antibody mediated rejection and gene expression profiles anatasia gangadin dr. mario c deng columbia...

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 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 GenesGENE NAME FUNCTION

CD28 CD28 molecule Essential for CD4 T cell proliferation

CD40 CD40 molecule Mediates CD40 responses, inflammation and other Immune responses

C4B 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 proliferation

IL10 Interleukin 10 Immunoregulation, 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 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