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Infection and Immunity, February 2006, p. 1419-1424, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1419-1424.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to Intact Streptococcus pneumoniae Is More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses

Abdul Q. Khan,¹ Goutam Sen,¹ Shuling Guo,² Owen N. Witte,² and Clifford M. Snapper^1*

Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814,¹ Howard Hughes Medical Institute, David Geffen School of Medicine at UCLA, 675 Charles E. Young Dr. S., 5-748 MRL, Los Angeles, California 90095-1662²

Received 18 August 2005/ Returned for modification 14 October 2005/ Accepted 23 November 2005

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The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btk^low mice exhibit reduced BCR signaling but largely restore B-cell development. Btk^low mice immunized with intact Streptococcus pneumoniae elicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btk^low mice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.

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Bruton's tyrosine kinase (Btk) plays a key role in B-cell receptor (BCR)-mediated signal transduction (3). Btk is critical for the normal development of B-1 and to a lesser extent B-2 B cells. Thus, CBA/N (xid) mice (2), which have a loss-of-function point mutation in the Btk gene (29, 42), exhibit a marked reduction in peritoneal B-1a cells, although a more modest decrease in B-1b cells, and a 30 to 50% reduction in splenic B-2 cells, including both marginal zone and follicular subsets (8, 9, 30). xid (6, 34) and Btk^–/– (15) mice also exhibit marked defects in Ig induction in response to soluble T-cell-independent type 2 (TI-2) antigens (e.g., polysaccharides). In contrast to soluble TI-2 antigens, the TI-1 antigens trinitrophenol (TNP)-lipopolysaccharide and TNP-Brucella abortus elicit normal immunoglobulin M (IgM) and IgG2 although reduced IgG3 responses in xid mice (24, 38), perhaps reflecting the adjuvant effect of the associated Toll-like receptor (TLR) activity intrinsic to the TI-1 but not TI-2 antigen. Ig responses to T-cell-dependent (TD) antigen, relative to those to TI-2 antigen, are variously and less severely affected in xid or Btk^–/– mice, with primary responses more defective than those following secondary immunization (4, 13, 15, 26, 33). Nevertheless, Btk appears to function as a BCR signal threshold modulator rather than as an essential component of the BCR signaling pathway (32). Thus, xid B cells can respond to particulate TI-2 antigens, such as TNP-sephadex or TNP-polyacrylamide (23). Additionally, defective TI-2 responses in xid mice can be corrected by coimmunization with a TLR agonist, such as 8-mercaptoguanosine (1, 21). Finally, TI-2 responses in xid mice can be partially reconstituted through provision of T-cell help (7, 18).

Defective humoral immune responses in xid or Btk^–/– mice could result from a combination of defective B-cell subset development and loss of Btk-mediated BCR signaling in the B cells that are present. In this regard, xid mice receiving one allele of a murine Btk transgene driven by the Ig heavy chain promoter and enhancer and expressing 25% of wild-type endogenous levels of Btk restore splenic B-2 cell development to wild-type levels and have a more modest decrease in peritoneal B-1a cells than xid mice (31). Nevertheless, these mice still have defective BCR signaling and weaker Ig responses to the soluble TI-2 antigen TNP-Ficoll than wild-type mice. Essentially similar observations were made with xid mice containing a transgene encoding the antiapoptotic protein Bcl-2 (43). Since B-1 cells do not participate in the TNP-Ficoll response (10), these data strongly suggest a direct role for Btk-dependent BCR signaling in Ig responses to soluble TI-2 antigens. The latter studies did not evaluate Ig responses to soluble TD antigens, which are also reduced, albeit less dramatically, in xid mice.

The studies discussed above collectively indicate that Ig responses, particularly to isolated polysaccharide (PS) antigens in xid or Btk^–/– mice, can vary dramatically depending upon the presence or absence of adjuvant, T-cell help, and/or antigen particulation and the level of restoration of B-cell subset development. In this regard, intact bacterial pathogens coexpress PS and protein antigens and TLR ligands within a particulate structure. Additionally, we previously demonstrated that IgG anti-PS and antiprotein responses to intact Streptococcus pneumoniae were both dependent upon CD4⁺ T-cell help, B7-dependent costimulation, and CD40-CD40 ligand interactions (14, 44). Thus, the relative role of Btk-dependent BCR signaling in directly regulating anti-PS versus antiprotein Ig responses to an intact bacterium in vivo remains an open and important question. In this study we determined PS- and protein-specific IgM and IgG responses to both intact S. pneumoniae and soluble TD conjugates of pneumococcal PS and protein antigens in xid and Btk^low mice. We demonstrate that Btk-dependent signaling plays a significantly greater role in stimulating anti-PS, versus anti-protein, responses to intact S. pneumoniae and to soluble pneumococcal conjugate in vivo following restoration of B-cell subset development. The relevance of these data in the context of anti-PS and antiprotein responses following natural pneumococcal infections in infants (28, 37, 40, 41) is discussed below.

Btk^low mice are Btk^–/– mice carrying a wild-type Btk transgene driven by the Ig heavy chain promoter and enhancer, as described previously (31), and backcrossed six generations onto the BALB/c background. These mice express 25% of endogenous levels of the Btk protein in splenic B cells. BALB/c mice (Jackson Labs, Bar Harbor, ME) were used as controls for Btk^low mice. CBA/CaHN-Btk^xid/J (xid) and control CBA/CaJ mice were also obtained from Jackson Labs.

Splenic and peritoneal B-cell subsets in both xid and Btk^low mice were enumerated, relative to those in wild-type mice, by flow cytometric analysis (six mice per group; cells from each mouse analyzed separately) (Table 1). For enumeration of marginal zone B (MZB) cells and follicular B (FB) cells, spleen cells were stained with rat IgG2b, anti-mouse CD21/CD35-phycoerythrin (PE) (clone 7G6) and rat IgG2a, anti-mouse CD23-biotin (clone B3B4) followed by streptavidin-PE-Texas Red. MZB and FB cells were identified as CD21^high CD23^low and CD21^intermediate CD23^high, respectively. For enumeration of splenic B-1 cells, spleen cells were stained with rat IgG2a, anti-mouse B220-PE (clone RA3-6B2) and rat IgG2a, anti-mouse CD5-biotin (clone 53-7.3), followed by streptavidin-PE-Texas Red, and B-1 cells were identified as B220⁺ CD5⁺. Peritoneal cells were stained with anti-B220-fluorescein isothiocyanate, rat IgG2b, antimouse CD11b-PE (clone M1/70), and anti-CD5-biotin followed by streptavidin-PE-Texas Red. B-1a cells were identified as B220⁺ CD11b⁺ CD5⁺, B-1b cells as B220⁺ CD11b⁺ CD5^–, and B-2 cells as B220⁺ CD11b^– CD5^–. All reagents were purchased from BD-Pharmingen.

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TABLE 1. B-cell subset development is largely restored in Btklow micea

Analysis of spleen cells from xid mice demonstrated a significant reduction (P 0.05) in the absolute numbers of FB (2.6-fold), MZB (2.2-fold), and B-1 (1.6-fold) cells, relative to wild-type (CBA/CaJ) mice (Table 1). A more profound reduction in peritoneal B-cell subsets was observed in xid mice relative to wild-type mice (for B-1a, 10-fold; for B1-b, 6-fold; for B-2, 10-fold). In contrast, spleen cells from Btk^low mice exhibited no reductions in the absolute numbers of FB, MZB, or B-1 cells relative to those of wild-type mice, with the numbers of MZB cells actually significantly higher (1.7-fold) in the Btk^low mice. Similarly, a partial or complete restoration in the numbers of peritoneal B-cell subsets was observed in Btk^low mice (for B-1a, threefold lower [P < 0.05]; for B1b, no difference; for B-2, 2.2-fold higher [P < 0.05]). Since the total numbers of spleen cells were comparable between xid and Btk^low mice relative to those of their wild-type counterparts, the absolute numbers of B-cell subsets reflected their relative percent representation (Table 1). Collectively, these data are consistent with those reported by others (15, 31) and confirm the ability of the single btk transgene to largely restore B-cell subset development in the xid mouse.

xid and Btk^low mice and their wild-type counterparts were immunized intraperitoneally (i.p.) with heat-killed intact Streptococcus pneumoniae, capsular type 14, in saline (2 x 10⁸ CFU S. pneumoniae serotype 14/mouse, six mice per group) and boosted in a similar fashion 14 days later. S. pneumoniae serotype 14 was prepared, heat killed, and stored, as described previously (14). Serum titers of IgM and IgG specific for the phosphorylcholine (PC) determinant of the cell wall C polysaccharide (C-PS), the capsular PS (PPS14), and the cell wall pneumococcal surface protein A (PspA) were determined on days 0, 7, 14, 21, and/or 28 as described previously (14). Whereas S. pneumoniae serotype 14 elicited a strong primary IgM and IgG anti-PC response in wild-type mice, xid mice were essentially unresponsive (Fig. 1), consistent with previous reports (22, 27). Secondary immunization with S. pneumoniae serotype 14 did result in a detectable IgM and IgG anti-PC response in xid mice, but this was still markedly below wild-type levels (for IgM, 56-fold; for IgG, 116-fold). In contrast, Btk^low mice elicited detectable, although still significantly reduced, primary IgM (3.9- to 7.5-fold) and IgG (5.8- to 6.1-fold) anti-PC responses relative to those of their wild-type controls. Secondary anti-PC titers for Btk^low mice showed reductions relative to titers for wild-type mice similar to those seen for the primary responses. In light of earlier data indicating that the anti-PC response to intact S. pneumoniae derives from both B-1 and MZB cells (20), these data suggest that the degree of reduction in anti-PC titers in Btk^low mice does not result solely from a reduced representation of responding B-cell subsets. A marked reduction in the primary IgM (46- to 76-fold) and IgG (29- to 75-fold) anti-PPS14 response, as well as the IgG anti-PspA response (>13-fold), was also observed in xid mice (Fig. 1). Of interest, secondary immunization of xid mice with S. pneumoniae serotype 14 resulted in a partial restoration in the IgM (18-fold reduced) and IgG (6.1-fold reduced) anti-PPS14 and IgG anti-PspA (4.6-fold reduced) responses relative to those of wild-type mice. In Btk^low mice, the reduction in the primary IgM (3.8- to 8.1-fold) and IgG (5.2- to 13-fold) anti-PPS14 response was significant but more moderate relative to results with xid mice, whereas the primary IgG anti-PspA response was comparable to that seen with wild-type mice. Again, secondary immunization of Btk^low mice with S. pneumoniae serotype 14 resulted in a substantial boost in the IgG, although not IgM, anti-PPS14 response, bringing the IgG titers up to wild-type levels. Comparable boosting of the IgG anti-PspA response was observed in both Btk^low and wild-type mice. Collectively, these data demonstrate that the degree of reduction in the anti-PC and anti-PPS14 responses to intact S. pneumoniae serotype 14 observed in Btk^low mice was significantly out of proportion to any alterations in B-cell subset development. This strongly suggests a role for btk-mediated BCR signaling in the responding B cells for induction of an anti-PS response that is greater than that required to elicit an antiprotein Ig response.

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FIG. 1. IgM and IgG anti-PC and anti-PPS14 responses, in contrast to the anti-PspA response, to intact S. pneumoniae serotype 14 are selectively defective for Btklow mice relative to those for wild-type mice. CBA/CaHN-Btkxid/J (xid) and control CBA/CaJ mice, as well as Btklow and control BALB/c mice (six mice per group), were immunized i.p. with intact heat-killed S. pneumoniae serotype 14 (2 x 108 CFU/mouse). Sera were obtained on the indicated days, and antigen-specific IgM and IgG serum titers were determined by enzyme-linked immunosorbent assay. Data are presented as geometric means plus standard errors of the means. Significance: *, P 0.05 by Student's t test.

As mentioned earlier, both adjuvant (1, 21) and particulation (23) of antigen as is present in intact S. pneumoniae serotype 14 can, at least partially, overcome the consequences of defective btk function in B cells. This might potentially obscure an even greater requirement for btk signaling in anti-PS as opposed to antiprotein Ig responses when using soluble antigens in the absence of an adjuvant. To test this hypothesis, we immunized xid and Btk^low mice and their wild-type counterparts with 1 µg each of soluble PPS14-PspA plus C-PS-PspA conjugates in saline, followed by boosting 14 days later. Serum titers of IgM and IgG anti-PC, anti-PPS14, and anti-PspA were measured on days 0, 7, 14, and 21. The conjugates were synthesized as described by us in detail elsewhere (14). Strikingly, in response to the soluble conjugates in saline, marked and nearly comparable reductions in primary and secondary IgM and IgG anti-PC and anti-PPS14 responses were observed for both xid and Btk^low mice, relative to results for their wild-type counterparts (Fig. 2), a defect more profound than that observed using intact S. pneumoniae serotype 14. In distinct contrast, the induction of serum titers of IgG anti-PspA for both xid and Btk^low mice were equivalent to that observed for wild-type mice. Of note, the IgG anti-PC, anti-PPS14, and anti-PspA responses to the soluble conjugates are all CD4⁺ T cell dependent in wild-type mice (14).

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FIG. 2. IgM and IgG anti-PC and anti-PPS14 responses, in contrast to the anti-PspA response, to PPS14-PspA plus C-PS-PspA in saline are selectively defective for Btklow mice relative to those for wild-type mice. CBA/CaHN-Btkxid/J (xid) and control CBA/CaJ mice and Btklow and control BALB/c mice (six mice per group) were immunized i.p. with 1 µg each of PPS14-PspA plus C-PS-PspA in saline. Sera were obtained on the indicated days, and antigen-specific IgM and IgG serum titers were determined by enzyme-linked immunosorbent assay. Data are presented as geometric mean plus standard errors of the means. Significance: *, P 0.05 by Student's t test.

In a final set of experiments, we wished to directly test the notion that the presence of an adjuvant could indeed restore the defective anti-PS responses to the soluble conjugates observed in the Btk^low mice and thus at least partially mimic the response to intact S. pneumoniae serotype 14. We therefore immunized Btk^low and wild-type mice with 1 µg each of soluble PPS14-PspA plus C-PS-PspA conjugates either in saline or adsorbed on 13 µg of alum (Allhydrogel, 2% [Brenntag Biosector, Denmark]) mixed with 25 µg of a 30-mer CpG-containing oligodeoxynucleotide (CpG-ODN) (35), a ligand for TLR9 (12). Mice were boosted in a similar fashion on day 14. Under these conditions, in which a subadjuvant amount of alum is used simply to facilitate the association of the conjugate with the CpG-ODN, the adjuvant activity was almost entirely due to the CpG-ODN (data not shown). In the absence of alum-CpG, the IgM and IgG anti-PC and anti-PPS14, but not IgG anti-PspA, responses were markedly reduced for Btk^low mice relative to those for wild-type mice, although partial restoration of IgG anti-PPS14 titers was observed following secondary immunization (Fig. 3), similar to observed results shown in Fig. 2. The addition of alum-CpG-ODN to the conjugates significantly enhanced both IgM and IgG anti-PC, anti-PPS14, and anti-PspA responses in wild-type mice, consistent with several previous reports (5, 16, 17). More remarkably, addition of alum-CpG-ODN almost completely restored the IgM and IgG anti-PC and anti-PPS14 responses in Btk^low mice to wild-type levels, confirming the notion that an adjuvant can overcome defects in Btk signaling for induction of Ig responses (1). The ability of adjuvant to restore anti-PS responses in Btk^low mice to the level of those observed in wild-type mice was delayed. Thus, a relatively greater effect of adjuvant was seen on day 14 than on day 7, with optimal effects observed after boosting.

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FIG. 3. Alum-CpG-ODN largely restores the IgM and IgG anti-PC and anti-PPS14 response to PPS14-PspA plus C-PS-PspA in Btklow mice. Btklow and control BALB/c mice (six mice per group) were immunized i.p. with 1 µg each of PPS14-PspA plus C-PS-PspA either in saline or adsorbed on 13 µg of alum (Allhydrogel, 2%) mixed with 25 µg of a 30-mer CpG-containing oligodeoxynucleotide (CpG-ODN). Sera were obtained on the indicated days, and antigen-specific IgM and IgG serum titers were determined by enzyme-linked immunosorbent assay. Data are presented as geometric means plus standard errors of the means. Significance: *, P 0.05 by Student's t test.

Further analyses of all serum samples obtained in the experiment illustrated in Fig. 3 demonstrated the following (data not shown). (i) The relative primary and secondary serum titers of all IgG isotypes specific for PC, PPS14, or PspA directly mirrored the corresponding serum titers for antigen-specific total IgG. Thus, no selective IgG isotype differences were observed between wild-type and Btk^low mice. Further, alum-CpG-ODN enhanced, to similar degrees, the serum titers of all IgG isotypes in wild-type and Btk^low mice. (ii). The idiotype of day 21 serum IgM anti-PC antibody in wild-type and Btk^low mice in the absence or presence of alum-CpG-ODN was overwhelmingly T15⁺. (iii) The ability of the serum samples to mediate opsonophagocytosis by macrophages in vitro directly mirrored the relative serum titers of PPS14-specific IgG. The opsonophagocytosis assay was performed as described previously (19) with minor modifications.

Collectively, these data strongly suggest that induction of anti-PS responses requires a greater degree of Btk-mediated BCR signaling than induction of antiprotein responses when tested under conditions where both responses are elicited in the presence of CD4⁺ T-cell help, equivalent levels of an adjuvant or its absence, and an antigen form (particulate or soluble) and where B-cell subset development is largely intact. Nevertheless, we show that the presence of a sufficiently strong adjuvant, in this case the TLR9 ligand CpG-ODN, can largely overcome this defect, whereas the degree of natural TLR adjuvant action provided by intact S. pneumoniae serotype 14 is only partially effective. Neonatal B cells, like xid or Btk^low B cells, exhibit defective signaling following BCR cross-linking (11, 36). In this regard, similar to what is observed in xid mice, TI-2 responses are also markedly defective in the immature host, whereas T cell-dependent antiprotein responses are less affected (24, 25). Thus, infants respond poorly to isolated PS vaccines but elicit protective anti-PS responses to PS conjugated to an immunogenic carrier protein, which effects recruitment of CD4⁺ T-cell help. As with xid B cells, the defective BCR-dependent function of neonatal B cells can be compensated for by TLR-dependent signaling (39). Surprisingly, however, only small minorities of infants less than 2 years of age elicit a detectable systemic or mucosal IgG anticapsular PS response following natural infection with S. pneumoniae, whereas a majority induce IgG specific for several S. pneumoniae proteins (28, 37, 40, 41). In light of our current data, we propose that this may be partly due to an insufficient level of TLR signaling, provided during natural S. pneumoniae infections, in order to overcome the defective BCR signaling that differentially impacts on the anti-PS response in the immature host.

 
Opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.

This study was supported by N.I.H. grants 1R01 AI49192 and the U.S.U.H.S. Dean's Research and Education Endowment Fund.

We thank Andy Lees (Biosynexus, Inc., Gaithersburg, Md.) for provision of PC-KLH, C-PS-PspA, and PPS14-PspA.

 
* Corresponding author. Mailing address: Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Phone: (301) 295-3490. Fax: (301) 295-1640. E-mail: csnapper{at}usuhs.mil.

Editor: J. N. Weiser

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Infection and Immunity, February 2006, p. 1419-1424, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1419-1424.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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