, Newcastle, Australia. Finalist for The Clinical Science Young Investigator Award


Graeme Russ, P. Toby H. Coates. Alloantibody involvement and risk factors in

transplant glomerulopathy. The 44th Annual Scientific Meeting of Australia and

New Zealand Society of Nephrology (ANZSN) 6-10 September 2008, Newcastle,

Australia.



Advisory Committee (RTAC). Predictive value of B-cell crossmatch and

Luminex® antibody analysis in well-matched highly sensitized patients from the

Australian National Interstate Exchange (ANIE). The 34th Annual Meeting of

American Society For Histocompatibility and Immunogentics (ASHI) 27-31

October 2008, Toronto, Canada.


Graeme Russ, P. Toby H. Coates. Donor specific anti-HLA antibodies in

transplant glomerulopathy. The 34th Annual Meeting of American Society For

Histocompatibility and Immunogentics (ASHI) 27-31 October 2008, Toronto,

Canada.

22

xii. Awards

1. Young Investigator Award of Amgen-The Transplantation Society of Australia and

New Zealand. The 31st Annual Scientific Meeting, 28-30 March 2007, Canberra,

Australia.

2. Kidney Health Australia For The Best Clinical Science Presentation Award. The

43rd Annual Scientific Meeting of The Australia and New Zealand Society of

Nephrology (ANZSN) 8-12 September 2007, Queensland, Australia.

3. Top 10 Abstracts Young Investigation Award of The XXII International Congress

of The Transplantation Society 10-14 August 2008, Sydney, Australia.

4. Young Investigator Award of Amgen-The Transplantation Society of Australia and

New Zealand. The XXII International Congress of The Transplantation Society

10-14 August 2008, Sydney, Australia.

5. Young Investigator Award of The Transplantation Society of Australia and New

Zealand. The XXII International Congress of The Transplantation Society 10-14

August 2008, Sydney, Australia.

6. Nominee for The President’s Award of The Transplantation Society of Australia

and New Zealand. The XXII International Congress of The Transplantation

Society 10-14 August 2008, Sydney, Australia.

7. Finalist for The Clinical Science Young Investigator Award The 44th Annual

Scientific Meeting of Australia and New Zealand Society of Nephrology (ANZSN)

6-10 September 2008, Newcastle, Australia.

Documents

Specificity and Properties of Anti-HLA Antibodies ... · Specificity and Properties of Anti-HLA Antibodies Associated with Renal Allograft Rejection . Hooi Sian Eng . Bachelor of