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The Journal of Immunology, 2003, 170: 781-787.
Copyright © 2003 by The American Association of Immunologists

Differential Requirements for Expression of CD80/86 and CD40 on B Cells for T-Dependent Antibody Responses In Vivo

Joanne M. Lumsden1,2,*, Joy A. Williams1,* and Richard J. Hodes*,{dagger}

* Experimental Immunology Branch, National Cancer Institute, and {dagger} National Institute on Aging, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulation of the immune response is marked by complex interactions among the cells that present or recognize Ags. These interactions are mediated in large part by receptor-ligand interactions involving molecules expressed on the surface of participating cells (1, 2). The activation of T cells for induction of proliferation and effector functions requires both signaling through the Ag-specific TCR and additional signals provided by costimulatory receptors on the T cell (3, 4). Similarly, the activation of B cells to proliferate and differentiate requires both Ag-specific signaling and additional costimulatory signals (5). Therefore, interactions between T cells and B cells involve a potentially complex network of signals mediated by communication through multiple receptors expressed by both populations of cells. One aspect of this complexity that is not well understood is the directionality, and possible bidirectionality, of signaling through specific "receptor-ligand" interactions. This question is particularly relevant in the case of two highly important sets of molecular interactions that can function in T cell-B cell communication: the interaction between CD28 on T cells and CD80 (B7.1) and CD86 (B7.2) on B cells, and that between CD40 ligand (CD40L3; CD154) on T cells and CD40 on B cells. Both of these interactions are essential for T-dependent (TD) B cell responses that generate high-affinity, class-switched Ab in vivo (6, 7, 8, 9, 10, 11, 12, 13), but the precise requirement for each interaction in signaling to T cells and/or B cells has not been established under physiologic in vivo conditions.

The essential role of CD28-CD80/86 interaction is demonstrated by the fact that mice lacking either in CD28 or in both CD80/86 family members are profoundly deficient in the ability to generate in vivo germinal center formation, Ig class switching, memory formation, and affinity maturation through somatic hypermutation (6, 8, 13). It has not been established, however, whether these defects reflect a requirement for CD28 signaling of Th cells and/or for CD80/86 signaling of B cells. The ability of CD28 to mediate signaling of T cells has been extensively demonstrated in a number of experimental systems, but evidence has also been reported for signaling of B cells through CD80/86 (14, 15, 16, 17). Thus, a role for CD80/86 signaling in costimulus-dependent activation of B cells is plausible but has not been directly assessed.

CD40-CD40L interaction is also essential for in vivo TD class-switched Ab responses, and mice lacking in either of these molecules manifest profound deficiencies similar to those observed in CD80/86- or CD28-deficient mice (9, 11, 12). Extensive evidence has been assembled for the ability of CD40 to transduce signals in B cells and a variety of other cell types (18, 19). Nevertheless, CD40-deficient B cells can produce class-switched Ab in response to certain T-independent Ag (20), demonstrating that induction of the class-switch machinery can occur without CD40 signaling. In addition, data indicating that CD40L can signal directly to T cells has been reported (21, 22, 23). Thus, it is again unclear whether signaling of T cells and/or of B cells explains the requirement for CD40-CD40L participation in TD B cell immune responses.

The experiments described here address the nature of the requirements for CD80/86- and CD40-dependent costimulation in TD IgG and IgE responses to in vivo antigenic challenge. The results demonstrate that the need for these two molecules is different. In a mixed bone marrow chimera environment only B cells that express CD40 are capable of responding, indicating a strict requirement for CD40 expression and potentially for CD40 signaling in in vivo TD B cell activation. In marked contrast, both wild-type (WT) B cells and CD80/86-deficient B cells respond efficiently in chimeric animals, indicating that although CD80/86 plays a critical role in these responses, direct CD80/86 signaling of a B cell is not essential for its efficient activation.


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

C57BL/6 (B6) mice were obtained from the Frederick Cancer Research and Development Center (Frederick, MD) and maintained at Bioqual (Rockville, MD). Congenic C57BL/6J-IghaThy1aGpi1a (B6.Igha) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained at the Frederick Cancer Research and Development Center. CD40-knockout (KO) mice on a B6 genetic background were obtained from The Jackson Laboratory and maintained at Bioqual. B6 mice deficient in both CD80 and CD86 (CD80/86 KO) were a generous gift from A. Sharpe (Brigham & Women’s Hospital, Boston, MA) (13).

Purification and in vitro activation of B cells

Enriched populations of splenic B cells were obtained using magnetic CD19 beads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Cells (2 x 105) were cultured in 24-well flat-bottom plates in 1 ml of complete medium consisting of RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Biofluids, Rockville, MD), sodium pyruvate (1%), nonessential amino acids (1%), L-glutamine (0.5%), 2-ME (5 x 10-5 M), penicillin (100 U/ml), and streptomycin (100 µg/ml) and incubated at 37°C in a humidified atmosphere containing 5% CO2. To induce specific isotype switching, B cells were stimulated either with 15 µg/ml LPS (Difco, Detroit, MI) and 1000 U/ml recombinant mouse IL-4 (prepared from a baculovirus expression system in our laboratory) or with 2.15 µg/ml mouse membrane CD40L (kindly provided by Dr. M. Kehry, IDEC Pharmaceuticals, San Diego, CA) and 1000 U/ml IL-4. Supernatants were harvested on day 6 for quantitation of Ig by ELISA.

Flow cytometric analysis

Single-cell suspensions were prepared from peripheral blood and spleen, and erythrocytes removed by treatment with ACK lysing buffer (Biofluids). Anti-FcR mAb 24G2 was added to prevent Fc receptor-mediated binding of mAb to cells. Cells were then incubated with FITC-labeled anti-IgMb, PE-labeled anti-B220, biotinylated anti-IgMa, and Cy5 conjugate (BD PharMingen, San Diego, CA) sequentially. Viable cells were analyzed by FACScan (BD Biosciences, San Jose, CA) using CellQuest software.

In vivo immunization

Mice were immunized i.p. with 100 µg trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) in Imject alum (Pierce, Rockford, IL), and serum was collected on day 0 before immunization and days 7, 14, and 21 after immunization. Mice were boosted in the same manner on day 27 or 28, and serum was collected on days 4, 8, and 12 after rechallenge.

Ig detection by ELISA

Total IgG1, IgM, or IgE was captured with purified goat anti-mouse IgG1, IgM, or IgE (Southern Biotechnology Associates, Birmingham, AL) and detected with HRP-conjugated goat anti-mouse {gamma}1, µ (Southern Biotechnology Associates) or with biotinylated rat anti-mouse IgE (BD PharMingen) and HRP-conjugated streptavidin (Southern Biotechnology Associates). To measure Ag-specific IgG1 or IgM, plates were coated with 2.5 µg per well trinitrophenyl-keyhole limpet hemocyanin-OVA, and Ig was detected with HRP-conjugated goat anti-mouse {gamma}1 or µ. Allotype-specific IgG1 and IgM were measured using biotinylated anti-mouse IgG1a, IgG1b, IgMa, or IgMb (BD PharMingen) and HRP-conjugated streptavidin. Allotype-specific IgE was captured with purified rat anti-mouse IgEa or IgEb and detected with biotinylated rat anti-mouse IgE (BD PharMingen) and HRP-conjugated streptavidin. Reagent specificity was confirmed by analysis of serum from immunized B6.Igha and B6.Ighb mice. In all cases, wells were developed with ABTS Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and OD was measured at 405 nm. Titers were determined by interpolation of the dilution that gave a 50% OD of the maximum absorbance achieved.

Chimeras

Radiation bone marrow chimeras were prepared as described previously (24). B6.Igha recipient mice were lethally irradiated with 1000 rad and reconstituted with 107 T cell-depleted bone marrow cells. CD80/86 WT/CD80/86 KO mixed chimeras were generated by combining equal numbers of bone marrow cells from CD80/86 WT (B6.Igha) and CD80/86 KO (Ighb) mice. CD40 WT/CD40 KO mixed chimeras received bone marrow cells from CD40 WT (B6.Igha) and CD40 KO (Ighb) mice. Control chimeras were generated by reconstituting lethally irradiated hosts with equal numbers of B6.Igha and B6.Ighb bone marrow cells. Six to 10 wk after reconstitution PBLs were stained with mAb specific for B220, IgMa, and IgMb, and the percentages of B220+IgMa+ and B220+IgMb+ cells were determined. Following completion of the immunization protocol, chimerism was reassessed by staining for B220+IgMa+ and B220+IgMb+ cells in the spleen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impaired IgG1 and IgE production in CD40- and CD80/86-deficient mice

A lack of CD80/86 or CD40 has been shown to profoundly reduce the Ab response to TD Ags in vivo, impairing germinal center formation, Ig class switching, memory formation, and affinity maturation through Ig hypermutation (9, 13). The need for CD80/86 and for CD40 was assessed here by immunizing WT, CD80/86 KO, and CD40 KO mice with the TD Ag TNP-KLH in alum adjuvant; boosting with the same Ag adjuvant at day 27 after the primary immunization; and measuring Ab levels at successive time points as indicated (Fig. 1). WT mice generated a robust Ag-specific primary response and a faster, amplified secondary response. There was no difference in Ag-specific IgM titers between CD80/86 or CD40 KO and WT animals, consistent with previous demonstrations that an unswitched IgM response can be generated in the absence of costimulatory molecules (8, 9, 11, 12, 13). In contrast, the Ag-specific IgG1 response was completely nullified in both CD80/86 KO and CD40 KO mice, even after a secondary immunization. Because it was not possible to measure Ag-specific IgE in response to immunization (data not shown), we measured total IgE serum levels. Levels of total IgE were low or undetectable in unimmunized mice but increased significantly after immunization of WT mice (Fig. 1). But unlike the controls, CD80/86 and CD40 KO mice were unable to generate an IgE response. These results are consistent with the conclusions of previous reports (6, 7, 8, 9, 10, 11, 12, 13) and demonstrate the need for both the CD40-CD40L and CD80/86-CD28 pathways in generating Ig class-switched primary and secondary Ab responses to TD Ags.



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  		FIGURE 1. Impaired IgG1 and IgE production in CD80/86- and CD40-deficient mice. WT, CD80/86 KO, and CD40 KO mice (4–5 per group) were immunized with TNP-KLH/alum on days 0 and 27 (as shown by arrows), and serum was collected at the indicated time points. Serum levels of Ag-specific IgG1 and IgM as well as total IgE were assayed by ELISA. Data points represent the geometric mean ± geometric SEM of the ELISA titers.

 
CD80/86- and CD40-deficient B cells do not have an intrinsic inability to make IgG1 and IgE

It was important to test the possibility that B cells maturing in the absence of CD80/86 or CD40 may have an intrinsic inability to make IgG1 or IgE. CD19+ splenic B cells were purified from WT, CD80/86 KO, and CD40 KO mice and cultured in vitro with the polyclonal activators LPS or mCD40L, plus IL-4 to facilitate class switching. Levels of IgG1, IgE, and IgM were measured in day 6 culture supernatants. There was no difference between CD80/86 KO and WT B cell responses to LPS (Fig. 2) or mCD40L (data not shown). As expected, CD40 KO cells did not respond to mCD40L (data not shown); however, responses to LPS were equivalent to those of WT B cells (Fig. 2). Thus, the observed in vivo deficiencies in the responses of CD80/86 KO and CD40 KO mice are not due to an inherent inability of their B cells to undergo activation and class switching to IgG1 and IgE as measured in vitro.



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  		FIGURE 2. Normal Ig production in CD80/86- and CD40-deficient B cells activated by LPS in vitro. CD19+ B cells were isolated from the spleens of CD80/86- and CD40-deficient mice and cultured with LPS and IL-4. Results are displayed as OD units from ELISAs of serially diluted supernatants after 6 days of culture and are representative of three independent experiments.

 
CD80/86 KO, but not CD40 KO, B cells participate in humoral immune responses in the presence of WT bone marrow-derived cells

Although previous studies have demonstrated that CD40-CD40L and CD80/86-CD28 costimulatory pathways are required for in vivo TD B cell responses, the mechanism mediating this requirement is not well established. To determine whether CD80/86 and CD40 are involved in the delivery of T cell signals directly to B cells, we developed a radiation bone marrow chimera strategy. The objective was to create a situation in which both WT and KO B cells matured and differentiated in the same mouse. CD80/86 WT/CD80/86 KO and CD40 WT/CD40 KO chimeric mice were generated in which WT (Igha)- and KO (Ighb)-derived B cells and Ig could be distinguished by allotype-specific reagents. Control chimeras were also generated in which irradiated WT mice were reconstituted with equal numbers of bone marrow cells from congenic WT Igha and WT Ighb mice. Mice were then immunized with TNP-KLH/alum, and Ab titers were measured at various time points using allotype-specific assays. The responses of control chimeras were analyzed first to determine whether Igha and Ighb production by WT cells could be detected in a chimeric environment (Fig. 3). While IgEa and IgEb titers were equivalent, in the TNP-specific IgG1 and IgM responses the Igha allotype titers consistently exceeded those of the Ighb allotype. This difference could reflect differential sensitivities of the allotype-specific assays used or a true difference in efficiency of response.



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  		FIGURE 3. Detection of allotype-specific IgG1, IgE, and IgM in mixed WT chimeras. Mixed chimeras were generated by reconstituting lethally irradiated B6.Igha host mice with equal numbers of bone marrow cells from congenic B6.Igha and B6.Ighb mice. Chimeric mice (n = 8) were immunized with TNP-KLH/alum on days 0 and 27 (as shown by arrows), and serum was collected at the indicated time points. Allotype-specific IgG1, IgE, and IgM were assayed by ELISA. Data points represent the geometric mean ± geometric SEM of the ELISA titers.

 
Similar analyses were conducted for CD80/86 WT/CD80/86 KO chimeras. IgMa and IgMb responses were detected in these chimeras, and IgMb titers were lower than IgMa titers, as was observed in control chimeras (Fig. 4). While the difference between IgMa and IgMb titers appeared to be greater in CD80/86 WT/CD80/86 KO chimeras than in control animals, variation among mice was large, and it is not clear whether this difference is significant. Interestingly, IgG1a and IgG1b responses as well as IgEa and IgEb responses were detected, indicating that both WT and CD80/86 KO B cells were capable of IgG1 and IgE responses in the chimeric environment. A difference in IgG1a and IgG1b titers was detected but was similar to that observed in control chimeras, suggesting that this may not reflect the absence of CD80/86.



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  		FIGURE 4. CD80/86-deficient B cells can class-switch in CD80/86 WT/CD80/86 KO mixed chimeras. Mixed chimeras were generated by reconstituting lethally irradiated B6.Igha host mice with equal numbers of bone marrow cells from congenic B6.Igha and CD80/86 KO (Ighb) mice. Chimeric mice (n = 7) were immunized with TNP-KLH/alum on days 0 and 28 (as shown by arrows), and serum was collected at the indicated time points. Allotype-specific IgG1, IgE, and IgM were assayed by ELISA. Data points represent the geometric mean ± geometric SEM of the ELISA titers.

 
Analysis of the CD40 WT/CD40 KO chimeras demonstrated that CD40 KO cells could respond to Ag challenge by producing IgM (Fig. 5). Engraftment of CD40 KO B cells was further evidenced by detection of IgMb+ cells in peripheral blood lymphocytes and splenocytes (Table I). The level of CD40 KO IgMb was lower than that of CD40 WT IgMa, a situation similar to that observed previously in CD80/86 WT/CD80/86 KO chimeras. However, in striking contrast to the earlier experiments, analysis of the CD40 WT/CD40 KO chimeras found only IgG1a and IgEa Ab (CD40 WT), and no IgG1b or IgEb (CD40 KO) responses. This was observed for both a primary and a secondary (memory) Ab response, in which the peak IgG1a titer was at least 103-fold higher than the undetectable IgG1b response. Thus, CD40 KO cells were unable to secrete IgG1 and IgE, even in a chimeric environment in which WT B cells were generating efficient responses. These data indicate that, in contrast to CD80/86-deficient B cells, CD40 deficiency on B cells cannot be overcome by supplying functional Th cells and other components of a competent response environment.



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  		FIGURE 5. CD40-deficient B cells cannot class-switch in CD40 WT/CD40 KO mixed chimeras. Mixed chimeras were generated by reconstituting lethally irradiated B6.Igha host mice with equal numbers of bone marrow cells from congenic B6.Igha and CD40 KO (Ighb) mice. Chimeric mice (n = 11) were immunized with TNP-KLH/alum on days 0 and 28 (as shown by arrows), and serum was collected at the indicated time points. Allotype-specific IgG1, IgE, and IgM were assayed by ELISA. Data points represent the geometric mean ± geometric SEM of the ELISA titers.

 

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  		Table I. Detection of IgMa+ and IgMb+ B cells in mixed chimeras

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies were designed to define the roles of CD80/86 and CD40 in B cell activation. Our goal was to establish whether cell-autonomous expression of CD80/86 and CD40 on B cells is required for B cell activation and Ig secretion in the context of a TD humoral immune response. Previous studies, as well as the experiments described in this study, demonstrated that CD80/86-CD28 and CD40-CD40L interactions are needed during TD B cell responses (6, 7, 8, 9, 10, 11, 12, 25, 26). However, it has been difficult in vivo to dissociate defects in T cell help from defects in B cell activation when either the CD80/86-CD28 or CD40-CD40L pathways have been disrupted. To address this issue, we created mixed chimeras in which CD80/86- or CD40-deficient B cells coexist with WT cells, thereby restoring an environment that is fully competent to support T cell- and costimulus-dependent B cell responses to Ag. Allotype-specific reagents were then used to determine whether CD80/86- or CD40-deficient bone marrow-derived cells were able to secrete levels of Ag-specific Ig comparable to the WT B cells in the same chimeras. Our results clearly demonstrated that in CD80/86 WT/CD80/86 KO chimeras, CD80/86 WT and CD80/86-deficient B cells respond comparably to a TD antigenic challenge. In contrast, in CD40 WT/CD40 KO chimeras only B cells expressing CD40 are capable of TD IgE and IgG1 responses (Fig. 6).



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  		FIGURE 6. Potential cell interactions regulating isotype switching in CD80/86 WT/CD80/86 KO and CD40 WT/CD40 KO chimeras. A, The presence of CD80/86 on WT cells restores an environment fully competent to induce class switching in CD80/86 KO cells. Thus, cognate interaction between CD80/86 on B cells and CD28 on Th cells is not required for class switching. B, The presence of CD40 on WT cells cannot restore the class-switching defect in CD40 KO cells. CD40 expression on responding B cells is essential for TD immune responses.

 
Our findings in the CD80/86 WT/CD80/86 KO mixed chimera indicated that direct CD80/86 signaling is not essential for B cell activation. Recent studies, however, have suggested that CD80/86 signaling can occur in B cells and can enhance the B cell immune response. Ligation of CD86 on human tonsillar cells (15) or on mouse TNP-specific B cells (16) has been shown to result in a modest increase in IgE and IgG4/IgG1 production, while cross-linking of CD86 on LPS-stimulated mouse B cells enhanced proliferation and production of IgG1 and IgG2a (17). Furthermore, cross-linking of CD80 on human B cell lines can induce protein phosphorylation, growth arrest, and morphological changes (14). While our findings do not preclude the possibility that direct signaling to B cells via CD80/86 may play a role under certain conditions, the CD80/86 WT/CD80/86 KO mixed chimera studies reported in this study demonstrate that CD80/86 signaling in B cells is not required for Ab production, Ig class switching, or memory B cell responses in a model of physiologic in vivo response to antigenic challenge.

CD40 is known to signal directly to cells on which it is expressed, and much about the mechanisms coupling CD40 to intracellular signaling pathways has been described (19, 27). Transgenic mice that express mutants of CD40 unable to associate with TNFR-associated factors, the adaptor molecules that couple CD40 to downstream kinases, are defective in Ab responses to TD Ags, illustrating the importance of CD40 signaling in these responses (28, 29). However, the requirement for CD40 expression by B cells participating in TD responses has not previously been characterized. The studies reported in this paper were designed to determine whether expression of CD40 by a B cell is essential for the ability of that B cell to respond in vivo, or whether CD40-dependent interactions in the immune environment can function in trans to support responses of CD40 KO B cells. Our CD40 WT/CD40 KO mixed chimeras allowed us to address this issue and demonstrated that CD40 must be expressed on B cells responding to TD antigenic challenge; expression in trans cannot rescue the response of CD40 KO B cells.

The need for cell-autonomous expression of CD40 on B cells responding to TD Ags most likely reflects a requirement for direct CD40 signaling to the B cell. CD40 signaling in B cells promotes a number of downstream effects, including up-regulation of cell surface molecules such as ICAM-1 (30), CD80/86 (31, 32, 33), and MHC class II (34); survival in germinal centers; and isotype switching. Any or all of these downstream effects of CD40 signaling may explain the necessity for cell-autonomous expression of CD40. But we cannot exclude the possibility that cell-autonomous expression of CD40 may play a role in increasing the affinity of cognate interactions with CD40L-expressing T cells, a role that would not necessarily require CD40 signaling.

Our finding that CD40 expression on B cells is necessary for B cell responses runs counter to a recent study (35) in which use of an agonistic Ab to CD28 was able to restore a Th2-dependent B cell response to an adenovirus vector in CD40L KO mice. The authors of this study concluded that direct CD40 signaling to B cells during a Th2-TD response is not required if Th cell function is activated. It is possible that the cross-linking of CD28 with agonistic anti-CD28 Ab may have distinctly different effects than ligation of CD28 by its natural ligand, CD80/86, in the course of physiologic cell interactions in vivo.

Although mice deficient in either the CD80/86-CD28 or CD40-CD40L costimulatory pathways display similar defects in response to challenge with TD Ags, we conclude that these pathways play very different roles in TD immune responses. Expression of CD80/86 molecules on responding B cells is not required, suggesting that the primary role of CD80/86 during TD responses is to facilitate events, such as the activation of T cells, that are necessary to drive the humoral response. In contrast, cell-autonomous expression of CD40 is required for B cell activation, indicating that ligation of CD40 on B cells elicits signals that are essential to the production of class-switched Ab in response to challenge with TD Ags and that cannot be provided by CD40 expressed on other cells in the immune environment.


   Acknowledgments
 
We thank Ron Schwartz, Jon Ashwell, Peter Lipsky, Karen Hathcock, and Melanie Vacchio for valuable comments and critical reading of this manuscript; Mark Hockenberry for excellent technical assistance; Genevieve Sanchez-Howard and staff at Bioqual for expert animal care and husbandry; and Arlene Sharpe for generously providing CD80/86 KO mice.


   Footnotes
 
1 J.M.L. and J.A.W. contributed equally to this work. Back

2 Address correspondence and reprint requests to Dr. Joanne M. Lumsden, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 4B10, MSC 1360, 10 Center Drive, Bethesda, MD; 20892-1360. E-mail address: lumsdenj{at}mail.nih.gov Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; TD, T-dependent; WT, wild type; KO, knockout; TNP-KLH, trinitrophenyl-keyhole limpet hemocyanin. Back

Received for publication August 16, 2002. Accepted for publication November 13, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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