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The function and differentiation state of many cell types ismore accurately defined by the molecules they secrete ratherthan by their surface phenotype. For example, the T-helper(CD4+) lymphocyte subsets, Th1 and Th2, are best defined bytheir production of interferon-γ (IFN-γ) and interleukin (IL)-4,respectively1. Moreover, recently described regulatory subsetsof T cells are characterized by the secretion of transforminggrowth factor (TGF)-β and IL-10 (refs. 2,3). Investigations intothe function of such cells and their potential uses in therapy re-quire cytokine-based identification methods that do not com-promise cell viability or physiological state. Here we describe anovel method that allows precise identification of cytokine se-cretion patterns in viable cells. The method, where the cells areencapsulated in a permeable antibody-capture matrix, is basedon technologies previously established to isolate secretorycells4,5 and have been used for the selection of antibody-secret-ing B-cell hybridomas6. The adaptations that we have made tothe technique allow it to be easily and conveniently used to de-tect cytokine production and sort cells on the basis of secretionpatterns. Here we used the new technique in conjunction withflow cytometry to distinguish lymphocytes secreting IFN-γ, IL-4, IL-10 or TGF-β. We present evidence that the method neitheralters the secretion patterns of the cells, nor affects their re-sponse to conventional stimulation following removal of theagarose matrix. The ability to combine this technique with cell-surface staining indicates its potential as a powerful tool in un-derstanding the role of T-cell subsets in regulating the immunesystem and also makes it applicable in isolating a broad rangeof other cell types.

Characterization of the capture matrix for secreted cytokinesAlthough small amounts of certain cytokines remain associatedwith the cell membrane of the producing cell7, most cytokinesare immediately released upon secretion. Therefore, in order toidentify the secreting cell, the cytokine must be maintained inthe vicinity of its source, where a fluorochrome-labeled anti-body can detect it. Normally, this is achieved by blocking cy-tokine secretion and permeabilizing the plasma membrane toallow entry of antibodies, but these processes prevent recoveryof living cells8. To measure cytokine production without com-promising cell viability, it is crucial to allow cellular activationand free exchange of nutrients between the cell and its envi-ronment. Effective new methods have been developed that an-chor anti-cytokine antibodies at the plasma membrane usingeither streptavidin as a linker after cell biotinylation9 or anti-body conjugates that also recognize cellular CD45 (refs. 7,10);however, use of these methods is limited. These processes oftenalter cell physiology and the internalization of membrane-asso-ciated antibodies prevents longer-term detection. In an at-

tempt to overcome these difficulties, we have investigated anextension of the use of a biotinylated agarose matrix for encap-sulation of living cells (Fig. 1a and b). Anti-cytokine capture-an-tibodies can be incorporated into the matrix using avidin tocross-link the biotinylated agarose to biotin-labeled antibodies.Upon culture of the encapsulated cells, secreted cytokine is re-tained in the matrix by the antibody. This can be readily detected using a second anti-cytokine antibody (fluorochrome-labeled). The process of cytokine detection is thus similar to aconventional sandwich ELISA where the assay is done in theimmediate vicinity of the secreting cell. Agarose was chosen be-cause: 1) it does not alter or bind the cell membrane (thusavoiding potential cell signaling effects); 2) produces a perme-able structure enabling exchange of growth factors; and 3) itproduces small microdrops that can be run through a flow cy-tometer, as previously shown4–6.

For entrapment into the gel microdrops, cells were sus-pended in FCS (fetal calf serum) and mixed at 37 °C withmolten biotinylated agarose with an ultra-low gelling point (gelpoint, 17 °C). In order to ensure sufficient gel strength, thefinal concentration of agarose was maintained at 2%.Microdrops were formed by vortexing the mixture into di-methylpolysiloxane (an inert silicone oil) and subsequently re-ducing the temperature to below the gelling point of theagarose. The use of a low concentration of cells in the agarosemixture (< 5 × 106/ml) and the small volume of the resultingmicrodrops ensures that very few microdrops contain morethan one cell (less than 1% as observed by Trypan blue stainingand light microscopy; as previously described4 and according tothe Poisson distribution statistics). After washing by standardcentrifugation in Hanks balanced salt solution (HBSS), the mi-crodrops were labeled with avidin and then a biotinylated cap-ture anti-cytokine antibody. Following overnight culture, themicrodrops are recovered and stained to detect the presence ofbound cytokine. The production of cytokine by cells wasclearly detectable using immunofluorescence (Fig. 1c and d).Importantly, empty agarose microdrops can be distinguishedfrom those that contain cells because they have low forward-and side-scatter and are not fluorescent despite the presence ofcapture-antibodies within the matrix. This indicates that onlythose microdrops containing cells that secrete the specific cy-tokine are labeled. The presence of empty microdrops in thepreparation thus acts as an internal control.

Analysis and sorting of micro-encapsulated cellsWe tested the applicability of micro-encapsulation for analyz-ing a number of key cytokines such as IL-4, TGF-β and IL-10 bypositive-control cell types (Fig. 2a and b). We analyzed produc-tion of TGF-β using a rat keratinocyte cell line that is trans-

Cell identification and isolation on the basis of cytokinesecretion: A novel tool for investigating immune responses

VICTOR TURCANU & NEIL A. WILLIAMS

University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol, UK

V.T. is now at Imperial College of Medicine, Department of Pediatrics, Norfolk PLace, London, UKCorrespondence should be addressed to N.A.W.; email: neil.a.williams@bristol.ac.uk

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fected with the human TGF-β gene (V69C5)11. IL-4 productionwas measured on human peripheral blood mononuclear cells(PBMCs) stimulated with phytohemagglutinin (PHA) in thepresence of IL-4, and IL-10 was measured on blood CD14+

monocytes following activation with lipopolysaccharide (LPS).In each case, entrapment into microdrops allowed the secre-tion of the cytokine to be clearly measured as an increase in flu-orescence above that seen using appropriate isotype-matchedcontrol antibodies. Our ability to detect TGF-β is of particularinterest as this cytokine cannot be detected in flow cytometryeven after cell permeabilization (Fig. 2a). The difficulty in de-tecting TGF-β in permeabilized cells is probably due to the factthat this cytokine is packaged into secretory granules withincells that are not accessible to the detecting antibody12. Thus,the micro-encapsulation method provides the first reliable ap-proach to detecting TGF-β–secreting cells.

After establishing the method with control cells, it was im-portant to determine whether it could be used to investigatecytokine production in a physiological immune response. Inorder to test this, we stimulated purified CD4+ T cells fromhuman peripheral blood in a mixed lymphocyte reaction

(MLR) using irradiated allogeneic CD14+ monocytes as stimula-tors. We determined the presence of putative regulatory T cellsby staining encapsulated cells recovered from the cultures forthe secretion of IL-10. This cytokine was chosen because it hasbeen widely implicated in the function of T-regulatory cellpopulations13,14. To test whether antibodies specific for cell-sur-face components could penetrate the gel microdrop matrix andstain efficiently, we co-stained cells for the expression of CD4.The results demonstrate that staining for both secreted cy-tokines and surface molecules occurs in microdrop gels (Fig.2c–e). Similar numbers of CD4+ cells were seen in samples ofunencapsulated and encapsulated cells (Fig. 2c). These data, aswell as a recent report describing the diffusion of phycoery-thrin conjugates (having relatively large molecules) for stainingagarose-encapsulated cells15, further support the fact that rapiddiffusional exchange is quite possible. The specificity of thestaining was checked using isotype-matched control antibodiesor by blocking before staining with an excess of unlabeled anti-body (Fig. 2e). Analysis of cytokine production in the allo-geneic MLR revealed the presence of IL-10-secreting CD3+ cells(Fig. 2d) that were not found in the syngeneic MLR, indicatingthat their differentiation results from the in vitro immune re-

Fig. 1 Cytokine detection using gel microdrop micro-encapsulation. aand b, The schematic diagrams illustrate the basis of the technique. Cellsare encapsulated in a biotinylated agarose matrix, which is then used tocapture biotin-labeled cytokine-specific antibodies using avidin as abridge. The encapsulated cells are cultured to allow cytokine release, andthen the captured cytokine is detected using a second cytokine specificantibody, which is fluorochrome-labeled. c and d, Photomicrographsshow human PBMCs contained within microdrops under phase contrast(c) or fluorescence microscopy (d). The paired images clearly demonstratethe fluorescence of a microdrop surrounding an IL-10 secreting cell to-gether with a lack of staining on both empty microdrops and those containing non-secreting cells.

Fig. 2 Microencapsulation allows the detection of cytokine production.a, V69C5 keratinocyte cells were stained for the production of TGF-βusing either standard permeabilization (left) or microencapsulation(right). b, Microencapsulation was used to reveal the production of IL-4and IL-10 by human PBMC. c, The ability of antibodies to the cell-surfacemolecule CD4 to reach the membrane of encapsulated cells was testedusing human PBMC. The cells were encapsulated or left non-encapsulatedbefore staining with anti-CD4 using standard flow cytometric techniques.d, Cytokine and cell-surface staining were combined to detect CD3, CD14and IL-10 production by cells in a mixed lymphocyte reaction. In all cases,staining using an appropriate isotype-matched control antibody is shownas an unfilled histogram or a separate dot plot. e, Specificity of stainingwas checked by blocking before staining with an excess of unlabeled anti-body. Also, IL-10 production by CD3+ cells is not found in the case of asyngeneic MLR, indicating that the differentiation of cytokine secretors re-sults from the in vitro immune response. f, The CD3+/IL-10+ cell popula-tion was sorted successfully and its increased production of IL-10 uponrestimulation was demonstrated using standard intracellular cytokinestaining in comparison with the CD3+/IL-10– subset (dotted line), the iso-type control (thin dotted line) and the unstained cells (filled histogram).

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sponse. Both CD3+/IL-10– and CD3+/IL-10+ cell populationswere sorted successfully and their increased production of IL-10upon restimulation was demonstrated using standard intracel-lular cytokine staining (Fig. 2f).

Recovery and functional assessment of sorted cellsAlthough the agarose matrix is permeable and does not directlyinteract with the plasma membrane, it is important that theprocess of encapsulation does not alter cytokine secretion. Toestablish this, we stimulated PBMC with PHA and then eitherencapsulated them or left them untreated. The ability of thecells to produce key cytokines during the following 24 hourswas tested using cell-based ELISA (celELISA) (ref. 16). We ob-served no significant differences between encapsulated andnon-encapsulated cells in the levels of secreted TGF-β, IL-10, IL-4 or IFN-γ (Fig. 3a). This indicates that encapsulation does notalter the activation or differentiation state of the cells over thelimited time period required for their analysis and sorting.

Another critical aspect of applying encapsulation to sorting

cytokine-secreting cells is the ability to recover the cells in a vi-able and functionally normal state. Thus, PBMC were encapsu-lated and then released from the agarose matrix using agarase.The recovered cells were viable, as indicated by their ability toexclude the vital dye Trypan blue (data not shown), and wereable to secrete similar quantities of TGF-β, IL-10, IL-4 or IFN-γ asunencapsulated cells following activation (Fig. 3b).

Summary and future perspectivesWe have shown that cell entrapment into microdrops can be

MethodsChemicals and antibodies. Chemicals were from Sigma unless

stated otherwise. Cytokine-specific antibodies were fromPharmingen (Cowley, UK). The clones used were for IFN-γ, 4S.B3and NIB42; for IL-4, MP4-25D2 and 8D4-8; for IL-10, JES3-12G8and JES-19F1; for TGF-β, A75-3.1 and A75-2.1. Anti-human CD3(clone UCHT-1) and CD4 (clone Q4120) were from Sigma andanti-human CD14 (clone RMO52) from Coulter Immunotech(Luton, UK).

Synthesis of biotin-agarose. Biotin-agarose was prepared bysodium periodate oxidation of type IX-A ultra-low gelling temper-ature agarose followed by coupling with biotinamidocaproyl hy-drazide17. The level of agarose biotinylation was measured using4′-hydroxyazobenzene-2-carboxylic acid (HABA)18.

Cell microencapsulation procedure. Cells were resuspendedin FCS at 5 × 106 cells/ml. An equal volume of biotin-agarose (4%in PBS) was melted in a microwave oven, cooled to 37 °C andmixed with the cells in a 50-ml tube before addition of prewarmeddimethylpolysiloxane (viscosity 50 cs). Cells were microencapsu-lated by vortexing vigorously using a Whirlimixer (FisonsScientific, Loughborough, UK) for 1 min and then incubated for10 min on ice to induce agarose gelling. Microencapsulated cellswere recovered by adding HBSS, vortexing for 20 s and centrifug-ing for 10 min at 650g. Large microdrops were excluded by filtra-tion through a 40-µm nylon cell strainer. Microdrops werepelleted and for up to 5 × 106 capsules, 250 µg avidin was added.After 15-min incubation at room temperature with roller mixing,cells were washed twice and 10 µl biotinylated antibody wasadded to the pellet. After further 15-min incubation at room tem-perature with roller mixing, the microdrops were washed and cul-tured in RPMI + 10% FCS for the required period.

Staining procedure for flow cytometric analysis and sortingof microencapsulated cells. Microdrops were collected, washedin HBSS and incubated with anti-cytokine antibodies for 1 h atroom temperature, then washed twice and analyzed using aFACScan or sorted using a FACS Vantage (Becton Dickinson,Cowley, UK).

Agarase digestion of the capsules. Agarase, dissolved in HBSScontaining 20 mM HEPES, was added (10 U/ml) to the micro-drops and these were incubated under shaking at 37 °C.Microdroplet disaggregation was observed after 2 h without lossof cell viability. At this point, cells could be transferred intomedium containing 1 U/ml agarase and left overnight to com-plete agarose digestion. Alternatively, complete microdrop disso-lution was observed after 6–8 h shaking with a minimal (< 5%)loss of cell viability.

Intracellular cytokine staining. For permeabilization, the cellswere cultured for 24 h in the presence of Brefeldin A to blockprotein secretion and then fixed and permeabilized using 2%formaldehyde and 0.1% saponin before staining (a range ofother fixatives and permeabilization agents gave identical results).

Stimulation of cell populations. For IL-4, human PBMC werestimulated with PHA and IL-4 and cultured for 7 days beforebeing restimulated with PHA alone. Thirty-six hours later the cellswere encapsulated and cultured overnight before staining for thepresence of cytokines. In the case of IL-10, human CD14+ mono-cytes were activated with LPS for 24 h before encapsulation.Staining the cells after overnight culture revealed IL-10 secretion.Human CD4+ T cells were cultured with irradiated allogeneicCD14+ monocytes for 7 days, after which they were restimulatedwith fresh monocytes and encapsulated after 24 h.

Fig. 3 Microencapsulation does not alter cytokine secretion by humanPBMCs. a, Cells were activated with phytohemagglutinin (10 µg/ml) for36 h and then either left untreated (�) or encapsulated ( ). Cytokine se-cretion was then measured upon subsequent culture for 24 h usingcelELISA. b, Cells were either left untreated (�) or encapsulated ( ).Encapsulated cells were then treated with agarase to disrupt the micro-drops and their cytokine secretion was compared with that of equal num-bers of non-encapsulated counterparts after activation withphytohemagglutinin (10 µg/ml) using celELISA.

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used for the quantification and isolation of cytokine-secretingcells by flow cytometry. This technique should be, as previ-ously suggested4,5, broadly applicable to the study of proteinsthat are secreted by a variety of cells and therefore of interestfor researchers from many areas of biomedical research. Ourdata show that: 1) the method can be readily combined withcell-surface staining; 2) it enables the visualization of cytokinesthat have until now been impossible to detect even after cellpermeabilization; and 3) the cells can be recovered still viableand able to function normally in subsequent assays. Our find-ings point toward the use of encapsulation as a tool for immu-nologists wishing to study the function of lymphocyte subsetswith particular cytokine secretion patterns. This ability will bekey to determining the precise roles of important cell subsetsand will allow the development of novel approaches to thetreatment of human disease.

AcknowledgmentsWe thank S. Sreckovic for assistance with cell sorting and Ian Patterson forthe V69C5 cells. N.A.W. is a Wellcome Trust Fellow.

1. Mossmann, T.R. & Coffman, R.L. Two types of mouse helper T-cell clone–implicationsfor immune regulation. Immunol. Today 8, 223–228 (1987).

2. Chen, Y. et al. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature376, 177–180 (1995).

3. Groux H. et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and pre-vents colitis. Nature 389, 737–742 (1997).

4. Powell, K.T. & Weaver, J.C. Gel microdroplets and flow cytometry: rapid determina-tion of antibody secretion by individual cells within a cell population. Biotechnology 8,333-337 (1990).

5. Weaver J.C., McGrath P. & Adams S. Gel microdrop technology for rapid isolation ofrare and high producer cells. Nature Med. 3, 583–585 (1997).

6. Gray F., Kenney J.S. & Dunne J.F. Secretion capture and report web: Use of affinity de-rivatized agarose microdroplets for the selection of hybridoma cells. J. Immunol. Methods 182, 155–163 (1995).

7. Scheffold, A., Assenmacher, M., Reiners-Schramm, L., Lauster R. & Radbruch A. High-sensitivity immunofluorescence for detection of the pro- and anti-inflammatory cy-tokines gamma interferon and interleukin-10 on the surface of cytokine-secreting cells. Nature Med. 6, 107–110 (2000).

8. Prussin, C. & Metcalfe, D.D. Detection of intracytoplasmic cytokine using flow cytom-etry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188,117–128 (1995).

9. Manz, R., Assenmacher, M., Pfluger, E., Miltenyi, S. & Radbruch, A. Analysis and sort-ing of live cells according to secreted molecules, relocated to a cell-surface affinity ma-trix. Proc. Natl. Acad. Sci. USA 92, 1921–1925 (1995).

10. Brosterhus, H., et al. Enrichment and detection of live antigen-specific CD4(+) andCD8(+) T cells based on cytokine secretion. Eur. J. Immunol. 29, 4053–4059 (1999).

11. Davies, M et al. Overexpression of autocrine TGFβ1 suppresses the growth of spindleepithelial cells in vitro and in vivo in the rat 4NQO model of oral carcinogenesis. Int. J.Cancer 73, 68–74 (1997).

12. Johnston, J.B. et al., Deposition of transforming growth factor-β in the marrow inmyelofibrosis and the intracellular localization and secretion of TGF-β by leukemiccells. Am. J. Clin. Pathol. 103, 574–582 (1995).

13. Powrie, F., Carlino, J., Leach, M.W., Mauze, S. & Coffman, R.L. A critical role for trans-forming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183, 2669–2674 (1996).

14. Groux, H. & Powrie, F. Regulatory T cells and inflammatory bowel disease. Immunol.Today 20, 442–446 (1999).

15. Gift, E.A. & Weaver, J.C. Simultaneous quantitative determination of electroporativemolecular uptake and subsequent cell survival using gel microdrops and flow cytom-etry. Cytometry 39, 243–249 (2000).

16. Beech, J.T., Bainbridge, T. & Thompson, S.J. Incorporation of cells into an ELISA sys-tem enhances antigen driven lymphokine detection. J. Immunol. Meth. 205, 163–168(1997).

17. Parikh, I., March, S. & Cuatrecasas, P. Topics in the methodology of substitution reac-tions with agarose. Meth. Enzymol. 34, 77–102 (1974).

18. Hermanson, G.T. in Bioconjugate Techniques. (Academic Press, San Diego, 1996).

A novel non-invasive, in vivo technique for thequantification of leukocyte rolling and extravasation at

sites of inflammation in human patientsJUHA KIRVESKARI1,2, MINNA H. VESALUOMA3, JUKKA A.O. MOILANEN3, TIMO M.T. TERVO3,

MATTHEW W. PETROLL4, EEVA LINNOLAHTI3 & RISTO RENKONEN1,2

1Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland2HUCH Laboratory Diagnostics, the Health Care Region of Helsinki and Uusimaa, Finland

3Department of Ophthalmology, University of Helsinki, Helsinki, Finland4Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Correspondence should be addressed to R.R.; email: risto.renkonen@helsinki.fi

Inflammatory lesions, regardless of whether they are causedby ischemia/reperfusion, trauma, infections or immunologi-cal stimuli, are characterized by a local accumulation ofleukocytes1,2. The extravasation of leukocytes from the bloodvessels into inflamed tissues is initiated by a complex cascadeof molecular interactions between the selectin family andtheir sialyl Lewis X-containing ligands, leading to rolling ofleukocytes3–5. We describe here the use of a novel non-inva-sive, in vivo technique based on reflected-light, tandem-scan-ning confocal microscopy of capillaries to observe leukocyterolling during lesional inflammation in human patients. Awell-characterized inflammation was induced by electivecataract surgery. We observed very few rolling or tissue-infil-trating leukocytes in preoperative analysis. However, at thesites of inflammation, we found a 40-fold increase in thenumber of rolling cells, with a concomitant significant de-crease in leukocyte velocity. At the same time, the number of

tissue-infiltrating cells was significantly elevated comparedwith control values. As the clinical inflammation resolved,the number of rolling and extravasated leukocytes decreasedback to normal levels. Our data indicate that this novel in vivotechnique, which can be applied to human patients repeat-edly, will allow the quantitative evaluation of inflammatoryconditions or their therapeutic intervention—a task that haspreviously only been approached invasively.

Leukocyte rolling has been extensively studied with intrav-ital microscopy in various experimental inflammatory modelsin animals6–8, but not yet in humans. The rolling and adhe-sion phases are important but do not predict the actual ex-travasation of leukocytes into tissues, and thus otherapproaches are needed for the analysis of inflammation9. Inanimal studies, cells that have migrated into the tissue havebeen quantified, for example, using histology from tissuebiopsies, local accumulation of leukocyte-specific myeloper-

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