Search

Hemokinin-1, encoded by the TAC4 gene, is a tachykinin most closely related to substance P. Previous studies have shown that TAC4 distinguishes itself from other tachykinins by its predominantly non-neuronal expression profile, particularly in cells of the immune system. Here we report for the first time that the highest levels of TAC4 expression are found in the olfactory epithelium. Furthermore, we identify olfactory neuron-specific transcription factor (Olf-1), also known as early B-cell factor (EBF), as a novel regulator of TAC4 expression. EBF present in the olfactory epithelium and in B cells binds to two sites in the TAC4 promoter and modulates expression in developing B cells. Our findings suggest a role for TAC4 in cell differentiation, and represent a regulatory bridge between the nervous system and the immune system.

Pathogenic common variable immunodeficiency diseases (CVID) are genetic, usually inherited diseases for which a limited number of genetic defects have been implicated. As CVID presents with a wide range of clinical characteristics, there are likely diverse and for the most part unidentified genetic causes. In some individuals, defects in somatic hypermutation (SHM) have been suggested as the underlying cause of CVID. To address the mechanisms of SHM defects in CVID, we conducted a comprehensive mutational analysis of immunoglobulin heavy chain sequences from CVID patients. We identified several remarkably specific alterations in the spectra of SHM in comparison to healthy individuals. We provide evidence that some CVID cases are associated with defective repair of AID-induced mutations by the DNA mismatch repair (MMR) machinery. Our findings together with reports of increased chromosomal radiosensitivity and associated lymphoproliferative disorders amongst CVID patients, suggest that altered DNA damage repair may be a cause of CVID.

Please cite this paper as: Duvvuri et al. (2010) Highly conserved cross-reactive CD4+ T-cell HA-epitopes of seasonal and the 2009 pandemic influenza viruses. Influenza and Other Respiratory Viruses 4(5), 249–258. Background The relatively mild nature of the 2009 influenza pandemic (nH1N1) highlights the overriding importance of pre-existing immune memory. The absence of cross-reactive antibodies to nH1N1 in most individuals suggests that such attenuation may be attributed to pre-existing cellular immune responses to epitopes shared between nH1N1 virus and previously circulating strains of inter-pandemic influenza A viruses. Results We sought to identify potential CD4+ T cell epitopes and predict the level of cross-reactivity of responding T cells. By performing large-scale major histocompatibility complex II analyses on Hemagglutinin (HA) proteins, we investigated the degree of T-cell cross-reactivity between seasonal influenza A (sH1N1, H3N2) from 1968 to 2009 and nH1N1 strains. Each epitope was examined against all the protein sequences that correspond to sH1N1, H3N2, and nH1N1. T-cell cross-reactivity was estimated to be 52%, and maximum conservancy was found between sH1N1 and nH1N1 with a significant correlation (P

Much attention has been paid to polymorphisms, germline mutations, copy number variations and other inherited forms of genetic disparity among individuals. Less attention, except in the area of tumor formation, has been given to somatic changes to the genome -- changes which have the potential to affect all areas of human health. Discussions of somatic mutations in disease must begin with an understanding of the underlying spontaneous mutation rate/frequency. Previous assays of spontaneous mutant frequency relied on peptide display or function -- a selective step that limits the type of mutations detected. In order to obtain mutation frequencies through unbiased means we used a purely genetic approach to quantitate spontaneous in vivo mutant frequency from human blood cells. Using the constitutively expressed, essential gene Dihydrolipoamide dehydrogenase (DLD) we found mutational frequencies on the order of one mutation per kilobasepair. This is 10-1000x higher than previously reported spontaneous mutant frequencies which depended on a selective step. Our genomic based methods also revealed a role for transcription levels in somatic mutation generation and/or accumulation.

The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005-2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.

Hemokinin-1, encoded by the TAC4 gene, is the most recent addition to the tachykinin family. Although most closely related to the neuropeptide Substance P, Hemokinin-1 distinguishes itself from other tachykinins by its predominantly non-neuronal expression pattern. Its expression in T and B lymphocytes, macrophages, and dendritic cells points to an important role for Hemokinin-1 in the immune system. To seek reasons for its preferential expression in the immune system and ultimately to provide clues to its function, we investigated the molecular mechanisms driving the differential expression pattern of this unique tachykinin. Our study provides the first analysis of the promoter region of the TAC4 gene, which reveals regulatory mechanism different from the Substance P promoter. We demonstrate for the first time that Hemokinin-1 initiates transcription from multiple start sites through a TATA-less promoter. Conservation of the 5′ non-coding region indicates the importance of the upstream regulatory region in directing expression of Hemokinin-1 in specific cell types, during cell differentiation and activation. Furthermore, NFκB, a transcription factor important in the activation of immune cells was shown to be involved in promoting increased TAC4 transcription during PMA induction of a T cell line. Our studies reveal that Hemokinin-1 is regulated by a unique transcription regulation system that likely governs its differential expression pattern and suggests a role for Hemokinin-1 distinct from Substance P .

The human T-cell receptor (TCR) alpha/delta variable loci are interspersed on the chromosome 14q11 and consist of 57 intergenic spaces ranging from 4 to 100 kb in length. To elucidate the evolutionary history of this locus, we searched the intergenic spaces of all TCR alpha/delta variable (TRAV/DV) genes for pseudogenes and potential protein-coding genes. We applied direct open reading frame (ORF) searches, an exon-finding algorithm and comparative genomics. Two TRAV/DV pseudogenes were discovered bearing 80 and 65% sequence similarity to TRAV14DV4 and TRAV9-1/9-2 genes, respectively. A gene bearing 85% sequence identity to B lymphocyte activation-related protein, BC-1514, upstream of TRAV26-2 was also discovered. This ORF (BC-1514tcra) is a member of a gene family whose evolutionary history and function are not known. In total, 36 analogs of this gene exist in the human, the chimpanzee, the Rhesus monkey, the frog and the zebrafish. Phylogenetic analyses show convergent evolution of these genes. Assays for the expression of BC-1514tcra revealed transcripts in the bone marrow, thymus, spleen, and small intestine. These assays also showed the expression of another analog to BC-1514, found on chromosome 5 in the bone marrow and thymus RNA. The existence of at least 17 analogs at various locations in the human genome and in nonsyntenic chromosomes of the chimpanzee suggest that BC-1514tcra, along with its analogs may be transposable elements with evolved function(s). The identification of conserved putative serine phosphorylation sites provide evidence of their possible role(s) in signal transduction events involved in B cell development and differentiation.

In mice, kappa light chains dominate over lambda in the immunoglobulin repertoire by as much as 20-fold. Although a major contributor to this difference is the recombination signal sequences (RSS), the mechanism by which RSS cause differential representation has not been determined. To elucidate the mechanism, we tested kappa and lambda RSS flanked by their natural 5' and 3' flanks in three systems that monitor V(D)J recombination. Using extra-chromosomal recombination substrates, we established that a kappa RSS and its flanks support six- to nine-fold higher levels of recombination than a lambda counterpart. In vitro cleavage assays with these same sequences demonstrated that single cleavage at individual kappa or lambda RSS (plus flanks) occurs with comparable frequencies, but that a pair of kappa RSS (plus flanks) support significantly higher levels of double cleavage than a pair of lambda RSS (plus flanks). Using EMSA with double stranded oligonucleotides containing the same kappa or lambda RSS and their respective flanks, we examined RAG/DNA complex formation. We report that, surprisingly, RAG-1/2 form only modestly higher levels of complexes on individual 12 and 23 kappa RSS (plus natural flanks) as compared to their lambda counterparts. We conclude that the overuse of kappa compared to lambda segments cannot be accounted for by differences in RAG-1/2 binding nor by cleavage at individual RSS but rather could be accounted for by enhanced pair-wise cleavage of kappa RSS by RAG-1/2. Based on the data presented, we suggest that the biased usage of light chain segments is imposed at the level of synaptic RSS pairs.

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2−/− and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.

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.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring VH81X and JH4 gene segments from JH+/− and λ5−/−mice. VH81X-JH4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Caspases are important for apoptosis but are also involved in mammalian cell survival and cell division. Here we report that caspase-3 is a negative regulator of B cell cycling. Mice deficient in caspase-3 (Casp3-/- mice) have increased numbers of splenic B cells that show normal apoptosis but enhanced proliferation in vivo and hyperproliferation after mitogenic stimulation in vitro. Cdkn1a encodes p21 (also called Waf1 or Cip1), a cyclin-dependent kinase (CDK) inhibitor. Although expression of p21 was increased, CDK activities and proliferating cell nuclear antigen (PCNA) were increased in Casp3-/- B cells. Using Casp3-/-Cdkn1a-/- mice, we show that the hyperproliferation of Casp3-/- B cells is abolished when Cdkn1a is also deleted. Our genetic and biochemical data demonstrate that caspase-3 is essential in the regulation of B cell homeostasis.

In this post-genomic era, it is necessary to formulate specific questions and develop bioinformatics tools to understand the vast amounts of information stored in DNA sequence. Using the combinatorial pattern discovery algorithm called Teiresias developed by the Bioinformatics and Pattern Discovery Group at IBM, we have identified novel conserved motifs present in the immunoglobulin loci. In the human VH promoter regions, two new putative regulatory elements have been implicated in basal transcriptional regulation of immunoglobulin. In the intergenic regions of the immunoglobulin constant region genes segments, elements were identified that are absent in similar regions of the Igbeta gene. Since the expression patterns of Igbeta and the Ig genes are similar such elements may have functions in activities specific to the Ig genes such as somatic hypermutation. These elements represent V gene-specific motifs identified through the use of a pattern discovery algorithm.

Immunization with superantigen in vivo induces transient activation of superantigen-specific T cells, followed by a superantigen-nonresponsive state. In this study, using a TCR α knock-in mouse in which the knock-in α-chain can be replaced with endogenous α-chain through secondary rearrangement, we show that immunization of superantigen changes the TCR α-chain expression on peripheral superantigen-specific T cells, induces expression of recombination-activating genes, and generates DNA double-strand breaks at the TCR α-chain locus. These results suggest that viral superantigens are capable of inducing peripheral TCR revision. Our findings thus provide a new perspective on pathogen-immune system interaction.

It is estimated that up to one in five individuals develop pituitary gland tumors. Despite the common occurrence of these tumors, the pathogenetic mechanisms underlying their development remain largely unknown. We report the identification of a novel pituitary tumor-derived, N-terminally truncated isoform of FGF receptor-4 (ptd-FGFR4). The corresponding mRNA results from alternative transcription initiation and encodes a polypeptide that lacks a signal peptide and the first two extracellular Ig-like domains. ptd-FGFR4 has a distinctive cytoplasmic residence, is constitutively phosphorylated, and is transforming in vitro and in vivo. Here we show that targeted expression of ptd-FGFR4, but not FGFR4, results in pituitary tumors that morphologically recapitulate the human disease.

Mutations in mitochondrial (mt) DNA accumulate with age and can result in the generation of neopeptides. Immune surveillance of such neopeptides may allow suboptimal mitochondria to be eliminated, thereby avoiding mt-related diseases, but may also contribute to autoimmunity in susceptible individuals. To date, the direct recognition of neo-mtpeptides by the adaptive immune system has not been demonstrated. In this study we used bioinformatics approaches to predict MHC binding of neopeptides identified from known deletions in mtDNA. Six such peptides were confirmed experimentally to bind to HLA-A*02. Pre-existing human CD4+ and CD8+ T cells from healthy donors were shown to recognize and respond to these neopeptides. One remarkably promiscuous immunodominant peptide (P9) could be presented by diverse MHC molecules to CD4+ and/or CD8+ T cells from 75% of the healthy donors tested. The common soil microbe, Bacillus pumilus, encodes a 9-mer that differs by one amino acid from P9. Similarly, the ATP synthase F0 subunit 6 from normal human mitochondria encodes a 9-mer with a single amino acid difference from P9 with 89% homology to P9. T cells expanded from human PBMCs using the B. pumilus or self-mt peptide bound to P9/HLA-A2 tetramers, arguing for cross-reactivity between T cells with specificity for self and foreign homologs of the altered mt peptide. These findings provide proof of principal that the immune system can recognize peptides arising from spontaneous somatic mutations and that such responses might be primed by foreign peptides and/or be cross-reactive with self.

Autoimmune disease is a critical health concern, whose etiology remains enigmatic. We hypothesized that immune responses to somatically mutated self proteins could have a role in the development of autoimmune disease. IFN-γ secretion by T cells stimulated with mitochondrial peptides encoded by published mitochondrial DNA was monitored to test the hypothesis. Human peripheral blood mononuclear cells (PBMCs) of healthy controls and autoimmune patients were assessed for their responses to the self peptides and mutated-self peptides differing from self by one amino acid. None of the self peptides but some of the mutated-self peptides elicited an immune response in healthy controls. In some autoimmune patients, PBMCs responded not only to some of the mutated-self peptides, but also to some of the self peptides, suggesting that there is a breach of self-tolerance in these patients. Although PBMCs from healthy controls failed to respond to self peptides when stimulated with self, the mutated-self peptide could elicit a response to the self peptide upon re-stimulation in vitro, suggesting that priming with mutated-self peptides elicits a cross-reactive response with self. The data raise the possibility that DNA somatic mutations are one of the events that trigger and/or sustain T cell responses in autoimmune diseases.

In 2013, a novel avian influenza H7N9 virus was identified in human in China. The antigenically distinct H7N9 surface glycoproteins raised concerns about lack of cross-protective neutralizing antibodies. Epitope-specific preexisting T-cell immunity was one of the protective mechanisms in pandemic 2009 H1N1 even in the absence of cross-protective antibodies. Hence, the assessment of preexisting CD4+ T-cell immunity to conserved epitopes shared between H7N9 and human influenza A viruses (IAV) is critical. A comparative whole proteome-wide immunoinformatics analysis was performed to predict the CD4+ T-cell epitopes that are commonly conserved within the proteome of H7N9 in reference to IAV subtypes (H1N1, H2N2, and H3N2). The CD4+ T-cell epitopes that are commonly conserved (∼ 556) were further screened against the Immune Epitope Database (IEDB) to validate their immunogenic potential. This analysis revealed that 45.5% (253 of 556) epitopes are experimentally proven to induce CD4+ T-cell memory responses. In addition, we also found that 23.3% of CD4+ T-cell epitopes have ≥ 90% of sequence homology with experimentally defined CD8+ T-cell epitopes. We also conducted the population coverage analysis across different ethnicities using commonly conserved CD4+ T-cell epitopes and corresponding HLA-DRB1 alleles. Interestingly, the indigenous populations from Canada, United States, Mexico and Australia exhibited low coverage (28.65% to 45.62%) when compared with other ethnicities (57.77% to 94.84%). In summary, the present analysis demonstrate an evidence on the likely presence of preexisting T-cell immunity in human population and also shed light to understand the potential risk of H7N9 virus among indigenous populations, given their high susceptibility during previous pandemic influenza events. This information is crucial for public health policy, in targeting priority groups for immunization programs.

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

CHARLES M. Steinberg (see pictures) began his scientific life as a geneticist and always said he felt more comfortable with that epithet than with “molecular biologist” or “immunologist,” although he was indeed those too. He was awarded a Ph.D. in 1961, at the California Institute of

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.

Estimates put the origin of V(D)J recombination at ∼450 million years ago (for reviews, see references 1, 2). It has been speculated that it all started with a chance occurrence, the integration of a mobile element into a gene encoding an Ig domain ([3][1]). The acquisition of the V(D)J

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.

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.

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.

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 DH 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 VH usage, with predominant expression of certain VH segments, such as VH81x, that are rarely evident during adult life. In this report, we examine the effect of enforced TdT expression on the neonatal repertoire of VH81xDJH rearrangements. We find that TdT synthesis abrogates DH RF bias during the fetal/neonatal period through a Ig-receptor-independent mechanism. These findings suggest that DH RF bias during neonatal life is determined largely by homology-directed joining. We also find that TdT synthesis alters the selection of productively rearranged VH81xDJH alleles in the neonatal spleen through a Ig-receptor-dependent mechanism. Analysis of predicted CDR3 amino acid sequences indicates that positive selection of VH81x-encoded H chains is correlated with the presence of a consensus sequence immediately adjacent to the VH segment. These data support the hypothesis that the CDR3 region is critical in determining the ability of VH81x-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.

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 μ 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.

Ig diversity is generated in large part by the combinatorial joining of the Ig gene segments, VH, D, and JH, that together encode the variable domain of Ig. The final Ig repertoire, however, not only reflects the diversity generated through V(D)J recombinatorial joining, but it is also the product of a number of developmental restraints and selections. To avoid such restrictions and assess the recombination potential of individual Ig gene segments, we constructed Ig heavy (H) chain microlocus plasmids, each of which contain germline coding, recombination signal, and flanking sequences of a VH, D, and JH gene segment. These plasmids allow us to assess the recombination potential of the segments in the context of their natural flanking DNA sequences, but in the absence of any higher order chromatin structure or cellular selection. We found that the frequency and extent of deletions and additions at the recombination breakpoints are similar to those observed at rearranged Ig H chain loci in intact animals. The relative frequencies of the types of rearrangements—VD-J, V-DJ, VinvD-J (invD = inverted D), and VDJ—however, differ strongly. Moreover, V81x, the most used VH gene segment in intact mice, also is overused in this plasmid assay, 15 to 30 times that of another VH segment. This result indicates that the overuse of V81x in the early B cell repertoire can be a consequence of its DNA sequence and not of cellular activities.

Genes encoding the heavy chain portion of immunoglobulin molecules arise from the combinatorial association of V, D and J gene segments, which occurs during discrete stages of B lineage development in the bone marrow. Recently, V(H) replacement, a form of receptor editing, has been described, in which the variable region of an existing VDJ(H) rearrangement is replaced by another V(H) gene segment in a recombination event believed to involve an embedded heptamer within the coding region of the V(H). Studies of transgenic mice with "knocked-in" VDJ(H) genes encoding anti-DNA specificity have demonstrated that receptor editing of the heavy chain is one mechanism by which autoreactive B cell receptors can be modified. Another mouse, the "quasi-monoclonal", which encodes a "knocked-in" VDJ(H) for the hapten NP also contains B lineage cells that undergo V(H) replacement. This suggests that V(H) replacement may play a role in the normal diversification of the antibody repertoire. Using a ligation-mediated PCR assay, we have identified V(QM) double-stranded DNA breaks indicative of V(H) replacement intermediates from bone marrow and splenic B lineage cells of quasi-monoclonal mice in the absence of immunization. V(QM) to J558 recombination deletion products consistent with V(H) replacement were also detected in both the bone marrow and spleen of non-immunized quasi-monoclonal mice. Moreover, RAG-1 transcripts were detected in the spleen. These data suggest that V(H) replacement can be part of the mechanism(s) used by B lineage cells to generate diversity throughout B lineage development, including later stages occurring in secondary lymphoid tissues.

The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic tissues depends on extracellular signals produced by stromal cells within the microenvironment. IL-7 is a stromal-derived growth factor that plays a crucial role in B lineage development. We have shown that in the presence of IL-7, pro-B cells proliferate and differentiate to a stage in which they are responsive to stromal cells and LPS, leading to terminally differentiated IgM-secreting plasma cells. In this report, we examine in detail the role of stromal cells in the transition from the IL-7-responsive pro-B cell stage to the mature LPS-responsive B cell stage. We demonstrate that this transition fails to occur, even in the presence of stromal cells and LPS, if constant exposure to IL-7 is maintained. The transition from the large pro-B cell stage to the small cμ+ pre-B cell stage occurs independent of stromal cells. Moreover, the “stromal cell-dependent” maturation that occurs subsequent to the expression of surface IgM leading to responsiveness to B cell mitogens can also be accomplished in the absence of stromal cells if pre-B cells are cultured in proximity to each other or at high cell concentrations. Together these results suggest that stromal cells mediate B cell differentiation by providing the necessary growth requirements (i.e., IL-7) to sustain the development of pre-B cells. The progeny of these pre-B cells can then differentiate through as yet unidentified homotypic interactions, leading to the production of LPS-responsive B cells.

The recombination activating genes, rag1 and rag2 are essential for the rearrangement of antigen receptor V, D, and J gene segments (Oettinger et al., 1990, Mombaerts et al., 1992; Sehatz and Oettinger, 1992; Shinkai et al., 1992). Both genes are found in all species that are known to rearrange their antigenspecific receptors. The coding regions as well as the genomic organization of the rag locus are highly conserved throughout evolution. Rag1 and rag2, which are convergently transcribed, are separated by an intergenic region of DNA that varies in size among species, being, for example, about 11 kb in the human (Homo sapiens), 8 kb in the mouse (Mus musculus), 5.2 kb in the frog (Xenopus laevis), 2.8 kb in the rainbow trout (Oncorhynchus mykiss) (Oettinger et al., 1990; Ichicara et al., 1992; Greenhalgh et al., 1993; Greenhalgh and Steiner., 1995; Hansen and Kaattari, 1996), and 2.6 kb in the zebrafish (Danio rerio)., National Institutes of Health (U.S.) (Grant 2R01 AI08054), National Institutes of Health (U.S.) (Grant 5T32 AI07436), National Institutes of Health (U.S.) (Grant 1F32 AI09072)

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.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3-5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic-treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

In this Review we will include studies on the development immunoglobulin repertoire. We will discuss the pattern of V, (D), and J rearrangement in both normal B cells and autoimmune disorders. We will define the role of the recombination signal sequences and the importance of the nucleotide sequence of these highly conserved motifs. Whether deviations from the consensus recombination signal sequence will be tolerated by the recombination mechanism and the importance of the recombination-activating genes are also discussed. We will address the issue of whether pathogenic autoantibodies are generated as part of the normal immune repertoire and the importance of receptor editing as a means by which the immune system deletes autoreactive B cells.

Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease.

The src family tyrosine kinase, Lck, transduces signals from the pre-TCR complex which regulate the development and expansion of CD4/CD8 double-positive (DP) thymocytes from CD25(+) CD4/CD8 double-negative progenitors. We and others have recently shown that sublethal gamma-irradiation bypasses the need for TCRbeta expression to promote the development and expansion of DP thymocytes in scid or recombinase-activating gene (RAG)-deficient mice. Here we demonstrate that gamma-irradiation activates an Lck-dependent signaling process in immature thymocytes similar to that initiated physiologically by the pre-TCR complex.

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.

Background Pre-existing cellular immunity has been recognized as one of the key factors in determining the outcome of influenza infection by reducing the likelihood of clinical disease and mitigates illness. Whether, and to what extent, the effect of this self-protective mechanism can be captured in the population dynamics of an influenza epidemic has not been addressed. Methods We applied previous findings regarding T-cell cross-reactivity between the 2009 pandemic H1N1 strain and seasonal H1N1 strains to investigate the possible changes in the magnitude and peak time of the epidemic. Continuous Monte-Carlo Markov Chain (MCMC) model was employed to simulate the role of pre-existing immunity on the dynamical behavior of epidemic peak. Results From the MCMC model simulations, we observed that, as the size of subpopulation with partially effective pre-existing immunity increases, the mean magnitude of the epidemic peak decreases, while the mean time to reach the peak increases. However, the corresponding ranges of these variations are relatively small. Conclusions Our study concludes that the effective role of pre-existing immunity in alleviating disease outcomes (e.g., hospitalization) of novel influenza virus remains largely undetectable in population dynamics of an epidemic. The model outcome suggests that rapid clinical investigations on T-cell assays remain crucial for determining the protection level conferred by pre-existing cellular responses in the face of an emerging influenza virus.

Objective To evaluate the impact of age-specific cross-reactive antibody protection levels on the outcomes of a pandemic outbreak of new variants of H3N2 influenza A viruses (H3N2v). Methods We calibrated a previously validated agent-based model of human-to-human transmission of influenza viruses to project the outcomes of various protection levels in a remote and isolated Canadian community, when demographics are drawn from the Statistics Canada census data. We then compared the outcomes with a scenario in which demographic variables were shifted to resemble an urban structure. This comparative evaluation was conducted using in-silico computer simulations, where the epidemiological data were drawn from relevant estimates in published literature. Results Simulations, using estimates of transmissibility for the 2009 H1N1 pandemic strain in the study population, show that the epidemic size is primarily affected by the cross-reactive protection levels of young children. A lower number of secondary infections at the early stages of an outbreak does not necessarily correspond to a lower epidemic size. Conclusions Demographic variables could play a significant role in determining the outcomes of an outbreak. The findings strongly suggest that, when an H3N2v-specific vaccine becomes available, children below the age of 17 should be prioritized for vaccination. This prioritization is essential in population settings with a low average age, including aboriginal communities in northern latitudes.

The 70Z/3 cell line is able to undergo lineage conversion from a pre-B to macrophage phenotype. Data presented here show that the transition from pre-B to macrophage follows a reproducible pathway via a stable intermediate stage. The cells in the intermediate stage are adherent and have lost the ability to respond to lipopolysaccharide or interferon-gamma by induction of immunoglobulin kappa light chains. However, these cells do not yet display the full range of macrophage-specific properties such as receptors for macrophage-colony stimulating factor or the beta 2 integrin CD11b/CD18. Subcloning experiments with the intermediate cells revealed that they retain the options of either persisting along the macrophage line of differentiation, acquiring additional macrophage traits, or reverting to the pre-B phenotype. Further differentiation to the macrophage stage is accompanied by the apparent loss of the ability to revert. Thus, these studies define relationships among lineage-specific traits, and begin to reveal critical stages in lineage commitment.

The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously, the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif. The heptamer and nonamer motifs are separated by a 'spacer' that was not thought to possess significant sequence conservation, however the length of the spacer could be either 12 +/- 1 bp or 23 +/- 1 bp long. In this report we have assembled and analyzed an extensive data base of published RSS. We have derived, through extensive consensus comparison, a more detailed description of the RSS than has previously been reported. Our analysis indicates that RSS spacers possess significant conservation of sequence, and that the conserved sequence in 12 bp spacers is similar to the conserved sequence in the first half of 23 bp spacers.

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

We describe an analysis of early B cell development in mice with X linked immunodeficiency (Xid). It was found that, compared with the normal CBA/J strain, CBA/N (Xid/Xid) pre-B cells show an increased proliferative response to IL-7 but a decreased ability for subsequent maturation on stromal cells. However, the addition of mast cell growth factor largely restored the ability to mature in the presence of stromal cells. No anomalies were found in the rate of Ig heavy or light chain gene rearrangement in CBA/N cells despite their failure to undergo maturation. This suggests that these two events may occur independently.