Immunological Tolerance to Microbial Antigens

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1 JouaNAL OF BACERIOLOOY, Oct., 1966 Copyright 1966 American Society for Microbiology Vol. 92, No. 4 Printed in U.S.A. Immunological Tolerance to Microbial Antigens II. Suppressed Antibody Plaque Formation to Shigella Antigen by Spleen Cells from Tolerant Mice HERMAN 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.). Imunological tolerance to microbial antigens. II. Suppressed antibody plaque formation to Shigella antigen by spleen cells from tolerant mice. J. Bacteriol. 92: An indirect, localized, antibody plaque procedure has been used to demonstrate a marked difference in the number of antibody plaques formed with spleen cell suspensions from normal and Shigella-tolerant mice. Whereas challenge with soluble 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 sheep erythrocytes, there was only a slight increase in plaque formation with spleen cell suspensions from similarly challenged mice which had been made tolerant to Shigella antigen during neonatal life. Apparently, the suppression of plaque formation to Shigella in tolerant animals was specific, since both Shigella-tolerant and normal mice responded equally well to untreated sheep red blood cells after challenge with sheep erythrocytes only. Spleen cells from nonchallenged Shigellatolerant mice did not form significant numbers of antibody plaques to SSA-treated red blood cells during an observation period of 4 to 30 weeks after neonatal administration of antigen. "Nonspecific" increases in plaque formation to untreated sheep red cells occurred with spleen cell suspensions from both normal and SSAtolerant mice after challenge injection with Shigella antigen, with or without sheep erythrocytes. Such a response suggested an adjuvant effect for the endotoxin-containing Shigella antigen even in mice tolerant to the agglutinogenic moiety of SSA. The results of these experiments support the view that specific antibody-forming cells are either absent or in low number in lymphoid tissue from mice specifically tolerant to Shigella antigens. It seems unlikely that the low postchallenge agglutinin titers of tolerant mice are due to suppressed antibody formation by normal numbers of individual antibody-producing cells, or due to "masking" of normal antibody production by persisting circulating antigen. Specific immunological unresponsiveness in present in Shigella-tolerant animals, but that each duced in neonatal mice by treatment at birth withi cell secretes subnormal quantities of antibody. a relatively large quantity of Shigella soluble anti - The sensitivity of immunofluorescence procedures gen (SSA) is specific and, depending on dose andi is relatively low and may be insufficient to detect route of inoculation at birth, may persist forr small quantities of antibody secreted by cells from several months (12). Such unresponsiveness iss tolerant animals (11, 28). A direct estimation of characterized by markedly suppressed agglutinir x the number of cells actively synthesizing antibody responses after challenge injection with Shigellai in situ by more sensitive serological or immuno- logical means has not been feasible until recently. antigen capable of inducing detectable levels off agglut in normal mice (12). As describedi Development of the localized hemolysis plaque previously, tolerant mice lack specific antibody-containing lymphoid cells as determined by the Bussard (16), has permitted use of a rapid and procedure by Jerne (17), and by Ingraham and indirect immunofluorescence technique (11). It iss sensitive immunological method for estimation possible, however, that normal numbers off of the number of lymphoid cells actively secreting specific immunologically competent cells may be antibody (6, 10, 23, 32). Studies utilizing the 820

2 VOL. 92, 1966 IMMUNOLOGICAL TOLERANCE TO MICROBIAL ANTIGENS antibody plaque procedure have been generally limited to the demonstration of cells secreting hemolytic antibody to mammalian or avian erythrocytes. Recently, however, the localized antibody plaque assay technique has been used to estimate the number of lymphoid cells forming antibody to soluble bacterial antigens. The plaquing test is accomplished with bacterial antigens coated onto isologous or heterologous erythrocytes (18, 19; I. Canellas and A. Intravastolo. Bacteriol. Proc., p. 53, 1965). Use of such indirect, or passive, plaque procedures with bacterial systems should permit direct comparison of the number of antibody-secreting cells in lymphoid tissue of normal and tolerant animals. This report is concerned with results of such a study designed to enumerate the number of anti- Shigella plaques formed in agar with spleen cell suspensions from Shigella-tolerant mice as compared with those from normal mice. MATERIALS AND METHODS Experimental animals, antigens, and induction of tolerance. Mice, SSA, route, and time of neonatal injection, and serological agglutination tests have been listed in the previous reports (5, 8, 12, 13). Hemolytic plaque assay. The direct immunoplaque assay for detection of localized zones of hemolysis with uncoated erythrocytes was performed essentially as described by Jeme, Nordin, and Henry (17). Mice were injected intraperitoneally with 0.1 ml of a 10% suspension of washed sheep erythrocytes. At the time of testing, each animal was bled by retro-orbital puncture and then sacrificed by cervical dislocation. Spleens were rapidly removed and placed immediately in cold sterile Hanks solution (ph 7.2). Cell suspensions were prepared aseptically by "teasing" with fine needles (5, 8). Tissue fragments and large aggregates of cells were removed by passing the suspensions through fine-mesh stainless-steel gauze, followed by two washings in cold (2 to 4 C) sterile Hanks solution by means of serial centrifugation. The number of nucleated cells in the suspensions was determined with a hemocytometer, and viability was estimated by trypan blue-staining technique. Suspensions of cells containing 5 X 104 to 10 X 106 nucleated cells per 821 milliliter were prepared, and plates were poured in duplicate or triplicate as follows: 0.1 ml of each cell suspension was quickly added to a tube filled with 2.0 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 a 3-mm thick layer of 1.5% Noble agar in Hanks solution (without phenol red), which had been previously prepared in petri plates (100 mm in diameter) and refrigerated for 24 to 48 hr. After solidification of the upper agar layer containing the spleen cells and sheep erythrocytes, the plates were incubated at 37 C for 1 hr, and then were treated with 5 ml of a 1:10 dilution of guinea pig serum as a source of complement (BBL). The plates were again incubated at 37 C for 30 min. Localized zones of hemolysis could be observed as clear areas against a background of unlysed red cells. The plaques were made more distinct and the plates were preserved for extended time periods by staining with cold, freshly prepared benzidine- H202-acetic acid solution (17). The total number of plaques formed per whole spleen or per 106 nucleated cells was calculated from the average plaque count of several plates prepared from each spleen when inocula that resulted in 50 to 300 plaques per plate were used. In most instances, plates were observed for nonspecific zones of hemolysis prior to addition of the complement. The number of such complementnondependent plaques, when present, was subtracted from the number of plaques appearing after treatment with complement. Generally, 5 to 20 nonspecific hemolytic areas appeared per plate when more than 5 X 106 spleen cells were used per plate. Indirect hemolytic plaque assay with erythrocytes coated with Shigella antigen. An indirect antibody plaque technique was used to determine the number of anti-shigella antibody-forming cells in spleens and lymph nodes of normal and tolerant mice before and after challenge injection with SSA. Freshly washed sheep erythrocytes were treated for 1 hr at 37 C with SSA 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 erythrocytes was optimal for sensitization. For the indirect plaque assay, 0.1 ml of a freshly prepared 10% suspension of SSA-treated erythrocytes was used in place of untreated erythrocytes. Otherwise, the plaque assay was performed exactly as described above. In addition, however, when relatively few plaques were formed with spleen cells from Shigella-tolerant mice, relatively large numbers of spleen cells were used per plate in attempts to obtain counts within the range of 50 to 300 hemolytic zones per plate. In these instances, there were often large numbers of nonspecific hemolytic areas, apparently due to the debris in the cell suspensions or to aggregated cells. Plates were poured in quadruplicate; one or two plates were not treated with complement, but were stained with benzidine. The number of hemolytic zones in these complement-free plates was subtracted from the number of plaques found in plates treated with complement. Serological hemagglutination procedure. Sera of normal and tolerant mice obtained at various intervals before or after challenge with SSA were tested for antibody to Shigella extract by passive hemagglutination and hemolysis procedures. Equal quantities of 10% suspensions of washed sheep erythrocytes and SSA (5 pg of N/ml) were incubated for 1 hr at 37 C and then were washed several times with cold sterile saline. The treated cells, in a concentration of 1.5%, were added in 0.05-ml quantities to 0.5 ml of serial twofold dilutions of the mouse sera, 1:10 through 1:10,240. After incubation at 37 C for 1 hr and in the cold for 24 hr, the hemagglutinin titer was recorded as the reciprocal ofthe highest dilution of serum resulting in complete agglutination of the treated red cells. For

3 822 FRIEDMAN J. BACTERIOL the hemolysis test, mouse serum was heated for 30 min at 56 C prior to titration. A 0.1-nml amount of a 1:20 dilution of guinea pig serum (as a source of complement) was added to each tube before the addition of the erythrocytes. Hemolysis was recorded after 1 hr of incubation at 37 C. All sera were absorbed with 1 volume of washed, packed, untreated red cells prior to dilution. In addition, all sera positive for hemaglutinins or hemolysins to SSA-treated red blood cells RBC) were tested with untreated RBC as a control. ESULTS Plaque formation with spleen cells from normal mice injected with Shigella antigen. Normal mice challenged 2 to 8 days previously with a known immunizing inoculum of Shigella antigen (SSA, 20,g of N) were sacrificed, and washed spleen cell suspensions were obtained for preparation of agar plates containing either untreated or SSAcoated erythrocytes. After incubation with complement, many localized zones of hemolysis appeared in plates containing SSA-treated erythrocytes (Tables 1 and 3). There was a maximum of several hundred complement-dependent plaques per 106 nucleated spleen cells from Shigella-immunized, nontolerant mice. Several dozen hemolytic zones per 106 spleen cells were formed when the same cell suspensions were tested with untreated sheep erythrocytes. Plaque formation with cells from Shigellatolerant mice. Shigella-tolerant mice, 5 to 6 weeks old, were sacrificed at various time intervals after challenge with SSA. Serum samples were obtained prior to sacrifice for determination of Shigella antibody levels through direct bacterial agglutination tests and through passive serological TABLE 1. hemolysis and hemagglutination tests with SSAtreated RBC. Spleen cell suspensions were tested for plaque-forming ability in agar plates containing SSA-treated erythrocytes. Table 1 indicates the plaque-forming ability of cell suspensions from tolerant mice at several time intervals after challenge injection with SSA. Whereas spleen cell suspensions from normal mice exhibited approximately a 40- to 100-fold increase in plaque formation over a 2- to 10-day period after challenge, there was only a slight increase in plaque formation with spleen cell suspensions from tolerant mice. The average plaque size was 0.05 mm with cells from either normal or tolerant mice. The number of plaques per spleen was the only observable difference between spleen cell suspensions from the two groups (Fig. 1). Plaque formation in plates containing untreated sheep erythrocytes increased 5- to 10-fold within a few days after challenge immunization with SSA, regardless of whether the test animal was a normal control or a tolerant mouse (Table 1). Effect ofage on antibody plaque formation with spleen cells from tolerant or normal mice. Mice injected at birth with a tolerance-inducing inoculum of SSA were sacrificed at various ages, and washed spleen cell suspensions were prepared and used for preparation of agar plates containing SSA-treated RBC. Although the number of plaques formed in agar containing SSA-treated sheep red cells increased slightly with the age of the SSA-tolerant animal (Table 2), it was not significantly different from that observed when the same spleen cell suspensions were tested with untreated sheep red cells. Spleen cell suspensions from normal, nontolerant animals also had a Serum antibody titers and antibody plaque formation with spleen cells of normal and Shigellatolerant mice obtained before and after SSA challenge Serum antibody (means) Hemolytic plaques/106 spleen cells Mouse group immunization No. of mice with SSA8 tested Shieila Tube hemol- SSA-treated Untreated agglutination ysis6 sheep RBC sheep RBC Normal 0 12 <1: 10 <1: :83 1: :365 1: :380 1: Shigella-tolerantc 0 10 <1:10 <1: <1:10 <1: :32 1: :30 1: 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.

4 VOL. 92, 1966 IMMUNOLOGICAL TOLERANCE TO MICROBIAL ANTIGENS 823 low, but readily detectable, increase in "background" count with increased age. Duration of tolerance as assessed by plaque formation. Shigella-tolerant and normal control mice, 4 to 30 weeks old, were challenged with SSA at various times. Groups of mice were sacrificed 5 to 6 days after each injection period, and spleen cell suspensions were tested in agar containing SSA-treated sheep erythrocytes (Table 3). The number of plaques formed with spleen cell suspensions from tolerant animals 4 to 12 weeks of age was less than 5 to 10% of that obtained with cell suspensions from normal control mice immunized with SSA at the same time. Plaque-forming ability of spleen cells from tolerant mice increased sharply when the mice were 15 to 20 weeks old and generally paralleled serum antibody responses. Tolerance to Shigella, as assayed both by circulating antibody and by plaque-forming ability, was generally lost by the time the mice were 3 to 5 months old, as observed in previous studies in which antibody formation was the only criterion used to determine loss of tolerance. Antibody plaque formation by spleen cell suspensions from tolerant and normal mice challenged with sheep erythrocytes. The ability of Shigellatolerant mice to form plaques to untreated sheep erythrocytes after specific immunization with RBC only was determined to demonstrate the specificity of plaque suppression to SSA-coated Downloaded from FIG. 1. Antibody plaques in agar gel containing Shigella antigen-coated sheep erythrocytes and spleen cell suspensions from 6-week-old mice immunized I week previously with SSA. Plate A contain 5.5 X 105 leukocytes from the 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 on April 7, 2019 by guest Age at Serum Plaques/lO6 spleen cells Serum Plaques/106 spleen cells No. of mice hemolysins No. of mice hemolysins tested to Shigella- Untreated tested to Shigella Untreated coatedrbc SA RBC RBC coated RBC SSA RBC UnReate weeks <1: <1: <1: <1: <1: <1: <1: <1: <1: a Tolerant mice injected at birth with SSA.

5 824 FRIEDMAN J. BACTrERIOL. erythrocytes. Groups of Shigella-tolerant and normal mice were injected with sheep erythrocytes only and sacrificed several days thereafter. Spleen cell suspensions were added to agar plates containing either SSA-treated or untreated sheep RBC. There was no discernible difference in the number of plaques formed to sheep RBC with spleen cells from either group injected with S-RBC only (groups B and F, Table 4). TABLE 3. Antibody plaque formation and hemolysin responses of Shigella-tolerant and normal mice challenged with SSA at various ages SSA-treated RBC Age at challenge No. of Mouse group with mice Mean peak Avgplaque SSA(e tested hemoly- count per (weeks) sin 106 spleen titer cells Shigella-tol- 4 4 <1:10 2 erantb 8 8 <1: : : : : Normal : : : : a Animals sacrificed 5 to 6 days after intraperitoneal challenge injection. btolerant mice injected at birth with SSA. TABLE 4. Antibody plaque formation and serum antibody response of Shigella-tolerant and normal mice challenged with SSA or sheep RBC Mouse group, Shigellatolerante A B C D Normal E F G H Challenged withb u C) No. of plaques/106 spleen cells u cn v) < <1 520 ~C) 419 < <1 611 Peak hemolysin titer vf) <1:10 1:240 <1:10 1:495 1:384 1:480 <1:10 1:480 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. Although plaque formation to SSA-treated sheep erythrocytes was low with spleen cells from Shigella-tolerant mice challenged with SSA only (group A, Table 4), there was an elevated number of antibody plaques to untreated S-RBC with spleen cell suspensions from both normal and tolerant mice challenged with both SSA and S-RBC (groups D and H, Table 4). There appeared to be an enhanced response to S-RBC in mice, either normal or tolerant, injected with both SSA and S-RBC (Table 4, D and H), as compared with normal mice receiving only S-RBC (Table 4, F). Sera from all mice injected with S-RBC, either normal or tolerant, agglutinated and hemolyzed both antigen-treated and untreated sheep erythrocytes in tube dilution tests. DISCUSSION The results indicate that injection of mice at birth with a tolerance-inducing inoculum of Shigella antigen not only inhibits subsequent appearance of circulating anti-shigella agglutinins, but also markedly suppresses the number of lymphoid cells secreting Shigella-specific antibody in agar gel. These findings support previous conclusions that cells capable of forming specific antibody either are absent or are present in low numbers in lymphoid tissue of mice tolerant to Shigella antigens (5, 8). Other investigators, using cell transfer procedures and other antigens, have similarly concluded that cells capable of forming specific antibody are not detectable in lymphoid tissue from specifically tolerant donors (1, 2, 4, 14, 28-30). The immunological nature of the "antibody plaques" described in this report is based on evidence similar to that obtained with direct plaque 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). Plaques to Shigella-coated erythrocytes occur in large numbers only with lymphoid cells from SSAchallenged animals. Plaques are complementdependent and are usually readily distinguishable from those occurring prior to addition of complement or those caused by cellular debris in the agar layer. Prior to injection of Shigella antigen, the anti-shigella plaque levels are low or negligible. Increase in plaque formation in normal animals after SSA challenge parallels, and usually precedes, the rise in circulating serum agglutinin titers. In additional experiments not reported here, it has been observed that several immunosuppressive agents, including X irradiation and

6 VOL. 92,1966 IMMUNOLOGICAL TOLERANCE TO MICROBIAL ANTIGENS 6-mercaptopurine, when administered prior to SSA challenge, suppress plaque formation. The use of the indirect antibody plaque procedure, first described by Landy et al. (18, 19), has permitted direct estimation of the number of antibody-secreting cells present in spleen cell suspensions from normal, immune, and Shigellatolerant mice. Previously, the antibody plaque procedure was limited to demonstration of antibody formation to antigens native to mammalian erythrocytes and, on occasion, to avian erythrocytes (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 complete than that obtained with other nonliving antigens, including Shigella extract, and usually requires repeated administration of red cells for persistence of the tolerant state (15, 20, 22). Once administration of red cell suspensions ceases, detectable antibody formation to the erythrocytes is usually restored. Despite this shortcoming, the direct antibody plaque technique has been successfully applied to the study of cellular aspects of tolerance to sheep erythrocytes in mice and rats (9, 24-26; T. Hraba, personal communication). Biweekly injections of large quantities of sheep red cell suspensions from birth through young adulthood have resulted in tolerance to sheep RBC in mice (9), demonstrable by marked reduction in the number of antibody plaques formed with spleen cell suspensions in agar, as compared with the number obtained with cells from normal immune mice. Rowley and Fitch (25, 26) have also observed a similar decrease in antibody plaque formation to sheep erythrocytes with spleen cells from rats treated, either as neonates or as adults, with multiple injections of sheep RBC. The results reported here extend the study of tolerance on the cellular level to a system using a soluble bacterial antigen. In previous studies, it was found that mice tolerant to Shigella antigen were not only markedly deficient in circulating agglutinin formation, but also lacked cells containing anti-shigella antibody identifiable by immunofluorescence procedures (11). Spleen cell suspensions from Shigella-tolerant mice also failed to transfer agglutinin-forming ability to normal or X-irradiated recipients (8). The results obtained in this study, from an extremely sensitive assay, further document the marked absence of specific antibody-forming cells in lymphoid tissue of Shigella-tolerant mice. However, once tolerance is lost, antibody plaque-forming ability is generally restored (Table 3). The specificity of tolerance to Shigella antigen 825 on the cellular level has been demonstrated in this study by the observation that spleen cells from Shigella-tolerant mice are capable of forming antibody plaques to untreated erythrocytes after challenge immunization of the animals with sheep RBC. It is of interest to note that the number of plaques with untreated sheep red cells is generally enhanced with spleen cell suspensions from both tolerant and normal mice injected with red cells and SSA simultaneously. This suggests that Shigella antigen exerts an adjuvant effect even in tolerant animals. Enhancing effects of gram-negative bacterial antigens on "background" and induced plaque formation to sheep RBC in normal and immune animals has also been noted by others (27; A. E. Hever and B. Pernis, Bacteriol. Proc., p. 44, 1964). It is assumed that the endotoxin moiety of gram-negative bacteria antigens "nonspecifically" stimulates antibody plaque formation towards sheep erythrocytes, even in animals receiving only endotoxin and no RBC. No attempts were made in this study to differentiate 7S from 19S agglutinins or hemolysins. However, it is possible that the low number of plaques produced with spleen cell suspensions from tolerant mice, as compared with normal mice, may reflect a difference in the molecular type of antibody secreted by the two groups of animals. It is known that hemolysins to sheep RBC are generally 19S (IgM) molecules during the early response to immunization, and usually 7S (IgG) molecules after secondary immunization. The 7S type of antibody is generally less efficient than 19S antibody in hemolytic reactions with similar numbers of erythrocytes and complement (3, 31). An indirect anti-y globulin modification of the Jerne antibody plaque technique has been used recently by others, to differentiate 19S from 7S antibody plaques with spleen cells from mice immunized with sheep RBC. This procedure was not used in the present study with spleen cells from normal or SSA-tolerant animals. It may be that the suppressed plaque formation with cells from RBC "tolerant" mice (9, 25, 26) could be due to lower efficiency of 7S antibody production after multiple injections with S-RBC. It is also possible that, in this study, antibody formation to SSA by individual cells from tolerant animals, which may have had an aborted or undetected primary antibody response after initial injection of Shigella antigen at birth, could have been predominantly of the 7S type which is less efficient in sensitizing antigen-coated RBC for the subsequent formation of visible plaques of hemolysis. Although most antibody in the circulation of Shigella-immune mice is of the 19S type, such an interpretation cannot be ruled out by the experi-

7 826 FRIEDMAN J. BACTERIOL. ments reported here. However, the results indicating a lack of antibody plaque-forming cells in spleens of tolerant mice are quite similar to those obtained from immunofluorescence, cell transfer, and direct serological agglutination tests. The similarity in the size of plaques produced with spleen cell suspensions from both tolerant and control mice also suggests that individual cells competent to form detectable amounts of antibody to Shigella-coated red blood cells are, in general, secreting the same quantity of antibody as that secreted by "normal cells" from control, nontolerant animals. The only significant difference observed was that the numbers of specific antibody-forming cells were greatly reduced in lymphoid tissue of tolerant animals. Antibody plaque-forming cells to an unrelated antigen, sheep erythrocytes, were present in normal numbers. AcKNowLEMENTrs We are grateful for the capable technical assistance of Gert Meloff, Phyliss Pivar, Barry Silver, Leony Mills, and Leslie Berger. This investigation was supported by the National Science Foundation. Some of the data in this report were presented at the 65th Annual Meeting, American Society for Microbiology, Atlantic City, N. J., April LITERATuRE CITED 1. ARGYRIS, B. F Adoptive tolerance; transfer of the tolerant state. J. Immunol. 90: BATTISrO, J. R., AND M. W. CHASE Immunologic unresponsiveness to sensitization with simple chemical compounds. A search for for antibody absorbing depots of allergen. J. Exptl. Med. 118: BoRsos, T., AND H. J. RAPP Complement fixation on cell surfaces by 19S and 7S antibodies. Science 150: BROOKE, M. S., AND M. J. KARNOWSKY Immunological paralysis and adoptive immunity. J. Immunol. 87: FRIEDMAN, H Transfer of antibody formation by spleen cells from immunologically unresponsive mice. J. Immunol. 89: FRIEDMAN, H Distribution of antibody forming cells in various tissues of several strains of mice injected with sheep erythrocytes. Proc. Soc. Exptl. Biol. Med. 117: FRIEDMAN, H Monospecific antibody plaque formation by spleen cells from mice immunized with sheep and chicken erythrocytes. Experientia 20: FRIEDMAN, H Adoptive tolerance to Shigella antigens in irradiated mice receiving spleen cell transplants from unresponsive donors. J. Immunol. 94: FRIEDMAN, H Failure of spleen cells from immunologically tolerant mice to form antibody plaques to sheep erythrocytes in agar gel. Nature 205: FRIEDMAN, H Absence of antibody plaque forming cells in spleens of thymectomized mice immunized with sheep erythrocytes. Proc. Soc. Exptl. Biol. Med. 118: FRIEDMAN, H Immunological tolerance to microbial antigens. I. Absence of specific antibody-containing cells in lymphoid tissue of mice injected at birth with Shigella soluble antigen. J. Bacteriol. 92: FRIEDMAN, H., AND W. L. GABY Immunologic unresponsiveness in mice following neonatal exposure to Shigella antigens. J. Immunol. 85: HARRIS, T. N., S. HARRIS, AND M. B. FARBER Studies on the transfer of lymph node cells. VII. Transfer of cells incubated in vitro with filtrates of trypsin-treated suspensions of Shigella paradysenteriae. J. Exptl. Med. 104: HASEK, M., A. LENGEROVA, AND T. HRABA Transplantation immunity and tolerance. Advan. Immunol. 1: HASEK, M., AND A. PUZA Induction of tolerance in adult life and reminiscence of tolerance, p In M. Haslk, A. Lengerova, and M. Vojtiska [ed.], Mechanisms of immunological tolerance. Academic Press, Inc., New York. 16. INGRAHAM, J. S., AND A. BUSSARD Application of a localized hemolysin reaction for specific detection of individual antibody-forming cells. J. Exptl. Med. 119: JERNE, N. K., A. A. NORDIN, AND C. HENRY The agar plaque technique for recognizing antibody-producing cells, p In B. Amos and H. Koprowski [ed.], Cell-bound antibodies. The Wistar Institute Press, Philadelphia. 18. LANDY, M., R. P. SANDERSON, M. T. BERNSrEIN, AND A. L. JACKSON Antibody production by leukocytes in peripheral blood. Nature 204: LANDY, M., R. P. SANDERSON, M. T. BERNSTEIN, AND E. M. LERNER Involvement of thymus in immune response of rabbits to somatic polysaccharides of gram-negative bacteria. Science 147: MITCHINSON, N. A Tolerance of erythrocytes in poultry: induction and specificity. Immunology 5: NETER, E Bacterial hemagglutination and hemolysis. Bacteriol. Rev. 20: NOSSAL, G. J. V Induction of immunological tolerance in rats to foreign erythrocytes. Australian J. Exptl. Biol. Med. Sci. 36: RICHARDSON, M., AND R. W. DUTTON Antibody synthesizing cells; appearance after secondary antigenic stimulation in vitro. Science 146: ROWLEY, D. A., AND F. W. FITCH Homeostasis of antibody formation in the adult rat. J. Exptl. Med. 120: ROWLEY, D. A., AND F. W. FITCH The

8 VOL. 92,1966 IMMUNOLOGICAL TOLERANCE TO MICROBIAL ANTIGENS 827 mechanism of tolerance produced in rats to sheep erythrocytes. I. Plaque-forming cell and antibody response to single and multiple injections of antigen. J. Exptl. Med. 121: ROWLEY, D. A., AND F. W. FITCH The mechanism of tolerance produced in rats to sheep erythrocytes. Il. The plaque forming cell and antibody response to multiple injections of antigen begun at birth. J. Exptl. Med. 121: SCHWARTZ, S. A., AND W. BRAUN Bacteria as an indicator of formation of antibody by single spleen cells in agar. Science 149: SERCARZ, E. E., AND A. H. CooNs Absence of antibody producing cells during unresponsiveness to bovine serum albumin (BSA) in the mouse. J. Immunol. 90: SMITH, R. T Immunological tolerance to non-livingantigens. Advan. Immunol. 1: STASTNY, P Persistence of acquired tolerance in cells transferred to an antigen-free environment. J. Immunol. 92: STELOS, P., AND D. W. TALMAGE The separation by starch electrophoresis of two antibodies to sheep red cells differing in hemolytic efficiency. J. Infect. Diseases 100: STERZL, J., AND L. MANDEL Estimation of the inductive phase of antibody formation by plaque technique. Folia Microbiol. (Prague) 9: Downloaded from on April 7, 2019 by guest