Flow cytometric enumeration of antigen‐specific T lymphocytes (2004)

Until the second half of the 1990s, quantification of MHC-restricted, peptide-specific T lymphocytes required cell culture–based techniques of mononuclear cell suspensions that were often laborious, cumbersome, and prone to bias towards cellular subsets with growth advantage. Initially, limiting dilution assays were used. These assays include multiple days of culturing T lymphocytes in the presence of antigen and antigen-presenting cells, and the functional read-outs produced were T-cell proliferation (1) or cytolytic activity (2). However, such assays may not accurately reflect the function and frequency of T cells in vivo, due to selective proliferation and apoptosis that occur over time in culture. Alternatively, assays for T-cell activation have been developed based on the upregulation of the early activation marker CD69 (3) and/or the production of cytokines upon specific stimulation. The cytokines thus being secreted by the T cells can be measured in the culture supernatant by ELISA (4, 5), or, after immobilization, on nitrocellulose plates coated with anti-cytokine mAbs (ELISPOT) (6, 7). The advent of three-color flow cytometry enabled the direct visualization of cytokine production by individual cells (8). Visualization was achieved either intracellularly (8, 9) or on the cell surface following retention of secreted cytokine in an affinity matrix allowing for subsequent staining with a fluorochrome-labeled mAb (10). This affinity matrix was later replaced with a bispecific mAb, specific for a cell surface marker (e.g., CD45) on the one hand, and for cytokine on the other (11). Alternatively, amplification of the fluorescence signal by fluorescent liposomes was shown to allow detection of surface-expressed IFN-γ on IFN-γ–producing cells (12). Among the flow cytometric techniques, the first approach, termed cytokine flow cytometry (CFC), is currently the most widely used. In all these functional assays, single peptides (13), mixtures of peptides (14), or more complex molecules (e.g., whole proteins or mixtures of proteins) (9), can be used as an antigen source. An alternative approach for the direct visualization of antigen-specific T cells has been established by the construction of multimers consisting of recombinant MHC molecules folded with the appropriate peptides. The low affinity and fast off-rates of MHC-peptide ligands to the T-cell receptor (15) precluded the direct detection of MHC-restricted, antigen-specific T cells using recombinant MHC-peptide monomers. This problem was overcome by the construction of soluble MHC-peptide multimers that were able to bind specifically to the TCR with higher affinity than the sum of the single monomeric affinities. Initially, tetrameric complexes of Class I MHC-peptide monomers were constructed (16), followed by the development of Class II MHC-peptide tetramers (17). Similar approaches have been taken to detect other types of antigen-specific lymphocytes such as NK cell subsets (18), NK-T lymphocytes (19, 20) and TCR-γδ+ T-cell subsets (21). Alternatively, MHC dimers have been developed, which are MHC-immunoglobulin fusion proteins that bind to antigen-specific T cells via the bivalent nature of the two binding sites available to interact with the TCR (22). Here, we discuss the principles and practice, and summarize clinical applications and relevance of antigen-specific T-cell enumeration using MHC-peptide tetramers, CFC, and a combination of both techniques. Extensive reviews of the methodological aspects of these assays and their performance in relation to other assays for detection of antigen-specific T lymphocytes have been published elsewhere (23, 24). For flow cytometric enumeration of antigen-specific T lymphocytes using tetramer technology, the HLA type of the test individual and the specific peptide epitope(s) to be studied must be known in advance. The Class I MHC transmembrane protein consists of a single heavy chain that contains the complete peptide-binding groove and is stable in soluble form when complexed with its essentially invariant light chain, β2-microglobulin (16). The β2-microglobulin and the recombinant Class I MHC heavy chain, on the COOH terminus of which a recognition tag for the BirA enzyme has been engineered, are synthesized using the Escherichia coli expression system. Following purification, the β2-microglobulin and heavy chains are then refolded in molar excess of the appropriate 8- to 10-mer peptide. Subsequently, the peptide-loaded Class I MHC monomers are connected by the addition of fluorochrome-labeled streptavidin; its four biotin-binding sites result in the formation of tetrameric complexes. For the enumeration of MHC-restricted, antigen-specific T cells, PE is commonly preferred for its high signal to noise ratio; alternatively, FITC or APC are used. For flow cytometric enumeration of Class I MHC-restricted CD8+ T cells, PBMC suspensions have been most commonly used, while bulk-lysed leukocyte suspensions can also be used (25), as well as the stain-lyse-wash method. Typically, tetramers are counterstained with CD8, after which lymphocytes are selected by gating on FSC and SSC, and the Class I MHC-restricted T cells are identified within the CD8bright subset of lymphocytes (16). As tetramers tend to bind nonspecifically to other cell types such as monocytes, we prefer to include CD3 in this assay in order to allow optimal identification of the T cells on the basis of SSC versus CD3 gating (26). Using this “T-gating” approach, CD3−,8dim NK lymphocytes can be excluded from analysis, providing more reliable assessment of the percentage of Class I MHC-restricted T cells as a fraction of the CD8+ T cells (25). Alternatively, cells other than CD8+ T lymphocytes can be excluded, by staining them with a mAb cocktail containing, e.g., CD4 (T-helper cells), CD14 (monocytes), CD16 (NK cells), and CD19 mAb (B cells) labeled with the same fluorochrome (so-called “dump gating”) (27). An example of the T-gating-based analysis is shown in Figure 1. Absolute numbers of Class I MHC-restricted, antigen-specific CD8+ T cells are subsequently calculated from the percentage within the CD8+ T cells and the simultaneously-obtained absolute CD8+ T-cell count (28, 29). Enumeration of CD8+ T lymphocytes specific for the CMV pp65-encoded peptide TPRVTGGGAM presented by HLA-B*0702. The subset of CD3+,8+ T cells are depicted in green, other CD3+ T cells are in red, and cells other than CD3+ T cells are in gray. Inadvertently acquired air bubbles are excluded by selecting only list mode data with a stable rate of events per second (time gate G4, not shown). T lymphocytes are identified by region R1 as CD3+ events with low sideward light scatter (A). Plots B and C serve to verify the positions of the total CD8+ T-cell population and the tetrameter-binding CD8+ cells, respectively. Among the CD3+ T lymphocytes, the CD8+ T cells are selected by region R2 (D). Events with low and high forward light scatter signals relative to the major CD8+ T-cell cluster, representing dead cells and cell aggregates, respectively, are excluded by placing region R3 as shown in (E). Within the viable single CD8+ T cells (gate G3), the percentage of tetramer-binding cells is assessed, while quadrant statistics allow the analysis of coexpression of a fourth marker by tetramer-binding CD8+ T cells (F). In this sample, the CD8+ subset constituted 68% of the CD3+ T cells; 8.1% of the CD8+ T cells were specific for TPRVTGGGAM presented by B*0702; and 69% of the latter cells coexpressed CD57. By adapting this technique for intracellular staining, coexpression of intracellularly expressed markers such as granzyme A or perforin by the tetramer-binding CD8+ T cells can be assessed (30). Depending on the pathogen or disease under study, the phase of the disease (e.g., acute versus chronic infection) or the timing relative to the clinical intervention (e.g., prior to or post vaccination) and the sampling site (e.g., peripheral blood versus biopsy), the proportions of antigen-specific CD8+ T cells as measured using Class I MHC-peptide tetramers may range from undetectable to several tens of percentages of total CD8+ T cells. The detection limit may vary between 0.01% and 0.1% of CD8+ T cells, depending on the method for sample processing, the tetramer used, and the number of CD8+ T cells acquired (23, 25). The functional status of the tetramer-binding CD8+ T cells can be assessed by stimulating the tetramer-labeled lymphocytes with the corresponding peptide and measuring intracellular cytokine production, as described below (30). The procedure for staining T lymphocytes with MHC dimers is very similar to that for MHC tetramers, as summarized above (22). The construction of Class II MHC-peptide tetramers has been technically more demanding, and successful use of these reagents was not shown until late 1999 (16). Class II MHC molecules are heterodimers of α and β chains. For the production of recombinant α and β chains, insect cells such as Drosophila S2 are used (16, 26). The α and β chains are cloned in separate expression vectors; both chains are extended with leucine zipper motifs and flexible linkers. Furthermore, a biotinylation site is added to the COOH terminus of the β chain. Following purification, biotinylation is performed. The leucine zippers allow stable pairing of the soluble α and β chains, allowing loading with the appropriate peptide. The flexible linkers prevent steric hindrance when the αβ heterodimers are complexed to the fluorochrome-linked streptavidin molecule. The use of Class II MHC-peptide tetramers to detect and monitor antigen-specific CD4+ T cells is less straightforward than the use of Class I tetramers for CD8+ T cells (27). CD4+ T cells specific for particular antigens, as detected by Class II tetramers, typically circulate at very low frequencies (i.e., <0.01% of CD4+ T cells). Such low frequencies are below the detection limit of flow cytometry (see above). In order to allow the detection of these low frequency CD4+ T cells, these T cells are stimulated with the study antigen so that following their proliferation, their frequencies are higher and their detection is facilitated (17). The incorporation of carboxyfluorescein diacetate succinimidyl ester, a fluorescent dye, into the cell surface membrane prior to culture allows the quantitation of the number of cell divisions (i.e., up to 10) during these expansion cultures. In this way, the original frequency of antigen-specific CD4+ T cells—which are capable of dividing—in the original sample can be extrapolated (17). CFC is based on the detection of intracellular cytokines with fluorochrome-conjugated mAb that occurs as a consequence of short-term stimulation of leukocytes with recall antigens (e.g., virus- or tumor-derived peptides or proteins) and polyclonal activators such as mitogens or inductors of intracellular signal cascades (i.e., phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Normally, unstimulated leukocytes produce little or no cytokines. PBMC suspensions or whole blood are typically used for this assay (31-33). An incubation time of 6 h is sufficient for inducing cytokine production by T cells using polyclonal activators and peptides. The peptides must have the appropriate format for direct loading of Class I and II MHC molecules in order to achieve efficient activation of CD8+ and CD4+ T cells, respectively. In contrast, whole proteins need to be taken up by antigen-processing cells for endosomal processing (34); the resulting peptides are mainly presented by Class II MHC molecules to CD4+ T cells. However, in a minority of individuals some proteins may enter the endogenous processing pathway so that the derived peptides are presented by Class I MHC molecules to CD8+ T cells (35, 36). However, T cells are much less efficiently stimulated via this route of presentation than via exogenously loaded peptides (36). Consequently, the use of complete proteins is recommended for stimulating CD4+ T cells, but not CD8+ T cells. In order to let the cytokines accumulate in the stimulated cells, protein transport inhibitors such as brefeldin A or monensin are added after 2 h of incubation. Following stimulation, the leukocytes are collected, washed, and fixed in paraformaldehyde. When whole blood has been used, simultaneous red cell lysis and fixation can be achieved by the use of FACS Lysing Solution (BD Biosciences, San Jose, CA). Subsequently, the cell membranes are permeabilized using mild nonionic detergents to allow intracellular staining. Staining is then performed using conjugated mAbs that have been selected and formulated to react with antigens in fixed and permeabilized cells. As with the tetramers, we recommend the use of CD3 to allow thresholding on total T cells during data acquisition, combined with the use of CD4 and CD8 to identify the helper and cytotoxic T cells, respectively. Alternatively, dump gating may be used to exclude unwanted cell populations from analysis (33). The third or fourth color is then used for cytokine detection. The analytical procedure of a sample stained with CD3, CD4, CD8 and anti-IFN-γ or mouse IgG1 isotype control mAb is shown in Figure 2. Cytokines are commonly counterstained with CD69 mAb in order to visualize the cytokine-producing cells as a 2D cluster (31-33). However, as we have rarely, if at all, observed CD69−,IFN-γ+ T cells after peptide or protein stimulations, we consider the use of CD69 staining redundant. Similarly, the use of isotype control mAb is controversial (37, 38), in particular when a negative procedure control (see below) is included that frequently yields similar information (T Bunde and F Kern, unpublished results). If an isotype control mAb staining is used, it must be appropriately matched and formulated, i.e., it should approximate the anti-cytokine mAb as much as possible (i.e., same concentration and fluorochrome-to-protein ratio), except for the very antigen binding portion (i.e., the paratope). Enumeration of CD4+ and CD8+ T lymphocytes producing IFN-γ after 6 h stimulation with a pool of overlapping 15-mer peptides spanning the CMV pp65 protein. In (A–C), CD4+ T cells are depicted in blue; among them, IFN-γ+ cells are highlighted in dark blue. CD8+ T cells are depicted in red; among them, IFN-γ+ cells are highlighted in violet. Other CD3+ T cells are shown in green; other cells than CD3+ T lymphocytes are in gray. During data acquisition, a generous threshold is set on CD3 PerCP in order to exclude most CD3− events; this threshold should not be set too tight on the CD3+ population to avoid exclusion of CD3dim T cells that have downregulated CD3 as a consequence of their activation. During list mode data analysis, viable T cells are identified by region R1 as CD3+ events with low sideward light scatter (A) combined with exclusion of dead cells with low forward light scatter (region R2) (B). The gated viable T cells are shown in (C); region R3 selects the CD4+ T cells and region R4 selects the CD8+ T cells. The reactivities of CD4+ and CD8+ T cells with the IgG1 isotype control mAb are shown in (D) and (E), and the reactivities of these subsets with the anti-IFN-γ mAb are shown in (F) and (G). The markers discriminating positive from negative events are placed on the isotype control histograms, and the proportions of cells exceeding these thresholds are subtracted from the respective percentages of IFN-γ+ cells. In this example, 0.65% of CD4+ T cells and 6.48% of CD8+ T cells are IFN-γ+. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinyl chlorophyllin. As with any functional negative and positive procedure are The negative control consists of the same blood sample stimulated with e.g., a from as control for cytokine production in is typically low (i.e., of CD4+ or CD8+ T cells) but it may be higher in with T cells. very low frequencies of antigen-specific T cells can be detected if a list mode data set is (23, The of the combination of Class I MHC-peptide tetramers, CFC, and these techniques, has and the analysis of CD8+ T cells. In with a between the of CD4+ and CD8+ T cells and was In on CD8+ T-cell in the of but when the was efficiently by the that the presence of antigens is to the proliferation of CD8+ T cells However, high of CD8+ T cells were to in to and in the combined use of tetramer staining and CFC that proportions of CD8+ T cells were as by the of these cells to accumulate IFN-γ after stimulation with the peptide An study the of CD4+ T cells in this being more in than in to the of CD4+ T cells was with a in functional (i.e., CD8+ T cells, of and to to the Class I MHC tetramers have been used to detect and monitor CD8+ T cells in and with CMV and In with and in such as and blood cell CMV and may and if not to CMV disease and The use of CFC the detection of CD4+ T cells in the blood of CMV their frequency was higher in than in individuals in T cells over time and in higher numbers than T cells specific for or The combined of these that CMV a and to the of its of and CD8+ T cells have their in under for both a combination of of and CD8+ T-cell the identification of at high for and disease The combination of tetramer staining with CFC CD8+ T cells (i.e., upon stimulation with the in (i.e., for acute the of these CD8+ T cells was with CMV By the same to were observed in with CD4+ T cell and (i.e., CD8+ T cells The of CD4+ T cells was in and In the first of CMV disease developed in CD4+ T cells, but not in with CD4+ T cells, while these cells in number after Furthermore, numbers of CD8+ T cells only in the of with CD4+ T cells the of CD4+ and CD8+ T cells was required for the of CMV in while the of these cells in at was with the development of late CMV disease As for CD8+ T cells specific for B or C were to during the acute phase of after its CD8+ T cells at higher in than in The summarized above have shown a between and the of their functional T cells of both CD4+ and CD8+ subsets are required to these under using Class I MHC tetramers and CFC to detect and monitor CD8+ T cells have been performed in with the detection and of CD8+ T cells from but not from such In these CD8+ T cells were not only in the of with but also in with an disease by the of and in individuals Other proteins specific CD8+ T-cell in are and the proteins Class I MHC tetramers are also to monitor specific CD8+ T-cell after CD8+ T cells, upon and can high of specific cytolytic activity cells in their in presence in is not with of these and functional of the CD8+ T cells their within and between a this was in individuals expressed an a or and were able to appropriately matched cells or to IFN-γ in to these cells The relative proportions of these subsets among CD8+ T cells in may vary over time one for the presence of CD8+ T cells in the of of be the of these T cells, e.g., due to the of the or of appropriate molecules on the cells. However, in this little if is known the of CD4+ T cells to and CD8+ T-cell CFC has also been used to in other CD8+ T-cell peptides derived from or both by was observed in of with of with using the peptide-specific CD8+ T cell were CD4+ T-cell were observed in with multiple after using cells with The use of Class II MHC-peptide tetramers has been to detect and monitor Class antigen-specific CD4+ T cells in and and and after The range of Class II MHC and the assay required to detect Class antigen-specific CD4+ T cells (see has thus their cytometric detection of individual antigen-specific T lymphocytes has and to has much into the between and and their major approaches i.e., MHC multimers (i.e., currently mainly Class I HLA and cytokine flow techniques can be used to monitor and antigen-specific T cells in a clinical can be combined to For detection and of antigen-specific T cells in a clinical Class I HLA tetramers have and the combination of Class I HLA tetramer staining and stimulation with the peptide in a single assay yields information on the functional status of the CD8+ T cells, which is in the The of Class I HLA tetramer staining are assay can be in 2 and and of are A in clinical is that the HLA type must be as well as a T-cell presented in the respective e.g., of T-cell a particular or are by the that the currently available HLA molecules only a minority of all Class I HLA and that peptides are not known for HLA it is to the CD8+ T-cell a protein by Class I HLA tetramers these reagents of the CD8+ T-cell which have thus very as the number of of Class I HLA tetramers is very and are high per flow cytometry is less than Class I HLA tetramer the test h if cells are stimulated with antigen for 6 The is and are (33). When peptide are used as of the HLA type only to be known in when of the T-cell have to be When using of 15-mer peptides that other by both CD4+ and CD8+ T-cell can be in a single this approach a much more of the T-cell specific proteins than Class I HLA of the of the studied protein must be and in peptide must be to This can be very depending on the of the protein and the synthesized per peptide. the peptides are peptide can be and at a per test in the range of Class I HLA tetramers and cytokine flow in combination with for cell have to T lymphocytes for CD8+ T cells have been using Class I HLA tetramers from the peripheral blood of and to with CMV in order to them of this T cells derived from have also been to with CMV not to using a cytokine assay flow cytometric detection of individual antigen-specific T cells has not only for but also for