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The Journal of Immunology, 2006, 176: 529-536.
Copyright © 2006 by The American Association of Immunologists

CD4+ T Lymphocytes Expressing CD40 Ligand Help the IgM Antibody Response to Soluble Pneumococcal Polysaccharides via an Intermediate Cell Type1

Axel Jeurissen*, An D. Billiau{dagger}, Leen Moens*, Li Shengqiao{dagger}, Willy Landuyt§, Greet Wuyts*, Louis Boon, Mark Waer{dagger}, Jan L. Ceuppens{ddagger} and Xavier Bossuyt2,*

* Laboratory of Experimental Laboratory Medicine, Department of Medical Diagnostic Sciences; {dagger} Laboratory of Experimental Transplantation, Department of Pathophysiology, Faculty of Medicine, and {ddagger} Laboratory of Experimental Immunology, Department of Pathophysiology, Faculty of Medicine, Catholic University, Leuven, Belgium; § Laboratory of Experimental Oncology, University Hospital Gasthuisberg, Leuven, Belgium; and Bioceros, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Streptococcus pneumoniae is a major human pathogen that frequently causes pneumonia, septicemia, and meningitis, particularly in young children, the elderly, and immunocompromised patients (1). In various animal species and in humans, protection against infection with S. pneumoniae is mediated by Abs against the pneumococcal capsular polysaccharides (caps-PS)3 (2). caps-PS are classified as T lymphocyte independent Ags (3) because neonatally thymectomized mice and athymic nude mice are able to mount an Ab response to these Ags. T lymphocytes increase the Ab response to caps-PS, and, therefore, caps-PS are categorized as T-independent (TI)-2 Ags (3). The mechanism of this T cell helper effect is unclear. Currently, it is a controversial issue whether the CD40-CD40L interaction, which plays an essential role in the immune response to protein Ags, is also important in the immune response to TI-2 Ags in general and caps-PS Ags in particular (4). Classically, it is believed that the immune response to TI-2 Ags is CD40L independent. Studies using the TI-2 Ags trinitrophenyl (TNP)-Ficoll or DNP-Ficoll (5, 6, 7) and one study using purified caps-PS serotype 6 (8) concluded that the Ab responses to these TI-2 Ags are independent of the CD40-CD40L interaction. By contrast, we more recently found that the Ab response to several caps-PS serotypes, including serotype 6, was dependent on the CD40-CD40L interaction (9, 10). This was demonstrated in a murine model by showing that the helper effect of CD4+ T lymphocytes and the inhibitory effect of CD8+ T lymphocytes on the anti-caps-PS immune response in vivo and in vitro was abrogated by MR1, a blocking anti-CD40L Ab (9). Also in humans, we were able to show in vitro and in vivo that CD4+ T lymphocytes strongly enhance the anti-caps-PS immune response and that this activation was inhibited by administering blocking anti-CD40 or anti-CD40L Abs (10). We found that polysaccharides induced the expression of CD40L on T cells (10). Moreover, CD4+ T lymphocytes obtained from patients with X-linked hyper-IgM syndrome were unable to stimulate the anti-caps-PS immune response, whereas CD4+ T lymphocytes obtained from a healthy individual were able to do so (10). The aim of this study was 2-fold. First, to use an alternative approach for proving that the murine Ab response to caps-PS is dependent on the CD40-CD40L interaction, thereby corroborating our previous observations (9, 10, 11). Second, to unravel in more detail the mechanisms that underlie the CD40L-dependent stimulation of the anti-caps-PS Ab response. T lymphocytes, once activated, could either directly or indirectly modulate B lymphocyte activity via CD40L. Direct ligation of CD40 on B lymphocytes, with subsequent stimulation, is one possibility. However, an indirect mechanism whereby T lymphocytes interact with APCs cannot be excluded. Contact of T lymphocytes with macrophages and/or dendritic cells through CD40-CD40L interaction could induce the release of multiple cytokines that can further stimulate B lymphocytes. To elucidate the question of whether the CD40L-dependent stimulation of the anti-caps-PS Ab response is the result of a direct or an indirect effect was the main goal of this study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Pneumovax, a 23-valent pneumococcal vaccine containing 25 µg of the caps-PS (Danish nomenclature) types 1–5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, was obtained from Aventis Pasteur MSD. Pneumococcal caps-PS were obtained from American Type Culture Collection. C-polysaccharide was obtained from Statens Serum Institute (Copenhagen, Denmark). MR1, a monoclonal blocking Ab to murine CD40L, was provided by Bioceros. Polyclonal hamster IgG were obtained from 10 P’s. PE-labeled anti-mouse CD4 (L3T4) and PE-labeled anti-mouse CD8a (Ly-2) were obtained from BD Biosciences. Peroxidase-conjugated goat-anti-mouse IgM and IgG were obtained from Nordic Immunological Laboratories. Goat serum and PBS were obtained from Invitrogen Life Technologies. 3,3'-5,5'-Tetramethylbenzidine was purchased from DAKO Diagnostics. HCl 1 N and H2SO4 3 N solution were obtained from Merck. N-(-3-dimethylaminopropyl)-N'-ethylcarbiimide hydrochloride, 5 g dissolved in 500 ml of AD, was obtained from Merck. Dynabeads were obtained from Dynal Biotech. Covalink ELISA plates were obtained from Nunc Brand Products (Nalge Nunc International). TNP-Ficoll was obtained from Biosearch Technologies, and TNP-Ficoll microbeads were obtained from Microbeads Bioscience. FITC-labeled goat-anti-mouse IgM was purchased from Serotec.

Mice

C57BL/6 mice were obtained from Janvier. CD40 knockout mice (background C57BL/6) were obtained from CDTA. CD40L knockout mice (background C57BL/6) were obtained from The Jackson Laboratory. Animals were used at the age of 6–8 wk and were kept under a standard protocol with free access to pelleted food and water. Six- to 8-wk-old SCID/SCID mice were provided by J. Mertens (REGA Institute, Leuven, Belgium). They were kept in sterilized plastic cages and were given sterilized tap water and sterilized pelleted food. The SCID/SCID mice were tested for leakiness by analyzing the level of mouse IgG Abs according to a previously described ELISA (12). Only mice with IgG concentrations <2 µg/ml were used in the experiments. All mice strains were held in a room with 12 h/12 h light/dark cycle. Approval of the study was granted by the local committee of the Catholic University Leuven.

Immunization of mice

Mice were immunized with Pneumovax. The vaccine was 1/25 diluted in 0.9% NaCl. Five hundred microliters of this diluted vaccine was given i.p. After 14 days, blood was drawn by intracardial punction and anti-caps-PS Abs were detected by ELISA (see infra). In experiments in which the effects of MR1 were studied, 500 µg of MR1 was injected i.p. 1 day before immunization with Pneumovax. Five hundred micrograms of hamster IgG control Ab was injected i.p. into the control group.

Mice were immunized with TNP-Ficoll using 100 µg of TNP-Ficoll through i.v. injection.

Detection of anti-caps-PS Abs and of anti-TNP-Ficoll Abs

ELISA for detection of anti-caps-PS Abs was performed as described previously (9). The immune response to four serotypes (3, 4, 14, 19F) was measured, including serotype 3 and 14, two serotypes that are highly bacteremic and associated with a high mortality. Moreover, serotype 3 is a highly immunogenic serotype.

Mouse anti-TNP-Ficoll serum IgM was determined by flow cytometry using TNP-Ficoll microbeads and FITC-labeled goat-anti-mouse IgM. Results are expressed as mean fluorescence intensity (MFI).

Transfer of spleen cells to SCID/SCID mice

C57BL/6 mice, CD40L knockout mice, or CD40 knockout mice were sacrificed under ether anesthesia and the spleens were removed. Mouse mononuclear cells were isolated from the spleen. When indicated, CD4+ and/or CD8+ lymphocytes were removed using Dynabeads. The percentage of B lymphocytes, CD4+ and CD8+ T lymphocytes in the lymphogate was determined by flow cytometry (FACSCalibur; BD Biosciences). The cells were dissolved in 500 µl of NaCl 0.9% and injected i.p. into SCID/SCID mice. On the same day, 500 µg of MR1 or hamster IgG was administered and the next day Pneumovax was given i.p. After 14 days, blood was drawn by heart puncture and IgM and IgG Abs to caps-PS measured.

Bone marrow transplantation

Bone marrow from 8-wk-old C57BL/6 mice was flushed from femurs and tibias with PBS and passed through sterile mesh filters. CD40 knockout recipient mice were conditioned with total body irradiation administered as a single exposure (9 Gy). The efficacy of the irradiation procedure was verified by irradiation of three mice without replacement of bone marrow. All three mice died within 3 wk, which confirmed the efficacy of the total body irradiation. Irradiated recipients received an i.v. injection of 5 x 106 bone marrow cells and were monitored daily for survival. Twelve weeks after bone marrow transplantation, mice received injections i.p. with Pneumovax. Fourteen days after injection with Pneumovax, blood was drawn and anti-caps-PS Abs were measured using ELISA. Engraftment was checked by flow cytometric analysis of CD40 expression on CD19+ cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4+ T lymphocytes from CD40L knockout mice fail to stimulate the anti-caps-PS Ab response

To investigate the role of the CD40-CD40L interaction in the stimulatory effect of CD4+ T lymphocytes, we examined whether CD4+ T lymphocytes obtained from CD40L knockout mice stimulated the anti-caps-PS Ab response to the same extent as CD4+ T lymphocytes obtained from wild-type mice. The results are shown in Fig. 1. SCID/SCID mice reconstituted with B lymphocytes obtained from wild-type mice without T lymphocytes did not mount an Ab response to caps-PS, whereas SCID/SCID reconstituted with B lymphocytes isolated from wild-type mice together with CD4+ T lymphocytes obtained from wild-type mice clearly generated anti-caps-PS Abs. Conversely, SCID/SCID mice reconstituted with B lymphocytes from wild-type mice together with CD4+ T lymphocytes isolated from CD40L knockout mice did not mount an Ab response to caps-PS. These results underscore the importance of the CD40-CD40L interaction in the CD4+ T lymphocyte-mediated modulation of the anti-caps-PS immune response and are consistent with our previous finding (9) that MR1 inhibits the stimulatory effect of CD4+ T lymphocytes in the murine anti-caps-PS immune response.



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  		FIGURE 1. CD4+ T lymphocytes from CD40L knockout mice fail to stimulate the anti-caps-PS Ab response. SCID/SCID mice were reconstituted with wild-type murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving B lymphocytes) ({blacktriangleup}), with wild-type murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving B lymphocytes) together with wild-type murine spleen cells depleted of B and CD8+ T lymphocytes (leaving CD4+ T lymphocytes) ({blacksquare}), or with wild-type murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving B lymphocytes) together with CD40L–/– murine spleen cells depleted of B and CD8+ T lymphocytes (leaving CD4+ T lymphocytes) ({square}). The B lymphocyte fraction contained >85% B lymphocytes and <1.5% CD4+ or CD8+ T lymphocytes. The CD4+ T lymphocyte fraction contained >80% CD4+ T lymphocytes and <2.5% B or CD8+ T lymphocytes. The cells were suspended in 500 µl of 0.9% NaCl and injected i.p. into SCID/SCID mice. The total amount of B lymphocytes was the same in all conditions and amounted to ± 10 x 10E6 cells. The total amount of CD4+ T lymphocytes was the same in all conditions and amounted to ± 4 x 10E6 cells. The IgM Ab response to caps-PS serotypes 3, 4, 14, and 19F was measured in duplicate 14 days after vaccination with Pneumovax. Results shown are from a representative experiment that was done three times with similar outcome.

 
To verify that B lymphocytes remained functional after transfer into SCID/SCID mice, we measured the Ab response of B cell alone reconstituted SCID/SCID mice to TNP-Ficoll, which is another TI Ag. In SCID/SCID mice reconstituted with B cells (in the absence of T lymphocytes), the anti-TNP-Ficoll Ab response 1 wk after immunization was 238 ± 65 MFI (mean ± SD) (n = 3). This was significantly higher than the Ab level measured in SCID/SCID mice in the absence of B lymphocytes, which was 4.4 ± 7.3 MFI (n = 7) (p = 0.016; Mann-Whitney U test). For comparison, the anti-TNP-Ficoll Ab response in wild-type mice amounted to 667 ± 40.7 MFI. These data indicated that B lymphocytes remained functional after transfer into SCID/SCID mice, even in the absence of T lymphocytes.

CD4+ T lymphocytes stimulate the anti-caps-PS Ab response by B lymphocytes through the CD40-CD40L interaction in an indirect way

To examine whether the stimulatory effect of CD4+ T lymphocytes on the anti-caps-PS Ab response is the result of a direct interaction between B and T lymphocytes or of an indirect effect, we studied the effect of CD4+ T lymphocytes on the Ab response of B lymphocytes obtained from CD40 knockout mice. If T lymphocytes directly interact with B lymphocytes through the CD40-CD40L interaction, then no effect of CD4+ T lymphocytes will be observed. If, however, CD4+ T lymphocytes exert their effect indirectly, e.g., via interaction with APCs, then CD4+ T lymphocytes will stimulate the anti-caps-PS Ab response of CD40 knockout B lymphocytes.

SCID/SCID mice were reconstituted with purified B lymphocytes obtained from the spleen of CD40 knockout mice in the presence or absence of purified CD4+ T lymphocytes obtained from wild-type mice. As a control condition, SCID/SCID mice were reconstituted with B lymphocytes of wild-type mice in the presence or absence of T lymphocytes from other wild-type mice. The SCID/SCID mice were immunized with Pneumovax and the Ab response to caps-PS serotypes 3, 4, 14, and 19F was measured after 14 days. Results are shown in Fig. 2. SCID/SCID mice reconstituted with B lymphocytes obtained from either CD40 knockout mice or wild-type mice did not mount an Ab response to caps-PS. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice clearly generated an augmented Ab response when compared with SCID/SCID mice reconstituted with only B lymphocytes from wild-type mice (p < 0.05 (Kruskal-Wallis) for all serotypes tested and for at least four serum dilutions), confirming the stimulatory effect of CD4+ T lymphocytes on the Ab response to caps-PS. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice together with CD4+ T lymphocytes from wild-type mice generated significantly higher (p < 0.05 (Kruskal-Wallis) for all serotypes tested and for at least four serum dilutions) anti-caps-PS Abs than SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice. In the presence of wild-type CD4+ T cells, the absence or presence of CD40 on the B cells did not make a statistically significant difference (p ≥ 0.5, Kruskal-Wallis). These results suggest that the stimulatory effect of CD4+ T lymphocytes on the Ab response to caps-PS was indirect.



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  		FIGURE 2. The stimulatory effect of CD4+ T lymphocytes on the Ab response to caps-PS is indirect. SCID/SCID mice were reconstituted with 1) wild-type murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving wild-type B lymphocytes) ({square}), 2) with CD40 knockout murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving CD40 knockout B lymphocytes) ({triangleup}), 3) with wild-type murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving wild-type B lymphocytes) together with wild-type murine spleen cells depleted of CD8+ T lymphocytes and B lymphocytes (leaving wild-type CD4+ T lymphocytes) ({blacksquare}), 4) or with CD40 knockout murine spleen cells depleted of CD8+ T lymphocytes and CD4+ T lymphocytes (leaving CD40 knockout B lymphocytes) together with wild-type murine spleen cells depleted of B lymphocytes and CD8+ T lymphocytes (leaving wild-type CD4+ T lymphocytes) ({blacktriangleup}). The B lymphocyte fraction contained >80% B lymphocytes and <1.5% CD4+ or CD8+ T lymphocytes. Depletion of B and CD8+ T lymphocytes resulted in a cell population consisting of >80% CD4+ T lymphocytes and <2.5% B lymphocytes and CD8+ lymphocytes. The cells were suspended in 0.9% NaCl and injected i.p. into SCID/SCID mice. The total amount of B lymphocytes was the same in all conditions and amounted to ± 10 x 10E6 cells. The total amount of CD4+ T lymphocytes was the same in all conditions and amounted to ± 4 x 10E6 cells. The IgM Ab response to caps-PS serotypes 3, 4, 14, and 19F was measured after 14 days. The results shown are means ± 1 SD of three independent experiments (representing mouse to mouse variation (n = 3)).

 
To exclude the possibility that a direct B lymphocyte-T lymphocyte interaction, other than via CD40-CD40L, was responsible for the stimulatory effect of CD4+ T lymphocytes, we studied the effect of blocking the CD40-CD40L interaction on the stimulatory effect of CD4+ T lymphocytes on the anti-caps-PS Ab response by B lymphocytes lacking CD40. SCID/SCID mice were reconstituted with B lymphocytes obtained from CD40 knockout mice and CD4+ T lymphocytes obtained from wild-type mice. The animals were then treated with MR1, a monoclonal anti-CD40L blocking Ab, or hamster IgG as an isotype control and immunized with Pneumovax. Administration of MR1 abolished the stimulatory effect of CD4+ T lymphocytes on the anti-caps-PS IgM Ab response by CD40 knockout B lymphocytes for all four serotypes measured (p < 0.05, Kruskal-Wallis) (Fig. 3). These data show that CD4+ T lymphocytes stimulate the anti-caps-PS Ab response via an effect of CD40L on non-B cells.



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  		FIGURE 3. Effect of MR1 on the stimulatory effect of CD4+ T lymphocytes on the anti-caps-PS Ab response. SCID/SCID mice were reconstituted with murine spleen cells depleted of CD4+ and CD8+ T lymphocytes (leaving B lymphocytes) from CD40 knockout mice together with murine spleen cells depleted of B and CD8+ T lymphocytes (leaving CD4+ T lymphocytes) from wild-type mice. The B lymphocyte fraction contained >80% B lymphocytes and <1.5% CD4+ or CD8+ T lymphocytes. The CD4+ T lymphocyte fraction contained >75% CD4+ T lymphocytes and <2.5% B or CD8+ T lymphocytes. The cells were suspended in 500 µl 0.9% NaCl and injected i.p. into SCID/SCID mice. The total amount of B lymphocytes was the same in all conditions and amounted to ± 10 x 10E6 cells. The total amount of CD4+ T lymphocytes was the same in all conditions and amounted to ± 4 x 10E6 cells. SCID/SCID mice received injections with 500 µg of MR1 ({blacksquare}) or polyclonal hamster IgG ({square}). The IgM Ab response to caps-PS serotype 3, 4, 14, and 19F was measured 14 days after vaccination with Pneumovax. The results shown are means ± 1 SD of four independent experiments (representing mouse to mouse variation (n = 4)).

 
To demonstrate that CD40L has to be occupied to enhance the anti-caps-PS Ab response, we transferred CD40L+ T cells into CD40 knockout mice. CD40L+ T cells failed to induce an IgM (Fig. 4) and IgG (data not shown) anti-caps-PS immune response (serotypes 3, 4, 14, 19F) into CD40 knockout mice, suggesting that CD40L has to interact with CD40 to activate the anti-caps-PS immune response.



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  		FIGURE 4. Effect of wild-type CD4+ T cells on the anti-caps-PS immune response in CD40-deficient mice. CD40 knockout mice ({square}) and CD40 knockout mice transferred with wild-type CD4+ T cells ({blacksquare}) were vaccinated with Pneumovax. The immune response to caps-PS serotypes 3, 4, 14, and 19F was measured after 2 wk. The results show the IgM Ab response. The CD4+ cell fraction was obtained from wild-type murine spleen cells after depletion of B lymphocytes and CD8+ T lymphocytes. Depletion of B and CD8+ T lymphocytes resulted in a cell population consisting of >80% CD4+ T lymphocytes and <2.5% B lymphocytes and CD8+ lymphocytes. The cells were suspended in 0.9% NaCl and injected i.p. into the CD40 knockout mice. The total amount of CD4+ T lymphocytes injected amounted to ± 6–8 x 10E6 cells. The results shown are means ± 1 SD of four independent experiments (representing mouse to mouse variation (n = 4)).

 
Lack of CD40 expression on endothelium does not play a role in the anti-caps-PS Ab response

In the above-described experiments, we showed that CD4+ T lymphocytes indirectly stimulate the anti-caps-PS Ab response via the CD40-CD40L interaction. This indicates that CD40L on T lymphocytes activates CD40 on non-B cells.

Next, we investigated whether the required CD40 is on hemopoietic cells or on nonhemopoietic cells, such as endothelium. Therefore, the anti-caps-PS Ab response was assessed in CD40 knockout mice that had been lethally irradiated and reconstituted with bone marrow from wild-type mice. Engraftment of bone marrow was controlled by measurement of the CD40 expression on CD19+ cells. In the transplanted mice, CD40 expression on CD19+ cells was 78, 69, 67, and 74%. In wild-type mice, CD40 expression on CD19+ cells was 67.5, 62, 61, and 63%. Previous studies using the same transplantation protocol as in this study have shown that total hemopoietic engraftment and APC repopulation was achieved (13, 14). Thus, transplanted mice have normal CD40 expression on hemopoietic cells but no CD40 expression on nonhemopoietic cells, such as endothelium. If CD40 expression on endothelium played a role in the anti-caps-PS Ab response, then the Ab response in CD40 knockout mice that received a bone marrow transplantation would be smaller than the Ab response in wild-type mice. Our results are shown in Fig. 5. There was no difference in the Ab response against all serotypes measured between the CD40 knockout mice that received a bone marrow transplantation and wild-type mice. This indicated that CD40 expression on endothelium did not play a role in the anti-caps-PS Ab response and that CD40 was required was on hemopoietic cells. The IgM immune response in CD40 knockout mice was lower than the immune response in wild-type mice. The OD values (mean ± SD) for wild-type mice and CD40 knockout mice at serum dilution of 1/400 were, respectively, as follows: 1.85 ± 0.59 (n = 6) and 0.93 ± 0.33 (n = 6) (p = 0.008, Kruskal-Wallis) for serotype 3; 0.48 ± 0.38 (n = 6) and 0.28 ± 0.16 (n = 6) (p = 0.22, Kruskal-Wallis) for serotype 4; 0.32 ± 0.28 (n = 6) and 0.15 ± 0.03 (n = 6) (p = 0.0006, Kruskal-Wallis) for serotype 14; and 0.20 ± 0.09 (n = 6) and 0.13 ± 0.04 (n = 6) (p = 0.0026, Kruskal-Wallis) for serotype 19F.



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  		FIGURE 5. Anti-caps-PS Ab response in lethally irradiated CD40 knockout mice that received wild-type bone marrow is similar to the anti-caps-PS immune response in wild-type mice. CD40 knockout mice (n = 4; {triangleup}) were lethally irradiated and reconstituted with bone marrow from wild-type mice. After 12 wk, mice received injections with Pneumovax and 2 wk thereafter, the IgM anti-caps-PS Ab response to several serotypes were measured and compared with the immune response in wild-type mice (n = 4; {blacksquare}). Results shown are mean ± 1 SD (representing mouse to mouse variation (n = 4)).

 
The role of the CD40-CD40L interaction in the immune response to TNP-Ficoll

Administration of a blocking anti-CD40L Ab (MR1; 500 µg) exerted no effect on the anti-TNP-Ficoll Ab response. The Ab response amounted to (mean ± SD) 399 ± 87 MFI (n = 4) and 421 ± 175 MFI (n = 4) in the absence and presence of MR1, respectively. In a separate experiment, we found that the Ab response to TNP-Ficoll was similar in wild-type mice (677 ± 41 MFI) (n = 8) and CD40 knockout mice (665 ± 100 MFI) (n = 9).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
S. pneumoniae is an organism that frequently causes serious infections (1). It has already been known for a long time that protection against infections with S. pneumoniae is mediated by Abs to the caps-PS. Vaccination with pneumococcal caps-PS is widely used to protect people against infection with S. pneumoniae (15). Besides, vaccination with caps-PS is also used to identify individuals with a selective immune defect in the immune response to polysaccharide Ags (16). Such individuals have an increased risk for infections with encapsulated bacteria.

The regulation of the Ab response to polysaccharide Ags is poorly understood. In the present paper, we demonstrate that the CD40-CD40L interaction plays a key role in the regulation of the IgM Ab response to soluble pneumococcal polysaccharides. Through adoptive transfer of B lymphocytes from wild-type mice with CD4+ T lymphocytes from either CD40L knockout mice or wild-type mice, we showed that CD4+ T lymphocytes stimulated the IgM anti-caps-PS Ab response in a CD40L-dependent manner. This excluded the possibility that the CD40L engaged in the anti-caps-PS immune reaction was from a non-T cell type, such as eosinophils, basophils, platelets, or dendritic cells. Through adoptive transfer of B lymphocytes from CD40 knockout mice with or without wild-type CD4+ T lymphocytes, and with or without MR1, a blocking anti-CD40L mAb, we showed that CD4+ T lymphocytes stimulated the anti-caps-PS immune response via an intermediate cell type. Wild-type CD4+ T lymphocytes did not enhance the anti-caps-PS immune response in CD40-deficient mice. This indicated that CD40L has to interact with CD40 to activate the anti-caps-PS immune response and excluded the possibility that there might be a second receptor for CD40L on B cells. Finally, through reconstitution of lethally irradiated CD40 knockout mice with bone marrow from wild-type mice, we established that the required CD40 was on hemopoietic cells. Taken together, our data indicate that the IgM anti-caps-PS Ab response is dependent on the interaction between CD40L on T lymphocytes and CD40 on non-B cell APCs. Such interaction could induce the release of multiple soluble factors (e.g., cytokines) that can further stimulate B lymphocytes.

Transfer experiments in which mice lymphocytes were transplanted to SCID/SCID mice resulted in very weak Ig Ab responses and did not allow us to reproducibly study the IgG immune response to caps-PS. Therefore, even though CD40 on B cells is not required for the IgM response, it is possible that a direct interaction between T cell CD40L and B cell CD40 might play a role in isotype switching.

The findings described in this report and in our earlier paper (9) are the first to implicate a role for endogenous CD40-CD40L interactions in the Ab response to purified polysaccharides. Many previous reports have stated that TI-2 responses are independent of endogenous CD40-CD40L interactions (5, 6, 7, 8). These studies typically used TNP-Ficoll and DNP-Ficoll, except for the report of Hwang et al. (8), who studied capsular polysaccharide serotype 6. It may be that the CD40-CD40L interaction is important for certain TI-2 Ags but not for others. For example, our data indicate that the CD40-CD40L interaction is not implicated in the immune response to TNP-Ficoll. We confirmed the observation reported by Foy et al. (5) that administration of a blocking anti-CD40L Ab (MR1) did not affect the anti-TNP-Ficoll Ab response. This is consistent with the finding of Xu et al. (17) that the Ab response to TNP-Ficoll was not reduced in CD40L knockout mice, but inconsistent with the observation of Van den Eertwegh et al. (18) that TNP-Ficoll induced Th cells to express CD40L. Finally, we confirmed the observation by Castigli et al. (19) that the Ab response in CD40 knockout mice was comparable to the response in wild-type mice.

The Ab response to polysaccharide Ags in general and pneumococcal caps-PS in particular is classically believed to be "independent" of T cells and the CD40-CD40L interaction (11, 15). This is in contrast to the Ab response to protein Ags, which is dependent on T lymphocytes and the CD40-CD40L interaction.

Although T lymphocytes are not required for the Ab response to caps-PS, they have regulatory effects. Caps-PS are therefore classified as TI type 2 Ags. The regulatory properties of T lymphocytes have been described by the extensive work from Baker and his group and others (20, 21, 22) in the 70 and 80s. They found that the Ab response to purified pneumococcal polysaccharide type III (SSS-III) in athymic nu/nu mice was comparable to the Ab response in conventional thymus-bearing mice. Moreover, they reported that treatment with a T lymphocyte-depleting agent resulted in a 20- to 40-fold increase in the Ab magnitude in thymus-bearing mice but not in athymic nu/nu mice. Based on these experiments and additional experiments, Baker et al. (20) proposed that the Ab response to SSS-III is controlled by two types of regulatory T lymphocytes: suppressor T lymphocytes (Ts) and amplifier T lymphocytes (Ta). They stated that "usually the effects produced by Ts and Ta are counterbalanced: this is why the Ab response to SSS-III is similar in magnitude in thymus-bearing and athymic mice and why Ts and Ta have escaped detection for so many years." The existence of stimulatory and inhibitory T cells has been confirmed by other groups (23, 24) and, more recently, we further substantiated that CD4+ T lymphocytes exert stimulatory, and CD8+ T lymphocytes exert inhibitory, effects, and that these effects depended on the CD40L-CD40 interaction (9, 10). In the present manuscript, we demonstrate that CD4+ T cells expressing CD40L help the IgM Ab response to soluble pneumococcal caps-PS via an intermediate cell type.

In adoptive transfer experiments, we found that murine B cells produced significantly less Abs to soluble caps-PS in the absence of CD4+ T lymphocytes than in the presence of CD4+ T lymphocytes. Similar findings were reported by Bondada et al. (25) who found that purified B cells were unable to produce Abs against TNP-Ficoll, a TI-2 Ag. This is at odds with previously published data that indicate that the Ab response in athymic nude mice is essentially unchanged relative to euthymic mice (20, 21), indicating that the Ab response to caps-PS can occur independent of T lymphocytes. Although T lymphocytes are not required for an Ab response to caps-PS in nude mice, T lymphocytes can modulate (positively and negatively) the anti-caps-PS immune response. This issue is currently under further investigation.

Our data strongly support the idea that APCs expressing CD40 interacting with T cells expressing CD40L play a role in the anti-caps-PS Ab response. Only scarce data is available on the role of APCs in the immune response to caps-PS. Colino et al. (26) reported that bone marrow-derived dendritic cells pulsed with intact S. pneumoniae could stimulate the anti-caps-PS Ab response when injected into naive mice and that endogenous T lymphocytes were critical for development of IgG Abs specific for both protein and polysaccharide Ags. Bone marrow dendritic cells obtained from mice genetically deficient in CD40 were defective in inducing polysaccharide-specific IgG1 (26). These studies were performed with intact S. pneumoniae in which proteins might modulate and influence the anti-caps-PS immune response. Much less information is available on the role of APCs in the immune response to soluble TI-2 Ags. Garcia de Vinuesa et al. (27) found that administration to mice of agonistic CD40 mAbs together with TNP-Ficoll, a TI-2 Ag, not only enhanced the Ab response, but also markedly increased the amount of APCs in the spleen. It was hypothesized that CD40 mAbs activate APCs, which then would activate T lymphocytes through cytokine secretion (27). This study, however, did not address the endogenous role of the role of CD40-CD40L interaction in the anti-caps-PS immune response. Garg et al. (28) showed that, in contrast to in vitro culture of spleen cells, in vitro culture of lymph node cells did not respond to caps-PS, and that addition of APCs isolated from spleen cells enabled the lymph node to respond to caps-PS. It was further put forward that defects in APC function might play a critical role in the failure of neonates to respond to caps-PS (29).

In conclusion, we provided evidence that T lymphocytes expressing CD40L modulate the immune response to soluble caps-PS by interacting with non-B cell APCs expressing CD40.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 X.B. is a senior clinical investigator and A.D.B. a post-doctoral fellow of the Fund for Scientific Research, Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO)-Vlaanderen). This work was supported by grants from the K.U. Leuven (Onderzoeksfonds) and the Fund for Scientific Research, Flanders (FWO-Vlaanderen). Back

2 Address correspondence and reprint requests to Dr. Xavier Bossuyt at the current address: Laboratory Medicine/Immunology, University Hospital Gasthuisberg, CDG 7th Floor, Herestraat 49, 3000 Leuven, Belgium. E-mail address: Xavier.bossuyt{at}uz.kuleuven.ac.be Back

3 Abbreviations used in this paper: caps-PS, capsular polysaccharide; TI, T independent; TNP, trinitrophenyl; MFI, mean fluorescence intensity; Ts, suppressor T lymphocyte; Ta, amplifier T lymphocyte. Back

Received for publication October 29, 2004. Accepted for publication October 24, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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