Chain Transcripts Research Articles

Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

Long-Term Follow-Up on Affinity Maturation and Memory B-Cell Generation in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) comprises a heterogeneous group of primary immunodeficiency disorders. Immunophenotyping of memory B cells at the time of diagnosis is increasingly used for the classification of patients into subgroups with different clinical prognoses. The EUROclass classification is a widely used method. Levels of somatic hypermutation (SHM) have proven useful as a prognostic marker for recurrent respiratory tract infections. As time of presentation and diagnosis is highly variable in CVID patients, and diagnostic delay is a common problem, it is important to know whether classification parameters are stable over time. The purpose of the study was to address this question in a cohort of 33 CVID patients followed from 3 to 19 years after diagnosis (average follow-up 8.8 years). Levels of class-switched memory B cells were analyzed using flow cytometric immunophenotyping, and patients were classified according to the EUROclass criteria. Affinity maturation of B cells was measured using Igκ-REHMA, which assesses somatic hypermutation in kappa light chain transcripts. Clinical manifestations in terms of splenomegaly, autoimmune disease and granulomatous disease were also determined. Switched memory B cells and levels of SHM were not consistently stable markers in a long-term follow-up setting. At a given time during follow-up, 60% of the patients were assigned to the EUROclass group SmB- (less than 2% switched memory B cells), but only 23% were consistently assigned to this group. Associations between clinical manifestations and levels of switched memory B cells or SHM were not observed in our study. Based on our findings, we suggest that immunologic characteristics in CVID patients should be evaluated several times after diagnosis using internationally standardized methods.

Analysis of the expressed porcine antibody repertoire in porcine and reproductive and respiratory syndrome virus infected pigs (P6077)

Abstract Adaptive humoral immunity is mediated by three separate loci (one heavy chain and two chains) that each encode multiple gene segments which recombine to generate a vastly diverse antigen-binding repertoire. We have characterized the diversity of the porcine heavy and light chain repertoires in pigs that were either infected or uninfected with porcine reproductive and respiratory syndrome virus (PRRSV) using amplicon-based 454 high-throughput sequencing. Bioinformatic analysis revealed that a small number of variable gene segments dominate the expression of the immunoglobulin repertoire. Among both heavy and light chain transcripts, several exceptionally common immunoglobulin sequences were identified in PRRSV-infected pigs which were rare in uninfected pigs, suggesting that we detected clonally expanded PRRSV-specific B-cells. The total richness was estimated to be approximately 3.5x10^5 for heavy chain, 1.5x10^5 for kappa light chain, and 2.3x10^5 for lambda light chain. Our calculated richness is similar to that reported for humans, suggesting that the swine antigen-binding repertoire is similarly complex, despite lack of diversity in the porcine IGHV framework regions. Genetic heterogeneity was detected among pigs in their lambda light chain loci, including the identification of a novel IGLV3 gene segment that is highly expressed in pigs possessing it. Our analysis suggests that this heterogeneity influences gene segment usage and thus, may affect humoral immunity.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

Comparison of two functional kappa light‐chain transcripts amplified from a hybridoma

Three heavy-chain and three kappa (κ)-chain transcripts were amplified from hybridoma cells secreting a monoclonal antibody (mAb) against transferrin receptor. Sequence analysis via IMGT/V-QUEST yielded the functional/aberrant prediction. Two functional κ-chain transcripts, Vκ2 and Vκ3, and one functional VH1 were revealed. Comprehensive bioinformatics analyses including sequence alignment, phylogenetic tree, somatic hypermutation prediction, and three-dimensional-molecular structure modeling were used to predict the origin of the two κ-chain transcripts. The results of bioinformatics analysis suggest that Vκ3 is derived from the myeloma partner of the hybridoma; Vκ2 is derived from B-cell. Functional transcripts VH1 and Vκ2 and Vκ3 were then used to construct two chimeric antibodies chi-C2 (Vκ2-VH1) and chi-C3 (Vκ3-VH1), respectively. Antigen-binding experiments showed that only chi-C2 remained the same affinity as its parental mAb. Possible explanations for the coexistence of two functional κ-chain transcripts and the different affinity of the two chimeric antibodies are discussed.

Perigranuloma Localization and Abnormal Maturation of B Cells

Recent observations of abnormal immunoglobulin responses and case reports describing successful B-cell ablative therapy suggest involvement of B cells in the pathogenesis of sarcoidosis. To investigate how abnormal B-cell maturation and function in patients with sarcoidosis contribute to disease. Patients with sarcoidosis (n = 32) were included for detailed analysis by immunohistochemistry of tissue, flow cytometry of blood B-cell subsets, and serum immunoglobulin levels. Vaccination responses in patients with sarcoidosis to influenza virus and encapsulated bacteria and molecular analysis of immunoglobulin heavy chain transcripts were studied for functional analysis of immunoglobulin responses. Perigranuloma localization of IgA-producing plasma cells and numerous B cells were found in affected tissues. Total blood B-cell numbers were normal, CD27(+) memory B cells were significantly reduced, and CD27(-)IgA(+) B cells were significantly increased; the results are normalized in patients treated with TNF-α blockers. Despite this, patients had normal serum immunoglobulin levels and normal antigen-specific immunoglobulin responses. IgA and IgG transcripts, however, showed high frequencies of somatic hypermutations and increased usage of downstream IgG subclasses, suggestive for prolonged or repetitive responses. The large B-cell infiltrates in granulomatous tissue and increased molecular signs of antibody maturation are indicative of direct involvement of B cells in local inflammatory processes in patients with sarcoidosis. Moreover, CD27(-)IgA(+) B cells could be a marker for treatment with TNF-α blockers. These findings of B cells as emerging key players provide a rationale for a systematic study on B-cell ablative therapy in patients with sarcoidosis.

IgA Response in Preterm Neonates Shows Little Evidence of Antigen-Driven Selection

After birth, contact to environmental Ags induces the production of IgA, which represents a first line of defense for the neonate. We sought to characterize the maturation of the repertoire of IgA H chain transcripts in circulating blood B cells during human ontogeny. We found that IgA H chain transcripts were present in cord blood as early as 27 wk of gestation and that the restrictions of the primary Ab repertoire (IgM) persisted in the IgA repertoire. Thus, B cells harboring more "mature" V(H) regions were not preferred for class switch to IgA. Preterm and term neonates expressed a unique IgA repertoire, which was characterized by short CDR-H3 regions, preference of the J(H) proximal D(H)7-27 gene segment, and very few somatic mutations. During the first postnatal months, these restrictions were slowly released. Preterm birth did not measurably accelerate the maturation of the IgA repertoire. At a postconceptional age of 60 wk, somatic mutation frequency of IgA H chain transcripts reached 25% of the adult values but still showed little evidence of Ag-driven selection. These results indicate that similar to IgG, the IgA repertoire expands in a controlled manner after birth. Thus, the IgA repertoire of the newborn has distinctive characteristics that differ from the adult IgA repertoire. These observations might explain the lower affinity and specificity of neonatal IgA Abs, which could contribute to a higher susceptibility to infections and altered responses to vaccinations, but might also prevent the development of autoimmune and allergic diseases.

Extensive Diversification of IgD-, IgY-, and Truncated IgY(ΔFc)-Encoding Genes in the Red-Eared Turtle (Trachemys scripta elegans)

IgY(ΔFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles. The duck IgY(ΔFc) is produced by the same υ gene that expresses the intact IgY form (CH1-4) using different transcriptional termination sites. In this study, we show that intact IgY and IgY(ΔFc) are encoded by distinct genes in the red-eared turtle (Trachemys scripta elegans). At least eight IgY and five IgY(ΔFc) transcripts were found in a single turtle. Together with Southern blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle δ gene are present in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound forms of the IgD H chain transcripts. Although a single μ gene was identified, the α gene was not identified in this species.

Abstract 5: Angiotensin II-induced Alterations of T Cell Receptor Vβ Chain Usage in Kidney and Mesenteric Vasculature

We have previously shown that T cells play a critical role in the genesis of hypertension, and that activated T cells infiltrate the vasculature and the kidney to promote vasoconstriction and sodium retention. Stimulation of the T cell receptor (TCR) with a specific antigen leads to clonal expansion of cells with identical TCRs. In contrast, if multiple antigens are presented, T cells with multiple TCRs proliferate. We sought to determine if hypertension promotes a mono- or polyclonal T cell response. TCR Vβ chain gene transcript length can be used to examine T cell clonality. We produced hypertension in C57Bl/6 mice by chronic (2 week, 490 ng/kg/min) infusion of angiotensin II and used vehicle infused mice as controls. PCR was employed to amplify the 24 TCR Vβ chain subfamilies in the kidney and mesenteric vasculature. PCR products were subjected to high-resolution capillary electrophoresis fragment analysis to determine TCR Vβ chain transcript length. Eleven of the 24 Vβ length profiles exhibited greater than 20% skewing with angiotensin II infusion in kidney and 6 of 24 exhibited greater than 20% skewing in mesentery. Dominant transcript lengths were present with angiotensin II infusion for Vβ 1 (30 ± 5 vs. 51 ± 7), 3 (29 ± 3 vs. 51 ± 12) and 20 (0 ± 0 vs. 41 ± 18) in kidney and 1 (30 ± 2 vs. 43 ± 4), 4 (27 ± 4 vs. 45 ± 3) and 20 (15 ± 3 vs. 46 ± 6) in mesentery (data are expressed as % ± S.E.M. of TCR transcripts of a given length within each Vβ family, sham vs. angiotensin II, n = 5 per group). These results suggest that hypertension results in oligoclonal T cell expansion in the kidney and mesentery. Alterations in Vβ 1 and 20 in both kidney and mesentery suggest a common TCR response between tissues.

In Vivo VL-Targeted Microbial Superantigen Induced Global Shifts in the B Cell Repertoire

To subvert host defenses, some microbial pathogens produce proteins that interact with conserved motifs in V regions of B cell Ag receptor shared by large sets of lymphocytes, which define the properties of a superantigen. Because the clonal composition of the lymphocyte pool is a major determinant of immune responsiveness, this study was undertaken to examine the in vivo effect on the host immune system of exposure to a B cell superantigen, protein L (PpL), a product of the common commensal bacterial species, Finegoldia magna, which is one of the most common pathogenic species among Gram-positive anaerobic cocci. Libraries of Vκ L chain transcripts were generated from the spleens of control and PpL-exposed mice, and the expressed Vκ rearrangements were characterized by high-throughput sequencing. A total of 120,855 sequencing reads could be assigned to a germline Vκ gene, with all 20 known Vκ subgroups represented. In control mice, we found a recurrent and consistent hierarchy of Vκ gene usage, as well as patterns of preferential Vκ-Jκ pairing. PpL exposure induced significant targeted global shifts in repertoire with reduction of Vκ that contain the superantigen binding motif in all exposed mice. We found significant targeted reductions in the expression of clonotypes encoded by 14 specific Vκ genes with the predicted PpL binding motif. These rigorous surveys document the capacity of a microbial protein to modulate the composition of the expressed lymphocyte repertoire, which also has broad potential implications for host-microbiome and host-pathogen relationships.

The ontogeny of New Zealand groper (Polyprion oxygeneios) lymphoid organs and IgM

This study investigates the ontogeny of New Zealand groper (Polyprion oxygeneios) immune system, a new species for aquaculture in the Southern Pacific Ocean. In the eggs, both lysozyme and IgM were detected. Egg IgM was found at 1.07–1.56μg/g wet weight and consisted of monomers compared to the polymerized IgM found in adult serum. In larvae, the head–kidney (HK) was first observed at 6dph, followed by the spleen at 16dph, and thymus at 20dph, and within these organs IgM+ cells were first detected in the HK (12dph), then the spleen (32dph) and finally in the thymus and the gastrointestinal tract (45dph). Low levels of Igμ heavy chain transcripts were detected at 2 and 3dph and they increased at 9dph. Igμ expression further increased from day 45 onwards. In juveniles (115dph), the HK and blood showed similar percentages of IgM+ cells as the adult groper. These results highlight the important maturation steps that occur during the development of the immune system in the marine teleost P. oxygeneios.

The role of ID3 in iNKT cell development (115.12)

Abstract Invariant NKT cells comprise an unconventional T cell population with innate characteristics. Like the conventional αβ T cell lineage, they develop in the thymus from DP precursors after receiving TCR-dependent positive selection signals, although utilizing different pathways. iNKT cells further mature through a series of differentiation stages and acquire their effector properties intrathymically. The ID3 protein is required to reduce E protein activity for proper selection of DP progenitors towards conventional thymocyte lineages. Here, we investigate the requirement for ID3 in the thymic development of iNKT lineage, in vivo. We show that the frequency of iNKT cells in the Id3-/- thymus is higher than in WT littermates, in a cell-intrinsic manner. Id3-deficient post-selected DP cells express higher levels of the canonical Vα14-Jα18 TCRα chain transcripts. Additionally, the transcription factor PLZF, a molecular determinant of the NKT lineage, is expressed in a subset of post-selected DP cells in the Id3-/- mice. However, further thymic iNKT differentiation is blocked at an immature stage. Taken together these results suggest that ID3 restricts the innate T cell potential of DP precursors thus contributing to the lineage fate decisions at the DP stage. However, ID3 is necessary for the completion of the innate T cell program during iNKT differentiation. Ongoing gene expression analysis will identify potent E protein target genes essential for NKT development

Highly diverse TCRα chain repertoire of pre-immune CD8+T cells reveals new insights in gene recombination

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

Immunoglobulin Analysis Tool: A Novel Tool for the Analysis of Human and Mouse Heavy and Light Chain Transcripts

Sequence analysis of immunoglobulin (Ig) heavy and light chain transcripts can refine categorization of B cell subpopulations and can shed light on the selective forces that act during immune responses or immune dysregulation, such as autoimmunity, allergy, and B cell malignancy. High-throughput sequencing yields Ig transcript collections of unprecedented size. The authoritative web-based IMGT/HighV-QUEST program is capable of analyzing large collections of transcripts and provides annotated output files to describe many key properties of Ig transcripts. However, additional processing of these flat files is required to create figures, or to facilitate analysis of additional features and comparisons between sequence sets. We present an easy-to-use Microsoft® Excel® based software, named Immunoglobulin Analysis Tool (IgAT), for the summary, interrogation, and further processing of IMGT/HighV-QUEST output files. IgAT generates descriptive statistics and high-quality figures for collections of murine or human Ig heavy or light chain transcripts ranging from 1 to 150,000 sequences. In addition to traditionally studied properties of Ig transcripts – such as the usage of germline gene segments, or the length and composition of the CDR-3 region – IgAT also uses published algorithms to calculate the probability of antigen selection based on somatic mutational patterns, the average hydrophobicity of the antigen-binding sites, and predictable structural properties of the CDR-H3 loop according to Shirai’s H3-rules. These refined analyses provide in-depth information about the selective forces acting upon Ig repertoires and allow the statistical and graphical comparison of two or more sequence sets. IgAT is easy to use on any computer running Excel® 2003 or higher. Thus, IgAT is a useful tool to gain insights into the selective forces and functional properties of small to extremely large collections of Ig transcripts, thereby assisting a researcher to mine a data set to its fullest.

Muscle-fiber transdifferentiation in an experimental model of respiratory chain myopathy

IntroductionSkeletal muscle fiber composition and muscle energetics are not static and change in muscle disease. This study was performed to determine whether a mitochondrial myopathy is associated with adjustments in skeletal muscle fiber-type composition.MethodsTen rats were treated with zidovudine, an antiretroviral nucleoside reverse transcriptase inhibitor that induces a myopathy by interfering with mitochondrial functions. Soleus muscles were examined after 21 weeks of treatment. Ten untreated rats served as controls.ResultsZidovudine induced a myopathy with mitochondrial DNA depletion, abnormalities in mitochondrial ultrastructure, and reduced cytochrome c oxidase activity. Mitochondrial DNA was disproportionally more diminished in type I compared with type II fibers, whereas atrophy predominated in type II fibers. Compared with those of controls, zidovudine-exposed soleus muscles contained an increased proportion (256%) of type II fibers, whereas neonatal myosin heavy chains remained repressed, indicating fiber-type transformation in the absence of regeneration. Microarray gene-expression analysis confirmed enhanced fast-fiber isoforms, repressed slow-fiber transcripts, and reduced neonatal fiber transcripts in the mitochondrial myopathy. Respiratory chain transcripts were diminished, whereas the enzymes of glycolysis and glycogenolysis were enhanced, indicating a metabolic adjustment from oxidative to glycolytic capacities. A coordinated regulation was found of transcription factors known to orchestrate type II fiber formation (upregulation of MyoD, Six1, Six2, Eya1, and Sox6, and downregulation of myogenin and ERRγ).ConclusionsThe type I to type II fiber transformation in mitochondrial myopathy implicates mitochondrial function as a new regulator of skeletal muscle fiber type.

Modulation of biallelic TCRα expression in influenza-specific CD8 T cells (62.6)

Abstract Our understanding of complementary T cell receptor (TCR)α and β chain utilization is very limited for pathogen-specific immunity. This problem has been solved with the development of a novel, multiplex nested RT-PCR method for the simultaneous amplification of TCRα and TCRβ chain transcripts from single cells. Using this technique, we found surprisingly, 25% of influenza-epitope-specific CD8+ cytotoxic T lymphocytes recovered directly from the pneumonic lung expressed a dominant, non-productively rearranged TCRα transcript, an effect that is never seen for TCRβ. T cells with these out-of-frame TCRα mRNAs also express an alternate, in-frame TCRα, while approximately10% have two productive TCRα transcripts. The proportion of cells with bi-allelic transcription increases over the course of a response; stayed high in the memory compartment but decreased in the recall response. However these bi-allelic cells appear to expand with low efficiency. Further, cells with out-of-frame expression appear to be specifically excluded from the recall response, a finding that has implications for immune memory and for autoimmunity.

Assessment of Bioactivity of a Recombinant Chicken Interferon-Gamma Expressed Using a Baculovirus Expression System

The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1β, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-β chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.

Characterization of Jk(a+weak): a new blood group phenotype associated with an altered JK*01 allele

The clinically important Kidd (JK) blood group system is considered to be relatively uncomplicated, both serologically and genetically. The JK*01 and JK*02 alleles give rise to Jk(a) and Jk(b) antigens, respectively, and silenced alleles result in Jk(a-b-). Other inherited variants analogous to Fy(x) and weak D phenotypes have not been characterized for JK, although recent abstracts indicate their presence. Six index samples from individuals whose RBCs reacted variably or weakly with different sources of anti-Jk(a) and 300 controls of the four known JK phenotypes were investigated by standard serology, flow cytometry, Western blotting, and the urea hemolysis test. Molecular analysis, including allele-specific polymerase chain reaction (PCR), DNA sequencing, and transcript analysis by real-time PCR, was performed. All Jk(a+(w)b-) and Jk(a+(w)b+) index samples were homo- or heterozygous for an altered JK*01 allele carrying 130G>A (Glu44Lys) and the JK*02-associated silent SNPs 588G and Intron 9 -46g. Blood donor screening indicated an allele frequency of 0.042. Titration and flow cytometry with anti-Jk(a) gave lower values in index samples compared to controls, as did anti-Jk3 titers. Donors with 130A also showed significantly decreased Jk(a) density by flow cytometry versus 130G. Western blotting with anti-UT-B demonstrated weaker reactivity with Jk(a+(w)) membranes while JK mRNA levels could not discriminate index samples from controls. The urea hemolysis test was only moderately affected in two Jk(a+(w)b-) samples. A new phenotype with weakened Jk(a) expression on RBCs is associated with a JK*01-like allele, which may constitute a risk for hemolytic transfusion reactions if antigen-positive units are missed by routine serology.

Antigen-dependent T lymphocytes (TcRβ +) are primarily differentiated in the thymus rather than in other lymphoid tissues in sea bass ( Dicentrarchus labrax, L.)

All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αβ/γδ chains. In this study, the TcRβ transcript expression and TcRβ + cell distribution during the ontogeny of the immune system of sea bass ( Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the β chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the β chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383–401]. The first thymus anlage was detectable at 32–33 days p.f. (Corresponding to about 27 days post-hatch). DLT15 + cells were detected at day 35 p.f. in the thymus whereas TcRβ + cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRβ + cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRβ + cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRβ + cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRβ + cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70–75 days p.f.), DLT15 + cells or TcRβ + cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRβ + cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRβ + cells in sea bass larvae and juveniles.

Regulation of platelet myosin light chain (MYL9) by RUNX1: implications for thrombocytopenia and platelet dysfunction in RUNX1 haplodeficiency

AbstractMutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region −729/−542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.