Functional analyses of polymorphic variants of human terminal deoxynucleotidyl transferase.

Human terminal deoxynucleotidyl transferase (hTdT) is a DNA polymerase that functions to generate diversity in the adaptive immune system. Here, we focus on the function of naturally occurring single-nucleotide polymorphisms (SNPs) of hTdT to evaluate their role in genetic-generated immune variation. The data demonstrate that the genetic variations generated by the hTdT SNPs will vary the human immune repertoire and thus its responses. Human TdT catalyzes template-independent addition of nucleotides (N-additions) during coding joint formation in V(D)J recombination. Its activity is crucial to the diversity of the antigen receptors of B and T lymphocytes. We used in vitro polymerase assays and in vivo human cell V(D)J recombination assays to evaluate the activity and the N-addition levels of six natural (SNP) variants of hTdT. In vitro, the variants differed from wild-type hTdT in polymerization ability with four having significantly lower activity. In vivo, the presence of TdT varied both the efficiency of recombination and N-addition, with two variants generating coding joints with significantly fewer N-additions. Although likely heterozygous, individuals possessing these genetic changes may have less diverse B- and T-cell receptors that would particularly effect individuals prone to adaptive immune disorders, including autoimmunity.

Coding joint diversity in mature and immature B-cell lines.

Antigen receptor gene rearrangement is regulated by many factors in B and T lymphocytes. The sequences of the gene segments themselves, their associated recombination signal sequences (RSS), expression of the RAG genes and the chromatin accessibility of the particular gene segments to be rearranged all influence the outcome of recombination and thus antigen receptor diversity. In the present study, we have evaluated the effect of variations in RAG activity level on the junctional diversity of coding joint sequences. Using the pre-B-like 204-1-8 and the mature B DR3 cell lines under different transfection conditions, we were able to investigate recombination activity levels that varied 100-fold. We evaluated the sequences of the coding joints for junctional diversity resulting from nucleotide addition or deletion. Surprisingly, we found that the sequence of coding joints of these recombinants did not exhibit significant variation despite the large difference in recombination frequency. Our results indicate that the fidelity of the joining phase of V(D)J recombination is not jeopardized by varying RAG activity.

Valpha gene replacement in a TCRalpha knock-in mouse.

Using a TCRalpha chain knock-in mouse, we demonstrate that V-gene replacement can operate in the T cell receptor alpha locus. Functional TCRalpha chain transcripts generated by Valpha-gene replacement at the site of the Valpha-embedded heptamer were identified in splenic T cells. This finding shows that Valpha-gene replacement can likely be used to shape the peripheral T cell repertoire. The conservation of the embedded heptamer in most Valpha segments adds support to the notion that V-gene replacement is a mechanism maintained to diversify the immune system and that argues that it is common to B and T cells.

VH gene replacement in thymocytes.

The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V(H) gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V(H) gene replacement. In normal mice, thymic DJ(H) rearrangements are common, but VDJ(H) rearrangements are not. We found intermediate products of V(H) replacement in double-positive CD4(+)CD8(+) cells of the QM thymus, demonstrating that the inserted V(H) gene was accessible and ruling out the possibility that a V(H) gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no mu H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V(H) gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V(H)-to-VDJ(H) joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V(H) joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.

Functional analysis of the human RAG 2 promoter.

Recombination activating genes RAG1 and RAG2 are essential components of V(D)J recombination, a process that generates the specific antigen receptors in lymphocytes. To understand the mechanisms underlying the lineage and developmental regulation of transcription of RAG2, we have characterized the human RAG2 exon 1A promoter. In this study, a series of deletion constructs were used to isolate the promoter while a linker scanning approach was taken to assess functionally relevant cis elements within the promoter. Two regulatory domains were identified. The -140 to -123 region is critical for promoter activity in all cell lines tested. Mutations to the putative Ets (-122 to -118) or to the C/EBP (-137 to -129) consensus core sequences did abrogate promoter activity, although specific DNA/protein interactions remained, as determined by EMSA. The -69 to -48 region demonstrates lineage specific promoter activity. Mutations to an overlapping, BSAP-myb-Ikaros-myb site (-65 to -39) resulted in differential promoter activity in human B and T cells. EMSA analysis of this region showed a B cell specific protein complex. Transfection of BSAP into cell lines trans-activates the human RAG2 promoter. We conclude that transcriptional regulation of the human RAG2 gene is complex, involving both tissue specific and ubiquitous factors, and both proximal and distal regulatory elements.

Evolution of the recombination signal sequences in the Ig heavy-chain variable region locus of mammals.

The Ig and T cell receptor (TCR) loci have an exceptionally dynamic evolutionary history, but the mechanisms responsible remain a subject of speculation. Ig and TCR genes are unique in vertebrates in that they are assembled from V, D, and J segments by site-specific recombination in developing lymphocytes. Here we examine the extent to which the V(D)J recombination in germline cells may have been responsible for remodeling Ig and TCR loci in mammals by asking whether gene segments have evolved as a unit, or whether, instead, recombination signal sequences (RSSs) and coding sequences have different phylogenies. Four distinct types of RSS have been defined in the human Ig heavy-chain variable region (Vh) locus, namely H1, H2, H3, and H5, and no other RSS type has been detected in other mammalian species. There is a well-supported discrepancy between the evolutionary history of the RSSs as compared with the Vh coding sequences: the RSS type H2 of one Vh gene segment has clearly become replaced by a RSS type H3 during mammalian evolution, between 115 and 65 million years ago. Two general models might explain the RSS swap: the first involves an unequal crossing over, and the second implicates germline activation of V(D)J recombination. The Vh-H2/RSS-H3 recombination product has likely been selected during the evolution of mammals because it provides better V(D)J recombination efficiency.

Specific antibody production by V(H)-gene replacement.

V(H)-gene replacement is a recombination event in which a pre-existing immunoglobulin heavy chain gene can be altered by the replacement of the rearranged V(H) gene segment with another V(H) gene segment. Although this event has been demonstrated in various model systems, its role in generating antibody diversity is still unsettled. We have used a genetically modified mouse strain, QM, with a quasi monoclonal primary B cell repertoire specific for NP to determine whether V(H) gene replacement can generate a new antigen specificity. Hybridomas generated from QM splenocytes after immunization with different antigens, gave rise to antibodies with specificity to the immunizing antigen or with new specificities. We found V(H)-gene replacement was used to change the original heavy chain gene rearrangement specific for NP into a heavy chain gene encoding the new antigen specificity. V(H)-gene replacement intermediates were detected both before and after the immunization, suggesting that the event was selective rather than instructive. These results demonstrate that V(H)-gene replacement can generate a new antibody heavy chain gene with a different functional and selectable antigen specificity.

Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis.

We report here the molecular cloning of a newly identified preprotachykinin gene, Pptc, which specifies the sequence for a new preprotachykinin protein and bioactive peptide designated hemokinin 1 (HK-1). PPT-C mRNA was detected primarily in hematopoietic cells in contrast to the previously described Ppta and Pptb genes, which are predominantly expressed in neuronal tissues. HK-1 has several biological activities that are similar to the most studied tachykinin, substance P, such as induction of plasma extravasation and mast cell degranulation. However, HK-1 also has properties that are indicative of a critical role in mouse B cell development. HK-1 stimulated the proliferation of interleukin 7-expanded B cell precursors, whereas substance P had no effect. HK-1, but not substance P, promoted the survival of freshly isolated bone marrow B lineage cells or cultured, lipopolysaccharide-stimulated pre-B cells. N-acetyl-L-trytophan-3,5-bistrifluromethyl benzyl ester, a tachykinin receptor antagonist, increased apoptosis of these cells and in vivo administration of this antagonist led to specific reductions of the B220lowCD43 population (the pre-B cell compartment) in the bone marrow and the IgMhighIgDlow population (the newly generated B cells) in the spleen. Thus, HK-1 may be an autocrine factor that is important for the survival of B cell precursors at a critical phase of development.

Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.

Hypothesis: genes which function in a stochastic lineage commitment process are subject to monoallelic expression.

The collection of genes which are now known to be monoallelically expressed in mammals is a diverse set. In the case of the genes which encode transducing receptors, such as immunoglobulins or odoront receptors, monoallelic expression ensures that cell activity is related to encountering a unique ligand. However, some monoallelically expressed genes do not encode receptors, and in these cases the physiological purpose of monoallelic expression is uncertain. Even more puzzling are the cases of imprinted genes, where only the maternal or only the paternal allele is expressed. In this article we consider the hypothesis that some of these cases of monoallelic expression reflect the unusual instances in development in which lineage commitment results from a selective rather than an instructive mechanism. These mechanisms are distinguished by their reliance on either external signals (instructive) or internal, cell autonomous events (selective) to cause the changes in gene expression which correspond to lineage commitment. While the instructive mechanism predicts that lineage commitment genes will be expressed or silenced biallelically, the selective mechanism predicts that commitment genes will be subject to monoallelic expression. Specifically, for the cases in which lineage commitment results from activating gene expression, the selective mechanism predicts that commitment genes will be monoallelically expressed following commitment, such as observed recently for some cytokine and transcription factor genes. For the cases in which extinction of gene expression causes commitment, the selective mechanism predicts that the commitment genes will be monoallelically expressed prior to commitment, as for X-linked and imprinted genes.

The role of components of recombination signal sequences in immunoglobulin gene segment usage: a V81x model.

It has long been appreciated that some immunoglobulin (and T-cell receptor) gene segments are used much more frequently than others. The VHsegment V81x is a particularly striking case of overusage. Its usage varies with the stage of B-cell development and with the strain of mice, but it is always high in B cell progenitors. We have found that the coding sequence and the recombination signal sequences (RSS) are identical in five mouse strains, including CAST/Ei, a strain derived from the species Mus castaneus. Thus, the strain differences cannot be attributed to sequences within V81x itself. V81x RSS mediated recombination at rates significantly higher than another VHRSS. Although the V81x nonamer differs at one base pair from the consensus sequence, an RSS with this nonamer and a consensus heptamer recombines as well as the consensus RSS. When the V81x spacer is replaced by that of VA1, the frequency of recombination decreases by approximately 5-fold; thus, the contribution of variation in natural spacers to variability in VHusage in vivo is likely to be more than has been previously appreciated. Furthermore, the contribution of the heptamer and nonamer to differential VHusage in our assay is correlated inversely with their conservation throughout the VHlocus.

Extensive junctional diversity in Ig light chain genes from early B cell progenitors of mu MT mice.

Nontemplated (N) nucleotide additions contribute significantly to the junctional diversity of all Ag receptor chains in adult mice except Ig light (L) chains, primarily because terminal deoxynucleotidyl transferase (TdT) expression is turned off at the time of their rearrangement in pre-B cells. However, because some Ig L chain gene rearrangements are detectable earlier during B cell ontogeny when TdT expression is thought to be maximal, we have examined the junctional processing of kappa- and lambda-chain genes of CD45(B220)+CD43+ pro-B cells from mu MT mice. We found that both kappa and lambda coding junctions formed in these B cell precursors were extensively diversified with N-region additions. Together, these findings demonstrate that Ig L chain genes are equally accessible to TdT in pro-B cells as Ig heavy chain genes. Surprisingly, however, the two L chain isotypes differed in the pattern of N addition, which was more prevalent at the lambda-chain locus. We observed the same diversity pattern in pre-B cells from TdT-transgenic mice. These results suggest that some aspects of TdT processing could be influenced by factors intrinsic to the sequence of Ig genes and/or the process of V(D)J recombination itself.

A mouse with a monoclonal primary immunoglobulin repertoire not further diversified by V-gene replacement.

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.

Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression.

The IL-7R and the pre-B cell receptor (pre-BCR) each provide critical signals during differentiation of B cell precursors. In this study we examine the interplay between signals dependent upon these receptors. We demonstrate that pre-BCR-deficient pro-B cells differ significantly from controls in their ability to use the IL-7R. We show that this difference, characterized by a failure to proliferate in response to IL-7, is narrowly restricted to IL-7 concentrations in the picogram per milliliter range and can be overcome with increasing amounts of IL-7. Restoration of Ig heavy chain to recombinase-activating gene-2-deficient pro-B cells leads to a restored response to picogram per milliliter levels of IL-7, providing strong evidence that modulation of the IL-7 dose-response threshold is dependent on pre-BCR. Culture of normal pro-B cells under low IL-7 conditions leads to selective outgrowth of cells expressing mu heavy chain, suggesting that modulation of IL-7 dose-response thresholds can allow for selective expansion of pre-BCR+ cells under conditions where IL-7 is limiting. We also provide evidence that expression of pre-BCR on pro-B cells limits the duration of IL-7 responsiveness by causing differentiation to an IL-7-unresponsive pre-B cell stage. Thus, the pre-BCR-dependent modulation of IL-7 responsiveness affects both the dose-response threshold and the duration of IL-7-induced clonal expansion. Our results suggest that positive selection of pre-BCR+ pro-B cells may be achieved through the fine tuning of IL-7 responses.

Terminal deoxynucleotidyl transferase expression during neonatal life alters D(H) reading frame usage and Ig-receptor-dependent selection of V regions.

During neonatal life, Ig diversity is limited in many respects. The absence of terminal deoxynucleotidyl transferase (TdT) expression with the consequent lack of nontemplated addition during the neonatal period, coupled with the predominant usage of a single D(H) reading frame (RF), leads to severe limitations of diversity in the CDR3 region of Ig heavy (H) chains. The neonatal Ig H chain repertoire is also characterized by restricted V(H) usage, with predominant expression of certain V(H) segments, such as V(H)81x, that are rarely evident during adult life. In this report, we examine the effect of enforced TdT expression on the neonatal repertoire of V(H)81xDJ(H) rearrangements. We find that TdT synthesis abrogates D(H) RF bias during the fetal/neonatal period through a Ig-receptor-independent mechanism. These findings suggest that D(H) RF bias during neonatal life is determined largely by homology-directed joining. We also find that TdT synthesis alters the selection of productively rearranged V(H)81xDJ(H) alleles in the neonatal spleen through a Ig-receptor-dependent mechanism. Analysis of predicted CDR3 amino acid sequences indicates that positive selection of V(H)81x-encoded H chains is correlated with the presence of a consensus sequence immediately adjacent to the V(H) segment. These data support the hypothesis that the CDR3 region is critical in determining the ability of V(H)81x-encoded H chains to form functional receptors that support positive selection of B lymphocytes. Together, our results demonstrate that TdT can indirectly influence the Ig repertoire by influencing both receptor-dependent and receptor-independent selection processes.

Sequence analysis of the mouse RAG locus intergenic region.

The recombination activating genes RAG-1 and RAG-2 are highly conserved throughout evolution and are necessary and essential for the DNA rearrangement of antigen-receptor gene segments. These convergently transcribed genes are expressed primarily by developing B and T lineage cells. In addition, recent data suggest that the RAG locus can be reactivated in mouse germinal center B cells. Despite these well-defined patterns of expression, little is known about mechanism(s) regulating transcription of the RAG locus. Experiments with a mouse fibroblast line stably transfected with a genomic fragment of the RAG locus suggest that the intergenic region between RAG-1 and RAG-2 may contain information modulating RAG transcription. In order to begin testing this hypothesis, we have sequenced the 7.0-kb RAG intergenic region of the mouse. The sequence did not contain open reading frames larger than 60 amino acids. Analysis with GCG software identified several potential transcription-factor binding sequences within this region. Many of these are associated with transcriptional regulation of the Ig locus.