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

Key Ideas Allograft Endomyocardial Biopsy Rejection CARGO Functional Genomics Patient Care

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

Rejection overall survival of heart transplant is 85% IncidenceMortality (Of those affected) Humoral17.6%17% Acute Cellular 1.4%23% Chronic13%18% Mild Severe

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

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

Basic Forms Of Rejection Chronic Rejection The movement of Smooth Muscle Tissue into the Coronary Arteries Causes Overtime narrowing of the Coronary Arteries Leads to lack of blood flow, Tissue Death, Heart Failure Cellular Rejection Inflammatory Response due to T cell infiltration in tissue Cardio Myocyte Damage Most Common Form Of rejection

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).

Antibody Mediated Rejection Introduction 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.

Diagnostic Criteria For Humoral Rejection Colvin RB, Smith RN, 2005 ClinicalAny Clinical Evidence of Graft Dysfunction SerologicalHLA specific antibodies or Donor specific antibodies HistologicalFibrinoid Necrosis, Injury, Infiltrates ImmunopathologicalC3 and C4d deposits, IG staining

How Humoral Rejection Works

T Cell receptor CD 4 CD 8 HLA Antigen Displayed

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.

Proposed Genes GENENAMEFUNCTION CD28CD28 moleculeEssential for CD4 T cell proliferation CD40CD40 moleculeMediates CD40 responses, inflammation and other Immune responses C4BComplement component 4B Provides interaction between antigen and complement components CD8Complement Component 8D Present on surface of T lymphocytes, Helps recognize Antigens displayed by Antigen Presenting Cells CCR6Chemokine receptor 6Helps B cell differentiation, and maturation. Expressed by CD4 and CD8 (natural killer cells) CCR4Chemokine (C-C motif) receptor 4 Development, homeostasis and function of Immune System CCr7Chemokine receptor 7Activation of B and T lymphocytes CCL2(C-C motif) ligandInflammatory response IL2Interleukin 2B and T cell proliferation IL10Interleukin 10Immunoregulation, inflammation and B cell survival

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

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

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).

Gene Microarray Analysis

C4D Staining Paraffin embedded sections. Antigens retrieval by a 10 minute bath at o 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

C4D Staining

Microarray Analysis 45 patients tested with gene microarrays (CUMC) 105 total samples obtained (CUMC) Immuno- pathological SerologicalHistologicalClinical Control (N= 12) No Humoral (N= 5) Yes Any criteria (N= 28) y/n

Humoral SAM

Humoral Clustered Genes (FDR 5%)

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.

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

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

Humoral HTGM results

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: Deng MC, Eisen HJ, Mehra MR. Noninvasive Discrimination of Rejection in Cardiac Allograft Recipients Using gene Expression Profiling. American Journal of Transplantation 2006; 6: Deng MC. Cardiac Transplantation. Heart 2002; 87: Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of Human organ Transplants. Springer Seminars in Immunopathology

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