T-bet regulates T-independent IgG2a class switching

Andrea J. Gerth1, Ling Lin1 and Stanford L. Peng1,2

Departments of 1 Internal Medicine and 2 Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA

Correspondence to: S. L. Peng, Department of Internal Medicine/Rheumatology, Campus Box 8045, CSRB 6617, 660 South Euclid Avenue, St Louis, MO 63110, USA. E-mail: speng{at}im.wustl.edu
Transmitting editor: K. M. Murphy


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The IgG2a Ig subclass plays a critical role in the pathogenesis of humoral autoimmunity and protection against pathogens. The T-box transcription factor T-bet has been implicated as a critical mediator of class-switch recombination (CSR) to IgG2a, but its relative importance to this process in various immune contexts remains incompletely defined. We report here that, surprisingly, T-bet is selectively required for IgG2a class switching in response to T-independent, but not T-dependent, stimuli. Specifically, T-dependent signaling through CD40, in contrast to T-independent signaling via lipopolysaccharide, can bypass a requirement for T-bet in IgG2a germline transcription and subsequent isotype switching. In contrast, T-bet-deficient B cells undergo class switching to other IgG isotypes at least as well as wild-type counterparts. Thus, T-bet is a class-specific regulator of IgG CSR and represents a unique regulator of B cell differentiation by participating in a T-independent, but not a T-dependent, activation pathway. T-bet-deficient B cells therefore represent a novel paradigm by which to investigate the regulation of humoral immune responses.

Keywords: antibody, B lymphocyte, cellular activation, rodent


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Via the process of class-switch recombination (CSR), activated B lymphocytes produce a single, specific Ig isotype, such as one of the IgG subclasses (1). Cytokines like IL-4, IFN-{gamma} and transforming growth factor-ß play particularly critical roles in B cell differentiation in part by directing the isotype specificity of CSR. IFN-{gamma}, in particular, appears to selectively stimulate IgG2a (2) and play a controversial role in the regulation of IgG3 (2,3). Such phenomena have remained of both scientific and clinical interest, since these IgG isotypes are often pathogenic in autoantibody-mediated disease states like lupus, particularly in relationship to IFN-{gamma} production (46), and at the same time play significant roles in the protection from pathogens (7). However, the context and specifics by which IgG2a is regulated during immune responses remain otherwise unknown.

Recently, the T-box transcription factor T-bet has been implicated in the induction of autoantibody production and IgG2a CSR by IFN-{gamma} (8): T-bet-deficient MRL/lpr animals developed greatly impaired IgG2a titers and autoantibodies, and were unable to initiate CSR to IgG2a in response to lipopolysaccharide (LPS) and IFN-{gamma} in vitro, as evidenced by a lack of germline {gamma}2a transcripts. Indeed, T-bet is induced in B cells by IFN-{gamma}, particularly in the context of polyclonal stimulators like LPS, CD40 ligation or BCR ligation (8). However, on a (129 x B6)F1 background, T-bet deficiency appeared to result in only a modest (~3-fold) reduction in antigen-specific IgG2a in response to immunization by 2,4,6-trinitrophenol-keyhole limpet hemocyanin (TNP-KLH) (9). Such observations clearly indicate the presence of T-bet-independent mechanisms to IgG2a production, such as the type I IFN (8), but at the same time such differences in phenotypes question the importance of this transcription factor in B cell activation in general.

Indeed, one explanation for such observations involves a context-specific role for T-bet during B cell effector differentiation. For example, T-bet may play a greater role in IgG2a CSR during autoreactive B cell activation, which contributes to the majority of the hypergammaglobulinemia in lupus-prone MRL/lpr mice (10,11), as opposed to conventional B cell responses. Alternatively, T-bet’s importance in IgG2a CSR may be strain-specific, being very important in some genetic backgrounds (e.g. MRL), but less important in others (e.g. 129). Regardless of the particular reason, a resolution of this discrepancy will likely shed substantial insight into the mechanism(s) of effector B cell activation and differentiation.

To address these issues, we examined B cell responses in T-bet-deficient animals backcrossed to defined genetic backgrounds (e.g. C57BL/6). Surprisingly, we found that T-bet was required for CSR to IgG2a during T-independent, but not T-dependent, processes and confirmed this phenomenon in CSR assays in vitro. Such findings reinforce the role of T-bet as a class-specific regulator of IgG class switching and uniquely identify this transcription factor as a regulator of T-independent, as opposed to T-dependent, immunity.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and immunizations
T-bet-deficient animals of the C57BL/6J and BALB/cJ backgrounds were generated by backcrossing a T-bet–/– allele (9) against each respective background (obtained from the Jackson Laboratory, Bar Harbor, ME) at least 10 times, followed by intercrossing and identification of wild-type and knockout littermates by PCR (8). Homozygosity of H-2 was confirmed by PCR as described (12) and homozygosity of IgH allotype was confirmed serologically by IgMa versus IgMb-specific ELISAs (data not shown). For immunizations, 25 µg 4-hydroxy-3-nitrophenylacetyl (NP)-chicken {gamma}-globulin (CGG), NP-KLH, TNP-KLH, TNP-CGG, NP-Ficoll, NP-LPS, TNP-Ficoll or TNP-LPS (Biosearch Technologies, Novato, CA) in 0.2 ml PBS were administered i.p. to mice aged 8–12 weeks and sera drawn at weekly intervals.

B cell cultures and ELISA
For in vitro analyses, B cells were purified by negative selection against CD43, and stimulated by LPS and/or anti-CD40, with or without recombinant IL-4 and IFN-{gamma} as described (8). Ig secretion was assessed on culture supernatants by ELISA. Of particular note, IgG2aa (BALB/c) antibodies were detected using goat F(ab')2 anti-mouse Ig and goat anti-mouse IgG2a–alkaline phosphatase as the capture and detection antibodies respectively (Southern Biotechnology Associates, Birmingham, AL). Since this system fails to detect IgG2a antibodies of the IgHb allotype [(13,14) and data not shown], IgG2ab (IgG2c, C57BL/6) was detected using goat anti-mouse IgG2a (Southern Biotechnology Associates) and biotin-5.7 (anti-mouse IgG2ab; BD PharMingen, San Diego, CA) as the capture and detection antibodies, respectively, followed by avidin–alkaline phosphatase (Sigma-Aldrich, St Louis, MO). Similarly, hapten-specific antibody activity was determined by Ig isotype-specific ELISA using plates pre-coated with NP-BSA or TNP-BSA (Biosearch Technologies) (8).

Flow cytometry
For flow cytometric analyses, splenocytes were cleared of erythrocytes by osmotic lysis, followed by staining at 0°C with the indicated combinations of TNP-fluorescein-aminoethylcarboxymethyl-Ficoll (Biosearch Technologies), FITC–1D3 (anti-mouse CD19), FITC– or phycoerythrin–M1/69 (anti-mouse CD24), phycoerythrin–R6-60.2 (anti-mouse IgM) and/or phycoerythrin– or biotin–B3B4 (anti-mouse CD23), followed by streptavidin–CyChrome (BD PharMingen). Cells were analyzed by flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA).

IgG2a CSR transcript analysis
Standard assessment of germline and post-switch IgG2a transcripts were assessed by RT-PCR as previously described (8,15). For quantification of these transcripts, real-time PCR analysis was performed in a GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA), using the following primer combinations: IgG2a germline, 5'-CTG GCAGTACCGATGCAGC and 5'-GCCAGTTGTATCTCCACA CACAG; IgG2a postswitch, 5'-GACCTCTCCGAAACCAGGC and 5'-GGGCCAGTGGATAGACCGA; and ß-tubulin, 5'-CAC ATCCAGGCCGGACA and 5'-TGTTCATCGCTTATGACCTC CC. Relative mRNA abundance of each transcript was normalized against tubulin, calculated as 2(Ct[tubulin] – Ct[IgG2a]), where Ct represents the threshold cycle for each transcript.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IgG2a responses to T-dependent antigens do not require T-bet
Because the importance of T-bet in IgG2a CSR has previously been best defined only in vitro (8), we assessed the ability of T-bet-deficient B cells to undergo CSR in vivo by immunizing T-bet+/+ and T-bet–/– animals of the BALB/c and C57BL/6 backgrounds with the T-dependent antigen, NP-CGG. As assessed by serology at day 28 after immunization, T-bet-deficient animals of either background were surprisingly capable of mounting appropriate hapten-specific responses of all isotypes, including IgG2a, sometimes with titers in excess of wild-type counterparts (Fig. 1, Table 1 and data not shown). Similarly, T-bet-deficient animals mounted hapten-specific IgG titers of all isotypes comparable to T-bet-sufficient counterparts when immunized with NP-KLH, TNP-CGG and TNP-KLH (Table 1 and data not shown). We were therefore left with the surprising observation that T-bet was not required for IgG2a responses, at least against these T-dependent antigens.



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Fig. 1. Antigen-specific IgG2a class switching against a T-dependent antigen is T-bet-independent. T-bet+/+ (open squares, solid lines) and T-bet–/– (solid circles, dotted lines) C57BL/6 animals were immunized i.p. with 25 µg of NP-CGG on day 0 and sera was assessed for isotype-specific anti-NP by ELISA on day 28. Activity was measured by serial dilution of sera at the amounts indicated (x-axis). Shown is one representative experiment of three, in which three animals of each genotype were immunized. Similar results were obtained from T-bet+/+ and T-bet–/– BALB/c animals (not shown). Horizontal dashed line on each graph indicates the threshold of positivity, corresponding to 3 SD above the background OD.

 

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Table 1. Immune responses to T-dependent antigens in T-bet-deficient micea
 
IgG2a responses to T-independent antigens require T-bet
Since previous assays, which demonstrated an impaired ability of T-bet-deficient B cells to class-switch to IgG2a, utilized only T cell-independent stimuli, like LPS and cytokines (8), we speculated that T-bet may play a more important role in T cell-independent CSR to IgG2a, thereby accounting for our findings above. We therefore immunized T-bet+/+ and T-bet–/– animals of the BALB/c and C57BL/6 backgrounds with the T-independent antigens, TNP-LPS and TNP-Ficoll, and observed the development of hapten-specific IgG antibodies (Fig. 2, Table 2 and data not shown). Strikingly, T-bet-deficient animals were able to mount normal, and often supernormal, anti-hapten responses of the IgG1, IgG2b and IgG3 isotypes, but were consistently defective in IgG2a anti-hapten responses. Similar findings were obtained with another T-independent type I (NP-LPS) and type II (NP-Ficoll) antigen (Table 2 and data not shown). Interestingly, two of nine BALB/c T-bet–/– animals developed low-titer IgG2a anti-TNP antibodies after immunization with TNP-LPS, suggesting that this IgG2a phenotype, while highly penetrant, may be modified by genetic (i.e. strain-related) factors and/or that some T-independent mechanisms may bypass a requirement for T-bet in IgG2a production. All the same, we conclude that T-bet plays a more critical role in IgG2a CSR in response to T-independent than T-dependent antigens.



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Fig. 2. Antigen-specific IgG2a class switching against a T-independent antigen is T-bet-dependent. T-bet+/+ (open squares, solid lines) and T-bet–/– (dark circles, dotted lines) C57BL/6 animals were immunized i.p. with 25 µg of TNP-LPS or TNP-Ficoll on day 0, and sera was assessed for isotype-specific anti-TNP by ELISA on day 28. Activity was measured by serial dilution of sera at the amounts indicated (x-axis). Shown is one representative experiment of three, in which three animals of each genotype were immunized. Similar results were obtained from T-bet+/+ and T-bet–/– BALB/c animals (not shown). Horizontal dashed line on each graph indicates the threshold of positivity, corresponding to 3 SD above the background OD.

 

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Table 2. Immune responses to T-independent antigens in T-bet-deficient micea
 
T-bet-deficient, hapten-specific B cells are of a normal B cell subtype
Since many T-independent B cell responses have been thought to be mediated by marginal zone and/or B1 B cells, we wondered if the T-independent CSR phenotype seen in T-bet-deficient animals might in some way be related to developmental defects in marginal zone and/or B1 B cells, especially since T-bet-deficient animals possess otherwise normal B cell populations as assessed by IgM, IgD and CD19 [(8) and data not shown]. Interestingly, the spleen of T-bet-deficient animals contained normal numbers of CD19+CD23CD24+ marginal zone B cells as assessed by flow cytometry (Fig. 3 and data not shown). Furthermore, immunized T-bet-deficient animals appropriately contained hapten-specific B cells predominantly of the IgM+CD23+ follicular phenotype, comparable to wild-type counterparts (Fig. 3 and data not shown). Finally, T-bet-deficient animals contained comparable numbers of B220+CD5+ peritoneal and splenic B cells (comparing wild-type to knockout: 385,000 ± 75,200 versus 300,000 ± 60,000 and 810,000 ± 110,000 versus 743,000 ± 80,000 respectively; n = 3). We therefore conclude that T-bet deficiency does not result in gross defects in mature, marginal zone or B1 B cell populations, suggesting that, similar to its role in the generation of IFN-{gamma} by Th cells (9), T-bet plays a role in a specific developmental program in mature B cells, i.e. differentiation into an IgG2a-secreting state, rather than in the maturation of immature B lymphocytes.



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Fig. 3. B cell subpopulations in T-bet-deficient mice. Top panels: splenocytes from 8- to 10-week-old T-bet+/+ and T-bet–/– C57BL/6 animals were analyzed by triple-color flow cytometry. Shown are CD19-gated cells, stained for CD23 and CD24. MZ indicates marginal zone B cells as defined by surface phenotype CD19+CD23CD24+, with percentage of gated cells indicated. Shown is one animal, representative of three animals similarly tested. Bottom panels: splenocytes from T-bet+/+ and T-bet–/– C57BL/6 animals, isolated on day 35 after primary immunization with TNP-Ficoll, were analyzed for the surface phenotype of TNP-binding B lymphocytes. Shown are TNP-binding cells, stained for CD23 and IgM; these dual-positive cells represent follicular (F0) B cells. Shown is one animal, representative of six animals similarly tested. Virtually identical results were obtained for animals immunized with TNP-LPS, as well as for BALB/c animals immunized with TNP-Ficoll or TNP-LPS (not shown).

 
T-dependent signals can overcome the IgG2a CSR defect in vitro
Given these observations, we sought to identify a potential mechanism by which T cells may bypass a requirement for T-bet in B cell differentiation into IgG2a secretion. We therefore examined the ability of T-bet-deficient B cells to undergo CSR in response to CD40 ligation, which has previously been shown to play significant roles in the generation of all IgG isotypes (1618). Strikingly, T-bet-deficient B cells were able to generate IgG2a responses when stimulated with anti-CD40 and IFN-{gamma}, albeit to a possibly lower degree than wild-type counterparts (Fig. 4). In contrast, T-bet-deficient B cells could not generate IgG2a in response to LPS and IFN-{gamma}. To determine the molecular basis of this phenomenon, we examined the ability of T-bet-deficient B cells to generate germline and post-switch IgG2a transcripts in response to LPS or CD40 ligation in the presence of IFN-{gamma} (Fig. 5). Indeed, CD40 ligation, but not LPS, could induce IgG2a germline transcription and subsequent switch recombination, as evidenced by post-switch IgG2a RNA transcripts, in the absence of T-bet. Although we cannot formally rule out the possibility that CD40 ligation, but not LPS stimulation, is able to expand selectively a B cell population pre-committed in vivo to IgG2a, these findings nonetheless suggest that CD40 ligation is at least one mechanism by which the T-bet requirement in IgG2a CSR may be bypassed.



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Fig. 4. In vitro class switching to IgG2a by T-bet-deficient B cells. Splenic B cells from T-bet+/+ and T-bet–/– C57BL/6 animals were stimulated in vitro by the indicated stimuli, supplemented with or without IL-4 for IgG1 or IFN-{gamma} for IgG2a, beginning on day 0, and culture supernatants were assessed for IgG1 and IgG2a production on day 6 by ELISA. Asterisk indicates below the limit of detection of the assay (<20 pg/ml). Unstimulated cells of both genotypes produced no detectable IgG1 or IgG2a (not shown). Error bars indicate SD. Shown is one experiment using a single animal for each genotype, with each stimulation performed in quadruplicate samples, representative of six separately performed experiments. Note the use of a logarithmic y-axis scale in the lower graph.

 


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Fig. 5. T-bet is required for IgG2a CSR in response to LPS, but not CD40, ligation. Splenic B cells from T-bet+/+ and T-bet–/– BALB/c animals were stimulated in vitro by the indicated stimuli, and their RNA was analyzed for germline and post-switch IgG2a transcripts on day 6 by (A) real-time PCR analysis and/or (B) standard RT-PCR. Asterisks indicate that transcripts were undetectable under the conditions indicated.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
These findings strongly suggest a primary yet specific role for T-bet in IgG2a CSR during T-independent B cell activation. In response to multiple signals, both T-dependent and -independent, T-bet-deficient B cells activate normally, as assessed by surface activation marker expression and most Ig isotype production, except for the production of IgG2a [(8,9) Figs 1, 2 and 4, and Table 2]. Most interestingly, though, this CSR defect appears to be capable of being overcome by T-dependent signals, as suggested by the ability of T-bet-deficient animals to mount IgG2a anti-hapten responses to T cell-dependent antigens like TNP-CGG and of T-bet-deficient B cells to produce IgG2a in vitro in response to anti-CD40. Such observations focus our understanding of T-bet in the context of effector B cell differentiation, at least vis-à-vis IgG2a production.

Interestingly, although T-bet affects a very specific aspect of B cell differentiation, it appears to occupy a rather unique position, at least with respect to T-dependent versus T-independent signals, since most currently described factors in B cell activation affect T-dependent responses at least as dramatically as T-independent responses. For instance, the octamer binding protein OBF-1/OCA-B/Bob (19) and members of the NF-{kappa}B/Rel family (20,21) play critical roles in antigen-specific B cell responses, but as such are required for both T-independent and -dependent responses, presumably by affecting a final common B cell activation pathway [reviewed in (22)]. In contrast, defects in genes that play a role in T-dependent B cell responses do not result in significant defects in T-independent B cell responses, e.g. as described for the CD40–CD154 system (1618). In this sense T-bet is a unique precedent of a specifically T-independent B cell transcriptional pathway.

At the same time, these present findings help reconcile previous studies, whose results differed as to T-bet’s importance in IgG2a production (8,9). In these studies, T-bet-deficient MRL/lpr animals developed dramatically reduced titers of serum IgG2a, while T-bet-deficient (129 x C57BL/6)F1 animals developed only a 3-fold reduction in antigen-specific IgG2a in response to TNP-KLH immunization. Now, these results can be explained, since the majority of the hypergammaglobulinemia in MRL/lpr mice likely results from T cell-independent B cell hyperactivity (10,11), while the IgG2a in (129 x B6)F1 mice was assayed in response to a T cell-dependent immunogen. At the same time, however, we cannot formally rule out the possibility that T-bet plays more important roles in IgG2a CSR in some strains (e.g. MRL), but not others (e.g. 129). Indeed, the lack of any apparent deficit of IgG2a responses to T-dependent antigens in the backcrossed animals of our present study, in contrast to the mild defect seen in (129 x B6)F1 animals, may reflect such effects. Alternatively, they may also reflect differences in the regulation of the IgG2aa and IgG2ab loci when interacting in tandem, as both IgH allotypes were likely segregating in (129 x B6)F1 animals (9). All the same, the somewhat similar importance of T-bet in T-independent IgG2a CSR in both BALB/c and C57BL/6 mice (Figs 1, 2 and 4, and Tables 1 and 2) suggests that such considerations are unlikely to have contributed significantly to the otherwise dramatic differences in T-independent versus -dependent IgG2a production seen in the present study.

The specific pathway(s) in B cell activation in which T-bet participates, however, remains incompletely understood. Although T-bet is rapidly induced by IFN-{gamma} in B cells, the presence of this cytokine is common to both T-dependent and -independent responses and seems unlikely to be the sole pathway in which T-bet participates [(8,23,24,25), and our unpublished data]. As such, it is interesting to note that T-bet is also induced by polyclonal, T-independent stimuli, such as LPS and BCR ligation [(8) and our unpublished data]. Perhaps T-bet mediates such specific responses as IgG2a CSR in response to such stimuli, in addition to its role in mediating the CSR function of IFN-{gamma} (8).


    Acknowledgements
 
We thank Laurie Glimcher for T-bet-deficient mice, Alec Cheng for NP-Ficoll and both for helpful comments regarding the manuscript during preparation. We also thank Wayne Yokoyama for the use of the GeneAmp 5700 system. This work was supported in part by the Siteman Cancer Center, as well as grants from the NIH (AI01803), the Lupus Research Institute and the Broad Medical Research Foundation.


    Abbreviations
 
CGG—chicken {gamma}-globulin

CSR—class-switch recombination

KLH—keyhole limpet hemocyanin

LPS—lipopolysaccharide

NP—4-hydroxy-3-nitrophenylacetyl

TNP—2,4,6-trinitrophenyl


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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