S8 Abstracts

BiomarkersFriday, June 122:45 pm-4:45 pm

OR.9. Detection of Islet-specific AutoreactiveT Cells by a Novel Robust and High ThroughputScreening Strategy in Stored Blood Samples ofType 1 Diabetic PatientsJoana Abreu1, Jurjen Velthuis1, Wendy Unger1, KeesFranken1, Ton Schumacher2, Bart Roep1. 1Leiden UniversityMedical Center, Leiden, Netherlands; 2The NetherlandsCancer Institute, Amsterdam, Netherlands

Type 1 diabetes (T1D) results from selective destructionof pancreatic β cells by cytotoxic T cells. In this process, βcells presenting islet-specific epitopes are recognized anddestroyed by autoreactive CD8+T cells. Therefore, tools toreliably detect and monitor β cell specific CD8+T cells areessential. Current approaches to screen blood of T1Dpatients suffer from the large number of candidate epitopes,their lower HLA binding affinity, low precursor frequencies ofcirculation autoreactive T-cells, the presence of regulatoryT-cells and the need for ex vivo culture. We developed a fastand high-throughput assay to determine HLA-binding proper-ties and monitor antigen-specific T cell responses in storedblood of T1D patients. In this assay, conditional HLA ligandsare cleaved upon UV-irradiation resulting in HLA moleculesthat are loaded with arrays of peptide ligands. Peptide-HLAmonomers are then labeled with distinct fluorochromes andthe blood of T1D patients is screened. We investigated theuse of quantum dots (Qdots) as fluorochromes to generatepeptide-HLA multimers instead of conventional tetramers.The use of Qdots allowed the simultaneous screening of eightdifferent epitopes derived from Insulin, Pre-pro-insulin, IA2,GAD65, IGRP and ppIAPP, with high specificity and sensitivity.We demonstrate that this assay is of particular interest whenmonitoring specific T cell responses during the course oftreatment trials and after clinical islet transplantation. Inaddition, the development of this technique allows thediscovery and detection of multiple islet epitopes requiringminimal amounts of patient material.

doi:10.1016/j.clim.2009.03.016

OR.10. Using Immune Response Data from aLymphoma Vaccine Clinical Trial to Define NovelEndpoints for an Immunotransplant TrialJoshua Brody1, Debra Czerwinski1, Wei Ai2, Ronald Levy1.1Stanford University Medical Center, Stanford, CA;2University of California San Francisco, San Francisco, CA

Background: Cancer vaccines need to bemore powerful andpractical. We have completed a clinical trial using thepractical approach of intratumoral injection of an boff-the-shelfQ TLR9-agonist for patients with lymphoma. Toincrease the power of vaccination, we have developed andimmunotransplantTmaneuver- the transfer of vaccine-primedlymphocytes to lymphodepleted recipients. The obstacle to

demonstrating the clinical benefit of immunotransplant is thatthe anti-lymphoma effect of dlymphodepletionT confoundsconventional clinical response measurements. However, cor-relating multiplex immune-response measurements withclinical response in the lymphoma vaccine trial could allowus to validate an immune endpoint to assess the benefit ofimmunotransplant. Results: Our lymphoma vaccine trialdemonstrated 1 CR, 2 PR, and 8 SD amongst 15 patients aswell induction of tumor-specific, memory CD8 T cellscorrelating temporally with tumor regression. In the super-natant of tumor-lymphocyte co-cultures,wehaveperformed amultiplex high throughput assaymeasuring dcytokine-responseprofilesT. We have tested these profiles for intra-patientreproducibility and inter-patient variability in the completedvaccine trial cohort. These, together with profiles of patientsin our ongoing iteration of that trial will be used to correlate aspecific signaturewith clinical outcome. Conclusions: An intra-tumoral TLR9-agonist-based vaccine for lymphoma is feasibleand induces tumor-specific CD8 T cell responses and clinicalresponses. Immunotransplant significantly increases the anti-tumor effect of this vaccine maneuver pre-clinically. Thevalidation of dcytokine-response profilesT in the lymphomavaccine trial may allow us to demonstrate the benefit ofimmunotransplant in early stage trials.

doi:10.1016/j.clim.2009.03.017

OR.11. Discovery of Novel Antigenic Targets andAutoantibody Markers in Rheumatoid ArthritisK. Somers, P. Geusens, P. Stinissen, V. Somers. HasseltUniversity and Transnationale Universiteit Limburg,Diepenbeek, Belgium

Rheumatoid arthritis (RA) is the most common auto-immune disease characterized by chronic inflammation ofsynovial joints. The aim of this study was to identify noveltarget (auto)antigens and antibody biomarkers for early RApatients and RA patients that are seronegative for the 2diagnostically applied serological RA markers, rheumatoidfactor (RF) and anti-CCP (cyclic citrullinated peptides)antibodies (ACPA), by applying an autoantibody profilingprocedure based on cDNA phage display. We report theidentification of 14 novel target antigens and autoantibodymarkers for RA. Inclusion of the detection of antibodiesagainst these antigens to the diagnostic testing for RF andACPA presence increased the sensitivity of serological testsfor RA by 25%. Moreover, antibodies against our panel weredetected in an early disease phase and were associated withhigher inflammatory disease activity, demonstrating thediagnostic and prognostic potential of the panel. Further-more, we identified several novel antigens with exclusiveimmunoreactivity in ACPA negative RA patients. An increasedexpression of several of the identified antigens in RA synovialtissue was demonstrated, providing evidence for a biologi-cally relevant role of the antigen-antibody systems in the RAdisease process. In conclusion, the identified markers areproven to be valuable for improved RA diagnosis andprognosis prediction, especially for RA patients that areseronegative based on current diagnostic laboratory tests.Moreover, the identified antigen-antibody systems provide