JouaNAL OF BACERIOLOOY, Oct., 1966Copyright © 1966 American Society for Microbiology

Vol. 92, No. 4Printed in U.S.A.

Immunological Tolerance to Microbial AntigensII. Suppressed Antibody Plaque Formation to Shigella Antigen

by Spleen Cells from Tolerant MiceHERMAN FRIEDMAN

Department ofMicrobiology, Division ofLaboratories, Albert Einstein Medical Center, Philadelphia, Pennsylvania

Received for publication 11 June 1966

ABSTRAcr

M rmm, HEaRAN (Albert Einstein Medical Center, Philadelphia, Pa.). Imuno-logical tolerance to microbial antigens. II. Suppressed antibody plaque formationto Shigella antigen by spleen cells from tolerant mice. J. Bacteriol. 92:820-827. 1966.-An indirect, localized, antibody plaque procedure has been used to demon-strate a marked difference in the number of antibody plaques formed with spleencell suspensions from normal and Shigella-tolerant mice. Whereas challenge withsoluble Shigella antigen (SSA) into normal mice, ranging in age from 4 to 40 weeks,resulted in a rapid rise in antibody plaque formation to Shigella-treated sheeperythrocytes, there was only a slight increase in plaque formation with spleen cellsuspensions from similarly challenged mice which had been made tolerant toShigella antigen during neonatal life. Apparently, the suppression of plaque forma-tion to Shigella in tolerant animals was specific, since both Shigella-tolerant andnormal mice responded equally well to untreated sheep red blood cells afterchallenge with sheep erythrocytes only. Spleen cells from nonchallenged Shigella-tolerant mice did not form significant numbers of antibody plaques to SSA-treatedred blood cells during an observation period of4 to 30 weeks after neonatal admin-istration of antigen. "Nonspecific" increases in plaque formation to untreatedsheep red cells occurred with spleen cell suspensions from both normal and SSA-tolerant mice after challenge injection with Shigella antigen, with or without sheeperythrocytes. Such a response suggested an adjuvant effect for the endotoxin-con-taining Shigella antigen even in mice tolerant to the agglutinogenic moiety of SSA.The results of these experiments support the view that specific antibody-formingcells are either absent or in low number in lymphoid tissue from mice specificallytolerant to Shigella antigens. It seems unlikely that the low postchallenge agglu-tinin titers of tolerant mice are due to suppressed antibody formation by normalnumbers of individual antibody-producing cells, or due to "masking" of normalantibody production by persisting circulating antigen.

Specific immunological unresponsiveness induced in neonatal mice by treatment at birth witha relatively large quantity of Shigella soluble antigen (SSA) is specific and, depending on dose androute of inoculation at birth, may persist forseveral months (12). Such unresponsiveness ischaracterized by markedly suppressed agglutinirresponses after challenge injection with Shigellaantigen capable of inducing detectable levels ofagglut in normal mice (12). As describedpreviously, tolerant mice lack specific antibody-containing lymphoid cells as determined by theindirect immunofluorescence technique (11). It ispossible, however, that normal numbers ofspecific immunologically competent cells may be

present in Shigella-tolerant animals, but that eachi cell secretes subnormal quantities of antibody.- The sensitivity of immunofluorescence proceduresI is relatively low and may be insufficient to detectr small quantities of antibody secreted by cells froms tolerant animals (11, 28). A direct estimation ofx the number of cells actively synthesizing antibodyi in situ by more sensitive serological or immuno-f logical means has not been feasible until recently.I Development of the localized hemolysis plaque* procedure by Jerne (17), and by Ingraham and

Bussard (16), has permitted use of a rapid ands sensitive immunological method for estimationf of the number of lymphoid cells actively secreting

antibody (6, 10, 23, 32). Studies utilizing the820

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antibody plaque procedure have been generallylimited to the demonstration of cells secretinghemolytic antibody to mammalian or avian eryth-rocytes. Recently, however, the localized anti-body plaque assay technique has been used toestimate the number of lymphoid cells formingantibody to soluble bacterial antigens. Theplaquing test is accomplished with bacterial anti-gens coated onto isologous or heterologouserythrocytes (18, 19; I. Canellas and A. Intra-vastolo. Bacteriol. Proc., p. 53, 1965). Use ofsuch indirect, or passive, plaque procedures withbacterial systems should permit direct compari-son of the number of antibody-secreting cells inlymphoid tissue of normal and tolerant animals.This report is concerned with results of such astudy designed to enumerate the number of anti-Shigella plaques formed in agar with spleen cellsuspensions from Shigella-tolerant mice as com-pared with those from normal mice.

MATERIALS AND METHODSExperimental animals, antigens, and induction of

tolerance. Mice, SSA, route, and time of neonatalinjection, and serological agglutination tests have beenlisted in the previous reports (5, 8, 12, 13).

Hemolytic plaque assay. The direct immunoplaqueassay for detection of localized zones of hemolysiswith uncoated erythrocytes was performed essentiallyas described by Jeme, Nordin, and Henry (17). Micewere injected intraperitoneally with 0.1 ml of a 10%suspension of washed sheep erythrocytes. At the timeof testing, each animal was bled by retro-orbital punc-ture and then sacrificed by cervical dislocation.Spleens were rapidly removed and placed immediatelyin cold sterile Hanks solution (pH 7.2). Cell sus-pensions were prepared aseptically by "teasing" withfine needles (5, 8). Tissue fragments and large aggre-gates of cells were removed by passing the suspensionsthrough fine-mesh stainless-steel gauze, followed bytwo washings in cold (2 to 4 C) sterile Hanks solutionby means of serial centrifugation. The number ofnucleated cells in the suspensions was determined witha hemocytometer, and viability was estimated bytrypan blue-staining technique. Suspensions of cellscontaining 5 X 104 to 10 X 106 nucleated cells permilliliter were prepared, and plates were poured induplicate or triplicate as follows: 0.1 ml of each cellsuspension was quickly added to a tube filled with2.0 ml of warm melted (48 to 52 C) 0.7% Noble agar(Difco) containing 1 mg of diethylaminoethyl-dextran(molecular weight, 10 X 106) and 0.1 ml of a 10%suspension of freshly washed sheep erythrocytes(10, 17). This mixture was carefully poured over a3-mm thick layer of 1.5% Noble agar in Hanks solu-tion (without phenol red), which had been previouslyprepared in petri plates (100 mm in diameter) andrefrigerated for 24 to 48 hr. After solidification of theupper agar layer containing the spleen cells and sheeperythrocytes, the plates were incubated at 37 C for1 hr, and then were treated with 5 ml of a 1:10 dilu-

tion of guinea pig serum as a source of complement(BBL). The plates were again incubated at 37 C for30 min. Localized zones of hemolysis could be ob-served as clear areas against a background of unlysedred cells. The plaques were made more distinct andthe plates were preserved for extended time periodsby staining with cold, freshly prepared benzidine-H202-acetic acid solution (17). The total number ofplaques formed per whole spleen or per 106 nucleatedcells was calculated from the average plaque countof several plates prepared from each spleen wheninocula that resulted in 50 to 300 plaques per platewere used. In most instances, plates were observedfor nonspecific zones of hemolysis prior to additionof the complement. The number of such complement-nondependent plaques, when present, was subtractedfrom the number of plaques appearing after treat-ment with complement. Generally, 5 to 20 nonspecifichemolytic areas appeared per plate when more than5 X 106 spleen cells were used per plate.

Indirect hemolytic plaque assay with erythrocytescoated with Shigella antigen. An indirect antibodyplaque technique was used to determine the numberof anti-Shigella antibody-forming cells in spleens andlymph nodes of normal and tolerant mice before andafter challenge injection with SSA. Freshly washedsheep erythrocytes were treated for 1 hr at 37 C withSSA in vitro to prepare the antigen-coated red cells(21). Preliminary block titrations indicated that I to 2,ug of SSA per ml ofa 10% suspension of sheep eryth-rocytes was optimal for sensitization. For the in-direct plaque assay, 0.1 ml of a freshly prepared 10%suspension of SSA-treated erythrocytes was used inplace of untreated erythrocytes. Otherwise, the plaqueassay was performed exactly as described above. Inaddition, however, when relatively few plaques wereformed with spleen cells from Shigella-tolerant mice,relatively large numbers of spleen cells were used perplate in attempts to obtain counts within the range of50 to 300 hemolytic zones per plate. In these instances,there were often large numbers of nonspecific hemo-lytic areas, apparently due to the debris in the cellsuspensions or to aggregated cells. Plates werepoured in quadruplicate; one or two plates were nottreated with complement, but were stained with benzi-dine. The number of hemolytic zones in these comple-ment-free plates was subtracted from the number ofplaques found in plates treated with complement.

Serological hemagglutination procedure. Sera ofnormal and tolerant mice obtained at various intervalsbefore or after challenge with SSA were tested forantibody to Shigella extract by passive hemagglutina-tion and hemolysis procedures. Equal quantities of10% suspensions of washed sheep erythrocytes andSSA (5 pg of N/ml) were incubated for 1 hr at 37 Cand then were washed several times with cold sterilesaline. The treated cells, in a concentration of 1.5%,were added in 0.05-ml quantities to 0.5 ml of serialtwofold dilutions of the mouse sera, 1:10 through1:10,240. After incubation at 37 C for 1 hr and in thecold for 24 hr, the hemagglutinin titer was recorded asthe reciprocal ofthe highest dilution of serum resultingin complete agglutination of the treated red cells. For

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the hemolysis test, mouse serum was heated for 30min at 56 C prior to titration. A 0.1-nml amount of a1:20 dilution of guinea pig serum (as a source ofcomplement) was added to each tube before the addi-tion of the erythrocytes. Hemolysis was recorded after1 hr of incubation at 37 C. All sera were absorbed with1 volume of washed, packed, untreated red cells priorto dilution. In addition, all sera positive for hemaglu-tinins or hemolysins to SSA-treated red blood cellsRBC) were tested with untreated RBC as a control.

ESULTS

Plaque formation with spleen cells from normalmice injected with Shigella antigen. Normal micechallenged 2 to 8 days previously with a knownimmunizing inoculum of Shigella antigen (SSA,20 ,g of N) were sacrificed, and washed spleencell suspensions were obtained for preparation ofagar plates containing either untreated or SSA-coated erythrocytes. After incubation with com-plement, many localized zones of hemolysisappeared in plates containing SSA-treated eryth-rocytes (Tables 1 and 3). There was a maximumof several hundred complement-dependentplaques per 106 nucleated spleen cells fromShigella-immunized, nontolerant mice. Severaldozen hemolytic zones per 106 spleen cells wereformed when the same cell suspensions weretested with untreated sheep erythrocytes.

Plaque formation with cells from Shigella-tolerant mice. Shigella-tolerant mice, 5 to 6weeks old, were sacrificed at various time intervalsafter challenge with SSA. Serum samples wereobtained prior to sacrifice for determination ofShigella antibody levels through direct bacterialagglutination tests and through passive serological

hemolysis and hemagglutination tests with SSA-treated RBC. Spleen cell suspensions were testedfor plaque-forming ability in agar plates contain-ing SSA-treated erythrocytes. Table 1 indicatesthe plaque-forming ability of cell suspensionsfrom tolerant mice at several time intervals afterchallenge injection with SSA. Whereas spleencell suspensions from normal mice exhibitedapproximately a 40- to 100-fold increase inplaque formation over a 2- to 10-day period afterchallenge, there was only a slight increase inplaque formation with spleen cell suspensionsfrom tolerant mice. The average plaque size was0.05 mm with cells from either normal or tolerantmice. The number of plaques per spleen was theonly observable difference between spleen cellsuspensions from the two groups (Fig. 1). Plaqueformation in plates containing untreated sheeperythrocytes increased 5- to 10-fold within a fewdays after challenge immunization with SSA,regardless of whether the test animal was a normalcontrol or a tolerant mouse (Table 1).

Effect ofage on antibody plaque formation withspleen cells from tolerant or normal mice. Miceinjected at birth with a tolerance-inducing inocu-lum of SSA were sacrificed at various ages, andwashed spleen cell suspensions were prepared andused for preparation of agar plates containingSSA-treated RBC. Although the number ofplaques formed in agar containing SSA-treatedsheep red cells increased slightly with the age ofthe SSA-tolerant animal (Table 2), it was notsignificantly different from that observed when thesame spleen cell suspensions were tested with un-treated sheep red cells. Spleen cell suspensionsfrom normal, nontolerant animals also had a

TABLE 1. Serum antibody titers and antibody plaque formation with spleen cells of normal and Shigella-tolerant mice obtained before and after SSA challenge

Serum antibody (means) Hemolytic plaques/106 spleen cellsMouse group immunization No. of mice

with SSA8 tested Shieila Tube hemol- SSA-treated Untreatedagglutination ysis6 sheep RBC sheep RBC

Normal 0 12 <1: 10 <1:10 2 3+2 7 1:83 1:60 76 6+5 8 1:365 1:490 242 24+8 8 1:380 1:742 188 11

Shigella-tolerantc 0 10 <1:10 <1:10 5 3+2 8 <1:10 <1:10 12 11+5 6 1:32 1:11 31 19+8 6 1:30 1:29 17 5

a All mice challenged at 5 to 6 weeks of age.b Shigella antigen treated sheep RBC.c Tolerant mice injected at birth with SSA.

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low, but readily detectable, increase in "back-ground" count with increased age.

Duration of tolerance as assessed by plaqueformation. Shigella-tolerant and normal controlmice, 4 to 30 weeks old, were challenged with SSAat various times. Groups of mice were sacrificed5 to 6 days after each injection period, and spleencell suspensions were tested in agar containingSSA-treated sheep erythrocytes (Table 3). Thenumber of plaques formed with spleen cell sus-pensions from tolerant animals 4 to 12 weeks ofage was less than 5 to 10% of that obtained withcell suspensions from normal control mice immu-nized with SSA at the same time. Plaque-formingability of spleen cells from tolerant mice in-

creased sharply when the mice were 15 to 20 weeksold and generally paralleled serum antibody re-sponses. Tolerance to Shigella, as assayed both bycirculating antibody and by plaque-formingability, was generally lost by the time the micewere 3 to 5 months old, as observed in previousstudies in which antibody formation was the onlycriterion used to determine loss of tolerance.

Antibody plaque formation by spleen cell sus-pensions from tolerant and normal mice challengedwith sheep erythrocytes. The ability of Shigella-tolerant mice to form plaques to untreated sheeperythrocytes after specific immunization withRBC only was determined to demonstrate thespecificity of plaque suppression to SSA-coated

FIG. 1. Antibody plaques in agar gel containing Shigella antigen-coated sheep erythrocytes and spleen cell sus-

pensions from 6-week-old mice immunized I week previously with SSA. Plate A contain 5.5 X 105 leukocytes fromthe spleen ofa nontolerant control mouse; plate B contains 2.3 X 106 leukocytes from a Shigella-tolerant mouse.Unlysed erythrocytes in each plate stained dark blue with benzidine-H202 (17).

TABLE 2. Effect ofage ofShigella-tolerant or normal mice on number ofantibody plaques formedper spleen

Shigella-toleranta Normal

Age at Serum Plaques/lO6 spleen cells Serum Plaques/106 spleen cellsNo. of mice hemolysins No. of mice hemolysins

tested to Shigella- Untreated tested to Shigella UntreatedcoatedRBC SA RBC RBC coated RBC SSA RBC UnReate

weeks

2-3 4 <1:10 0.5 0.4 6 <1:10 0.1 0.33-6 10 <1:10 1.4 0.1 15 <1:10 0.6 0.18-10 6 <1:10 1.2 0.6 10 <1:10 1.1 0.912-15 4 <1:10 2.5 1.1 _ _20-25 7 <1:10 3.9 1.3 6 <1:10 2.1 1.4

a Tolerant mice injected at birth with SSA.

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erythrocytes. Groups of Shigella-tolerant andnormal mice were injected with sheep erythrocytesonly and sacrificed several days thereafter. Spleencell suspensions were added to agar plates con-taining either SSA-treated or untreated sheepRBC. There was no discernible difference in thenumber of plaques formed to sheep RBC withspleen cells from either group injected withS-RBC only (groups B and F, Table 4).

TABLE 3. Antibody plaque formation and hemolysinresponses of Shigella-tolerant and normalmice challenged with SSA at various ages

SSA-treated RBCAge at

challenge No. ofMouse group with mice Mean peak Avgplaque

SSA(e tested hemoly- count per(weeks) sin 106 spleen

titer cells

Shigella-tol- 4 4 <1:10 2erantb 8 8 <1:10 11

12 6 1:37 1615 6 1:82 6020 6 1:173 8330 8 1:346 131

Normal 4 15 1:238 1688 10 1:310 192

15 10 1:490 18530 10 1:380 145

a Animals sacrificed 5 to 6 days after intra-peritoneal challenge injection.

bTolerant mice injected at birth with SSA.

TABLE 4. Antibody plaque formation and serumantibody response of Shigella-tolerant and

normal mice challenged with SSA orsheep RBC

Mousegroup,

Shigella-toleranteABCD

NormalEFGH

Challengedwithb

+

+

+

u

C)

+

+

+

No. ofplaques/106spleen cells

u

cnv)

9332<1643

240385<1520

~C)

419<1531

26402<1611

Peak hemolysintiter

vf)

<1:101:240

<1:101:495

1:3841:480

<1:101:480

Although plaque formation to SSA-treatedsheep erythrocytes was low with spleen cells fromShigella-tolerant mice challenged with SSA only(group A, Table 4), there was an elevated numberof antibody plaques to untreated S-RBC withspleen cell suspensions from both normal andtolerant mice challenged with both SSA andS-RBC (groups D and H, Table 4). There ap-peared to be an enhanced response to S-RBC inmice, either normal or tolerant, injected with bothSSA and S-RBC (Table 4, D and H), as com-pared with normal mice receiving only S-RBC(Table 4, F). Sera from all mice injected withS-RBC, either normal or tolerant, agglutinatedand hemolyzed both antigen-treated and un-treated sheep erythrocytes in tube dilution tests.

DISCUSSION

The results indicate that injection of mice atbirth with a tolerance-inducing inoculum of Shi-gella antigen not only inhibits subsequent ap-pearance of circulating anti-Shigella agglutinins,but also markedly suppresses the number oflymphoid cells secreting Shigella-specific anti-body in agar gel. These findings support previousconclusions that cells capable of forming specificantibody either are absent or are present in lownumbers in lymphoid tissue of mice tolerant toShigella antigens (5, 8). Other investigators, usingcell transfer procedures and other antigens, havesimilarly concluded that cells capable of formingspecific antibody are not detectable in lymphoidtissue from specifically tolerant donors (1, 2, 4,14, 28-30).The immunological nature of the "antibody

plaques" described in this report is based onevidence similar to that obtained with directplaque procedures by Jerne and co-workers (17),by Ingraham and Bussard (16), and by others (23,32, D. Syiklocha, Federation Proc. 24:614, 1965;R. J. Trapani, G. S. La Fontaine, and L. D.Carter, Federation Proc. 24:252, 1965). Plaquesto Shigella-coated erythrocytes occur in largenumbers only with lymphoid cells from SSA-challenged animals. Plaques are complement-dependent and are usually readily distinguishablefrom those occurring prior to addition of com-plement or those caused by cellular debris in theagar layer. Prior to injection of Shigella antigen,the anti-Shigella plaque levels are low or neg-ligible. Increase in plaque formation in normalanimals after SSA challenge parallels, and usuallyprecedes, the rise in circulating serum agglutinintiters. In additional experiments not reportedhere, it has been observed that several immuno-suppressive agents, including X irradiation and

a At least four mice per immunization group.6Mice, 5 to 6 weeks old, injected with challenge agent;

sacrificed 4 to 8 days later.C Tolerant mice injected at birth with SSA.

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6-mercaptopurine, when administered prior toSSA challenge, suppress plaque formation.The use of the indirect antibody plaque proce-

dure, first described by Landy et al. (18, 19), haspermitted direct estimation of the number ofantibody-secreting cells present in spleen cellsuspensions from normal, immune, and Shigella-tolerant mice. Previously, the antibody plaqueprocedure was limited to demonstration of anti-body formation to antigens native to mammalianerythrocytes and, on occasion, to avian erythro-cytes (7, 16).The direct plaque procedure with uncoated

erythrocytes was used in earlier experiments (9)to study tolerance to red cell antigens. However,tolerance to erythrocytes is generally less com-plete than that obtained with other nonliving anti-gens, including Shigella extract, and usually re-quires repeated administration of red cells forpersistence of the tolerant state (15, 20, 22). Onceadministration of red cell suspensions ceases, de-tectable antibody formation to the erythrocytes isusually restored. Despite this shortcoming, thedirect antibody plaque technique has been suc-cessfully applied to the study of cellular aspects oftolerance to sheep erythrocytes in mice and rats(9, 24-26; T. Hraba, personal communication). Bi-weekly injections of large quantities of sheep redcell suspensions from birth through young adult-hood have resulted in tolerance to sheep RBC inmice (9), demonstrable by marked reduction inthe number of antibody plaques formed withspleen cell suspensions in agar, as compared withthe number obtained with cells from normalimmune mice. Rowley and Fitch (25, 26) havealso observed a similar decrease in antibodyplaque formation to sheep erythrocytes withspleen cells from rats treated, either as neonates oras adults, with multiple injections of sheep RBC.The results reported here extend the study of

tolerance on the cellular level to a system using asoluble bacterial antigen. In previous studies, itwas found that mice tolerant to Shigella antigenwere not only markedly deficient in circulatingagglutinin formation, but also lacked cells con-taining anti-Shigella antibody identifiable byimmunofluorescence procedures (11). Spleen cellsuspensions from Shigella-tolerant mice alsofailed to transfer agglutinin-forming ability tonormal or X-irradiated recipients (8). The resultsobtained in this study, from an extremely sensitiveassay, further document the marked absence ofspecific antibody-forming cells in lymphoid tissueof Shigella-tolerant mice. However, once toler-ance is lost, antibody plaque-forming ability isgenerally restored (Table 3).The specificity of tolerance to Shigella antigen

on the cellular level has been demonstrated in thisstudy by the observation that spleen cells fromShigella-tolerant mice are capable offorming anti-body plaques to untreated erythrocytes afterchallenge immunization of the animals with sheepRBC. It is of interest to note that the number ofplaques with untreated sheep red cells is generallyenhanced with spleen cell suspensions from bothtolerant and normal mice injected with red cellsand SSA simultaneously. This suggests that Shi-gella antigen exerts an adjuvant effect even intolerant animals. Enhancing effects of gram-nega-tive bacterial antigens on "background" andinduced plaque formation to sheep RBC innormal and immune animals has also been notedby others (27; A. E. Hever and B. Pernis, Bac-teriol. Proc., p. 44, 1964). It is assumed that theendotoxin moiety of gram-negative bacteria anti-gens "nonspecifically" stimulates antibody plaqueformation towards sheep erythrocytes, even inanimals receiving only endotoxin and no RBC.No attempts were made in this study to differ-

entiate 7S from 19S agglutinins or hemolysins.However, it is possible that the low number ofplaques produced with spleen cell suspensionsfrom tolerant mice, as compared with normalmice, may reflect a difference in the moleculartype of antibody secreted by the two groups ofanimals. It is known that hemolysins to sheepRBC are generally 19S (IgM) molecules duringthe early response to immunization, and usually7S (IgG) molecules after secondary immuniza-tion. The 7S type of antibody is generally lessefficient than 19S antibody in hemolytic reactionswith similar numbers of erythrocytes and comple-ment (3, 31). An indirect anti-y globulin modifica-tion of the Jerne antibody plaque technique hasbeen used recently by others, to differentiate 19Sfrom 7S antibody plaques with spleen cells frommice immunized with sheep RBC. This procedurewas not used in the present study with spleen cellsfrom normal or SSA-tolerant animals. It may bethat the suppressed plaque formation with cellsfrom RBC "tolerant" mice (9, 25, 26) could bedue to lower efficiency of 7S antibody productionafter multiple injections with S-RBC. It is alsopossible that, in this study, antibody formation toSSA by individual cells from tolerant animals,which may have had an aborted or undetectedprimary antibody response after initial injectionof Shigella antigen at birth, could have been pre-dominantly of the 7S type which is less efficient insensitizing antigen-coatedRBC for the subsequentformation of visible plaques of hemolysis. Al-though most antibody in the circulation of Shi-gella-immune mice is of the 19S type, such aninterpretation cannot be ruled out by the experi-

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ments reported here. However, the results indicat-ing a lack of antibody plaque-forming cells inspleens of tolerant mice are quite similar to thoseobtained from immunofluorescence, cell transfer,and direct serological agglutination tests.The similarity in the size of plaques produced

with spleen cell suspensions from both tolerantand control mice also suggests that individualcells competent to form detectable amounts ofantibody to Shigella-coated red blood cells are, ingeneral, secreting the same quantity of antibodyas that secreted by "normal cells" from control,nontolerant animals. The only significant differ-ence observed was that the numbers of specificantibody-forming cells were greatly reduced inlymphoid tissue of tolerant animals. Antibodyplaque-forming cells to an unrelated antigen,sheep erythrocytes, were present in normal num-bers.

AcKNowLEMENTrsWe are grateful for the capable technical assistance

of Gert Meloff, Phyliss Pivar, Barry Silver, LeonyMills, and Leslie Berger.

This investigation was supported by the NationalScience Foundation.Some of the data in this report were presented at

the 65th Annual Meeting, American Society forMicrobiology, Atlantic City, N. J., 25-29 April 1965.

LITERATuRE CITED1. ARGYRIS, B. F. 1963. Adoptive tolerance; transfer

of the tolerant state. J. Immunol. 90:29-34.2. BATTISrO, J. R., AND M. W. CHASE. 1963. Im-

munologic unresponsiveness to sensitizationwith simple chemical compounds. A search forfor antibody absorbing depots of allergen. J.Exptl. Med. 118:1021-1035.

3. BoRsos, T., AND H. J. RAPP. 1965. Complementfixation on cell surfaces by 19S and 7S anti-bodies. Science 150:505-506.

4. BROOKE, M. S., AND M. J. KARNOWSKY. 1961.Immunological paralysis and adoptive immu-nity. J. Immunol. 87:205-208.

5. FRIEDMAN, H. 1962. Transfer of antibody forma-tion by spleen cells from immunologically un-responsive mice. J. Immunol. 89:257-263.

6. FRIEDMAN, H. 1964. Distribution of antibodyforming cells in various tissues of several strainsof mice injected with sheep erythrocytes. Proc.Soc. Exptl. Biol. Med. 117:526-530.

7. FRIEDMAN, H. 1964. Monospecific antibodyplaque formation by spleen cells from miceimmunized with sheep and chicken erythrocytes.Experientia 20:564-566.

8. FRIEDMAN, H. 1965. Adoptive tolerance to Shigellaantigens in irradiated mice receiving spleen celltransplants from unresponsive donors. J.Immunol. 94:352-357.

9. FRIEDMAN, H. 1965. Failure of spleen cells fromimmunologically tolerant mice to form anti-

body plaques to sheep erythrocytes in agar gel.Nature 205:508-509.

10. FRIEDMAN, H. 1965. Absence of antibody plaqueforming cells in spleens of thymectomizedmice immunized with sheep erythrocytes.Proc. Soc. Exptl. Biol. Med. 118:1176-1180.

11. FRIEDMAN, H. 1966. Immunological tolerance tomicrobial antigens. I. Absence of specific anti-body-containing cells in lymphoid tissue ofmiceinjected at birth with Shigella soluble antigen.J. Bacteriol. 92:390-397.

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13. HARRIS, T. N., S. HARRIS, AND M. B. FARBER.1956. Studies on the transfer of lymph nodecells. VII. Transfer of cells incubated in vitrowith filtrates of trypsin-treated suspensions ofShigella paradysenteriae. J. Exptl. Med. 104:645-661.

14. HASEK, M., A. LENGEROVA, AND T. HRABA. 1961.Transplantation immunity and tolerance.Advan. Immunol. 1:1-66.

15. HASEK, M., AND A. PUZA. 1962. Induction oftolerance in adult life and reminiscence oftolerance, p. 257. In M. Haslk, A. Lengerova,and M. Vojtiska [ed.], Mechanisms of im-munological tolerance. Academic Press, Inc.,New York.

16. INGRAHAM, J. S., AND A. BUSSARD. 1964. Applica-tion of a localized hemolysin reaction for speci-fic detection of individual antibody-formingcells. J. Exptl. Med. 119:667-684.

17. JERNE, N. K., A. A. NORDIN, AND C. HENRY.1963. The agar plaque technique for recognizingantibody-producing cells, p. 105. In B. Amosand H. Koprowski [ed.], Cell-bound antibodies.The Wistar Institute Press, Philadelphia.

18. LANDY, M., R. P. SANDERSON, M. T. BERNSrEIN,AND A. L. JACKSON. 1964. Antibody productionby leukocytes in peripheral blood. Nature204:1320-1321.

19. LANDY, M., R. P. SANDERSON, M. T. BERNSTEIN,AND E. M. LERNER. 1965. Involvement ofthymus in immune response of rabbits tosomatic polysaccharides of gram-negative bac-teria. Science 147:1591-1592.

20. MITCHINSON, N. A. 1962. Tolerance of erythro-cytes in poultry: induction and specificity.Immunology 5:359-369.

21. NETER, E. 1956. Bacterial hemagglutination andhemolysis. Bacteriol. Rev. 20:166-188.

22. NOSSAL, G. J. V. 1958. Induction of immuno-logical tolerance in rats to foreign erythrocytes.Australian J. Exptl. Biol. Med. Sci. 36:235-244.

23. RICHARDSON, M., AND R. W. DUTTON. 1964. Anti-body synthesizing cells; appearance aftersecondary antigenic stimulation in vitro. Science146:655-656.

24. ROWLEY, D. A., AND F. W. FITCH. 1964. Home-ostasis of antibody formation in the adult rat.J. Exptl. Med. 120:987-1005.

25. ROWLEY, D. A., AND F. W. FITCH. 1965. The

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