Published online before print June 5, 2007
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* Laboratory of Experimental Laboratory Medicine, Department of Molecular Cell Biology, and
Laboratory of Experimental Immunology, Department of Pathophysiology, Faculty of Medicine, Catholic University Leuven, Leuven, Belgium;
Department of Surgery and Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA; and
Bioceros, Utrecht, The Netherlands
1 Correspondence: Laboratory of Experimental Laboratory Medicine, Department of Molecular Cell Biology, Faculty of Medicine, Catholic University Leuven, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. E-mail: xavier.bossuyt{at}uz.kuleuven.ac.be
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Key Words: capsular polysaccharides • pneumococcal surface protein A • costimulatory molecule
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In addition to caps-PS, there are some streptococcal surface proteins, such as pneumococcal surface protein A (PspA), which are considered virulent [2 ]. Proteins are thymus-dependent (TD) antigens, whereas caps-PS are considered thymus-independent Type 2 (TI-2) antigens [3 ], which are able to stimulate B lymphocytes directly. T lymphocytes are not required for induction of the antibody production against TI-2 antigens, but they exert a regulatory effect during the antibody response [4 ]. It has been reported that CD4+ T lymphocytes exert a positive effect, whereas CD8+ T lymphocytes exert a negative effect [5 ]. The molecular mechanisms of the T lymphocyte-mediated modulation of the anti-caps-PS immune response are not fully understood, although there is growing evidence indicating that the T lymphocyte-mediated regulation of the antibody response to the classic TI-2 antigens is dependent on "costimulatory" interactions. Recently, we showed that the antibody response to caps-PS is dependent on the CD40-CD40 ligand (CD40L) interaction [6 ]. Furthermore, it has been reported that the immune response to the cell wall polysaccharide phosphorylcholine is dependent on the CD40–CD40L interaction and the CD28–B7 interaction [7 ].
In addition to these interactions, the antibody response to classic TI-2 antigens may be dependent on other costimulatory interactions, such as the 4-1BB and 4-1BB ligand (4-1BBL) interaction.
4-1BB (CD137), a 30-KDa glycoprotein, is a member of the TNFR family, a group of cysteine-rich cell surface molecules, and is expressed rapidly on the surface of CD4+ T, CD8+ T, and NK1.1 cells after activation [8
]. 4-1BB is also expressed on activated B cells, on resting monocytes, and on dendritic cells (DC) [9
]. 4-1BBL, a Type II transmembrane glycoprotein, is a member of the TNF ligand family and is highly expressed on activated APC such as mature B lymphocytes, macrophages, and DC [10
]. 4-1BBL is also expressed on monocytes and activated T cells [9
]. The 4-1BB–4-1BBL interaction provides strong costimulatory signals to T lymphocytes, especially to CD8+ T cells, resulting in enhanced proliferation, survival, and secretion of cytokines (IL-2, IL-4, IFN-
, and TNF-
). The 4-1BB–4-1BBL interaction also amplifies the effector functions of APC [11
, 12
], such as B cell proliferation, IL-8, IL-6, and TNF- secretion, by macrophages and increased adhesion by monocytes [13
].
The question whether the 4-1BB–4-1BBL interaction plays a role in the immune response to TI-2 antigens in general and pneumococcal caps-PS in particular remains unsolved. There is contradictory data about the role of the 4-1BB–4-1BBL interaction in the antibody response to TI-2 antigens. Mittler et al. [14 ] reported that administration of anti-4-1BB mAb did not affect the antibody response to TNP-Ficoll, whereas Wu et al. [15 ] reported that the antibody response to the cell wall polysaccharide phosphorylcholine of a heat-killed, nonencapsulated S. pneumoniae Serotype 2 strain was reduced strongly in 4-1BBL–/– mice. Whether these differences are a result of immunization with different antigens or the use of 4-1BBL–/– mice versus anti-4-1BB mAb treatment is unknown.
The present study was undertaken to determine the role of the 4-1BB–4-1BBL interaction in the antibody response to a TI-2 and TD antigen, being pneumococcal caps-PS Serotype 3 (caps-PS3) and PspA, respectively.
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Mice
Balb/c and C57BL/6 mice were bought at Elevage Janvier (France). 4-1BBL–/– mice, on a C57BL6 background, were kindly provided by Dr. J. Peschon (Amgen, Thousand Oaks, CA, USA). The 4-1BBL–/– offspring was tested by PCR to distinguish the 4-1BBL+/+, +/–, and –/–mice as described by DeBenedette et al. [16
]. The animals were maintained under a standard protocol with free access to food and water. All mice strains were held in a room with a 12-h/12-h light/dark cycle. The permission to perform mice experiments was given by the local ethics committee.
Immunization of Balb/c, C57BL/6, and 4-1BBL–/– mice
Balb/c, C57BL/6, and 4-1BBL–/– mice were immunized with Pneumovax, heat-killed S. pneumoniae Serotype 3 (2x108 CFU/200 µL PBS) [7
], or OVA (20 µg/500 µL 0.9% NaCl). The vaccine was 1/25 diluted in 0.9% NaCl. This diluted vaccine, heat-killed S. pneumoniae Serotype 3, or OVA was given i.p. After 14 days, blood was drawn by intracardial puncture in isofluran-anesthetized mice, which were killed after isofluran inhalation by cervical dislocation. In experiments in which the effect of agonistic anti-4-1BB mAb (3H3) was studied, 500 µg anti-4-1BB mAb was injected i.p. 1 day before immunization. In the control group, 500 µg rat IgG was injected i.p. 1 day before immunization.
ELISA for detection of anti-caps-PS and anti-PspA antibodies
Anti-caps-PS antibodies were measured as described previously [6
]. Serum was treated at room temperature for a minimum of 30 min with pneumococcal C-polysaccharide (5 µg/mL PBS containing 2% goat serum) and PS-22F (5 µg/mL PBS containing 2% goat serum) to remove anti-C-polysaccharide antibodies and non-S. pneumoniae-specific antibodies, respectively.
PspA was prepared as described previously [17 ]. Anti-PspA antibodies were measured as follows: 96-well plates were coated with 100 µL 0.5 µg PspA/mL in 0.5 M carbonate/bicarbonate buffer (pH 9.6). The plates were incubated at room temperature for 1 h and stored at 4°C overnight. Between all steps, the plates were washed four times with PBS containing 0.05% Tween 20. The plates were blocked with PBS containing 10% goat serum (37°C, 1 h), incubated with 100 µL PBS, 1% BSA-diluted serum (37°C, 2 h), and incubated with peroxidase-labeled goat anti-mouse IgG/M (1/5000 dilution) in PBS, 1% BSA (37°C, 1.5 h). After washing, the plates were incubated with 3,3',5,5'-tetramethylbenzydin (100 µL/well, room temperature, 30 min) and 100 µL 0.5 M H2SO4. The OD was measured at 450 nm (A450).
Statistical analysis
The nonparametric Mann-Whitney statistics (P<0.05; Analyze-It for Microsoft Excel) was used to evaluate differences between groups.
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Figure 1. The in vivo IgM and IgG antibody response to pneumococcal caps-PS in anti-4-1BB mAb-treated (a) or 4-1BBL –/– (b) mice compared with corresponding control mice along with Pneumovax® administration. Balb/c, C57BL/6, and 4-1BBL–/– mice were immunized with Pneumovax® on day 0. Mice belonging to the anti-4-1BB mAb-treated groups were injected i.p. with 500 µg anti-4-1BB ( , n=4) or 500 µg rat IgG ( amp;, n=4, control group) on day –1. The IgM and IgG antibody response to pneumococcal caps-PS3 was measured 14 days after immunization. The results (mean±SD) show the absorbance values at various serum dilutions. Data are representative of three (a) and two (b) experiments with similar outcomes, respectively.
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Table 1. Anti-caps-PS3, Anti-OVA, and Anti-PspA Antibody Responses Obtained in Anti-4-1BB mAb-Treated or 4-1BBL–/– Mice Compared with the Corresponding Control Mice Groups after Immunization with Pneumovax®, OVA, or Heat-Killed, Intact S. pneumoniae Serotype 3
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Figure 2. The in vivo IgM and IgG immune response to caps-PS and PspA in anti-4-1BB mAb-treated mice compared with rat IgG-treated, control mice after immunization with heat-killed, encapsulated S. pneumoniae Serotype 3. Balb/c mice were immunized with heat-killed, encapsulated S. pneumoniae Serotype 3 on day 0 and treated i.p. with 500 µg anti-4-1BB (, n=5) or 500 µg rat IgG (amp;, n=4) on day –1. The IgG and IgM antibody response to caps-PS3 and PspA was measured 14 days after immunization. The results (mean±SD) show the absorbance values at various serum dilutions (P<0.02 for IgG and IgM anti-PspA). Data are representative of two experiments with similar outcome.
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Caps-PS antibody response in 4-1BBL–/– mice
The experiments described above suggest that the 4-1BB–4-1BBL interaction does not play a role in the antibody response to caps-PS. In a second approach to evaluate the role of the endogenous 4-1BB–4-1BBL interaction in the anti-caps-PS antibody response to soluble antigen, 4-1BBL–/– mice and C57BL6 wild-type mice were immunized with soluble caps-PS (Pneumovax®). After 14 days, the IgG and IgM antibody response to caps-PS3 was determined. There was no significant difference between 4-1BBL–/– mice and wild-type mice (Fig. 1b
and Table 1
). The IgG subclass antibody response also showed no significant differences between 4-1BBL–/– mice and wild-type mice (data not shown).
It is surprising, however, that when we immunized C57BL/6 and 4-1BBL–/– mice with heat-killed, encapsulated S. pneumoniae Serotype 3, a significant decrease of the IgG anti-caps-PS production in the 4-1BBL–/– mice compared with the control mice was observed (P<0.008). The IgM anti-caps-PS response showed a nonsignificant, down-regulated trend in the 4-1BBL–/– mice compared with the control mice.
Contradictory to our results in mice treated with the anti-4-1BB mAb, there was no difference of the IgG and IgM anti-PspA production between the 4-1BBL–/– mice and the C57BL/6 mice (Fig. 3 and Table 1 ). Similar data were observed after immunization with OVA as control TD antigen (data not shown).
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Figure 3. The in vivo IgM and IgG pneumococcal caps-PS and PspA antibody response in 4-1BBL–/– mice compared with C57BL/6 control mice along with heat-killed, encapsulated S. pneumoniae Serotype 3 administration. C57BL/6 wild-type mice (amp;, n=4) and 4-1BBL –/– mice (, n=4) were immunized i.p. with heat-killed, encapsulated S. pneumoniae Serotype 3 on day 0, and the IgM and IgG antibody response to pneumococcal caps-PS3 and PspA was measured 14 days after immunization. The results (mean±SD) show the absorbance values at various serum dilutions (P<0.008 for IgG anti-PS, Mann-Whitney statistics). Data are representative of two experiments with similar outcome.
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Treatment with agonistic anti-4-1BB mAb has been demonstrated previously to abrogate the TD antibody response to sheep RBCs or human IgG in mice through the induction of anergy of CD4+ T cells [14 ]; to suppress the humoral response to OVA in nonhuman primates [20 ]; to have no effect on the antibody response to the TI-2 antigen TNP-Ficoll [14 ]; to inhibit the in vivo primary IgG anti-PspA response and the generation of PspA-specific memory; and to have no effect on the antiphosphorylcholine response to intact, heat-killed, nonencapsulated S. pneumoniae Serotype 2 [15 ]. 4-1BBL–/– mice have been reported to have no apparent defects in in vivo antigen-specific Ig responses to a number of viruses such as vesicular stomatitis virus, lymphocytic choriomeningitis virus, or influenza A virus, suggesting that endogenous 4-1BBL-dependent costimulation plays little, if any, physiologic role in humoral immunity [15 ]. However, immunization of 4-1BBL–/–mice with a nonencapsulated variant of S. pneumoniae Serotype 2 resulted in a strong reduction of the in vivo IgM and IgG antiphosphorylcholine response. No significant effect on the IgG anti-PspA response was observed [15 ].
In the present study, the effect of the 4-1BB–4-1BBL interaction on the antibody response to pneumococcal caps-PS was studied by immunizing anti-4-1BB-treated, wild-type mice or 4-1BBL–/– mice with Pneumovax® or with heat-killed, encapsulated S. pneumoniae Serotype 3, which is more virulent than the nonencapsulated variant strains.
As neither 4-1BB mAb treatment nor the 4-1BBL–/– approach affected the IgG and IgM anti-caps-PS antibody response to soluble caps-PS (Pneumovax®), we conclude that the 4-1BB–4-1BBL interaction is not involved in the antibody response to soluble caps-PS. This was confirmed for the antibody responses to other serotypes, such as Serotypes 4 and 9N (data not shown). By contrast, our data indicate that the endogenous 4-1BB–4-1BBL interaction supports the in vivo anti-caps-PS immune response to killed S. pneumoniae Serotype 3. Our results indicate that distinct pathways are involved in the humoral immune response to pneumococcal antigens, depending on the nature of the antigen and the context in which the different antigens are presented.
Treatment with anti-4-1BB mAb had no effect on the IgG and IgM anti-caps-PS response but inhibited the IgG anti-PspA response strongly in Balb/c wild-type mice. In contrast to our observations with the anti-4-1BB mAb, the IgG anti-caps-PS immune response was reduced strongly in 4-1BBL–/– mice immunized with heat-killed, encapsulated S. pneumoniae Serotype 3. The IgG anti-PspA antibody response in the 4-1BBL–/– mice was comparable with the immune response in the wild-type mice.
The observed, contradictory data obtained with the mAb treatment and the knockout mice are most probably a result of differences in the two used approaches, as our results are in agreement with previous studies using similar experimental approaches but other TI-2 antigens (TNP-Ficoll and phosphorylcholine) [14 , 15 ]. We hypothesize that the two approaches (mAb treatment vs. knockout mice) do not create identical experimental conditions. DeBenedette et al. [16 ] found no differences between 4-1BBL–/– and wild-type mice with respect to the expression of cell surface markers on T cells, B cells, and CD8+ DC, an observation that we confirmed (data not shown). The mechanisms, which lie behind the differential effects between the two experimental approaches, are not understood. A number of issues, which should be taken into account, follow.
(i) Injection of anti-4-1BB mAb induces one or more signaling events [21 ]. Whether the signals result in a positive or negative outcome is at present unclear [21 ]. The binding of anti-4-1BB mAb to the 4-1BB receptor partially, but not completely, blocks the 4-1BB binding to 4-1BBL. Thus, administration of anti-4-1BB mAb in vivo can induce signaling in cells expressing 4-1BB, and these targets could be T cells, NK cells, NKT cells, neutrophils, or mast cells. Furthermore, anti-4-1BB mAb binding may diminish, but not preclude, receptor interaction with the ligand. In the case of the 4-1BBL-deficient mice, it is clear that in the absence of alternative receptor ligand partners, neither 4-1BB nor 4-1BBL signaling can occur.
(ii) Mittler et al. [14 ] reported that anti-4-1BB mAb abrogate TD humoral immune responses in vivo through the induction of Th cell anergy. Sun et al. [22 ] suggested that anti-4-1BB mAb treatment deletes follicular DC specifically, thus preventing subsequent events, leading to germinal center formation and antibody production [22 ]. One could hypothesize that in 4-1BBL–/– mice, T cell anergy is not induced, and follicular DC are not deleted. This could explain the differential effects between the results obtained in the knockout mice and the results obtained with the agonistic anti-4-1BB antibody for TD antigens.
(iii) CD25+CD4+ regulatory T cells (Tregs) are capable of specifically suppressing the functional response of other lymphocyte subsets [23 ]. 4-1BB is constitutively expressed on Tregs and is up-regulated upon stimulation [24 ]. It has been shown that activated CD25+CD4+ T cells suppress B cell proliferation in response to polyclonal B cell activators by inducing death of the responding B cell in a granzyme- and partially perforin-dependent way [25 ]. Signaling through the 4-1BB receptor, e.g., through administration of agonistic anti-4-1BB, inhibits the suppressive function of Tregs [26 ]. So, the lack of 4-1BB in the knockout mice could lead to excessive Treg effects on B cells.
(iv) C57BL/6 mice tend to produce Th1 responses, whereas Balb/c mice tend to produce Th2 responses [27 , 28 ]. Therefore, treatment with agonistic anti-4-1BB mAb was done in Balb/c mice and C57BL/6 mice to exclude the possibility that the observed effect of the antibody treatment was strain-dependent.
(v) When heat-killed S. pneumoniae or soluble caps-PS is given i.p., peritoneal B1 cells respond. However, there is evidence that when these antigens are administered at high concentration, then these antigens also reach the spleen [29 ], which contains a B1 cell, marginal zone B cells, and follicular B cells. Marginal zone B and B1 B cells are generally considered to be important for the immune response to TI antigens, whereas follicular B cells are generally considered to be important for the immune response to TD antigens [30 , 31 ].
We conclude that distinct pathways are involved in the humoral antibody response to pneumococcal antigens, depending on the nature of the antigen and the context in which the different antigens are presented. The 4-1BB–4-1BBL interaction is not involved in the antibody response to soluble caps-PS. The role of the 4-1BB–4-1BBL interaction in the immune reaction to S. pneumoniae depends on the experimental system used. Anti-4-1BB treatment strongly inhibited the IgG response to PspA but not to caps-PS. By contrast, the IgG anti-caps-PS antibody response, but not the anti-PspA antibody response, was reduced strongly in 4-1BBL–/– mice immunized with S. pneumoniae Serotype 3.
Received October 11, 2006; revised April 4, 2007; accepted April 30, 2007.
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