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The Journal of Immunology, 2007, 179, 6783 -6789
Copyright © 2007 by The American Association of Immunologists, Inc.
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Localization and Differential Expression of Activation-Induced Cytidine Deaminase in the Amphibian Xenopus upon Antigen Stimulation and during Early Development1

Shauna Marr2,*, Heidi Morales2,*, Andrea Bottaro*,{dagger}, Michelle Cooper{ddagger}, Martin Flajnik{ddagger} and Jacques Robert2,*

* Department of Microbiology and Immunology, and {dagger} Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642; and {ddagger} Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As in mammals, B cell maturation in the amphibian Xenopus involves somatic hypermutation (SHM) and class switch recombination to diversify the B cell receptor repertoire in response to Ag stimulation. Unlike mammals, however, the resulting increase in Ab affinity is poor in Xenopus, which is possibly related to the absence of germinal centers and a suboptimal selection mechanism of SHM. In mammals, both SHM and class switch recombination are mediated by the activation-induced cytidine deaminase enzyme and under Ag-dependent regulation. Given its evolutionary conservation in jawed vertebrates, we used activation-induced cytidine deaminase as a marker to monitor and localize B cell maturation in Xenopus upon immune responses and during early development. In adult, Xenopus laevis AID (XlAID) was detected mainly in the spleen, where cells expressing XlAID were preferentially distributed in follicular B cell zones, although some XlAID+ cells were also found in the red pulp. XlAID was markedly up-regulated in the spleen with different kinetics upon bacterial stimulation and viral infection. However, during secondary anti-viral response XlAID was also noticeably expressed by PBLs, suggesting that XlAID remains active in a subset of circulating B cells. During ontogeny, XlAID expression was detected as early as 5 days postfertilization in liver before the first fully differentiated B cells appear. Concomitant with appearance of mature B cells XlAID was up-regulated upon bacterial stimulation or viral infection at later larval stages. This study highlights the conserved involvement of XlAID during Ag-dependent B cell responses in Xenopus but also suggests another role in B cell differentiation earlier in ontogeny.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In mammals, during the elaboration of T-dependent Ab responses in germinal centers (GC)3, Ag-experienced B cells further increase Ab specificity by a process of somatic hymermutation (SHM) that introduces point mutations into assembled IgH (Ig H chain) and IgL (Ig L chain) V region exons. In addition, the initial Cµ exons can be replaced by one of the several downstream sets of CH exons through IgH class-switch recombination (CSR), which alters the effector function of the Ab without changing its Ag-binding specificity. Despite their differences both SHM and CSR are catalyzed by the B cell-specific activation-induced cytidine deaminase (AID), which is a multifunctional enzyme that belongs to the APOBEC-1-related family of cytidine deaminases (1). AID was discovered by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma cells (2), and has been shown to be required for SHM, CSR, and gene conversion (3, 4, 5, 6). Increasing evidence suggests that AID acts by deaminating cytidine residues in genomic DNA, inducing DNA repair cascades that ultimately result in SHM, CSR, and gene conversion (7).

The capacity to generate a large rearranged Ig gene repertoire and further diversify this repertoire by SHM during affinity maturation is present in all gnathostomes (reviewed in Ref. 8). Consistent with this, AID homologues have been identified by sequence prediction or cDNA cloning in shark (9), several teleost fish (10, 11, 12), and amphibian (13). However, the ability to modify the Ig effector functions by CSR is first observed in the amphibians whose common ancestor with mammals dates back ~350 million years ago.

Therefore, amphibians such as the laboratory model Xenopus, occupy a pivotal position in the evolution of the vertebrate immune system. Xenopus and mammals have similar organization and usage of their Ig gene loci with RAG-dependent somatic combinatorial joining of V, D, and J elements (reviewed in Ref. 14). During the course of an immune response, Xenopus Ig’s can also switch from IgM to IgY, the functional equivalent of mammalian IgG (15), by mechanisms that require T-B cell collaboration (16, 17). Although the CSR substrate S region sequences are divergent between mammals and Xenopus (i.e., Xenopus Sµ is rich in AT and not prone to form R-loops), a Xenopus S region can functionally replace mouse equivalents to mediate CSR in vivo (18), and Xenopus AID can induce CSR in AID-deficient murine B cells (13).

Despite these fundamental similarities, however, affinity maturation in Xenopus is poor when compared with mammals. For example, the affinity of IgY Ab against DNP-KLH proteins increases by <10 times during a humoral response in contrast to more than a 10,000-fold affinity increase in mammals (19). Sequence analysis of VH1 gene family during Ab response to DNP-KLH has revealed that the rate of somatic mutations is not very different between Xenopus and mice (20). However, the relatively low ratio of replacement to silent mutation ratio in the CDRs (20), suggests that in Xenopus the selection mechanism is not optimal. This could be related to the simpler organization of the lymphoid organs in Xenopus, with neither lymph nodes nor GC (14), ultimately resulting in poor affinity maturation.

Another peculiarity of amphibians such as Xenopus is the early development of immunocompetence. Unlike mammals, the immune system of Xenopus is under pressure to develop quickly and produce a heterogeneous repertoire because larvae hatch 2 days following fertilization. In contrast to mammalian embryos development in the uterus, Xenopus larvae hatch in water that contains microorganisms. Early stages of immune development must occur in the absence of a spleen, leaving differentiation events that take place in the immune system to depend either solely or essentially on intrinsic factors (14).

Taking advantage of AID as a marker of Ag-driven B cell selection, Xenopus provides therefore a unique opportunity to study B cell immunity in the natural absence of GC and during ontogeny. We show here that although XlAID is involved in Ag-dependent B cell maturation in adults and larvae, it may have another role in B cell differentiation earlier in ontogeny.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents

Production of mAbs specific to Xenopus IgY (11D5;21) and anti-IgM (10A9;21), as well as the breeding of adult outbred (OB) Xenopus were done in our X. laevis research resource for immunobiology at the University of Rochester (www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). Developmental stages were according to (22).

Immune stimulation and viral infection

Adult frogs were injected i.p. with 200 µg/ml heat killed bacteria. E. coli (XL1-blue; Strategene) cultured overnight at 37°C were boiled for 1 h, spun, and resuspended in 0.1 volume (~108 bacteria/ml) of Xenopus cell culture medium (23). Larvae were injected with 5 µl of the same preparation of bacteria. Frog virus 3 (FV3; Iridoviridae) was produced at high titer by infecting the Xenopus A6 epithelial cell line according to a published protocol (24). Viral titer was determined on A6 cells by the end-point dilution method (25). Xenopus larvae were injected i.p. with 5 µl containing 10,000 PFU of FV3 in sterile amphibian PBS (APBS); adults were injected i.p. with 200 µl containing 1 million PFU of FV3 in sterile APBS.

Northern blotting

Total RNA was isolated from Xenopus tissue using the TRIzol reagent (Invitrogen Life Technologies). A total of 10–20 µg of RNA was separated on 1% glyoxal-agarose gel according to standard protocol, transferred to {zeta}-probe GT Membrane (Bio-Rad) and UV crosslinked. A 32P-labeled probe derived from a 606 bp AID cDNA fragment was used for hybridization under stringent conditions (64°C, and 0.2% SSC and 0.2% SDS).

RT-PCR

RNA from Xenopus cells was isolated by the TRIzol method following the supplier protocol (Invitrogen Life Technologies). For each PCR (30 µl total volume), 3 µl of 1.25 mM dNTPs, 3 µl of 10x PCR buffer, 1 µl of each primer, 2 U of TaqDNA polymerase (Invitrogen Life Technologies), and 1 µl of cDNAs. Tubes were then set for 35 cycles of denaturation for 45 s at 95°C, annealing for 45 s at 58°C, and extension for 1 min at 72°C. The primers used were the following: XlAID, 5'-TCACGACCCCCATAGGAACTAC-3' (forward) and 5'-TTAGGAGACTTTGCCTCAAGG-3' (reverse); Ig chain {gamma}, constant domain (Ig-C{gamma}), 5'-ATCCAGCAGCCAACTCACCATC-3' (forward) and 5'-AAGGTGGCAGCAAGGTGATTG-3' (reverse); Xenopus β2M, 5'-CCCTTGTGGTGTAACTGTGCTC-3' (forward) and 5'-GCACACACCAATCAGAAAAAGGAC-3' (reverse). Possible contamination by genomic DNA was controlled by using primers recognizing sequences on different exons (i.e., a larger product is amplified on genomic DNA). In addition, RT-minus controls were routinely performed.

Magnetic cell sorting

Splenocytes were harvested from 15 larvae 7 days after infection by ip injection of 2 x 104 PFU FV3 and sorted with magnetic microbeads as already described (23). Cells were first incubated with the Xenopus-specific anti-IgM 10A9 mAbs (IgG1 isotype; Ref. 21) for 30 min on ice and Ab-coated cells were positively selected using MACS microbeads (Miltenyi Biotec) coupled to mouse-specific anti-{gamma} following the manufacturer’s instructions. The cell fraction depleted of IgM+ B cells was then washed and incubated with the Xenopus-specific anti-CD8 AM22 mAb (IgM isotype; Ref. 26) to isolate CD8 T cells using MACS coupled to mouse-specific anti-µ. RNA was isolated from 1.5 x 105 cells from each fraction for RT-PCR.

In situ hybridization

A 606 nucleotide fragment of the Xenopus AID cDNA sequence was inserted into pPCR-Script Amp SK+ using restriction enzymes. A total of 1 µg of linearized template was used to synthesize the riboprobe using a RNA-labeling kit from Roche. Tissues were fixed in 4% paraformaldehyde (PFA) followed by 20% sucrose, before sectioning. Sections were postfixed in 4% PFA/PBS at room temperature, and digested with 20 µg/ml Proteinase K at 37°C. Slides were then acetylated in 0.1 M Tris-Acetate-EDTA buffer, and refixed in 4% PFA. Slides were also washed, and dehydrated in 70 and 95% ethanol before prehybridization. Approximately 0.2 µg of probe was denatured and hybridized to the slides at 55°C overnight. Slides were washed in high stringency wash buffers, 50% formamide plus 2 x SSC at 65°C for 30 min. They were then incubated with 20 µg/ml RNase A at 37°C, and then washed in RNase buffer. The high stringency wash was repeated 2x at 65°C for 30 min. DIG-labeled probes were detected using anti-DIG-alkaline phosphatase Fab (Roche) and 5-bromo-4-chloro-3-indolyl phosphate/NBT (Promega) substrate.

Histology

Frozen 5-µm sections of various tissue (embedded in O.C.T) were fixed for 30 s in acetone and incubated in a humidified chamber for 30 min with 1% normal Xenopus serum, 0.5% Tween 20, and 2% BSA. Biotin and Avidin were used for blocking. Sections were then washed in buffer (1x TBS, 0.1% Tween 20, 1% BSA). A total of 200 µl of the 10A9 hybridoma supernatant (specific for Xenopus IgM) was added overnight at 4°C. After washing biotinylated horse anti-mouse IgG-HRP was added for 1 h at room temperature. Slides were incubated with Vectastatin Elite ABC reagent mix from kit (Vector), washed, developed with diaminobenzidine substrate (Vector), and counterstained, dried, and mounted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression pattern of XlAID in Xenopus adults

Despite the lack of GC, typical primary and secondary Ab response involving SHM and CSR occur in adult Xenopus (review in Ref. 14). Because AID mediates these molecular events, we determined XlAID expression pattern to further characterize the site and the level of Ag-dependent B cell maturation in Xenopus. Northern blot analysis (Fig. 1A) revealed that XlAID is expressed at low levels as two transcripts of 2 and 1.3 Kb in the spleen, which in adult is the main site of B cell activation (i.e., there are no lymph nodes in Xenopus; Ref. 14). Little or no signal was detected in the thymus, intestine (Fig. 1A) or any other tissues tested (data not shown). Similarly, using RT-PCR XlAID was found expressed primarily in spleen, although occasionally expression was also detected in intestine, liver, kidney, and lung (Fig. 1B and Table I).


Figure 1
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  		FIGURE 1. Expression patterns of XlAID in Xenopus adults. A, Northern blot of total RNA (10 µg) from adult OB Xenopus thymus (T) spleen (S) and intestine (I), probed with a 606 bp 32P-labeled Xenopus AID cDNA fragment. An Ef-1{alpha} probe was used for reference (lower panel). B, RT-PCR of various adult tissues. Equal amounts of total RNA (1 µg) were reverse transcribed and amplified with primers specific for XAID or β2M as control. No genomic DNA contamination was detected (see Material and Methods). T, T hymus; Li, liver; I, intestine; K, kidney; Lu, lung. C, Differential expression of XlAID upon bacterial stimulation assayed by RT-PCR of spleen cDNA from three adults 4 days after i.p. injection of 200 µl (2 x 107) of heat-killed E. coli (2 x 107 immunized), and from three untreated frog as negative controls.

 

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  		Table I. Summary of XAID expression in different tissues at different developmental stages in untreated and immunized animalsa

 
There was variation in basal XlAID expression level between individuals for the different tissues, including the spleen (Fig. 1C and data summarized in Table I), which presumably reflects ongoing or unpredictable immune responses in our frog colony.

To further determine whether XlAID expression is also up-regulated during an immune response, we used a recently established system of bacterial B cell stimulation in vivo (23) and tested tissue expression by RT-PCR. Although XlAID is often present at low levels in spleen of untreated adults, there was a considerable increase in expression in the spleen as early as day 4 following immunization with heat killed E. coli (Fig. 1C). In contrast, bacterial stimulation did not result in consistent change of XlAID expression in other adult tissues, including thymus and intestine and bone marrow (Table I).

Localization of AID-expressing cells in Xenopus spleen

In mammals, low levels of AID expression is detected mainly in lymphoid tissues before stimulation, but it is rapidly up-regulated in GC B cells in response to LPS and inflammatory cytokines (27, 28). Although CSR also occurs in Xenopus, no structures resembling GChave been found in Xenopus lymphoid tissues (14). In addition, studies with DNP-KLH immunization and BrdU incorporation have revealed a scattered distribution of proliferating Ag-specific B cells in the spleen (14). To determine whether B cells undergoing SHM and/or CSR are located in GC-equivalent microenvironments or scattered within the whole lymphoid tissue, in situ hybridization was performed on frozen spleen sections. As shown in Fig. 2, specific signals were detected with sense but not anti-sense probes throughout the splenic tissue, and AID+ cells were detected in both red and white pulp. However, examination at higher magnification revealed that AID+ cells were found more frequently in the white pulp, especially in the follicular area where IgM+ B cell reside (Figs. 2, a and b and 3, a and b) (29, 30). AID+ cells in the red pulp appeared to be secretory cells (e.g., large cells with noticeable cytoplasm; Figs. 2a and 3a, black arrows). Compared with untreated animals, spleens from animals immunized to E. coli displayed more AID+ cells distributed mainly in the white pulp (Figs. 2–4, blue arrows). Some groups of AID+ cells were localized around large blood vessels. However, no organized clusters of AID+ cells reminiscent of GCs were observed.


Figure 2
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  		FIGURE 2. Localization of XlAID expressing cells by in situ hybridization. Frozen sections of paraformaldehyde-fixed spleen from untreated OB adult Xenopus were hybridized with sense (1 ) or anti-sense (3a, 3b, and 4) DIG-conjugated XlAID riboprobe. After development with alkaline phosphatase substrate, slides were counterstained with methyl green. Spleen from untreated OB frog at 200x magnification (3a); follicular B cell area of the same spleen at higher 600x magnification (3b); (4 ) spleen from a frog 4 days after injection of heat-killed E. coli with many cluster of AID+ cells (blue arrows); (2 ). Spleen immunostained with the Xenopus-specific anti-IgM 10A9 mAb followed with biotinylated goat anti-mouse and HRP-conjugated streptavidin, showing the B cell area. BV, Blood vessel; RP, red pulp; F, follicular area; blue arrows, AID+ cells in the follicular area; black arrows AID+ cells in the red pulp.

 

Figure 3
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  		FIGURE 3. XlAID differential expression upon viral infection. Adults OB were infected once (primary infection) or twice at 1 mo interval (secondary infection) by i.p. injection of 106 pfu FV3. RT-PCR was performed on spleen (A), kidney tissues (B), and PBLs (C) obtained from day 3, 6, 9, and 14 or 15 postinfection with primer specific for XAID, the major capsid protein of FV3 (MCP), IgY, or either Ef-1{alpha} or β2M as reference.

 

Figure 4
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  		FIGURE 4. XlAID expression in bone marrow. Bone marrow from femurs were flushed using APBS from an untreated frog (N) or three separate frogs and stimulated for 4 days with heat killed E. coli bacteria (A), or following secondary FV3 infection (B). Total RNA was extracted from cells and reverse transcribed for RT-PCR analysis using primers specific for XlAID, Xenopus RAG-1, and β2M for reference.

 
XlAID differential expression during response to viral infection

The ranavirus FV3 is a natural amphibian pathogen (reviewed in Ref. 31). We have shown that Xenopus adults resist high doses of FV3 infection and generate specific anti-FV3 IgY Abs 10–14 days after a secondary but not upon primary infection (32, 33). The failure to detect FV3 specific Abs during a primary infection is probably due to their low levels because the titer remains low even after multiple infections (limit of detection at 1/200 dilution; Ref. 32). To further assess anti-viral B cell responses we determined the expression kinetics of XlAID during primary and secondary FV3 infection in the spleen.

A marked increase of XlAID mRNA expression was consistently detected in the spleen 9 days after primary infection (Fig. 3A), whereas during a secondary infection, a strong XlAID up-regulation was detected as early as 3 days postinfection. In addition, augmented XlAID expression in the memory response persisted up to 14 days postinfection. Note that the lower level of XlAID expression at day 9 postsecondary infection was observed in three independent experiments. Because XlAID expression has been detected occasionally in kidney (Table I), the major site of FV3 infection (34), we next determined the level of XlAID mRNA expression during primary and secondary infections in this organ. Low or no XlAID mRNA expression was detected during primary infection, but a higher signal was consistently observed at day 9 postsecondary infection (Fig. 3B). Moreover, noticeable XlAID expression was also detected in peripheral blood lymphocytes during secondary but not primary FV3 infection (Fig. 3C).

In mammals, the bone marrow is the main site for lymphopoiesis. It also is a reservoir for Ab-secreting B cells, some of which express AID (27). Little is known about the function of bone marrow in Xenopus, although cells with blastoid morphology typical of lymphocyte precursor have been described (35), and RAG is expressed (36). Because XlAID was consistently found expressed in peripheral blood lymphocytes during secondary FV3 infection, we assessed its expression in bone marrow following secondary FV3 infection or after exposure to heat killed bacteria, and no consistent expression of XlAID in stimulated or unstimulated animals was detected (Fig. 4).

Early developmental AID expression

Although in Xenopus RAG is first expressed 4 days postfertilization, and B cell precursors expressing IgH chain are detectable 5 days postfertilization, it is only from day 10 onwards that IgL expression is initiated and mature B cells appear (14). These early differentiation events are postulated to occur independent of extrinsic Ag. To determine whether AID expression is restricted to Ag-dependent B cell maturation, we assessed AID mRNA levels during early ontogeny. Using RNA extracted from whole embryos or early larval stages (5 pooled individuals), XlAID was consistently found expressed as early as 2.5 days postfertilization corresponding to stage 39 (Fig. 5A, left panel), approximately one week before the detection of productive rearrangements of the L chain locus and consequently of mature B cells (37). In three different experiments, level of XlAID expression peaked between day 5 and 6 postfertilization. The PCR product of day 6 postfertilization was confirmed to be XlAID after cloning and sequencing (data not shown), and by analysis of the PCR products with two restriction enzymes expected to digest the XlAID cDNA at particular sites (Fig. 5A, right panel).


Figure 5
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  		FIGURE 5. XlAID expression during early larval and premetamorphic development. A, Left panel, RT-PCR analysis of whole unfertilized eggs (0), embryos 1 to 60 h, larvae 4 to 6 days, and larval liver from 6 days postfertilization. Equal amounts of total RNA (100 ng) obtained form 5 pooled embryos, 3 pooled larvae, or livers were reverse transcribed and amplified with primers specific for XAID, XRAG1, or B2M as control. RT, RT minus control. Right panel, XlAID identity of PCR product from d5 and d6 larvae was controlled by digestion with XhoI or Pst-1 restriction enzymes, Southern blotting and hybridization with a radiolabeled XlAID cDNA probe. RT, RT minus control. B, RT-PCR of isolated head (H), liver (L), intestine (I), tail (T), and spleen (S) pooled from 3 larvae on day 5, 6, and 7 post fertilization. W, Water control. Upper panel, PCR products were probed using a fragment specific for XlAID. Middle panel, RT minus controls were probed using the same XlAID fragment. Lower panel, β2M expression control.

 
Further analysis was performed on isolated tissues pooled from three individuals from day 5 to 7 postfertilization. The identity of the amplified AID product was confirmed by hybridization with a radiolabeled XlAID cDNA probe under stringent conditions (Fig. 5B, upper panel). This probe did not hybridize to other PCR products (e.g., β2-M, IgY; data not shown), and no XlAID specific signal was detected in RT-minus negative controls (Fig. 5B, middle panel). At day 5 postfertilization XlAID was mainly expressed in the liver, whereas on day 6 strong XlAID expression was also detected in intestine and the spleen, as well as in the head.

These data clearly indicate that XlAID is expressed in tissues containing lymphocyte precursors at development stages preceding the differentiation of mature B cells and Ag-driven SHM and CSH.

At later developmental stages (stage 50 or 12 days postfertilization onward) larvae are immunocompetent, display a diverse Ig repertoire distinct from adult, and are able to undergo SHM (14). Compared with adults, however, larvae have a lower V region diversity, and reduced class switch and Ab affinity (14, 19, 20). We asked therefore whether this somewhat immature state of B cell maturation was reflected in the expression pattern of XlAID. As in adults, Northern blot analysis of premetamorphic larvae (stage 58, 4 wk postfertilization) revealed that XlAID is expressed at low levels in the spleen but not in thymus as two transcripts of 2 and 1.3 Kb (Fig. 6A). By RT-PCR we detected XlAID in the spleen, thymus, kidney, and to a lesser degree in the liver of premetamorphic larvae (stage 56, 3 wk postfertilization; Table I). As in adults, there was a considerable variation in XlAID expression between individuals, possibly reflecting their immune status. AID expression in the larval thymus is consistent with early data showing a significant population of B cells in this organ, mainly expressing IgY (38). No XlAID expression was detected in intestine, which is consistent with earlier reports absence of B cell in larval intestine (14).


Figure 6
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  		FIGURE 6. XlAID differential expression upon bacterial stimulation or viral infection in larvae. A, Northern blot of total RNA (10 µg) from spleen and thymus of Xenopus premetamorphic larvae (stage 58, 4 wk postfertilization), probed with a 606 bp 32P-labeled Xenopus AID cDNA fragment. 28S and 18S RNA stained by Ethidium bromide before transfer is shown as loading control. B, RT-PCR analysis of spleen from premetamorphic (st 56) larvae untreated (N) or 3, 6, and 10 days after ip injection with 5 µl (~5 x 105) of heat-killed E. coli suspension. Equal amounts of total RNA (100 ng) were reverse transcribed and amplified with primers specific for XlAID or β2M as control. No genomic DNA contamination was detected (see Material and Methods). C, Premetamorphic larvae (st 56) were infected once by ip injection of 2 x 104 PFU FV3. RT-PCR was performed on spleen and kidney RNA 7 days postinfection with primers specific for XlAID, IgY, or β 2M as control. D, RT-PCR was performed as in C with RNA from spleen and kidney of FV3-infected larvae. XlAID identity was controlled by digestion with XhoI or Pst-1 restriction enzymes, Southern blotting and hybridization with a radiolabeled XlAID cDNA probe.

 
We next assessed whether in larvae as in adult XlAID expression is up-regulated during an immune response. In three different experiments (one representative is shown in Fig. 6B) significant up-regulation of both XlAID and IgY in larval spleen was detected compared with APBS-injected controls starting on day 3 and continuing until day 10. Up-regulation of XlAID concomitant with IgY expression was also observed at 7 day following FV3 infection in the spleen (Fig. 6C). Interestingly, XlAID and IgY expression were also detected in the kidney of infected larvae. Analysis with two restriction enzymes expected to digest the XlAID cDNA at particular sites provided an additional control that the product amplified from spleen and kidneys of infect larvae was indeed XlAID (Fig. 6D).

To show that XlAID expression induced during FV3 infection is due to B cells, surface IgM+ cells were isolated by magnetic sorting from splenocytes of larvae infected for 7 days with FV3. CD8 T cells were also isolated from the same splenocytes after B cell separation. XlAID expression was mainly found in the isolated IgM+ B cell fraction, whereas CD8 T cells were negative (Fig. 7). Low levels of XlAID expression were detected in the remaining IgM/CD8-depleted splenocytes fraction, which is likely due to residual IgM B cells that were not retained during the sorting as indicated by the expression of IgM H chain, or by IgM-negative B cells. Interestingly, IgY H chain was found expressed only in IgM/CD8 depleted cells, suggesting the presence of plasma cells and/or IgY-expressing memory B cells in this fraction.


Figure 7
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  		FIGURE 7. Induced XlAID expression by larval IgM+ B cells upon viral infection. RT-PCR of sorted IgM+ B (B1, B2), CD8+ T (8 ), and IgM/CD8-depleted (Dpl) splenocytes from larvae infected with FV3 for 7 day (15 individuals pooled). Total RNA was amplified with primers specific for Xenopus AID, IgY, IgM, and B2M as described in Materials and Methods section. RT negative controls revealed no genomic contamination in any of the samples tested. (B2) Control for XlAID identity of the B cell RT-PCR sample by digestion with the PstI restriction enzyme, Southern blotting, and hybridization with a radiolabeled XlAID cDNA probe.

 
We conclude that, similar to adults, XlAID expression can be induced in immunocompetent larvae upon exposure to bacteria and results in potent humoral response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this study provide evidence of the evolutionarily conserved role of AID in Ag-dependent B cell maturation in vertebrates. Amphibians are the most primitive vertebrates where B cell response is associated with both SMH and CSR, two AID-dependent molecular events. Correlation between different kinetics of B cell responses and AID up-regulation, as well as localization of splenic AID+ cells in B cell follicles by in situ hybridization, strongly support the involvement of AID in Ag-selection of B cells in adult and larval Xenopus. However, our finding that XlAID is expressed earlier in development than RAG and before the emergence of mature B cells, suggests that AID may have other roles in early ontogeny or in ectothermic vertebrates.

Tight association of XlAID expression with Ag-driven B cell response in adult Xenopus

Immune responses elicited by bacterial stimulation and ranaviral infection indicate a strong correlation between XlAID differential expression and B cell maturation in adults. During secondary FV3 viral infection involving typical immunological memory (32), a significant increase of splenic B cells was observed from day 3 to day 10 postinfection, and specific anti-FV3 IgY Abs were detected in the serum 12–14 days postinfection (31, 32). The rapid up-regulation of XlAID three days postsecondary infection is in agreement with an active Ag-dependent B cell response. Interestingly, during ranavirus responses a fraction of B cells undergoing active Ag-dependent maturation appears to be leaving the spleen, because substantial XlAID expression is detected in PBLs and in kidneys. Several nonexclusive possibilities may explain this phenomenon. First, XlAID expression may be perpetuated in B cells after they have undergone CSR. The detection of XlAID+ B cells in the red pulp of the spleen and in the kidney is consistent with this possibility. Alternatively, egress of XlAID+ B cells from the spleen could be related to the lack of bona fide GC and a concomitant deficiency in selection (14, 19, 20). It would be interesting to determine the frequency of abortive or defective B cell receptors in PBLs during secondary FV3 infection. The up-regulation of XlAID expression during primary FV3 infection, despite the fact that anti-FV3 specific Ab cannot be detected in serum, suggests that anti-FV3 Abs are produced during primary infection but at a titer below the ELISA detection level. Interestingly, no XlAID expression was found in PBLs during primary infection. It is tempting to speculate that the smaller number of Ag-specific B cells undergoing maturation during primary infection are less likely to escape selection in the spleen. In contrast, recent evidence in mice suggests that induction of AID expression is not limited to GCbut can also occur following a variety of cell interactions and activation conditions. For example, epithelial cells lining tonsillar crypts have shown to induce AID expression by B cells to trigger frontline Ig class switching in human (39). In mouse, immature and transitional B cells mobilized in the spleen following inflammation constitutively express low but sufficient level of AID independently of T cells to promote Ig class switch (40). It is quite possible that the inflammatory response during FV3 infection in Xenopus is more potent and sustained than stimulation with heat killed bacteria. Unfortunately, no B cell differentiation markers are available in Xenopus. In addition, the spleen in Xenopus is also a major site for B cell differentiation. Therefore, it is difficult at this stage to determine whether inflammation resulting from FV3 infection causes or contributes to XlAID expression in the blood and other tissues like kidneys.

In mammals and birds, GC are critically involved in the generation and selection of B cells displaying BCR with increased affinity for Ag. Accordingly, AID is expressed in GC (26, 41). In chicken, in contrast, AID is also involved in Ig gene conversion earlier in B cell ontogeny, before Ag exposure (5, 42), which may suggest a distinct regulation of AID expression during development and during immune response. This in turn would imply that beside its requirement for SHM and CSR, AID has acquired additional functions such as GC during evolution from amphibians to mammals. It has been proposed that one of the main difference in the immune system between cold-blooded vertebrates such as Xenopus and warm-blooded vertebrates is lymphocyte networking (14). In the case of Ab response, it is generally considered that GCs provide an optimal site to display a localized high level of Ag and T cell help to select B cells with improved BCR affinity. In Xenopus, the architecture of the spleen in the white pulp shows a clear thymus-dependent area, with cells labeled with anti-CD8 mAbs surrounding the B cell area around the central arteriole (28, 29). However, histological studies in ectothermic vertebrates including Xenopus (43), suggest that ongoing B cell responses occur in the absence of organized GC-like structure. In addition, experiments using a model Ag (DNP) and a marker of proliferation (BrdU) showed that both specific Ag-reactive B cells and proliferating Ag-specific B cells were scattered in the white pulp, and were not organized in clusters (14). Consistent with these studies, we found AID+ cells scattered both in the white and red pulp of Xenopus spleen by in situ hybridization, and we did not find appreciable organized clusters of AID+ cells. One can speculate as to whether the noticeable number of AID+ cells localized in the B cell area close to the central arteriole can be view as an ancestral site of B cell selection. The number of these AID+ cells in the follicular B cell area increased upon bacterial stimulation. Although follicular dendritic cells have not yet been characterized in Xenopus, the close location of B cells undergoing Ag-driven maturation (AID+) around a central blood vessel and surrounded by T cells might viewed as an ancestral site of B cell selection. In such a view, Ag-specific B cell proliferation would be dispersed in the whole white pulp, whereas selection would be more localized. Alternatively, this preferential distribution of AID+ cells may just be the result of the convergence of mature B cells that leave the spleen.

Association of XlAID expression with Ag-driven B cell response in larval Xenopus

Although SMH in Xenopus larvae occurs at a rate not significantly lower than that of Xenopus adults or mammals (44), some evidence suggests that larvae have poorer CSR than adults (14, 20). First, whereas the level of IgM protein is comparable between adult, the level of IgY protein is very low in the serum of unimmunized stage 50 larvae (45). Second, although specific IgY Abs can be detected following immunization with DNP-KLH, they are produced in lower amounts than in adult (45). Finally, the transfer of irradiated splenic T cells from DNP-KLH primed isogenetic adults in isogenetic larvae one day before immunization significantly increased the amount of larval anti-DNP IgY produced (45). Our study clearly indicates that in larvae, as in adults XlAID, expression is Ag-driven, because XlAID expression is up-regulated following either injection of heat killed bacteria or FV3 infection. Moreover, this up-regulation is accompanied by an increase in total IgY expression in the spleen. These results suggest that XlAID-mediated CSR and SHM is functional, and further support the proposition that the poor generation of Ag-specific IgY in larvae results from inadequate T cell help and not an inherent B cell deficiency.

Interestingly, increased XlAID expression in larval kidney and intestine occurring during FV3 infection suggests that as in adult AID remains actively produced in some Ag-activated B cells outside the spleen. In contrast to adults, however, XlAID expression outside the spleen occurs even during primary FV3 infection. It is possible that in larvae B cell undergoing maturation are even more likely to escape selection in the spleen due to insufficient T cell help. Alternatively, as discussed before, inflammation and/or other type of cell interaction may contribute to induce and maintain XlAID expression outside the spleen. More experiments will be needed to determine the kinetics of B cell maturation and whether as in adult circulating AID+ B cells express a productive Abs.

Expression of XlAID at early developmental stage

Although RAG activity and rearrangement of the Ig H chain is detected as early as 5 days after fertilization in the liver, mature competent B cells that have also rearranged IgL genes and expressing a complete BCR are not detected until 10 days postfertilization or stage 42 (35, 37). The immune repertoire at this early stage is subject to particular constraints, because it has to be built rapidly (the larvae hatch two days after fertilization and must be immunocompetent) and with a very small number of cells available (5 to 200 IgM+ cells on day 5–12 after fertilization, respectively). Nevertheless, SHM in larvae and adults is not significantly different from what has been observed in mammals (20).

Although all these early differentiation events occur without the need for Ag selection and before the development of the spleen, our data indicate consistent AID expression as early as 2.5 days postfertilization, more than a day before RAG is first expressed. XlAID mRNA is also clearly detected in the liver at day 6 postfertilization, several days before any mature B cells can be found (37). This suggests a possible role of XlAID in early B cell development, analogous to the diversification of Ig genes by AID-dependent gene conversion during bursal development in the chicken (42). A notable difference however is that in Xenopus the expression of AID occurs before the appearance of the first IgM-positive B cell, whereas in the chicken bursa it occurs in IgM-positive cells. Whether XlAID is involved in the diversification of the Ig repertoire of minor more differentiated B cell population or perform a yet unknown function during early B cell differentiation remains to be determined. Although the possibility of XlAID expression by other embryonic cell types other than B cell precursors cannot presently be ruled out, the early detection of XlAID mRNA in Xenopus larvae liver, the first site of hematopoiesis (46), suggests that developmental, rather than immune response-dependent regulation, and a role in Ag-independent receptor diversification may be the rule rather than exception for AID in land vertebrates.


   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 research was supported by National Institutes of Health R25-GM64133 (to S.M.), National Institutes of Health R24-AI-059830 (to J.R.) and NSF MCB-0136536 (to J.R. and H.M.), National Institutes of Health AI45012 (to A.B.), and National Institutes of Health RR06603 (to M.F.). Back

2 S.M. and H.M. are equally contributing authors. Back

3 Address correspondence and reprint requests to Dr. Jacques Robert, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642. E-mail address: robert{at}mail.rochester.edu Back

4 Abbreviations used in this paper: GC, germinal center; SHM, somatic hymermutation; AID, activation-induced cytidine deaminase; CSR, class-switch recombination; APBS, amphibian PBS; PFA, paraformaldehyde. Back

Received for publication March 13, 2007. Accepted for publication September 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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