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Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides
119,222-232
CELLULARIMMUNOLOGY
(1989)
Regulation of IgG Responses by Helper and Suppressor T Cells Activated by Pneumococcal Capsular Polysaccharides’ GREGG N.MILLIGAN
ANDHELENBRALEY-MULLEN
Departments of Microbiology and Medicine, University of Missouri School of Medicine, Columbia. Missouri 65212 Received October 13,198s; accepted November 21.1988 Type 2 antigens are usually unable to prime the helper T cells (Tn) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate Tn. Low doses of S 19 or S3 (subimmunogenic for a primary IgM response) activated Tn capable of providing help to S 19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses ofthese antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (Ts). Removal of these Ts prior to transfer of T cells to recipient mice resulted in expression of Tn function. Therefore, the preferential activation of Tn versus Ts was dependent on the dose of antigen used for priming. Tn activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S 19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if Ts precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S 19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific Tn. o 1989 Academic Press. Inc.
INTRODUCTION Pneumococcal capsular polysaccharides type 3 (S3)’ and 19 (S 19) are type 2 antigens which elicit primary antibody responses composed almost entirely of IgM antibodies ( 1-3).3 Subsequent challenge of S3- or S 19-immunized mice with these antigens does not result in the elicitation of secondary IgG responses (3, 4). However, priming of mice with S3 or S 19 coupled to erythrocytes has been shown to induce an IgG memory response which can be detected upon secondary immunization with the ’ Supported in part by NIH Grant CA25054 and by NIH Training Grant T32AI07276. ’ Abbreviations used: S3, type 3 pneumococcal capsular polysaccharide; S19, type 19 pneumococcal capsular polysaccharide; PVP, polyvinylpyrrolidone; T n , helper T cell; PVP-HRBC, PVP-coupled horse erythrocytes; Ts, suppressor T cell; S3-CRBC, S3-coupled chicken erythrocytes; SIPCRBC, Sl9-coupled chicken erythrocytes; S6, type 6 pneumococcal capsular polysaccharide; Cy, cyclophosphamide; PFC, plaque-forming celk SRBC, sheep red blood cell. ’ G. M. Mill&an, R. L. Fairchild, K. E. Sterner, and H. Braley-Mullen, submitted for publication.
222 0008-8749/89$3.00 Copy+& Q 1989 by Academic Pm% Inc. All rights of reproduction in any form reserved
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appropriate polysaccharide-erythrocyte conjugate ( 5-7).3 The inability of S3 or S19 to prime mice for secondary IgG responses has been suggested (7,8) to be due to the inability of these antigens to prime helper T cells (Tu) which are required for the differentiation of B cells to IgG-producing B memory cells (8- 11). Previous studies from this laboratory indicated that very low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could prime mice for a secondary IgG response which was elicited following challenge with PVP-coupled horse erythrocytes (PVPHRBC), a T cell-dependent (TD) form of the antigen ( 12). It was subsequently shown that this dose of PVP activated PVP-specific TH as well as PVP-specific IgG memory B cell precursors (13). Interestingly, doses of PVP which were optimally immunogenic for a primary IgM response did not prime mice for a secondary IgG response but interfered with the induction of IgG memory B cells by PVP-HRBC ( 12). These doses of PVP were shown to activate PVP-specific suppressor T cells (Ts) which prevented the functional expression of the Tu cells capable of providing help for a secondary IgG response (13). It was of interest to determine if the activation of TH by doses of antigen that were suboptimal for a primary IgM response was unique to PVP or if other type 2 antigens might also activate TH under similar conditions. The present study was therefore undertaken to determine if the type 2 antigens S19 and S3 could activate Tu cells. It was demonstrated that doses of S 19 or S3 which are subimmunogenic for primary IgM responses could activate antigen-specific Tn capable of providing help for a polysaccharide-specific secondary IgG response. In contrast, T cells from mice primed with doses of S3 or S 19 which are optimally immunogenic for primary IgM responses activated Ts which inhibited the helper function of the polysaccharide-specific TH . Interestingly, doses of S 19 which primed Tn could neither prime memory B cells nor elicit capsular polysaccharide-specific IgG responses from B cells primed with the TD form of the antigen. However, high but not low doses of S3 were capable of priming IgG memory B cells if Ts precursors were first removed by treatment of mice with cyclophosphamide (Cy) at the time of priming. MATERIALS
AND METHODS
Mice. CAF, mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Balb/c mice were obtained through Mr. Clarence Reeder at the National Institutes of Health, Bethesda, Maryland. Female mice, 8- 12 weeks old, were used for all experiments. CAF, mice were used for all experiments with S3 while Balb/c mice were used for experiments with S19. These strains of mice have been shown to generate optimal secondary IgG responses to these antigens (unpublished data). Antigens. Pneumococcal capsular polysaccharides type 6 (S6) (Danish type 6A) and type 19 (S19) (Danish type 19F) were obtained from Merck and Co., Inc., Rahway, New Jersey. Pneumococcal capsular polysaccharide type 3 (S3) was obtained from Eli Lilly and Co., Indianapolis, Indiana. Polyvinylpyrrolidone (360 kDa) was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin. S3 and S 19 were coupled to chicken erythrocytes (S3-CRBC, Sl9-CRBC) by a modification of the chromic chloride method described previously ( 14). Priming and immunization. Mice to be used as B cell donors were primed intravenously 2-3 months before adoptive cell transfer with 2.0 X lo8 S3- or SIPcoupled chicken erythrocytes (S3-CRBC, S 19-CRBC) (5,6).’ Mice to be used as T cell donors were primed ip with 5.0 pg S 19,O.Ol pg S 19,0.6 fig S3,0.006 pg S3,0.0025 pg PVP,
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0.0 1 pg S6, or intravenously with 2.0 X lo* S3- or S 19-CRBC 2-3 months before cell transfer. These doses are optimally immunogenic (0.6 pg S3,5.0 pg S 19) suboptimal (0.0025 pg PVP), or subimmunogenic (0.01 pg S6, 0.01 pg Sl9, 0.006 pg S3) for primary IgM responses to these antigens (3,4, 13, 15, 16). Adoptive transfer and secondary challenge. Mice to be used as T and B cell recipients received 650 R from a 6oCo source at the University of Missouri Dalton Research Center. Mice were repopulated 4-6 hr after irradiation with B cells alone ( 1.4- 1.8 X lO’/mouse) or with B cells plus T cells (6.0-8.0 X lo6 T cells/mouse) and challenged with 2.0 X 10’ S19- or S3-CRBC iv. Preparation of B and T cells. Spleen cells were depleted of T cells by treating spleen cells from S3- or SI9-CRBC-primed mice with monoclonal anti-Thy 1.2 (HO 13.4) and anti-L3T4 (monoclonal antibody GK1.5 originally obtained from Dr. John Kemp, University of Iowa) and complement (C) as described (17). Enriched T cell populations were prepared by passage of spleen cells from primed or unprimed mice over nylon wool as previously described ( 13). For some experiments, T cell populations were treated with anti-Lyt 2.2, GK1.5, or anti-Thy 1.2 plus complement (Low-Tox Rabbit complement, Cedarlane Laboratories, Ontario, Canada) before transfer (17). Plaque-forming cell (PFC) assay. Capsular polysaccharide-specific PFC were enumerated 7 days after cell transfer and antigen challenge of recipient mice using S3SRBC ( 14) or S19-SRBC (3) as indicator cells. SRBC-specific PFC (always less than 1000 PFC/spleen) were subtracted from the total number of PFC. IgM and IgG PFC were detected as described previously in detail (6). RESULTS Activation
of TH by Low Doses of Type 2 Antigens
To determine if the type 2 antigen S 19 could activate Tn, T cells from mice primed with 5.0 pg S 19 (optimally immunogenic for a primary IgM response to S19) (3) or 0.0 1 S 19 pg (subimmunogenic for a primary IgM response to S 19) (16) were tested for their ability to provide help for an S 19-specific secondary IgG response. S19-CRBCprimed B cells were transferred to irradiated mice alone or with antigen-primed or unprimed T cells. Mice were challenged with S 19-CRBC and IgM and IgG PFC were enumerated 7 days later (Fig. IA). The number of IgM PFC produced by each group of mice receiving T cells was similar. Therefore, for simplicity, IgM PFC are omitted from the figures but are included in the figure legends. S19-CRBC-primed B cells (Group A) did not produce significant IgG antibody upon challenge with S 19-CRBC. Naive T cells (unprimed) provided little, if any, help to the same population of B cells (Group B) whereas S19-CRBC-primed T cells (Group C) provided help for an IgG response. Interestingly, the helper activity of T cells from mice primed with 0.0 1 pg S19 (Group D) was essentially equivalent to that provided by S19-CRBC-primed T cells. Treatment of T cells from mice primed with 0.0 1 pg S 19 with anti-Lyt 2.2 and C prior to transfer did not further increase the level of help (Group G). T cells from mice primed with 5.0 pg S 19 provided minimal help to primed B cells (Group E vs Groups A and B) unless these T cells were treated with anti-Lyt 2.2 plus C (Group F). To determine if another type 2 antigen might also be able to activate Tut T cells from mice primed with 0.006 pg (subimmunogenic for a primary IgM response to S3) or 0.6 pg S3 (optimally immunogenic for a primary IgM response to S3) ( 15) were
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FIG. 1. Activation of TH by low doses of S 19 and S3. (A) Irradiated Balb/c mice received 1.2 X 10’ B cells from S19-CRBC-primed Balb/c mice. Group A received no T cells. Groups B-G also received 8 X lo6 T cells as follows: B, unprimed T cells; C, S 19-CRBC-primed T cells; D, 0.0 1 wg S 19-primed T cells; E, 5.0 pg S 19-primed T cells; F, 5.0 pg Sl9-primed T cells (anti-Lyt 2.2 plus C treated); and G, 0.0 1 pg S19-primed T cells (anti-Lyt 2.2 plus C treated). (B) Irradiated CAF, mice received I .2 X 10’ B cells from S3-CRBC-primed mice with or without 6 X lo6 T cells as follows: A, no T cells; B, SZCRBC-primed T cells; C, 0.006 pg S3-primed T cells; D, 0.006 rg S3-primed T cells (anti-Lyt 2.2 plus C treated); E, 0.6 wg S3-primed T cells; and F, 0.6 pg S3-primed T cells (anti-Lyt 2.2 plus C treated). All recipients were challenged with S19-CRBC (A) or S3-CRBC (B). Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen values for the experiment shown in (A) were as follows: A, 1500 + 197; B, 2975 + 467; C, 2963 k 769; D, 3080 + 664; E, 1940 f 182; F, 3040 + 490; and G, 2300 f 223. IgM PFC/spleen values for(B) were as follows: A, 462 f 78; B, 1760 + 443; C, 936 k 103; D, 1230 -+ 297; E, 1150 f 252; and F, 970 f 95.
tested for their ability to provide help for an S3-specific secondary IgG response. As shown in Fig. lB, T cells from mice primed with S3-CRBC (Group B) or 0.006 pg S3 (Group C) provided help to S3-CRBC-primed B cells for an IgG response. T cells from mice primed with doses of S3 optimally immunogenic for a primary IgM response (0.6 pg) provided minimal help to S3-CRBC-primed B cells (Group E) unless the T cells were treated with anti-Lyt 2.2 and C (Group F). Treatment of low dose S3-primed T cells with anti-Lyt 2.2 and C only slightly increased their helper activity (Group D).
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The fact that the helper function of T cells of mice primed with 0.6 pg S3 or 5.0 pg S19 was detected only when T cells were treated with anti-Lyt 2.2 and C suggests that optimally immunogenic doses of these antigens might activate Ts which prevent the expression of Tn function. In contrast, subimmunogenic doses of S3 or S19 activate Tn which are not masked by Ts .
Optimally Immunogenic Doses of S3 and S19 Preferentially Activate Suppressor T Cells The presence of Ts in T cell populations from mice primed with 5 pg S 19 or 0.6 /lg S3 was directly demonstrated by cell mixing experiments (Fig. 2). As shown in Fig. 2A, secondary IgG responses were again obtained in mice receiving S 19-CRBCprimed B cells plus S 19-CRBC-primed or 0.0 1 pg S 19-primed T cells (Groups A and B). T cells from mice primed with 5.0 pg S19 did not provide significant help for IgG responses (Group C versus F). When equal numbers of 5.0 pg S 19-primed and 0.0 1 pg S19-primed T cells were mixed prior to transfer the IgG response was markedly suppressed (Group D). Similarly, T cells from S3-CRBC- or 0.006 pg S3-primed mice supported S3-specific secondary IgG responses by S3-CRBC-primed B cells (Fig. 2B, Groups A and B). In contrast, T cells of mice primed with 0.6 pg S3 did not provide help for a secondary IgG response (Group C) and suppressed the IgG response when mixed with 0.006 pg S3-primed T cells and S3-CRBC-primed B cells (Group D). The suppression was due to an S34nduced T cell as naive T cells which also did not provide help to B cells (Group F) had no effect on the secondary response of S3-CRBC-primed B cells when mixed with 0.006 pg S3-primed T cells (Group E). Therefore, priming of mice with 5.0 pg S19 or 0.6 hg S3 resulted in preferential activation of Ts which inhibited the function of Tn from mice primed with low doses of these antigens.
Antigen Specificity of THActivated by Sl9 To determine if the Tu providing help for a secondary IgG response were activated in an antigen-specific manner or could be activated by priming with any antigen, T cells from S 19-, S6-, and PVP-primed mice were tested for their ability to provide help for S 19-specific secondary IgG responses. T cells from mice primed with 0.0 1 pg S19 or S19XRBC again provided help to S19-CRBC-primed B cells for IgG responses (Fig. 3, Groups A and B). Groups receiving S19-CRBC-primed B cells and T cells from mice primed with 0.01 pg S6 or 0.0025 pg PVP, doses of antigen known to activate Tu which provide help to S6-CRBC-primed or PVP-HRBC-primed B cells ( 13, 16), had only minimal IgG responses (Groups C and D) which were no greater than those of mice receiving B cells alone (Fig. 3, Group F) or naive T cells plus primed B cells (Fig. 3, Group E). The inability of T cells from 0.0 1 pg S6-primed mice to provide help for S 19-specific secondary IgG responses apparently was not due to activation of antigen-nonspecific Ts as treatment of these T cells with anti-Lyt 2.2 and C did not increase their helper function (data not shown). Thus, priming of mice with suboptimal doses of S19 results in the activation of antigen-specific Tu.
Surface Phenotypeof Sl9-Specijic TH The expression of cell surface markers on the T cells from low dose S19-primed mice was examined to ensure that a typical Tn cell was responsible for the develop
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FIG. 2. Doses of S3 and S19 which are optimally immunogenic for a primary IgM response activate suppressor T cells. (A) Irradiated Balb/c mice received 1.8 X 10’ S19-CRBC-primed B cells alone or with 8 X lo6 T cells as follows: A, SlB-CRBC-primed T cells; B, 0.01 pg Sl9-primed T cells; C, 5.0 wcgS19primed T cells; D, 0.0 1 pg S 19 plus 5.0 pg S 19-primed T cells; E, unprimed T cells; and F, B cells alone. (B) Irradiated CAF, mice received 1.4 X 10’ S3-CRBC-primed B cells alone or with 6 X lo6 T cells as follows: A, S3-CRBC-primed T cells; B, 0.006 pg S3-primed T cells; C, 0.6 pg S3-primed T cells; D, 0.006 pg S3-primed T cells plus 0.6 pg S3-primed T cells; E, 0.006 pg S3-primed T cells plus unprimed T cells; F, unprimed T cells; and G, B cells alone. All recipients were challenged with S 19-CRBC (A) or S3-CRBC (B). Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/ spleen for the experiment shown in (A) were as follows: A, 4 138 f 496; B, 3490 f 706; C, 4090 + 778; D, 5180 k 1152; E, 2075 k 244; and F, 1813 f 428. IgM PFC/spleen for (B) were as follows: A, 1988 + 157; B,2230?361;C, 1900+375;D,2690+543;E,2210+297;F, 1500+61;andG,700+63.
ment of S 19-specific secondary IgG responses. The surface phenotype of S19-specific Tu was determined by treatment of Tu populations with monoclonal antibodies specific for Thy 1 and for the classical Tu cell marker L3T4 (CD4) followed by C treatment. T cells from mice primed with 0.01 pg S19 which were treated with C alone (Fig. 4, Group B) provided help for an S19-specific secondary IgG response which was comparable to that provided by S 19-CRBC-primed T cells (Group A). Treatment of these T cells with either anti-L3T4 monoclonal antibody GKl.5 and C (Group C) or anti-Thy 1.2 and C (Group D) eliminated Tn activity. Therefore, Thy l+, L3T4+
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ABCDEF RECIPIENT
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FIG. 3. Antigen-specific Tu are activated by low doses of S19. Irradiated Balb/c mice received 1.8 X 10’ S19-CRBCprimed B cells alone or with 8 X lo6 T cells as follows: A, S 19-CRBC-primed T cells; B, 0.0 1 pg S19-primed T cells; C, 0.01 pg S6-primed T cells; D, 0.0025 pg PVP-primed T cells; E, unprimed T cells; and F, B cells alone. All recipients were challenged with S19-CRBC. Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen were as follows: A, 3850 f 1395; B, 2550 + 307; C, 3020 +- 723; D, 2660 + 470; E, 1750 + 154; and F, 1213 k 447.
cells activated by suboptimal doses of S 19 are capable of providing S 19-specific secondary IgG responses.
help to B cells for
Inability of S3 or SI 9 to Prime IgG Memory B Cells Primary immunization of mice with TD antigens induces differentiation of B cells to memory cells. Upon a second exposure to antigen, memory B cells produce a
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FIG. 4. Surface phenotype of S19-specific Tu. Irradiated Balb/c mice received 1.8 X 10’ S19-CRBCprimed B cells with or without 8 X lo6 T cells as follows: A, S19-CRBC-primed T cells; B, 0.0 1 pg S19primed T cells (C treated); C, 0.0 1 pg S 19-primed T cells (anti-L3T4 plus C treated); D, 0.0 1 pg Sl9-primed T cells (anti-Thy 1.2 plus C treated); E, unprimed T cells; and F, B cells alone. All recipients were challenged with S 19-CRBC. Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen were as follows: A, 1550 rt 446; B, 2240 f 212; C, 2410 + 391; D, 1425 + 468; E, 1575 +497;andF, 1713+356.
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TABLE 1 Effect of Priming with S3 or S19 on the Generation of B Memory Cells PFC/spleen b B cells from donors primed with” Nothing S3-CRBC 0.006 fig S3 Cy + 0.006 pg S3 0.6 pg S3 Cy + 0.6 fig
Expt 1
Expt 2 kG
1550+ 1850+ 1638 f 2120* 21252 1470?
175 297 269 411 347 98
300 4550 -675 620 363 --3210
+ 164 + 575 + 452 + 332 + 200 f 388
W 930 + 162 1280 + 128 763 + 136 ND 950 Y!z175 1160+222
W 850 f 308 --7260+768 813&302 ND 141Ok464 --3880 X!I640
Expt 3 Nothing S19-CRBC 5crgs19 cy+spgs19
2563 + 7420 + 7650 + 4410+
446 566 1193 546
88 + 88 w+ -173 300 + 174 310+ 121
LICAF, (Experiments 1 and 2) or Balb/c (Experiment 3) mice were primed with S3 (Experiments 1 and 2) or S19 (Experiment 3) as indicated. Some mice received Cy (100 mg/kg) 2days before immunization. Two months later B cells (anti-Thy 1.2 + C-treated spleen) from these mice were assessedfor their ability to produce polysaccharide-specific IgG PFC after transfer to irradiated syngeneic recipients with T cells from S3-CRBC (Experiments 1 and 2)- or S19-CRBC (Experiment 3)-primed donors and challenge with S3-CRBC (Experiments 1 and 2) or S 194XBC (Experiment 3). b Mean IgM and IgG PFC/spleen + SEM of four to five mice determined 7 days after challenge. S3SRBC were used as indicator cells for Experiments 1 and 2 and Sl9-SRBC for Experiment 3.
response which is characterized by the production of increased amounts of antibody of the IgG class ( 18). Type 2 antigens generally do not induce differentiation of B cells to memory cells (4, 9, 19). However, low doses of the type 2 antigen PVP which activate TH could also prime mice to produce IgG antibody upon secondary challenge with a TD form of PVP, PVP-HRBC (12,20). It was therefore of interest to determine if S3 and S19 could also prime B cells for a secondary IgG response. To remove Ts precursors which may mask Tn activity and thus prevent priming of IgG memory B cells, (20) some mice were given cyclophosphamide ( 100 mg/kg) 2 days prior to priming with S19 or S3 (21). (&pretreated or untreated mice were primed with 0.6 or 0.006 pg of S3. Two months later B cells from these mice and from S3-CRBC-primed and normal mice were mixed with S3-CRBC-primed T cells and tested for their ability to develop a secondary IgG response after challenge with S3-CRBC. As shown in Table 1, Experiments 1 and 2, recipients of S3-CRBC-primed B cells produced significantly more IgG antibody than did recipients of normal unprimed B cells. B cells from mice primed with a low (0.006 pg) or optimally immunogenic (for a primary IgM response) dose of S3 (0.6 pg) produced little, if any, IgG after challenge with S3-CRBC. In contrast, B cells from Cy-treated mice primed with an optimally immunogenic dose of S3 produced IgG in response to S3-CRBC challenge whereas B cells from Cy-treated mice primed with a low dose of S3 produced very little IgG. Apparently removal of Ts precursors by Cy treatment resulted in the priming of IgG
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memory B cells by the 0.6-pg dose of S3. In contrast, 0.006 pg of S3 is apparently insufficient to prime IgG memory B cells in untreated or Cy-treated mice even though this amount of S3 does activate S3-specific Tn (Fig. 1). Similar experiments involving adoptive transfer of S 19-CRBC-primed T cells and B cells from Cy-treated or control mice primed with optimally immunogenic (5.0 pg) doses of S19 were performed. Whereas B cells from mice primed with Sl9-CRBC produced IgG antibody, B cells from Cy-pretreated or control mice primed with 5.0 pg S19 did not produce capsular polysaccharide-specific IgG antibody (Table 1, Experiment 3). These results are in contrast to the ability of low doses of PVP to prime B cells in normal mice and high doses of S3 or PVP to prime IgG memory B cells in Cy-treated mice. Neither high (5.0 pg) nor low (0.0 1 pg) doses of S 19 could prime IgG memory B cells in control or Cy-treated mice (Table 1 and data not shown).
Challenge of S3- or Sl9-CRBC-Primed Mice with S3 or Sl9 The ability of low doses of S3 or S 19 to activate Tn and the inability of any dose of S 19 to prime IgG memory B cells raised the question as to whether either of these antigens could elicit a secondary IgG response in mice which had been reconstituted with S3- or S 19-CRBC-primed spleen cells. Mice receiving S3- or S 19-CRBC-primed spleen cells developed S3- and S 19-specific secondary IgG response upon challenge with the appropriate antigen (S3- or S 19-CRBC, respectively). In contrast, challenge of S3- or S 19-CRBC-primed spleen cell recipients with a dose of S3 or S 19 known to activate S3- or S 19-specific Tn (0.006 or 0.0 1 pg) or with a dose of antigen optimally immunogenic for a primary IgM response (0.6 pg S3 or 5.0 pg S19) failed to elicit significant capsular polysaccharide-specific secondary IgG responses (data not shown). DISCUSSION It is now known that help provided by T cells or T cell products is required for antibody responses to type 2 antigens (22-26). In addition, T cells have been known to influence the isotype expression of antibodies elicited by type 2 antigens (27). Although antigen-specific Tn were not directly demonstrated in any of these studies, previous studies from our laboratory demonstrated that priming mice with subimmunogenic doses of PVP activated PVP-specific TH which could provide help for a PVP-specific secondary IgG response ( 13, 17). Therefore, under appropriate conditions, antigen-specific TH can be activated by a type 2 antigen. In the present study, the ability of other type 2 antigens to activate Tn was examined. As is true for PVP, very low doses of S3 and S 19 were required in order to activate Tn capable of providing help for secondary IgG responses (Fig. 1). Priming mice with doses of these antigens which are optimally immunogenic for induction of primary IgM responses resulted in the preferential activation of Ts which prevented the ability of unfractionated T cells from such mice to provide help to polysaccharide-primed B cells for a secondary IgG response (Fig. 2). Removal of Ts by treatment of T cells with anti-Lyt 2.2 and complement unmasked Tn function. Therefore, the antigen dose dependence of Tn vs Ts activation by S3 and S 19 is very similar ‘to that described for PVP ( 13, 20). These results suggest that the ability of low doses of type 2 antigens to activate Tn is a general phenomenon rather than a unique property of the type 2 antigen PVP. Furthermore, the activation of Ts by doses of these antigens which are optimally
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immunogenic for a primary IgM response prevents the functional expression of polysaccharide-specific Tn. These results may explain the reported inability of optimally immunogenic doses of these antigens to activate TH and to induce IgG memory responses in B cells. The antigen specificity and surface phenotype of the S 19-specific TH were also examined. Only S 19-CRBC- or S19-primed T cells provided help for S 19-specific secondary IgG responses. T cells from mice primed with doses of S6 or PVP which are known to provide help to S6-CRBC- or PVP-HRBC-primed B cells (( 13) and unpublished observations) provided no detectable help to S 19-CRBC-primed B cells (Fig. 3). The S19-specific TH (Fig. 4) and the S3-specific TH (data not shown) expressed typical TH cell markers Thy 1 and L3T4 (CD4). S3 and S19 differ from PVP in terms of their ability to prime IgG memory B cells. Although low doses of PVP could prime IgG memory B cells ( 12) priming mice with low doses of S3 or S19 failed to prime IgG memory B cells (Table 1). Removal of putative Ts precursors by treating mice with Cy prior to priming with S19 (Table 1 and data not shown) also did not result in priming of capsular polysaccharide-specific IgG memory B cells. In contrast, immunogenic doses of S3 (0.6 pg) could prime IgG memory B cells but only when Ts precursors had been removed by Cy treatment. It may be that 0.0025 pg PVP is sufficient to prime memory B cells, since this dose of PVP is capable of inducing a low but detectable primary IgM response (13). In contrast, neither 0.006 pg S3 nor 0.01 pg S19 can induce a significant primary IgM response (15, 16). It is possible that although these doses of S3 and S 19 are capable of activating Tu, they are insufficient for activating immunoglobulin-secreting B cells or IgG memory B cell precursors. Lite and Braley-Mullen ( 12) also showed that challenge of PVP-HRBC-primed mice with various doses of PVP did not result in elicitation of a secondary IgG response. In fact, challenge of PVP-HRBC-primed mice with an optimally immunogenic dose of PVP interfered with the elicitation of a secondary IgG response by PVP-HRBC. S3 and S 19 apparently are similar to PVP in this regard since they could not elicit capsular polysaccharide-specific secondary IgG responses (data not shown) and challenge with optimally immunogenic doses of these antigens interfers with elicitation of a secondary IgG response by S3-HRBC (28) or S19-CRBC (manuscript in preparation). The results presented here and in our previous studies with PVP ( 13, 17) clearly establish that several type 2 antigens are able to activate antigen-specific L3T4+ T cells which can provide help to B cells for the production of IgG antibody responses. However, in contrast to TD antigens, the ability to detect TH activity in T cells from mice primed with type 2 antigens is highly dependent on the dose of antigen used for priming. This is apparently due to the fact that these antigens also readily activate Ts (Fig. 2) which can prevent the expression of Tn activity (( 13) and Fig. 1) and the priming of memory B cells (Table 1 and (20)). Preferential activation of Ts which mask TH function has also been observed for several immune response gene-controlled responses in which a TD antigen is unable to elicit a response in low or nonresponder strains of mice (29-32). The reason(s) why type 2 antigens seem to preferentially activate Ts rather than TH even in “high-responder” strains of mice is unknown and is currently under investigation. It is, however, clear that the antigen-specific helper activity described here and in previous studies ( 13, 17) is required for the production of IgG but not IgM responses to these type 2 antigens. Thus masking of TH activity by Ts can explain why commonly employed doses (i.e., immunogenic for
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IgM responses) of type 2 antigens have not usually been found to activate TH or to induce memory in B cells. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3 1. 32.
Baker, P. J., and Stashak, P. W., J. Immunol. 103, 1342, 1969. Braley, H., and Freeman, M. J., Cell. Immunol. 2,73, 1971. Fairchild, R. L., and Braley-Mullen, H., Infect Immun.39,615, 1983. Baker, P. J., Stashak, P. W., Amsbaugh, D. F., and Prescott, B., Immunology 20,469, 197 1. Braley-Mullen, H., J. Immunol. 113, 1909, 1974. Braley-Mullen, H., J. Zmmunol. 114, 1184, 1975. Braley-Mullen, H., J. Immunol. 116,904, 1976. Braley-Mullen, H., Eur. J. Immunol. 7,775, 1977. Braley-Mullen, H., hmunology 40,52 1, 1978. LaFrenz, D. E., and Feldbush, T. L., ImmunoIogy44,177,1981. Davie, J. M., and Paul, W. E., J. Immunol. 113, 1436, 1974. Lite, H. S., and Braley-Mullen, H., J. Immunol. 126,928, 198 1. Braley-Mullen, H.,J. Exp. Med. 161, 1357, 1985. Baker, P. J., Stashak, P. W., and Prescott, B., Appl. Microbial. 17,422, 1969. Braley-Mullen, H., J. Immunol. 136,296, 1986. Milligan, G. N., Ph.D. dissertation, University ofMissouri, 1988. Van Buskirk, A. M., and Braley-Mullen, H., J. Zmmunol. 138,103 1,1987. Katz, D. H., and Benacerraf, B., In “Advances in Immunology” (W. H. Taliaferro and J. H. Humphrey, Eds.), Vol. 15, p. 1. Academic Press, New York, 1972. Howard, J. G., Trunsplunt. Rev. 8,50, 1972. Van Buskirk, A. M., and Braley-Mullen, H., GUI. Immunol. 107, 121, 1987. Germain, R. N., and Benacerraf, B., &and. J. Immunol. 13,1, 198 1. Endres, R. O., Kushnir, E., Kappler, J. W., Marrack, P., and Kinsky, S. G., J. Zmmunol. 130, 781, 1983. Mond, J. J., Mongini, P. K. A., Sieckmann, D., and Paul, W. E., J. Zmmunol, 125, 1066, 1980. Letvin, N. L., Benacetraf, B., andGennain, R. N., hoc. Nat. Acad. Sci. USA 78,5113, 1981. Bretscher, P. A., Wilkins, J., and Diner, U. E., Cell. Immunol. 85,396, 1984. Murray, P. D., and Kagnoff, M. F., J. Immunol. X38,2439,1987. Mongini, P. K. A., Stein, K. E., and Paul, W. E., J. Exp. Med. 153,1, 198 1. Wilson, D. A., and Braley-Mullen, H., Cell. Immunol. 64, 177, 1981. Baxevanis, C. N., Ishii, N., Nagy, Z. A., and Klein, J., J. Exp. Med. 156,822, 1982. Sercan, E. E., Yowell, R. L., Turkin, D., Miller, A., Araneo, B. A., and Adorini, L., Immunol. Rev. 39, 108, 1978. Kapp, J. A., Pierce, C. W., Schlossman, S., and Benacerraf, B., J. Exp. Med. 140,648, 1974. Jensen, P. E., Kapp, J. A., and Pierce, C. W., J. Mol. Cell. Immunol. 3,267, 1987.
CELLULARIMMUNOLOGY
(1989)
Regulation of IgG Responses by Helper and Suppressor T Cells Activated by Pneumococcal Capsular Polysaccharides’ GREGG N.MILLIGAN
ANDHELENBRALEY-MULLEN
Departments of Microbiology and Medicine, University of Missouri School of Medicine, Columbia. Missouri 65212 Received October 13,198s; accepted November 21.1988 Type 2 antigens are usually unable to prime the helper T cells (Tn) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate Tn. Low doses of S 19 or S3 (subimmunogenic for a primary IgM response) activated Tn capable of providing help to S 19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses ofthese antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (Ts). Removal of these Ts prior to transfer of T cells to recipient mice resulted in expression of Tn function. Therefore, the preferential activation of Tn versus Ts was dependent on the dose of antigen used for priming. Tn activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S 19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if Ts precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S 19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific Tn. o 1989 Academic Press. Inc.
INTRODUCTION Pneumococcal capsular polysaccharides type 3 (S3)’ and 19 (S 19) are type 2 antigens which elicit primary antibody responses composed almost entirely of IgM antibodies ( 1-3).3 Subsequent challenge of S3- or S 19-immunized mice with these antigens does not result in the elicitation of secondary IgG responses (3, 4). However, priming of mice with S3 or S 19 coupled to erythrocytes has been shown to induce an IgG memory response which can be detected upon secondary immunization with the ’ Supported in part by NIH Grant CA25054 and by NIH Training Grant T32AI07276. ’ Abbreviations used: S3, type 3 pneumococcal capsular polysaccharide; S19, type 19 pneumococcal capsular polysaccharide; PVP, polyvinylpyrrolidone; T n , helper T cell; PVP-HRBC, PVP-coupled horse erythrocytes; Ts, suppressor T cell; S3-CRBC, S3-coupled chicken erythrocytes; SIPCRBC, Sl9-coupled chicken erythrocytes; S6, type 6 pneumococcal capsular polysaccharide; Cy, cyclophosphamide; PFC, plaque-forming celk SRBC, sheep red blood cell. ’ G. M. Mill&an, R. L. Fairchild, K. E. Sterner, and H. Braley-Mullen, submitted for publication.
222 0008-8749/89$3.00 Copy+& Q 1989 by Academic Pm% Inc. All rights of reproduction in any form reserved
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appropriate polysaccharide-erythrocyte conjugate ( 5-7).3 The inability of S3 or S19 to prime mice for secondary IgG responses has been suggested (7,8) to be due to the inability of these antigens to prime helper T cells (Tu) which are required for the differentiation of B cells to IgG-producing B memory cells (8- 11). Previous studies from this laboratory indicated that very low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could prime mice for a secondary IgG response which was elicited following challenge with PVP-coupled horse erythrocytes (PVPHRBC), a T cell-dependent (TD) form of the antigen ( 12). It was subsequently shown that this dose of PVP activated PVP-specific TH as well as PVP-specific IgG memory B cell precursors (13). Interestingly, doses of PVP which were optimally immunogenic for a primary IgM response did not prime mice for a secondary IgG response but interfered with the induction of IgG memory B cells by PVP-HRBC ( 12). These doses of PVP were shown to activate PVP-specific suppressor T cells (Ts) which prevented the functional expression of the Tu cells capable of providing help for a secondary IgG response (13). It was of interest to determine if the activation of TH by doses of antigen that were suboptimal for a primary IgM response was unique to PVP or if other type 2 antigens might also activate TH under similar conditions. The present study was therefore undertaken to determine if the type 2 antigens S19 and S3 could activate Tu cells. It was demonstrated that doses of S 19 or S3 which are subimmunogenic for primary IgM responses could activate antigen-specific Tn capable of providing help for a polysaccharide-specific secondary IgG response. In contrast, T cells from mice primed with doses of S3 or S 19 which are optimally immunogenic for primary IgM responses activated Ts which inhibited the helper function of the polysaccharide-specific TH . Interestingly, doses of S 19 which primed Tn could neither prime memory B cells nor elicit capsular polysaccharide-specific IgG responses from B cells primed with the TD form of the antigen. However, high but not low doses of S3 were capable of priming IgG memory B cells if Ts precursors were first removed by treatment of mice with cyclophosphamide (Cy) at the time of priming. MATERIALS
AND METHODS
Mice. CAF, mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Balb/c mice were obtained through Mr. Clarence Reeder at the National Institutes of Health, Bethesda, Maryland. Female mice, 8- 12 weeks old, were used for all experiments. CAF, mice were used for all experiments with S3 while Balb/c mice were used for experiments with S19. These strains of mice have been shown to generate optimal secondary IgG responses to these antigens (unpublished data). Antigens. Pneumococcal capsular polysaccharides type 6 (S6) (Danish type 6A) and type 19 (S19) (Danish type 19F) were obtained from Merck and Co., Inc., Rahway, New Jersey. Pneumococcal capsular polysaccharide type 3 (S3) was obtained from Eli Lilly and Co., Indianapolis, Indiana. Polyvinylpyrrolidone (360 kDa) was purchased from Aldrich Chemical Co., Milwaukee, Wisconsin. S3 and S 19 were coupled to chicken erythrocytes (S3-CRBC, Sl9-CRBC) by a modification of the chromic chloride method described previously ( 14). Priming and immunization. Mice to be used as B cell donors were primed intravenously 2-3 months before adoptive cell transfer with 2.0 X lo8 S3- or SIPcoupled chicken erythrocytes (S3-CRBC, S 19-CRBC) (5,6).’ Mice to be used as T cell donors were primed ip with 5.0 pg S 19,O.Ol pg S 19,0.6 fig S3,0.006 pg S3,0.0025 pg PVP,
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0.0 1 pg S6, or intravenously with 2.0 X lo* S3- or S 19-CRBC 2-3 months before cell transfer. These doses are optimally immunogenic (0.6 pg S3,5.0 pg S 19) suboptimal (0.0025 pg PVP), or subimmunogenic (0.01 pg S6, 0.01 pg Sl9, 0.006 pg S3) for primary IgM responses to these antigens (3,4, 13, 15, 16). Adoptive transfer and secondary challenge. Mice to be used as T and B cell recipients received 650 R from a 6oCo source at the University of Missouri Dalton Research Center. Mice were repopulated 4-6 hr after irradiation with B cells alone ( 1.4- 1.8 X lO’/mouse) or with B cells plus T cells (6.0-8.0 X lo6 T cells/mouse) and challenged with 2.0 X 10’ S19- or S3-CRBC iv. Preparation of B and T cells. Spleen cells were depleted of T cells by treating spleen cells from S3- or SI9-CRBC-primed mice with monoclonal anti-Thy 1.2 (HO 13.4) and anti-L3T4 (monoclonal antibody GK1.5 originally obtained from Dr. John Kemp, University of Iowa) and complement (C) as described (17). Enriched T cell populations were prepared by passage of spleen cells from primed or unprimed mice over nylon wool as previously described ( 13). For some experiments, T cell populations were treated with anti-Lyt 2.2, GK1.5, or anti-Thy 1.2 plus complement (Low-Tox Rabbit complement, Cedarlane Laboratories, Ontario, Canada) before transfer (17). Plaque-forming cell (PFC) assay. Capsular polysaccharide-specific PFC were enumerated 7 days after cell transfer and antigen challenge of recipient mice using S3SRBC ( 14) or S19-SRBC (3) as indicator cells. SRBC-specific PFC (always less than 1000 PFC/spleen) were subtracted from the total number of PFC. IgM and IgG PFC were detected as described previously in detail (6). RESULTS Activation
of TH by Low Doses of Type 2 Antigens
To determine if the type 2 antigen S 19 could activate Tn, T cells from mice primed with 5.0 pg S 19 (optimally immunogenic for a primary IgM response to S19) (3) or 0.0 1 S 19 pg (subimmunogenic for a primary IgM response to S 19) (16) were tested for their ability to provide help for an S 19-specific secondary IgG response. S19-CRBCprimed B cells were transferred to irradiated mice alone or with antigen-primed or unprimed T cells. Mice were challenged with S 19-CRBC and IgM and IgG PFC were enumerated 7 days later (Fig. IA). The number of IgM PFC produced by each group of mice receiving T cells was similar. Therefore, for simplicity, IgM PFC are omitted from the figures but are included in the figure legends. S19-CRBC-primed B cells (Group A) did not produce significant IgG antibody upon challenge with S 19-CRBC. Naive T cells (unprimed) provided little, if any, help to the same population of B cells (Group B) whereas S19-CRBC-primed T cells (Group C) provided help for an IgG response. Interestingly, the helper activity of T cells from mice primed with 0.0 1 pg S19 (Group D) was essentially equivalent to that provided by S19-CRBC-primed T cells. Treatment of T cells from mice primed with 0.0 1 pg S 19 with anti-Lyt 2.2 and C prior to transfer did not further increase the level of help (Group G). T cells from mice primed with 5.0 pg S 19 provided minimal help to primed B cells (Group E vs Groups A and B) unless these T cells were treated with anti-Lyt 2.2 plus C (Group F). To determine if another type 2 antigen might also be able to activate Tut T cells from mice primed with 0.006 pg (subimmunogenic for a primary IgM response to S3) or 0.6 pg S3 (optimally immunogenic for a primary IgM response to S3) ( 15) were
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5000, 400030002000lOOO-
2 nABCDEF
FIG. 1. Activation of TH by low doses of S 19 and S3. (A) Irradiated Balb/c mice received 1.2 X 10’ B cells from S19-CRBC-primed Balb/c mice. Group A received no T cells. Groups B-G also received 8 X lo6 T cells as follows: B, unprimed T cells; C, S 19-CRBC-primed T cells; D, 0.0 1 wg S 19-primed T cells; E, 5.0 pg S 19-primed T cells; F, 5.0 pg Sl9-primed T cells (anti-Lyt 2.2 plus C treated); and G, 0.0 1 pg S19-primed T cells (anti-Lyt 2.2 plus C treated). (B) Irradiated CAF, mice received I .2 X 10’ B cells from S3-CRBC-primed mice with or without 6 X lo6 T cells as follows: A, no T cells; B, SZCRBC-primed T cells; C, 0.006 pg S3-primed T cells; D, 0.006 rg S3-primed T cells (anti-Lyt 2.2 plus C treated); E, 0.6 wg S3-primed T cells; and F, 0.6 pg S3-primed T cells (anti-Lyt 2.2 plus C treated). All recipients were challenged with S19-CRBC (A) or S3-CRBC (B). Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen values for the experiment shown in (A) were as follows: A, 1500 + 197; B, 2975 + 467; C, 2963 k 769; D, 3080 + 664; E, 1940 f 182; F, 3040 + 490; and G, 2300 f 223. IgM PFC/spleen values for(B) were as follows: A, 462 f 78; B, 1760 + 443; C, 936 k 103; D, 1230 -+ 297; E, 1150 f 252; and F, 970 f 95.
tested for their ability to provide help for an S3-specific secondary IgG response. As shown in Fig. lB, T cells from mice primed with S3-CRBC (Group B) or 0.006 pg S3 (Group C) provided help to S3-CRBC-primed B cells for an IgG response. T cells from mice primed with doses of S3 optimally immunogenic for a primary IgM response (0.6 pg) provided minimal help to S3-CRBC-primed B cells (Group E) unless the T cells were treated with anti-Lyt 2.2 and C (Group F). Treatment of low dose S3-primed T cells with anti-Lyt 2.2 and C only slightly increased their helper activity (Group D).
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The fact that the helper function of T cells of mice primed with 0.6 pg S3 or 5.0 pg S19 was detected only when T cells were treated with anti-Lyt 2.2 and C suggests that optimally immunogenic doses of these antigens might activate Ts which prevent the expression of Tn function. In contrast, subimmunogenic doses of S3 or S19 activate Tn which are not masked by Ts .
Optimally Immunogenic Doses of S3 and S19 Preferentially Activate Suppressor T Cells The presence of Ts in T cell populations from mice primed with 5 pg S 19 or 0.6 /lg S3 was directly demonstrated by cell mixing experiments (Fig. 2). As shown in Fig. 2A, secondary IgG responses were again obtained in mice receiving S 19-CRBCprimed B cells plus S 19-CRBC-primed or 0.0 1 pg S 19-primed T cells (Groups A and B). T cells from mice primed with 5.0 pg S19 did not provide significant help for IgG responses (Group C versus F). When equal numbers of 5.0 pg S 19-primed and 0.0 1 pg S19-primed T cells were mixed prior to transfer the IgG response was markedly suppressed (Group D). Similarly, T cells from S3-CRBC- or 0.006 pg S3-primed mice supported S3-specific secondary IgG responses by S3-CRBC-primed B cells (Fig. 2B, Groups A and B). In contrast, T cells of mice primed with 0.6 pg S3 did not provide help for a secondary IgG response (Group C) and suppressed the IgG response when mixed with 0.006 pg S3-primed T cells and S3-CRBC-primed B cells (Group D). The suppression was due to an S34nduced T cell as naive T cells which also did not provide help to B cells (Group F) had no effect on the secondary response of S3-CRBC-primed B cells when mixed with 0.006 pg S3-primed T cells (Group E). Therefore, priming of mice with 5.0 pg S19 or 0.6 hg S3 resulted in preferential activation of Ts which inhibited the function of Tn from mice primed with low doses of these antigens.
Antigen Specificity of THActivated by Sl9 To determine if the Tu providing help for a secondary IgG response were activated in an antigen-specific manner or could be activated by priming with any antigen, T cells from S 19-, S6-, and PVP-primed mice were tested for their ability to provide help for S 19-specific secondary IgG responses. T cells from mice primed with 0.0 1 pg S19 or S19XRBC again provided help to S19-CRBC-primed B cells for IgG responses (Fig. 3, Groups A and B). Groups receiving S19-CRBC-primed B cells and T cells from mice primed with 0.01 pg S6 or 0.0025 pg PVP, doses of antigen known to activate Tu which provide help to S6-CRBC-primed or PVP-HRBC-primed B cells ( 13, 16), had only minimal IgG responses (Groups C and D) which were no greater than those of mice receiving B cells alone (Fig. 3, Group F) or naive T cells plus primed B cells (Fig. 3, Group E). The inability of T cells from 0.0 1 pg S6-primed mice to provide help for S 19-specific secondary IgG responses apparently was not due to activation of antigen-nonspecific Ts as treatment of these T cells with anti-Lyt 2.2 and C did not increase their helper function (data not shown). Thus, priming of mice with suboptimal doses of S19 results in the activation of antigen-specific Tu.
Surface Phenotypeof Sl9-Specijic TH The expression of cell surface markers on the T cells from low dose S19-primed mice was examined to ensure that a typical Tn cell was responsible for the develop
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FIG. 2. Doses of S3 and S19 which are optimally immunogenic for a primary IgM response activate suppressor T cells. (A) Irradiated Balb/c mice received 1.8 X 10’ S19-CRBC-primed B cells alone or with 8 X lo6 T cells as follows: A, SlB-CRBC-primed T cells; B, 0.01 pg Sl9-primed T cells; C, 5.0 wcgS19primed T cells; D, 0.0 1 pg S 19 plus 5.0 pg S 19-primed T cells; E, unprimed T cells; and F, B cells alone. (B) Irradiated CAF, mice received 1.4 X 10’ S3-CRBC-primed B cells alone or with 6 X lo6 T cells as follows: A, S3-CRBC-primed T cells; B, 0.006 pg S3-primed T cells; C, 0.6 pg S3-primed T cells; D, 0.006 pg S3-primed T cells plus 0.6 pg S3-primed T cells; E, 0.006 pg S3-primed T cells plus unprimed T cells; F, unprimed T cells; and G, B cells alone. All recipients were challenged with S 19-CRBC (A) or S3-CRBC (B). Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/ spleen for the experiment shown in (A) were as follows: A, 4 138 f 496; B, 3490 f 706; C, 4090 + 778; D, 5180 k 1152; E, 2075 k 244; and F, 1813 f 428. IgM PFC/spleen for (B) were as follows: A, 1988 + 157; B,2230?361;C, 1900+375;D,2690+543;E,2210+297;F, 1500+61;andG,700+63.
ment of S 19-specific secondary IgG responses. The surface phenotype of S19-specific Tu was determined by treatment of Tu populations with monoclonal antibodies specific for Thy 1 and for the classical Tu cell marker L3T4 (CD4) followed by C treatment. T cells from mice primed with 0.01 pg S19 which were treated with C alone (Fig. 4, Group B) provided help for an S19-specific secondary IgG response which was comparable to that provided by S 19-CRBC-primed T cells (Group A). Treatment of these T cells with either anti-L3T4 monoclonal antibody GKl.5 and C (Group C) or anti-Thy 1.2 and C (Group D) eliminated Tn activity. Therefore, Thy l+, L3T4+
228
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ABCDEF RECIPIENT
GROUPS
FIG. 3. Antigen-specific Tu are activated by low doses of S19. Irradiated Balb/c mice received 1.8 X 10’ S19-CRBCprimed B cells alone or with 8 X lo6 T cells as follows: A, S 19-CRBC-primed T cells; B, 0.0 1 pg S19-primed T cells; C, 0.01 pg S6-primed T cells; D, 0.0025 pg PVP-primed T cells; E, unprimed T cells; and F, B cells alone. All recipients were challenged with S19-CRBC. Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen were as follows: A, 3850 f 1395; B, 2550 + 307; C, 3020 +- 723; D, 2660 + 470; E, 1750 + 154; and F, 1213 k 447.
cells activated by suboptimal doses of S 19 are capable of providing S 19-specific secondary IgG responses.
help to B cells for
Inability of S3 or SI 9 to Prime IgG Memory B Cells Primary immunization of mice with TD antigens induces differentiation of B cells to memory cells. Upon a second exposure to antigen, memory B cells produce a
wz 4000-
!!I El \ 2 a a
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FIG. 4. Surface phenotype of S19-specific Tu. Irradiated Balb/c mice received 1.8 X 10’ S19-CRBCprimed B cells with or without 8 X lo6 T cells as follows: A, S19-CRBC-primed T cells; B, 0.0 1 pg S19primed T cells (C treated); C, 0.0 1 pg S 19-primed T cells (anti-L3T4 plus C treated); D, 0.0 1 pg Sl9-primed T cells (anti-Thy 1.2 plus C treated); E, unprimed T cells; and F, B cells alone. All recipients were challenged with S 19-CRBC. Results are expressed as the mean IgG PFC/spleen f SEM for groups of four to five mice. IgM PFC/spleen were as follows: A, 1550 rt 446; B, 2240 f 212; C, 2410 + 391; D, 1425 + 468; E, 1575 +497;andF, 1713+356.
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POLYSACCHARIDES
TABLE 1 Effect of Priming with S3 or S19 on the Generation of B Memory Cells PFC/spleen b B cells from donors primed with” Nothing S3-CRBC 0.006 fig S3 Cy + 0.006 pg S3 0.6 pg S3 Cy + 0.6 fig
Expt 1
Expt 2 kG
1550+ 1850+ 1638 f 2120* 21252 1470?
175 297 269 411 347 98
300 4550 -675 620 363 --3210
+ 164 + 575 + 452 + 332 + 200 f 388
W 930 + 162 1280 + 128 763 + 136 ND 950 Y!z175 1160+222
W 850 f 308 --7260+768 813&302 ND 141Ok464 --3880 X!I640
Expt 3 Nothing S19-CRBC 5crgs19 cy+spgs19
2563 + 7420 + 7650 + 4410+
446 566 1193 546
88 + 88 w+ -173 300 + 174 310+ 121
LICAF, (Experiments 1 and 2) or Balb/c (Experiment 3) mice were primed with S3 (Experiments 1 and 2) or S19 (Experiment 3) as indicated. Some mice received Cy (100 mg/kg) 2days before immunization. Two months later B cells (anti-Thy 1.2 + C-treated spleen) from these mice were assessedfor their ability to produce polysaccharide-specific IgG PFC after transfer to irradiated syngeneic recipients with T cells from S3-CRBC (Experiments 1 and 2)- or S19-CRBC (Experiment 3)-primed donors and challenge with S3-CRBC (Experiments 1 and 2) or S 194XBC (Experiment 3). b Mean IgM and IgG PFC/spleen + SEM of four to five mice determined 7 days after challenge. S3SRBC were used as indicator cells for Experiments 1 and 2 and Sl9-SRBC for Experiment 3.
response which is characterized by the production of increased amounts of antibody of the IgG class ( 18). Type 2 antigens generally do not induce differentiation of B cells to memory cells (4, 9, 19). However, low doses of the type 2 antigen PVP which activate TH could also prime mice to produce IgG antibody upon secondary challenge with a TD form of PVP, PVP-HRBC (12,20). It was therefore of interest to determine if S3 and S19 could also prime B cells for a secondary IgG response. To remove Ts precursors which may mask Tn activity and thus prevent priming of IgG memory B cells, (20) some mice were given cyclophosphamide ( 100 mg/kg) 2 days prior to priming with S19 or S3 (21). (&pretreated or untreated mice were primed with 0.6 or 0.006 pg of S3. Two months later B cells from these mice and from S3-CRBC-primed and normal mice were mixed with S3-CRBC-primed T cells and tested for their ability to develop a secondary IgG response after challenge with S3-CRBC. As shown in Table 1, Experiments 1 and 2, recipients of S3-CRBC-primed B cells produced significantly more IgG antibody than did recipients of normal unprimed B cells. B cells from mice primed with a low (0.006 pg) or optimally immunogenic (for a primary IgM response) dose of S3 (0.6 pg) produced little, if any, IgG after challenge with S3-CRBC. In contrast, B cells from Cy-treated mice primed with an optimally immunogenic dose of S3 produced IgG in response to S3-CRBC challenge whereas B cells from Cy-treated mice primed with a low dose of S3 produced very little IgG. Apparently removal of Ts precursors by Cy treatment resulted in the priming of IgG
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memory B cells by the 0.6-pg dose of S3. In contrast, 0.006 pg of S3 is apparently insufficient to prime IgG memory B cells in untreated or Cy-treated mice even though this amount of S3 does activate S3-specific Tn (Fig. 1). Similar experiments involving adoptive transfer of S 19-CRBC-primed T cells and B cells from Cy-treated or control mice primed with optimally immunogenic (5.0 pg) doses of S19 were performed. Whereas B cells from mice primed with Sl9-CRBC produced IgG antibody, B cells from Cy-pretreated or control mice primed with 5.0 pg S19 did not produce capsular polysaccharide-specific IgG antibody (Table 1, Experiment 3). These results are in contrast to the ability of low doses of PVP to prime B cells in normal mice and high doses of S3 or PVP to prime IgG memory B cells in Cy-treated mice. Neither high (5.0 pg) nor low (0.0 1 pg) doses of S 19 could prime IgG memory B cells in control or Cy-treated mice (Table 1 and data not shown).
Challenge of S3- or Sl9-CRBC-Primed Mice with S3 or Sl9 The ability of low doses of S3 or S 19 to activate Tn and the inability of any dose of S 19 to prime IgG memory B cells raised the question as to whether either of these antigens could elicit a secondary IgG response in mice which had been reconstituted with S3- or S 19-CRBC-primed spleen cells. Mice receiving S3- or S 19-CRBC-primed spleen cells developed S3- and S 19-specific secondary IgG response upon challenge with the appropriate antigen (S3- or S 19-CRBC, respectively). In contrast, challenge of S3- or S 19-CRBC-primed spleen cell recipients with a dose of S3 or S 19 known to activate S3- or S 19-specific Tn (0.006 or 0.0 1 pg) or with a dose of antigen optimally immunogenic for a primary IgM response (0.6 pg S3 or 5.0 pg S19) failed to elicit significant capsular polysaccharide-specific secondary IgG responses (data not shown). DISCUSSION It is now known that help provided by T cells or T cell products is required for antibody responses to type 2 antigens (22-26). In addition, T cells have been known to influence the isotype expression of antibodies elicited by type 2 antigens (27). Although antigen-specific Tn were not directly demonstrated in any of these studies, previous studies from our laboratory demonstrated that priming mice with subimmunogenic doses of PVP activated PVP-specific TH which could provide help for a PVP-specific secondary IgG response ( 13, 17). Therefore, under appropriate conditions, antigen-specific TH can be activated by a type 2 antigen. In the present study, the ability of other type 2 antigens to activate Tn was examined. As is true for PVP, very low doses of S3 and S 19 were required in order to activate Tn capable of providing help for secondary IgG responses (Fig. 1). Priming mice with doses of these antigens which are optimally immunogenic for induction of primary IgM responses resulted in the preferential activation of Ts which prevented the ability of unfractionated T cells from such mice to provide help to polysaccharide-primed B cells for a secondary IgG response (Fig. 2). Removal of Ts by treatment of T cells with anti-Lyt 2.2 and complement unmasked Tn function. Therefore, the antigen dose dependence of Tn vs Ts activation by S3 and S 19 is very similar ‘to that described for PVP ( 13, 20). These results suggest that the ability of low doses of type 2 antigens to activate Tn is a general phenomenon rather than a unique property of the type 2 antigen PVP. Furthermore, the activation of Ts by doses of these antigens which are optimally
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immunogenic for a primary IgM response prevents the functional expression of polysaccharide-specific Tn. These results may explain the reported inability of optimally immunogenic doses of these antigens to activate TH and to induce IgG memory responses in B cells. The antigen specificity and surface phenotype of the S 19-specific TH were also examined. Only S 19-CRBC- or S19-primed T cells provided help for S 19-specific secondary IgG responses. T cells from mice primed with doses of S6 or PVP which are known to provide help to S6-CRBC- or PVP-HRBC-primed B cells (( 13) and unpublished observations) provided no detectable help to S 19-CRBC-primed B cells (Fig. 3). The S19-specific TH (Fig. 4) and the S3-specific TH (data not shown) expressed typical TH cell markers Thy 1 and L3T4 (CD4). S3 and S19 differ from PVP in terms of their ability to prime IgG memory B cells. Although low doses of PVP could prime IgG memory B cells ( 12) priming mice with low doses of S3 or S19 failed to prime IgG memory B cells (Table 1). Removal of putative Ts precursors by treating mice with Cy prior to priming with S19 (Table 1 and data not shown) also did not result in priming of capsular polysaccharide-specific IgG memory B cells. In contrast, immunogenic doses of S3 (0.6 pg) could prime IgG memory B cells but only when Ts precursors had been removed by Cy treatment. It may be that 0.0025 pg PVP is sufficient to prime memory B cells, since this dose of PVP is capable of inducing a low but detectable primary IgM response (13). In contrast, neither 0.006 pg S3 nor 0.01 pg S19 can induce a significant primary IgM response (15, 16). It is possible that although these doses of S3 and S 19 are capable of activating Tu, they are insufficient for activating immunoglobulin-secreting B cells or IgG memory B cell precursors. Lite and Braley-Mullen ( 12) also showed that challenge of PVP-HRBC-primed mice with various doses of PVP did not result in elicitation of a secondary IgG response. In fact, challenge of PVP-HRBC-primed mice with an optimally immunogenic dose of PVP interfered with the elicitation of a secondary IgG response by PVP-HRBC. S3 and S 19 apparently are similar to PVP in this regard since they could not elicit capsular polysaccharide-specific secondary IgG responses (data not shown) and challenge with optimally immunogenic doses of these antigens interfers with elicitation of a secondary IgG response by S3-HRBC (28) or S19-CRBC (manuscript in preparation). The results presented here and in our previous studies with PVP ( 13, 17) clearly establish that several type 2 antigens are able to activate antigen-specific L3T4+ T cells which can provide help to B cells for the production of IgG antibody responses. However, in contrast to TD antigens, the ability to detect TH activity in T cells from mice primed with type 2 antigens is highly dependent on the dose of antigen used for priming. This is apparently due to the fact that these antigens also readily activate Ts (Fig. 2) which can prevent the expression of Tn activity (( 13) and Fig. 1) and the priming of memory B cells (Table 1 and (20)). Preferential activation of Ts which mask TH function has also been observed for several immune response gene-controlled responses in which a TD antigen is unable to elicit a response in low or nonresponder strains of mice (29-32). The reason(s) why type 2 antigens seem to preferentially activate Ts rather than TH even in “high-responder” strains of mice is unknown and is currently under investigation. It is, however, clear that the antigen-specific helper activity described here and in previous studies ( 13, 17) is required for the production of IgG but not IgM responses to these type 2 antigens. Thus masking of TH activity by Ts can explain why commonly employed doses (i.e., immunogenic for
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IgM responses) of type 2 antigens have not usually been found to activate TH or to induce memory in B cells. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3 1. 32.
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