Tetramer Staining T Cells with Optimized HLA Class II + CD4

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

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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. PBS/formaldehyde. At least 30,000 cells were acquired on a FACSCalibur PBMC were isolated by density gradient centrifugation of blood layered flow cytometer (BD Biosciences), and data were analyzed using CellQuest over Lymphoprep (Axis Shield). High-resolution HLA class II genotypes software (BD Biosciences). were determined for each study participant by PCR using sequence-specific Cell surface tetramer decay assay primers (31). CD4ϩ T cells were preincubated on ice for 5 min with chilled azide buffer Cell lines and clones ␮ (0.1% NaN3, 0.5% BSA in PBS) and incubated with 1 g/ml tetramer in

Fresh PBMC were depleted of CD8ϩ cells using anti-CD8-conjugated azide buffer for2honice. Stained cells were washed twice in chilled azide Downloaded from ␮ buffer, split into two aliquots, and placed at room temperature. To one magnetic beads (Dynal) and stimulated with 1–2 M peptide in RPMI ␮ 1640 medium containing 10% heat-inactivated human ABϩ serum. When aliquot, 100 g/ml anti-HLA-DR Ab (clone L243; BD Biosciences) was lines were cultured from HIV-infected patients, 0.4 ␮M indinavir (Merck) added, and cells were taken at time points 0, 1, 2, 5, 10, and 30 min, and 0.5 ␮M zidovudine (GlaxoSmithKline) were added to the medium. resuspended in PBS, and acquired on a ﬂow cytometer. The plates were incubated at 37°C in 5% CO2, and after 3 days, the me- Microscopy dium was supplemented with 100 U/ml IL-2 (Proleukin; Chiron) and 5% ϩ

human T cell culture supplement (T-STIM) (BD Biosciences). Medium For microscopy, CD4 T cells were stained at 37°C or on ice in azide http://www.jimmunol.org/ was exchanged as necessary. Cell lines were restimulated with peptide, buffer as described for each experiment and washed with PBS/0.5% BSA. IL-2, human T cell culture supplement (T-STIM), and irradiated autolo- For imaging, stained cells were suspended in PBS/0.5% BSA in eight-well gous PBMC every 12–14 days. For generation of CD4ϩ T cell clones, cell chambers (Labtek; Nunc). A Zeiss 200M/Universal Imaging system with a lines were stimulated with 4 ␮M peptide for 4 h, and IFN-␥- and IL-2- ϫ63 objective was used for wide-field fluorescence microscopy. PE fluo- producing cells were selected using a cytokine detection and enrichment rescence was detected using a 535/50 excitation, 610/75 emission, and method (Miltenyi Biotec). Ag-specific (IFN-␥ϩ and IL-2ϩ cells) CD4ϩ 565LP dichroic filter set (Chroma). To cover the entire three-dimensional cells were plated at limiting dilution in medium containing 10% human surface of the cell, z-stack fluorescent images were taken (21 individual ABϩ serum, 2 ϫ 105 irradiated mixed lymphocytes/ml, 100 U/ml IL-2, 5% planes; 1 mm apart). Data were analyzed using Metamorph software. Data T-STIM, and 1 ␮g/ml PHA (Sigma-Aldrich). Cell lines and clones were were evaluated for at least 20 cells in each experimental condition. tested for Ag specificity by IFN-␥ ELISPOT, intracellular cytokine staining

(ICS), or HLA class II tetramer staining. ELISPOT and ICS assays were Detection of rare cells by magnetic bead enrichment and by guest on September 24, 2021 performed with either peptides or recombinant proteins as Ag as described phenotypic staining (20). The cell lines and clones used in this study are listed in Table I. All Magnetic bead enrichment of tetramer-positive CD4ϩ cell lines were used for experiments after two or three rounds of antigenic T cells was done as described previously (20). Briefly, the cells were incubated with 5 ␮g/ml stimulation, and all experiments were done at least 7 days after restimu- ϩ HLA class II tetramer (to compensate for large cell numbers). Anti-CD4- lation of cell lines and clones. The influenza HA307-319-specific CD4 T cell clone HA1.7 (32) was kindly donated by J. Lamb (Medical Research allophycocyanin, anti-CD19-PerCP, and anti-CD14-PerCP Abs and Council Center for Inflammation Research, University of Edinburgh, Ed- 7-AAD (all from BD Biosciences) were added during the last 20 min for inburgh, U.K.). the 120-min staining at 23°C and for 20 min after a wash for the 20-min staining at 37°C. For phenotypic staining of tetramer-positive CD4ϩ cells, MHCII tetramers anti-CCR7-FITC (R&D Systems) and anti-CD27-FITC and anti- CD28-FITC (both from BD Biosciences) were added with the other Abs. DRB1*0101 and DRB1*0401 iTAg MHCII tetramers were purchased The cells were washed and labeled with anti-PE microbeads according to from Beckman Coulter. DRB1*0101 tetramers were complexed to the HIV the manufacturer’s protocol (Miltenyi Biotec), and 10% of the cells were p24 Gag peptide p24.17 (aa 294–313; FRDYVDRFYKTLRAEQASQD), reserved for FACS analysis while 90% were subjected to magnetic bead the p24 Gag peptide p24.14 (aa 264–283; KRWIILGLNKIVRMYSPTSI), enrichment of PE-conjugated tetramer-positive cells using a single MACS the TT830–843 peptide (QYIKANSKFIGITE), the influenza hemagglutinin column (Miltenyi Biotec). All cells were acquired on a FACSCalibur flow Ϫ Ϫ (HA)307–319 peptide (PKYVKQNTLKLAT) and the CMV pp65108–127 cytometer (BD Biosciences), gated on CD14 , CD19 , and 7-AAD-neg- peptide (LPLKMLNIPSINVH). These tetramers are referred to as p24.17- ative lymphocytes, and analyzed using CellQuest software (BD Bio- DR1, p24.14-DR1, TT830-DR1, HA307-DR1, and pp65-DR1, respec- sciences). The frequency of tetramer-positive cells was calculated by di- tively. The DRB1*0401 tetramer p24.4-DR4 was complexed to the p24 viding the number of postenrichment CD4ϩtetramerϩ cells by the number

Table I. Ag-speciﬁc CD4ϩ T cell lines and clones

HLA-DRB1* Cell Line/Clone Line/Clone Typea Epitope Speciﬁcity Speciﬁc Tetramer Epitope Sequence

Ox24-p24.17 Line *0101, *0102 HIV Gag p24294–313 p24.17-DR1 FRDYVDRFYKTLRAEQASQD Ox97-clone10 Clone *0101, *0401 HIV Gag p24294–313 p24.17-DR1 FRDYVDRFYKTLRAEQASQD Ox24-p24.14 Line *0101, *0102 HIV Gag p24264–283 p24.14-DR1 KRWIILGLNKIVRMYSPTSI K37-p24.4 Line *0401 HIV Gag p24164–183 p24.4-DR4 AFSPEVIPMFSALSEGATPQ JF-TTox Line *0101, *1401 TT830–843 TT830-DR1 QYIKANSKFIGITE HA1.7 Clone *0101 Influenza HA307–319 HA307-DR1 PKYVKQNTLKLAT JF-HA Line *0101, *1401 Influenza HA307–319 HA307-DR1 PKYVKQNTLKLAT LA-pp65 Line *0101, *1301 CMV pp65108–127 pp65-DR1 LPLKMLNIPSINVH a HLA restriction of epitope specificity is in boldface type. 6336 ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4ϩ T CELLS of CD4ϩ cells in the pre-enrichment sample multiplied by 9 (to account for to label Ag-specific cells within their respective cell lines and to the fact that 90% of the cells were used for the enrichment). label true clones as well. At 5–10 ␮g/ml and higher concentrations, background staining increased for all tetramers as nonspecific cells Results were also stained (Fig. 2A and data not shown). Staining of Ag-specific CD4ϩ T cells with HLA class II tetramers is highly specific HLA class II tetramer staining is rapid We studied the labeling of Ag-specific CD4ϩ T cells with HLA CD8ϩ T cells can readily be labeled with HLA class I tetramers by class II tetramers at different experimental conditions. Cell lines incubation at 37°C for 10 min (29, 35) and staining can be detected and clones specific for antigenic peptides encoded by HIV type 1 as early as after 30 s (29). To test the rate at which HLA class II (HIV-1) p24 Gag, influenza HA, TT, or human CMV pp65 were tetramers stained CD4ϩ T cells, we incubated all cell lines and generated and tested for specificity by IFN-␥ ELISPOT assay or clones with 2 ␮g/ml HLA class II tetramer at 37 and 23°C for ICS (Fig. 1A). Clones and CD4ϩ T cell lines were stained with 1–120 min. We found efficient staining of CD4ϩ T cells of all their corresponding peptide-HLA class II tetramer (Fig. 1B). Al- specificities within 10 min at 37°C. This was observed with the though not identical, the frequencies of HLA class II tetramer- TT, the influenza HA and CMV pp65 DR1 tetramers as well as the positive CD4ϩ cells were comparable with the frequencies of IFN- HIV Gag p24 DR1 and DR4 tetramers when used to stain all ␥-producing cells as measured by ICS (Fig. 1 and data not shown). CD4ϩ cell lines and clones (Fig. 3, A and B). Although all cells Staining of all cell lines and clones with peptide-mismatched, were stained after 10 min, the mean fluorescence intensity (MFI) DRB1-allele-matched tetramers yielded CD4ϩtetramerϩ popula- continued to increase up to 120 min after starting incubation with tions that never exceeded 0.2% (Fig. 1B). The cell lines and clones the tetramer (data not shown). Notably, the proportion of Downloaded from and peptide-matched HLA class II tetramers are shown in Table I. tetramerϩCD4Ϫ cells also increased with time, suggesting that internalization of pMHCII-TCR complexes was taking place (data Optimizing the use of HLA class II tetramers for staining T cells not shown). At 23°C, tetramer staining was slower for most spec- A wide range of HLA class II tetramer concentrations are reported ificities, although the HIV-specific Ox24-p24.17 and K37-p24.4 to be required for adequate staining of specific CD4ϩ T cells. cell lines and the influenza-specific HA1.7 clone were fully stained ϩ Some investigators have used tetramer concentrations of 20 ␮g/ml after 10 min. At 23°C, complete staining of all CD4 T cells was http://www.jimmunol.org/ or more (8–10, 12, 13, 15–18, 21, 23–26). Others have used lower seen only after 30–60 min for the other cell lines and clones (Fig. concentrations (11, 14, 20, 22, 33, 34). Meyer et al. (17) found that 3C), and the MFI was generally lower than when cells were stained certain Borrelia-specific CD4ϩ T cell clones could be stained with at 37°C (data not shown). as little as 0.2 ␮g/ml. To assess the optimal HLA class II tetramer staining concentrations for a range of cell lines and clones, we HLA class II tetramer staining does not require internalization incubated CD4ϩ T cells with tetramer ranging from 0.01 to 10 Labeling of Ag-specific CD8ϩ T cells with HLA class I tetramers ␮g/ml in a total volume of 100 ␮l (Fig. 2) for 30 min at 37°C. can be efficient at 4°C (30, 35). However, some earlier investiga- Although some variation was seen, 2 ␮g/ml tetramer was sufficient tors failed to label Ag-specific CD4ϩ T cells with HLA class II by guest on September 24, 2021

FIGURE 1. HLA class II tetramer staining of Ag-specific CD4ϩ T cell lines and clones. A, Testing the frequency of Ag-specific CD4ϩ T cells by intracellular IFN-␥ staining. FACS dot plots showing Th cell lines Ox24-p24.17 and K37-p24.4 either unstimulated (No peptide) or stimulated with 2 ␮M peptide. The percentage of CD4ϩ cells producing IFN-␥ is indicated in each plot. B, FACS dot plots showing the cell lines stained with their matched (upper plots) and unmatched (lower plots) HLA class II tetramer. The percentage of tetramerϩCD4ϩ cells is indicated in each plot. The histograms represent the HIV-p24-specific Ox97-clone10 and the influenza HA-specific clone HA1.7 stained with matched (in red) and three unmatched HLA class II tetramers. The Journal of Immunology 6337

FIGURE 2. Concentration titration of HLA class II tetramers. A, FACS dot plots of the CD4ϩ T cell line Ox24-p24.17 stained with the indicated concentrations of p24.17-DR1 tetramer at 37°C for 30 min. The percentage of cells falling into the upper right quadrant is indicated in each plot. B, Graphical representation of the MFI obtained from concentration titrations of ﬁve HLA class II tetramers. C, Graphical representation of the percentage CD4ϩtetramerϩ cells recorded for each tetramer. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. HLA class II tetramer staining is rapid. FACS density plots (A) and histograms (B) showing efficient HLA class II tetramer staining of Ag-specific CD4ϩ cell lines and clones after a 10-min incubation at 37°C. C, Graphical representation of the percent maximum CD4ϩtetramerϩ cells measured when cells were stained at 37°C (upper chart) and 23°C (lower chart) for the indicated time periods. 6338 ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4ϩ T CELLS tetramers at 4°C (12, 21, 25). By contrast, we stained CD4ϩ T cell of HLA-DR1-restricted, HIV p24-specific Ox97-clone10 stained lines and clones with tetramer at 4°C (Fig. 4). The HIV p24-spe- with tetramer at 37°C or in azide buffer on ice. When cells were cific cell lines Ox24-p24.17 and K37-p24.4 and Ox97-clone10 labeled at 37°C for 60 min, clear PE-clusters were visible that were could be stained at 4 and at 0°C. Successful HLA class II tetramer not associated with the cell membrane when the mid-cell z-section staining was also seen when the TT-specific cell line JF-TT830 of PE-fluorescence and differential interference contrast (DIC) im- and the influenza-specific cell line JF-HA were incubated with ages were overlaid (Fig. 6). These clusters typically localized to their cognate tetramers at 4°C. Staining at 4°C was markedly compartments within the cytoplasm, suggesting internalized TCR/ slower than at 23 or 37°C and resulted in considerably lower flu- tetramers. In contrast, when azide-treated cells were stained on ice, orescence intensities (Fig. 4C). ϩ PE-clusters of lower fluorescence intensity than those seen at 37°C Successful staining at 4°C suggests that CD4 T cells can be were only visible along the outer perimeter of the cells. These labeled with surface-bound tetramer and that this process does not clusters aligned with the plasma membrane when the DIC and require receptor internalization. To assess this further, we stained mid-cell z-section of PE-fluorescence images were overlaid, sug- prechilled, azide-treated cells with HLA class II tetramer in azide gesting that visible tetramers were membrane-bound and not buffer on ice. Following removal of unbound tetramer and block- internalized. ing of tetramer rebinding, the decay of cell surface-bound tetramer was followed by monitoring the decrease of the PE-fluorescence. ϩ Azide-treated CD4 T cells from the HIV-Gag p24-specific Ox97- Sensitive detection of rare CD4ϩ T cells clone10 and JF-TT830 were labeled with HLA class II tetramer on ϩ ice (Fig. 5 and not shown). When rebinding of unbound tetramer The frequencies of most CD4 Th cell populations are markedly Downloaded from ϩ was blocked with anti-HLA-DR Ab, the fluorescence of tetramer- lower than those seen for CD8 T cells (36). Because the lower labeled cells fell, suggesting tetramer dissociation from the cell detection limit of flow cytometric techniques is ϳ0.02% (17, 37, ϩ surface. This effect was seen to a lesser degree when no competitor 38), confident detection of many Ag-specific CD4 T cell re- Ab was added (Fig. 5). sponses with conventional HLA class II tetramer staining and flow To confirm that HLA class II tetramer can bind CD4ϩ T cells cytometry is impossible. Recently, the use of HLA class I and II

without being internalized, we performed ﬂuorescence microscopy tetramers and a magnetic bead enrichment technique has improved http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Staining of cell lines and clones with HLA class II tetramer at 4°C. A, FACS density plots showing staining of cell lines for 120 min at 4°C. B, Histograms of the DR1-restricted CD4ϩ clones Ox97-clone10 and HA1.7 showing staining for 120 min at 4°C. C, Graphical representation of the percent CD4ϩtetramerϩ cells (upper chart) and the MFI (lower chart) when cells were stained at 4°C for the indicated time periods expressed as a percentage of the maximum recorded at 37°C. The Journal of Immunology 6339 Downloaded from

FIGURE 5. HLA class II tetramer can dissociate from the cell surface. http://www.jimmunol.org/ Azide-treated JF-TTox cells were stained with the TT830-DR1 tetramer in ice. The fluorescence of surface-bound tetramer was then measured 1, 2, 5, FIGURE 6. 10, 20, and 30 min later in the presence or absence of a competitor Ab HLA class II tetramers are internalized at 37°C but remain A (anti-HLA-DR). A, FACS density plots showing TT830-DR1 staining of surface bound at 0°C. , FACS histogram representing the fluorescence the JF-TTox cell line 30 min after the competitor was added. The percent- intensity seen when Ox97-clone10 cells were stained with p24.17-DR1 ϩ ϩ B age of CD4 tetramer cells is indicated in the top right quadrant of each tetramer for 60 min at 37°C or on ice in the presence of azide. , Fluo- plot. B, Graphical representation of the percent CD4ϩtetramerϩ cells de- rescence microscopy of Ox97-clone10 cells stained with the PE-labeled tected at each time point. p24.17-DR1 tetramer at 37°C or on ice in the presence of azide. The DIC and mid-cell z section of PE-fluorescence recordings as well as an overlay of the two are shown. Images were recorded at a ϫ63 magnification. by guest on September 24, 2021 the sensitivity of detection considerably (18–20, 37). This technique enriches the cells of interest and reduces background stain- Direct ex vivo HLA class II tetramer staining of Ag-specific ϩ ing by ensuring that only cell surface-stained cells are enriched CD4 cells from PBMC at frequencies that fall below the typical (thereby eliminating nonspecific tetramer internalization). We pre- threshold of detection (0.02%) are shown in Fig. 8. Although a few ϩ ϩ viously demonstrated that combination of these techniques yielded CD4 tetramer cells can be identified by normal flow cytometric a linear enrichment recovery from human PBMC (20). The appli- detection, the very low frequency does not allow confident quan- cation of the optimized tetramer labeling techniques developed in tification of such responses. However, after magnetic bead enrich- ϩ ϩ this report to the study of CD4ϩ T cell populations directly ex vivo ment, the number and proportion of CD4 tetramer cells is in- may allow HLA class II tetramers to become more useful. To creased, greatly enhancing the detection sensitivity and reducing optimize the combination of HLA class II tetramer staining and background staining (Fig. 8A). We performed cross-sectional magnetic bead enrichment, a balance must be met between obtain- staining of HIV-infected individuals with the HIV-specific tetram- ing tetramer staining intense enough to allow discrimination be- ers p24.4-DR4, p24.14-DR1, and p24.17-DR1, as well as the ϩ ϩ tween tetramer-positive and -negative populations. Internalization CMV-specific pp65-DR1 tetramer. Patients with CD4 tetramer of tetramers must also be prevented so that magnetic beads can T cell responses Ͻ0.02% were studied (Fig. 8). Tetramer staining bind to cell surface-associated tetramers. To address this, we com- combined with magnetic bead enrichment detected Ag-specific ϩ pared the detection of Ag-specific CD4ϩ T cells by HLA class II CD4 responses reliably at frequencies as low as 0.0004% (1 in ϩ tetramer staining and magnetic bead enrichment with two meth- 250,000 CD4 cells; Fig. 8B). No HLA class II tetramer staining ods: staining at 23°C for 120 min and 37°C for 20 min. The mean of HLA-mismatched or HLA-matched, HIV-negative and CMV- and SEs obtained for triplicate stainings at two defined spike fre- negative control PBMCs was seen with the p24.17-DR1 and quencies (0.8 and 1.6%) before and after magnetic bead enrich- p24.4-DR4 or pp65-DR1 tetramers, respectively (data not shown ment are shown in Fig. 7. The mean pre- (Fig. 7A) and posten- and Ref. 20). ϩ ϩ richment (B) CD4 tetramer frequencies obtained with the ϩ different staining protocols were virtually identical. When the en- Phenotypic analysis of virus-specific CD4 T cells richment recovery was determined (expected postenrichment fre- Memory virus-specific CD4ϩ T cells have different functions and quency of CD4ϩtetramerϩ cells as a percentage of observed different phenotypes that are thought to be associated with virus postenrichment frequency), the two methods were also indistin- persistence and re-exposure to Ag. In infections such as influenza guishable (Fig. 7C). Notably, the recovery of tetramerϩCD4ϩ cells where virus is cleared from the host, virus-specific CD4ϩ T cells was Ͻ50%, indicating that a considerable number of tetramer- have been shown to predominantly produce IL-2 and display a stained cells are lost during the enrichment process. CCR7ϩ phenotype. In contrast, the persistent Ag exposure seen in 6340 ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4ϩ T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. Comparison of direct tetramer staining and magnetic bead by guest on September 24, 2021 enrichment methods. PBMC were spiked at 0.8 and 1.6% with Ox97- clone10 cells and stained with p24.17-DR1 tetramer for 120 min at 23°C (f) or 20 min at 37°C (Ⅺ). The mean frequency of tetramerϩCD4ϩ cells obtained from three repeat analyses before (A) and after (B) magnetic bead enrichment is shown. Error bars represent SEM. C, The bars represent the mean and SEM of the observed CD4ϩtetramerϩ cell frequency recovered following magnetic bead enrichment expressed as a percentage of the expected frequency calculated from the pre-enrichment sample.

FIGURE 8. Detection of rare Ag-specific CD4ϩ T cell populations with chronic, viremic HIV infection is most often associated with HLA class II tetramer staining and magnetic bead enrichment. A, Repre- CCR7Ϫ HIV-specific CD4ϩ T cells that produce IFN-␥ (39, 40). sentative FACS dot plots showing normal tetramer staining (left plots) and Most studies that have compared phenotypes of virus-specific tetramer staining in combination with magnetic bead enrichment (right CD4ϩ T cells have relied on techniques that identify Ag-specific plots). The percentage of cells falling into the top right quadrant is indicated for each plot. B, Cross-sectional quantification of Ag-specific CD4ϩ cells by the detection of cytokines in response to Ag. These meth- T cell frequencies by HLA class II tetramer staining and magnetic bead ods may fail to detect all of the cells with the defined specificity (5, enrichment from patients for whom normal tetramer staining yielded re- 6). These methods may also alter the phenotype of the specific sponses below the accepted limit for detection (0.02%). The data points in cells during Ag-induced activation (41, 42). By using the sensitive B that correspond to the FACS plots shown in A are demarcated by match- combination of HLA class II tetramer staining and magnetic bead ing shapes. enrichment, we compared the phenotype of rare CD4ϩ T cells specific for HIV p24 Gag, CMV pp65, influenza HA, and HCV by costaining with HLA class II tetramers and surface-marker Abs. and the DR1-restricted, CMV pp65-specific pp65-DR1 tetramer in The phenotypes of CD4ϩ T cells were previously analyzed with HIV-negative individuals (Fig. 9). Memory CD4ϩ T cells of all the DR1-restricted, HIV p24 Gag-specific p24.17-DR1 tetramer in specificities expressed CD28 (HCV NS3- and NS4-specific tet- viremic HIV infection (20); the DR1-restricted, influenza HA-spe- ramer data were not available). CD4ϩ Th cells specific for both cific HA307-DR1 tetramer in resolved influenza infection (19); HIV p24 epitopes (p24.17-DR1 and p24.4-DR4), the influenza HA and the DR4-restricted, HCV nonstructural protein 3- and 4-spe- epitope 307–319, and the HCV NS3 and NS4 epitopes were pre- cific DR4-HCV 1248, 1579, 1770 tetramers in resolved HCV in- dominantly CD27-positive. However, a lower proportion of CMV fection (18). In this study, we measured the expression of CCR7, pp65-specific CD4ϩ Th cells expressed CD27. Interpatient varia- CD27, and CD28 on CD4ϩ T cells with the DR4-restricted, HIV tion of CD27 expression on CMV-specific CD4ϩ T cells (as in- p24 Gag-specific tetramer p24.4-DR4 in viremic HIV infection, dicated by error bars representing SEM) was also markedly higher The Journal of Immunology 6341

class II tetramer staining of Ag-specific CD4ϩ T cells should be rapid. Yet, most studies which report HLA class II tetramer staining used long incubation times (1–5 h) (8–10, 13–17, 21–27, 33). We find efficient and rapid staining of CD4ϩ T cells with HLA class II tetramers. Specifically, incubation at 37°C for 10 min was sufficient to allow complete labeling of all Ag-specific cells. At 23°C, staining was slower, and although some lines and clones were stained after 10 min, complete staining for all cell specificities required 30–60 min. Ag-specific CD8ϩ T cells can also be labeled with HLA class I tetramers at 4°C (30, 35). Moreover, MHCI tetramers bind Ag- specific T cells independent of a cellular response and do not require an active cellular process (4, 43, 44). However, little or no HLA class II tetramer staining of CD4ϩ T cell clones was observed at 4°C in previous studies (12, 21, 25). These findings led to the hypothesis that HLA class II tetramer staining is dependent on active cellular processes. This was supported by experiments that showed blockade of tetramer staining of the HA1.7 clone

when endocytosis and cytoskeletal rearrangements were disrupted Downloaded from (25). In contrast to these studies, we demonstrate successful staining of the TT-, influenza HA-, and HIV Gag-specific cell lines and the HIV Gag-specific Ox97-clone10 at 4°C. The JF-TTox cell line and Ox97-clone10 were also labeled with tetramer in the presence of azide on ice as detected by flow cytometry and wide-field mi-

croscopy. Furthermore, when the JF-TTox cell line and Ox97- http://www.jimmunol.org/ clone10 were prestained with HLA class II tetramer, PE-fluorescence decreased upon addition of the competitor Ab anti-HLA- DR, suggesting dissociation of cell surface-bound tetramer. These ϩ FIGURE 9. Phenotypic analysis of rare virus-specific CD4 T cells de- results show that HLA class II tetramer can be surface-bound and tected with HLA class II tetramers. PBMC were stained ex vivo with HLA that active cellular processes such as TCR/pMHCII internalization class II tetramer and surface marker Abs and subjected to magnetic bead are not an absolute requirement for HLA class II tetramer staining enrichment. A, Representative postenrichment FACS dot plots showing of Ag-specific CD4ϩ T cells. costaining of PBMC with anti-CD27 Ab and the HLA class II tetramers Fluorescence microscopy showed a clear accumulation of PE-

p24.4-DR4 (left plot) and pp65-DR1 (right plot). The percentage of by guest on September 24, 2021 CD4ϩtetramerϩ cells expressing CD27 is indicated in parentheses in the conjugated HLA class II tetramer clusters that are internalized at upper right quadrant. B, Phenotypic profile of tetramer-stained CD4ϩ T 37°C. This finding is in agreement with Cameron et al. (25), who cells specific for different viruses. The number of subjects analyzed (n) and reported tetramer clusters that colocalized with endocytic compart- infection state are indicated for each HLA class II tetramer. Data for the ments. At 4°C or on ice, bound tetramer remained on the cell p24.17-DR1, HA307-DR1, and HCV NS3,4-DR4 tetramers have been surface. These surface-bound tetramers clustered. This suggested published previously (18–20). Error bars represent SEM. Statistical dif- that the TCRs were in close apposition before the tetramers were ferences were calculated with the Mann-Whitney U test, and only signif- added. This is in agreement with previous work, demonstrating icant differences are shown. nd, Not done. that TCRs are asymmetrically localized to distinct regions of the plasma membrane that contain the highest concentration of lipid rafts (45). than that measured for other specificities and surface markers (Fig. We were unable to completely stain some CD4ϩ T cells at 4°C. 9). A comparison of CCR7 expression on tetramer-positive cells Specifically, the HA-specific clone HA1.7 could only be partially ϩ revealed significant differences. Virtually all HA-specific CD4 T stained and the CMV-specific line LA-pp65 could not be stained. cells from individuals with cleared influenza infection and HCV- These findings are in accordance with previous studies (15, 25, specific Th cells from patients with resolved HCV infection were 27). The differential staining at low temperatures may be due to ϩ ϩ CCR7 . In contrast, HIV-specific CD4 Th cells in persistently differences in the binding affinities of the TCR/pMHC complexes infected patients expressed significantly lower levels of CCR7. as suggested by Reichstetter et al. (27). Data supporting the hy- ϩ Approximately 30% of CMV-specific CD4 T cells expressed pothesis that only “high”-affinity TCRs are bound by tetramers was CCR7 (Fig. 9). published by Falta et al. (46). They reported a strong correlation between functional avidity and intensity of HLA class II tetramer Discussion staining in human cartilage-specific T cell hybridomas. In this study, we optimized the conditions required to label Ag- Direct flow-cytometric detection of cell populations is too in- specific CD4ϩ T cells with HLA class II tetramers and developed sensitive to reliably identify cell frequencies of 0.02% or lower techniques for detection of HLA class II staining in direct ex vivo (17, 37, 38). HLA class II-restricted Ag-specific CD4ϩ T cells are samples. Many investigators have reported HLA class II tetramer- rare, so confident detection of these cells with conventional HLA staining methods that differ substantially from those used to stain class II tetramer staining and flow cytometry is impossible. When HLA class I-restricted CD8ϩ T cells. CD8ϩ T cells can readily be HLA class I or II tetramer staining is combined with a magnetic labeled with HLA class I tetramers by incubation with them at bead enrichment technique, the sensitivity of detection is consid- 37°C for 5–10 min as shown by FACS analysis and confocal mi- erably improved (18–20, 37). This advance has made possible the croscopy (29, 30, 35). Because the association rates of tetrameric study of very rare Ag-specific cells without reliance on functional TCR/pMHCI and TCR/pMHCII interactions are similar (28), HLA readouts or cellular responses that may detect only a subset of 6342 ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4ϩ T CELLS cells. We visualized HIV-specific and CMV-specific CD4ϩ T cells fined, so the utility of HLA class II tetramers is expanding. This that were undetectable with direct tetramer staining when we com- technique will advance our knowledge of the Th immune response. bined tetramers with magnetic bead enrichment. This technique This form of immunity can be critical as a means of curbing in- was sufficiently sensitive to detect reliably 1 in 250,000 CD4ϩ fections but remains poorly defined. It is certainly part of antiviral cells, corresponding to a frequency in PBMC populations of al- immune control and should be evaluated when protective immu- most 1 in a million). Previous experiments also demonstrated that nity is the aim of vaccination. this technique offered a quantitative readout when a range of low cell frequencies were stained (20). The enrichment technique also Acknowledgments reduces background staining. However, this may reduce detection We thank Jonathan Lamb for the HA1.7 clone. of low-avidity cells, because the HLA class II tetramer may not adhere strongly enough to remain bound after magnetic bead en- Disclosures richment. Evidence for such an effect is suggested by the posten- The authors have no financial conflict of interest. richment preference for cells with high fluorescence intensity, which probably represent high-avidity T cells. References 1. Sykulev, Y., A. Brunmark, M. Jackson, R. J. Cohen, P. A. Peterson, and We then used this sensitive technique to compare the pheno- H. N. Eisen. 1994. Kinetics and affinity of reactions between an antigen-specific types of Ag-specific Th cells with different Ag exposure in viral T cell receptor and peptide-MHC complexes. Immunity 1: 15–22. infections of varying persistence. These analyses might, in princi- 2. Matsui, K., J. J. Boniface, P. Steffner, P. A. Reay, and M. M. Davis. 1994. Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the ple, allow further insights into the function, survival, and in vivo dissociation rate with T-cell responsiveness. Proc. Natl. Acad. Sci. USA 91: recirculation patterns of different antiviral T cell populations. The 12862–12866. Downloaded from proportion of HLA class II tetramer-positive cells that expressed 3. Corr, M., A. E. Slanetz, L. F. Boyd, M. T. Jelonek, S. Khilko, B. K. al-Ramadi, Y. S. Kim, S. E. Maher, A. L. Bothwell, and D. H. Margulies. 1994. T cell the surface markers CCR7, CD27, and CD28 was measured on receptor-MHC class I peptide interactions: affinity, kinetics, and specificity. Sci- HIV-specific CD4ϩ cells in viremic HIV infection and CMV-spe- ence 265: 946–949. cific CD4ϩ cells in HIV-negative individuals. These characteris- 4. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, ϩ M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, and M. M. 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