The CDR-H3 repertoire from TdT-deficient adult bone marrow is a close, but not exact, homologue of the CDR-H3 repertoire from perinatal liver.

Compared with adult bone marrow (BM), the composition of the perinatal liver CDR-3 of the Ig H chain (CDR-H3) repertoire is marked by a paucity of N nucleotides and by enrichment for use of J(H) proximal DQ52 and D(H) proximal V(H) and J(H) gene segments. To test the extent to which these differences reflect limited perinatal TdT activity versus differences in the fetal/adult environment, we used the Hardy scheme to sort fractions B-F B lineage cells from TdT-deficient BALB/c adult BM. V(H)7183-containing VDJCµ transcripts from these cells were amplified, cloned, sequenced, and compared with transcripts from wild-type perinatal liver and adult BM. The pattern of V(H)DJ(H) usage in TdT-deficient BM largely matched that of TdT-sufficient adult cells. What minor differences were detected in the pro-B cell stage tended to diminish with B cell maturation, suggesting strong environmental or Ag-driven pressure to achieve a specific range of V(H)DJ(H) usage regardless of the extent of N nucleotide addition. However, although the patterns of V(H)DJ(H) usage in the TdT-deficient B lineage cells paralleled that of wild-type adult cells, the length distribution, global amino acid composition, and charge distribution of the CDR-H3 repertoire proved to be a close, although not exact, homologue of the CDR-H3 repertoire first expressed by late pre-B cells in the TdT-insufficient perinatal liver. Thus, although differing in V(H) content, TdT-deficient mice appear to represent a good, although not perfect, model for testing the role of perinatal CDR-H3 limitations on late B cell development and Ab responses.

Terminal deoxynucleotidyl transferase is required for an optimal response to the polysaccharide α-1,3 dextran.

An understanding of Ab responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of Ags. In this study, we have investigated the role of the lymphoid-specific enzyme TdT in generating B cell clones responsive to alpha-1,3 dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte AgRs during V(D)J recombination. In this study, we show that the DEX-specific Ab response is lower, and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT(-/-) mice when compared with wild-type (WT) BALB/c mice. Nucleotide sequencing of H chain CDR3s of DEX-specific plasmablasts, sorted postimmunization, showed that TdT(-/-) mice generate a lower frequency of the predominant adult molecularly determined clone J558. Complementation of TdT expression in TdT(-/-) mice by early forced expression of the short splice variant of TdT-restored WT proportions of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 d after birth in IgM-negative B cell precursors in the liver and spleen of WT and TdT-transgenic mice but not in TdT(-/-) mice. These data show that TdT is essential for the generation of the predominant higher-affinity DEX-responsive J558 clone.

Altered immunodominance hierarchies of influenza A virus-specific H-2(b)-restricted CD8+ T cells in the absence of terminal deoxynucleotidyl transferase.

Immunodominance is considered an obstacle to successful T cell-based vaccination, and constant efforts are made to uncover the underlying mechanisms for this phenomenon. We have examined the contribution of terminal deoxynucleotidyl transferase (TdT), whose function accounts for approximately 90% of T cell receptor diversity, to dominance hierarchies of H-2(b)-restricted flu-specific T(CD8+). Using intracellular cytokine staining to quantitatively detect epitope-specific T(CD8+), we demonstrate that TdT-deficient mice exhibit a distinct hierarchical pattern in their primary and recall T(CD8+) responses to influenza A viruses, which results from skewed responsiveness towards select influenza epitopes. Our data establish a link between TdT and immunodominance in H-2(b)-restricted antiviral T(CD8+) responses.

Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses.

Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.

Terminal deoxynucleotidyl transferase establishes and broadens antiviral CD8+ T cell immunodominance hierarchies.

The action of TdT on mouse TCR genes accounts for approximately 90% of T cell repertoire diversity. We report that in TdT-/- mice, total T(CD8+) responses to influenza and vaccinia viruses are reduced by approximately 30% relative to wild-type mice. We find that T(CD8+) responses to three subdominant influenza virus determinants are reduced to background values in TdT-/- mice while responses to three immunodominant determinants undergo a major reshuffling. A similar reshuffling occurs in T(CD8+) responses to immunodominant vaccinia virus determinants, and is clearly based on broad differences in TCR family usage and CDR3 length between wild-type and TdT-/- mice. These findings demonstrate that TdT plays a critical role in the magnitude and breadth of anti-viral T(CD8+) responses toward individual determinants and suggests that germline TCR repertoire bias toward the most dominant determinants is a major factor in establishing immunodominance hierarchies.

Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire.

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT(-/-)) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D(H)) with an altered D(H) segment (namely, Delta D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT(-/-) mice compared to those in Delta D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.

Paucity of V-D-D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and TdT+/+ mice.

CDR3 regions containing two D segments, or containing the footprints of V(H) replacement events, have been reported in both mice and humans. However, the 12-23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for V(H) replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential V(H) replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the "cryptic heptamer" but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and V(H) replacement are rare occurrences in the mouse.

Valpha and Vbeta public repertoires are highly conserved in terminal deoxynucleotidyl transferase-deficient mice.

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Valpha-Jalpha and Vbeta-Dbeta-Jbeta rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the alphabeta TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT(o/o) mice, even for the CDR3 loops which are shorter than those found in TdT(+/+) animals. Moreover, the 10- to 20-fold decrease in alphabeta T cell diversity in TdT(o/o) mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3beta in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3beta. We interpret our findings as suggesting that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHCalpha helices are involved in the selection of public repertoires.

Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice.

The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Fas(lpr) mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT(-/-) mice, and reduced glomerulonephritis and increased life span in MRL-Fas(lpr) TdT(-/-) mice. Using tetramer staining, TdT(-/-) and TdT(+/+) NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4(+) T cells. We found no increase in CD4(+)CD25(+) regulatory T cells in NOD TdT(-/-) mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.

The M603 idiotype is lost in the response to phosphocholine in terminal deoxynucleotidyl transferase-deficient mice.

The majority of anti-phosphocholine (PC) antibodies induced by the PC epitope in Proteus morganii (PM) express the M603 idiotype (id), which is characterized by an invariant Asp to Asn substitution at the V(H):D(H) junction. To elucidate the molecular basis by which M603-like B cells acquire the mutations resulting in this invariant substitution, we analyzed the immune response to PC-PM in terminal deoxynucleotidyl transferase (TdT) gene knockout (KO) mice. In the absence of TdT, T15-id antibodies comprised 80-100% of the primary response to PC-PM. Less than 10% of the response in wild-type mice is T15-id(+). In TdT KO mice, the secondary response to PC-KLH was higher than in wild-type mice and was dominated by the germ-line T15-id. About 10% of this response, in both TdT KO and wild-type mice, comprised M167-id(+) antibodies. Additionally, none of the functionally rearranged V1/DFL16.1/J(H)1 cDNA isolated from PC-PM-immunized TdT KO mice showed the Asp/Asn substitution characteristic of PC-binding, PC-PM-induced M603-like antibodies. These data indicate that production of M603-id antibody is TdT dependent, while generation of M167-id antibody is TdT independent, and that in the absence of competition from M603-like B cells, T15-id B cells can respond to PC-PM.

Terminal deoxynucleotidyl transferase deficiency decreases autoimmune disease in MRL-Fas(lpr) mice.

The neonatal Ab and TCR repertoires are much less diverse, and also very different from, the adult repertoires due to the delayed onset of terminal deoxynucleotidyl transferase (TdT) expression in ontogeny. TdT adds nontemplated N nucleotides to the junctions of Igs and TCRs, and thus its absence removes one of the major components of junctional diversity in complementarity-determining region 3 (CDR3). We have generated TdT-deficient MRL/lpr, Fas-deficient (MRL-Fas(lpr)) mice, and show that they have an increased lifespan, decreased incidence of skin lesions, and much lower serum levels of anti-dsDNA, anti-chromatin, and IgM rheumatoid factors. The generalized hypergammaglobulinemia characteristic of MRL-Fas(lpr) mice is also greatly reduced, as is the percentage of CD4(-)CD8(-)B220(+) (double-negative) T cells. IgG deposits in the kidney are significantly reduced, although evidence of renal disease is present in many mice at 6 mo. CDR3 regions of both IgH and TCR from peripheral lymphocytes of MRL-Fas(lpr) mice are shorter in the absence of TdT, and there is a paucity of arginines in the IgH CDR3 regions of the MRL-Fas(lpr) TdT(-/-) mice. Because the amelioration of symptoms is so widespread, it is likely that the absence of N regions has more of an affect than merely decreasing the precursor frequency of anti-dsDNA B cells. Hence, either the T or B cell repertoires, or more likely both, require N region diversity to produce the full spectrum of autoimmune lupus disease.

Evidence that terminal deoxynucleotidyltransferase expression plays a role in Ig heavy chain gene segment utilization.

TdT is a nuclear enzyme that catalyzes the addition of random nucleotides at Ig and TCR V(D)J junctions. In this paper we analyze human IgH rearrangements generated from transgenic minilocus mice in the presence or absence of TdT. In the absence of TdT, the pseudo-VH gene segment present in the minilocus is rearranged dramatically more frequently. Additionally, JH6 gene segment utilization is increased as well as the number of rearrangements involving only VH and JH gene segments. Thus, the recombination of IgH gene segments that are flanked by 23-nt spacer recombination signal sequences may be influenced by TdT expression. Extensive analysis indicates that these changes are independent of antigenic selection and cannot be explained by homology-mediated recombination. Thus, the role played by TdT may be more extensive than previously thought.

Effect of a genetic deficiency of terminal deoxynucleotidyl transferase on autoantibody production by C57BL6 Fas(lpr) mice.

Terminal deoxynucleotidyl transferase (TdT) adds nontemplate coded nucleotides (N additions) between the recombining ends of immunoglobulin and T cell receptor genes. These nucleotides add significant diversity to the Ig and TCR repertoires. Amino acids coded for by these nucleotides play a key role in the binding of self antigens by autoantibodies and autoreactive T cells. To determine the effect of a lack of N additions on autoantibody production, we bred the TdT knockout genotype onto the autoimmune C57BL/6-Fas(lpr) background. TdT-deficient mice had significantly lower sera anti-DNA and rheumatoid factor activity than their TdT-producing littermates. C57BL/6-Fas(lpr) TdT-deficient mice had shorter VH CDR3 regions and fewer VH CDR3 arginines [0.6% versus 4. 7%] than their TdT-producing littermates. These data indicate that the absence of TdT limited the production of anti-DNA antibodies and rheumatoid factors in C57BL/6-Fas(lpr) mice, likely due to constraints on Ig diversity secondary to the lack of TdT-derived N additions.

Terminal deoxynucleotidyl transferase deficiency reduces the incidence of autoimmune nephritis in (New Zealand Black x New Zealand White)F1 mice.

Terminal deoxynucleotidyl transferase (TdT) enzyme activity in lymphocytes generates diversity in the Ag receptor repertoires by adding template-independent N nucleotides and disrupting homology-directed rearrangements. The importance of this diversity in vivo and the significance of the suppression of TdT during fetal life remain uncertain. Previous studies have shown that in TdT knockout mice (TdT(0)) 1) the T cell repertoire is less peptide oriented; and 2) natural autoantibody, particularly anti-DNA autoantibodies, are less polyreactive, and their mean affinities are reduced. Consequently, the suppression of TdT during early T/B cell ontogeny may participate in controlling autoimmunity. To study the impact of TdT suppression in autoimmune-prone mice, we introduced the TdT null mutation into the (NZB x NZW)F1 (B/W) mouse strain. We show that TdT deficiency significantly reduces the incidence of autoimmune nephritis and prolongs survival compared with those in control mice. Surprisingly, the long-term survivor TdT(0) mice produced amounts of anti-ADN and anti-histone autoantibodies similar to those of their TdT+ littermates. However, these TdT(0) mice showed no evidence of renal inflammation, and the immune deposits were restricted to the mesangium, whereas basal membrane deposits were clearly correlated with overt renal disease. The present study supports the idea that the absence of TdT enzyme activity in lymphocytes protects mice against autoimmunity and could offer a therapeutic approach to autoimmune diseases. Moreover, our results may help to unravel the mechanisms of lupus nephritis.

Autoantibodies in mice lacking terminal deoxynucleotidyl transferase: evidence for a role of N region addition in the polyreactivity and in the affinities of anti-DNA antibodies.

The generation of terminal deoxynucleotidyl transferase knockout mice (TdT0) has demonstrated that TdT is the only major activity involved in N region addition. This enzyme generates diversity by adding random nucleotides at the V-D-J junctions and by disrupting the formation of repetitive "homology-directed" junctions. Several studies have demonstrated that the Ig heavy chain third complementarity-determining region (H-CDR3) and the N region play a critical role: 1) in distinguishing between polyreactive and monospecific combining sites in natural and Ag-induced Abs; and 2) in the specificity and polyreactivity of natural autoantibodies (autoAbs) and in particular of anti-DNA Abs. To examine the impact of the lack of TdT on the natural autoAb repertoire in adult mice, we have stimulated TdT0 and TdT+ littermates with LPS. Serum studies demonstrate that TdT is not critical for the generation of B cells expressing autoAbs including anti-DNA Abs and rheumatoid factors. However, the generation of a large collection of hybridomas indicates that the frequencies of these cells are reduced in TdT0 mice mainly due to a lower incidence of polyreactivity; also, the lack of N region diversity seems to negatively affect the affinity of anti-DNA Abs. The physiologic relevance of these data is discussed.

Repertoires of antigen receptors in Tdt congenitally deficient mice.

Tdt deficient mice show lack of N region in V(D)J junctions of immunoglobulin and T cell receptor genes and revealed that "immature recombinase" in fetal stage would boil down to no more than a lack of Tdt. Although particular junctions which are thought to be created by homology-mediated joining are frequently observed, one fourth of junctions lacked even one bp of overlap, indicating the existence of a V(D)J joining pathway that is homology independent. Lymphocyte repertoire which express VH81X gene without N region is negatively selected, which shows that the repertoire of Tdt deficient mice is not a truly fetal repertoire. Positive selection of thymocytes is more efficient in Tdt deficient mice. Furthermore Tdt-/- mice produce significant amounts of anti-dsDNA antibodies as Tdt+/+ mice, indicating that increased diversity of the third complementarity-determining region (CDR3) by Tdt is not essential for the expansion of precursor B cells programmed to produce anti-DNA antibodies.

Increased peptide promiscuity provides a rationale for the lack of N regions in the neonatal T cell repertoire.

Making use of mice deficient for terminal deoxynucleotidyl transferase (TdT) expression and a random peptide library, we have examined the diversity and peptide specificity of the neonatal T cell repertoire specific for a single H-2Db-restricted peptide. Consistent with the predicted decrease in repertoire diversity, polyclonal CTL lines and individual clones from different TdTo mice are more similar to each other than those from different wild-type mice in terms of their fingerprints of cross-reactivity to the library and their TCR sequences. We have also found that several TdTo CTL clones cross-react with many more library peptides than wild-type CTL clones. In a few instances, the degree of peptide promiscuity correlates with TCR sequence characteristics such as N region addition and homology-directed recombination, but not CDR3 loop length. Based on epitope titrations for each clone, TCR affinity for antigen is consistently high; thus, this reduced specificity for peptide may coincide with an accentuated affinity for the alpha helices of the MHC. Peptide promiscuity in the neonate may allow the relatively small numbers of T cells in the periphery to protect against a broader range of pathogens.

Mice lacking terminal deoxynucleotidyl transferase: adult mice with a fetal antigen receptor repertoire.

TdT knock-out mice have established the role of this enzyme in vivo: TdT mediates the transition from the relatively limited fetal to the highly diverse adult antigen receptor repertoire by adding template independent "N" nucleotides and disrupting homology-directed recombination. Lacking this source of diversity, TdT degree mice harbor essentially fetal antigen receptor repertoires. In alpha beta TCRs, the TdT null mutation affects the length and diversity of the CDR3 loops thought to be important in "directing" MHC/peptide recognition. N- CDR3 loops appear to wield less influence than do their N+ counterparts--positive selection is more efficient in the TdT degree animals and the peripheral repertiore is more polyreactive and less peptide-oriented than is the N+ repertoire. However, this loss of specificity does not markedly diminish the response to specific peptides. Overall, mice harboring essentially fetal repertoires are robust and effectively respond to a wide variety of challenges to the immune system.

Coding end processing is similar throughout ontogeny.

During the recombination process, extensive processing of the coding ends provides tremendous potential diversity to the joint of any two gene segments. However, the diversity of the newborn B and T cell repertoires is greatly reduced compared with that of the adult. At the mechanistic level, this difference is primarily due to the absence of terminal deoxynucleotidyltransferase expression until the first week after birth. Additionally, one direct consequence of the lack of N regions early in ontogeny is the more frequent occurrence of homology-directed recombination, reducing even further the potential of diversity. Other enzymatic factors could also contribute to this ontogenic difference. However, the use of the homology-directed recombination pathway early in life obscures the analysis of the coding end processing. In this study we compared the coding end processing throughout ontogeny, in normal and terminal deoxynucleotidyltransferase -/- mice in the presence of minimal homology-directed recombination. The analysis of partial D-J joints allowed us to avoid potential bias by early selection events. Our results show that the extent of nucleotide deletion of a given end is consistent throughout ontogeny in the presence or absence of terminal deoxynucleotidyltransferase. However, a distinctive processing pattern is observed for each coding end.