Immuno-Magnetic T Cell and Alloreactive T Cell
Total Page：16
File Type：pdf, Size：1020Kb
IMMUNO-MAGNETIC T CELL DEPLETION FOR ALLOGENEIC HEMATOLOGICAL STEM CELL TRANSPLANTATION DISSERTATION Presented in Partical Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ying Xiong, B.S. ***** The Ohio State University 2008 Dissertation Committee: Professor Jeffrey J. Chalmers, Adviser Approved By Professor Andre F. Palmer Professor James C. Lang Professor Maciej Zborowski Advisor Graduate Program in Chemical and Biomolecular Engineering ABSTRACT Bone marrow transplantation or stem cell transplantation has become standard treatment for patients with hematological malignant diseases such as acute leukemia, chronic leukemia and refractory non-Hodgkin’s lymphoma, which involves the transfer of autologous or allogeneic stem cells depending on the availability of HLA-matched donor. As only 25-30% of patients can have a completely HLA-matched sibling donor, allogeneic stem cell transplantation is the only curative option for most of the patients with hematological malignancies or inherited disorders of hematopoietic system. However, the application of allogeneic stem cell transplantation is restricted by the development of severe graft-versus-host disease (GVHD), which is caused by donor T cells. Although researches have shown that high depletion of donor T cells eliminates GVHD, the increased chance of viral infection and leukemia relapse results in the un- improved post-transplant mortality and morbidity. As a result, selective depletion of donor’s allo-reactive T cells toward the patient has been suggested as a compensation strategy of T cell depleted stem cell transplantation, which is expected to protect the patient from opportunistic infections and shorten the post-graft immunodeficiency without affecting GVHD reduction. ii In this dissertation, a complete strategy for safe and efficient hematopoietic stem cell transplantation was studied: first, the donor’s T cells were as completely depleted as possible; then, the depleted T cells were stimulated by the patient’s white blood cells and depleted. The depletion was achieved by immuno-magnetic separation. Different monoclonal antibody conjugated magnetic particles were utilized. Based on the experiment results and statistical analysis, it was determined that antiCD3 dynabeads would be employed for T cell depletion in QMS system, by which 4.0 log10 T cell depletion and >99% recovery of CD34+ cells could be achieved for the separation of >108 leukocytes. For alloreactive T cell depletion, since MACS nanobeads were demonstrated to non-specifically connect to the cells and MACS separation system did not have consistent performance, Stemcell Technologies’ customized tetrameric antibody and magnetic microparticles were employed alternatively along with DQMS system. Studies are in process to verify the improvement of alloreactivity elimination by employing increased alloreactive antigens in the separation. iii Dedicated to my parents iv ACKNOWLEDGMENTS I wish to thank Dr. Jeffrey Chalmers and Dr. Sherif Farag for their guidance, support and enthusiasm during the whole development of my research. In addition, I would like to thank the research group members, especially Dr. Xiaodong Tong, for his contribution, and Dr. Mei Shao, for her help and suggestions to the completion of this work. I am grateful to Yang Zhao for his help in CTV adjustment and DQMS construction. I also wish to thank Mr. Bryan McElwain at the Ohio State University Analytical Cytometry Laboratory for his expertise on flow cytometry analysis. v VITA July 06, 1981…………………………Born- Sichuan, China 2003…………………………………..B.S. Chemical Engineering, Tsinghua University. 2003-present………………………….Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Tong X., Xiong Y., et al. A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation. Experimental Hematology. 2007; 35: 1613-1622. FIELDS OF STUDY Major Field: Chemical and Biomolecular Engineering vi TABLE OF CONTENTS Abstract……………………………………………………………………………..……..ii Dedication…………………………………………………………………………….......iv Acknowledgments………………………………………………………………………..v Vita………………………………………………………………………………………..vi List of Tables……………………………………………………………………………xiii List of Figures…………………………………………………………………………...xvi Chapters: 1. Introduction……………………………………………………………………………1 1.1 Stem cell transplantation…………………………………………………………..1 1.2 Graft-versus-host disease (GVHD)………………………………………………..3 1.3 T cell depletion (TCD)…………………………………………………………….6 1.4 Alloreactive T cell depeletion……………………………………………………..9 1.5 Immunomagnetic cell separation technology……………………………………13 1.5.1 Immuno-magnetic cell labeling methods………………………………...16 1.5.2 MACS separation system………………………………………………...18 1.5.3 Quadrupole magnetic flow sorter (QMS)………………………………..19 1.6 Definition and measurement of magnetophoretic mobility……………………...20 1.7 Dissertation organization………………………………………………………...26 2. CTV histogram modeling……………………………………………………………29 2.1 Motivation………………………………………………………………………..29 2.2 Modeling………………………………………………………………………....31 vii 2.2.1 Distribution analysis……………………………………………………..31 2.2.2 Distribution simulation…………………………………………………..33 2.3 Material and methods……………………………………………………………35 2.3.1 Cell sources………………………………………………………………35 2.3.2 Sample preparation………………………………………………………36 2.3.3 Cell analysis……………………………………………………………...36 2.3.4 Model simulation………………………………………………...………37 2.4 Results and discussions………………………………………………….……….38 2.4.1 Distribution simulation with different bin numbers……………………...38 2.4.2 Mobility of different cell species………………………………………...40 2.4.3 Mobility of unlabeled PBLs…………………………………...…………43 2.5 Conclusion………………………...……………………………………………..47 3. Cell labeling for immuno-magnetic T cell depletion…………………………….…..48 3.1 Motivation………………………………………………………………………..48 3.2 Material and methods…………………………………………………………….52 3.2.1 Cell sources……………………………………………………………....52 3.2.2 Cell endurance study……………………………………………………..53 3.2.3 Immuno-magnetic cell labeling……………………………………….....54 3.2.4 Immuno-magnetic cell separation in QMS………………………………56 3.2.5 Flow analysis………...…………………………………………………..57 3.3 Results and discussions…………………………………………………………..60 3.3.1 Cell endurance study……………………………………………………..60 Effect of time and initial cell concentration……………………………..61 Effect of centrifugation……………………………...…………………..63 Effect of EDTA component in PBS buffer…………………………...…66 Effect of BSA component in PBS buffer………………………………..66 viii Effect of labeling tubes………………………………………………….67 3.3.2 CD3 antibody clone determination………………………………………68 3.3.3 Immuno-magnetic labeling reagent trial…………………………………69 One-step labeling methods……………………………………………….70 One-step labeling vs. two-step labeling………………………………….72 Stemcell Technologies reagents………………………………………….74 Combination of different magnetic particles…………………………….75 3.3.4 Effect of separation sample size on T cell depletion…………………….77 3.4 Conclusion……………………………………………………………………….79 4. Optimization of T cell depletion and scale up……………………………………….81 4.1 Motivation………………………………………………………………………..81 4.2 Material and methods…………………………………………………………….84 4.2.1 Cell sources………………………………………………………………84 4.2.2 Immuno-magnetic cell labeling………………………………………….85 4.2.3 Immuno-magnetic cell separation………………………………………..86 4.2.4 Flow analysis…………………………………………...………………..87 4.2.5 Limiting dilution assay…………………...……………………………...90 4.2.6 Statistical analysis………………………………………………………..92 4.3 Results and discussions…………………………………………………………..93 4.3.1 Flow cytometry instruments……………………………...……………...93 4.3.2 Comparison between non-T cell and CD34+ cell recovery……………..96 4.3.3 Labeling reagent usage determination……………………………...……97 The two-step labeling method……………………………………………98 Dynabeads labeling method……………………………………………..99 Stemcell tetrameric antibody method…………………………………..104 4.3.4 QMS procedure optimization…………………………………………...106 ix T cell depletion……………………………...………………………….109 Non-T cell recovery……………………………………………...……..111 Effect of labeling strategy…………………………………………...….113 Effect of initial T cell number……………………………………...…...114 4.3.5 Large scale TCD using optimized protocol………………………….....117 4.4 Discussion……………………………………………………………………....121 5. Alloreactive T cell depletion using MACS system………………………………..124 5.1 Motivation……………………………………………………………………...124 5.2 Material and methods…………………………………………………………..126 5.2.1 Cell sources……………………………………………………………..126 5.2.2 Primary one-way MLR setup…………………………………………...127 5.2.3 One-way MLR harvest and cell labeling……………………………….128 5.2.4 Alloreactive T cell depletion and secondary MLR setup………………129 5.2.5 Flow cytometry sample preparation……………………………………129 5.3 Results and discussions…………………………………………………………131 5.3.1 Flow cytometry analysis………………………...……………………...131 5.3.2 Alloreactive cell fraction comparison…………………………………..138 5.3.3 Multiantibody cell labeling optimization……………………………….143 Primary labeling optimization…………………………………………..143 Secondary labeling optimization………………………………………..144 5.3.4 Cell depletion study……………………...……………………………..146 3-antibody depletion……………………………………………………147 4-antibody depletion………...………………………………………….148 5.4 Conclusion…………...…………………………………………………………149 x 6. Non-specific binding of MACS nanobeads and alternative method for alloreactive T cell depletion………………………………………………………………………..153 6.1 Motivation………………………………………………………………………153 6.2 Material and methods…………………………………………………………...160 6.2.1 Cell sources……………………………………………………………..160 6.2.2 One-way mixed lymphocyte