Scund. J. Immunol. 32, 687 694. 1990

Immunological Marker Analysis of Mitogen-Induced Proliferating Lymphocytes Using BrdUIncorporation or Screening of MetaphasesStaphylococcal Protein A is a Potent Mitogen for

^ Lymphocytes

H. J. ADRIAANSEN. C. OSMAN, J. J. M. VAN DONGEN,J. H. F. M. WIJDENES-DE BRFSSER.C. M. J. M. KAPPETIJN-VAN TILBORG & H. HOOIJKAAS

Department of Immunology. University Hospital Dijkzigt and Erasmus University, Rotterdam.The Netherlands

Adriaansen, [|,J.. Osman, C , van Dongen. J.J.M.. Wijdcnes-dc Bresser, J.H.F.M.. Kappctijn-van Tilborg. C.M.J.M. & Hooijkaas, H. Immunological Marker Analysis of Mitogen-InducedProliferating Lymphocytes Using BrdU Incorporation or Screening of Metaphases.Staphyiococcal Protein A is a Potent Mitogen for CD4' lymphocytes. Stand. J. Immunol. 32,687-694. 1990

The proliferalive ctlects of the mitogens phytohaemaggliitiniri (PHA). concanavalin A (Con A),pokewced mitogen (PWM). and slaphylococcal protein A (SpA) were invcstigalcd using twodifferent methods which enable immunologies I marker analysis of proliferating eells: eilhersurface marker labelling followed by BrdU incorporation or screening of metaphases aftersurface marker labelling. Therefore peripheral blood mononuclear cells from si\ healthyvolunteers were stimulated with these four mitogens. Botli PHA and t'on A gave rise to moreCD8 ' than CD4 ' proliferating cells. PHA, but not Con A, induced B-eell proliferation as well.PWM mainly caused T-cell proliferation. SpA also appeared lo be a poleiit T-cell mitogen inaddition to its capacity to induce B-cell proliferation. However, in contrast to the other milogensSpA predominantly stimulated CD4^ eells.

H. J. Adriaansen. MD. Deptirinwni of Immunology. Erasmus I'nitersiry. PO Bo.\ I73H. 300(1 DRRotterdam. The Netherlands

In vitro mitogenic activation is widely used toinvestigate the functiona! properties of humanperipheral blood celts. The evaluation of mito-genic responsiveness is especially of interest incases of immunodeticiency. Most often mitogenssuch as the plant lectins phytohaemagglutinin(PHA), pokeweed mitogen (PWM). and concana-valin A (Con A) are used. Staphylococcus proteinA (SpA). a protein present in the cell wall oC manySiaphylococcu.s aureus strains, can also be used tostudy mitogenic responsiveness [18. 28].

Incorporation of PH]thymidine by the prolifer-ating cells is often used to measure the responsive-ness of mononuclear cells (MNC) to these mito-gens. Using this technique, it is not easy toinvestigate which specific cell subset responds.

Approaches that have been used to determinewhich cells proliferate include separation of sub-populations either before or after in vitro cultureby cell sorting, panning, or lysis [19. 25. 28, 32].Other approaches make use of double-markeranalysis of single cells. In this way the simulta-neous presence of both certain differentiationmarkers and cell activation markers, such asHLA-DR. the transferrin receptor, and the IL-2receptor, can be demonstrated [6, 17. 30]. How-ever, these activation markers are probably notrestricted to proliferating cells [20].

Definite markers lor cell proliferation are thosemarkers which arc only expressed during specificphases of the cell cycle, e.g. the Ki-67 antigen andPCNA [8. 9. 21]. DNA content can also be

687

688 H. J. Adriaansen ct a!.

regarded as a marker for eell cycle phase [5, 22],Furthermore, cells in S phase of the cell cycle canincorporate the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU). which can be detected byanti-BrdU monoclonal antibodies (MoAb) [4. 11.24). Finally, metaphase cells can be regarded asthe best marker for the M phase of cell cycle.

We here describe two methods to determine theimmunoiogical phenolype of proliferating cells:BrdU incorporation and metaphases as markersfor prolileration. We investigated which cellsproliferate after stimulation with PHA. PWM.Con A. and SpA.

MATERIALS AND METHODS

Isolation iijnwmnnulear (.ells. Periphcfitl blood (PB)MNC were obtained from hep;irinized venous blood ofsix healthy volunteers, aged 25 .15 years, by FieollPaque (1,077 g/cm"; Pharmacia. Uppsala. Sweden)density cenlrifugation. Cells were washed twice inRPMI 1640 cuhure medium wilhoul Hepes. supple-mented wiih L-glutamincH mM) penicillin (100 lU/ml).streplomycin(50/jg/ml).and \5"'<-< heat-inaclivated fetalcalFserum (FCS), The KCS was specifically selected forits growth-supporting properties and low endogenousmitogenic activity.

Iniiminohgiiul marker analysis. To detect surfacemembrane markers, the cells were incubated withoptimally titrated relevant MoAb as described [33], Theimmurolonica! markers used were CD20 (Bl; CoulterClone. Hialeah. Flii USA). CD37 (Y2'J,.«; Dr H.K-Forster. Basel, Switzerland) to label B cells. CD3 (Leu-4; Becton Dickinson. San Jose. Calif,. USA). CD4 (Leu-3: Becton Dickinson), and CD8 (Leu-2; Beclon Dickin-son) to label T cells; CD14 (My4; Coulter Clone) andCD15 (VIM-fX'̂ ; Dr W, Knapp. Vienna. Austria) tolabel monocylic eells and myeloid cells respectively.Natural killer (NK) cells were labelled by useofCD16(Leu-l Ib; Becton Dickinson)andCD57(Leu-7; BectonDickinson); in addition, CD25 (2A3. anti-IL-2-recep-tor; Becton Dickinson); CD71 (66IGI0. anti-transferrinreceptor; Dr M. van Rijn. Amsterdam. The Nether-lands), and anti-HLA-DR (L243; Becton Dickinson)were used to label cells. The reactivity ofthc MoAb wasvisualized by use of a fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse immunoglobulin(Ig) antiserum (Centra! Laboratory of the Blood Trans-fusion Service, Amsterdam. The Netherlands). Thefluorescence stainings were evaluated with Zeiss micro-scopes equipped with phase-eontrast facilities (CarlZeiss, Oberkochen. FRG) [33],

Ciihure systems. For all cell culture experimentsMNC weie adjusted to a final concentralion of 5 x 10''cells per ml. Cells were cultured in the same RPM! 1640medium as was used for the isolation of MNC,Mitogens added in optimal concentrations were: PHA(100 ;jg;ml; Wellcome Diagnostics. Dartford. UK).Con A (100/jg/ml; Sigma Chemical Co,. St Louis. Mo,.USA). PWM (10 /(g/ml: Gibco Laboratories. GrandIsland. NY. USA). SpA (25 /ig,.ml; Pharmacia), As a

control, cells were cultured without mitogens. For themeasurement of ['H]thymidine incorporation MNCwere cultured in 9(i-well llat-bottom tissue culture plates(lO'̂ cells per well; Costar. Cambridge. Mass,. USA),For the other experiments cells were cultured in tubes(,ix lO*" cells per tube; Corning. New York. USA),Incubation was performed at 37 C. al lOO'l̂ ) relativehumidity, and at a P((>: of 5"-..,

l^H jlhymidine incorporation. MNC from fourdonors were cultured for 2. 3. 4. 5. 6. and 7 days,[^H]thymidine (specific activity 6,7 Ci/mmol; Amer-sham International. Amersham. UK) pulsing was for 6h using 0.5 /iCi per well. After the 6-!i pulse the cells wereharvested using an automatic cell harvester (Skatron.Lier. Norway), ['H]lhymidine incorporation was mea-sured in a liquid scintillation counter (Packard,Downers Grove. III.. tJSA). Each determination wasperformed in triplicate. The results are given as mediansof ihe results obtained in the four donors,

Immtinolo^icLiI marker analysis of nwlupbase cells.MNC from twodonors were cultured wiih PHA or SpAfor 2. 3. 4. 5. and 6 days. Cells were centrifuged andresuspended at a concentration of lO' cells/ml, Fiflymicrolitres of the cell suspensions was incubated atroom icmpcrature with 50 /il of the relevant MoAb,After 15 min the cells were washed twice and 50 /d ofatetramethylrhodamine isothiocyanate (TRITC)-conju-gated goat anti-mouse Ig antiserum (Central Labora-tory of the Blood Transfusion Service) was added; toarrest the cells in melaphase 0,25 /ig Colcemid (GibcoLaboratories) was added. After another 15 min the eellswere washed in a hypotonic KCl solution (O,O75M)andcytocentrifuge preparations were made (Nordic Immu-nological Laboratories. Tilburg. The Netherlands), Theslides were put in atebrinc (Gurr. Poole. UK) dissolvedinethanol 70"ii (vol/vol) for 10 min to fix the cells and tostain DNA, The alebrine staining was evaluated byusing the same filter combination as used for KITC. andthe expression of (he immunoiogical markers wasevaluated by using a TRITC filler combination [33],When possible, up to 100 metaphase cells were evalu-ated for each immunoiogical marker. The percentage ofceiis in metaphase was determined by screening 2000MNC,

BrclV incorptiration mul immunnlogiialmarker analy-sis iif BrdU^ cells. MNC from ihe same four donors aswere used for the [ni]thymidine incorporation experi-ment were cultured with PlIA. Con A. PWM. or SpAfor 2. 3. 4. 5. 6. and 7 days. To label ihe cells in ihe Sphase of the cell cycle. BrdU (Sigma) was added to afinal concentration of 10 /iM. 30 min before harvesting.After harvesting, cells were washed twiee. incubatedwith MoAb for 30 min to label a surface membranemarker, and washed again. Subsequently 50 ^1 of agoal-anti-mouse Ig antiserum conjugated with colloidalgold particles of 5 nm size was added (Janssen Pharma-ceulica. Beerse. Belgium), After 30 min the cells werewashed twice and a cytocentrifuge preparation wasmade. The slides were air-dried. fl,xed in ethanol 70%(vol/vol) for 30 min. and denatured in NaOH 0,07 Mfor2 min. After washing in Na;H>BO,i. the slides wereincubated with a FITC-conjugated anti-BrdU MoAb(Beclon Dickinson) for 30 min in a moisl chamber.After two washings the immunogold-labelled slideswere subjected to a silver enhancement procedure as

Marker Analvsi.s of Milogen-Iiiiluveil Prolifer(tlitii> Lymplmcyle.s 689

specified by Jans.sen Pharniiicciitica. We used a silverdevelopment lime or25 min, lor ihe visualization oflhesilver granules we used diii-illuminatioii or. in ihe caseof weak slaining. epi-illuminaliori wiih polari/aliorifillers. The percentage of cells having incorporatedBrdU was determined by screening 400 MNC. Fordouble-marker analyses with ditTerentlaiion markers200 BrdU^ cells were evaluated.

After 6 days of culture cells were tested for cyiophis-mic Ig (Cylg) expression as previously described |3.'̂ ].Cytocentrifuge preparations were simultaneously incu-bated wiih an FITC-conjugated burro anti-human Ig-lambdii antiserum and a TRITC-coiijugaled goat anti-human Ig-kappa antiserum (Kallestad Laboratories,Austin, Tex,, USA).

RESULTS

Immunological marker analysis

The MNC Iraclion from Ihe PB of all donorscontained 5-10% B cells (CD20\ CD37*). 506O'M> T cells (CO3 +) with a CD4/CD8 ratio whichvaried from I.I to 1.8. <\'Vu granulocytic cells(CD I 5 * K and I 5-25% monocytic cells (CD 14 ' ) .In addition, about lO";. of (he cells were positivefor NK markers such as CD16 (Leu-llb) andCD57 (Leu-7), The percentages of positivity forHLA-DR reflected the sum of B cells and mono-eytic cells. Cells expressing CD7I (translerrinreceptor) and/or CD25 (IL-2 reeeplor) were onlydetected at levels of < l"/.i.

f'HJlhymidine imoiporalion

The results of ['Hjthymidine ineorporation onmitogenic stimulation are summarized in Fig, 1.left. Each point represents the median count fromvalues obtained in four donors. The kineticsfound in the four donors were similar lo thekinetics shown in the compilation. The maximumPHA-indueed ['HJthymidine incorporation wasfound on days 3-5 (71.000-84,000 cpm). For ConA, maximum incorporation was found on day 4in all donors (56.000-71.000 cpm). After PWMstimulation the maximum incorporation (23,00052.000 cpm) was found on day 4 in one donor andon days 6 and 7 in the other three donors. ForSpA the maximum incorporation was found ondays 5 and 6 (63,000 113.000 cpm). Without theaddition oi mitogens, ['H]thymidine ineorpora-tion was < 1000 cpm up today 5 and < 3000cpmon days 6 and 7.

di^icul marker analysis of melaphase

The results of the immunological markeranalysis of metaphases after stimulation withPHA and SpA are summarized in Fig. 2. Eachpoint represents the mean value of the countsobtained in two donors. The kinetics were similarin both donors. The maximum of PHA stimula-

days in culture days in culture

FIG. I, ['H]thymidine incorporation (left) and BrdU incorporation (right) of PBMNC" uponmilogenicstimulation with PHA ( ), Con A (••:;• v,:0, PWM ( ).andSpA(::-:-:wx ). Each pointrepresents the median count from values obtained in four donors.

690 H. J. .'idrhmn.sen ct al.

IS

SpA

10

5-

2 3 4 5

days in culture

2 3 4 5

days in culture

fiti, 2, Immunological marker analysis of metaphases after stimulation of PBMNC(left) and SpA (right). The total number of metaphases (-xw.-) is shown as well as theCD4+ metaphases (••••), CD8' melaphascs ( )andCD20+ metaphases("••point represents the average of two donors.

with PHAnumber of

). Each

tion was found on day 4. On this day about 35"4>of the metaphases were CD4 ' , about 55'V,. wereC D 8 \ and about 10% were CD20*. Uponstimulation with SpA the majority of the prolifer-ating eells was CD4 '. The maximum percentageof cells in mctaphase was found on day 5. At thispoint about 65".. of the metaphases were CD4'and about 35% were C D 8 \ A small proportionof proliferating B eells was detected on days 3 and4.

BrdU incorporalion and immunological markeranalysis of BrdU' cells

The results of the incorporation of BrdU andthe immunological marker analysis of BrdU*cells after stimulation with PHA. Con A, PWM.and SpA are summarized in Fig. I, right, and Fig.3 respeetively. Eaeh point represents the medianof the results obtained in four donors. Thekinetics found in the four donors were quitesimilar. Maximum stimulation, i.e. the highestpercentage of cells in S phase of the cell cyele, wasfound for Con A on day 2. for PHA on day 3. forPWM on day 7. and for SpA on day 6 (Fig. 1,right). First we tried to label the surface mem-brane differentiation markers with a TRITC-conjugated antibody, however, probably becauseof the denaturation and fixation procedure

necessary for visualization of BrdU, the TRITCsignal was too weak. Therefore immunogoldlabelling with silver enhancement was used forlabelling the surfaee membrane markers.

In all but one donor PHA and Con A gave riseto more CD8 • than CD4- proliferating eells. Inaddition, distinct proliferation of B cells wasobserved on days 3 and 4 of PHA culture, whileB-eell proliferation after Con A stimulation wasminimal (Fig. 3. upper part). Stimulation withPWM resulted mainly in proliferation of T cells,whieh were slightly more often CD4 • thanCD8 ' , and to a lesser extent in proliferation of Bcells (Fig. 3. lower left). Upon stimulation withSpA inalldonors the majority of the proliferatingcells was CD4 ' . B-cell proliferation was observedon days 2 and 3 only (Fig, 3. lower right).

Determination of other markers revealed thaton all days of culture NK markers such as CDI6and CD57 were expressed on about 10'!'., of theproliferating eells after PHA or Con A stimula-tion and about 5% of those eells after stimulationwith PWM or SpA.

Activation markers were expressed by theBrdU' eells as well as BrdU" eells. Thesemarkers were not expressed by all BrdU"^ cells.Positivily for HLA-DR on Ihe BrdU^ eells wasfound in roughly the same pereentages as positi-vity for CD20. indieating that these HI_A-DR\

Marker Analy.sis of Mito^en-fiulticcd ProUlerating Lymphocytes 691

30

25

b 20•aQQ

10

0

30

25

20

15

l O '

5-

PHA

2 3 4 5 6 7

PWM

days in culture

25

20

15

10

30

25-

20

15

10

Con A

s

2 3 4 5 6 7

SpA

3 4 5 6

days in culture

FKJ. 3. Immunological marker analysis of BrdU ^ cells after stimulation of PBMNC with PHA(upper left). Con A (tipper righ!). PWM (lower left), and SpA (lower right). The percentage of lotalBrdU ' cells (. yw ) is shown as well as the percentages of C D 4 ' . BrdU' cells (-•••). CDS*.BrdU " cells ( ), and CD2()'", BrdU"^ cells ( }. Each point represents Ihe mediancount from values obtained in four donors.

BrdU ' cells were probably proliferating B cells.CD71 (transferrin receptor) atid CD25 (IL-2receptor) were found on 60-80'^ of the BrdU ^cells on day 3 and 30-60"n of the BrdU ' cells onday 7.

Without the addition oK mitogens <^\"As ofBrdU ' cells were seen on days 2-6. On day 7 ofthe cultures. 2'Mi of BrdU ' eells were found in twodonors, while in the other two donors about 7".>of the cells had incorporated BrdU. Labelling o'ithe eells revealed that on dav 3 about 35"ii of the

BrdU+ eells were B cells and aboul 50";> were Tcells. In addition some monocytic cells (CDI4^)and NK cells {CDI6^ CD57 + } were proliferat-ing. On day 7 the majority of the proliferatingeells was CD4 T cells.

To investigate whether cells coexpressed CD4and CDS. as has been reported by others [2, 30].double-marker analysis for these two markerswas performed in two donors. It was not possibleto perform a triple immunological marker analy-sis for BrdU and these two T-cell markers. In all

692 H. J. Adriaan.ien el al.

cultures CD4 ' . CD8' cells were seen, howevermostly the percentages were I "<i or lower. In onedonor, about 2'''i) of CD4 ' CDS* eells were seen 6days after stimulation with PHA.

After 6 days of stitnulation with PWM about1-5% of the MNC expressed Cylg. in the culturesstimulated with PHA. ConA. or SpA, and in theeultures without a mitogen. the proportion o{Cylg* cells was less than 0.1 "'';i.

DISCUSSION

We here describe two diiferent methods to deter-mine the immunological phenotype of proliferat-ing eells. The method which enables itnmunologi-cal marker analysis of metaphase cells wasoriginally developed to combine cytogeneticanalysis and immunologieal marker analysis atthe single-eel! level [13]. Using this method wefound that PHA is a better mitogen for CD8 *PBMNC Ihan for CD4' PBMNC. while SpAmainly stimulates CD4-^ eells. To confirm thisfinding and to eompare the kineties of PHA andSpA with those of Con A and PWM. we used atechnique whieh was less laborious and whiehenables the analysis of eells in S phase of the eeilcyele, i.e. a double-marker analysis for BrdUineorporation and a differentiation marker.

Using the BrdU method, maximum percent-ages of cells in S phase were found on day 2 forCon A and on day 3 for PHA {Fig i. right). On theother hand, using ['H]thymidine incorporation.the kineties for these two mitogens seemed to hedifferent. The maximum incorporation for Con Aand PHA was found on day 4 and day 5respectively (Kig 1. left). This discrepancy isreadily explained, since [*H]thymidine incorpora-tion is measured over the whole population ofeells, while in the BrdU technique the percentageof cells in S phase is determined. Owing to the risein the tolal cell number during culture, theabsolute number of proliferating eells is maximalon day 4 or day 5 in Con A and PHA cultures,while the maximum proportion of proliferatingcells is already found by day 2 or day 3 respeet-ively. For SpA and PWM this discrepancy wasnot observed, probably because of the fact thatthe proliferation peak oeeurs at a later stage (day5-6), when the culture medium becomesexhausted.

The results of the immunological markeranalysis of BrdU' eells after PHA and SpA

stimulation confirmed the results obtained by themetaphase technique. The preponderance ofCD8^ proliferating eells over CD4*^ proliferating

others using different methods [3. 6. 30]. Con Aalso appeared to stimulate more CD8+ eells thanCD4 ' cells. This is in contrast with the tindings ofPersson & Johansson [25]. However, they separ-ated the T-cell subsets prior to mitogenic stimula-tion. A major difference between PHA and Con Awas the finding of a signifieant B-eell proliferationupon PHA stimulation, whieh was minimal afterCon A stimulation. B-eell proliferation by PHAhas previously been reported [4. 22, 23]. Interest-ingly this PHA-indueed B-eell proliferation tookplace early (days 3 and 4). which suggests a directeffect of PHA on B eells and whieh supports theapplication of PHA to the culture of malignant Beells [26], On the other hand, this early stimula-tion of B cells might be induced by early release ofeytokines.

PWM is often used as a mitogen to test T-cellproliferation as well as T cell-dependent B-cellproliferation, but it turned out to stimulateprimarily Teells. This eonfirms previous reporteddata [30]. In contrast to PHA and Con A, slightlymoreCD4* cells than CDS'" eells were proliferat-ing in the PWM eultures.

SpA appeared to be a potent T-cell mitogen.accompanied by some B-cell proliferation on day3. Jaeoh & Rose [18] suggested using SpA as amitogen for studying human T-eell funetion,however they did not test whieh T-eell subsetswere proliferating. The consistent preponderanceof CD4^ over CD8* proliferating eells is incontrast to the results obtained with the othermitogens. Based on discontinuous bovine serumalbumin density-gradient fractionation experi-ments, Sakane & Green [28] found that SpA-responsive T eells in human PB were differentfrom the eells that respond to PHA. However.they did not perform immunologieal markeranalysis.

Since SpA is such a potent mitogen for CD4"'celU. it is interesting to use SpA for in vitrofunetional studies aimed at testing the prolifera-tive capacity of this T-eell subset. It is well knownthat HIV type I infects and destroys CD4 • cells[7. 12], Deereased in vitro lymphocyte prolifera-tive responses are well known in HIV-1-infectedindividuals and AIDS patients [29, 31]. The poorresponse of MNC from AIDS and pre-AIDSpatients is not only aseribed to a deereased

Marker Analy.Kis of Milogcn-Induced Proliferating Lymphocytes 693

number of CD4* cells, but the responsiveness ofthese eeils also seems to be deficient [10. 16].Publications on the use of SpA in funetionalstudies of the MNC from HIV-infeeted personsare stilt laeking. Recently. Hofmann c/ al. [\5\described the application of the mitogen-stimu-lated lymphoeyte transformation response as apredictive marker for the development of AIDSand AlDS-related symptoms in persons infectedwith HIV. They found that, by comparing PHA,Con A. and PWM as mitogens, the risk of aworsened clinical condition was 55 times higher inseropositive men with a decreased PWM responsethan in other seropositive men. Later on, reduc-tion of the proliferative responses to PHA andCon A took place. This sequential decrease inresponsiveness to PWM, Con A, and PHA wasalso observed by Radvany ei al. [27], but nol byAlcoeer-Varela ci al. [1]. Hofmann el al. [14]explained this phenomenon by an at least partlypreserved funetion of the PHA CD2-dependentpathway. On the other hand, our data revealedthat, in eontrast to PHA and Con A, PWMstimulates more CD4' eells than CD8' eells.However. SpA turned out to be an even bettermitogen for CD4* cells. Therefore, it may berewarding to inelude SpA in funetional studies ofHIV-infeeted persons.

ACKNOWLEDGMENTS

We thank Mr M. W. M. van den Beemd and MsK. Benne. W. M. Comans-Bitter. E. G. M-Cristen, A. A. van der Linde-Preesman, P. W. C.Soeting. A. F. Wierenga-Wolf, and I. L. M.Wolvers-Tettero for teehnieal assistance, Mr T.M. van Os for graphies. Professor Dr, R. Bennerfor his continuous support, and Ms J. de Goey-van Dooren and D. Heinsius for their seeretarialsupport. This work was financially supported bythe Sophia Foundation tor Medieal Research,

REFERENCES

1 Alcocer-Varela, J., Alarcon-Segovia. D. & Abud-Mendoza. C. Immunoregulatory circuits in theacquired immune deficiency syndrome and relatedcomplex. Production of and response to inlcrleukinsI and 2. NK function and iis enhancement byinterleukin-2 and kinetics of the autologoiis mixedlymphocyte reaction, Clin. E.\p. Immunol. 60, H.1985.

2 Blue, M.L., Daley. J.F., Levine. H. & Schlossman.S.F. Coexpression ofT4 and T8 on peripheral bloodT cells demonstrated by two-color fluorescence flowcytometry. J. Immunol. \M, 2281. 1985.

3 Boyd. A.W. & Burns. G.F. Changes in cell surfacemarkers during T lymphocyte mitogenesis. Pp. 737738 in Bernard. A . Boumscll. I... Dausset. 1 ,Milstein, C. & Schlossmann, S.F. (eds) EeueocyleTyping. Springer Verlag, Heidelberg, 1984.

4 Campana. D., Coustan-Smith. F. & Janossy, G.Double and triple staining methods for studying theproliferative activity of human B and T lymphoidcells. J. Immunol. Melhod.s 107. 79, 198K.

5 Coiner. T.. Williams, J.M.. Christenson, L.,Shapiro, H.M.. Strom, T.B. & Stromingcr. J,Simultaneous flow cylomctric analysis of humanT cell activation antigen expression and DNAcontent. J. Exp. Med. 157. 461, 1983.

6 Creeniers. P.C. Determination of co-expression ofactivation antigens on proliferating CD4 ' . CD4*"CD8' and CD8* lymphocyte subsets by dualparameter flow cytometry. / . Immunol. Methods 97.165. 1987.

7 Diilglcish. A.G., Bcvcrlcy, P.C.L., Clapham. P.R.,Crawford. D.H., Greaves. M.F- & Weiss. R.A. TheCD4 (T4) antigen is an essential component of thereteplor for the AIDS retrovirus. Nature 312, 763.1984.

8 Gerdes, J., Lemke, H., Baisch, H.. Wacker, H.H.,Schwab, tJ. & Stein. H. Cell cycle analysis of a cellprolifcralion-associated human nuclear antigendefined by ihc monoclonal antibody Ki-67. J.Immunol. 133, 1710, 1984.

9 Gcrdcs. J.. Schwab. U.. Lemke. H. & Stein. H.Production of a mouse monoclonal antibody reac-tive with a human nuclear antigen associated withcell proli feral ion. Inl. J. CancerM, 1.1. 1983-

10 Gluckman, J .C. Klatzmann. D.. Cavaille-Coll. M..Brisson, E., Messiah. A.. Lachiver, D & Rozen-baiim, W. Is there correlation of T cell proliferalivefuiiclionsand surface marker phcnotypes in paiientswith acquired immune deficiency syndrome or lym-phadenopathy syndrome? Clin. E.xp. Immunol. 60.8.!985.

! I Gratzner. H.G. Monoclonal antibody to 5-bromo-and 5-iododcoxyuridine: a new reagent for detectionof DNA-replication. Scieuce 218. 474. 1982.

12 Ho.D.D.,Pomcrantz.R.J.& Kaplan, J.C. Pathoge-nesis of infection with human immunodeficiencyvirus. :V. Enf-t. J. Med. 317, 278. 1987,

n HoOiuis. WJ.D.. Adriaansen. H.J.. Smit. E.M.E..Dongen. van J.J.M., Zanen. van G,E. & Hage-meijer. A.M. Monosomy-7 syndrome: hematologicand cytogenetic course in two children. Eur. J. Ped.144. l'O6. 1985.

14 Hofmann, B.. Jakobscn, K.D., Odum. N.. Dick-meiss, E., Platz. P., Ryder, L.P., Pedersen. C ,Mathicscn. L., Bygbjerg. I.B.. Faber. V, & Svej-gaard, A. IllV-induccd immunodeliciency. Rela-tively preserved phytohemagglutin as opposed todecreased pokcwccd mitogen responses may be dueto possibly preserved responses via CD2/phytohe-magglulin pathway. J. Immunol. 142. 1874. 1989,

15 Hofmann, B.. Lindhardt, B.O.. Gerstofl, J., Peter-sen. C.S.. Platz, P.. Ryder, L.P., Odum, N., Dick-

694 H. J. Adriaansen ct al.

meiss. E., Nielsen, P.B., Ullman. S. & Svcjgaard. A.Lymphocyte triinsformation response lo pokeweedmitogen as a predictive marker for development ofAIDS and AIDS relaied symptoms in homosexualmen with HIV antibodies. Br. Med. J. 295, 293,1987.

16 Hofmann. B.. Odum, N.. Platz, P-. Ryder. L,P..Svejgaard. A. & Nielsen, J.O. Immunoiogicalstudies in acquired immunodeficiency syndrome.Functional studies oi lymphocyte subpopulations.Scamt. J. Immunol. 21, 235 1985.

17 Holter. W,. Majdic. O.. Liszka, K.. Stockinger. H, &Knapp. W. Kinetics of activation antigen e.\-pression by in viiro-stimulaled human T lympiio-cytes. Cell Imnmitol. 90, 322. 1985.

18 Jacob, M.C. 8i Rose. M.L. Slaphylococcal protein-A is a potent T-cell mitogen of tonsillar lympho-eyles. Immunol. Lell. 7, 77, 198.1.

19 Keightley, R.G.. Cooper, M.D. & Lawton, A.R.The T cell dependetice of B cell differentiationinduced by pokeweed mitogen, J. Immunol. 117,i538,1976-

20 Ko. H.S.. Fu. S-M.. Winchester, R.J., Yu, D.T.Y. &Kunkel. H.G. Ia determinants on stimulated humanT lymphocytes. Occurrence on milogen- and anli-gen-activaled T cells. J. Exp. Med. 150. 246, 1979.

21 Kurki, P , Ogata. K. & Tan, F.M. Monoclonalantibodies lo prolilcrating ccii nuclear antigen(PCNA),cyclin as probes for proliferating cells byimmunolluorescence microscopy and How cylo-meiry. J. Immunol. Methods 109, 49. I98S.

22 Lakhanpal.S.Gonchorofr, N.J..Katzmanii,J.A.&Handwerger, B.S. A flow cytofluorometrie doublestaining technique lor simuUaneous determinalionof human mononuclear cell surface phenotype andcell cycle phase. J. Immunol. Methods 96. 35. 1987.

23 Lea, T., Vartdal, F., Davies, C. & Ugelstad. J.Magnetic monosized polymer particles for fast andspecific fractionation of human mononuclear cells.Scamt. J. Immunol. 11, 207, 1985.

24 Magaud, J.P.. Sargent, 1. & Mason, D.Y. Detectionof human while cell proliferalive responses by

immunoenzymatic measurement of bromodeoxyur-idine uptake. J. Immunol. Methods 106, 95, 1988-

25 Persson, U. & Johansson. S.G.O. Concatiavalin A-induced activation of human T cell subpopulations.hn. .Arch. Allergy Appl. Immunol. 75, 337, 1984.

26 Radnay. J.. Goldman, 1. & Rozenszajn. L,A.Growth of human B-lymphocyte colonies in vitro.Nature 27H,i5\. 1979.'

27 Radvany. R.M., Schuberl, M.S., Kiamar, M.. Jar-zabek, V., Koehler-Carone. K.. Kuban. D. & Hecht.F. Lymphocyte subpopulation profiles and mitogenkinetics in immunoiogical deficiency testing. J. Clin.//HHiH/w/. 7, 312, 1987.

28 Sakane, T. & Green, I. Protein A from Staphylococ-cus aurcus—a mitogen for human T lymphocytesand B lymphocytes but not L lymphocytes. J.Immunol. 120, 302, 1978.

29 Schellekens. P.Th.A., Roos. M.Th.L.. Wolf, de F..Lange. J.M.A. & Miedema. F. Low T-cell respon-siveness to activation via CD.^.'TCR is a prognosticmarker for acquired immunodeficiency syndrome(AIDS) in human immunodeficiency virus-I (HIV-l)-mfected men. J. Clin. Immunol. 10, 121. 1990.

30 Serke, S.. Scrke. M. & Brudler. O. Lymphocyteactivation by phytohaemagglutinin and pokeweedmitogen. J. Immunol. Methods 99., 167, 1987.

31 Spickclt, G.P. &. Dalgleish. A.G. Cellular mimuno-logy of MlV-infeclion. Clin. E.\p. Immunol. 71, I,1988.

32 Suez. D. & Hayward, A.R. Phenolyping of prolifer-ating cells in cultures of human lymphocytes. J.Immunol. Methods 7H. 49. 1985.

33 Van Dongcn. J.J.M., Adriaansen, H.J, & Hooij-kaiis.!!. Immunoiogical marker analysis of cells inthe various hematopoietic differentiation stages andtheir malignant counlerparts. Pp. 87-116 in Ruiter.D.J., Fleuren. G.J. & Warnaar, S.O. (eds) Appli-cation of Monoclonal Antibodies in Tumor Patho-logy. M. Nijhoff Publ.. Dordrecht, 1987.

Received 29 January 1990Accepted in revised form 30 July 1990