Terminal Deoxynucleotidyl Transferase Is Required for an Optimal Response to the Polysaccharide α-1,3 Dextran

Polysaccharides serve as important virulence factors for many pathogenic microorganisms (1) and induction of polysaccharide-specific Abs is a major factor in successful vaccination against pathogens, such as Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis (2–4). An understanding of the cellular and molecular events involved in the generation of B cell clones that give rise to protective polysaccharide-specific Abs will provide clues as how the immune system can be induced to make such Abs.

Many Ab responses to polysaccharides in mice are T cell independent and characterized by the rapid production of IgM and IgG3 (5), oligoclonality, and low affinity (6–8). Polysaccharides are generally poor inducers of memory, although features of memory Ab responses to polysaccharides have been recently demonstrated (9, 10).

Polysaccharides induce poor Ab responses in neonatal humans and mice (11–14), and several mechanisms have been proposed to account for this relative unresponsiveness compared with adults (reviewed in Refs. 15 and 16). One possible mechanism is that the neonate, in contrast to the adult, does not contain B cells with the appropriate polysaccharide-reactive IgRs (8, 14). The neonatal B cell repertoire differs significantly from that of the adult with respect to Ig V_H, V_L, D_H, and J_H gene usage (17–21). One notable difference between neonatal versus adult Ig repertoire is that H chain CDR3 lengths are shorter in the neonate because of the lack of, or lower TdT activity in, mice (21, 22) and humans (23), respectively. In addition, in-frame rearrangements predominate as a result of enhanced homology-mediated recombination, leading to increased representation of certain CDR3 sequences (24–26).

TdT is a lymphoid-specific DNA polymerase that plays a major role in the generation of B and T cell AgR diversity (27–29). TdT is conserved among vertebrate species (30, 31) and of the TdT alternative splice variants; the short form of TdT (TdTS) has been shown to exert its diversifying activity by adding nontemplated nucleotides (N-addition) at the V(D)J junctions of rearranging B and TCRs (28, 29, 32–34).

The presence or absence of TdT functional activity has been shown to play a significant role in mouse Ab responses to T-independent Ags. The germline-encoded T15 Ab specific for phosphorylcholine (PC), expressed on the surface of S. pneumoniae, is generated early in life in the absence of TdT (24) and protects against infection with this pathogen (35, 36). Forced expression of TdT during this period leads to the loss of the canonical T15 Ab in adulthood and hence loss of protection (37). In contrast, the activity of TdT is required for the generation of the M603 idiotype+ (Id+) B cell clone, responsive to PC expressed on Proteus morganii (38). Both of these studies provide examples of the significant role that TdT plays in modulating the B cell repertoire.

In this study, we investigated the role of TdT during the generation of B cell clones involved in the Ab response to the polysaccharide α-1,3 dextran (DEX) (39–41). DEX is a branched polymer containing α-1,3 glucose epitopes, which are also expressed in glucans associated with a variety of organisms, such as Enterobacter cloacae, Histoplasma capsulatum yeast cell wall (42), and Aspergillus fumigatus (B. L. Dizon and J. F. Kearney, unpublished observations). The Ab response of adult BALB/c mice to DEX is oligoclonal and consists almost entirely of Abs bearing the λ L chain (40), and the majority of anti-DEX Abs possess idiotypic determinants cross-reactive with the BALB/c plasmacytoma proteins J558 and M104E (40, 41, 43). Amino acid sequence analysis of DEX-binding hybridoma proteins showed V_H region homology, with most diversity existing in the putative D_H region of the H chain CDR3. It has been shown previously that this region contributes to the individual Id identity expressed by distinct B cell clones (44). Now, by comparing the anti-DEX Ab response of TdT-deficient and TdT-transgenic mice, we show that TdT activity is required for the generation of the optimal anti-DEX Ab response in adult BALB/c mice and the accompanying dominance of the J558 Id.

BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TdT^−/− were originally obtained from Dr. D. Mathis (Harvard Medical School, Boston, MA) (28) and crossed for 10 generations or more to the BALB/c background. TdTS-transgenic mice were developed in our laboratory (37) and were crossed to TdT^−/− mice in this study (TdTS × TdT^−/−). All mice were bred and housed within the specific pathogen-free facility at the University of Alabama and used at 8–12 wk of age according to protocols approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

Mice were immunized i.v. with 10⁸ DEX-expressing heat-killed E. cloacae (strain MK7). ELISA was used to quantify serum Abs by methods described previously (39). Plates were coated with either DEX at 2 μg/ml (Dr. M. Slodki, U.S. Department of Agriculture, Peoria, IL) or the anti-J558 Id Ab EB3–7 at 1 μg/ml (45). Bound serum Igs were developed with alkaline-phosphatase–conjugated anti-IgM for DEX-coated plates or anti-λ for EB3-7–coated plates (Southern Biotechnology Associates, Birmingham, AL).

Single cell suspensions of spleen cells or peritoneal cavity cells were treated with Ab 93 (46) to block FcRs then surface stained with CD19 PE, DEX Ag labeled with Alexa 488, and anti-λ (JC5-1) for staining DEX-binding B cells. The “dump” channel included anti-CD3, anti-CD4, anti-CD8, anti-CD11c, and anti–Gr-1 biotin-labeled Abs, which were then developed with streptavidin-PerCP. DEX⁺λ⁺ plasmablasts (B220^+/−Syndecan-1⁺) were sorted 7 d postimmunization with E. cloacae and genomic DNA was extracted. Cells were sorted into RPMI 1640 medium supplemented with 10% FCS, and the collection tubes were maintained at 4°C throughout the sort. The sorted cells were then centrifuged, and digestion buffer (50 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, and 1% SDS [pH 8.0]) plus proteinase K (0.5 mg/ml) was added to the cell pellet and incubated overnight at 50°C. DNA was precipitated using isopropanol, and the DNA pellet was washed with 70% ethanol, air-dried, and resuspended in molecular grade water. All samples were analyzed using a FACSCalibur flow cytometer or FACSAria cell sorter (BD Biosciences, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR). All Abs were purchased from BD Biosciences, except for Ab 93 and JC5-1, which were developed in our laboratory.

V(D)J rearrangements were amplified by PCR from genomic DNA extracted from sorted plasmablasts using primers specific for gene segments J558.3, V_H 5′-AGCTGCAACAATCTGGACCT-3′, and J_H1, 5′-CCCCAGACATCGAAGTACCA-3′. PCR conditions were 95°C for 3 min then 95°C for 1 min, 58°C for 1 min and 72°C for 1 min for 35 cycles then 72°C for 7 min. RT-PCR for detection of TdTS, and actin was done as described previously (33).

PCR products were subcloned into TOPO-TA vector and transformed into TOP10 competent bacteria (Invitrogen, Carlsbad, CA). Templiphi Amplification Kits were used to isolate plasmid DNA from colonies (Amersham Biosciences, Piscataway, NJ). Plasmid DNA was sequenced at the Center for AIDS Research sequencing facility at the University of Alabama at Birmingham.

Any nucleotides that could not have been derived from a coding sequence or P addition were considered as non-germline nucleotides. In the case of D_H–J_H homology joining, where nucleotides could be assigned to either D_H or J_H, nucleotides were assigned to D_H except when P-addition to J_H gene was identified, in which case the nucleotides were assigned to J_H. HCDR3 was identified as the region between the 3′ V_H encoded conserved cysteine (TGT) at Kabat position 92 (IMGT 104) and the 5′ J_H-encoded conserved tryptophan (TGG) at Kabat position 103 (IMGT 118). Duplicate sequences (identical V_H, D_H, and J_H segments as well as junctional sequences) derived from individual PCR reactions were only counted once. Nucleotide sequences and deduced amino acid translations reported in this article are available at GenBank (www.ncbi.nlm.nih.gov/Genbank/) under the accession numbers GQ478711–GQ478848.

Data comparing three or more groups were analyzed by a one-way ANOVA test, followed by Tukey’s posttest, for data with normal distribution and Kruskal–Wallis test, followed by Dunn’s posttest, for data that did not distribute normally. Data comparing two groups were analyzed by a two-tailed unpaired t test. To determine statistical significance for data comparing proportions, we calculated the 95% confidence interval of the mean for each group. Statistical significance was determined by a p value < 0.05.

In previous ontogenetic studies, we showed that DEX-responsive B cells were first detected in BALB/c mice between 5 and 11 d of age and gradually increased to adult frequencies, which peaked at ≈30 d of age. By using idiotypic markers, it was determined that the M104E Id+ clone predominated during the emergence of the anti-DEX repertoire but was eventually largely replaced by the J558 Id+ clone which normally dominates the adult anti-DEX repertoire (47) (Fig. 1A, 1B). The cross-reactive Id (IdX) was consistently expressed by the majority of DEX-specific clones including J558 and M104E. The J558 and M104E B cell clones were originally defined by the amino acid sequences from corresponding plasmacytomas and have identical H and L chain amino acid sequences, except for two amino acids constituting the H chain CDR3 region (Fig. 1C) (40, 43, 48, 49). Nucleotide sequence alignment of the two clones show that the difference lies in five nucleotides, where TTACGAC (encoding amino acids YD) in the M104E H chain CDR3 is substituted by CCGCTAC, or four nucleotides, where TACGAC is replaced by AGGTAC (encoding amino acids RY) in the J558 H chain CDR3 (50). The differences in the H chain CDR3 of these clones are mostly encoded by D_H gene segment DSP2.2 usage by M104E, DSP2.10 usage (AGGTAC), or J_H1 usage (CCGCTAC) by J558. Both J558 clones show evidence of junctional diversity (P- and N-nucleotides) in contrast to the M104E clone. In summary, the presence of clones with the ability to respond to DEX as well as the pattern of Id expression on anti-DEX Abs is age-dependent, with the major responding clones differing only in their H chain CDR3 regions.

Because the amino acid content of the H chain CDR3 is the major contributor to differences between J558 Id and M104E Id, we examined the Ab response to DEX in wild-type (WT) versus TdT^−/− BALB/c mice. TdT^−/− mice showed a marked reduction in the total IgM anti-DEX response compared with WT mice, at day 7 postimmunization (Fig. 2A) and J558 Id⁺ Ab was barely detectable (Fig. 2B).

To determine whether J558 Id⁺ Abs could be restored by the expression of TdTS, we next immunized transgenic TdTS × TdT^−/− mice. Although the total DEX-specific Ab response elicited in TdTS × TdT^−/− mice was lower than in WT mice (Fig. 3A), J558 Id+ anti-DEX Ab levels were restored to that of the WT (Fig. 3B). We suspect that the lower anti-DEX Ab level may be partly due to N-additions in the λ1 L chain of these TdTS transgenic mice, thus contributing to the partial loss of J558 Id expression. In addition, early forced expression of TdTS may have prevented the generation of the CDR3 that gives rise to the M104E Id, which is expressed by ∼25% of anti-DEX spleen B cell precursors detected in the adult by the splenic focus assay (Fig. 1B). Taken together, these results suggest that the activity of TdTS is involved in the formation of the H chain CDR3 expressed by J558 Id+ anti-DEX Abs.

We next asked whether the frequency of DEX-specific B cells in adult mice is dependent on the expression of TdTS. If this were true then we would expect TdT^−/− animals to have a lower number of DEX-specific B cells than WT mice. Flow cytometric analysis showed that, within the limits of detection of this assay, the absolute cell number of λ L chain expressing B cells that bind to DEX was similar in the spleens and peritoneal cavity cells of TdT^−/− (mean ± SEM, 670 ± 317 and 364 ± 211 cells, respectively) compared with WT mice (475 ± 124 and 171 ± 45 cells, respectively) (Fig. 4A, 4B). As a negative control, we showed that B cells from the C57BL/6 strain, which is a DEX nonresponder strain, did not stain with DEX and anti-λ L chain Ab (Fig. 4A). These results show that the lack of TdTS does not appear to affect the frequency of DEX-binding B cells in the spleen and peritoneal cavity.

To compare molecular associations with Id expression in DEX-responding B cells of WT, TdT^−/−, and TdTS × TdT^−/− mice, we amplified and sequenced H chain V(D)J rearrangements from genomic DNA of DEX⁺λ⁺ plasmablasts sorted from the spleens of E. cloacae-immunized animals. As expected, WT mice expressed mostly the J558 V(D)J rearrangement, giving rise to the amino acids arginine and tyrosine (RY) in HCDR3. In agreement with serum Ab Id expression, TdT^−/− animals expressed a much lower proportion of this V(D)J rearrangement in DEX⁺λ⁺ plasmablasts (Fig. 5A). The frequency of RY-expressing DEX⁺λ⁺ plasmablasts was completely restored in TdTS × TdT^−/− animals. The frequency of M104E CDR3 sequences (YD in HCDR3) detected in TdT^−/− DEX⁺λ⁺ plasmablasts was similar to that of the WT but barely detectable in TdTS × TdT^−/− DEX⁺λ⁺ plasmablasts (Fig. 5A). WT sequences showed a greater number of non-J558 non-M104E, but J558.3-J_H1 expressing, DEX-responsive rearrangements than sequences from TdT^−/− mice. The frequency of non-J558, non-M104E sequences in TdTS × TdT^−/− is also reduced, which, in addition to the absence of M104E clone, could partially explain the lower total DEX Ab response of these animals. These data show that the altered DEX-responsive repertoire in TdT^−/− mice, as shown by the decreased expression of J558 and decreased frequencies of non-J558, non-M104E sequences, is an important factor in the overall decreased Ab response to DEX.

Although TdTS × TdT^−/− DEX⁺λ⁺ plasmablasts showed a restoration of the WT frequency of the dominant J558 sequence, examination of the deduced amino acid sequences of the V(D)J rearrangements amplified from DEX⁺λ⁺ plasmablasts shows that diversity at the H chain CDR3 region was lower than the WT (Fig. 5B). Comparison of nucleotide sequences from WT and TdTS × TdT^−/− H chain CDR3 regions showed that although some of the WT sequences require one or two N-additions for their formation, overexpression of TdTS in TdTS × TdT^−/− mice led to the abolition of these sequences and the emergence of other sequences with longer N-additions. As expected, the diversity in TdT^−/− sequences was also lower compared with WT sequences with the M104E sequence being the most frequently used (Fig. 5C).

To further confirm the requirement for TdTS in the generation of the J558 sequence and to rule out any role for AgR-mediated selection, we examined the expression of TdTS in WT BALB/c day 7 neonates at the time when DEX-responsive B cells are first beginning to appear and correlated it with J558 sequence generation. TdTS mRNA message levels were detected in B220⁺IgM⁻ B pro-B cells in the liver, spleen, and bone marrow (Fig. 6A). We then amplified Ab H chain V(D)J rearrangements that use V_H J558.3 and J_H1, the gene segments used in both M104E and J558 and other reported DEX-specific sequences, from these pro-B cell populations. With the exception of the bone marrow compartment, the expression of the J558 sequence correlated with the expression of TdTS as shown in Fig. 6B. V(D)J rearrangements that give rise to DEX-responsive clones were barely detected in TdT^−/− neonates; however, they were readily detectable in neonates from TdTS × TdT^−/− mice with J558 being the dominant sequence amplified at 7 d of age. In these B220⁺IgM⁻ cells no other (non-J558) sequences that give rise to known DEX-responsive B cells were detected at this time point. These data show that the expression of TdTS coincides with the expression of the J558 V(D)J rearrangement when DEX-responding clones start to emerge. They also show that selection for J558 could be taking place independently of AgR-mediated signals.

Immune protection against encapsulated bacteria, such as S. pneumoniae, N. meningitidis, and H. influenzae, is provided by Abs against the bacterial capsular polysaccharides. We investigated the molecular mechanisms involved in generating an oligoclonal response to a typical type 2 T cell-independent Ag (DEX) and demonstrated that TdTS is important for the optimal Ab responses to DEX. We show that TdTS is required to generate a diverse DEX-responsive repertoire including optimal numbers of the J558 DEX-responsive clone involved in the adult response to DEX.

Earlier studies suggested that TdT is not required for the generation of efficient polyclonal T cell and Ab responses to selected complex protein Ags (29, 51). However, there is mounting evidence that the advantage offered by the diversifying activity of TdT is subtle and is better measured at the level of clonal populations. Heterosubtypic immunity to influenza A virus infection, which affords protection against a virus subtype differing from that used for immunization, is impaired in TdT^−/− mice (52). This observation may be explained in part by the finding that TdT is required for the generation of private influenza virus-specific CD8⁺ repertoires that contribute to enhanced diversity in TCRs across the population of a species as a whole (53). Yewdell and colleagues (54) have also shown that TdT^−/− mice mount a total CD8⁺ response to influenza and vaccinia viruses that is lower in magnitude and breadth compared with WT and that responses to immunodominant viral determinants are reshuffled. From these studies, as well as our findings, it is clear that TdT plays a significant role in the expression of a fully immunocompetent B and T cell repertoire in a given host population.

Alternatively, it has been shown that the absence of TdT expression in neonatal life and the window of limited diversity that ensues are required to generate certain Ab clones with protective, germline-encoded specificity. This was clearly demonstrated in TdTS-transgenic mice, where forced TdT expression exerted its diversifying activity in fetal and neonatal repertoires, with the result that the mice were incapable of establishing the dominant T15 Id+ anti-PC Ab that is protective against S. pneumoniae (37). TdT^−/− mice have also been shown to generate a more robust T15 Ab response than WT animals (38, 55). However, there are clearly other clones that require the activity of TdT for their generation, such as the M603 anti-PC Ab (38). In this study, it was shown that TdT is responsible for the Asn/Asp substitution at the V-D junction of the T15 Id to generate the M603 Id+ clones. In our present study, we reveal another example of the requirement for TdT to generate the J558 Id instead of the germline M104E Id, both of which are responsive to the polysaccharide DEX.

The finding that TdT is required for the expression of the J558 Id may partially explain the observation in our earlier study that: 1) the frequency of anti-DEX spleen precursors, as detected by the splenic focus assay, gradually increases to adult levels subsequent to the onset of TdTS expression; and 2) the Id pattern of the DEX-responding precursors gradually changes from a high M104E/J558 ratio to a much lower one. The difference between the two Ids lies in the H chain CDR3 region, where TdT exerts its diversifying activities. Our data confirm that the M104E sequence is germline as evidenced by its absence in TdTS × TdT^−/− mice, where TdT is expressed constitutively in fetal and neonatal B cell precursors. The J558 Id+ H chain CDR3 could be encoded by DSP2.10 (AGGTAC) or by the nucleotides (CCGCTAC), which could be encoded by the first four nucleotides of the J_H1 segment, a P-nucleotide, an N-nucleotide, and a D_H that cannot be assigned to a known D_H segment. We recover predominantly J558 Id+ sequences that express the latter CDR3 from our DEX⁺λ⁺ plasmablasts after immunization with E. cloacae. This CDR3 region is GC rich, which is also suggestive that TdT is involved in its formation. However, the generation of the J558 Id CDR3 is not absolutely dependent on TdT, because we recover some sequences from TdT^−/− mice that express this Id. These could be generated by the activity of other DNA polymerases, such as DNA polymerase λ (Pol λ), that has been shown to be implicated in modifying gene segment ends during H chain V(D)J rearrangement (56). Examination of Ab responses by Pol λ^−/− × TdT^−/− would test this possibility. However, the two genes are on the same chromosome, and it would be very difficult to obtain double-knockout animals. The alternative would be to test Pol λ^−/− neonates before TdTS is expressed; however, B cells responsive to DEX in BALB/c mice do not emerge until 5 d of age, at which time point TdT is already beginning to be expressed (21, 22). Other members of the DNA Pol X family, which play a significant role in DNA repair, may also be playing a considerable role in V(D)J junctional diversity in the absence of TdT to contribute to these nongermline nucleotides. It is important to note that in Fig. 5A we show that the overall frequency of DEX-specific plasmablasts of TdT^−/− mice postimmunization is lower than that of the WT. This may account for our observation that J558 Id-expressing Abs in the serum of immunized TdT^−/− mice are very low (3–58 μg/ml) or not detected by ELISA. It is possible that the higher affinity of the rare J558 clones that we find in TdT^−/− mice could lead to their preferential enrichment in the pool of DEX-specific cells in our FACS sort. The frequency of DEX⁺ plasmablasts in Fig. 5A suggests that the baseline numbers of J558 Id+ B cells in TdT^−/− are much lower than WT. We have attempted to determine these baseline numbers by direct FACS staining of J558 Id+ B cells using EB3-7 (anti-J558 Id); however, the background staining was too high to make any meaningful interpretation of the data. Alternatively, our method of sequence analysis, which excludes repeat sequences, may have led to an underrepresentation of the M104E germline sequences obtained from TdT^−/− plasmablasts. Regardless of the mechanism by which the J558 sequence is generated in the absence of TdTS, it is not as efficient in generating this clone as in WT mice.

The possibility exists that the absence of TdT alters the repertoire of other B cells that would have otherwise contributed to the selection of the J558 clone. There is evidence supporting a role for B cell idiotypic interactions early in life that modulate the development of B cell clones responsive to DEX and to other T-independent Ags (57, 58). Neonatal injection of the anti-PC Abs, such as the T15 Ab that is presumably more abundant in TdT^−/− mice, leads to a reduction in J558 Id expression when these mice were immunized with DEX as adults (59). Therefore, the mechanism by which J558 Id expression requires TdTS may be indirect and involves the selection of J558-expressing clones as a result of B cell interactions with other B cells or other immune cells.

However, the most likely explanation of our findings is that the J558 clone, along with HDEX9 (K_a = 2.0 × 10⁴) and HDEX37 (K_a = 5.3 × 10⁵) (all of which use RY amino acids at their CDR3 regions) show the highest affinity to DEX (60, 61). HDEX9 and HDEX37 sequences were not detected in our PCR assays because they use different V_H and J_H segments, respectively. The difference in Ab affinity between J558 and the germline M104E to DEX is ∼100-fold (K_a = 1.4 × 10⁵ versus 1.3 × 10³) (60). This illustrates the importance of TdT in the generation of clones of higher affinity and which are likely involved in the higher Ab response in WT versus TdT^−/− mice. The higher affinity of the J558 clone may also be responsible for its selective expansion in the splenic marginal zone and the peritoneal B1b cell compartment under the influence of environmental or self-Ags (10).

The diversity of the anti-DEX Ab response contributed by TdT is also evident in the increased number of non-J558 and non-M104E sequences recovered from immunized WT versus TdT^−/− animals. The activity of TdT promotes a more diverse usage of D_H segments and less homology-mediated joining that is characteristic of fetal and neonatal sequences. This is still evident despite the fact that we only amplified J558.3V_H− J_H1 rearrangements with the important limitation that TdT^−/− mice may be forced to generate DEX-specific rearrangements that do not use J558.3V_H− J_H1 because of their limited ability to generate the J558 rearrangement. However, the response in BALB/c mice is known to be pauciclonal, and the two major anti-DEX clones (J558 and M104E) use these specific gene segments. Overexpression of TdTS in TdTS × TdT^−/− mice did not restore this element of Ab diversity. However, TdTS × TdT^−/− mice are capable of harboring N-additions in their L chains, which in turn might compromise the ability of clones to bind to DEX, thus decreasing the frequency of overall DEX-binding plasmablasts that were sorted. Therefore, sequences recovered from these plasmablasts might not show the same level of diversity as those from WT mice. An examination of the nucleotide sequences of V(D)J rearrangements amplified postimmunization from DEX-specific plasmablasts of these mice reveals that it is also possible that supra-physiological levels of TdTS activity might obliterate the generation of some of the DEX-specific sequences normally recovered from WT animals.

Examination of neonatal pro-B cells in the liver and spleen revealed a correlation between TdTS and J558 expression. This correlation did not hold true for neonatal bone marrow cells, perhaps because the bone marrow has not been colonized with precursors capable of generating J558 at that point. Our assay did not detect other non-J558 sequences that would give rise to DEX-specific B cells in all neonatal pro-B cells examined, perhaps because the ontogenetic development of these clones is different, and they may be generated at a later time point than 7 d after birth when the samples were taken. However, J558 contributes to ∼70% of the adult response and detection of other DEX-responsive non-J558 sequences may require a very large sample size.

Most of the neonatal sequences we examined from spleen and liver pro-B cells could potentially give rise to J558 Id-expressing Ab. This suggests that selection, independent of BCR-mediated signaling, is taking place in these pro-B cells. J558-expressing H chain could be associating with the surrogate L chain in such a manner that leads to efficient pre-BCR assembly, followed by rapid proliferation. Wasserman et al. (62) showed by transfecting different H chains in pro-B cell lines, that bias in V_H usage in the B cell repertoire can depend on the nature of the interactions between the H chain V(D)J rearrangement and the surrogate L chain which leads to different outcomes in fetal versus adult pro-B cells.

This report sheds light on the role of TdT on the host Ab response to a polysaccharide Ag. Abs vary in their protective abilities and our findings highlight another factor that may be considered to explain the molecular mechanism of these differences. A number of DNA polymerases have been shown to play a role in modulating H and L chain junctional diversity (56, 63). It would be of interest to examine the role of these factors in modulating the Ab response to polysaccharides. These findings have further implications for the observed differences in the ability of neonatal versus adult B cell repertoires to respond to polysaccharide Ags. The results lend support to the hypothesis that in some instances the poor neonatal response to polysaccharides may be due to the lack of B cell clones expressing the appropriate BCR.

We thank Jeremy Foote for very helpful discussions and Dr. Nicholas Kin for critically reading the manuscript.

Disclosures The authors have no financial conflicts of interest.