Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses Xinhui Ge 1 , John A. Gebe 1 , Paul L. Bollyky 1 , Eddie A. James 1 , Junbao Yang 1 , Lawrence J. Stern 2 , William W. Kwok 1 * 1 Benaroya Research Institute at Virginia Mason, Seattle, Washington, United States of America, 2 Department of Pathology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Abstract Background: Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation. Methodology/Principal Findings: We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag- specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells. Conclusions/Significance: The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings. Citation: Ge X, Gebe JA, Bollyky PL, James EA, Yang J, et al. (2010) Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses. PLoS ONE 5(6): e11355. doi:10.1371/journal.pone.0011355 Editor: Douglas F. Nixon, University of California San Francisco, United States of America Received March 26, 2010; Accepted June 7, 2010; Published June 28, 2010 Copyright: ß 2010 Ge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants to W.K from American Diabetes Association (ADA 7-06-RA-75, www.diabetes.org) and National Institutes of Health (R21 DK077525-02; HHSN272200900043C, www.nih.gov), and a grant to LJ.S from National Institutes of Health (U19 AI57319, www.nih.gov). X.G is currently supported by Juvenile Diabetes Research Foundation International Postdoctoral Fellowship Award (3-2008-548, www.jdrf.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bkwok@benaroyaresearch.org Introduction As important initiators of adaptive immunity, CD4 T cells express highly specific T-cell receptors that recognize epitopes derived from protein antigens in the context of Class II MHC complexes. In the last decade, epitopes associated with a variety of infectious diseases, allergies, tumors and autoimmune diseases have been extensively explored. The advent of the Class II MHC tetramer and its related applications such as tetramer-guided epitope mapping have substantially increased the efficiency and accuracy of the epitope discovery process [1,2]. As adequate epitope information has been generated, one of the next challenges is to integrate this knowledge to examine overall cellular immune responses in relevant disease settings. Undoubtedly, tetramer technology continues to provide a promising approach for ex vivo and in vitro Ag-specific T cell analysis. However, technical issues such as the number of fluorochoromes that are distinguishable by current flow cytometry technology limit the simultaneous analysis of multiple antigen specificities. Although the latest peptide:MHC multi-color tetramer staining protocol managed to examine up to 15 Ag-specific T cell populations [3], most often, the detection is restricted to one or two epitopes at a time. Cellular microarrays provide an alternative solution [4,5,6,7,8]. This approach uses a high-precision robot arrayer to spot peptide:MHC complex onto a glass slide with high density. Much like artificial antigen presenting cells, the immobilized pepti- de:MHC activates Ag-specific T cells and triggers cytokine secretion. By co-immobilizing cytokine capture antibody with the peptide:MHC complex on the glass surface, the cytokines secreted by the activated T cells are retained in situ and probed with fluorescence-conjugated cytokine detection antibody for quantification. Unlike tetramer staining, which provides superb single-cell-level characteristics, the aim of peptide:MHC cellular microarrays is to achieve a high-throughput detection solution. PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11355