Ultrasensitive Detection and Phenotyping of CD4 + T Cells with Optimized HLA Class II Tetramer Staining This information is current as Thomas J. Scriba, Marco Purbhoo, Cheryl L. Day, Nicola of September 24, 2021. Robinson, Sarah Fidler, Julie Fox, Jonathan N. Weber, Paul Klenerman, Andrew K. Sewell and Rodney E. Phillips J Immunol 2005; 175:6334-6343; ; doi: 10.4049/jimmunol.175.10.6334 http://www.jimmunol.org/content/175/10/6334 Downloaded from References This article cites 52 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/175/10/6334.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Ultrasensitive Detection and Phenotyping of CD4؉ T Cells with Optimized HLA Class II Tetramer Staining1 Thomas J. Scriba,* Marco Purbhoo,† Cheryl L. Day,* Nicola Robinson,* Sarah Fidler,‡ Julie Fox,‡ Jonathan N. Weber,‡ Paul Klenerman,* Andrew K. Sewell,* and Rodney E. Phillips2* HLA class I tetramers have revolutionized the study of Ag-specific CD8؉ T cell responses. Technical problems and the rarity of Ag-specific CD4؉ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4؉ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4؉ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4؉ cells) in human PBLs analyzed Downloaded from directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4؉ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques. The Journal of Immunology, 2005, 175: 6334–6343. lymphocytes recognize antigenic peptides presented by the ability of cells to respond to synthetic Ags; a quality that may MHC molecules on the surface of APCs with their unique define only a subset of the relevant cell population (5, 6). http://www.jimmunol.org/ TCR. The interaction between the TCR complex and pep- The application of this powerful technology to the study of Ag- T 3 ϩ tide-MHC (pMHC) is weak and has a fast dissociation rate (1–3). specific CD4 T cells has not been fully realized due to numerous As a result, soluble recombinant monomeric pMHC molecules do technical issues. Production of MHC class II (MHCII) complexes not stably adhere to the surface of cognate T cells. This limitation has been more difficult than MHCI. The MHCI H chain can be can be overcome by increasing the valency of interactions by mul- expressed in Escherichia coli as a soluble product by truncation of timerization of biotinylated pMHC molecules. Multimerized the membrane-spanning domain and folded readily around the an- ␤ pMHC can be conjugated to fluorochromes to allow detection by tigenic peptide in the presence of 2-microglobulin (4). pMHCII ␣ ␤ FACS (4). Such multimeric pMHC complexes were able to bind molecules consist of two polymorphic chains ( and ), which by guest on September 24, 2021 multiple TCRs on Ag-specific cells and thus lowered the dissoci- have been more difficult to refold around the antigenic peptide. E. ation rate sufficiently to be used as an immunological stain. The coli expression of the of MHCII alleles has been inefficient (7). development of these peptide-HLA tetrameric complexes marked However, correctly folded molecules can be manufactured in in- the beginning of a new era in the study of Ag-specific T cells, and sect and mammalian cells (8, 9). These efforts allowed successful HLA class I complexes have revolutionized the study of MHC staining of in vitro-stimulated CD4ϩ T cell lines and clones (9– ϩ class I (MHCI)-restricted CD8 T cell responses. Specifically, the 16). Despite these advances, the direct detection of Ag-specific capacity to physically detect Ag-specific T cells in diverse states of CD4ϩ T cells in PBMC remained difficult, mainly because the activation or function makes the use of HLA class I, and poten- cells are rare in peripheral blood (17–20). tially class II tetramers, more powerful than techniques that rely on Virtually all studies reporting MHCII tetramer staining used high concentrations of tetramer (20 ␮g/ml or more), long incuba- *The Peter Medawar Building for Pathogen Research, Nuffield Department of Clin- tion times (1–5 h) and temperatures of 23 or 37°C (8–10, 13–17, ical Medicine, University of Oxford, Oxford, United Kingdom; †Avidex Ltd., Abing- 21–27). In these studies, HLA class II tetramers stained cognate T don, United Kingdom; and ‡Department of Medicine, Imperial College, St. Mary’s Hospital, London, United Kingdom cell clones poorly or not at all at 4°C (12, 21, 25). Cameron et al. (25) further suggested that HLA class II tetramer staining was Received for publication July 8, 2005. Accepted for publication September 12, 2005. dependent on active cellular processes by demonstrating blockade The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of tetramer staining when endocytosis and cytoskeletal rearrange- with 18 U.S.C. Section 1734 solely to indicate this fact. ments were disrupted. Because the association rates of tetrameric 1 This work was supported by SPARTAC, a Wellcome Trust clinical trial, and a TCR/pMHCII interactions fall within a similar range to those seen for programme grant from the Wellcome Trust (to R.E.P. and J.N.W.). T.J.S. is funded TCR/pMHCI (28), the kinetics of HLA class II tetramer staining by the Fogarty Foundation and the South African National Research Foundation. C.L.D is a Royal Society Research-funded fellow. P.K. is a Wellcome Trust Senior should resemble those of HLA class I tetramers. Efficient staining of Clinical Research Fellow, and A.K.S. is a Wellcome Trust Senior Basic Research CD8ϩ T cells was reported at 4°C, and incubation of cells with tet- Fellow. R.E.P and P.K. are foundation investigators of the James Martin 21st Century School, University of Oxford. ramer for 10 min is sufficient for complete staining (29, 30). 2 In this study, we determine the conditions required for optimal Address correspondence and reprint requests to Prof. Rodney E. Phillips, The Peter ϩ Medawar Building for Pathogen Research, South Parks Road, Oxford, OX1 3SY, HLA class II tetramer staining of DR1- and DR4-restricted CD4 U.K. E-mail address: [email protected] T cells specific for HIV, CMV, tetanus toxoid (TT), and influenza 3 Abbreviations used in this paper: pMHC, peptide-MHC; MHCI, MHC class I; virus. We find rapid and efficient staining of CD4ϩ T cells in MHCII, MHC class II; ICS, intracellular cytokine staining; HCV, hepatitis C virus; conditions similar to those reported for HLA class I staining of 7-AAD, 7-aminoactinomycin D; HA, hemagglutinin; TT, tetanus toxoid; MFI, mean ϩ fluorescence intensity; DIC, differential interference contrast. CD8 T cells. Active cellular processes such as internalization are Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6335 not a requirement for staining. We test and apply an optimized Gag peptide p24.4 (aa 164–183; AFSPEVIPMFSALSEGATPQ), and a method in combination with a magnetic bead enrichment tech- DRB1*0401 tetramer complexed to the hepatitis C virus (HCV) NS3 pep- nique to detect HIV- and CMV-specific CD4ϩ T cells at frequen- tide (aa 1248–1261; GYKVLVLNPSVAATL) was used as control. These tetramers are referred to as p24.4-DR4 and HCV1-DR4, respectively. All cies below 1 in 100,000 in direct ex vivo human PBMC samples. tetramers were PE-conjugated and supplied at a concentration of 100 This technology allowed us to compare the phenotypic profile of ␮g/ml. rare virus-specific CD4ϩ T cells from different infections. Tetramer staining and flow cytometry Materials and Methods Unless stated otherwise, cell lines and clones were incubated with 2 ␮g/ml Patients and HLA typing HLA class II tetramer for 30 min at 37°C in PBS/1% FCS or PBS/1% BSA buffer. The cell surface marker Abs CD4-allophycocyanin or CD4-FITC Heparinized blood was obtained from HIV-infected patients who were re- and 7-aminoactinomycin D (7-AAD) (all BD Pharmingen) were added for cruited at St Mary’s Hospital, London, as described elsewhere (20), the the last 20 min or for 20 min after the cells were washed with cold PBS/1% SPARTAC Trial or from healthy, HIV-negative donors. Informed consent FCS. Stained cells were washed with cold PBS/1% FCS and fixed in 1% was obtained from all participants, and the study had full ethical approval.