INFECTION AND IMMUNrrY, Feb. 1975, p. 265-272Copyright 0 1975- American Society for Microbiology

Vol. 11, No. 2Printed in U.SA.

Deregulation of Mouse Antibody-Forming Cells In Vivo and inCell Culture by Streptococcal Pyrogenic Exotoxin

EDGAR E. HANNA* AND MARTHA HALE

Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda,Maryland 20014

Received for publication 12 September 1974

An unregulated, elevated rebound of antibody levels in rabbits was shown tofollow late (10 to 15 days) after streptococcal pyrogenic exotoxin (SPE)-inducedimmunosuppression. Because of that result we have suggested that SPE acts bypreferentially inhibiting a regulatory cell which normally limits the extent of fullexpression of antibody formation by B-cells. We are currently testing thishypothesis in mice. NIH (Swiss Webster) mice (+/+) or NIH (Swiss Webster)mice heterozygous (+/nu) for the mutant athymic nude gene and phenotypicallynormal showed an elevated plaque-forming cell (PFC) response to sheeperythrocytes (SE) late (10 to 15 days) after immunosuppressive SPE treatmentsimilar to that described in rabbits. Homozygous nude mice (nu/nu) that are

phenotypically athymic normally show a reduced early (4 day) PFC response toSE (a T-cell-dependent antigen) as compared with +/nu littermates or +/+parent strain mice. This cryptic early 4-day response was improved by injectionof purified endotoxin (a B-cell mitogen), but these relatively elevated nude PFCresponses had decreased to normal control (SE only) nude PFC levels before 10days. In similar SE-injected nude mice treated instead with SPE, no elevation at4 days was observed and, more pertinently, the late (10 to 15 day) elevatedrebound of PFC levels observed in normal response controls (+/nu or +/+) was

not observed. Similar experiments were subsequently conducted in Marbrook-type spleen PFC cultures during periods of 12 days. The results of theseexperiments paralleled the in vivo results above, and in addition showed thatSPE induced a large proliferation of either +/+ or +/nu cells (T- and B-cells) inculture but had no such effect on nu/nu cells (B-cells) in culture. Purifiedendotoxin, the B-cell mitogen, had a better sparing effect on nu/nu cells in thisrespect. These results are consistent with our premise that SPE inhibitspreferentially the function of a regulator of the antibody response. The regulatorappears to be a T-cell and is likely a suppressor T-cell.

The finding of an elevated antibody level to asingle antigen exposure in rabbits late afterstreptococcal pyrogenic exotoxin (SPE)-induced immunosuppression has been docu-mented recently (12). This result led us tospeculate that SPE may preferentially inacti-vate a regulatory cell. Since elevated plaque-forming cell (PFC) levels were measured, thiseffect is not thought to be mediated at thecatabolic level of immunoglobulin (Ig) degrada-tion. We showed earlier that SPE effects phago-cytosis in the intact rabbit (10) and that it issuppressive for early 4-day response PFC (11).Current data obtained with the mouse and withcongenitally athymic mice (25) that respondpoorly to T-dependent antigens (1, 19) haveconcentrated our attention on a regulatory cell,possibly a suppressor T-cell (3, 8, 9, 24), as the

preferential site of action of the toxin, as previ-ously speculated (12).

MATERIALS AND METHODSMice. National Institutes of Health (NIH) strain,

Swiss-Webster [NIH(SW)], NIH(SW)nu/nu ("nude"),and NIH(SW) +/nu mice, 6 to 12 weeks old and ofeither sex, were obtained from the NIH Small AnimalSection. The nu/nu mice were kept in disposablecages on sterilized bedding. Food holders and waterbottles were presterilized. The mice were given acidi-fied, chlorinated water; otherwise they were handledroutinely while in our animal rooms. Injections weremade intravenously (i.v.) by tail vein using 27-gaugeneedles. Littermates of nu/nu mice with coats are+/nu and the parent strain NIH (SW) mice are des-ignated +/+.

Sheep erythrocytes. Sheep erythrocytes (SE) wereprepared from sterile sheep blood which was received

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weekly in donor bottles containing acid citrate solu-tion from the Ungulate Unit, NIH Animal Center. ForSE immunogen, gravity-packed SE were resuspendedin cold, sterile 0.1 M tris(hydroxymethyl)aminometh-ane-buffered saline (TBS), resedimented by centrif-ugation two times at 1,000 x g and finally suspendedin a volume of TBS to give the desired number of SEper milliliter. The injection volume for mice was 0.2ml. For culture immunogen, Hanks balanced saltsolution (HBSS) was substituted for TBS as thewashing and suspension medium. All other SE sus-pensions were prepared by the first procedure, exceptthat the final suspension of reagent SE was inmodified TBS (TBS + 0.0007 M CaCl2.2H20, 0.004M MgCl2 6H20, and 0.056 M dextrose).

Spleen cell suspension. There were two types ofspleen cell suspension (SC). Spleens were obtained byaseptic surgery and immersed immediately into coldHBSS held on ice. When obtained from immunizedmice for immediate assay of PFC, the spleens werequartered and pressed against 0.45-,gm stainless-steel,wire-mesh cloth (W. S. Tyler Co., Mentor OH 44060).The single-cell suspensions were obtained by washingthe cells through with HBSS and collecting them in abeaker. The SC were then sedimented by centrifuga-tion at 1,000 x g and resuspended in HBSS to anappropriate cell number for plating. To obtain cul-ture-seeding SC suspensions, spleens obtained asabove were sectioned, and the cells were gently teasedfrom the membrane in HBSS held on ice in anappropriate size sterile plastic petri dish using irisscissors and forceps. The cells were dispersed furtherby jetting through the orifice of Pasteur pipettes. Thecells were sedimented once by centrifugation at 1,000x g and resuspended in a volume of nonsupplementedculture medium for counting.

Cell counting. SE and SC counts were performedelectronically in the Autocytometer-II (Fisher Scien-tific, Pittsburgh, Pa.).

Cell cultures. SC prepared as described abovewere cultured in Marbrook vessels (21) (Bio-ResearchGlass, Inc., Vineland, N.J.) on a dialyzing membranesuspended in excess culture medium. The culturemedium was alpha minimal essential medium (27),modified Eagle minimal essential medium (FlowLaboratories, Inc., Rockville, Md.) supplementedwith 10% fetal bovine serum obtained from eitherGray Industries, Inc., Ft. Lauderdale, Fla., Microbio-logical Associates, Bethesda, Md., or Rheis ChemicalCo., Kankakee, Ill. Cell cultures were seeded usuallyat 20 x 106 acid-resistant cells (leukocytes) per 1 ml ofculture. Inoculated cultures received 1.5 x 101 to 2.5x 106 SE as a 0.04-ml drop in HBSS at the start ofcultures. Other additions, e.g., toxins, were added atthe concentrations given in the text in volumes of 0.1or 0.2 ml immediately after cells were seeded. Penicil-lin (100 U/ml) and streptomycin (100 gg/ml) wereadded to all cultures. Cultures were incubated in ahumidified tissue culture incubator at 37 C in acontinuously flowing atmosphere of 83% N2, 7% 02,and 10% CO2. The cultures were rocked continually ona rocker platform at 5 to 7 cycles per min, sincecontrary to our expectations (21), preliminary testsshowed that the rocking procedure was beneficialwhen compared with static cultures. Cells were har-

vested from the membrane and inner cylinder by useof Pasteur pipettes, sedimented by centrifugation at1,000 x g, resuspended in 1.0 ml of HBSS, assayed forPFC, and counted; thick smears were prepared andfixed for staining. The smears were stained withGiemsa or Wright stain.PFC assays. Molten agarose (pretested lot no.

81C-2680, Sigma Chemical Co., St. Louis), 2.5 ml of a0.8% solution in 0.1 M tris(hydroxymethyl)amino-methane-buffered Eagle medium held at 47 C, wasmixed with 0.2 ml of a washed 20 to 50% suspension ofSE in modified TBS and portions (e.g., 0.1 and 0.2ml) of appropriate dilutions of the SC suspension.This mixture was poured immediately into plastictissue culture dishes (20 mm by 100 mm), held atroom temperature on a leveling board, and allowed togel. The plates were then incubated for 1.5 h at 37 C.To estimate IgG-secreting PFC by the indirect assay(7, 28), designated plates were flooded with 4.0 ml ofan appropriate dilution in modified TBS of hyperim-mune, SE-absorbed, rabbit anti-mouse hyperimmuneanti-SE antibodies and incubated for an additional 1h. Those plates designated for estimation of 19S Igsecreting PFC by the direct assay (13, 14) wereflooded with modified TBS and incubated concur-rently. For plaque development all plates were rinsedbriefly with modified TBS, flooded with 4.0 ml of 10%guinea pig complement (BBL, BioQuest, Cockeys-ville, Md.), and incubated at 37 C for an additional 1h. Plates were cooled at 4 C and stored briefly orscored immediately for plaques. The plaques werecounted at x 7 to x15 magnification, using a dark-field stereomicroscope (Olympus Optical Co., Tokyo).IgG plaque counts represent the total PFC per spleenas estimated from antiglobulin-facilitated indirectplates.SPE. This was the group A streptococcal exotoxin

isolated and purified by Kim and Watson (18) andused previously (11,12). It was produced by type 10,strain NY-5, group A streptococcus. It is an acidicprotein with an average molecular weight of 29,000. Itsrelative biological activity, using the minimal pyro-genic dose (MPD-3), was 0.07 gg/kg i.v. in rabbits (11,12, 18). SPE was dissolved in TBS for injection or infetal bovine serum-free culture medium when addedto SC cultures.

Et. Endotoxin (Et) was the purified hot phenol-water-extracted (17, 30) lipopolysaccharide from Sal-monella typhimurium Lt2, wild type, a gift from Y. B.Kim. Relative biological activity using the MPD-3was 0.0024 ag/kg i.v. in rabbits (17).

Et was dispersed in water at 100 C as a concen-trated solution and then diluted appropriately in TBSfor injection or in fetal bovine serum-free culturemedium for addition to SC cultures. The mean lethaldoses of Et and SPE in mice were 350 and 3,600 ;&g,respectively (17, 18).

RESULTSEffect of SPE on the mouse anti-SE PFC

response. We observe that SPE affects mousePFC as well as those of rabbits (Fig. 1). SPEinjected into mice 3, 24, and 48 h after a single

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FIG. 1. Effect of SPE (50 ,ug, i.v., days 0, 1, 2) onthe 4-day and the 12-day anti-SE PFC responses to asingle suboptimal immunizing dose of SE (3.5 x 10"SE, i.v.) in NIH (SW) mice. PFC responses of miceinjected similarly with Et (1.7 Mg) were measured incomparison as a representation of a direct B-celleffect. Symbols: open bars, 19S Ig PFC estimated bythe direct assay; cross-hatched bars, IgG PFC esti-mated by the indirect assay. PFC/spleen were deter-mined from the pooled spleen cell suspensions of fiveto eight mice at each point. The graph was con-structed from the means i standard errors of threeseparate experiments.

injection of SE caused a slight (- twofold)suppression at 4 days of the number of anti-SE PFC developed as compared with the num-ber of such PFC developed in control mice in-jected with SE alone (10,017 4 923 PFC/spleenversus 18,810 ± 877 PFC/spleen, respectively).This level of PFC suppression in mice was lessthan that observed in similar previous experi-ments conducted in rabbits (11). Nevertheless,the 19S Ig PFC response measured at 12 days inSPE-treated mice was maintained 10-fold higherthan that of control mice injected with SEalone. Moreover, mice treated similarly withthe B-cell mitogen, Et, showed an early eleva-tion of 19S Ig PFC, a response consistent withexpectations for one modified by a B-cell mito-gen and the reverse of the response observedafter SPE treatment.IgG PFC in SPE-treated mice were markedly

elevated in comparison with those in eitherEt-treated mice or the nontreated control miceat 12 days. Thus, the "unregulated" increase ofPFC which occurs late (11) can be studied in themouse as well as in the rabbit. Moreover, theT-cell, B-cell synergy was originally observed in

mice (6) and more elementary information andmethodology is available in this species. Ofparticular interest here was that nude, congeni-tally athymic mice were available.

Effect of SPE on the mouse anti-SE PFCresponse in culture. Figure 2 shows resultsobtained in Marbrook-type (21) PFC cultures,which were inoculated with SE and treated withSPE or with the B-cell mitogen Et (2) at thetime of culture initiation. Et elevated the early4-day peak ofPFC slightly above SE-inoculatedcontrols, consistent with its B-cell stimulatingactivity (2). The early 4-day response in SE-inoculated, SPE-treated cultures was sup-pressed (: threefold) but was greatly elevated( eightfold) late at 12 days, a time whenSE-inoculated controls and Et-treated, SE-inoculated, control culture PFC levels had de-creased. Thus, the effect characterized by anearly suppression followed by recovery and alate increase above controls can be observed

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FIG. 2. Effect of SPE (100 Mig/culture) on theanti-SE (1.5 x 10' SE/culture) PFC response in SC(2.75 x 107 seeded) cultures from NIH (SW) mice.Symbols: (--O--), (uninoculated) SE alone for 12days assayed for anti SE, PFC at 4, 8, or 12 days;(-0-) SE inoculated, assayed for anti-SE, PFC at4, 8 or 12 days; (-A-), SE inoculated, Et treated (10Mg/culture), assayed for anti-SE, PFC at 4, 8, or 12days; (-), SE inoculated, SPE treated, assayedfor anti-SE, PFC at 4, 8, or 12 days. Each pointrepresents the PFC in a pool of SC from duplicate ortriplicate cultures in a representative experiment.Distinctive IgG PFC could not be ascertained fromculture.

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even more clearly in cell culture. Moreover, avery similar effect (Fig. 3) was exhibited bySPE- and Et-treated uninoculated cultures.This delayed SPE effect on background PFCmakes it apparent that the background anti-SE-PFC clones can also be released from anormal regulatory influence. The early (4 day)background PFC elevation in Et-treated cul-tures is consistent with previous observations inthe rabbit (11), an effect originally described byJohnson et al. (15, 22). Since it is now held thatEt is a preferential B-cell mitogen (2), the effectof Et here is probably due to a direct stimula-tory effect on existing B-cell background clones.Increased cell number in SPE-treated cul-

tures. SPE-treated cultures showed a 120%increase in cell number at 4 days (Fig. 4); thenumber was essentially maintained for 8 daysand was 85 to 90% maintained at 12 days in bothSE-inoculated and uninoculated SPE-treatedcultures. This effect was not observed in Et-treated cultures, although Et-treated cultureswere better maintained (60 to 70%) than SE-inoculated control cultures and cells alone inculture. The appearance and relative number ofthese cells at 12 days are shown in Fig. 5.Uninoculated cultures (lower left panel) andSE-inoculated, nontreated cultures (lower rightpanel) showed degenerating nondividing cellsonly, and is consistent with the data of Fig. 4.Cultures treated with SPE or with Et showed amuch larger number of basophilic intact lym-phocytes with only a few degenerating cells.Similar morphological criteria were presentedrecently as evidence of cell proliferation (26).Consistent with the data of Fig. 4 was the

5 NIH (SW)

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FIG. 3. Effect of SPE (100 ,g/culture) on non-SE-inoculated PFC numbers in SC (2.75 x 107 SEseeded) cultures from NIH (SW) mice assayed at 4, 8,or 12 days. Symbols: (0), cells alone; (A), Et treated(10 ug/culture); (0), SPE treated. Each point rep-

resents the number of PFC in the SC pool of duplicateor triplicate cultures in a representative experiment.

DAYS IN CULTURE

FIG. 4. Effect of SPE on SC survival in the cul-tures of Fig. 3 and 4. Symbols: (0), cells alone; (A), Ettreated (10 Ag/culture), (-), SPE treated. Each pointrepresents the number of PFC in the SC pool ofduplicate or triplicate cultures in a representativeexperiment. Cells were counted electronically imme-diately after plating for PFC assays.

observation (Fig. 5) that SPE-treated culturesshowed the greatest number of intact lympho-cytes with good basophilia at 12 days and theyappeared to be a more homogeneous popula-tion.

Effect of SPE on the anti-SE PFC responseof athymic nude mice. The anti-SE PFC re-sponse of nude mice (congenitally lackingT-cells) (Fig. 6, top panel) was 10- to 15-fold(1.0 to 1.5 logs) lower than the +/nu, T- andB-cell control response (botton panel) in thosegroups injected with SE alone at all assaypoints. Whereas Et stimulated an increasedPFC level in nude mice (top panel, 4 and 8days), it was surprising that SPE did not inducesuppression of the early (4 day) nude PFCresponse. This observation is being investigatedfurther. More relevant to our current interpreta-tion, however, was that the characteristic de-layed "unregulated" increase of PFC at 10 to 15days did not occur in nude mice (Fig. 6, toppanel). Nude mice did not exhibit IgG PFCregardless of the stimulus in these experiments,whereas the unregulated response, post-SPE-treatment, was accentuated in the IgG PFCcompartment of +/+ mice (Fig. 1). The re-sponses of +/nu littermate mice (Fig. 6, bottompanel), in a concurrent control experiment, werecharacteristic of SPE-treated parent strain +/+mouse responses. These characteristics asshown by +/nu mice (Fig. 6, bottom panel)were: SPE suppression or delay at 4 days andelevation of IgG PFC which occurred late (12 to14 days) in the SPE-treated group.

Effect of SPE on the athymic nude mouseanti-SE PFC response in culture. Et, a B-cellmitogen, stimulated the 4-day peak response of

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FIG. 5. Appearance of SC from the cultures shown in Fig. 3 and 4 at 12 days. Upper left, uninoculated, SPEtreated; upper right, SE inoculated and SPE treated; mid-left, uninoculated, Et treated; mid-right, SE inocu-lated and Et treated; lower left, SC alone; lower right, SE inoculated. x 100 magnification of Wright-stained por-

tion preparations.

nude (B-cells) PFC (Fig. 7). This was evident inboth SE-inoculated (SE + Et) and background(uninoculated) (Et only) cultures. Consistentwith previous data, SPE-treated (SE inoculatedand uninoculated) nude culture responses werelower than the control inoculated-culture re-

sponse and much lower than those stimulated inaddition with the B-cell stimulant Et. The more

important finding in this experiment was thatthe unregulated delayed PFC increase in SPE-treated cultures did not occur, thus contrastingsharply with the counterpart experiment in+/+ mouse SC cultures (Fig. 2). Moreover,SPE-treated cultures did not show cell prolifer-ation at 4 or 12 days, but rather a steady decline

in cell number (Fig. 8). It is pertinent, however,that Et-treated nude (B-cell) cultures were

maintained at a higher cell number at bothpoints tested.

DISCUSSIONWe have previously documented an unex-

pected finding of elevated antibody formationafter suppressed antibody formation inducedwith streptococcal pyrogenic exotoxin (12). Thispaper presents recent data obtained in a mouse

model system in support of the speculation thatSPE might have a preferential effect on a

regulator cell. Temporary elimination of theregulator cell or inhibition of its activity would

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DAYS AFTER A SINGLE SE INJECTIONFIG. 6. Effect of SPE (50 gg, i.v., =700 MPD-3

days 0, 1, 2) on the anti-SE (4.7 x 105 SE, i.v., day 0)PFC response in control +/nu, NIH (SW) mice (lowerpanel) and in test nu/nu mice (upper panel). (-O-),19S Ig PFC responses to SE alone; (--- -), IgGPFC response to SE alone; (-O-), 19S Ig PFCresponse to SE as modified by SPE; (- -O - -), IgGPFC responses to SE as modified by SPE; (-A-),19S Ig PFC responses to SE as modified by Et (1.7Mg i.v., oe700 MPD-3, days 0, 1, 2); (- -A- -), IgGPFC response to SE as modified by Et. IgG PFCcould not detected in nude PFC populations. Eachpoint was derived from a five to eight mouse SC poolin a representative experiment.

allow clonal expansion of B-cells, among whichwould be greater elevated levels of antigen-stimulated descendent PFC (Fig. 1, 2, and 6). Itmight be expected that background PFC popu-lations (unstimulated B-cells) may also be un-leashed by SPE. Results consistent with thiswere obtained in spleen cell cultures of +/+mice containing both B- and T-cells (Fig. 3). Thecounterpart experiment involving nu/nu cells(Fig. 7) moreover, supports this conclusion sinceEt-treated cultures showed that these cells werecapable of being stimulated (i.e., direct B-cellstimulation), but SPE with suggested preferen-tial antisuppressor T-cell activity was withouteffect (i.e., no regulator available). In addition,this experiment showed that SPE had only littleovert effect on B-cells alone, at the dose used inthese experiments, and whether the early (4 to 8day) suppressive effect of SPE is mediated

through an effect on helper T-cells (6) has notyet been determined. Baker et al. (4) haverecently offered strong evidence in a helperT-cell-independent antigen (SSS-III) systemthat suppressor T-cells modulate B-cell matu-ration and clonal expansion. We have not yettested the effect of SPE on the response toT-independent antigens nor on a synthetichapten-carrier immunogen, two systems whichshould provide interesting future data relativeto any effect of SPE on helper T-cells (6, 23, 29).However, available data predict that the re-sponse to these antigens would have similar

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FIG. 7. Effect of SPE (100 ug/culture) on theanti-SE (1.5 x 10. SE/culture) PFC response ininoculated and uninoculated cultures (2.26 x 107 SCseeded) from lu/fu, NIH (SW mice. (0), cells alonein uninoculated culture; (0), uninoculated culturestreated with SPE; (A), uninoculated cultures treatedwith Et (10 ug/culture); (0), SE-inoculated cultures.(@), SE inoculated and SPE treated; (A), SE inocu-lated and Et (10 aggculture) treated; each pointrepresents the number of PFC in the SC pool ofduplicate or triplicate cultures in a representativeexperiment.

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FIG. 8. Effect of SPE on nu/nu SC survival in thecultures of Fig. 7. Solid lines, SE-inoculated cultures;broken lines, uninoculated cultures; (0) cells alone;(A), uninoculated, Et treated; (0), uninoculated SPEtreated; (O), SE inoculated; (A), SE inoculated, Ettreated; (O), SE inoculated, SPE treated.

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characteristics to that shown currently. SPEwould act to retard the regulator step, leavingB-cells unregulated or available for amplifica-tion by amplifier cells as suggested by Baker etal. (3), in the case of T-independent antigens, orby T-helper cells (6), in the case of synthetichapten-carrier, T-dependent immunogens (23).The mode of action of SPE being tested (Fig. 9)would require only a slight modification shouldit be shown that suppressor activity is anotheractivity of the T-helper cell or of this cell at adifferent developmental stage (5, 16).The finding of a neutral or negative effect of

SPE in nude mice, which are T-cell negativebut not known to be macrophage deficient,argues against a remotely possible suppressor-macrophage as the SPE target. This, however,is important to be ruled out in the future sinceSPE in a different form was shown in our veryearly studies of its biological activity to beantiphagocytic (10), although the type of phag-ocyte effected, polymorphonuclear or mononu-clear, is not known. It is, nevertheless, lessremotely possible that an early coordinate sup-pressive effect could be mediated by macro-phages possibly by withholding effective anti-gen and that this suppression could be slowlyrelieved by an antiphagocytic effect of SPE.Because of a current limited amount of puri-

fied SPE, we have not yet determined how longthe unregulated PFC levels are retained. In therabbit experiments, however, the persistence ofthe unregulated PFC level was dose dependent,and a high-dose-treated group (70 jg/day, 3days) showed elevated levels which were main-tained longer than 30 days (12). This is consist-ent with our suggestion (12) that the suppressorcell population recovers more slowly than does

suppressed B-cells and possibly therefore maybe sufficiently affected so as to interfere withinduced and constituent states of tolerance. Inthis respect, we do not yet know whether asimilar sustained "unregulated" antibody re-sponse to a second or a battery of antigenswould be simultaneously deregulated or not.This important possibility is being tested. Rela-tive to the attributes of SPE to the disease-pro-ducing process of group A pathogenic strepto-cocci, we have previously suggested that theearly transient suppression may be relevant toacute streptococcal disease by creating a para-site-preferring, low-antibody environment forthe streptococcus (3). Secondly, a number of thelate streptococcus-associated sequelae have notbeen consigned to immediate direct effects ofthe streptococcus but are considered in therealm of autoimmunity (20) somehow associ-ated with ox initiated by the streptococcus. Inthis respect, whether SPE can effect self-toler-ance adversely may be tested in specific animalmodel systems in the future.Presented in Fig. 9 is a diagram illustrating

how SPE might act. B-cells, when stimulatedby antigen and amplified by helper T-cells,initiate maturation and proliferation of a cloneof B-cells. Clone size normally would be regu-lated by a suppressor cell. SPE would affectinhibition of the suppressor event, thus allowingfull unregulated expression of B-cell clonalpotential possibly with additional amplificationby helper cells. The diagram allows for thepossibility that the suppressor cell and thehelper cell may be different stages of the samecell line (suggestions contained in references 5and 16). This speculated mechanism by whichSPE may deregulate antibody formation, by

n-T Helper T-cell

antigen

B-cellPrecurser s

Suppressor T- cell Bc

FIG. 9. Illustration of how SPE might deregulate antibody formation by interference with a normalregulator of B-cell clonal expansion. The suppressor cell (s-7) would normally limit the extent of B-cell pro-liferation. SPE would either preferentially inactivate or inhibit the suppressor cell. The B-cell would then befree, stimulated by specific antigen, and amplified by the helper T-cell into an enlarged clone ofantibody-secreting, mature antibody-forming cells (B-c).

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affecting deregulation of progenitor antibodysecreting B-cell clones, if further supported, willdefine important heretofore unrecognized attri-butes of SPE. Furthermore, SPE may be avaluable probe for further studies on regulationof the antibody response.

ACKNOWLEDGMENTS

We thank D. W. Watson and Y. B. Kim for additionalsamples of purified SPE and Et; B. Merchant for an initialgift of nude mice and advice on their unique care; M. Cashel,P. Leder, B. Merchant, J. B. Robbins, and D. W. Watson forreading the manuscript; and C. Kunkle for expert secretarialpreparations.

LITERATURE CITED

1. Aden, D. P., N. D. Reed, and J. W. Jutila. 1972.Reconstitution of the in vitro immune response ofcongenitally thymusless (nude) mice. Proc. Soc. Exp.Biol. Med. 140:548-552.

2. Andersson, J., 0. Sjoberg, and G. Moller. 1972. Mitogensas probes for immunocyte activation and cellularcooperation. Transplant. Rev. 11:131-177.

3. Baker, P. J., P. W. Stashak, D. F. Amsbaugh, B. Pres-cott, and R. F. Barth. 1970. Evidence for the existenceof two functionally distinct types of cells which regulatethe antibody response to type III pneumococcal polysac-charide. J. Immunol. 105:1581-1583.

4. Baker, P. J., P. W. Stashak, D. F. Amsbaugh, and B.Prescott. 1974. Regulation of the antibody response totype-III pneumococcal polysaccharide II. Mode of ac-

tion of thymic-derived suppressor cells. J. Immunol.112:404-409.

5. Basten, A., J. F. A. P. Miller, J. Sprent, and C. Cheers.1974. Cell-to-cell interaction in the immune response.X. T-cell-dependent suppression in tolerant mice. J.Exp. Med. 140:199-217.

6. Claman, H. N., E. A. Chaperon, and R. F. Triplett. 1966.Thymus-marrow combinations. Synergism in antibodyproduction. Proc. Soc. Exp. Biol. Med. 122:1167-1171.

7. Dresser, D. W., and H. H. Whortis. 1965. Use of an-

tiglobulin serum to detect cells producing antibodywith low hemolytic efficiency. Nature (London) 208:858-861.

8. Gershon, R. K., and K. Kondo. 1970. Cell interactions inthe induction of tolerance: The role of thymic lympho-cytes. Immunology 18:723-737.

9. Ha, T. Y., and B. H. Waksman. 1973. Role of the thymusin tolerance. X. "Suppressor" activity of antigen-stimulated rat thymocytes transferred to normal recipi-ents. J. Immunol. 110:1290-1299.

10. Hanna, E. E., and D. W. Watson. 1965. Host-parasiterelationships among group A streptococci. III. Depres-sion of reticuloendothelial function by streptococcalpyrogenic exotoxins. J. Bacteriol. 89:154-158.

11. Hanna, E. E., and D. W. Watson. 1968. Host-parasiterelationships among group A streptococci. IV. Suppres-sion of antibody response by streptococcal pyrogenicexotoxin. J. Bacteriol. 95:14-21.

12. Hanna, E. E., and D. W. Watson. 1973. Enhancedimmune response after immunosuppression by strep-tococcal pyrogenic exotoxin. Infect. Immun.7:1009-1011.

13. Ingraham, J. S. 1963. Identification individuelle des

cellules productrices d'anticorps par une reaction he-molytique locale. C. R. Acad. Sci. 256:5005-5008.

14. Jerne, N. K., A. A. Nordin, and C. Henry. 1963. The agarplaque technique for recognizing antibody producingcells, p. 105-109. In B. Amos and H. Koprowski (ed.),Cell-bound antibodies. Wistar Institute Press, Phila-delphia.

15. Johnson, A. G., S. Gaines, and M. Landy. 1956. Studieson the "O" antigen of Salmonella typhosa. V. En-hancement of antibody response to protein antigens bythe purified lipopolysaccharide. J. Exp. Med.103:225-246.

16. Kapp, J. A., C. W. Pierce, and B. Benacerraf. 1974.Genetic control of immune responses in vitro. III.Tolerogenic properties of the terpolymer L-glutamicacid"-L-alanine30-L-tyrosine"0 (GAT) for spleen cellsfrom non-responder (H-2 and H-2q) mice. J. Exp.Med. 140:172-184.

17. Kim, Y. B., and D. W. Watson. 1967. Biologically activeendotoxins from Salmonella mutants deficient in 0-

and R-polysaccharides and heptose. J. Bacteriol.94:1320-1326.

18. Kim, Y. B., and D. W. Watson. 1970. A purified group Astreptococcal pyrogenic exotoxin. Physiochemical andbiological properties including the enhancement ofsusceptibility to endotoxin lethal shock. J. Exp. Med.131:611-628.

19. Kindred, B. 1971. Immunological unresponsiveness ofgenetically thymus-less (nude) mice. Eur. J. Immunol.1:59-61.

20. McCarty, M. 1972. Theories of pathogenesis of strep-tococcal complications, p. 517-526. In L. W. Wan-namaker and J. M. Matsen (ed.), Streptococci andstreptococcal diseases. Academic Press, Inc., NewYork.

21. Marbrook, J. 1967. Primary immune response in culturesof spleen cells. Lancet ii:1279-1281.

22. Merritt, K., and A. G. Johnson. 1963. Studies on theadjuvant action of bacterial endotoxins on antibodyformation. V. The influence of endotoxin and 5-FUDRon the primary antibody response of the BALB mouseto a purified antigen. J. Immunol. 91:266-272.

23. Mitchison, N. A. 1971. The carrier effect in the secondaryresponse to hapten-protein conjugates. II. Cellularcooperation. Eur. J. Immunol. 1:18-27.

24. Okumura, K., and T. Tada. 1971. Regulation of homocy-totropic antibody formation in the rat. VI. Inhibitoryeffect of thymocytes on the homocytotropic antibodyresponse. J. Immunol. 107:1682-1689.

25. Pantelouris, E. M. 1968. Absence of thymus in a mouse

mutant. Nature (London) 217:370-371.26. Pessac, B., and G. Calothy. 1974. Transformation of chick

embryo neuroretinal cells by Rous sarcoma virus invitro:, induction of cell proliferation. Science185:709-710.

27. Stanners, C. P., G. L. Eliceiri, and H. Green. 1971. Twotypes of ribosome in mouse-Hamster hybrid cells.Nature (London) New Biol. 230:52-54.

28. Sterzl, J., and I. Riha. 1965. A localization hemolysis ingel method for the detection of cells producing 7Santibody. Nature (London) 208:858-859.

29. Uzunova, A. D., and E. E. Hanna. 1973. B-cell amplifica-tion by dibutyryl cyclic AMP in mice. Cell. Immunol.7:507-511.

30. Westphal, O., 0. Luderitz, and F. Bister. 1952. tOber dieExtraktion von Bakterien mit Phenol/Wasser. Z. Na-turforsch. 7b: 148-155.

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