Antibody Mediated Rejection and
Gene Expression ProfilesAnatasia Gangadin
Dr. Mario C DengColumbia University
College of Physicians and Surgeons
Key Ideas• Allograft
• Endomyocardial Biopsy
• Rejection• CARGO
• Functional Genomics
• Patient Care
Patient Care
Endomyocardial biopsy• Currently Only
way to test for rejection
• Risk Factors associated
Rejection overall survival of heart transplant is 85%
Incidence
Mortality(Of those affected)
Humoral 17.6% 17%
Acute Cellular
1.4% 23%
Chronic 13% 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
DesignDesign
HypothesisHypothesis
ResultResult
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
MethodMethod
7370 Gene microarray and PCR analysis taken at time of biopsies
Basic Forms Of RejectionChronic 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
Antibody Mediated 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 CriteriaFor Humoral Rejection
Colvin RB, Smith RN, 2005
Clinical Any Clinical Evidence of Graft Dysfunction
Serological HLA specific antibodies or Donor specific antibodies
Histological Fibrinoid Necrosis, Injury, Infiltrates
Immunopathological
C3 and C4d deposits, IG staining
How Humoral Rejection Works
T Cell receptor CD4
CD8
HLA
Antigen Displayed
Gene ExpressionWhen 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 GenesGENE NAME FUNCTION
CD28 CD28 molecule Essential for CD4 T cell proliferationCD40 CD40 molecule Mediates CD40 responses, inflammation and
other Immune responsesC4B Complement
component 4B Provides interaction between antigen and complement components
CD8 Complement Component 8D
Present on surface of T lymphocytes, Helps recognize Antigens displayed by Antigen Presenting Cells
CCR6 Chemokine receptor 6
Helps B cell differentiation , and maturation. Expressed by CD4 and CD8 (natural killer cells)
CCR4 Chemokine (C-C motif) receptor 4
Development, homeostasis and function of Immune System
CCr7 Chemokine receptor 7
Activation of B and T lymphocytes
CCL2 (C-C motif) ligand
Inflammatory response
IL2 Interleukin 2 B and T cell proliferationIL10 Interleukin 10 Immunoregulation, inflammation and B cell
survival
ExperimentAim: 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 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
C4D Staining
Microarray Analysis45 patients tested with gene microarrays
(CUMC)105 total samples obtained (CUMC)
Immuno-pathological
Serological
Histological
Clinical
Control (N= 12)
No No No No
Humoral(N= 5)
Yes Yes Yes Yes
Any criteria(N= 28)
y/n y/n y/n 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.
• http://discover.nci.nih.gov/gominer/htgm.jsp
• 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; 5:1553-1558
• 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.
Acknowledgements• Dr. Mario Deng• Martin Cadeiras• Manuel Prinz von Bayern • Sarfaraz Memon• Dr. Sat Bhattacharya• Columbia College of Physicians and
Surgeons
Harlem Children Society