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The Journal of Immunology, 2006, 177: 5414-5419.
Copyright © 2006 by The American Association of Immunologists, Inc.

Induced Expression of Murine {gamma}2a by CD40 Ligation Independently of IFN-{gamma}1

John T. Collins*, Jian Shi*, Bryna E. Burrell{dagger},{ddagger}, D. Keith Bishop*,{dagger} and Wesley A. Dunnick2,*

* Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; {dagger} Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109; and {ddagger} Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IgG2a, with {gamma}2a H chains, is important for protection against viruses and other intracellular pathogens. Although a large portion of IgG2a expression is dependent upon IFN-{gamma}, some germline transcription and switch recombination to the murine {gamma}2a H chain gene expression are independent of IFN-{gamma}. We found that agonistic anti-CD40 Abs injected into IFN-{gamma}-deficient mice induce a >200-fold increase in the amount of serum Ig2a, while other Ig isotypes are increased by 16-fold or less. In vitro, ligation of CD40 on B cells, without the addition of other B cell activators or cytokines, results in germline transcription and switch recombination that are largely restricted to the {gamma}2a gene. These results suggest that some immune responses to infectious agents can result in large amounts of IgG2a expression through ligation of CD40, without the expression of IFN-{gamma} by Th1 or other cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of the optimal class and subclass of Ab is important for immunity against pathogens. In mice, IgG2 Abs are the best complement activators, and are readily bound by Fc{gamma}Rs (1). Thus, IgG2 Abs are optimal for protection against intracellular infections, and, in fact, Ab immunity to viruses is dominated by IgG2a with a significant contribution by IgG2b (2). The regulation of expression of the {gamma}2 H chains found in IgG2 is, to some extent, understood. {gamma}2b H chains are induced in tissue culture by LPS, and are enhanced further by treatment with TGFbeta (3, 4). Expression of the murine {gamma}2a gene in vitro is induced only weakly by LPS, but robustly by LPS + IFN-{gamma} (5, 6). In vivo and in vitro, induction of the {gamma}2a gene by T cell-independent Ags depends on the transcription factor T-bet (7, 8). Although induction of the {gamma}2a H chain genes after B cell activation via a TLR (by LPS or CpG) has been investigated thoroughly (5, 6, 9), the induction of the {gamma}2a in the context of stimulation via T cell help (by CD40 ligation) has been relatively understudied (4). Induction of {gamma}2a to T cell-dependent Ags is independent of T-bet (8), implying that T cell-independent and T cell-dependent switch recombination to {gamma}2a differ. To investigate the effect of T cell help on B cell expression of {gamma}2a H chain genes, we studied the effect of CD40 ligation on B cells, with or without IFN-{gamma}.

Agents other than IFN-{gamma} are likely to induce the expression of {gamma}2a. Although serum IgG2a is reduced in mice deficient in the IFN-{gamma} receptor, these mice continue to express significant amounts of IgG2a (10, 11). One candidate for this alternative agent would be type I IFN; mice deficient in the receptors for both IFN-{gamma} and IFN-{alpha}beta fail to express Ag-specific IgG2a, make tiny amounts of IgG2b, but produce abundant IgG1 (12). A second candidate for this alternative agent would be interaction of B cells with NK cells. B cell:NK cell interaction induces {gamma}2a germline transcripts, independently of IFN-{gamma} (13). Mice that lack NK cells make poor IgG2a responses to protein Ags, even though their T cells produce large amounts of IFN-{gamma} (14). However, B cell:NK cell interaction alone is insufficient for switch recombination to {gamma}2a (13), and so it is likely that at least one additional agent must affect the expression of the {gamma}2a gene. We propose that CD40 ligation is an additional factor in switch recombination to the {gamma}2a gene, in that CD40 ligation alone induces the {gamma}2a gene robustly, independently of IFN-{gamma}.

There are abundant data to indicate that the {gamma}2aa gene and the {gamma}2ab gene are not alleles, but two different genes (15, 16, 17, 18, 19). Hence, Jouvin-Marche and colleagues (15, 19) have suggested that the {gamma}2ab gene be renamed {gamma}2c. As we used mice with both Igha and Ighb loci, we studied both genes. Even though the {gamma}2a and the {gamma}2c genes may not be true alleles, we have found that both genes are apparently regulated by IFN-{gamma} and by CD40 ligation. For simplicity of presentation, in this publication we will refer to both genes as {gamma}2a.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
B cell cultures

C57BL/6, IFN-{gamma}–/– (20), and CD40–/– (21) mice were purchased from The Jackson Laboratory. STAT1–/– mice (22) were purchased from Taconic Farms. After lysis of red cells with ammonium chloride, T-depleted splenocytes were prepared by treatment with anti-Thy-1 (mAb 30H12) and guinea pig complement (23). The surviving cells were washed twice with HBSS/1% BSA and cultured at 60,000 cells/ml for the analysis of Ab secretion (7 days) or at 1.5 million cells/ml for the preparation of RNA (1–3 days) in RPMI 1640 supplemented with 10% FBS, penicillin, streptomycin, glutamine, and 2-ME. LPS from Salmonella typhimirium (Sigma-Aldrich; L7261) was added at 25 µg/ml, murine rIFN-{gamma} (BioSource International) was added at 20 ng/ml, and murine rIL-4 (BioSource International) was added at 35 ng/ml. One Sf21 cell expressing CD40 ligand (CD40L)3 (24) was added for every 10 T-depleted lymphocytes.

Flow cytometry and ELISA for Ab isotype expression

For flow cytometric analysis, live, T cell-depleted splenic lymphocytes from 4-day cultures were purified on Lympholyte gradients. Biotinylated anti-IgG2ab (5 µg/ml; BD Pharmingen) was bound to the cells in PBS with 1% FBS, 15% rabbit serum, and 2.5 µg/ml anti-Fc{gamma}RII (BD Biosciences). Secondary staining used 4 µg/ml FITC avidin (BD Pharmingen). Live cells were also stained with R-PE anti-B220 (60 ng/ml; Southern Biotechnology Associates); virtually all cells were B220+. A FACScan was used for analysis, gating on lymphocytes.

The amount of various Ig isotypes was determined by sandwich ELISA, using coating Abs and alkaline phosphatase-conjugated goat polyclonal Abs from Southern Biotechnology Associates. For culture supernatants or sera from mice with an Ighb locus, anti-IgG2c Abs were used (25); for culture supernatants from mice with an Igha locus, anti-IgG2a Abs were used. Appropriate dilutions were determined by preliminary experiments so that absorbance readings were in the linear part of the dose-response curve, as constructed using dilutions of unmanipulated C57BL/6 serum. Amounts of IgG or IgA isotypes (from the average of three ELISA wells) were normalized to the amount of IgM in the same culture conditions. C57BL/6, CD40–/–, or IFN-{gamma}–/– mice were injected i.p. with 0.5 mg of anti-CD40 rat mAb (FGK45) (26) on days 0 and 1, and bled via the saphenous vein on days 0, 3, 6, 9, 12, and 15. The average amount of a given Ig isotype on days 3–15 is reported relative to the amount of that isotype in day 0 sera of the same mouse.

mRNA analysis

RNA was prepared from cultures (27), and cDNA was prepared from that RNA. Aliquots of cDNA were tested by PCR using primers for hypoxanthine phosphoribosyltransferase, specific I exons (5'-most exons of H chain germline transcripts), and specific CH regions, as reported (28). All PCR cross an intron. For each cDNA, 5 times more cDNA was used to measure I exon-containing transcripts than hypoxanthine phosphoribosyltransferase transcripts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
During a study of the role of CD40:CD40L interactions in transplantation, two of us (B. Burrell and D. Bishop) found that injection of an agonistic anti-CD40 Ab resulted in ~100-fold increases in the amount of serum IgG2a in C57BL/6 mice, but 10-fold increases in the amounts of other isotypes. To document this observation further, and to test the IFN-{gamma} dependence of the IgG2a expression, we injected IFN-{gamma}–/– mice with 0.5 mg of an agonistic anti-CD40 Ab (FGK45) (26) on days 0 and 1, and then monitored the expression of various Ig isotypes over time (Fig. 1A). Anti-CD40 treatment resulted in a 244-fold increase (on average, ranging from 61- to 495-fold) in serum IgG2a 9 days after anti-CD40 injection compared with the levels in unmanipulated serum. The second largest increase was for IgG2b (an average of 16.2-fold on day 9). Other isotypes were increased by 2- to 9-fold compared with serum from unmanipulated mice (Fig. 1A). We used mice deficient in CD40 to demonstrate that almost all of the IgG2c induction observed in IFN-{gamma}–/– mice (Fig. 1A) was dependent on CD40 expression. We injected CD40–/– mice with FGK45, and tested these mice for various Ig isotypes from 0 to 15 days. The induction of IgG2c by FGK45 injection in CD40–/– mice was <12% that of mice that could express CD40 (data not shown). The FGK45-induced expression of other IgG and IgA isotypes in CD40–/– mice (data not shown) was similar to or less than the IgG and IgA expression in IFN-{gamma}–/– mice (Fig. 1A).


Figure 1
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  		FIGURE 1. A, Expression of Ig isotypes after injection of anti-CD40. Anti-CD40 Ab was injected i.p. on days 0 and 1. Isotype expression was determined by sandwich ELISA. Data are presented as the average fold increase of a given isotype compared with the day 0 value for each of three mice. *, The pooled increases in IgG2a expression at days 9 and 12 were statistically greater than the increases for any other isotype; p < 0.035. We performed the same experiment in wild-type C57BL/6 mice. The increase in IgG2a expression was smaller (50- to 112-fold; data not shown); the greater fold increase in IFN-{gamma}–/– mice is due to the reduced level of IgG2a in the serum from unmanipulated IFN-{gamma}–/– mice. The amounts of IgG2a in the three C57BL/6 sera at the peak of the response were 1726, 926, and 759 µg/ml; the amounts of IgG2a in the three IFN-{gamma}–/– sera at the peak of the response were 792, 205, and 88 µg/ml. In C57BL/6 sera from mice injected with anti-CD40, the increases in expression of other isotypes were similar to those of IFN-{gamma}–/– mice. B, IFN-{gamma}–/– B cells were cultured for 7 days with the indicated additions. Culture supernatants were tested for isotype expression by sandwich ELISA. The Ig isotype analyzed is indicated below the data for experiment 1 and above the data for experiment 2. Relative expression from two independent sets of cultures was calculated by dividing the amount of a given Ig isotype by the amount of IgM secreted in the same culture. In experiment 2, CD40L+IL-4 cultures were not tested. C, C57BL/6 T-depleted lymphocytes were cultured with LPS (thin line in left panel), CD40L+IL-4 (thin line in middle panel), CD40L+IFN-{gamma} (thin line in right panel) or CD40L only (bold line in all three panels) for 4 days. Live, small lymphocytes were then analyzed by flow cytometry for surface IgG2a expression.

 
This massive increase in IgG2a expression in IFN-{gamma}–/– mice implies that a large portion of B cells exposed to CD40 ligation switches to {gamma}2a expression, independently of IFN-{gamma}. To explore this phenomenon further, we tested the ability of IFN-{gamma}–/– T-depleted splenocytes to be activated by CD40 ligation in tissue culture. We activated T-depleted splenocytes with combinations of LPS, various cytokines, and Sf21 insect cells expressing CD40L on their cell surface. We cultured the cells for 7 days and determined the amount of Ig secreted (normalized to IgM secretion in the same culture). Consistent with results published by Snapper et al. (5), the amount of IgG2a expressed by T-depleted splenocytes stimulated with LPS + IFN-{gamma} was increased 4- to 10-fold compared with the amount expressed by cells stimulated by LPS alone. The increase in IgG2a expression by T-depleted splenocytes activated by CD40L and IFN-{gamma} was much greater: 60 (experiment 2) to 1000 (experiment 1) times more IgG2a than cells cultured in LPS alone (Fig. 1B). Parallel to the results in vivo, IgG2b was also increased by treatment of T-depleted splenocytes by CD40L + IFN-{gamma}, but only 3- to 9-fold over LPS values. IgA expression was also increased slightly by culture in CD40L + IFN-{gamma} in experiment 1, but not in experiment 2; IgG3 and IgG1 expression decreased in both experiments. CD40 ligation, in the absence of cytokines, does not provide a signal for Ig secretion (29, 30), and we detected background levels of all Ig isotypes in supernatants of T-depleted splenocytes treated with CD40L alone (data not shown). The errors intrinsic to this small amount of secreted Ig preempted any analysis of switch recombination in cells treated with CD40L only.

If CD40 ligation induces switch recombination to the {gamma}2a gene, but not secretion of IgG2a, then one might expect CD40L-cultured B cells to express surface IgG2a. We tested this by flow cytometry. Relative to the negative control, T-depleted lymphocytes cultured in LPS, CD40L-cultured cells become IgG2a surface positive (Fig. 1C, left). Although there is extensive overlap in anti-IgG2a binding of the LPS- and CD40L-cultured cells, the entire CD40L-cultured population is shifted to the right, suggesting that a significant portion of the cells becomes IgG2a+. The culture of T-depleted splenocytes in CD40L + IL-4 should shift expression from IgG2a to IgG1. Consistent with this notion, IgG2a expression on CD40L + IL-4-cultured cells is very similar to LPS-cultured cells. The profile of IgG2a expression is virtually identical with that of cells cultured in CD40L + IFN-{gamma} (Fig. 1C, right).

To further test for a role of CD40 ligation alone in switch recombination, we turned to RNA-based tests. We tested RNA from cultures of BALB/c T-depleted splenocytes for expression of pre- and postswitch transcripts (Fig. 2). As expected, LPS + IFN-{gamma} treatment of T-depleted splenocytes induced more {gamma}2a germline transcripts than did LPS treatment alone (Fig. 2A). More postswitch IµC{gamma}2a transcripts were expressed after treatment with LPS + IFN-{gamma} than after LPS alone, consistent with the induction of switch recombination to the {gamma}2a gene (5, 6). In a novel finding, we observed that treatment with CD40L alone induced {gamma}2a germline transcripts and postswitch IµC{gamma}2a transcripts (Fig. 2A). The amounts of {gamma}2a germline transcripts and postswitch IµC{gamma}2a transcripts were not increased further by treatment with CD40L + IFN-{gamma}. Part of the action of IFN-{gamma} on CD40L-treated cells might be to increase switch recombination. However, the data in Fig. 2A are also consistent with the interpretation that CD40 ligation leads to most of the switch recombination to {gamma}2a, and that IFN-{gamma} leads to its secretion, a known activity of IFN-{gamma} (31, 32).


Figure 2
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  		FIGURE 2. Germline and postswitch transcripts. T-depleted splenocytes from BALB/c mice were cultured with combinations of LPS, CD40L, IFN-{gamma}, and IL-4, as indicated. A–E, Transcripts, indicated along the right side of the figure, were analyzed by RT-PCR. In this, and subsequent figures, "water" indicates the RT-PCR control without cDNA. The faster migrating I{gamma}2aC{gamma}2a fragment is the result of alternative splicing of the I{gamma}2a exon to C{gamma}2a (43 ).

 
These effects by CD40L were largely specific for the {gamma}2a gene. The amount of {gamma}3 germline transcripts was the same in the various LPS and CD40L treatments, and the amount of postswitch IµC{gamma}3 transcripts was reduced by CD40L relative to LPS (Fig. 2C). As expected, only small amounts of germline or postswitch {gamma}1 transcripts were produced by CD40L or by additional treatment with IFN-{gamma} (Fig. 2D), as expression of {gamma}1 is inhibited by IFN-{gamma} and increased by IL-4 (3, 4). As observed for Ig secretion in vivo and in vitro (Fig. 1), the pattern of {gamma}2b expression shared some similarity to {gamma}2a. {gamma}2b germline transcripts were increased by CD40L compared with LPS alone. Again, the induction of switching by the {gamma}2a gene was quantitatively greater than the induction of the {gamma}2b gene. The amount of postswitch IµC{gamma}2b transcripts expressed by cells cultured in CD40L was similar to that expressed by cells cultured in LPS alone, while the amount of IµC{gamma}2a transcripts was much greater in cells cultured with CD40L than in cells cultured in LPS (Fig. 2, A and B). Similar results for {gamma}1, {gamma}3, and {gamma}2b transcripts have been reported by Ström et al. (33) using anti-CD40 as the B cell stimulator.

The induction of {gamma}2a in T-depleted splenocytes by CD40L could be due, in part, to IFN-{gamma} induced in other cells (e.g., NK cells) contaminating the T-depleted splenocytes. We tested the induction of {gamma}2a germline and postswitch transcripts in IFN-{gamma}–/– and in C57BL/6 (to which the IFN-{gamma}–/– mice were backcrossed) T-depleted splenocytes (Fig. 3A). Both germline and IµC{gamma}2a transcripts were induced by CD40L as efficiently in IFN-{gamma}–/– T-depleted splenocytes as in control C57BL/6 T-depleted splenocytes. Hence, expression and switch recombination to the {gamma}2a gene, induced by CD40L, are independent of IFN-{gamma}. At the same time, we tested T-depleted splenocytes from BALB/c mice. The amount of germline transcripts induced in BALB/c T-depleted splenocytes by CD40L was dramatically increased compared with C57BL/6 T-depleted splenocytes. Over several experiments, the amounts of {gamma}2a germline transcripts were 3- to 6-fold greater in BALB/c T-depleted splenocytes than in C57BL/6 T-depleted splenocytes, as induced by both CD40L (Fig. 3A) and LPS + IFN-{gamma} (data not shown). Using cloned versions of the BALB/c and C57BL/6 {gamma}2a germline transcripts, we verified that both versions are amplified equally in PCR (data not shown). We have reported elsewhere that this increased expression of {gamma}2a germline transcripts is observed for a transgene of the BALB/c-type H chain locus (28). Hence, the increased expression is due to some cis-acting element in the locus. It is likely that the promoter for germline transcripts in the {gamma}2a gene found in BALB/c DNA (Igha) results in more efficient production of germline transcripts than does the version of this gene ({gamma}2c) found in C57BL/6 DNA (Ighb). There are many differences in nucleotide sequences throughout these two genes, any of which might be responsible for improved transcription or posttranscriptional processing of the germline transcripts. The increased germline transcription of the BALB/c {gamma}2a gene does not apparently result in increased switch recombination; the amount of IµC{gamma}2a transcripts was increased slightly or not at all compared with C57BL/6 or IFN-{gamma}–/– T-depleted splenocytes.


Figure 3
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  		FIGURE 3. {gamma}2a gene expression in B cells with deficiencies in the IFN-{gamma} signaling pathway. T-depleted splenocytes were cultured with combinations of LPS, CD40L, and IFN-{gamma}, as indicated. Transcripts, indicated along the left side of the figure, were analyzed by RT-PCR. A, IFN-{gamma}–/– B cells. B, STAT1–/– B cells.

 
As STAT1 is the first transcription factor activated by binding of IFN-{gamma} to its receptor (34), we tested whether STAT1 was necessary for CD40L induction of the {gamma}2a gene. As reported by Xu and Zhang (35), the {gamma}2a gene was not induced by LPS + IFN-{gamma}, relative to LPS treatment alone, in STAT1-deficient T-depleted splenocytes (Fig. 3B). However, both germline and postswitch {gamma}2a transcripts were induced by CD40 ligation in STAT1-deficient T-depleted splenocytes. Therefore, CD40L-induced germline transcription and switch recombination of the {gamma}2a gene depend on neither IFN-{gamma} nor one of its most important mediators, STAT1.

I{gamma}2aCµ transcripts are produced from the deletion circles resulting from switch recombination to {gamma}2a (36). Because these deletion circles are eventually lost from the B cell after switching, they are an indication of recent switch events (36). In parallel to the results for postswitch IµC{gamma}2a transcripts, we found that I{gamma}2aCµ circle transcripts were increased in IFN-{gamma}–/– T-depleted splenocytes cultured with LPS + IFN-{gamma} compared with cells cultured with LPS alone, and were also increased in T-depleted splenocytes cultured in CD40L (Fig. 4). Apparently, the majority of the switch recombination to {gamma}2a had not occurred in vivo; if it had occurred in vivo, the I{gamma}2aCµ circle transcripts would have been detected at 5 h, but lost after 2 or 3 days of culture. The presence of circle transcripts in RNA from cells cultured in CD40L for 2 or 3 days indicates that the switch recombination was induced by CD40 ligation. Furthermore, because the T-depleted splenocytes were derived from IFN-{gamma}–/– mice, this switch recombination did not depend on IFN-{gamma}. Consistent with results in Figs. 1 and 2, switch recombination to the {gamma}2b gene was induced by CD40L, in that I{gamma}2bCµ circle transcripts were detected after 2 and 3 days of culture. Apparently, CD40L-induced switch recombination to {gamma}2b preceded LPS-induced switch recombination; at day 2, more I{gamma}2bCµ transcripts were detectable in RNA from cells cultured in CD40L than in cells cultured in LPS. Consistent with the observations on IµC{gamma}2b transcripts (Fig. 3), by day 3, I{gamma}2bCµ transcripts were equally abundant in cells treated with CD40L and in cells treated with LPS (Fig. 4). Consistent with results presented in Figs. 1–3, the effects of CD40 ligation on switch recombination did not extend to the {gamma}1 and {gamma}3 genes. Some small amount of I{gamma}3Cµ transcripts was detectable at 5 h, indicating that they preexisted from switch recombination in vivo. Small amounts of I{gamma}3Cµ transcripts were also detected in LPS- and LPS + IFN-{gamma}-treated cells at later time points, in which they may represent new switch recombination. The small amount of I{gamma}1Cµ transcripts detected at 5 h (from in vivo switching?) disappears at later time points, consistent with data that none of the activation regimens used in this experiment leads to switch recombination to {gamma}1 (3, 4).


Figure 4
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  		FIGURE 4. Expression of deletion circle transcripts. T-depleted splenocytes from IFN-{gamma}–/– mice were cultured with combinations of LPS, CD40L, and IFN-{gamma} for 5 h, 1 day, 2 days, or 3 days. Transcripts, indicated along the left side of the figure, were analyzed by RT-PCR.

 
We would like to identify the transcription factor/DNA interactions that distinguish IFN-{gamma}- and CD40L-induced expression of the {gamma}2a gene. Examination of DNA sequences revealed several potential IFN regulatory factor (IRF)-binding motifs in the promoter region for {gamma}2a germline transcripts (data not shown). Because IRFs have multiple transcriptional regulatory roles for immune system genes, and because some IRFs are induced by IFN-{gamma} (37), we investigated the transcriptional status of IRFs in B cells treated with LPS, with LPS + IFN-{gamma}, and with CD40L (Fig. 5). To avoid induction of the IRFs in vivo, we used B cells from IFN-{gamma}–/– mice. Transcripts from the IRF1 gene were induced transiently by LPS + IFN-{gamma}, with levels significantly above the LPS levels at 3.5 h, and perhaps slightly elevated after 1 day of culture (Fig. 5). Thus, the transcriptional activity of the IRF1 gene precedes that of the {gamma}2a gene, which is detectable at day 1, and peaks at day 2 or 3. Transcripts from the IRF2 and IRF8 genes may have been induced marginally by LPS + IFN-{gamma} at 3.5 h. None of the other IRFs tested was induced by LPS + IFN-{gamma} treatment relative to LPS. None of the IRFs was induced by CD40L relative to LPS (Fig. 5).


Figure 5
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  		FIGURE 5. Expression of IRF transcripts. T-depleted splenocytes from IFN-{gamma}–/– mice were cultured with combinations of LPS, CD40L, and IFN-{gamma} for 3.5 h, 1 day, or 2 days. Transcripts, indicated along the left side of the figure, were analyzed by RT-PCR. The kinetics of induction of {gamma}2a germline transcripts was variable, initiating earlier in this experiment than in most others (Fig. 4, for example).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It seems likely that much of IgG2a expression by murine B cells is the result of CD40 ligation via an interaction with CD40L (CD154) on Th cells, followed by exposure to IFN-{gamma} from Th or other cells. However, the {gamma}2a gene can be induced by other mechanisms, including interactions with NK cells (13, 14). Some portion of {gamma}2a expression must be independent of IFN-{gamma}, as mice deficient in the IFN-{gamma} receptor express significant amounts of IgG2a (10, 11). Type I IFN may substitute for IFN-{gamma} (12, 38); however, in tissue culture, a direct effect of type I IFN on switch recombination by B cells to {gamma}2a cannot be detected (5). We have identified an alternative mechanism for this IFN-{gamma}-independent induction of {gamma}2a. The ligation of CD40 on the B cell surface leads to germline transcription and switch recombination of the {gamma}2a H chain gene (Figs. 2–4). This does not require IFN-{gamma} expression by any cell, as it occurs in IFN-{gamma}–/– T-depleted splenocytes (Figs. 3 and 4). CD40L induction of the {gamma}2a gene can occur in tissue culture in the absence of exogenous stimulation of the B cells by other cytokines (Figs. 1C, 2, 3, and 4). Perhaps cytokines produced by B cells or other cells contaminating the preparation play a role in the activation of the {gamma}2a gene. In vivo, we would propose that exposure to cytokines (one of which may be type I or type II IFN) would cause a B cell whose CD40 is ligated to differentiate to a cell that can secrete IgG2a. Costimulation by cross-linking of Ig receptors on B cells might also contribute to induce B cells to secrete IgG2a (39, 40). This scenario for the production of IgG2a by B cells following CD40 ligation is supported by the results of Gurunathan et al. (41). These authors showed that coinjection of the Ag beta-galactosidase and DNA encoding the expression of trimers of CD40L results in dramatic increases in beta-galactosidase-specific IgG2a (41).

The induction of the {gamma}2a gene by CD40 ligation is different from the induction by IFN-{gamma} in that the former is independent of both IFN-{gamma} and STAT1 (Fig. 3). We tested whether differential transcriptional regulation of IRFs might account for the differential induction of the {gamma}2a gene by CD40L (Fig. 5). (For most IRFs, expression in LPS, in LPS + IFN-{gamma}, and in CD40L is induced relative to that in resting T-depleted splenocytes, in which tiny amounts of transcripts are detected by our RT-PCR; data not shown.) We could not identify a candidate IRF whose transcriptional expression differed in cells cultured in LPS + IFN-{gamma} vs CD40L. If IRFs are involved at all in the CD40L-mediated induction of the {gamma}2a gene, then the effects are likely to be posttranscriptional. Because the transcriptional induction of IRF1 precedes that of {gamma}2a germline transcripts, IRF1 would be a strong candidate for a regulator of IFN-{gamma}-induced expression (Fig. 5). Nevertheless, both IFN-{gamma} and CD40L induction of {gamma}2a germline transcripts and postswitch transcripts are unaffected in IRF1–/– B cells (data not shown). B cells deficient in other IRFs (particularly IRF8) should be tested for their induction of the {gamma}2a gene.

Our results using CD40L-expressing Sf21 cells as a B cell activator in tissue culture differ somewhat from results using agonistic anti-CD40 Abs (33). Anti-CD40 Abs have not been found to induce germline or postswitch transcripts of the {gamma}2a gene (29, 30, 33). Importantly, expression of the {gamma}2a isotype as a result of CD40 ligation was not investigated in most previous studies. In addition, there are multiple technical explanations for these differences. First, other studies have not used RNA analysis, which is the most sensitive way to detect the effects of CD40 ligation on H chain expression (comparing Figs. 1 and 2). Second, some studies have used more highly purified, resting B cells than we used. It is possible that the effects we observe depend on prior activation of some B cells, or expression of additional cytokines by B cells or contaminating cells in our cultures. However, even if B cell activation does prepare the B cell to switch to {gamma}2a after CD40 ligation, it does not include transcriptional changes in the {gamma}2a gene. The germline transcription and switch recombination of the {gamma}2a gene do not preexist in our T-depleted splenocyte preparations (Fig. 4), and it depends on CD40 ligation. We have also prepared resting B cells with a density >68% Percoll (but <100% Percoll), and found that the induction of {gamma}2a germline and postswitch transcripts was virtually identical with that of our typical B cell preparation (data not shown). Third, it seems likely that expression of CD40L on Sf21 cells leads to more extensive or different signal transduction compared with soluble anti-CD40 Abs. We compared anti-CD40 Abs and Sf21 cells expressing CD40L directly, and found that the former reagent led to less cell division and expression of the activation-induced cytidine deaminase. Consistent with the results reported by Strom et al. (33), we found that anti-CD40 did not induce germline or postswitch transcripts of the {gamma}2a gene above the level in LPS-treated T-depleted lymphocytes (data not shown). Snapper et al. (31) have reported that IFN-{gamma} treatment does not increase Ig secretion by B cells cultured with CD40L, whereas we suggested that IFN-{gamma} results in IgG2a secretion by B cells cultured with CD40L (see above). One explanation for the difference is that Snapper et al. (31) use resting B cells, and some activation in our B cell preparation may allow secretion by cells cultured in CD40L + IFN-{gamma}. Another explanation is that our CD40L on Sf21 cells might result in different or more potent signaling through CD40 than does the membrane preparation used by Snapper et al. (31).

The CD40 ligation pathway for IgG2a expression may play an important role in rapid immunity to intracellular pathogens. We suggest that ligation of CD40 on B cells (with costimulation by receptor cross-linking or yet to be identified cytokines) would lead to switch recombination to {gamma}2a. This ligation might be due to CD40L expression on NK or monocytes (29) even before T cell help or a high level of IFN-{gamma} is available. IFN-{gamma}-independent induction of the {gamma}2a gene may result in the significant levels of {gamma}2a germline and postswitch transcripts that we detected in STAT1–/– cells cultured in LPS only (Fig. 3B). STAT1-deficient mice are susceptible to viral infection (22, 42). Such infections may lead to increased induction of {gamma}2a by CD40 ligation or interaction with NK cells (13, 14). This switch recombination in vivo would result in detectable IµC{gamma}2a transcripts in STAT–/– cells cultured in LPS (Fig. 3B).


   Acknowledgments
 
We thank Dr. Dorothy Yuan for insightful comments on the manuscript.


   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 This work was supported by the National Institutes of Health (R01 CA39068 to W.A.D. and RO1 AI061469 to D.K.B.) and by the Midwest Affiliate of the American Heart Association (0151127Z to W.A.D.). B.E.B. was supported by the Research Training in Experimental Immunology Grant (T32AI007413). Back

2 Address correspondence and reprint requests to Dr. Wesley A. Dunnick, Department of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0620. E-mail address: wesadunn{at}umich.edu Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; IRF, IFN regulatory factor. Back

Received for publication April 4, 2006. Accepted for publication July 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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