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Ex vivo analysis of T-cell function
Ex vivo analysis of T-cell function Maria A Suni, Vernon C Maino and Holden T Maecker Our ability to analyze T-cell function in vitro has progressed in recent years to include analysis of early signaling events, such as specific protein phosphorylation, intermediate functions, such as degranulation and cytokine production, and later functions, such as proliferation. Many assays are now available to monitor these events, and comparative studies of some of these assays have been published. Major recent developments in this area include the ability to measure T-cell degranulation via cell surface exposure of CD107 and the use of polychromatic flow cytometry to examine multiple phenotypes and functions of responding T cells. Addresses Becton, Dickinson and Company Biosciences, 2350 Qume Drive, San Jose, California 95131, USA Corresponding author: Maecker, Holden T ([email protected])
Current Opinion in Immunology 2005, 17:434–440 This review comes from a themed issue on Immunological techniques Edited by Daniel Speiser Available online 13th June 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.05.002
Introduction CD4+ and CD8+ T cells possess a variety of overlapping functions that are modulated by signals from their environment (antigen, co-stimulation, cytokines) as well as by the stimulation history of the cells. T-cell responses can be roughly grouped into early, intermediate and late functions (Figure 1). Early functions include intracellular calcium flux and phosphorylation of key signaling proteins on serine, threonine, or tyrosine residues. Intermediate functions include degranulation, cytotoxicity and cytokine production. The array of cytokines produced by T cells is a function of their differentiation and can include IL-2 as well as Th1 cytokines (e.g. IFNg, TNF-a), Th2 cytokines (e.g. IL-4, IL-5, IL-13), or regulatory cytokines (e.g. TGF-b, IL-10). Late functions include proliferation, as well as apoptosis or activationinduced cell death. Many assays are available for ex vivo monitoring of T-cell function. Calcium flux can be detected fluorometrically as well as by flow cytometry using calcium-sensitive dyes. Phosphorylation can be measured using western blotting, Current Opinion in Immunology 2005, 17:434–440
and more recently using phosphospecific antibodies in flow cytometry [1]. Degranulation can be measured using a novel flow cytometry assay detecting the cellsurface expression of CD107 from cytotoxic granules [2]. Classic cytotoxicity assays measure cell lysis via release of 51 Cr from labeled target cells; modifications of this method have been developed to measure the loss of dye-labeled target cells by flow cytometry [3]. Cytokines can be quantitated by a wide variety of techniques. These include bulk culture assays (ELISA for cytokine protein, and PCR or RNase protection for cytokine mRNA) and single-cell assays (ELISPOT and cytokine flow cytometry). Proliferation can be measured by uptake of 3Hthymidine or bromodeoxyuridine (BrdU), or by dilution of a dye such as 5(6)-carboxylfluorescein diacetate succinimidyl ester (CFSE); when using flow cytometry as a readout, these latter two methods can be combined with analyses of cytokine production or differentiation marker expression. Finally, apoptosis via activation-induced cell death can be measured by a variety of methods, but is not within the scope of this review. Functional T-cell responses can be evaluated following stimulation by polyclonal activation using mitogens, such as phorbol myristate acetate (PMA) plus ionomycin, phytohemagglutinin (PHA), anti-CD3 and Staphylococcal enterotoxin B (SEB). A major application of functional assays, however, is the monitoring of antigen-responsive cells, so the ability to detect responses using specific antigenic peptides is often critical. In this respect, great advances have been made in recent years, especially with the advent of single-cell assays such as ELISPOT and cytokine flow cytometry. Because these assays have sufficient sensitivity to detect rare populations of positive cells, it is possible to quantitate responses to specific antigens without in vitro expansion in many clinical settings. In this review, we will examine assays of early, intermediate and late T cell function. Particular attention will be given to novel assays (such as CD107 expression) or technologies that have undergone significant improvement in the past year (such as polychromatic cytokine flow cytometry). The relationship of these developments to clinical findings will be emphasized and reviewed in the context of what they can teach us about T-cell responses in particular diseases or vaccination strategies.
Analyzing early T-cell signaling events Calcium flux
Calcium plays a critical role in several early T-cell functions, including regulation of signal transduction protein www.sciencedirect.com
Ex vivo analysis of T-cell function Suni, Maino and Maecker 435
Figure 1
T cell activation
Early
Intermediate
IL-2
Late
IL-4
CD107 ∗ ∗
∗ ∗∗
IFN-γ
T cell – APC interaction
Ca2+ flux phosphorylation
TNF-α
Degranulation cytokine production
Proliferation apoptosis
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Stages of T cell activation upon interaction of T cells with antigen-presenting cells. Specific activation of T-cell receptors and co-stimulatory molecules on T cells leads to a cascade of signaling events. Early changes can be detected in a matter of seconds-to-minutes; intermediate changes in the order of several hours; and late events after several days. Abbreviations: APC, antigen-presenting cell.
phosphorylation, and also in subsequent events, including cytokine expression and proliferation. Recent studies using cytometric methods have demonstrated a broad variety of Ca2+ signals in T cells, ranging from infrequent spikes to sustained oscillations and plateaus [4]. These events occur asynchronously in T cells within seconds to minutes following engagement of functional receptors. Single cell measurements of increased calcium concentration as a consequence of activation usually rely on calcium-complexing fluorescent dyes, including indo-1 and fluo-3, which can be measured by flow or image cytometry [5,6]. More recently, Tsien and co-workers [7] introduced real-time analysis of Ca2+ in endoplasmic storage vesicles in single cells using calcium dyes and image cytometry. This technology enables the analysis of calcium changes on a subcellular, not just single-cell, level. Phosphorylation of signal transduction proteins
Enhanced phosphorylation of signal transduction proteins is linked to changes in calcium metabolism. Recently, several investigators detected intracellular phosphoprotein alterations in single primary T cells following activation [8–10]. The measurement of specific protein phosphorylation states has been made possible by the recent availability of antibodies with specificities for unique phosphorylated determinants [11,12]. Using flow cytometry, multiparametric analysis can be performed to detect phosphorylation events within unique T-cell subsets following the introduction of functional signals www.sciencedirect.com
[13,14–16]. In this way information can be obtained about the heterogeneity of early events in T-cell activation, which were previously overlooked by traditional bulk analysis methods [14]. Flow cytometric analysis of phosphoproteins has been employed to evaluate both MAP kinase pathways in activated T cells [17,18] and disease state profiling in leukemias and lymphomas [13], and also in autoimmune diseases [19]. Other potential applications include evaluation of pharmacodynamic effects of drugs and immunomodulatory agents and inhibitors [14]. It is important to note that, although flow cytometry is an extremely powerful approach for assessing phosphorylation events in single cells, the permeabilization and staining techniques for detection of intracellular phosphorylated proteins are technically challenging. For the evaluation of each set of phosphodeterminants and cell surface antigens, both the protocol and antibodies must be optimized. A more complete review of this technology has been published recently [15].
Analyzing intermediate T-cell signaling events Degranulation of cytotoxic T cells 51
Cr release assays measure the lysis of target cells, following cytotoxic cell stimulation, but offer no direct information about the identity and frequency of the effector cells that perform the killing. Recently, two new techniques have been developed that directly examCurrent Opinion in Immunology 2005, 17:434–440
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ine the degranulation of CD8+ T cells, one by the flow cytometric measurement of CD107 surface expression, and the other by the detection of secreted granzyme B (GrB) in an ELISPOT assay.
that responded to cytomegalovirus (CMV) or HIV were found to produce IFN-g (MA Suni, et al., unpublished observations; [2]). In these studies, brefeldin A was added in addition to monensin to optimize the detection of IFNg and CD107.
Flow cytometric measurement of CD107 surface expression
Following conjunction of a CD8+ T cell with an appropriate target cell, lytic granules are transported to the point of contact with the target cell and the granule contents are released into the immune synapse between the effector and the target [20–22]. These lytic granules contain perforin and a set of serine proteases called granzymes [20,23,24]. The granule core is surrounded by a lipid bilayer containing lysosomal associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1), CD107b (LAMP-2) and CD63 (LAMP-3) [2,20,25]. These proteins become transiently expressed on the T cell surface upon granule exocytosis (Figure 2; [20,25]). Betts et al. [2,26] measured the mobilization of CD107 to the surface of activated T cells in peripheral blood mononuclear cell (PBMC) cultures stimulated in the presence of the secretion inhibitor monensin. Because of the transient surface expression and rapid internalization of CD107 by the endocytic pathway [25], staining for CD107 is maximized by addition of the antibody during cell stimulation and by the addition of monensin. By virtue of their parallel kinetics, CD107 and intracellular cytokines can be assessed at the same time using 4–6 hours of stimulation. Most, but not all, CD107+ cells
Using the CD107 assay to study degranulation and cytotoxic activity
Because the degranulation of cytotoxic T cells and killing of target cells are correlated, the CD107 mobilization assay can be used as an alternative to 51Cr release assays. Betts et al. [2] demonstrated a good correlation between degranulation and cytotoxic activity of CD8+ T cells, as measured in a flow-cytometry-based killing assay. Rubio et al. [27] also demonstrated a strong correlation between the killing activity of tumor-specific CD8+ T-cell clones, as measured by 51Cr release, and the level of CD107 induction on those cells. One group, however, showed that degranulation and cytotoxicity are not correlated at all stages of T cell differentiation in mice (the previous two references used human cells). Antigen-experienced T cells can be classified according to their expression of various differentiation markers. So-called ‘central memory’ T cells express CCR7, CD62L and CD28, but not CD45RA [28]. These cells are thought to be long-term, renewable precursors of cells that can develop cytotoxic effector function (socalled ‘effector memory’ and ‘effector’ T cells). Wolint et al. [29] showed that effector, central memory, and effector memory CD8+ T cells all have similar kinetics of
Figure 2
Granule membrane expressing CD107a, CD107b and CD63
Cytotoxic granule Granule core with enzymes (e.g. perforin and granzyme B)
Cell membrane
Granule is transported to cell membrane
Degranulation and release of granule contents; cell surface exposure of CD107a, CD107b and CD63 Current Opinion in Immunology
Diagrammatic representation of the principle underlying the CD107 degranulation assay. CD107a, CD107b and CD63 are expressed on the inner leaflet of granule membranes, and are thus transiently exposed on the cell surface upon granule fusion with the plasma membrane. The presence of antibodies to CD107 in the stimulation culture allows for labeling of these transiently exposed molecules. Current Opinion in Immunology 2005, 17:434–440
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Ex vivo analysis of T-cell function Suni, Maino and Maecker 437
degranulation following antigen encounter. Only effector T cells, however, possess strong immediate cytolytic activity against target cells, whereas effector memory cells show low, and central memory cells showed no, cytolytic activity. These findings correlated with the levels of cytolytic effector molecules, such as GrB, in the lytic granules of the various CD8+ T cell subsets. Because the CD107 mobilization assay does not require cell fixation and permeabilization, it can also be used for the sorting of live cells. Rubio et al. [27] sorted cells on this basis, and showed that only a minority of vaccine-elicited melanoma-specific CD8+ T cells possess degranulation activity when stimulated by relevant tumor target cells. This study emphasized the importance of recognition efficiency in T-cell function, as tetramer+ T cells with low recognition efficiency could degranulate in response to target cells pulsed with excess cognate peptide, but not in response to tumor cells (which present much lower concentrations of peptide–MHC). Degranulation and cytokine production are also differentially sensitive to the concentration of stimulating peptide [30]. Both intracellular cytokine expression and CD107 cell surface exposure were observed at high peptide concentrations, whereas at low peptide concentrations degranulation but not cytokine production were detected. Thus, depending upon the level of presented antigen, an effector response might be both cytolytic and cytokineproducing, or only cytolytic. Detection of granzyme B by ELISPOT assay
Recently, investigators have shown good correlation between ELISPOT assays that measure secreted (GrB) and 51Cr release assays [31,32]. The GrB ELISPOT was found to be highly sensitive and required fewer effector cells for accurate assessment of T-cell cytotoxicity. In addition, the GrB ELISPOT provided a direct estimation of cytotoxic T lymphocyte (CTL) frequencies, which is not possible with 51Cr release. Comparisons were also carried out between GrB ELISPOT and IFN-g ELISPOT and, in many cases, these two assays correlated well [31]. By measuring a protein that directly participates in target cell killing, however, the GrB ELISPOT is a more direct assessment of cytotoxic activity [31,33]. Correlations of GrB ELISPOT with CD107 degranulation have not yet been reported. Measurement of cytokine production
The detection of IFN-g production by ELISPOT is standard for demonstrating a cellular immune response to vaccination. In the case of HIV disease, however, recent work suggests that an IFN-g response alone is unlikely to be sufficient for determining protection, and is unlikely to be useful for predicting progression [34]. Rather, the production of IL-2, either alone or in combiwww.sciencedirect.com
nation with IFN-g by antigen-specific CD4+ T cells, correlates with control of viremia and non-progression [35,36]. Proliferation of CD4+ T cells, which is frequently suppressed in HIV+ individuals, can also be restored by exogenous IL-2 [37]. Investigators have begun to use polychromatic flow cytometry (up to 12 colors) to examine antigen-specific immune responses [38]. Using up to four anti-cytokine (macrophage inhibitory protein [MIP]-1b, TNF-a, IFNg and IL-2) antibodies plus anti-CD107, Betts and colleagues have demonstrated a loss of polyfunctional CD8+ T cells in patients with progressive HIV infection (MR Betts et al., personal communication; see Update). Longterm non-progressive HIV patients, however, demonstrated a consistent presence of CD8+ T cells that were polyfunctional; that is, they could produce all four cytokines investigated (MIP-1b, TNF-a, IFN-g and IL-2) and also degranulate in response to antigen. This study demonstrates the power of multiparameter analysis to discriminate between groups of patients in a way that would not be possible using a series of single-cytokine assays. Central memory T cells specific for recall antigens are depleted in chronic HIV infection [39], and the HIVspecific response is largely composed of CD4+ and CD8+ T cells that have an intermediate differentiation state [40,41]. Such work suggests that the phenotype of cytokine-producing T cells also influences their longevity and function, and might be helpful in predicting the course of disease.
Analyzing later T-cell signaling events T-cell proliferation
Methods have been developed using the membraneassociated fluorescent dye CFSE to detect dividing cells by flow cytometry through reduction in CFSE intensity [42,43]. This technique enables the visualization of six or more discrete cycles of cell division by flow cytometry, both in vitro and in vivo. It is compatible with detection of multiple cell-surface and intracellular markers on the proliferating cells [35,44], as well as with live-cell sorting of proliferating populations. The technique has been used in immune monitoring of vaccine potency [44] and in monitoring immune competence during disease progression [35,45–47]. In addition, Dion et al. [46] combined a biomarker assay to detect T-cell receptor excision circles with CFSE to demonstrate that HIV infection results in a substantial reduction in intrathymic proliferation.
Increasing assay standardization and throughput As the number and size of immunomonitoring studies increases, issues of standardization and throughput are becoming increasingly apparent. One methodology for Current Opinion in Immunology 2005, 17:434–440
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Update
Figure 3
PBMC or whole blood
Plate with lyophilized peptides + brefeldin A
Betts et al. [51] have recently shown that the phenotypes and cytokine profiles of vaccine-induced responses to HIV change upon breakthrough infection with HIV. It is sobering that the patient described in this report made a polyfunctional T-cell response to multiple epitopes, and was nevertheless still susceptible to infection and underwent rapid disease progression.
References and recommended reading • 6 hours @ 37C • EDTA treatment • Fixation • Permeabilization
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Plate with lyophilized antibodies
To flow cytometer
1. Perez OD, Krutzik PO, Nolan GP: Flow cytometric analysis of kinase signaling cascades. Methods Mol Biol 2004, 263:67-94. This definitive chapter highlights methods for multiparametric flow cytometry in the analysis of intracellular kinase activity. Methods for simultaneous cell-surface and intracellular kinase staining are detailed, together with examples of the types of information that can be obtained. 2.
Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA: Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 2003, 281:65-78.
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Lesinski GB, Badgwell B, Zimmerer J, Crespin T, Hu Y, Abood G, Carson WE III: IL-12 pretreatments enhance IFNalpha-induced Janus kinase-STAT signaling and potentiate the antitumor effects of IFN-alpha in a murine model of malignant melanoma. J Immunol 2004, 172:7368-7376.
Current Opinion in Immunology
Protocol for the use of lyophilized antigen and antibody plates in a cytokine flow cytometry study. Such technology makes complex studies more feasible and increases standardization between assays. PBMCs or whole blood are added directly to a plate containing preconfigured lyophilized stimuli (e.g. peptides) and brefeldin A. Following incubation and processing, a second plate with preconfigured lyophilized antibodies is hydrated, and the antibodies transferred to the cell plate. After staining and washing, the plate can be loaded directly onto a plate-compatible flow cytometer. Abbreviations: EDTA, ethylene diamine tetraacetic acid.
minimizing set-up time and potential errors is the use of pre-configured assay plates with lyophilized activation and staining reagents (Figure 3; [48]). These reagents promise to provide superior stability as well as utility for large studies. The performance of lyophilized reagents also compares favorably to liquid antigens and antibodies [48,49]. Together with analytical tools, such as dynamic gating and batched data analysis [50], considerable time savings and standardization of large studies can be achieved.
Conclusions Assays of ex vivo T-cell function are beginning to contribute significantly to the fields of vaccine design and clinical monitoring. Early analysis of single-cell phosphorylation states and antigen-specific detection of degranulation, cytokine production, differentiation markers and proliferation, can all be combined using polychromatic flow cytometry. Further advances in throughput and standardization of such assays will make them invaluable tools to the immunologist and clinician of the future. In particular, the use of polychromatic flow cytometry is likely to assume a role in defining the correlates of protection for vaccine efficacy as well as in monitoring immunotherapies in diseases such as HIV and cancer. Current Opinion in Immunology 2005, 17:434–440
10. Chow S, Patel H, Hedley DW: Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 2001, 46:72-78. 11. Blaydes JP, Vojtesek B, Bloomberg GB, Hupp TR: The development and use of phospho-specific antibodies to study protein phosphorylation. Methods Mol Biol 2000, 99:177-189. 12. Mandell JW: Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 2003, 163:1687-1698. 13. Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP: Single cell profiling of potentiated phosphoprotein networks in cancer cells. Cell 2004, 118:217-228. www.sciencedirect.com
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A powerful application of single-cell phosphoprotein analysis by flow cytometry to the profiling of cancer cells, reflecting heterogeneity within and between tumors. 14. Krutzik PO, Irish JM, Nolan GP, Perez OD: Analysis of protein phosphorylation and cellular signaling events by flow cytomery: techniques and clinical applications. Clin Immunol 2004, 110:206-221. 15. Krutzik PO, Nolan GP: Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 2003, 55:61-70. 16. Perez O, Nolan GP: Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotech 2002, 20:155-162. 17. Zell T, Jenkins MK: Flow cytometric analysis of T cell receptor signal transduction. Sci STKE 2002, 128:PL5 (doi: 10.1126/stke.2002.128.pl5). 18. Zell T, Khoruts A, Ingulli E, Bonnevier JL, Mueller DL, Jenkins MK: Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo. Proc Natl Acad Sci USA 2001, 98:10805-10810. 19. Perez OD, Mitchell D, Jager GC, Nolan GP: LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56+CD8+ natural killer cells. Blood 2004, 104:1083-1093. 20. Peters PJ, Borst J, Oorschot V, Fukuda M, Krahenbuhl O, Tschopp J, Slot JW, Geuze HJ: Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med 1991, 173:1099-1109. 21. Podack ER: Execution and suicide: cytotoxic lymphocytes enforce Draconian laws through separate molecular pathways. Curr Opin Immunol 1995, 7:11-16. 22. Stinchcombe JC, Bossi G, Booth S, Griffiths GM: The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 2001, 15:751-761. 23. Smyth MJ, Trapani JA: Granzymes: exogenous proteinases that induce target cell apoptosis. Immunol Today 1995, 16:202-206. 24. Jenne DE, Tschopp J: Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation. Immunol Rev 1988, 103:53-71. 25. Fukuda M: Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem 1991, 266:21327-21330. 26. Betts MR, Koup RA: Detection of T-cell degranulation: CD107a and b. Methods Cell Biol 2004, 75:497-512. This chapter gives detailed methods for the detection of CD107 expression during T cell degranulation, with important hints for the optimization of such assays in parallel with intracellular cytokine and other staining. 27. Rubio V, Stuge TB, Singh N, Betts MR, Weber JS, Roederer M, Lee PP: Ex vivo identification, isolation and analysis of tumor-cytolytic T cells. Nat Med 2003, 9:1377-1382. 28. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999, 401:708-712. 29. Wolint P, Betts MR, Koup RA, Oxenius A: Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells. J Exp Med 2004, 199:925-936. This paper highlights the fact that CD107 expression measures the act of degranulation, but not cytotoxicity. These authors show that different types of antigen-experienced CD8+ T cells can all degranulate, but their cytotoxic potential is dependent upon expression of effector molecules such as granzyme B. 30. Betts MR, Price DA, Brenchley JM, Lore K, Guenaga FJ, Smed Sorensen A, Ambrozak DR, Migueles SA, Connors M, Roederer M et al.: The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration. J Immunol 2004, 172:6407-6417. An interesting study of the triggering threshold for cytokine production versus degranulation. The authors suggest that, at limiting antigen density, cytotoxicity in the absence of IFN-g production may predominate, whereas at high antigen density, both responses are triggered. www.sciencedirect.com
31. Shafer-Weaver K, Sayers T, Strobl S, Derby E, Ulderich T, Baseler M, Malyguine A: The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity. J Transl Med 2003, 1:14 (doi:10.1186/1479-5876-1-14). 32. Shafer-Weaver KA, Sayers T, Kuhns DB, Strobl SL, Burkett MW, Baseler M, Malyguine A: Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay. J Transl Med 2004, 2:31 (doi:10.1186/1479-5876-2-31). 33. Waterhouse NJ, Sedelies KA, Clarke CJ: Granzyme B; the chalk-mark of a cytotoxic lymphocyte. J Transl Med 2004, 2:36 (doi:10.1186/1479-5876-2-36). 34. Pantaleo G, Koup RA: Correlates of immune protection in HIV-1 infection: what we know, what we don’t know, what we should know. Nat Med 2004, 10:806-810. 35. Younes SA, Yassine-Diab B, Dumont AR, Boulassel MR, Grossman Z, Routy JP, Sekaly RP: HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity. J Exp Med 2003, 198:1909-1922. 36. Harari A, Petitpierre S, Vallelian F, Pantaleo G: Skewed representation of functionally distinct populations of virus-specific CD4 T cells in HIV-1-infected subjects with progressive disease: changes after antiretroviral therapy. Blood 2004, 103:966-972. This group shows the importance of IL-2-producing CD4+ T cells specific for HIV, and contrasting CMV-specific versus HIV-specific CD4+ T cells. 37. Iyasere C, Tilton JC, Johnson AJ, Younes S, Yassine-Diab B, Sekaly RP, Kwok WW, Migueles SA, Laborico AC, Shupert WL et al.: Diminished proliferation of human immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-2 (IL-2) production and is recovered by exogenous IL-2. J Virol 2003, 77:10900-10909. 38. Roederer M, Brenchley JM, Betts MR, De Rosa SC: Flow cytometric analysis of vaccine responses: how many colors are enough? Clin Immunol 2004, 110:199-205. 39. Elrefaei M, McElroy MD, Preas CP, Hoh R, Deeks S, Martin J, Cao H: Central memory CD4+ T cell responses in chronic HIV infection are not restored by antiretroviral therapy. J Immunol 2004, 173:2184-2189. 40. Yue FY, Kovacs CM, Dimayuga RC, Parks P, Ostrowski MA: HIV-1-specific memory CD4(+) T cells are phenotypically less mature than cytomegalovirus-specific memory CD4(+) T cells. J Immunol 2004, 172:2476-2486. By multiparametric flow cytometry, these authors phenotype the CD4+ T-cell response to HIV in a number of patients, and find a preponderance of CD27+CD28– cells after HAART therapy, as well as a skewing of the CMV response toward less differentiated cells in the presence of high HIV viral loads. 41. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GM, Papagno L, Ogg GS, King A, Lechner F, Spina CA et al.: Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002, 8:379-385. 42. Parish CR: Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 1999, 77:499-508. 43. Lyons AB, Parish CR: Determination of lymphocyte division by flow cytometry. J Immunol Methods 1994, 171:131-137. 44. Putz T, Ramoner R, Gander H, Rahm A, Bartsch G, Holtl L, Thurnher M: Monitoring of CD4+ and CD8+ T-cell responses after dendritic cell-based immunotherapy using CFSE dye dilution analysis. J Clin Immunol 2004, 24:653-663. 45. Stebbing J, Patterson S, Portsmouth S, Thomas C, Glassman R, Wildfire A, Gotch F, Bower M, Nelson M, Gazzard B: Studies on the allostimulatory function of dendritic cells from HCV-HIV-1 co-infected patients. Cell Res 2004, 14:251-256. 46. Dion ML, Poulin JF, Bordi R, Sylvestre M, Corsini R, Kettaf N, Dalloul A, Boulassel MR, Debre P, Routy JP et al.: HIV infection rapidly induces and maintains a substantial Current Opinion in Immunology 2005, 17:434–440
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plate-based cytokine flow cytometry with automated data analysis. BMC Immunol 2003, 4:9 (doi: 10.1186/1471-2172-4-9). 51. Betts MR, Exley B, Price DA, Bansal A, Camacho ZT, Teaberry V, West SM, Ambrozak DR, Tomaras G, Roederer M et al.: Characterization of functional and phenotypic changes in antiGag vaccine-induced T cell responses and their role in protection after HIV-1 infection. Proc Natl Acad Sci USA 2005, 102:4512-4517. These authors have recently shown that the phenotypes and cytokine profiles of vaccine-induced responses to HIV change upon breakthrough infection with HIV. It is sobering that the patient described in this report made a polyfunctional T-cell response to multiple epitopes, and was nevertheless still susceptible to infection and underwent rapid disease progression.
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Current Opinion in Immunology 2005, 17:434–440 This review comes from a themed issue on Immunological techniques Edited by Daniel Speiser Available online 13th June 2005 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.05.002
Introduction CD4+ and CD8+ T cells possess a variety of overlapping functions that are modulated by signals from their environment (antigen, co-stimulation, cytokines) as well as by the stimulation history of the cells. T-cell responses can be roughly grouped into early, intermediate and late functions (Figure 1). Early functions include intracellular calcium flux and phosphorylation of key signaling proteins on serine, threonine, or tyrosine residues. Intermediate functions include degranulation, cytotoxicity and cytokine production. The array of cytokines produced by T cells is a function of their differentiation and can include IL-2 as well as Th1 cytokines (e.g. IFNg, TNF-a), Th2 cytokines (e.g. IL-4, IL-5, IL-13), or regulatory cytokines (e.g. TGF-b, IL-10). Late functions include proliferation, as well as apoptosis or activationinduced cell death. Many assays are available for ex vivo monitoring of T-cell function. Calcium flux can be detected fluorometrically as well as by flow cytometry using calcium-sensitive dyes. Phosphorylation can be measured using western blotting, Current Opinion in Immunology 2005, 17:434–440
and more recently using phosphospecific antibodies in flow cytometry [1]. Degranulation can be measured using a novel flow cytometry assay detecting the cellsurface expression of CD107 from cytotoxic granules [2]. Classic cytotoxicity assays measure cell lysis via release of 51 Cr from labeled target cells; modifications of this method have been developed to measure the loss of dye-labeled target cells by flow cytometry [3]. Cytokines can be quantitated by a wide variety of techniques. These include bulk culture assays (ELISA for cytokine protein, and PCR or RNase protection for cytokine mRNA) and single-cell assays (ELISPOT and cytokine flow cytometry). Proliferation can be measured by uptake of 3Hthymidine or bromodeoxyuridine (BrdU), or by dilution of a dye such as 5(6)-carboxylfluorescein diacetate succinimidyl ester (CFSE); when using flow cytometry as a readout, these latter two methods can be combined with analyses of cytokine production or differentiation marker expression. Finally, apoptosis via activation-induced cell death can be measured by a variety of methods, but is not within the scope of this review. Functional T-cell responses can be evaluated following stimulation by polyclonal activation using mitogens, such as phorbol myristate acetate (PMA) plus ionomycin, phytohemagglutinin (PHA), anti-CD3 and Staphylococcal enterotoxin B (SEB). A major application of functional assays, however, is the monitoring of antigen-responsive cells, so the ability to detect responses using specific antigenic peptides is often critical. In this respect, great advances have been made in recent years, especially with the advent of single-cell assays such as ELISPOT and cytokine flow cytometry. Because these assays have sufficient sensitivity to detect rare populations of positive cells, it is possible to quantitate responses to specific antigens without in vitro expansion in many clinical settings. In this review, we will examine assays of early, intermediate and late T cell function. Particular attention will be given to novel assays (such as CD107 expression) or technologies that have undergone significant improvement in the past year (such as polychromatic cytokine flow cytometry). The relationship of these developments to clinical findings will be emphasized and reviewed in the context of what they can teach us about T-cell responses in particular diseases or vaccination strategies.
Analyzing early T-cell signaling events Calcium flux
Calcium plays a critical role in several early T-cell functions, including regulation of signal transduction protein www.sciencedirect.com
Ex vivo analysis of T-cell function Suni, Maino and Maecker 435
Figure 1
T cell activation
Early
Intermediate
IL-2
Late
IL-4
CD107 ∗ ∗
∗ ∗∗
IFN-γ
T cell – APC interaction
Ca2+ flux phosphorylation
TNF-α
Degranulation cytokine production
Proliferation apoptosis
Current Opinion in Immunology
Stages of T cell activation upon interaction of T cells with antigen-presenting cells. Specific activation of T-cell receptors and co-stimulatory molecules on T cells leads to a cascade of signaling events. Early changes can be detected in a matter of seconds-to-minutes; intermediate changes in the order of several hours; and late events after several days. Abbreviations: APC, antigen-presenting cell.
phosphorylation, and also in subsequent events, including cytokine expression and proliferation. Recent studies using cytometric methods have demonstrated a broad variety of Ca2+ signals in T cells, ranging from infrequent spikes to sustained oscillations and plateaus [4]. These events occur asynchronously in T cells within seconds to minutes following engagement of functional receptors. Single cell measurements of increased calcium concentration as a consequence of activation usually rely on calcium-complexing fluorescent dyes, including indo-1 and fluo-3, which can be measured by flow or image cytometry [5,6]. More recently, Tsien and co-workers [7] introduced real-time analysis of Ca2+ in endoplasmic storage vesicles in single cells using calcium dyes and image cytometry. This technology enables the analysis of calcium changes on a subcellular, not just single-cell, level. Phosphorylation of signal transduction proteins
Enhanced phosphorylation of signal transduction proteins is linked to changes in calcium metabolism. Recently, several investigators detected intracellular phosphoprotein alterations in single primary T cells following activation [8–10]. The measurement of specific protein phosphorylation states has been made possible by the recent availability of antibodies with specificities for unique phosphorylated determinants [11,12]. Using flow cytometry, multiparametric analysis can be performed to detect phosphorylation events within unique T-cell subsets following the introduction of functional signals www.sciencedirect.com
[13,14–16]. In this way information can be obtained about the heterogeneity of early events in T-cell activation, which were previously overlooked by traditional bulk analysis methods [14]. Flow cytometric analysis of phosphoproteins has been employed to evaluate both MAP kinase pathways in activated T cells [17,18] and disease state profiling in leukemias and lymphomas [13], and also in autoimmune diseases [19]. Other potential applications include evaluation of pharmacodynamic effects of drugs and immunomodulatory agents and inhibitors [14]. It is important to note that, although flow cytometry is an extremely powerful approach for assessing phosphorylation events in single cells, the permeabilization and staining techniques for detection of intracellular phosphorylated proteins are technically challenging. For the evaluation of each set of phosphodeterminants and cell surface antigens, both the protocol and antibodies must be optimized. A more complete review of this technology has been published recently [15].
Analyzing intermediate T-cell signaling events Degranulation of cytotoxic T cells 51
Cr release assays measure the lysis of target cells, following cytotoxic cell stimulation, but offer no direct information about the identity and frequency of the effector cells that perform the killing. Recently, two new techniques have been developed that directly examCurrent Opinion in Immunology 2005, 17:434–440
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ine the degranulation of CD8+ T cells, one by the flow cytometric measurement of CD107 surface expression, and the other by the detection of secreted granzyme B (GrB) in an ELISPOT assay.
that responded to cytomegalovirus (CMV) or HIV were found to produce IFN-g (MA Suni, et al., unpublished observations; [2]). In these studies, brefeldin A was added in addition to monensin to optimize the detection of IFNg and CD107.
Flow cytometric measurement of CD107 surface expression
Following conjunction of a CD8+ T cell with an appropriate target cell, lytic granules are transported to the point of contact with the target cell and the granule contents are released into the immune synapse between the effector and the target [20–22]. These lytic granules contain perforin and a set of serine proteases called granzymes [20,23,24]. The granule core is surrounded by a lipid bilayer containing lysosomal associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1), CD107b (LAMP-2) and CD63 (LAMP-3) [2,20,25]. These proteins become transiently expressed on the T cell surface upon granule exocytosis (Figure 2; [20,25]). Betts et al. [2,26] measured the mobilization of CD107 to the surface of activated T cells in peripheral blood mononuclear cell (PBMC) cultures stimulated in the presence of the secretion inhibitor monensin. Because of the transient surface expression and rapid internalization of CD107 by the endocytic pathway [25], staining for CD107 is maximized by addition of the antibody during cell stimulation and by the addition of monensin. By virtue of their parallel kinetics, CD107 and intracellular cytokines can be assessed at the same time using 4–6 hours of stimulation. Most, but not all, CD107+ cells
Using the CD107 assay to study degranulation and cytotoxic activity
Because the degranulation of cytotoxic T cells and killing of target cells are correlated, the CD107 mobilization assay can be used as an alternative to 51Cr release assays. Betts et al. [2] demonstrated a good correlation between degranulation and cytotoxic activity of CD8+ T cells, as measured in a flow-cytometry-based killing assay. Rubio et al. [27] also demonstrated a strong correlation between the killing activity of tumor-specific CD8+ T-cell clones, as measured by 51Cr release, and the level of CD107 induction on those cells. One group, however, showed that degranulation and cytotoxicity are not correlated at all stages of T cell differentiation in mice (the previous two references used human cells). Antigen-experienced T cells can be classified according to their expression of various differentiation markers. So-called ‘central memory’ T cells express CCR7, CD62L and CD28, but not CD45RA [28]. These cells are thought to be long-term, renewable precursors of cells that can develop cytotoxic effector function (socalled ‘effector memory’ and ‘effector’ T cells). Wolint et al. [29] showed that effector, central memory, and effector memory CD8+ T cells all have similar kinetics of
Figure 2
Granule membrane expressing CD107a, CD107b and CD63
Cytotoxic granule Granule core with enzymes (e.g. perforin and granzyme B)
Cell membrane
Granule is transported to cell membrane
Degranulation and release of granule contents; cell surface exposure of CD107a, CD107b and CD63 Current Opinion in Immunology
Diagrammatic representation of the principle underlying the CD107 degranulation assay. CD107a, CD107b and CD63 are expressed on the inner leaflet of granule membranes, and are thus transiently exposed on the cell surface upon granule fusion with the plasma membrane. The presence of antibodies to CD107 in the stimulation culture allows for labeling of these transiently exposed molecules. Current Opinion in Immunology 2005, 17:434–440
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Ex vivo analysis of T-cell function Suni, Maino and Maecker 437
degranulation following antigen encounter. Only effector T cells, however, possess strong immediate cytolytic activity against target cells, whereas effector memory cells show low, and central memory cells showed no, cytolytic activity. These findings correlated with the levels of cytolytic effector molecules, such as GrB, in the lytic granules of the various CD8+ T cell subsets. Because the CD107 mobilization assay does not require cell fixation and permeabilization, it can also be used for the sorting of live cells. Rubio et al. [27] sorted cells on this basis, and showed that only a minority of vaccine-elicited melanoma-specific CD8+ T cells possess degranulation activity when stimulated by relevant tumor target cells. This study emphasized the importance of recognition efficiency in T-cell function, as tetramer+ T cells with low recognition efficiency could degranulate in response to target cells pulsed with excess cognate peptide, but not in response to tumor cells (which present much lower concentrations of peptide–MHC). Degranulation and cytokine production are also differentially sensitive to the concentration of stimulating peptide [30]. Both intracellular cytokine expression and CD107 cell surface exposure were observed at high peptide concentrations, whereas at low peptide concentrations degranulation but not cytokine production were detected. Thus, depending upon the level of presented antigen, an effector response might be both cytolytic and cytokineproducing, or only cytolytic. Detection of granzyme B by ELISPOT assay
Recently, investigators have shown good correlation between ELISPOT assays that measure secreted (GrB) and 51Cr release assays [31,32]. The GrB ELISPOT was found to be highly sensitive and required fewer effector cells for accurate assessment of T-cell cytotoxicity. In addition, the GrB ELISPOT provided a direct estimation of cytotoxic T lymphocyte (CTL) frequencies, which is not possible with 51Cr release. Comparisons were also carried out between GrB ELISPOT and IFN-g ELISPOT and, in many cases, these two assays correlated well [31]. By measuring a protein that directly participates in target cell killing, however, the GrB ELISPOT is a more direct assessment of cytotoxic activity [31,33]. Correlations of GrB ELISPOT with CD107 degranulation have not yet been reported. Measurement of cytokine production
The detection of IFN-g production by ELISPOT is standard for demonstrating a cellular immune response to vaccination. In the case of HIV disease, however, recent work suggests that an IFN-g response alone is unlikely to be sufficient for determining protection, and is unlikely to be useful for predicting progression [34]. Rather, the production of IL-2, either alone or in combiwww.sciencedirect.com
nation with IFN-g by antigen-specific CD4+ T cells, correlates with control of viremia and non-progression [35,36]. Proliferation of CD4+ T cells, which is frequently suppressed in HIV+ individuals, can also be restored by exogenous IL-2 [37]. Investigators have begun to use polychromatic flow cytometry (up to 12 colors) to examine antigen-specific immune responses [38]. Using up to four anti-cytokine (macrophage inhibitory protein [MIP]-1b, TNF-a, IFNg and IL-2) antibodies plus anti-CD107, Betts and colleagues have demonstrated a loss of polyfunctional CD8+ T cells in patients with progressive HIV infection (MR Betts et al., personal communication; see Update). Longterm non-progressive HIV patients, however, demonstrated a consistent presence of CD8+ T cells that were polyfunctional; that is, they could produce all four cytokines investigated (MIP-1b, TNF-a, IFN-g and IL-2) and also degranulate in response to antigen. This study demonstrates the power of multiparameter analysis to discriminate between groups of patients in a way that would not be possible using a series of single-cytokine assays. Central memory T cells specific for recall antigens are depleted in chronic HIV infection [39], and the HIVspecific response is largely composed of CD4+ and CD8+ T cells that have an intermediate differentiation state [40,41]. Such work suggests that the phenotype of cytokine-producing T cells also influences their longevity and function, and might be helpful in predicting the course of disease.
Analyzing later T-cell signaling events T-cell proliferation
Methods have been developed using the membraneassociated fluorescent dye CFSE to detect dividing cells by flow cytometry through reduction in CFSE intensity [42,43]. This technique enables the visualization of six or more discrete cycles of cell division by flow cytometry, both in vitro and in vivo. It is compatible with detection of multiple cell-surface and intracellular markers on the proliferating cells [35,44], as well as with live-cell sorting of proliferating populations. The technique has been used in immune monitoring of vaccine potency [44] and in monitoring immune competence during disease progression [35,45–47]. In addition, Dion et al. [46] combined a biomarker assay to detect T-cell receptor excision circles with CFSE to demonstrate that HIV infection results in a substantial reduction in intrathymic proliferation.
Increasing assay standardization and throughput As the number and size of immunomonitoring studies increases, issues of standardization and throughput are becoming increasingly apparent. One methodology for Current Opinion in Immunology 2005, 17:434–440
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Update
Figure 3
PBMC or whole blood
Plate with lyophilized peptides + brefeldin A
Betts et al. [51] have recently shown that the phenotypes and cytokine profiles of vaccine-induced responses to HIV change upon breakthrough infection with HIV. It is sobering that the patient described in this report made a polyfunctional T-cell response to multiple epitopes, and was nevertheless still susceptible to infection and underwent rapid disease progression.
References and recommended reading • 6 hours @ 37C • EDTA treatment • Fixation • Permeabilization
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Plate with lyophilized antibodies
To flow cytometer
1. Perez OD, Krutzik PO, Nolan GP: Flow cytometric analysis of kinase signaling cascades. Methods Mol Biol 2004, 263:67-94. This definitive chapter highlights methods for multiparametric flow cytometry in the analysis of intracellular kinase activity. Methods for simultaneous cell-surface and intracellular kinase staining are detailed, together with examples of the types of information that can be obtained. 2.
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Current Opinion in Immunology
Protocol for the use of lyophilized antigen and antibody plates in a cytokine flow cytometry study. Such technology makes complex studies more feasible and increases standardization between assays. PBMCs or whole blood are added directly to a plate containing preconfigured lyophilized stimuli (e.g. peptides) and brefeldin A. Following incubation and processing, a second plate with preconfigured lyophilized antibodies is hydrated, and the antibodies transferred to the cell plate. After staining and washing, the plate can be loaded directly onto a plate-compatible flow cytometer. Abbreviations: EDTA, ethylene diamine tetraacetic acid.
minimizing set-up time and potential errors is the use of pre-configured assay plates with lyophilized activation and staining reagents (Figure 3; [48]). These reagents promise to provide superior stability as well as utility for large studies. The performance of lyophilized reagents also compares favorably to liquid antigens and antibodies [48,49]. Together with analytical tools, such as dynamic gating and batched data analysis [50], considerable time savings and standardization of large studies can be achieved.
Conclusions Assays of ex vivo T-cell function are beginning to contribute significantly to the fields of vaccine design and clinical monitoring. Early analysis of single-cell phosphorylation states and antigen-specific detection of degranulation, cytokine production, differentiation markers and proliferation, can all be combined using polychromatic flow cytometry. Further advances in throughput and standardization of such assays will make them invaluable tools to the immunologist and clinician of the future. In particular, the use of polychromatic flow cytometry is likely to assume a role in defining the correlates of protection for vaccine efficacy as well as in monitoring immunotherapies in diseases such as HIV and cancer. Current Opinion in Immunology 2005, 17:434–440
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suppression of thymocyte proliferation. Immunity 2004, 21:757-768. 47. Becker MD, Dullforce PA, Martin TM, Smith JR, Planck SR, Rosenbaum JT: Immune mechanisms in uveitis. What can be learned from in vivo imaging? Ophthalmol Clin North Am 2002, 15:259-270. 48. Dunne JF, Maecker HT: Automation of cytokine flow cytometry assays. J Assoc Lab Automation 2004, 9:5-9. 49. Ghanekar SA, Maecker HT, Maino VC: Immune monitoring using cytokine flow cytometry. In Manual of Clinical Laboratory Immunology, edn 7. Edited by Rose NR. ASM Press; 2005. 50. Suni MA, Dunn HS, Orr PL, deLaat R, Sinclair E, Ghanekar SA, Bredt BM, Dunne JF, Maino VC, Maecker HT: Performance of
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plate-based cytokine flow cytometry with automated data analysis. BMC Immunol 2003, 4:9 (doi: 10.1186/1471-2172-4-9). 51. Betts MR, Exley B, Price DA, Bansal A, Camacho ZT, Teaberry V, West SM, Ambrozak DR, Tomaras G, Roederer M et al.: Characterization of functional and phenotypic changes in antiGag vaccine-induced T cell responses and their role in protection after HIV-1 infection. Proc Natl Acad Sci USA 2005, 102:4512-4517. These authors have recently shown that the phenotypes and cytokine profiles of vaccine-induced responses to HIV change upon breakthrough infection with HIV. It is sobering that the patient described in this report made a polyfunctional T-cell response to multiple epitopes, and was nevertheless still susceptible to infection and underwent rapid disease progression.
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