Direct Demonstration of Cytokine Synthesis Heterogeneity Among Human Memory/Effector T Cells by Flow Cytometry

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1 Diret Demonstration of Cytokine Synthesis Heterogeneity Among Human Memory/Effetor T Cells by Flow Cytometry By Louis J. Piker, Manoj K. Singh, Zoran Zdraveski, John R. Treer, Sharr L. Waldrop, Paul R. Bergstresser, and Vernon C. Maino The array of ytokines produed by T ells in effetor sites is a primary means by whih these ells mediate host defense. It is well reognized that loned T ells are heterogeneous with regard to ytokine synthesis and, thus, in their abiliity to mediate speifi immune responses, but the extent to whih the patterns of ytokine seretion observed in loned ells reflet atual populations of memoryleffetor T ells existing in vivo is largely unknown. Here, we report our findings using a multiparameter flow ytometri assay that allows simultaneous determination of an individual Tell's ability to produe multiple ytokines and its phenotype after only short (4 to 8 hours) in vitro inubation with an ativating stimulus and the seretion inhibitor Brefeldin A. This assay shows a rapid aumulation of interleukin2 (IL2). IL4, and yinterferon (yifn) in the ytoplasm of CD4+ ells after stimulation with either aessory ellindependent (phorbol 12 myristate 1aetate [PMA] + ionomyin [I]) or aessory elldependent (staphylooal enterotoxins [SE] A and B) Tellativating stimuli. Further analysis showed that prodution of yifn and IL4 is predominantly, if not exlusively, restrited to the CD45ROhigh memoryleffetor Tell subset, whereasil2may be produed by both the CD45ROh'gh T HE MANIFESTATION OF immunity, either protetive or pathologi, ritially depends on the funtional ativity of the soalled memory/effetor Tell subset, haraterized in the human by its CD45RA'0wiROhigh phenotype. Indeed, only these previously ativated T ells appear to have the apability of effiiently homing to and loalizing within sites of Tell effetor funtion, inluding all extralymphoid sites of inflammation (eg, lung, skin, synovium, and so on) and the unique lymphoid miroenvironment of the germinal enter.'.' However, memoryleffetor T ells are not uniform in their funtional potential. Similar to the immune reations they initiate, these ells manifest remarkable heterogeneity, both in terms of their ability to extravasate in a partiular site and to manifest effetor funtion From the Laboratories of Experimental Pathology and Clinial Immunology, Department of Pathology, and the Department of Dermatology, University of Texas Southwestem Medial Center, Dallas, TX; and Beton Dikinson Immunoytometry Systems, San Jose, CA. Submitted Deember 22, 1994; aepted April, Supported by National Institutes of Health Grants No. A11545 and AR L.J.P. is an awardee of the President's Researh Counil of the University of Texas Southwestern Medial Center and of the Advaned Tehnology Program of the Texas Higher Eduation Coordinating Board. M.K.S. was supported by a fellowship grant from the World Health Organization. Address reprint requests to Louis J. Piker, MD, Department of Pathology. The University of Texas Southwestern Medial Center, 52 Harry Hines Blvd, Dallas, TX The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be herebv marked "advertisement" in aordane with 18 U.S.C. setion 174 solely to indiate this fat. Q 1995 by The Amerian Soiety of Hematology I/95/ $.00/0 and CD45RO'"" subsets. Simultaneous determination of IL 2 and yifn prodution among CD45ROMBh/CD4+ T ells showed distint subsets that produe eah of these ytokines alone (an average of 0% for IL2 alone, 8% for yifn alone), both (16%). or neither (46%). Similar analyses with the small IL4produing memory/effetor Tell subset (only 4.% of total CD4+/CD45ROhbh T ells) showed that an average of 5150 of these IL4produing ells also synthesize IL2.2% synthesize only IL4,16% synthesize all three ytokines, and 9.6% synthesize IL4 and ylfn. These patterns of ytokine synthesis were found to be similar with both PMA + I and SEA/SEB stimulation and were observed in both peripheral blood memory/effetor CD4+ T ells and in T ells of similar phenotype obtained from utaneous delayedtype hypersensitivity sites. Taken together, these data strongly support the in vivo existene of human memory/effetor T ell subsets with "preprogrammed ytokine synthesis potential, although they suggest that these subsets may be more omplex than originally proposed in the THI/TM hypothesis by The Amerian Soiety of Hematology. therein." For example, we have desribed and haraterized a subset of CD45RA'0w/R0hi8h T ells that are seletively targeted to skin:4 and subsets thought to be targeted to other tissues have been desribed as well.'4 However, perhaps the most investigated funtional heterogeneity shown by memory/effetor T ells is their ytokine synthesis potential.'.''" Detailed studies of loned CD4+ T ells in both mie and humans have suggested that the ability to synthesize and serete many key effetor ytokines, inluding interleukin 2 (IL2), IL4, IL5, and yinterferon (?EN), is restrited to a fration of the overall memory/effetor population. Indeed, the finding of predominant patterns of ytokine seretion among Tell lones has led to the proposal that CD4+ effetor T ells may be generally separated into TH1 (IL 2 and yifnproduing) and TH2 (IL4 and L5produing) subsets.''" Even though the ytokine synthesis potential of individual, genuine (ie, nonloned) effetor T ells is largely unknown, the THl/TH2 terminology has beome so well aepted that it is routinely extended to omplex in vivo immune responses (so that responses with a predominane of yifn or IL4 produing T ells are termed "TH1" or "TH2" responses, respetively). Beause reent evidene has strongly suggested that the ability of memory/effetor T ells to produe a given ytokine is heavily influened by the miroenvironmental onditions present at the time of Tell ativation,""itis plausible that the longterm in vitro ulture required for loning T ells may shape the ytokine synthesis phenotype of the resulting lones. Thus, the patterns of ytokine seretion observed in suh lones may not be representative of all physiologially relevant memory/effetor T ell subsets. Given the lear importane of understanding the entire physiologi range of Tell funtional heterogeneity, we sought to assess the ytokine (?EN, IL2, and IL 1408 Blood, VOI 86, NO 4 (August 15). 1995: pp

2 CYTOKINE SYNTHESISDEFINED TCELL SUBSETS ) synthesis apabilites of normal, freshly isolated, human memory/effetor T ells on a singleell basis. These studies were based on the pioneering methodologial report of Jung et who first showed the feasibility of using intraytoplasmi staining and flow ytometry to assess Tell ytokine prodution. Here, we improve on these methods and ombine them with sophistiated multiparameter analysis tehniques to simultaneously determine the ytokine prodution apabilities of preisely defined subsets of human peripheral blood (PB) and inflammatory site T ells after only short periods (4 to 8 hours) of in vitro ativation with mitogen or superantigen. Our results onfirm an extensive heterogeneity in the ability of memory/effetor T ells to produe these three ytokines in response to both aessory elldependent and aessory ellindependent stimuli and show reproduible patterns of ytokine seretion by these ells. The diret visualization of Tell effetor responses afforded by this flow ytometri approah allows an unparalleled ability to determine the funtional potential of phenotypially distint Tell subsets and, thus, the opportunity to evaluate the partiipation of these subsets in human immune responses. MATERIALS AND METHODS Monolonal antibodies (MoAbs). MoAbs Leu4 (CD; fluoresein isothioyanate [FITC], phyoerythrin [PE], peridinin hlorophyll protein [PerCP]), Leua (CD$ FITC, PE, PerCP), Leu2a (CD% FITC, PE, PerCP), Leu45RO (CD45RO; unonjugated, PE), Leu18 (CD45RA; unonjugated), Leu2 (CD69; FITC, PE), GlCL (mouse IgGl ontrol; FITC, PE, PerCP), and G2GL (mouse IgG2 ontrol; FITC, PE, PerCP) were obtained from Beton Dikinson Immunoytomehy Systems (BDIS; San Jose, CA). Purified MoAbs BG5 (antihuman IL2; mouse IgG1) and BB1 (antihuman yifn; IgGl) were purhased from Biosoure International (Camarillo, CA) and were onjugated to PE or FITC by the reagent laboratories at BDIS. The antihuman IL4 MoAb E41 (mouse IgGI) and a mouse IgGl ontrol reagent were purhased from Pharmingen (San Diego, CA). A series of mouse antihuman ytokine MoAbs (all IgGl) from the researh labs of R & D Systems (Minneapolis, MN) were also used in this study (largely to onfirm the staining speifiity of the abovelisted reagents), inluding lones no and (both antiil4), no (antiyifn), and no ,58.111, and (all antiil2). The unonjugated antiytokine MoAbs were visualized with a Trioloronjugated goat antimouse IgGl seond stage obtained from Caltag (South San Franiso, CA). Cell preparation. PB mononulear ells (PBMCs) obtained from normal donors were isolated from heparinized venous blood by densitygradient sedimentation over FiollHypaque (Histopaque; Sigma Chemial CO, St Louis, MO). Cells were then washed times in Hank's balaned salt solution (HBSS; GIBCO, Grand Island, NY) and resuspended in medium appropriately for ell ulture, immunofluoresene staining of freshly isolated ells, or futher purifiation. Purified T ells (>95% CD+) were isolated from the PBMC preparations by negative seletion using R & D Systems Tell purifiation olumns, as desribed by the manufaturer. Purified whole virgin (295% CD45RO), whole memory/effetor (2595% CD45RO+), or CD4' memory/effetor (295% CD4+ and 295% CD45RO+) T ells were isolated from Tell preparations via 2 to rounds of negative panning using the Leu45R0, Leu 18, and Leu2a + Leu18 MoAbs, respetively, as previously de~ribed.'~ In some experiments, aessory ell (monoyte) reonstitution was required to allow purified Tell preparations to respond to superantigens (see below). For these experiments, the ulture wells to be used with the purified populations were preseeded with PBMCs at 0.5 X lo6 ells/ml in omplete media and allowed to inubate for 1 hour at 7 C after whih the ulture wells were extensively washed to remove nonadherent ells (leaving >95% monoytes) and then were seeded with purified Tell populations (see below). Leukoyte preparations from delayedtype hypersensitivity (DTH) sites in skin were obtained as previously de~ribed.'~ Briefly, ommon skintest antigens (Ags; eg, mumps Ag) were injeted intradermally into the normal forearm skin of sensitized volunteers before the plaement of sution blisters 18 to 24 hours later. (Clinial evidene of a delayed hypersensitivity reation usually appeared at about 24 hours, peaking at 40 to 50 hours.) Leukoyteontaining blister fluid was withdrawn at suessive 24hour intervals after blister plaement for up to 92 hours. (Fluids were ompletely evauated at eah time point, with natural refilling ourring between samplings.) Colleted leukoytes were washed by entrifugation and proessed immediately for the immunofluoresent ytokine prodution assays. The results presented in this study represent the haraterization of T ells taken from the blister fluid sample orrelating with the peak of the observed DTH response. Flow ytometri ytokine prodution assays. Flow ytometri assessment of Tell ytokine prodution is based on the stimulation of T ells in the presene of a pharmaologi inhibitor of seretion, followed by ell fixation and permeabilization, and then intraytoplasmi staining of aumulated ytokines. This tehnique was originally desribed by Jung et all but has been signifiantly modified in these studies. These hanges have both simplified the staining proedure and onsiderably enhaned the sensitivity and reproduibility of the ytokine detetion (data not shown). In our protool, Brefeldin A (BFA), a relatively nontoxi but potent inhibitor of intraellular mnsport,i6 was used to blok the ytokine seretion stimulated by both aessory ellindependent (phorbol 12myristate 1aetate [PMA] + ionomyin [I]) and aessory elldependent (the baterial superantigens, staphylooal enterotoxin [SE] A and SEB)17,18 Tell agonists. In preliminary experiments, BFA ( pg/ ml final onentration) ompletely bloked the surfae appearane of the ativation Ag CD69I9 on 4hour PMA + Istimulated T ells without any loss of viability (viability maintained for for up to 18 hours). Importantly, BFA did not interfere with early ativation events, beause the level of intraytoplasmi CD69 staining (see below) shown by PMA + Iativated, BFAtreated T ells was almost idential to the level of surfae CD69 staining on nonbfatreated, similarly ativated T ells (data not shown). Preliminary experiments also indiated that maximal aumulation of intraellular ytokine ourred after 4 hours of BFAinhibited PMA + I stimulation and after 6 to 8 hours of BFAinhibited superantigen stimulation. (Cytokine aumulation plateaued and then dereased with longer periods of BFA exposure.) Thus, T ells (without aessory ells for PMA + I stimulation; with aessory ells for superantigen stimulation) were ultured in RPM (GIBCO) supplemented with % heatinativated fetal alf serum (Hylone Sterile Systems, In, Logan UT), 20 mmovl HEPES buffer (GIBCO), 0 Uld peniillin (GIBCO), 0 pg/ml streptomyin (GIBCO), and 2 mmovl Lglutamine (GIBCO) with or without the Tell stimulants PMA + I (5 ng/ml and 1 pmovl, respetively; Sigma), or the superantigens SEA and SEB (0 ng/ml of eah; Toxin Tehnology, Sarasota, FL). BFA was added 4 hours (PMA + I) or 6 to 8 hours (SEA + SEB) before harvest so that, in kineti experiments, the period of seretion inhibition and, thus, ytokine aumulation was for 4 or 6hour windows of time (eg, 0 to 4 hours, 14 to 18 hours, 40 to 44 hours, and so on). After harvesting, ells (0.1 to 0.5 x 6/test) were washed with old HBSS, stained (if appropriate) on the ell surfae with an MoAbfluorohrome onjugate (IgG2 MoAbs only, in this ase Leu45RO), and then were simultaneously fixed and permeabilized

3 14 PICKER ET AL Purified Peripheral Blood T Cells: Unstimulated 2 hl d C e S x L E 2 Q) Q) ki 0 L. L 0 Q) Q) C L L 1 o1 C C ; PMA + I Stimulated 4 hrs. CD8 u C m S e (0 L P Q) f 0 L f C C % CD8 Fig 1. Flow ytometri detetion of ytokine synthesis among purified PB T ells stimulated with PMA + 1. Purified PB T ells were stimulated with PMA + I for 4 hours in the presene of the seretion inhibitor BFA and then ompared with unstimulated T ells for their orrelated expression of intraellular ytokine (IL2, IL4, yifn) or the ativation Ag CD69 versus CD8. A total of,000 events, gated on viable ells using light satter riteria, are shown in eah plot. The perentages in the upper left and right orners of eah plot represent the net perentage (96) of positive (+) ells (ie, after subtrating bakground) among the CD8 and CD8' subsets, respetively. As disussed in the Materials and Methods setion, the CD8 population is essentially equivalent to the CD4' subset in these experiments. Note that the vast majority of the PMA + ltreated T ells are highly.. positive for the ativation As CD69, indiating that the lak of prodution of a partiular ytokine by a given ell was not likely due to inomplete ativation. in HBSSwith mmoll HEPESbuffer, 4% paraformaldehyde, and 0.1% saponin (all from Sigma) for IO to 15 minutes at4 C. After washing 2 times witholddulbeo'sphosphatebuffered saline (dpbs) ontaining 0.1% bovine serum albumin, ells were stained as previously desribed's,'n for surfae Ag immunofluoresene. Continued or additional permeabilization was not neessary for optimal staining. Briefly, ells were inubated suessively with the following reagents (all at 0 to 4 C) with washing in dpbslbovine serum albumin in between: ( I) an appropriately titred unonjugated antiytokine MoAb (usually 0.1 to 0.4 &test for 0 minutes); (2) the Trioloronjugated goat antimouse IgGl seond stage in dpbs with 5% normal human serum (0 minutes); () 0.4 mutest of % normal mouse serum ( minutes; for bloking); and (4) I to 2 onjugated MoAbs against additional ytokines or other Ags (CD4. CD8, and/or CD69). The CD4 and CD8 MoAbs delineated idential populations whether they were used before or after permeabilization. However, beause of the previously doumented rapid and omplete downmodulation of CD4 in response to phorbol esters," CD4 MoAbs ould not be used to delineate the CD4' Tell subset after more than 4 hours of PMA + I stimuluation. Beause CD8 expression diminishes only slightly after this treatment, and beause multiparameter staining of the unstimulated purified Tell preparations used in our study showed that the vast majority of CD8 ells were indeed CD4+ (not shown), CD8 MoAbs alone ould effetively differentiate the CD4' and CD8' subsets. Therefore, in the experiments examining PMA + lstimulatedpurified T ells, the CD8 subsetwasonsidered equivalent to the CD4' subset. CD4 downmodulation does not our with superantigen stimulation; therefore, the CD4 subset ould be diretly identified in these experiments. CD69 expression was used in these studies as an indiator of ativated ells." In BFAtreated ells, CD69 upregulation is onfined to the ytoplasm, requiring that CD69 staining be performed after permeabilization (see above). In ontrast, BFA proteted the ell surfae from ativationindued hanges in CD4SRO expression, allowing delineation of the original virgin and memory/effetor subsets only if staining was performed before permabilization. Beause ontinuous BFA was required for this effet and beause ontinuous BFA for longerthan 8 hours results in diminshing ytokine aumulation, this tehnique ould only be used for the 0 to 4hour or 0 to 6hour time points. Fiveparameter analysis was performed on a FACSort flow ytometer (BDIS) using FITC, PE, and PerCP or Triolor (PWCyS tandem) as the three fluoresent parameters. Methods of ytometer setup and data aquisition have been desribed previously.*" Listmode multiparameter data files (eah file with forward satter, side satter, and three fluoresent parameters) were analyzed using the PAINT AGATE""' program (BDIS). Negative ontrol reagents were used to verify the staining speifiity of experimental antibodies and as a guide for setting markers to delineate positive and negative populations. All perentageslisted in thefiguresandtextrepresent net perent positive, after subtration of bakground. AI1 analyses were performed using a light satter gate designed to inlude only viable lymphoytes. In some analyses, additional live gating based on CD4 or CD8 reativity or CD69 reativity was performed to enhane the sampling of small populations. Preliminary experiments using this flow ytometri ytokine assayontwowellharaterizedhuman THI lones andonil4 (versus ontrol) transfetants showed an exellent qualitative orrelation between the flow ytometri results and the level of overall ytokines measured by onventional tehniques (immunoassay, analysis of mrna; data not shown), similar

4 CMOKINE SYNTHESISDEFINEDTCELLSUBSETS 1411 CD4+ T Cells to previously reported res~1ts.l~ Moreover, idential patterns of reativity were observed with at least two independent MoAb lones for eah ytokine. The data presented in the figures are representative of to greater than separate experiments. RESULTS Initial studies foused on the Row ytometri haraterization of the ytokine prodution apabilities of purified PB human T ells ativated by the aessory ellindependent stimulus of PMA + I in the presene of BFA. Signifiant ytokine reativity was not deteted within fresh, unstimulated T ells but was dramatially upregulated (along with the ativation Ag CD69) after as little as 4 hours of inubationwith this potent ativating stimulus (Fig 1). This response was not apparent or was markedly diminished with (1) in vitro inubation in the absene of an ativating stimulus (with or without BFA), (2) appropriate ativation in the absene of seretion inhibition (ie, the inlusion of BFA with the PMA + I), or () the lak of ell permeabilization before staining (data not shown). The absolute requirement for permeabilization (ie, the lak of speifi signal after ell surfae staining), along with the inability of exogenously added ytokine to produe a signal using our staining protools (data not shown), indiates that Tell binding to soluble ytokine in the ulture medium did not ontribute to the observed responses. Cytokine synthesis learly preeded blastogenesis, beause, at the 4hour time point, the vast majority of ytokineproduing ells still manifested the light satter properties of small lymphoytes (not shown). Signifiantly, despite the fat that essentially all PMA + Itreated T ells showed marked upregulation of CD69 (see Fig l), whih is onsistent with universal ativation, only a subset of T ells produed detetable ytokine at any given time point (Figs 1 and 2). Both the CD4+ (CD87 and CD8+ subsets partiipated in this ytokine response, with more CD4+ T ells produing IL2 than CD8+ T ells, and vie versa for y IFN. Among CD4+ ells, IL2 prodution usually peaked in the 0 to 4hour time period, with a sharp partial derease at the 14 to 18hour time points and a slow derease thereafter. Peak levels of yifn by CD4+ T ells and of both y IFN and L 2 by CD8+ T ells were ahieved in the first 4 hours of stimulation, with a very slow derease in the numbers of produing ells over the next days. Among CD4' T ells, IL4 prodution was transient (usually identified in 0 to 4hour time period only) and was only observed in a small subset of these ells (overall, mean 2 SEM = 2.7% 2 0.6% for 18 different experiments). E4 was usually deteted in even fewer (usually <l%) CD8+ T ells at the 4 hour time point but, in some experiments, reappeared in a slightly larger subset of CD8+ ells after 40 hours of stimulation (Fig 2). We next assessed the ability of the aessory elldependent baterial superantigens SEA and SEB to stimulate detetable Tell ytokine responses. Beause these superantigens are known to stimulate T ells in a V@speifi manner (eg, Vpl.1, Vp5, Vp6, Vp7., and Vp9.l for SEA; Vp, Vp 12, and Vpl7 for SEB)" and, thus, only ativate a subset of the overall Tell population, we simultaneously evaluated CD69 expression along with ytokine synthesis and CD418 phenotype so as to determine the perentage of ytokineproduing ells within the ativated CD4+ or CD8+ Tell populations. As shown in Fig, only the CD69' T ells synthesized and aumulated signifiant intraellular ytokine, allowing us to gate on the CD69+ ells to determine the extent to whih SEA/SEBresponsive ells produe these ytokines. A representative experiment omparing the partiipation of (CD69gated) CD4+ versus CD8+ T ells in an SEAISEBindued ytokine response is shown in Fig 4. Similar to what was observed with PMA + I, more CD4+ T ells produe L2 than CD8+ T ells, whereas the reverse was true for yifn. However, the overall number of ells manifesting an L2 or yifn response to SENSEB was less than that observed for PMA + I (even with analysis restrited to the ativated population). This was not apparent for IL2produing CD4+ ells, whih were twofold to threefold fewer in number (see below). In ontrast, the perentage

5 I g 1412 PICKER ET AL SuperAg Stimulated CD4+ Peripheral Blood T Cells (6 hrs.) 0) x V l0 lo1 1 o1 1 lo1 l0 ontrol lnterleukln 2 lnterleukln yinterferon lnterleukln 2 lnterleukln 4 1 2,Interferon Fig. Flow ytometri haraterization of ytokine synthesis by superantigenstimulated PB T ells; evaluation per total ativated T ells. PBMCs were stimulated with the baterial superantigens SEA + SEB for 6 hours in the presene of BFA and then stained intraellularly for (1) IL2, IL4, yifn, or an isotypemathed ontrol; (2) CD4; and () the rapid ativation Ag CD69. A total of 15,000 events gated on CD4 expression (top panel) or 7,500 to 8,000 events gated on CD4 and CD69 expression (bottom panel) are shown. Note that ytokine synthesis is restrited to the ativated CD69' subset (whih in this experiment, omprise of CD4' ells at 6 hours). Thus, gating on CD69 allows determination of the ytokineproduing ells as a fration of the ativated population only (16.8% , and 11.2% for 1L2, IL4, and yifn, respetively, in this experiment). Suh gating greatly inreases the auray and reproduibility of ytokineproduing ell quantitation after stimulation with subsetrestrited ativating agents (suh as superantigenl, beause it (1) orrets for differenes in the number of T ells responding to these agents and (2) allows olletion of more positive events (ie, ytokineproduing ells). of IL4produing T ells indued by SEA/SEB (after CD69 perentage (5 SEM; n = 4) of CD69' ells inreased from gating) was as large or larger than that indued by PMA % t I.7% after 6 hours of SEA/SEB stimulation to I (note partiularly the relatively inreased IL4 response in 51.1% 2 5.5% after 18 hours of stimulation. the CD8' subset). SEA/SEBstimulated responses for all Previous studies have established that the soalled virgin three ytokines were transient (peak responses always in the and memory/effetor subsets of CD4' T ells, delineated by 0 to 6hour time period), despite the fat that the mean differential expression of the CD45RA and R0 isoforms, SuperAg Stimulated Peripheral Blood T Cells* (CD69 gated) 0 6 hrs. N r al : L 2 l hrs. N E Y Y : L L CD4 ' Aessory Cell Reonstituted the Fig 4. Flow ytometri haraterization of ytokine synthesis among patterns superantigenstimulated PB T ells. Purified PB T ells were reonstituted with monoytes and stimulated with the baterial superantigens SEA + SEB for 6 and 18 hours (with BFA added during the final 6 hours of ulture). These ells were then assessed for their orrelated expression of intraellular ytokine (IL2, IL 4, yifn), CD4, and CD69 and analyzed after gating on CD69' Tell as subset, in shown Fig (,000 events in eah plot). The perentages in the upper left and right orners of eah plot represent the net perentage (%l of positive I +) ells among the CD4 and CD4' subsets, respetively. Beause the original T ells are highly purified, the CD4 population is essentially equivalent to the CD8' subset in these experiments (the few nonadherent monoytes present at the time of harvest are eliminated by appropriate gating on light satter parameters).

6 CYTOKINESYNTHESISDEFINEDTCELL SUBSETS 141 differ markedly in ytokine gene expression and seretion. In general, the putative virgin Tell subset expresses IL 2 only after appropriate stimulation, whereas the putative memory/effetor subset expresses all three ytokines.2''" Our multiparameter flow ytometri analysis onfirms these data on a singleell basis. Figure 5 shows a representative experiment for PB CD4'T ells after 4 hours of stimulation with PMA + I. Note that CD4' T ells with a surfae CD45ROhigh phenotype inlude the vast majority of T ells produing detetable levels of yifn and IL4. About half of this subset produes IL2 as well, but 1L2 is also produed by about 25% of CD45RO'"" putative virgin T ells. Signifiantly, among SEA/SEBstimulated CD4' T ells, prodution of all three of these ytokines was restrited to the CD45R0'ligh subset (data not shown), likely aounting for the above noted pronouned derease in IL2produing CD4' ells after this stimulus, as ompared with PMA + I stimulation. It is possible that the differenes observed in PMA + I indued yifn prodution by the CD45ROdefined, CD4' Tell subsets in Fig 5 are a funtion of delayed kinetis of y IFN prodution by the CD45RO'"" subset. (This possibility annot explain the observed differential IL4 prodution, beause signifiant IL4 prodution is only present in the 0 to 4hour time period.) To address this issue, we determined yifn and IL2 prodution by CD45RNROdefined CD4' Tells subsets during the 14 to 18hour and 6 to 40hour time periods. Beause ontinuous BFA annot be used for these longer inubation periods (see Materials and Methods setion), we purified CD45RA'(RO) and CD45(RA)RO' CD4' T ells before in vitro stimulation for these studies. As shown in Fig 6, both the CD45RNROdefined subsets responded to the PMA + I at these later time points with a marked upregulation of CD69 and substantial prodution of IL2, yet only the CD45(RA)RO' subset produed signifiant yifn. To examine the degree of overlap between the CD4' memory/effetor Tell populations synthesizing yifn, IL 2, and IL4 after shortterm PMA + I stimulation, we used our flow ytometri proedure to simultaneously analyze pairs of these ytokines among purified memory/effetor T ells, with gating on the CD8 (CD47 subset (Fig 7). The results indiate a strikingly omplex, yet onsistent. pattern of ytokine synthesisdefined, CD4' memory/effetor Tell subsets. In all experiments, the most ommon memory/ef fetor phenotype was the prodution of IL2 alone (0.4% 2 2.0%of ells; n = 5), followed by the IL2 + yifnproduing phenotype (16.4% 2 2.6%), the yifn aloneproduing phenotype (7.7%? 1 S%), and, finally, the phenotype haraterized by 1L4 prodution (4.% 2 0.8%). either alone or in ombination with other ytokines (see below). Kineti studies (Fig 8) indiated the IL2 + yifnproduing subset was relatively shortlived, ompared with the 1L 2 alone or yifn aloneproduing subsets, pratially disappearing by the 22hour time point. Interestingly, the IL4 and yifnproduing CD4' T subsets were predominantly nonoverlapping, whereas. in ontrast, the majority of IL4 produers simultaneously produed IL2 (Fig 7). These results were onfirmed and extended in experiments simultaneously analyzing all three ytokines on PMA + Istimulated Tell populations gated on the IL4produing subset (Fig 9). A total of 51.l% Ir: 1.9% (n = ) of IL4produing T ells also produed IL2; 2.0% 2 4.% of these T ells produed IL4 alone. 16.% 2.5% produed IL4 in ombination with both IL2 + yifn, whereas only 9.6%? 05% produed IL4 in ombination with yifn but not IL 2. To determine whether the ability of CD4' memory/effetor T ells to heterogeneously produe the ytokines under study was a funtion of the nature of the ativating stimulus, the presene or absene of aessory ells, or the site from whih the T ells were obtained, we performed similar multiple intraellular ytokine analyses on superantigenstimulated CD4'/CD45RO' T ells from both PB and from skin blisters overlying utaneous DTH sites. The PB CD4'/CD4SRO' T ells were prepurified by negative seletion, reonstituted with monoytes, and, after SEA+SEB stimulation, analyzed onomitantly for intraellular ytokine and CD69 expression, with ytokine responses measured on the ativated (CD69gated) population. The skin PMA + I Stimulated CD4+ Peripheral Blood T Cells (4 hrs.) g l0 2 g 2 2 n l4 lo1 lo1 lo1 1 2 lnterleukin 2 lnterleukin 4 y Interferon Fig 5. Charaterization of the CD45RO phenotype of ytokineproduing CD4' PB T ells after shortterm PMA + I stimulation. Purified PB T ells were stimulated with PMA + I for 4 hours in the presene of BFA and then assessed for their orrelated expression of ell surfae CD45RO versus intraellular ytokine (IL2, IL4, yifn) and CD8. Representative profiles (.000 events are shown in eah plot) are shown, gated on the CD8 (CD4') subset. The perentages in the upper right and lower right orners of eah plot represent the net perentage (%) of positive 1+1 ells among the CD45ROhigh and CD45RO'"" subsets, respetively. As disussed in the Materials and Methods setion, BFA prevents ativationindued hanges in surfae CD45RO lscd45ro) expression, leaving the scd45ro expression pattern essentially idential to that observed among fresh T ells (data not shown).

7 1414 PICKERET AL A. CD4+/CD45RA+ T Cells U z 1418 hrs hrs. l lnterleukln 2 B. CD4+/CD45RO+ T Cells lo1 2 1 L Interleukln y Interferon.,l I 1 1d2 d y Interferon Fig 6. Charaterization of the CD45RO phenotype of ytokineproduing CD4' PB T ells after longer term PMA + I stimulation. CD45RAhiph/ROi"" and CD45RA"/ROhigh T ells were prepurified by negative seletion, stimulated with PMA+ I for 18 and 40 hours (with BFA inluded the last 4 hours), and then analyzed for their orrelated expression of intraellular ytokine (IL2 and yifn only; 114 is not signifiantly produed by CD4' T ells at these time points), CD8, and CD69. Representative profiles of ytokine versus CD69 (5,000 events in eah plot) are shown, gated on the CD8 (CD4') population. The perentages in the upper right orners of eah plot represent the net perentage (46) of ytokinepositive (+l ells among the ells shown. Note the high CD69 expression by all populations at both time points, onsistent with nearly universal ativation, and the strong prodution of 112 by both the CD45RAhiwL/ROioW and CD45RAtow/ROhigh subsets; yet, only the CD45RAioW/ROhioh (CD4') T ells pro due signifiant yifn. blister T ells were simultaneously analyzed for two ytokines and CD4, with gating on the CD4' population. These skinderived T ells are essentially all CD45RO' to begin Withl.lS and show an intrinsi twofold to fourfold higher response to SEA + SEB, obviating the need for CD69 gating (data not shown). As shown in Figs IO and 1 I, the pattern of ytokinedefined Tell subsets generated by superantigen stimulation of PB and utaneous T ells are remarkably similar to eah other and to the pattern observed with PMA + I, showing in eah irumstane a similar relative proportion of ells synthesizing eah of the three studied ytokines, alone and in ombination. DISCUSSION The onept of ytokine synthesis heterogeneity among memory/effetor Tell populations was initially suggested by data in the mouse indiating that otherwise similar, loned CD4' T ells differed in their ytokine prodution apabilities S.8IO.. Two general patterns were observed: (I) the TH 1 pattern haraterized by IL2, yifn, and tumor nerosis fator 0 (TNF0) prodution; and (2) the TH2 pattern haraterized by IL4, IL5, IL6, ILIO and (later) ILl prodution. The physiologi relevane of these THI and TH2 T ell lones was suggested by the demonstration of polarized THI and TH2like ytokine responses in different in vivo situations that frequently orrelated with immune protetion against a variety of infetious agent^.".'^*'^ More reently, analysis of Tell lones in the human have shown an analogous, although not idential, ytokine synthesis heterogeneit^.".^ In addition, bulk ytokine analysis tehniques (measurement of sereted ytokines and ytokine mrna from omplex, nonlonal populations), as well as enzymelinked immunospot assays that allow determination of the number of ells sereting a single ytokine, have indiated that in vivoderived human Tell effetors from different disease states or immune situations are often assoiated with different relative ratios of ytokine gene expression and/or seretion.?9' However, despite the wealth of data on polarized ytokine responses in both mie and humans, methodologial limitations have preluded a detailed understanding of the ytokine synthesis potential of single in vivoderived T ells in either speies. The vast majority of studies addressing the ability of individual ells to simultaneously produe two or more ytokines have relied on the study of Tell lones that have, by neessity, spent weeks to months in vitro before analysis. Although it is unlear the extent to whih bias is introdued by this long period of ell ulture, it is possible that true in vivo Tell phenotypes may be modified during Tell loning by both seletion (only those ells apable of extensive ell division are evaluated) and the inadvertent regulation of ytokine synthesis phenotype. The latter would potentially be mediated by inidental ytokines that may have been added to the ulture media to failitate Tell outgrowth or produed by the ativated T ells themselves and/or their aessory ells during the loning proess. We reasoned that one feasible methodologial approah to this problem was multiparameter immunofluoresent analysis of seretioninhibited, shorttermativated, normal T ells. This tehnique allows simultaneous assessment of the synthesis of multiple ytokines by T ells that have been ultured in vitro as few as 4 hours. Both the limited time spent in vitro and the seretion inhibition (eg, BFA treatment) greatly diminishes the possibility that Tell or aessoryell ytokines ould modulate the ytokine response patterns of the T ells. Moreover, beause proliferation isnot a requirement for a responding ell, and beause in most experiments there is little or no ell death during the shortterm ativation period (L.J. Piker, unpublished observations), seletion is unlikely to

8 CYTOKINESYNTHESISDEFINEDTCELLSUBSETS 1415 CD4+/CD45RO+ Peripheral Blood T Cells (PMA + I Stimulated; 4 hrs.) Experiment #l l0 g z 2 ( Y r S E: 1 lnterleukln 2 lnterleukln 4 lnterleukln 4 Fig 7. Charaterization of patterns of mukiytokine prodution by PMA + lstimulated CD4+l Experiment #2 CD45ROhIQh PB T ells. CD45RA"""lROh'Qh T ells were prepurified by negative seletion, stimulated with PMA+ I for 4 hours in the presene of BFA, and then ( Y analyzed for their orrelated expression of intraellu 2 5 lar pokines (yifn versus IL2; yifn versus IL4, f 2 r: 2 and IL2 versus IL4) and CD8. Two representative 7 2 Q) e I experiments are shown with,000 events in eah + l 0. I lo1 E 1 plot, gated on the CD8 (CD4') population. The perentages in the orners of eah plot represent the net 1 perentage (96) of positive (+l ells in the appropriate 2 quadrants (ie, the +I. +I+, subsets). I+ lnterleukln 2 lnterleukln 4 lnterleukln 4 signifiantly bias the observed results. Finally, this tehnique allows simultaneous orrelation of ytokine synthesis apabilities with other surfae or ytoplasmi phenotypi markers, allowing determination of the ytokine synthesis potential of preisely defined Tell subsets. In the first part of this report, we showed the ability of multiparameter flow ytometri ytokine analysis to determine the ytokine synthesis apabilities of the CD4', CDS', CD4'/CD45RO'"", and CD4'/CD45ROhigh Tell subsets. Our results indiate that the relative ytokine prodution apabilities of CD4' and CDS' T ells are in the order of IL2 > yifn > IL4 and yifn > IL2, respetively. In some experiments, we also identified a small IL4produing CDS' Tell subset, onsistent with reent reports indiating the existene of suh ells."".4 Both the absolute and relative perentages of IL2, IL4, and yifnproduing CD4' versus CDS+ T ells found in our study were remarkably similar to the perentages reported by Lewis et al.'' using in situ hybridization tehniques. Also onsistent with previous reports is our observation that, among CD4' T ells, the CD45ROhiPh putative memory/effetor subset aounts for the preponderane of IL4 and yifn prodution, whereas both the CD45ROhiPh and CD45RO'"" virgin subset an produe 1L2.2~2"s" Both our study and the few others in the literature using tehniques apable of measuring ytokine expression/prodution on a perell strongly suggest that these subsetbased differenes in ytokine prodution are largely attributable to differenes in the frequeny of ells apable of produing signifiant levels of these ytokines, as opposed to differenes in either the amount of ytokine produed per ell or the kinetis of prodution. For example, the higher yifn prodution by CD4+/CD45ROhigh T ells as ompared with that by CD4'/CD45R01"" T ells2' was not due to homogeneous high prodution of yifn by the former versus homogeneous low prodution by the latter subset, but rather to the ratio of yifnproduing and nonproduing ells in eah population. Indeed, for any given T ell subset (CD4 versus CD8; CD45ROhiph ), the number of IL2, yifn, and IL4produing ells generated in response to either PMA + I or superantigen was a onsis 2 CD4+/CD45RO+ Peripheral Blood T Cells (PMA + I Stimulated) 04 hrs hrs. ( Y 2 2 ti E lo1 I, 1 21d lo1 y Interferon Fig 8. Kinetis of mukiytokineproduing (yifn versus IL2) CD4+/CD45ROhigh PB T ells after PMA + I stimulation. CD45RA'owlROh'Qh T ells were prepurified by negative seletion, stimulated with PMA+ I for 4,22, and 46 hours with BFA added during the final 4 hours of ulture, and then analyzed for their orrelated expression of yifn, IL2, and CD8. Representative profiles of ylfn versus IL2 (,000 events in eah plot) are shown, gated on the CD8 (CD4') population. The perentages in the orners of eah plot represent the net perentage (%) of positive (+) ells in the appropriate quadrants lie, the +I, +I+, I+ subsets).

9 1416 PICKER ET AL Forward Light Satter Experiment #2 g E Forward Light Satter y Interferon gate Forward Forward Satter Light Satter Llght 1 2 y Interferon Fig 9. Charaterization of yifn and IL2 prodution by IL4 produing PB T ells. Purified PB T ells were stimulated with PMA + I for 4 hours in the presene of BFA and then analyzed for their orrelated expression of yifn, IL2, and 114. Two representative experiments are shown. In the left and middle panels (forward light satter versus 114 or isotypemathed ontrol), the overall Tell subset is shown with the positive ells olored blak, and the remaining T ells gray (28,000 to 0,000 events shown). Speifi IL4 staining (after subtrating bakground) was 1.2% and 1.% for experiments no. 1 and 2, respetively. The right panels show the yifn versus 112 profiles of the IL4' ells only (2.000 events shown; gated on the blakolored ells in the middle panel). As previously shown (Figs 1 and 5; and onfirmed in parallel analyses in these experiments for CD41, under these onditions, a signifiant 114 response is only found in the CD4/CD45ROhigh Tell subset, obviating the need for subset prepurifiation or gating on these parameters in these experiments. The perentages in the orners of the gated plots represent the perentage I%) of positive (+I ells in the appropriate quadrants (ie, the +I, +I+, /+ subsets for IL2 and yifn, with the I population onstituting the remaining ells). Note (as previously indiated in the text) that the vast majority of IL4produing ells have the light satter harateristis of small lymphoytes (low forward light satter), onsistent with ytokine synthesis predominantly preeding blastogenesis during the ativation response. tent fration of the overall ativated (CD69') Tell population. These ytokineproduing T ells manifested harateristi levels of intraellular ytokine, and they appeared and disappeared with preditable kinetis. The differenes in the overall number of CD4'/CD45ROh'Fh T ells produing IL2 versus yifn in these initial studies strongly suggested a divergene from the predited IL2'/ yifn+ phenotype of THI memory/effetor T ells. Simultaneous evaluation of two to three ytokines within the same ells onfirmed this divergene and showed the existene of distint subsets of memory/effetor T ells that manifested synthesis of all possible ombinations of the three ytokines assessed. Most ommon were ells produing IL2 alone (0%). Classi THI (IL2'/yIFN'/IL4) and TH2 (IL2/ yifn/ll4') patterns were displayed by only a fration (16% and <I%, respetively) of the PMA + Iativated, memory/effetor T ells. Interestingly, the TH llike IL2'/ yifn' subset generated by PMA + I appeared only transiently at the 0 to 4hour time point; by 18 hours, most of this subset either disappeared or onverted to single IL2 or yifnproduing ells. Among the small, transientlyappearing IL4produing subset, 75% also produed IL2, yifn, or both. Taken together, these data learly indiate that there is no obligate pairing of the ability to produe these three ytokines during the development of memory/ effetor Tell funtion and strongly suggest that the expression potential of eah of these three ytokines is independently regulated. Similar onlusions were drawn by Assenmaher et al" who used multiparameter flow ytometry to analyze ytokine expression patterns among murine spleni CD4+ T ells stimulated with SEB. Beause ytokine synthesis phenotypes are thought to initially develop during the Agindued differentation of virgin to memory/effetor T ells in seondary lymphoid myriad the ytokine synthesisdefined Tell subsets observed in this study are onsistent with a highly omplex spatial and/or temporal regulation of this differentiation proess. The relative rarity of IL4/yIFN doubleproduing memory/effetor T ells suggests a relatively nonoverlapping, independent regulation of these two ytokines, as opposed to a likely more promisuous regulation of IL2. We further showed that the pattern of ytokinedefined subsets was qualitatively similar after (l) PMA + I stimulation of aessory elldepleted PB memory/effetor T ells, (2) superantigen stimulation of monoytereonstituted PB memory/effetor T ells, and () superantigen stimulation of skin blisterderived memory/effetor T ells thatwere stimulated in the ontext of skinderived aessory ells. Preliminary data also indiate that Tell stimulation with anticd plus anticd28 yields analogous ytokineprodu

10 CYTOKINE SYNTHESISDEFINEDTCELL SUBSETS 1417 CD4+/CD45RO+ Peripheral Blood T Cells* (SuperAg Stimulated; 6 hrs.; CD69 gated) A. Experiment #l N E 7 2 E 1 Fig. Charaterization of patterns of mukiytokine prodution by superantigenstimulated CD4'1 CD45ROhiph PB T ells. CD4'ICD45RA"/ROhigh T ells were prepurifed by negative seletion, reonstituted with monoytes, and then stimulated with the baterial superantigens SEA + SEB for 6 hours in the presene of BFA. These ells were then assessed for their orrelated expression of intraellular ytokines (y IFN versus IL2, yifn versus IL4, and IL2 versus IL 4) and CD69. Two representative experiments are shown with,000 events in eah plot, gated on the CD69' population, as shown in Fig 2. The perentages in the orners of eah plot represent the net perentage (YO) of positive (+) ells in the appropriate quadrants (ie, the +I, +I+, I+ subsets) $ Interleukin 2 Interleukin 4 lnterleukln 4 B. Experiment #2 't l02 Q) + l 1 2 Interleukin 2 Interleukin 4 lnterleukln 4 Aessory Cell Reonstituted ing subsets (L. Piker and B. FergusonDarnell, unpublished portant beause ytokine synthesisdefined memory/effetor observations). These findings strongly suggest that, in the Tell subsets have been shown to differ in their ativation setting of a fully ativating stimulus, the observed heteroge requirements,'*.." making it possible that some threshold neity of memory/effetor Tell ytokine prodution is an stimuli may differentially ativate a partiular subset and not intrinsi harateristi of the T ell and not a funtion of the another, thus giving the false appearane of a shortterm, regulatory influenes of the ativating signal, or the ativat funtional plastiity among memory/effetor T ells.) The ing miroenvironment. (The aveat "fully ativated" is im stability of this memory/effetor Tell ytokine program Experiment #l CD4+ Skin Blister T Cells: DTH Site (SuperAg Stimulated; 8 hrs.) Interleukin 2 Experiment #2 5 2 C E 5 2 L : : F lo1 x lnterleukln lnterleukln b Interleukin lnterleukln lnterleukln 4 Fig 11. Charaterization of patterns of mukiytokine prodution by superantigenstimulated CD4'1 CD45ROhiSh utaneous T ells. Leukoytes obtained from skin blisters overlying DTH reations (against mumps virus Ag) were stimulated with the baterial superantigens SEA + SEB for 8 hours in the presene of BFA. These ells were then assessed for their orrelated expression of intraellular ytokines (yifn versus IL2, yifn versus IL4, and IL2 versus IL4) and CD4. Two representative experiments are shown with,000 and 5,600 events in eah plot in experiments no. 1 and 2, respetively; both gated on the CD4' T ells population. Beause of the twofold to threefold inreased reativity to baterial superantigens manifested by these skinderived T ells versus that of PB T ells, it was not neessary to use CD69 gating to effetively visualize the ytokine response of this predominantly (>95%) CD45ROhigh skin Tell population. The perentages in the orners of eah plot represent the net perentage (YO) of positive (+) ells in the appropriate quadrants (ie, the +I. +I+. I+ subsets).

11 1418 PICKER ET AL ming has reently been attested to in experiments showing that the speifi ytokine synthesis phenotype of adoptively transferred murine Tell lones are retained for prolonged periods in vivo.4o It is important to note that, although we feel that our data suggest that the ability of memory/effetor T ells to synthesize IL2, IL4 and yifn is independently preprogrammed, they do not impiy that a11 the ytokinedefined T ell subsets identified in this report are independent of eah other. Beause it has been suggested that mouse TH1 and TH2 memory/effetor T ells differentiate via THO (IL2+/ IL4'IyIFN+) or, in some instanes, via intermediates that produe IL2 alone:' it is possible that the analogous human subsets reported here may represent intermediate forms that will terminally differentiate at some future enounter with Ag t other relevant miroenvironmental onditions. However, the presene of these putative memory/ effetor Tell intermediates among irulating resting T ells and among T ells in inflammatory sites suggests that they an be fully ontributory to Tell mediated immune responses, and, thus, their putative intermediate status should not be onfused with lak of funtionality. The tehnial simpliity and rapidity of the flow ytometri intraellular ytokine detetion tehnique desribed in this report, as well as the widespread availability of appropriate flow ytometers and Tell phenotyping antibodies in linial laboratories, suggest the possiblity that this tehnique ould be broadly appliable to the linial evaluation of immune status. For example, previous studies (using more umbersome immunologi assays) have suggested that alteration of Tell effetor funtion may be an earlier marker of progressing human immunodefiieny virusassoiated immune dysfuntion than the absolute loss of whole Tell subsets (eg, the CD4+ s~bset).~' Our observation of qualitatively and quantitatively onsistent patterns of memoryleffetor Tell ytokine expression in normal subjets offers the possibility that hanges in these patterns may preede and predit the linial manifestations of progressive immunodefiieny. Therefore, flow ytometri quantitation of the ytokinedefined memory/effetor Tell subsets desribed in this report may prove to be an invaluable aid in both the diagnosis and monitoring of pathologi immunodefiieny and therapeuti immunosuppression. ACKNOWLEDGMENT The authors gratefully aknowledge the invaluable assistane of the following individuals: Drs A. Levine (Monsanto CO, St Louis, MO), H. Yssel (DNAX Researh Institute, Palo Alto, CA), and H. Gaylord (R&D Systems, Minneapolis, MN) for providing L4 transfetants, Tell lones, and antiytokine MoAbs, respetively; A. Yin, K. Teall, B. FergusonDarnell, andj. Bass for tehnial assistane, J. White, B. Reynolds, and Dr K. Houpt (UT Southwestem Medial Center, Dallas, TX) for assistane with the olletion of skin blister leukoytes; and Dr K. Davis (BDIS, San Jose, CA) for the preparation of diretly fluorohromeonjugated antiytokine MoAbs. REFERENCES 1. Piker LJ, Siegelman MH: Lymphoid tissues and organs, in Paul WE (ed): Fundamental Immunology (ed ). New York, NY, Raven, 199, p 145 (hapter 6) 2. Makay CR Migration pathways and immunologi memory among T lymphoytes. Semin Immunol 451, Piker W, Buther EC: Physiologial and moleular mehanisms of lymphoyte homing. Annu Rev Immunol :561, Piker LJ: Control of lymphoyte homing. 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