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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.

Neisseria meningitidis is the agent of invasive meningococcal disease, including cerebral meningitis and septicemia. Because the diseases caused by different clonal groups (sequence types) have their own epidemiological characteristics, it is important to understand the differences among the genomes of the N. meningitidis clonal groups. To this end, a novel interpretation of a structural dot plot of genomes was devised and applied; exact nucleotide matches between the genomes of N. meningitidis serogroup A strain Z2491 and serogroup B strain MC58 were identified, leading to the specification of various structural regions. Known and putative virulence genes for each N. meningitidis strain were then classified into these regions. We found that virulence genes of MC58 tend more to the translocated regions (chromosomal segments in new sequence contexts) than do those of Z2491, notably tending towards the interface between one of the translocated regions and the collinear region. Within the collinear region, virulence genes tend to occur within 16kb of gaps in the exact matches. Verification of these tendencies using genes clustered in the cps locus was sufficiently supportive to suggest that these tendencies can be used to focus the search for and understanding of virulence genes and mechanisms of pathogenicity in these two organisms.

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

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 .

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.

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.

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.

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.

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.

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and aλ1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a “knocked-in” VHDJHrearrangement. 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 41λlight-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.

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.

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.

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.

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.

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.

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.

The primary immunoglobulin (Ig) repertoire in the mouse develops during fetal life in the liver. The first Ig gene rearrangement--the joining of a DH to a JH gene segment--contributes largely to the diversity found in CDR3, as well as potentially encoding the D mu protein which is believed to function in the development of a B cell. In this report, the number of DJH joins in two mouse strains, C57BL/6 and BALB/c, were enumerated from days 12 to 16 of fetal development. It was found that the number of DJH structures increased from less than 300 per liver on day 12 to greater than 700,000 (C57BL/6) and 300,000 (BALB/c) on day 16. Each JH gene segment was used approximately equally on each day examined. When the DJH structures were examined by cloning and sequencing it was found that the DJH reading frame (RF) usage (with respect to JH) was not random--RF1 was used 70% of the time. Moreover, a single D gene segment, DFL16.1, was used in greater than 50% of all joins reinforcing the notion that the fetal repertoire is restricted in its antigen binding potential.

The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (λ5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with λ5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.

Previous studies on c-Abl-deficient mice have shown high post-natal mortality and lymphopenia. However, the mechanisms by which c-Abl may influence B lymphopoiesis remain obscure. In this study, we analyzed B cell sub-populations at various differentiation stages in the bone marrow (BM) of c-Abl-deficient mice. Phenotypic analyses revealed that c-Abl(-/-) pro-B cells were reduced to half of normal incidence and absolute number, while pre-B cells showed an even greater reduction. Both c-Abl(-/-) pro-B and pre-B cell populations showed considerably elevated apoptosis ex vivo and in short-term culture but their cell cycle progression was not impaired. In contrast, apoptosis of immature IgM(+)IgD(-) B lymphocytes remained at normal control levels. Inhibition of c-Abl activity by STI571 in normal BM cultures significantly increased apoptosis in B cell precursors while the survival of immature B cells was not affected. To determine whether c-Abl deficiency affects Ig heavy-chain rearrangement, we found that the frequency of V(D)J recombination was markedly reduced by 15-fold in c-Abl(-/-) pro-B cells compared with the control values. However, no perturbation in the levels of signal-end recombination intermediates was found. Taken together, we propose that c-Abl mediates a stage-specific anti-apoptotic response in precursor B cells and is required for efficient V(D)J recombination during B cell development.

Background The 2009 pandemic influenza was milder than expected. Based on the apparent lack of pre-existing cross-protective antibodies to the A (H1N1)pdm09 strain, it was hypothesized that pre-existing CD4+ T cellular immunity provided the crucial immunity that led to an attenuation of disease severity. We carried out a pilot scale study by conducting in silico and in vitro T cellular assays in healthy population, to evaluate the pre-existing immunity to A (H1N1)pdm09 strain. Methods Large-scale epitope prediction analysis was done by examining the NCBI available (H1N1) HA proteins. NetMHCIIpan, an eptiope prediction tool was used to identify the putative and shared CD4+ T cell epitopes between seasonal H1N1 and A (H1N1)pdm09 strains. To identify the immunogenicity of these putative epitopes, human IFN-γ-ELISPOT assays were conducted using the peripheral blood mononuclear cells from fourteen healthy human donors. All donors were screened for the HLA-DRB1 alleles. Results Epitope-specific CD4+ T cellular memory responses (IFN-γ) were generated to highly conserved HA epitopes from majority of the donors (93%). Higher magnitude of the CD4+ T cell responses was observed in the older adults. The study identified two HA2 immunodominant CD4+ T cell epitopes, of which one was found to be novel. Conclusions The current study provides a compelling evidence of HA epitope specific CD4+ T cellular memory towards A (H1N1)pdm09 strain. These well-characterized epitopes could recruit alternative immunological pathways to overcome the challenge of annual seasonal flu vaccine escape.

When immunoglobulin (Ig) gene rearrangements are analyzed, several patterns emerge. The rearrangements at the various loci generally appear in a specific temporal order. In addition, within a given locus the frequency of rearrangement of the various gene segments is not equal but is skewed towards preferential rearrangement of particular gene segments. Understanding the reasons for these patterns will shed light on the mechanism of recombination, the regulation of rearrangement, and the relationship between Ig gene rearrangement and B cell development. In this review, we discuss evidence that the observed patterns of Ig gene rearrangement are due to a combination of factors including the recombination signal sequences, sequences in the coding termini, the accessibility of genetic elements to V(D)J recombinase, and cellular selection.

The VDJ recombination potential of a number of Abelson murine leukemic virus transformed fetal liver cell lines derived from (C57BL/6 x BALB/c) F1 mice was measured. The specific developmental stage of each line was determined using Southern blot analysis to ascertain their rearrangement status at the immunoglobulin heavy chain locus (DJ/DJ, VDJ/DJ or VDJ/VDJ). It was observed that DNA from DJ/DJ lines gave many more 'subhaploid' bands hybridizing with JH than did DNA from VDJ/VDJ lines. While the lack of appropriate substrate (VDJ/VDJ lines have exhausted the normal IgH substrate) contributes to the decrease in 'subhaploid bands', this result indicates that the rate of ongoing immunoglobulin heavy chain gene rearrangement was higher in the DJ/DJ lines. However, when the lines were examined using the assay developed by Hesse et al. (4) to measure VDJ recombinase activity, it was found that although all lines had recombinase activity, the DJ/DJ lines had four times more VDJ recombinase activity than did the VDJ/VDJ lines.

An adherent cell line was established from the much studied pre-B cell line 70Z/3. The adherent cell line had the morphological features of a macrophage and stained positive for non-specific esterase. In addition, this line expressed high levels of RNA for the macrophage specific enzyme, lysozyme. Although the 70Z/3 macrophage variant has lost the ability to respond to lipopolysaccharide and interferon-gamma by expressing RNA transcripts for immunoglobulin light chain, it exhibited a new response characterized by increased levels of I-A RNA transcripts. Both the pre-B and macrophage variants also expressed RNA transcripts which hybridize to a Hox 2.3 probe. In contrast, transcripts which hybridize to Hox 1.1, 2.1, and 6.1 probes are expressed in the pre-B line but could not be detected in the macrophage.

Somatic mutations have a role in the pathogenesis of a number of diseases, particularly cancers. Here we present data supporting a role of mitochondrial somatic mutations in an autoimmune disease, rheumatoid arthritis (RA). RA is a complex, multifactorial disease with a number of predisposition traits, including major histocompatibility complex (MHC) type and early bacterial infection in the joint. Somatic mutations in mitochondrial peptides displayed by MHCs may be recognized as non-self, furthering the destructive immune infiltration of the RA joint. Because many bacterial proteins have mitochondrial homologues, the immune system may be primed against these altered peptides if they mimic bacterial homologues. In addition, somatic mutations may be influencing cellular function, aiding in the acquirement of transformed properties of RA synoviocytes. To test the hypothesis that mutations in mitochondrial DNA (mtDNA) are associated with RA, we focused on the MT-ND1 gene for mitochondrially encoded NADH dehydrogenase 1 (subunit one of complex I – NADH dehydrogenase) of synoviocyte mitochondria from RA patients, using tissue from osteoarthritis (OA) patients for controls. We identified the mutational burden and amino acid changes in potential epitope regions in the two patient groups. RA synoviocyte mtDNA had about twice the number of mutations as the OA group. Furthermore, some of these changes had resulted in potential non-self MHC peptide epitopes. These results provide evidence for a new role for somatic mutations in mtDNA in RA and predict a role in other diseases.

A sensitive dot blot procedure for detecting RNA transcripts with radiolabelled cDNA probes in small numbers of cells in suspension culture is described. For a frequent mRNA species such as that for immunoglobulin in an IgM secretor hybridoma, as few as 30 cells can be detected using a C mu probe; for a less frequent mRNA species such as transcripts coding for alpha, beta or gamma genes of the T cell receptor in T cell tumors or CTL clones, as few as 3 X 10(3) to 10(4) cells can be detected using C alpha, C beta or C gamma probes. High sensitivity is achieved by confining the cells to a very small area on the filter on which they are probed, and by treating the filter with formaldehyde.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V–J joining in vitro. The DNA from a hybrid phage (λ VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J–CK sequence was incubated with lymphoid cell extracts and packaged in vitro. Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance. Although no site-specific V–J fusion events were detected, the packaging efficiency of λ VJCK DNA was 102- to 103-fold lower than that of λ DNA. This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences. To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots. Double-stranded cleavages were observed to occur near the 5′ end of each one of the five JK gene segments and near the 3′ end of a VK gene segment. A plasmid containing the mouse I-Aβ gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments.

Clonal analyses of hematopoietic progenitor cells using in vitro semisolid cultures have been used extensively to identify and characterize unipotent, oligopotent, and pluripotent precursors. We have applied these techniques to the study of the B lymphocyte developmental pathway. We describe a method which detects early members of the B lineage, designated clonable pre-B cells. Evidence is presented which shows that some of these are uncommitted regarding immunoglobulin light chain isotype expression. Further, analysis of RNA produced by the progeny of these cells suggests that clonable pre-B cells derived from early fetal liver are not yet committed to heavy chain variable region expression.

The hallmark of the immune system is its ability to produce a seemingly infinite variety of antigen-binding receptors. This is made possible by molecular and cellular mechanisms uniquely suited to continuously generate a large number of individual receptor molecules and to select some for further expansion. The well-studied genetic rearrangement that results in the juxtaposition of germ line-encoded variable, diversity, and joining elements remains the foundation for diversification on which the repertoire is built. Many of the rules that regulate this phenomenon have been described, although the underlying enzymatic machinery responsible for these events remains to be elucidated. Recent progress in categorizing the immunoglobulin heavy-chain variable region genes into families as well as studies establishing their utilization in both fetal and adult life is helping to further refine these rules. Subsequent cellular interactions 1) permit the discriminant expansion of clones expressing relevant antibody molecules, 2) allow the active affinity alterations needed for effective ongoing immune responses, and 3) limit the potential deleterious effect of autoreactive cells.

Publisher Summary This chapter presents the double-layer modification that was developed to facilitate the detection of antibodies secreted by the progeny of clonable B cells. This allows a more complete analysis of B cell colonies, especially regarding questions of antigen responsiveness, repertoire, and isotype switch. It allows the unequivocal identification of B cells, which arise under conditions that are not themselves selective for B cell growth alone. It is also useful for preservation of colonies for subsequent morphological analysis and more recently is used to monitor RNA transcripts in developing colonies. Clonal assays based on limiting dilution liquid cultures, limiting dilution fragment cultures, and semisolid cultures are extensively utilized. Subsequent studies appeared that examined the characteristics of the B cells, which initiated colony formation based upon physical properties, organ distribution, cell surface phenotype, and other criteria.

The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma. We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain. This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene. The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied. The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining. In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions. Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing.