Monitoring the T-Cell Receptor Repertoire at Single- Clone Resolution Hendrik P. J. Bonarius 1 , Frank Baas 2 , Ester B. M. Remmerswaal 3 , Rene ´ A. W. van Lier 3 , Ineke J. M. ten Berge 4 , Paul P. Tak 1 , Niek de Vries 1 * 1 Division of Clinical Immunology and Rheumatology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands, 2 Department of Neurogenetics, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands, 3 Department of Experimental Immunology, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands, 4 Division of Nephrology, Department of Internal Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands The adaptive immune system recognizes billions of unique antigens using highly variable T-cell receptors. The ab T-cell receptor repertoire includes an estimated 10 6 different rearranged b chains per individual. This paper describes a novel micro- array based method that monitors the b chain repertoire with a resolution of a single T-cell clone. These T-arrays are quantitative and detect T-cell clones at a frequency of less than one T cell in a million, which is 2 logs more sensitive than spectratyping (immunoscope), the current standard in repertoire analysis. Using T-arrays we detected CMV-specific CD4+ and CD8+ T-cell clones that expanded early after viral antigen stimulation in vitro and in vivo. This approach will be useful in monitoring individual T-cell clones in diverse experimental settings, and in identification of T-cell clones associated with infectious disease, autoimmune disease and cancer. Citation: Bonarius HPJ, Baas F, Remmerswaal EBM, van Lier RAW, ten Berge IJM, et al (2006) Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution. PLoS ONE 1(1): e55. doi:10.1371/journal.pone.0000055 INTRODUCTION T cells are key players in the antigen specific immune responses. Antigen specificity is provided by the T-cell receptor (TCR), which is unique for each T-cell clone. Upon antigen recognition, individual T cell clones generally expand and acquire differential effector properties. Although the number of potential TCRs has been estimated at 10 15 different a/b combinations [1], the actual ab TCR repertoire per individual is estimated to include 10 6 different b chains [2], each pairing with a limited number of a chains [3]. There is no rapid technology available that can sensitively and quantitatively monitor this highly diverse T-cell receptor repertoire. Current technology for screening the TCR repertoire for expanded T-cell clones relies on ‘spectratyping’ [4], often referred to as immunoscope, and/or individual cloning and sequencing of a sample of the T-cell population [2,5–9]. In spectratyping analysis, PCR amplified TCR DNA is separated on size of the CDR3 region. This approach separates the TCRb repertoire in approximately 230 fractions, resulting from the use of ,23 primers for all functional Vb families and about 8 different CDR3 lengths per Vb [10]. A higher resolution can be attained when V- and J-region primers are used; however, this requires 23 ? 13 individual PCR reactions, and results in a resolution of approximately 23 ? 13 ? 8 peaks (Table S2). Spectratyping itself generally does not identify individual T cell clones, and is therefore often followed by repetitive cloning and sequencing. Clonal peaks identified in the spectratype patterns are sequenced, typically 10 2 clones and maximally 10 4 clones per sample [2,5–9] in previous publications. The sensitivity of this combined approach depends on the sensitivity of spectratyping for identification of clonal peaks, and on the number of T-cell- receptor rearrangements cloned and sequenced. Thus, although the combination of spectratyping with sequencing can attain sufficient resolution to analyze TCR diversity, the approach is laborious and time consuming as it requires PCR amplification, isolation of individual bands based on DNA size, purification, followed by repeated cloning and sequencing. Here, we explore a novel approach which exploits the high capacity of DNA microarrays to monitor the expression of many T-cell receptor rearrangements in parallel. At present, it can be used to follow T cell responses in cases where type of Vb/Jb and length of Jb-gene segment are available, e.g. from prior immunoscope (spectratyping) experiments. The feasibility of this approach is shown, and validated both in vitro and in vivo. We show that T arrays quantitatively monitor the expansion of T-cell clones after viral infection with high sensitivity (1 in 10 6 cells), and with sufficient resolution to identify single clones in a background of polyclonal peripheral blood T-cells. While at present it allows monitoring a Vb/Jb-specific fraction of 0.03% of the T-cell receptor repertoire on a single 4000-spot slide, the microarray- based method can be scaled up to monitor and screen a large pool of the T-cell repertoire for dominant clonotypes. We envision that this sensitive and rapid technology will be useful for monitoring and screening of clonal T-cell expansions for many applications in medical research. Academic Editor: Etienne Joly, Universite ´ de Toulouse, France Received September 12, 2006; Accepted October 13, 2006; Published December 20, 2006 Copyright: ß 2006 de Vries 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: The Netherlands Genomics Initiative (NGI: www.genomics.nl) funded the position of H.P.J.B. in a Horizon Breakthrough Grant to N.V. The NGI did not play a role in the conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript. There are no other financial or other relations that might be relevant to the submission of this manuscript. Competing Interests: The AMC at the University of Amsterdam, the employer of all authors, filed for a patent application listing N.V. as inventor. In case the system becomes profitable, personal revenues are maximized at J50.000 a year; the department may receive up to 10% of the financial rewards. Neither the AMC nor the transfer office were involved in the conception, editing or the decision to submit the manuscript. * To whom correspondence should be addressed. E-mail: Niek.deVries@amc. uva.nl PLoS ONE | www.plosone.org 1 December 2006 | Issue 1 | e55