There are many complex relationships between tu-

mour cells and effector cells in the immune system.

These interactions are controlled predominantly by cy-

tokines, either within the tumour environment, or sys-

temically where the effector cells may be stimulated as

a response to the presence of the tumour. Favourable

clinical responses in cancer patients have been shown

to be associated with enhanced cell-mediated immunity

as well as T cell infiltration in tumours. This status

is controlled in part by a predominantly Th1 cytokine

profile e.g. IFN γ , TNF α and IL-12. Conversely, pa-

tients with advancing cancer may have impaired cell-

mediated immunity as a result of an imbalance of Th1

and Th2 cytokines e.g. IL-4 and IL-10 [6,9,15]. Whilst

cytokines have long been known to orchestrate the im-

mune system by allowing communication between reg-

ulatory and effector cells, the pleiotropic nature of these

molecules results in a very complex environment in

which to study any single molecule’s properties. Several

in vitro protocols have been developed,which aim to closely reflect cytokine production and T cell function in vivo. However, these assays have been developed in artificial settings and as such only allow conclusions to be drawn within a defined context [11]. The aim of this report is to outline the basic proto-cols and applications for the detection of intracellular cytokines by flow cytometry, in the context of disease monitoring

Dalgleish, AG

John, J

Pandha, H

Disease Markers

J. John

H. Pandha

A. G. Dalgleish

Crossref

71ReviewDetection of T cell cytokine production as atool for monitoring immunotherapyJ. John∗, H. Pandha and A.G. DalgleishSt. George’s Hospital Medical School, London SW17ORE, UK1. IntroductionThere are many complex relationships between tu-mour cells and effector cells in the immune system.These interactions are controlled predominantly by cy-tokines, either within the tumour environment, or sys-temically where the effector cells may be stimulated asa response to the presence of the tumour. Favourableclinical responses in cancer patients have been shownto be associated with enhanced cell-mediated immunityas well as T cell infiltration in tumours. This statusis controlled in part by a predominantly Th1 cytokineprofile e.g. IFNγ, TNFα and IL-12. Conversely, pa-tients with advancing cancer may have impaired cell-mediated immunity as a result of an imbalance of Th1and Th2 cytokines e.g. IL-4 and IL-10 [6,9,15]. Whilstcytokines have long been known to orchestrate the im-mune system by allowing communication between reg-ulatory and effector cells, the pleiotropic nature of thesemolecules results in a very complex environment inwhich to study any single molecule’s properties.Several in vitro protocols have been developed,which aim to closely reflect cytokine production and Tcell function in vivo. However, these assays have beendeveloped in artificial settings and as such only allowconclusions to be drawn within a defined context [11].The aim of this report is to outline the basic proto-cols and applications for the detection of intracellularcytokines by flow cytometry, in the context of diseasemonitoring.∗Corresponding author: Tel.: +44 208 7255841; Fax: +44 2087250158; E-mail: rjohn@sghms.ac.uk.2. MethodologyJunge and colleagues [7] detailed a method to studythe intracellular cytokine production of single cellsby flow cytometry, which has become the basis ofmany studies. This assay has become an indispens-able means of evaluating immunotherapy in the patient,where cytokines responses are postulated to influenceclinical outcome. The basic protocol for the detectionof mitogen-induced cytokine production is outlined inProtocol 1. A variation on this method, which allowsthe detection of cytokines from antigen-specific cells,has been optimised and is outlined in Protocol 2.2.1. Protocol 1Mitogen-induced cytokine production can be de-tected by flow cytometry as described by Junge et al. [7]with slight modification [17]. Sodium heparinizedvenous blood was diluted with an equal volume ofstimulation medium (RPMI 1640 containing 2 mM L-glutamine; 10% FCS; 100 µg/ml penicillin; 100 µg/mlstreptomycin; 1 µg/ml Iomomycin; 2.5 ng/ml phorbol12-myristate 13-acetate (PMA); 10 µg/ml Brefeldin A(BFA)). 1 ml of diluted blood was aliquoted into 15 mlconical polypropylene tubes and incubated in a hu-midified incubator at 37◦C 5% CO2 for 4.5 hr. 1 mlof unstimulated blood incubated with BFA alone wasalso set up as a control. 100 µl aliquots of stimulatedblood were then surfaced stained with CD3-PerCP andCD8-FITC antibodies at room temperature for 15 min.Erythrocytes were lysed and leukocytes fixed by theaddition of 2 ml of 1xFACS Lysing Solution (BectonDickinson, Oxford, UK) and incubated at room tem-perature for 10 min. Remaining cells were washed in2 ml wash buffer (PBS/0.5% BSA/0.1% NaN3), beforepermeabilising with 500 µl of 1xFACS permeabilisa-tion solution (Becton Dickinson, Oxford, UK) and in-cubating at room temperature for 10 min. The cellswere then washed in 2 ml wash buffer before stain-Disease Markers 16 (2000) 71–75ISSN 0278-0240 / $8.00  2000, IOS Press. All rights reserved72 J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapying cells with the appropriate anti-cytokine antibodiesfor 30 min at room temperature. Cells were washedwith 2 ml wash buffer and fixed with 200 µl of CellFix(Becton Dickinson, Oxford, UK) before analysis on aFACScan (Becton Dickinson, Oxford, UK). Typically10,000 events were collected.2.2. Protocol 2Antigen-specific cytokine production can also be de-tected by flow cytometry as described by Waldrop etal. [16] for PBMC and more recently by Dr. V.C. Maino(Personal communication) for whole blood. Sodiumheparinized venous blood was aliquoted into 15 ml con-ical polypropylene tubes at 1 ml per tube. The appro-priate antigen was added at the optimal concentrationalong with anti-CD28 (Becton Dickinson, Oxford, UK)and anti-CD49d (Becton Dickinson, Oxford, UK) anti-bodies both at 1 µg/ml final concentration. The bloodwas mixed gently before being incubated in 5%CO2 for2 hr. BFA (10 µg/ml final) was added and the dilutedblood incubated at 37◦C for a further 4 hours. EDTA(20 mM final) was added and vortexed vigorously to re-move adherent cells and incubated at room temperaturefor 15 min. 100 µl aliquots of stimulated blood weretaken, erythrocytes lysed and leukocytes fixed by theaddition of 2 ml of 1×FACS Lysing Solution (BectonDickinson. Oxford, UK) and incubated at room tem-perature for 10 min. Remaining cells were washed in2 ml wash buffer (PBS/0.5% BSA/0.1% NaN3), beforepermeabilising with 500 µl of 1xFACS permeabilisa-tion solution (Becton Dickinson. Oxford, UK) and in-cubated at room temperature for 10 min. The cells werewashed in 2 ml of wash buffer before being stainedfor surface CD69-PE, CD4-PerCP and intracellular cy-tokines simultaneously at room temperature for 30 minin the dark. The cells were then washed and fixed foranalysis as outlined in the mitogen-stimulated protocolabove. Typically, 50,000 events were collected.3. ResultsThe following examples comprise of cytokine dataobtained from 2 patients with malignant melanomawho received two different immune treatments overvarying time schedules. Patient 1 was injected twicewith BCG (Bacille-Calmette-Gue´rin), which is a non-specific adjuvant commonly used in the treatment ofbladder cancer. Whilst its application in immunother-apy has been shown to correlate with cell mediated re-sponses, there have been marked differences in clinicaloutcome of individual patients [15]. Figure 1 representsthe mitogen-induced cytokine (protocol 1) profiles ofthe Th1-associated cytokine interferon-γ (IFNγ) andthe Th2-associated cytokine interleukin-4 (IL-4) beforeand after the administration of BCG. It is apparent thatthere is a significant alteration in the general cytokineprofiles with an increase in the number of IFN( produc-ing cells and a decrease in IL-4 producing cells, this istypical of a Th2 to Th1 switch.Patient 2 was treated with a HLA-A2* binding pep-tide, specific for a melanoma-associated antigen, overa period of 6 weeks. Blood samples were tested be-fore and after treatment using the same peptide in anantigen-specific cytokine assay (protocol 2). The pres-ence of peptide, as well as anti-CD28 and anti-CD49dantibodies, has induced activation of some CD4+ Tcells and the production of IFNγ from a low frequencyof responding cells (Fig. 2).4. DiscussionTo date, the majority of published studies evaluatingcytokine production after immunotherapy have cou-pled mitogenic stimulation of lymphocytes to responsesat either the mRNA level using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, or at theprotein level by ELISA. Significant disadvantages areassociated with both of these techniques. Firstly, RT-PCR requires isolation of peripheral blood mononu-clear cells (PBMC), rendering the analysis of smallblood volumes impossible. Secondly, translocationalcontrol methods involved in the regulation of cytokinegene expression mean that the levels of cytokine RNAmay not accurately reflect the amount of protein syn-thesised [17]. Thirdly, RT-PCR gives rise to many falsepositive results within the context of detecting cytokineproduction.The detection of cytokines at the protein level re-quires their specific isolation and concentration fromserum or supernatants of experimental cell cultures.This can also be achieved by the use of specific mono-clonal anti-cytokine antibodies employed in an ELISA.However, many molecules, such as β-2-microglobulin,produced by cells are capable of interfering with thedetection of cytokines by binding to cytokines them-selves, thus inhibiting their detection by ELISA [3].Whilst ELISA studies are informative, they can onlygive an impression of the bulk cytokine production froma heterogeneous population of cells. They assume thatJ. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy 73Pre BCG Post BCG24%19% 31% 31%49% 8% 33% 5%15.5% 0.5% 3.5% 0.5%59% 25% 57% 39%CD8 FITCIFNγ PEIL-4 PEABFig. 1. Intracellular flow cytometry detection of cytokine synthesis from peripheral blood T lymphocytes stimulated following protocol 1 (seeMethods). Cells were selected from a ‘tight’ lymphocyte gate set on the cells forward and side scatter properties, and T cells selected by stainingpositive for CD3. The percentages in each quadrant represent percentage of CD8+ (LR, UR) or CD4+ (i.e. CD3+/CD8- (LL, UL) T cellsproducing IFN( (A) or IL-4 (B).all cells of a given phenotype respond in a similar wayunder experimental conditions, which is not an accuratereflection of cytokine expression by individual effectorcells [2,8].Assays have therefore been developed to studycytokine production by single effector T cells –these include immunofluorescence microscopy [14];ELISpot [1] and limiting dilution analysis [13]. How-ever, even though all three assays offer additional ben-efits they involve labour intensive techniques requiringconsiderable technical expertise, the result of whichis that the routine examination of single cells is stillgreatly restricted. The levels of cytokine production byindividual T cells stimulated following these protocolsis relatively low. This makes the subsequent discrim-ination between cytokine positive and negative cellsprone to subjective discrepencies. The advent of flowcytometry has allowed the automated detection of manythousands of cytokine-secreting cells simultaneously.The employment of golgi transport inhibitors such asmonensin and brefeldin A allow the concentration ofcytokines within the cell amplifying the detection ofcytokine producing cells. Furthermore, individual cellsmay be assessed for secretion of more than 1 cytokineat once.In patients receiving inert adjuvants such as BCG,there may be little point in trying to examine the re-sponse of lymphocytes to specific antigens. The verylow frequencies of antigen-specific T cells comparedto the vast heterogeneous T cell repertoire mobilised inresponse to adjuvants means that there is little chanceof being able to detect subtle changes in their cytokineprofile. A more appropriate assessment of the im-mune response to immunotherapy may be to examinealterations in the balance between Th1 and Th2 cy-tokine production as determined by mitogen-inducedprotocol. Likewise, where specific effector cells havebeen targeted by antigen-specific immunotherapy, sim-ply screening for gross changes in the balance betweenTh1 and Th2 cytokine production gives us very littleinformation, as the responding T cells occur at such lowfrequencies. The frequency and cytokine profiles ofresponding T cells can only be detected and comparedwhen examined by antigen-specific intracellular flowcytometry, as the assay is designed to look for changesat the minute level of individual cells.74 J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy100%0%0%0%0%0.26%90.64%9.1%IFNγ FITCCD69 PEA BFig. 2. Intracellular flow cytometry detection of cytokine synthesis from peripheral blood T lymphocytes following protocol 2 (see Methods).Cells were selected from a ‘tight’ lymphocyte gate set on the cells forward and side scatter properties and T helper cells selected by stainingpositive for CD4. The percentages in each quadrant represent percentage of CD69+ (activated) T cells producing IFNγ of unstimulated (A) andpeptide/CD28/CD49d stimulated cells.When selecting which protocol for detecting cy-tokine production to use consideration must be madewith respect to the heterogeneity of responding cells.Many researchers have concluded that, in response toselected antigens, cytokine synthesis varies betweenstimulated cells. They postulate that even these sub-tle differences may hold the key to detecting positiveimmunomodulation [12].However, there are disadvantages associated with thedetection of intracellular cytokines common to bothprotocols. Firstly, the necessity of blocking golgi trans-port prevents the ability to quantitate cytokine produc-tion at the single cell level. Secondly, the processof fixing and permeabilising cells prevents subsequentfunctional investigation.Peptide-MHC tetramers have been developed whichallow the detection and isolation of viable antigen-specific T cells by flow cytometry. This method canalso be used in conjunction with cytokine detection [4]however, this approach is limited by the need for HLAand specific immunogenic peptide determination. An-other recently developed technology is that of antibody-antibody conjugates as a cell surface matrix technology.The conjugates are specific for cell surface CD45 andcytokine and as such allow the detection of secreted cy-tokine from specific viable cells which can be isolatedand characterised further [10].In conclusion, there are now several establishedmethods for the detection of cytokine production at thesingle cell level. Each assay has its own advantagesand disadvantages making the selection of the most ap-propriate application dependant on the resources andexpertise available. However, the major considerationin the selection should be the specificity of the assayin relation to that of the stimulation regime if cytokineproduction is to reflect positive clinical outcome, e.g.vaccination.References[1] U. Andersson, J. Andersson, A. Lindfors, K. Wagnder, G.Moller and C.H. Heusser, Simultaneous production of IL-2,IL-4 and Interferon-γ by activated human blood lymphocytes,Eur. J. Immunol. 20 (1990), 1591–1596.[2] R.P. Bucy, A. Panoskaltsis-Mortari and G.Q. Huang, Hetero-geneity of single cell cytokine gene expression in clonal T cellpopulations, J. Exp. Med. 180 (1994), 1251–1262.[3] S. De Kossodo, V. Houba and G.U. Granu, WHO CollaborativeStudy Group. Assaying TNF concentrations in human serum,a WHO collaborative study, J. Immunol. Methods 282 (1995),107–112.[4] N. Gerovois, N. Labarriere, S. Le Guiner, M.-C. Pandolfino,J.-F. Fonteneau, Y. Guilloux, E. Diez, B. Dreno and F. Jotereau,High avidity melanoma-reactive cytotoxic T lymphocytes areefficiently induced from peripheral blood lymphocytes onstimulation by peptide-pulsed melanoma cells, Clin. Can. Res.6 (2000), 1459–1467.[5] J.M. Grange, J.L. Stanford and G.A.W. Rook, Tuberculosisand cancer: parallels in host response and therapeutic ap-proaches? Lancet 345 (1995), 1350–1352.[6] N. Ito, H. Nakamura, Y. Tanaka and S. Ohqi, Lung carci-noma: analysis of T helper type 1 and 2 cells and T cytotoxictype 1 and 2 cells by intracellular cytokine detection with flowcytometry, Cancer 85 (1999), 2359–2367.[7] T. Junge, U. Schauer, C. Heusser, C. Neumann and C. Reiger,Detection of intracellular cytokines by flow cytometry, J. Im-munol. 159 (1993), 197–207.[8] C.E. Lewis, Detecting cytokine production at the single cellslevel, Cytokine 3 (1991), 184.J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy 75[9] D.R. Lucey, M. Clerici and G.M. Shearer, Type 1 and Type 2cytokine dysregulation in human infections, Clin. Microbiol.Rev. 9 (1996), 532–562.[10] R. Manz, M. Assenmacher, E. Pfluger, S. Miltenyi and A.Radbruch, Analysis and sorting of live cells according to se-creted molecules, relocated to a cell-surface affinity matrix,Proc. Natl. Acad. Sci. USA 92 (1995), 1921–1925.[11] C. Nathan and M. Sporn, Cytokines in context, J. Cell Biol.133 (1991), 981–986.[12] L.A. Pekarek, R.R. Weichselbaum, M.A. Beckett, J. Nachmanand H. Schreiber, Footprinting individual tumours and theirvariants by constitutive cytokine expression patterns, CancerRes. 53 (1993), 1978–1981.[13] G.D. Powers, A.K. Abbas and R.A. Miller, Frequencies ofIl-2 and Il-4 secreting T cells in na¨ıve and antigen stimulatedlymphocyte populations, J. Immunol. 140 (1988), 3352–3359.[14] B. Sander, J. Andersson and U. Andersson, Assessment ofcytokines by immuofluorescence and the paraformaldehyde-saponin procedure, Immunol. Rev. 119 (1991), 65–93.[15] M. Sato, S. Goto, R. Kaneko, M. Ito, S. Soto and S. Takeuchi,Impaired production of Th1 cytokines and increased frequencyof Th2 subsets in PBMCs from advanced cancer patients,Anticancer. Res. 18 (1998), 3951–3955.[16] S.L. Waldrop, C.J. Pitcher, D.M. Peterson, V.C. Maino and L.J.Picker, Determination of antigen-specific memory/effectorCD4+ T cells frequencies by flow cytometry, J. Clin. Invest.99 (1997), 1739–1750.[17] M. Westby, J.B. Marriott, M. Guckian, S. Cookson, P. Hay andA.G. Dalgleish, Abnormal intracellular IL-2 and interferon-gamma (IFNγ) production as HIV-1-associated markers ofimmune dysfunction, Clin. Exp. Immunol. 111 (1998), 257–263.Submit your manuscripts athttp://www.hindawi.comStem CellsInternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014MEDIATORSINFLAMMATIONofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Behavioural NeurologyEndocrinologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Disease MarkersHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014BioMed Research InternationalOncologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Oxidative Medicine and Cellular LongevityHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014PPAR ResearchThe Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal ofObesityJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Computational and  Mathematical Methods in MedicineOphthalmologyJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Diabetes ResearchJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Research and TreatmentAIDSHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Gastroenterology Research and PracticeHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Parkinson’s DiseaseEvidence-Based Complementary and Alternative MedicineVolume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

English

Detection of T Cell Cytokine Production as a Tool for Monitoring Immunotherapy

St George's Online Research Archive

71ReviewDetection of T cell cytokine production as atool for monitoring immunotherapyJ. John∗, H. Pandha and A.G. DalgleishSt. George’s Hospital Medical School, London SW17ORE, UK1. IntroductionThere are many complex relationships between tu-mour cells and effector cells in the immune system.These interactions are controlled predominantly by cy-tokines, either within the tumour environment, or sys-temically where the effector cells may be stimulated asa response to the presence of the tumour. Favourableclinical responses in cancer patients have been shownto be associated with enhanced cell-mediated immunityas well as T cell infiltration in tumours. This statusis controlled in part by a predominantly Th1 cytokineprofile e.g. IFNγ, TNFα and IL-12. Conversely, pa-tients with advancing cancer may have impaired cell-mediated immunity as a result of an imbalance of Th1and Th2 cytokines e.g. IL-4 and IL-10 [6,9,15]. Whilstcytokines have long been known to orchestrate the im-mune system by allowing communication between reg-ulatory and effector cells, the pleiotropic nature of thesemolecules results in a very complex environment inwhich to study any single molecule’s properties.Several in vitro protocols have been developed,which aim to closely reflect cytokine production and Tcell function in vivo. However, these assays have beendeveloped in artificial settings and as such only allowconclusions to be drawn within a defined context [11].The aim of this report is to outline the basic proto-cols and applications for the detection of intracellularcytokines by flow cytometry, in the context of diseasemonitoring.∗Corresponding author: Tel.: +44 208 7255841; Fax: +44 2087250158; E-mail: rjohn@sghms.ac.uk.2. MethodologyJunge and colleagues [7] detailed a method to studythe intracellular cytokine production of single cellsby flow cytometry, which has become the basis ofmany studies. This assay has become an indispens-able means of evaluating immunotherapy in the patient,where cytokines responses are postulated to influenceclinical outcome. The basic protocol for the detectionof mitogen-induced cytokine production is outlined inProtocol 1. A variation on this method, which allowsthe detection of cytokines from antigen-specific cells,has been optimised and is outlined in Protocol 2.2.1. Protocol 1Mitogen-induced cytokine production can be de-tected by flow cytometry as described by Junge et al. [7]with slight modification [17]. Sodium heparinizedvenous blood was diluted with an equal volume ofstimulation medium (RPMI 1640 containing 2 mM L-glutamine; 10% FCS; 100 µg/ml penicillin; 100 µg/mlstreptomycin; 1 µg/ml Iomomycin; 2.5 ng/ml phorbol12-myristate 13-acetate (PMA); 10 µg/ml Brefeldin A(BFA)). 1 ml of diluted blood was aliquoted into 15 mlconical polypropylene tubes and incubated in a hu-midified incubator at 37◦C 5% CO2 for 4.5 hr. 1 mlof unstimulated blood incubated with BFA alone wasalso set up as a control. 100 µl aliquots of stimulatedblood were then surfaced stained with CD3-PerCP andCD8-FITC antibodies at room temperature for 15 min.Erythrocytes were lysed and leukocytes fixed by theaddition of 2 ml of 1xFACS Lysing Solution (BectonDickinson, Oxford, UK) and incubated at room tem-perature for 10 min. Remaining cells were washed in2 ml wash buffer (PBS/0.5% BSA/0.1% NaN3), beforepermeabilising with 500 µl of 1xFACS permeabilisa-tion solution (Becton Dickinson, Oxford, UK) and in-cubating at room temperature for 10 min. The cellswere then washed in 2 ml wash buffer before stain-Disease Markers 16 (2000) 71–75ISSN 0278-0240 / $8.00  2000, IOS Press. All rights reserved72 J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapying cells with the appropriate anti-cytokine antibodiesfor 30 min at room temperature. Cells were washedwith 2 ml wash buffer and fixed with 200 µl of CellFix(Becton Dickinson, Oxford, UK) before analysis on aFACScan (Becton Dickinson, Oxford, UK). Typically10,000 events were collected.2.2. Protocol 2Antigen-specific cytokine production can also be de-tected by flow cytometry as described by Waldrop etal. [16] for PBMC and more recently by Dr. V.C. Maino(Personal communication) for whole blood. Sodiumheparinized venous blood was aliquoted into 15 ml con-ical polypropylene tubes at 1 ml per tube. The appro-priate antigen was added at the optimal concentrationalong with anti-CD28 (Becton Dickinson, Oxford, UK)and anti-CD49d (Becton Dickinson, Oxford, UK) anti-bodies both at 1 µg/ml final concentration. The bloodwas mixed gently before being incubated in 5%CO2 for2 hr. BFA (10 µg/ml final) was added and the dilutedblood incubated at 37◦C for a further 4 hours. EDTA(20 mM final) was added and vortexed vigorously to re-move adherent cells and incubated at room temperaturefor 15 min. 100 µl aliquots of stimulated blood weretaken, erythrocytes lysed and leukocytes fixed by theaddition of 2 ml of 1×FACS Lysing Solution (BectonDickinson. Oxford, UK) and incubated at room tem-perature for 10 min. Remaining cells were washed in2 ml wash buffer (PBS/0.5% BSA/0.1% NaN3), beforepermeabilising with 500 µl of 1xFACS permeabilisa-tion solution (Becton Dickinson. Oxford, UK) and in-cubated at room temperature for 10 min. The cells werewashed in 2 ml of wash buffer before being stainedfor surface CD69-PE, CD4-PerCP and intracellular cy-tokines simultaneously at room temperature for 30 minin the dark. The cells were then washed and fixed foranalysis as outlined in the mitogen-stimulated protocolabove. Typically, 50,000 events were collected.3. ResultsThe following examples comprise of cytokine dataobtained from 2 patients with malignant melanomawho received two different immune treatments overvarying time schedules. Patient 1 was injected twicewith BCG (Bacille-Calmette-Gue´rin), which is a non-specific adjuvant commonly used in the treatment ofbladder cancer. Whilst its application in immunother-apy has been shown to correlate with cell mediated re-sponses, there have been marked differences in clinicaloutcome of individual patients [15]. Figure 1 representsthe mitogen-induced cytokine (protocol 1) profiles ofthe Th1-associated cytokine interferon-γ (IFNγ) andthe Th2-associated cytokine interleukin-4 (IL-4) beforeand after the administration of BCG. It is apparent thatthere is a significant alteration in the general cytokineprofiles with an increase in the number of IFN( produc-ing cells and a decrease in IL-4 producing cells, this istypical of a Th2 to Th1 switch.Patient 2 was treated with a HLA-A2* binding pep-tide, specific for a melanoma-associated antigen, overa period of 6 weeks. Blood samples were tested be-fore and after treatment using the same peptide in anantigen-specific cytokine assay (protocol 2). The pres-ence of peptide, as well as anti-CD28 and anti-CD49dantibodies, has induced activation of some CD4+ Tcells and the production of IFNγ from a low frequencyof responding cells (Fig. 2).4. DiscussionTo date, the majority of published studies evaluatingcytokine production after immunotherapy have cou-pled mitogenic stimulation of lymphocytes to responsesat either the mRNA level using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, or at theprotein level by ELISA. Significant disadvantages areassociated with both of these techniques. Firstly, RT-PCR requires isolation of peripheral blood mononu-clear cells (PBMC), rendering the analysis of smallblood volumes impossible. Secondly, translocationalcontrol methods involved in the regulation of cytokinegene expression mean that the levels of cytokine RNAmay not accurately reflect the amount of protein syn-thesised [17]. Thirdly, RT-PCR gives rise to many falsepositive results within the context of detecting cytokineproduction.The detection of cytokines at the protein level re-quires their specific isolation and concentration fromserum or supernatants of experimental cell cultures.This can also be achieved by the use of specific mono-clonal anti-cytokine antibodies employed in an ELISA.However, many molecules, such as β-2-microglobulin,produced by cells are capable of interfering with thedetection of cytokines by binding to cytokines them-selves, thus inhibiting their detection by ELISA [3].Whilst ELISA studies are informative, they can onlygive an impression of the bulk cytokine production froma heterogeneous population of cells. They assume thatJ. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy 73Pre BCG Post BCG24%19% 31% 31%49% 8% 33% 5%15.5% 0.5% 3.5% 0.5%59% 25% 57% 39%CD8 FITCIFNγ PEIL-4 PEABFig. 1. Intracellular flow cytometry detection of cytokine synthesis from peripheral blood T lymphocytes stimulated following protocol 1 (seeMethods). Cells were selected from a ‘tight’ lymphocyte gate set on the cells forward and side scatter properties, and T cells selected by stainingpositive for CD3. The percentages in each quadrant represent percentage of CD8+ (LR, UR) or CD4+ (i.e. CD3+/CD8- (LL, UL) T cellsproducing IFN( (A) or IL-4 (B).all cells of a given phenotype respond in a similar wayunder experimental conditions, which is not an accuratereflection of cytokine expression by individual effectorcells [2,8].Assays have therefore been developed to studycytokine production by single effector T cells –these include immunofluorescence microscopy [14];ELISpot [1] and limiting dilution analysis [13]. How-ever, even though all three assays offer additional ben-efits they involve labour intensive techniques requiringconsiderable technical expertise, the result of whichis that the routine examination of single cells is stillgreatly restricted. The levels of cytokine production byindividual T cells stimulated following these protocolsis relatively low. This makes the subsequent discrim-ination between cytokine positive and negative cellsprone to subjective discrepencies. The advent of flowcytometry has allowed the automated detection of manythousands of cytokine-secreting cells simultaneously.The employment of golgi transport inhibitors such asmonensin and brefeldin A allow the concentration ofcytokines within the cell amplifying the detection ofcytokine producing cells. Furthermore, individual cellsmay be assessed for secretion of more than 1 cytokineat once.In patients receiving inert adjuvants such as BCG,there may be little point in trying to examine the re-sponse of lymphocytes to specific antigens. The verylow frequencies of antigen-specific T cells comparedto the vast heterogeneous T cell repertoire mobilised inresponse to adjuvants means that there is little chanceof being able to detect subtle changes in their cytokineprofile. A more appropriate assessment of the im-mune response to immunotherapy may be to examinealterations in the balance between Th1 and Th2 cy-tokine production as determined by mitogen-inducedprotocol. Likewise, where specific effector cells havebeen targeted by antigen-specific immunotherapy, sim-ply screening for gross changes in the balance betweenTh1 and Th2 cytokine production gives us very littleinformation, as the responding T cells occur at such lowfrequencies. The frequency and cytokine profiles ofresponding T cells can only be detected and comparedwhen examined by antigen-specific intracellular flowcytometry, as the assay is designed to look for changesat the minute level of individual cells.74 J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy100%0%0%0%0%0.26%90.64%9.1%IFNγ FITCCD69 PEA BFig. 2. Intracellular flow cytometry detection of cytokine synthesis from peripheral blood T lymphocytes following protocol 2 (see Methods).Cells were selected from a ‘tight’ lymphocyte gate set on the cells forward and side scatter properties and T helper cells selected by stainingpositive for CD4. The percentages in each quadrant represent percentage of CD69+ (activated) T cells producing IFNγ of unstimulated (A) andpeptide/CD28/CD49d stimulated cells.When selecting which protocol for detecting cy-tokine production to use consideration must be madewith respect to the heterogeneity of responding cells.Many researchers have concluded that, in response toselected antigens, cytokine synthesis varies betweenstimulated cells. They postulate that even these sub-tle differences may hold the key to detecting positiveimmunomodulation [12].However, there are disadvantages associated with thedetection of intracellular cytokines common to bothprotocols. Firstly, the necessity of blocking golgi trans-port prevents the ability to quantitate cytokine produc-tion at the single cell level. Secondly, the processof fixing and permeabilising cells prevents subsequentfunctional investigation.Peptide-MHC tetramers have been developed whichallow the detection and isolation of viable antigen-specific T cells by flow cytometry. This method canalso be used in conjunction with cytokine detection [4]however, this approach is limited by the need for HLAand specific immunogenic peptide determination. An-other recently developed technology is that of antibody-antibody conjugates as a cell surface matrix technology.The conjugates are specific for cell surface CD45 andcytokine and as such allow the detection of secreted cy-tokine from specific viable cells which can be isolatedand characterised further [10].In conclusion, there are now several establishedmethods for the detection of cytokine production at thesingle cell level. Each assay has its own advantagesand disadvantages making the selection of the most ap-propriate application dependant on the resources andexpertise available. However, the major considerationin the selection should be the specificity of the assayin relation to that of the stimulation regime if cytokineproduction is to reflect positive clinical outcome, e.g.vaccination.References[1] U. Andersson, J. Andersson, A. Lindfors, K. Wagnder, G.Moller and C.H. Heusser, Simultaneous production of IL-2,IL-4 and Interferon-γ by activated human blood lymphocytes,Eur. J. Immunol. 20 (1990), 1591–1596.[2] R.P. Bucy, A. Panoskaltsis-Mortari and G.Q. Huang, Hetero-geneity of single cell cytokine gene expression in clonal T cellpopulations, J. Exp. Med. 180 (1994), 1251–1262.[3] S. De Kossodo, V. Houba and G.U. Granu, WHO CollaborativeStudy Group. Assaying TNF concentrations in human serum,a WHO collaborative study, J. Immunol. Methods 282 (1995),107–112.[4] N. Gerovois, N. Labarriere, S. Le Guiner, M.-C. Pandolfino,J.-F. Fonteneau, Y. Guilloux, E. Diez, B. Dreno and F. Jotereau,High avidity melanoma-reactive cytotoxic T lymphocytes areefficiently induced from peripheral blood lymphocytes onstimulation by peptide-pulsed melanoma cells, Clin. Can. Res.6 (2000), 1459–1467.[5] J.M. Grange, J.L. Stanford and G.A.W. Rook, Tuberculosisand cancer: parallels in host response and therapeutic ap-proaches? Lancet 345 (1995), 1350–1352.[6] N. Ito, H. Nakamura, Y. Tanaka and S. Ohqi, Lung carci-noma: analysis of T helper type 1 and 2 cells and T cytotoxictype 1 and 2 cells by intracellular cytokine detection with flowcytometry, Cancer 85 (1999), 2359–2367.[7] T. Junge, U. Schauer, C. Heusser, C. Neumann and C. Reiger,Detection of intracellular cytokines by flow cytometry, J. Im-munol. 159 (1993), 197–207.[8] C.E. Lewis, Detecting cytokine production at the single cellslevel, Cytokine 3 (1991), 184.J. John et al. / Detection of T cell cytokine production as a tool for monitoring immunotherapy 75[9] D.R. Lucey, M. Clerici and G.M. Shearer, Type 1 and Type 2cytokine dysregulation in human infections, Clin. Microbiol.Rev. 9 (1996), 532–562.[10] R. Manz, M. Assenmacher, E. Pfluger, S. Miltenyi and A.Radbruch, Analysis and sorting of live cells according to se-creted molecules, relocated to a cell-surface affinity matrix,Proc. Natl. Acad. Sci. USA 92 (1995), 1921–1925.[11] C. Nathan and M. Sporn, Cytokines in context, J. Cell Biol.133 (1991), 981–986.[12] L.A. Pekarek, R.R. Weichselbaum, M.A. Beckett, J. Nachmanand H. Schreiber, Footprinting individual tumours and theirvariants by constitutive cytokine expression patterns, CancerRes. 53 (1993), 1978–1981.[13] G.D. Powers, A.K. Abbas and R.A. Miller, Frequencies ofIl-2 and Il-4 secreting T cells in na¨ıve and antigen stimulatedlymphocyte populations, J. Immunol. 140 (1988), 3352–3359.[14] B. Sander, J. Andersson and U. Andersson, Assessment ofcytokines by immuofluorescence and the paraformaldehyde-saponin procedure, Immunol. Rev. 119 (1991), 65–93.[15] M. Sato, S. Goto, R. Kaneko, M. Ito, S. Soto and S. Takeuchi,Impaired production of Th1 cytokines and increased frequencyof Th2 subsets in PBMCs from advanced cancer patients,Anticancer. Res. 18 (1998), 3951–3955.[16] S.L. Waldrop, C.J. Pitcher, D.M. Peterson, V.C. Maino and L.J.Picker, Determination of antigen-specific memory/effectorCD4+ T cells frequencies by flow cytometry, J. Clin. Invest.99 (1997), 1739–1750.[17] M. Westby, J.B. Marriott, M. Guckian, S. Cookson, P. Hay andA.G. Dalgleish, Abnormal intracellular IL-2 and interferon-gamma (IFNγ) production as HIV-1-associated markers ofimmune dysfunction, Clin. Exp. Immunol. 111 (1998), 257–263.

Detection of T cell cytokine production as a tool for monitoring immunotherapy

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Detection of T cell cytokine production as a tool for monitoring immunotherapy

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