Antibody Mediated Rejection and Gene Expression Profiles

1. Antibody Mediated Rejection and Gene Expression Profiles Anatasia Gangadin Dr. Mario C Deng Columbia University College of Physicians and Surgeons

2. Key Ideas • Allograft • Endomyocardial Biopsy • Rejection • CARGO • Functional Genomics • Patient Care

3. Patient Care

4. Endomyocardial biopsy • Currently Only way to test for rejection • Risk Factors associated

5. Rejection Mild Severe

6. CARGO Study • To Reduce the Number of Biopsies by taking samples of blood as opposed to heart Tissue • Being able to predict rejection after a transplant • Establishing a pattern of Genes which could ultimately predict Rejection

7. CARGO Clinical Study Design • Prospective, multicenter, 4 year observational study on 629 patients Hypothesis Gene expression profiling of peripheral blood mononuclear cells discriminate quiescence (ISHLT Grade 0) from moderate/severe rejection (Grade ≥ 3A/2R) Method • 7370 Gene microarray and PCR analysis taken at time of biopsies Result • Validated 20 gene classifier to distinguish rejection from quiescence Deng/Eisen/Mehra et al. Am J Transplant 2006;6:150

8. Basic Forms Of Rejection

9. Humoral Rejection • Accounts for 20-30% of rejections in allografts. • Common Risk Factors • Causes hemodynamic dysfunction (shock, hypertension, decreased cardiac output, and rise in pulmonary artery pressure).

10. Antibody Mediated RejectionIntroduction • Antibodies produced by B lymphocytes recognize the allograft and attempt to destroy it. • Characterized by IgM, IgG, IgA and IgE. • Increased secretion of cytokines and up-regulation of HLA molecules in the arteries and capillaries makes the allograft hypersensitive and antibodies bind to the graft endothelium more frequently. • Either May Occur: • Lysis occurs when the membrane is attacked and the cell lyses. • Activation occurs when the complement components trigger growth factors, such as extra tissue production.

11. Diagnostic CriteriaFor Humoral Rejection Colvin RB, Smith RN, 2005

12. How Humoral Rejection Works

13. HLA Antigen Displayed CD4 T Cell receptor CD8

15. Gene Expression When Lymphocytes release specific cytokines and plasma cells, They are doing so because of the patient’s gene Expression, which is why some patients experience rejection or no Rejection.

16. Proposed Genes

17. Experiment Aim: To find genes related to the entity “Antibody Mediated Rejection” which can predict Present/Future Humoral Rejection and give light to pathophysiological mechanisms. Inclusion: • Patients included in the CARGO study that have been tested with gene microarrays. Exclusion: • Patients with Oversensitive Immune Systems • Patients > 1 transplant

18. Methods • Peripheral Blood Mononuclear cells • Gene Microarray Analysis • C4d Staining of endomyocardial biopsies • Statistical Analysis of Clinical data • SPSS 11.5 • T-Test, Chi-square test, Kaplan Meier • Statistical Analysis of gene expression data • Significance analysis of microarrays (SAM) • Gene Ontology analysis

19. Gene Microarray Analysis • RNA of a patient is added to a solution of hybridization buffer and fragmentation buffer, along with cyanine 3 and 5. • This solution is put into a gasket slide and into a hybridization chamber. • After washing, these are now gene chips, which are read by a microarray scanner, which detects fluorescent molecules (by the cyanine 3 and 5).

20. Gene Microarray Analysis

21. C4D Staining • Paraffin embedded sections. • Antigens retrieval by a 10 minute bath at 95-100o C in a pH 6.0 buffer. • Biotin blockage by Biotin Blocking kit. • Incubation 30 minutes with C4dpAb and anti-C4d antibody. • Prevention of peroxidase activity: by washing with buffer and hydrogen peroxide or methanol

22. C4D Staining

23. Microarray Analysis45 patients tested with gene microarrays (CUMC)105 total samples obtained (CUMC)

24. Humoral SAM

25. Humoral Clustered Genes (FDR 5%)

26. Gene Ontology Analysis • The Gene Ontology project classify genes into specific categories according to their: • Cellular component • Biological process • Molecular function • High-Throughput GoMiner organizes lists of 'interesting' genes for biological interpretation in the context of the Gene Ontology. • http://discover.nci.nih.gov/gominer/htgm.jsp

27. Apoptosis • Programmed cell death • Cell death • Regulation of cell death • Regulation of apoptosis • Response to other organism • Humoral immune response • Antimicrobial humoral response • Humoral defense mechanism • mRNA metabolism • RNA metabolism • Negative regulation of apoptosis • Negative regulation of programmed cell death • Regulation of cell differentiation • Lymphocyte differentiation • T cell differentiation • Positive regulation of T cell activation • Regulation of immune response • Immune cell activation • Lymphocyte activation

28. These clusters of genes are very up-regulated or down regulated in the humoral patients The Genes in these clusters relate to the gene categories previously

29. Humoral HTGM results

30. References • Marboe C, Deng MC, Billingham M. Nodular Endocardial Infiltrates (Quilty Lesions) Cause A Significant Variability in Diagnosis of ISHLT Grade 2 and 3A Rejection in Cardiac Allograft Recipients. Journal of Heart and Lung Transplantation July 2005; 24:s219-s226. • Evans R, Williams G, Deng MC. The Economic Implications of Noninvasive Molecular Testing for Cardiac Allograft Rejection. American Journal of Transplantation 2005; 5:1553-1558 • Deng MC, Eisen HJ, Mehra MR. Noninvasive Discrimination of Rejection in Cardiac Allograft Recipients Using gene Expression Profiling. American Journal of Transplantation 2006; 6:150-160 • Deng MC. Cardiac Transplantation. Heart 2002; 87:177-184 • Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of Human organ Transplants. Springer Seminars in Immunopathology 2003 119-140.

31. Acknowledgements • Dr. Mario Deng • Martin Cadeiras • Manuel Prinz von Bayern • Sarfaraz Memon • Dr. Sat Bhattacharya • Columbia College of Physicians and Surgeons Harlem Children Society