Novel Tools for Immune Quiescence Monitoring in Kidney TransplantationS.Roedder1, L.Li2, M.Alonso2, S.Hsieh1, H.Dai1,T.Sigdel1,I.Bostock3,C.Macedo4,D.Metes4,A.Zeevi4,R.Shapiro4,O.Salvatierra2,J.Scandling2,J.Alberu3,E.Engleman2 & M.Sarwal1

1 Department of Surgery, UCSF, San Francisco, United States; 2School of Medicine, Stanford University, United States;

3Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico, and 4UPMC, Pittsburgh, United States.

Table 1

BACKGROUNDOrgan transplant patients face life-long immunosuppression (IS) with increased morbidity. Currently unknown numbers of kidney transplant recipients develop a state of targeted immune quiescence (Operational Tolerance, TOL) allowing them to withdraw IS while retaining stable graft function and continuing immune responses against 3rd party antigens. Transcriptional Profiling Peripheral Blood is a means to provide a gene signature to monitor this state of TOL, to titrate IS in patients with this signature, and to better understand the underlying biology.

STUDY DESIGN

Figure 1: 4-Stage Study Design utilizing to define a 3-gene TOL Assay in a total of 571 peripheral blood-/cell- and tissue samples.

Cell-TypeCommon targets

fc≥3

# mapped probes in

TOL

p-value

DC 3922 7 0.013B-lymphocyte 5028 7 0.047CD8+ T-cell 3278 3 0.259CD4+ T-cell 3157 3 0.254CD56+ NK 3802 6 0.042Monocyte 3241 3 0.258CD34+ 3898 3 0.265CD33+ 11218 4 0.212

Figure 4: FACS in PBMC from 5 TOL, 6 SI and 5 HC showed confirmed enrichment of DC (CD11c+, CD14lo ) in TOL compared to HC and SI; ratio to CD4- T-cells)

Stage 4: TOL Biology Analysis

METHODSMicroarray Discovery (n=31) (Agilent 4x44k), cross platform validations I (n=29) (Lymphochip), II (n=58) (Affymetrix HgU133, GSE22229) (Stage 1); QPCR-validation (n=59) (SABioscience, Stage 2); QPCR-modeling and- prediction (n=220) (Fluidigm, Stage 3); TOL Biology Analysis (n=174) (FACS, Gene Expression, Stage 4). Total of 574 unique peripheral blood samples (Figure 1 Study Design).

Figure 3: Post Stage 2 QPCR validation of the 21 TOL gene signature in 65 independent samples (not shown), a 3-gene logistic regression model predicted TOL with an AUC=0.95 (95%CI: 0.97-0.92); 84.6% Sensitivity and 90.2% Specificity (ROC Curve); threshold for TOL prediction was Θ=25%)

Stage 2,3: QPCR Validation & 3-Gene Modeling

TOL (n=43)A SI (n=78)A SI (n=150)B

RecipientsGender (% males) 68.4% 74.0% 63.3%Age (mean±StdD) 28 ± 20 15 ± 13 33.3±19.2Months post Tx (mean/median±StdD)

216.8/195.7±139.2

47.6/23.5±71.7

25.5/29.0±11.4

Rx (Induction)C N.A. Dac./Thymo. Dac./Camp./Bas.

Rx (Maintenance)C -CNI, ±Steroids/

MMF, ±AZACNI, Bela.,

±Steroids/MMF Serum Creatinine (mg/dL)

0.95 ± 0.2 2.92 ± 2.9 1.27 ± 0.3

DonorsSource (%LRD) 32% 67% 59.3%HLA mismatch (x/6; mean±StdD)

0.75 ± 1.5 2.92 ± 2.9 2.93 ± 1.7

Gender (% males) 50% 42% 48%Age (mean±StdD) 39.8±16.6 42.9±10.8 37.7±13.1

PATIENTS

A utilized for novel Discovery (Stage 1), QPCR-Validation (Stage 2) & TOL Modeling (Stage 3A) (n=121 unique patients); B utilized for independent prediction (Stage 3B) (n=150 unique patients); C Dac.=Daclizumab, Thymo=Thymoglobulin, Camp.=Campath, Bas=Basiliximab, CNI= Calcineurin Inhibitor (Cyclosporin, Tacrolimus); MMF= Mycophenolate Mofetil; AZA=Azathioprin

Stage 3: Frequency of predicted TOL Phenotype in stable Patients on standard Immunosuppression

Figure 3: Application of the 3-Gene assay to 150 patients on standard IS identified 11 patients (7.33%) with a TOL signature (TOL-phenotype prediction Θ=60%); following anti CD25 induction, more patients on Belatacept revealed a TOL signature (n=5 vs. =2 on CNI).

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Predicted TOLPredicted Non-TOL

CONCLUSIONIn conclusion, this study provides a highly validated, peripheral blood, 3 gene assay for an operationally tolerant patient phenotype and infers underlying mechanisms related to myeloid derived cell types. The assay offers a potential means to monitor for donor specific hypo responsiveness and graft accommodation in immunosuppressed transplant recipients, to segregate patients who maybe on a larger burden of chronic immunosuppression than “immunologically” necessary.

Statement of Competing Financial Interests: The authors of this study have no competing financial interests; References.: Brouard et al., PNAS 2007, Newell et al., JCI 2010

Figure 2: Nearest shrunken centroid (PAM, FDR5%) defined a 21 TOL gene signature in 31 patients to predict TOL on the Agilent Platform (y-axis: % predicted probability); validated in 2 publicly available data-sets on Lymphochip (n=29) and Affymetrix (n=58) platforms. Θ (TOL prediction) = 50% Probability .

RESULTSStage 1: Cross Platform Microarray analysis for a 21 Gene TOL-Signature

Table 2: Hypergeometric Enrichment analysis of 174 cell/tissue samples identified Dendritic Cells (DC) as most significant cell type for with enrichment of 7/21 TOL genes