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Programmed cell death ligand-1 (PD-L1) expression levels in patients' tumors have demonstrated clinical utility across many cancer types and are used to determine treatment eligibility. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available and have demonstrated different levels of staining between assays, generating interest in understanding the similarities and differences between assays. Previously, we identified epitopes in the internal and external domains of PD-L1, bound by antibodies in routine clinical use (SP263, SP142, 22C3, and 28-8). Variance in performance of assays utilizing these antibodies, observed following exposure to preanalytical factors such as decalcification, cold ischemia, and duration of fixation, encouraged additional investigation of antibody-binding sites, to understand whether binding site structures/conformations contribute to differential PD-L1 IHC assay staining. We proceeded to further investigate the epitopes on PD-L1 bound by these antibodies, alongside the major clones utilized in laboratory-developed tests (E1L3N, QR1, and 73-10). Characterization of QR1 and 73-10 clones demonstrated that both bind the PD-L1 C-terminal internal domain, similar to SP263/SP142. Our results also demonstrate that under suboptimal decalcification or fixation conditions, the performance of internal domain antibodies is less detrimentally affected than that of external domain antibodies 22C3/28-8. Furthermore, we show that the binding sites of external domain antibodies are susceptible to deglycosylation and conformational structural changes, which directly result in IHC staining reduction or loss. The binding sites of internal domain antibodies were unaffected by deglycosylation or conformational structural change. This study demonstrates that the location and conformation of binding sites, recognized by antibodies employed in PD-L1 diagnostic assays, differ significantly and exhibit differing degrees of robustness. These findings should reinforce the need for vigilance when performing clinical testing with different PD-L1 IHC assays, particularly in the control of cold ischemia and the selection of fixation and decalcification conditions., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

Background: Several anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand-1 (PD-L1) therapies have shown encouraging safety and clinical activity in a variety of tumor types. A potential role for PD-L1 testing in identifying patients that are more likely to respond to treatment is emerging. PD-L1 expression in clinical practice is determined by testing one tumor section per patient. Therefore, it is critical to understand the impact of tissue sampling variability on patients' PD-L1 classification., Methods: Resected non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC) tissue samples (five samples per tumor type) were obtained from commercial sources and two tumor blocks were taken from each. Three sections from each block (~ 100 μm apart) were stained using the VENTANA PD-L1 (SP263) assay, and scored based on the percentage of PD-L1-staining tumor cells (TCs) or tumor-infiltrating immune cells (ICs) present. Each section was categorized as PD-L1 high or low/negative using a variety of cut-off values, and intra-block and intra-case (between blocks of the same tumor) concordance (overall percentage agreement [OPA]) were evaluated. An additional 200 commercial NSCLC samples were also analyzed, and intra-block concordance determined by scoring two sections per sample (≥70 μm apart)., Results: Concordance in TC PD-L1 classification was high at all applied cut-offs. Intra-block and intra-case OPA for the 15 NSCLC, HNSCC or UC samples were 100% and 80-100%, respectively, across all cut-offs; intra-block OPA for the 200 NSCLC samples was 91.0-98.5% across all cut-offs. IC PD-L1 classification was less consistent; intra-block and intra-case OPA for the 15 NSCLC, HNSCC or UC samples ranged between 70 and 100% and between 60 and 100%, respectively, with similar observations in the intra-block analysis of the 200 NSCLC samples., Conclusions: These results show the reproducibility of TC PD-L1 classification across the depth of the tumor using the VENTANA PD-L1 (SP263) assay. Practically, this means that treatment decisions based on TC PD-L1 classification can be made confidently, following analysis of one tumor section. Although more variable than TC staining, consistent IC PD-L1 classification was also observed within and between blocks and across cut-offs.

Purpose: Immunotherapies targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) demonstrate encouraging antitumor activity and manageable tolerability in non-small cell lung cancer (NSCLC), especially in patients with high tumor PD-L1 expression, as detected by companion or complementary diagnostic assays developed for individual agents. A laboratory is unlikely to use multiple assay platforms. Furthermore, commercially available diagnostic assays are not standardized, and different assay methods could lead to inappropriate treatment selection. This study establishes the extent of concordance between three validated, commercially available PD-L1 IHC diagnostic assays for NSCLC patients [Ventana SP263 (durvalumab), Dako 22C3 (pembrolizumab), and Dako 28-8 (nivolumab)]. Experimental Design: Five hundred formalin-fixed, paraffin-embedded archival NSCLC samples were obtained from commercial sources. Stained slides were read in batches on an assay-by-assay basis by a single pathologist trained in all methods, in a Clinical Laboratory Improvements Amendments program-certified laboratory. An additional pathologist performed an independent review of 200 stained samples for each assay. Results: PD-L1 expression was evaluable with all assays in 493 samples. The three assays showed similar patterns of tumor membrane staining, with high correlation between percent PD-L1 staining. An overall percentage agreement of >90% was achieved between assays at multiple expression cutoffs, including 1%, 10%, 25%, and 50% tumor membrane staining. Conclusions: This study builds optimism that harmonization between assays may be possible, and that the three assays studied could potentially be used interchangeably to identify patients most likely to respond to anti-PD-1/PD-L1 immunotherapies, provided the appropriate clinically defined algorithm and agent are always linked. Clin Cancer Res; 23(14); 3585-91. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Analysis of tumour samples for mutations is becoming increasingly important in driving personalised therapy in cancer. As more targeted therapies are developed, options to survey mutations in multiple genes in a single tumour sample will become ever more attractive and are expected to become the mainstay of molecular diagnosis in non-small cell lung cancer (NSCLC) in the future., Materials and Methods: 238 non-small cell lung cancer (NSCLC) tumour samples were analysed using a custom panel of 82 mutation assays across 14 oncogenes including KRAS and EGFR using Sequenom iPlex Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF). We compared the data generated for KRAS mutations to those detected by Amplification Refractory Mutation System (ARMS) based DxS TheraScreen K-RAS Mutation Kit., Results: The ARMS detected mutations in 46/238 tumour samples. For samples with mutations detected by both approaches, 99.1% overall agreement was observed. The MALDI-TOF method detected an additional 6 samples as KRAS mutation positive and also provided data on concomitant mutations including PIK3CA and TP53., Conclusions: The Sequenom MALDI-TOF method provides a sensitive panel-based approach which makes efficient use of patient diagnostic samples. This technology could provide an opportunity to deliver comprehensive screening of relevant biomarkers to the clinic earlier in disease management, without the need for repeat biopsy and allow for additional downstream analysis in NSCLC where available tissue may have been exhausted.

Although the negative selection of self-reactive B cells in the bone marrow of mammals has been clearly demonstrated, it remains unclear in models of gut-associated B cell lymphopoiesis, such as that of the chicken (Gallus gallus). We have generated chicken surface IgM-related receptors in which the diversity region of the lamprey variable lymphocyte receptor (VLR) has been fused to the C region of chicken surface IgM (Tμ). Expression of a VLR:Tμ receptor with specificity for PE supported normal development of B cells, whereas a VLR:Tμ receptor specific to hen egg lysozyme (a self-antigen with respect to chicken B cells) induced, in vivo, complete deletion of VLR(HEL)Tμ-expressing B cells. In ovo i.v. injection of PE resulted in deletion of VLR(PE)Tμ-expressing Β cells in the embryo spleen, demonstrating that negative selection was independent of the bursal microenvironment. Although chickens transduced with a murine CD8α:chicken Igα fusion protein contained B cells expressing mCD8α:chIgα, cotransfection of the mCD8α:chIgα construct, together with thymus leukemia Ag (a natural ligand for mCD8α), resulted in reduced levels of mCD8α:chIgα-expressing B cells in inverse proportion to the levels of thymus leukemia Ag-expressing cells. Deletion of mCD8α:chIgα-expressing cells was specific for B cells and required active signaling downstream of the mCD8α:chIgα receptor. Ag-mediated negative selection of developing chicken B cells can therefore occur independently of the bursal microenvironment and is dependent on signaling downstream of the BCR.

In many mammals and birds, B cell lymphopoiesis takes place in GALT, such as the avian bursa of Fabricius. Although BCR expression is sufficient for bursal colonization, the role of BCR ligation in the later stages of bursal B cell lymphopoiesis remains elusive. To address this directly, we introduced a surface Ig-related construct with defined Ag specificity containing the Ag-binding portion of a lamprey variable lymphocyte receptor specific for PE fused to a truncated chicken μ-chain (VLR(PE)Tμ) into developing chick embryos. VLR(PE)Tμ expression supports bursal follicle colonization, clonal expansion, and Ig V gene diversification. VLR(PE)Tμ-expressing B cells migrate to the periphery in the absence of the Ag starting from day 18 of embryogenesis. VLR(PE)Tμ-expressing B cells declined rapidly in the bursa and periphery in the absence of Ag after hatch; however, intrabursal injection of PE prolonged survival of VLR(PE)Tμ(+) bursal and peripheral B cells. Intrabursal introduction of Ag increased emigration of short-lived LT2(+) B cells. Peripheral VLR(PE)Tμ(+) B cells were maintained following intrabursal PE application and contained both short-lived LT2(+) and long-lived LT2(-) B cells. In the chicken bursa, the later stages of B cell development occur in the presence of gut-derived Ag; therefore, we conclude that Ag-mediated ligation of BCR in bursal B cells acts to positively select bursal B cells into both short-lived and long-lived peripheral B cell populations.

Chronic Obstructive Pulmonary Disease (COPD) is a disease characterized by a largely irreversible airflow obstruction and a persistent, excessive inflammatory response. Alveolar macrophages (AMs) are increased in the lungs of COPD patients, and act as orchestrators of the inflammatory response, releasing a range of mediators to coordinate recruitment and activation of leukocytes. Attempts to treat the inflammatory component of COPD with anti-inflammatory drugs such as steroids has met with limited success. In this study, we compared the ability of the phosphodiesterase IV (PDEIV) inhibitor Cilomilast, the steroid Budesonide, and the p38 mitogen activated protein kinase inhibitor BIRB-796 to inhibit tumour necrosis factor alpha (TNFα) and interleukin 6 (IL-6) releases from AMs isolated from COPD lung transplant tissue. All studies were carried out with appropriate ethical approval and written, informed consent was obtained from each subject. Cilomilast had little effect on cytokine release from AMs. There was considerable variability in the responsiveness of AMs to Budesonide, with a subset of AMs responding poorly to Budesonide. BIRB-796 inhibited TNFα release from all AM donors, including those that responded poorly to steroids. Treatment with BIRB-796 and Budesonide together gave an additive decrease in TNFa release. These results suggest that a p38 inhibitor may provide advantages over existing anti-inflammatory treatments for COPD, either as an add-on to existing therapy, or to treat patients who respond poorly to steroids.

Memory lymphocytes support inflammatory and immune responses. To do this, they enter tissue via blood vascular endothelial cells (BVEC) and leave tissue via lymphatic vascular endothelial cells (LVEC). In this study, we describe a hierarchy of signals, including novel regulatory steps, which direct the sequential migration of human T cells across the blood and the lymphatic EC. Cytokine-stimulated (TNF and IFN) human BVEC preferentially recruited memory T cells from purified PBL. Lymphocyte recruitment from flow could be blocked using a function-neutralizing Ab against CXCR3. However, a receptor antagonist directed against the PGD(2) receptor DP2 (formerly chemoattractant receptor-homologous molecule expressed on Th2 cells) inhibited transendothelial migration, demonstrating that the sequential delivery of the chemokine and prostanoid signals was required for efficient lymphocyte recruitment. CD4(+) T cells recruited by BVEC migrated with significantly greater efficiency across a second barrier of human LVEC, an effect reproduced by the addition of exogenous PGD(2) to nonmigrated cells. Migration across BVEC or exogenous PGD(2) modified the function, but not the expression, of CCR7, so that chemotaxis toward CCL21 was significantly enhanced. Thus, chemokines may not regulate all stages of lymphocyte migration during inflammation, and paradigms describing their trafficking may need to account for the role of PGD(2).

Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selective agonists of human peroxisome proliferator-activated receptor δ (PPARδ. Compound 18 demonstrated efficacy in a biomarker for increased fatty acid oxidation, with upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) in human primary myotubes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Chickens provide an important model for developmental biology as well as phylogenetic studies of the immune system. In many species cytokines are important regulators of immune functions and recently we identified the chicken homologue of BAFF (B cell activating factor of the TNF family), shown to be an essential survival factor for B cells in mammals. Characterisation of BAFF function in the phylogenetically distant chicken requires in vivo studies, but despite considerable progress to date no efficient transgene or knockout technology for chickens is available. Thus the retroviral vector system RCAS (replication-competent ASLV long terminal repeat (LTR) with a Splice acceptor) was used to generate chickens stably expressing chBAFF or a soluble chBAFF neutralizing decoy receptor. Expression of RCAS based proteins was demonstrated in ovo and for the complete observation period of 2 month after hatch. In contrast to injections with recombinant proteins the prolonged RCAS based cytokine expression allowed assessment of BAFF function not only in newly hatched chicks but also in more mature birds. We demonstrate that RCAS based secreted products were biologically active, with chBAFF overexpression leading to a drastic and continuous increase in B cell numbers and neutralisation of chBAFF by expression of a soluble decoy receptor leading to severely reduced B cell numbers in transduced birds. This increase in B cell numbers was matched by increased immunoglobulin levels. Using RCASBP transduced birds we provide evidence that the RCAS system provides a fast and highly efficient gene transfer system for analysis of secreted protein function in the chicken and we show that in the chicken BAFF is necessary for B cell survival but in contrast to mammals, not only mature B cells but also immature B cells in the bursa of Fabricius are maintained by BAFF., (Copyright 2010 Elsevier Ltd. All rights reserved.)

CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.

Background: Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates., Results: We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence., Conclusions: There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring.

Prostaglandin (PG)E(2) has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs. MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients. In MDMs, PGE(2) potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-alpha release (p[A](50) 8.51+/-0.11, maximum inhibition 95.9+/-4.8%). In human AMs, PGE(2) also inhibited TNF-alpha release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE(2) curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP(4)-receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE(2) in both AMs and MDMs were mediated by activation of EP(4) and EP(2) receptors. These data indicate that both E-prostanoid(4) and E-prostanoid(2) selective agonists may have anti-inflammatory properties in lung diseases where macrophages play a role.

Surface immunoglobulin (sIg) expression has been conserved as a critical checkpoint in B lymphocyte development. In the chicken embryo, only sIg+ B cells are selectively expanded in the bursa of Fabricius, a primary lymphoid organ unique to the avian species. We have previously demonstrated that an interaction between the antigen- binding sites of sIg and a specific bursal ligand(s) is not required to regulate this developmental checkpoint. Rather, the requirement for sIg expression can be attributed to the surface expression of the Igalpha/beta heterodimer associated with sIg. More specifically, ligand-independent signaling downstream of the Igalpha cytoplasmic domain drives all bursal stages of B cell development during embryogenesis. We discuss here a site-directed mutagenesis approach to identify the critical membrane proximal events involved in ligand-independent signaling during B cell development.

The bursa of Fabricius is critical for the normal development of B lymphocytes in birds. It is productively colonized during embryonic life by a limited number of B cell precursors that have undergone the immunoglobulin gene rearrangements required for expression of cell surface immunoglobulin. Immunoglobulin gene rearrangement occurs in the absence of terminal deoxynucleotidyl transferase and generates minimal antibody diversity. In addition, observations that immunoglobulin heavy and light chain variable gene rearrangement occur at the same time and that allelic exclusion of immunoglobulin expression is regulated at the level of variable region gene rearrangement provide a striking contrast to rodent and primate models of immunoglobulin gene assembly. Following productive colonization of the bursa, developing B cells undergo rapid proliferation and the immunoglobulin V region genes that generate the specificity of the B cell surface immunoglobulin receptor undergo diversification. Immunoglobulin diversity in birds is generated by somatic gene conversion events in which sequences derived from upstream families of pseudogenes replace homologous sequences in unique and functionally rearranged immunoglobulin heavy and light chain variable region genes. This mechanism is distinct from and much more efficient than mechanisms of antibody diversification seen in rodents and primates. While the bursal microenvironment is not required for immunoglobulin gene rearrangement and expression, it is essential for the generation of antibody diversity by gene conversion. Following hatch, gut derived antigens are taken up by the bursa. While bursal development prior to hatch occurs in the absence of exogenous antigen, chicken B cell development after hatch may therefore be influenced by the presence of environmental antigen. This review focuses on the differences between B cell development in the chicken as compared to rodent and primate models.

The bursa of Fabricius is critical for the normal development of B lymphocytes in avian species. Productive colonization of bursal follicles by B cell precursors requires surface immunoglobulin expression. We have shown using retroviral gene transfer that expression of chimeric receptors containing the extracellular and transmembrane domains of murine CD8alpha and CD8beta fused to the cytoplasmic domains of chicken Igalpha and Igbeta can support productive bursal colonization in the chicken embryo in bursal cells lacking the expression of endogenous sIgM. We show here that chimeric receptor expression does not support continued bursal cell development after hatch. However intrabursal administration of anti-CD8 antibodies that ligate the CD8alpha:Igalpha chimeric receptor results in maintained numbers of bursal cells that express the chimeric receptor in the absence of endogenous sIgM. These results support a model in which sIgM receptor expression is required for productive bursal colonization in the chick embryo but sIgM receptor ligation is required to support later B cell development after hatch.

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.

Background: Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined., Results: We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein., Conclusions: These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/beta-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus.

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.

The bursa of Fabricius has evolved in birds as a gut-associated site of B-cell lymphopoiesis that is segregated from the development of other hematopoietic lineages. Despite differences in the developmental progression of chicken as compared to murine B-cell lymphopoiesis, cell-surface immunoglobulin (sIg) expression has been conserved in birds as an essential checkpoint in B-cell development. B-cell precursors that express an sIg complex that includes the evolutionarily conserved Igalpha/beta heterodimer colonize lymphoid follicles in the bursa, whereas B-cell precursors that fail to express sIg due to non-productive V(D)J recombination are eliminated. Productive retroviral gene transfer has allowed us to introduce chimeric receptor constructs into developing B-cell precursors in vivo. Chimeric proteins comprising the extracellular and transmembrane regions of murine CD8alpha fused to the cytoplasmic domain of chicken Igalpha efficiently supported B-cell development in precursors that lacked endogenous sIg expression. By contrast, expression of an equivalent chimeric receptor containing the cytoplasmic domain of Igbeta actively inhibited B-cell development. Consequently, the cytoplasmic domains of Igalpha and Igbeta play functionally distinct roles in chicken B-cell development.

Expression of surface immunoglobulin (sIg) related receptors has been conserved in phylogenetically distinct species as a critical checkpoint in B cell development. The sIg receptor comprises extracellular IgM heavy and light chains, with the potential for ligand binding, complexed to the Igalpha/Igbeta heterodimer that is responsible for signal transduction through sIg. Experimental systems, from both avian and murine models of B cell development, have been designed to identify the function of individual receptor components in B cell development. In this review, we assess the regulatory functions of different components of the sIg receptor complex during early development in experimental systems from evolutionarily distinct species.

B lymphocyte development can occur in a variety of anatomical sites. While typically considered to be a process that occurs in the bone marrow throughout life, it is becoming clear that gut associates sites of B cell development are critically important in many species of veterinary importance. Among these sites, the bursa of Fabricius in chickens and the ileal Peyer's patches of sheep are among the best studied. In these organs, it has become clear that many of the properties associated with B cell development in rodent and primate bone marrow do not apply. Thus while bone marrow B cell development typically involves an ongoing maturation of mature B cells from immature B lineage precursors that lack the expression of a surface immunoglobulin complex, gut associated lymphoid tissues (GALTs) may be colonized by a single wave of precursor cells during embryo development. Nonetheless, molecular analysis of the requirements for B lymphocyte development in GALTs reveals some striking parallels with requirements identified for B cell development in bone marrow. This article will discuss differences between B cell development in the bone marrow and GALTs and recent evidence emerging that yields insights into how these processes are regulated.

Allelic, or haplotype, exclusion of immunoglobulin gene expression ensures that the products of a single allele or light chain isotype are expressed on the B cell surface. Evidence has accumulated in rodent and primate models to indicate that the products of successful rearrangement regulate this process. In contrast, haplotype exclusion of chicken immunoglobulin gene expression is regulated at the level of variable region gene rearrangement. We discuss here alternative models for ensuring haplotype exclusion that may operate in the chicken and extend the discussion to address the issue as to how two apparently distinct mechanisms may have evolved to yield the same outcome.

Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. Using early Xenopus embryos, we studied how PP2A functions in Wnt signal transduction. The catalytic subunit of PP2A (PP2A-C) potentiated secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibited Dishevelled-mediated signal transduction, as did the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C did not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin was sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, we propose that PP2A regulates the Wnt pathway at multiple levels.

The bursa of Fabricius is critical to normal B-lymphocyte development in birds. During embryonic life, B-cell precursors migrate to the bursal rudiment and those which have undergone productive V(D)J recombination colonize lymphoid follicles and undergo immunoglobulin V gene diversification by gene conversion. The chicken surface IgM complex appears structurally and functionally equivalent to its mammalian counterpart, with homologs to CD79a and CD79b. Expression of a truncated Igmu chain is sufficient to drive the early stages of B-cell development in the embryo bursa. Bursal cells expressing the truncated mu receptor complex proliferate in bursal follicles, and those which contain V gene rearrangements undergo V gene diversification by gene conversion. The bursa is a gut-associated organ and antigen is focused to bursal lymphoid follicles after hatch. While expression of the truncated mu chain is sufficient to support B-cell development in the embryo, B cells expressing this receptor are rapidly eliminated after hatch. We suggest the possibility that B-cell development in the bursa after hatch is driven by encounter with antigen leading to redistribution of B cells within the lymphoid follicle, B-cell proliferation and V gene repertoire development by gene conversion.

During embryonic development, the avian bursa of Fabricius selects B cell precursors that have undergone productive V(D)J recombination for expansion in oligoclonal follicles. During this expansion, Ig diversity is generated by gene conversion. We have used retroviral gene transfer in vivo to introduce surface Ig molecules that lack V(D)J-encoded determinants into B cell precursors. This truncated mu heavy chain supports both B cell expansion within embryo bursal lymphoid follicles and gene conversion. We show that individual follicles can be colonized exclusively by cells expressing the truncated mu chain and lacking endogenous surface IgM, ruling out a requirement for V(D)J-encoded determinants in the establishment of bursal lymphoid follicles. In striking contrast to their normal development in the embryo, bursal cells expressing the truncated mu-chain exhibit reduced rates of cell division and increased levels of apoptosis after hatching. The level of apoptosis in individual follicles reflects the proportion of cells within the follicle that express the truncated mu-chain. In particular, high levels of apoptosis are associated with follicles containing exclusively cells expressing the truncated micro receptor. Thus, apoptotic elimination of such cells is not due to competition within the follicle by cells expressing endogenous surface IgM receptors. This provides the first direct demonstration that the regulation of B cell development in the avian bursa after hatching differs fundamentally from that seen in the embryo. The requirement for intact IgM expression when the bursa is exposed to exogenous Ag implicates a role for Ag in avian B cell development after hatching.

Signaling by the Wnt family of secreted proteins plays an important role in animal development and is often misregulated in carcinogenesis. Wnt signal transduction is controlled by the rate of degradation of beta-catenin by a complex of proteins including glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli, and Axin. Dishevelled is required for Wnt signal transduction, and its activation results in stabilization of beta-catenin. However, the biochemical events underlying this process remain largely unclear. Here we show that Xenopus Dishevelled (Xdsh) interacts with a Xenopus Axin-related protein (XARP). This interaction depends on the presence of the Dishevelled-Axin (DIX) domains in both XARP and Xdsh. Moreover, the same domains are essential for signal transduction through Xdsh. Finally, our data point to a possible mechanism for signal transduction, in which Xdsh prevents beta-catenin degradation by displacing GSK3 from its complex with XARP.

The bursa of Fabricius is critical for the development of B lymphocytes in avian species. Despite considerable advances in our understanding of the molecular mechanisms by which avian antibody diversity is generated, many stages of B-cell development in the bursa and the means by which they are regulated remain unclear. Here we discuss the use of productive chicken retroviral vectors which allow gene transfer in vitro or in vivo as tools to probe the requirements for bursal B-cell development. Expression of a truncated form of bursal cell surface IgM, lacking variable region encoded determinants, is sufficient to promote the initial colonization and clonal expansion of B-cells within the bursa. Expression of this truncated IgM does not, however, protect developing bursal cells against the apoptosis that occurs within the bursa after hatch. Conversely, over-expression of the proto-oncogene bcl-2, following retroviral gene transfer, protects cells against apoptotic cell death but is not sufficient to allow B lineage progression in the absence of sIgM expression. Finally we discuss the use of regulated promoters within the retroviral gene transfer system to show that while bursal cells are susceptible to transformation by the v-rel oncogene in vitro, this oncogene preferentially targets mature peripheral cells in vivo.

Antibody diversification in the bursa of Fabricius occurs by gene conversion: pseudogene-derived sequences replace homologous sequences in rearranged immunoglobulin genes. Bursal cells expressing a truncated immunoglobulin mu heavy chain, introduced by retroviral gene transfer, bypass normal requirements for endogenous surface immunoglobulin expression. Immunoglobulin light chain rearrangements in such cells undergo gene conversion under conditions where the products are not selected based on their ability to encode a functional protein. The efficiency with which gene conversion maintains a productive reading frame exceeds 97% under such non-selective conditions. By analysis of donor pseudogene usage we demonstrate that bursal cell development is not driven by a restricted set of antigenic specificities. We further demonstrate that gene conversion can restore a productive reading frame to out-of-frame VJ(L) junctions, providing a rationale for the elimination of cells containing non-productive VJ(L) rearrangements prior to the onset of gene conversion in normal bursal cell development.

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor.

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120(cas)/p120(ctn), and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.

A series of ascorbic acid and isoascorbic acid derivatives has been evaluated as inhibitors of malt, bacterial, fungal, pancreatic and salivary alpha-amylases using a simple and quick assay procedure. The results demonstrate that the enediol moiety of ascorbic acid is essential for alpha-amylase inhibition. Acylation of the primary and secondary alcohols, and the absolute configuration of the secondary alcohol, do not affect the potency of inhibition.

Protein kinase C signaling pathways have been implicated in the disruption of intercellular junctions, but mechanisms are not clear. p100 and p120 are members of the Armadillo family of proteins and are localized to cellular adherens junctions. In strain I Madin-Darby canine kidney cells, protein kinase C activation leads to disruption of tight junctions and an increase in permeability of cell monolayers. We show that this permeability increase is accompanied by dephosphorylation of p100/p120 on serine and threonine residues. The dephosphorylation of these proteins can also be induced by the kinase inhibitors staurosporine, KT5926, and Gö 6976. Treatment of cells with phosphatase inhibitors induced hyperphosphorylation of p100 and p120. Thus, p100 and p120 participate in a regulatable cycle of serine/threonine phosphorylation and dephosphorylation. Protein kinase C must act, directly or indirectly, by perturbing this phosphorylation cycle, by inhibition of a p100/p120 kinase and/or activation of a phosphatase. These data clearly show that p100 and p120 are targets of a novel protein kinase C signaling pathway. Dephosphorylation of these proteins precedes the permeability increase across epithelial cell monolayers seen in response to phorbol esters, raising the possibility that this pathway may play a role in the modulation of intercellular junctions.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.

Expression screening has been used to clone cDNAs encoding the alpha- and beta-chains of chicken CD8. Amino acid sequence similarities with the mammalian sequences were about 30%. Many amino acid residues of structural or functional importance were more highly conserved, as were the overall structures of both chains. Like human CD8 alpha, the chicken alpha-chain lacked sites for N-linked glycosylation, but the beta-chain contained three such sites. In COS cells transfected with CD8 beta cDNA, surface expression of the beta-chain was dependent on co-transfection of the alpha-chain cDNA, indicating that, as in mammals, chicken CD8 can be expressed as a CD8 alpha alpha homodimer or as a CD8 alpha beta heterodimer. Immunofluorescence analysis with mAbs that were shown to identify the CD8 alpha- and CD8 beta-chains revealed that the vast majority of the CD8+ cells in the thymus, spleen, and blood of adult chickens express both CD8 alpha- and CD8 beta-chains. However, a relatively large proportion of the CD8+ TCR-gamma delta cells in the spleens of embryos and young chicks express only the alpha-chain of CD8. Among intestinal epithelial lymphocytes the major CD8+ T cell populations present in mice are conserved, but there is a population of TCR-gamma delta CD8 alpha beta cells that is not found in rodents. This observation is important in interpretation of experiments examining the pathways of development of intestinal intraepithelial lymphocytes in chickens.

The vast majority of lymphocytes generated daily in the chicken bursa of Fabricius do not emigrate to the periphery but die in situ. Apoptotic cells in the bursa can be readily detected by the presence of fragmented DNA and by the large numbers of condensed cellular nuclei observed by electron microscopy. Consequently, most newly generated lymphocytes die by programmed cell death. We show that bursal cells divide rapidly and apoptotic cells are derived from rapidly dividing precursors. Analysis of the phenotype of bursal cells undergoing apoptosis demonstrated that cell death does not occur in the most mature bursal cell population and is therefore not random. High levels of surface Ig are expressed on bursal cells entering S phase of the cell cycle. In contrast, bursal cells in the early stages of apoptosis in vivo express very low to undetectable levels of surface Ig but were unequivocally confirmed as being of the B lineage by polymerase chain reaction (PCR) detection of rearranged Ig genes. Bursal cells induced to undergo apoptosis in vitro express high levels of surface Ig demonstrating that induction of apoptosis does not in itself induce a loss of surface Ig expression. Consequently, loss of surface Ig expression precedes bursal cell death by apoptosis in vivo, suggesting that maintenance of a threshold level of surface Ig may be a requirement for the continued progression of chicken B lymphocyte development in the bursa.

The bursa of Fabricius is the site of primary B cell generation in the chicken. Analysis of the rate of bursal cell emigration demonstrated that about 1% of the blood B cell pool was replaced per hour by bursal emigrants. Surgical bursectomy distinguished three populations of blood B cells. About 60% of blood B cells in 3-wk-old chickens were short lived, with a lifespan of 2 to 3 d, and included most bursal emigrants. These cells migrate directly from the bursal follicular cortex to the periphery, express the LT2 antigen, and are proposed to represent a diversified repertoire of B cell specificities that have emigrated from the bursa in the absence of interaction with environmentally derived antigens in the follicular medulla. About 35% of blood B cells were much longer lived cells, having a lifespan exceeding 2 wk. These cells represent about 10% of bursal emigrants, which do not express the LT2 antigen and do not divide in the peripheral blood following emigration from the bursa. This population may represent cells that have undergone positive selection in the bursa by interacting with environmentally derived antigens and should, therefore, contain a restricted repertoire of B cell specificities. The third population, about 5% of blood B cells, were short-lived cells that represent the progeny of postbursal B cell production. The frequency of these cells progressively increases with time, taking over from the short-lived bursal emigrants as the bursa involutes, likely as a diversified repertoire of B cell specificities.

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.

Immunoglobulin gene rearrangement in the chicken has evolved not to generate antibody diversity per se but to generate an immunoglobulin variable region which can be diversified by subsequent somatic gene conversion events. While the molecular mechanism of V(D)J recombination in chickens cannot be distinguished from that seen in other species, the way in which this recombination is regulated during chicken B lymphocyte development does differ from the more widely known models of gene rearrangement in humans and rodents. In this review we focus on these differences, relating V(D)J recombination to the progression of chicken B cell development in the bursa of Fabricius.

In the peripheral blood (PBL) of juvenile chickens three populations of B cells have previously been distinguished based on life-span and origin of cells within each population. In this report we show that the largest PBL B cell subset, population 1 B cells, which are short-lived cells corresponding to about 60% of PBL B cells and the vast majority of bursal emigrants, exit from the bursa directly from the follicular cortex. This conclusion is based on the specific labeling of rapidly dividing cortical lymphocytes with bromodeoxyuridine, followed by their detection in the periphery prior to the appearance of bromodeoxyuridine labeled cells in the bursa medulla. Furthermore, the rate of emigration of cortical lymphocytes, 1.00 +/- 0.1% of PBL B cells per hour, is indistinguishable from the emigration rate of B cells from the bursa as a whole. The anatomical organization of the bursa has evolved to focus gut-derived antigens from the bursal lumen into the lymphoid follicles. The emigration of cortical bursal cells is discussed in relation to the exposure of bursal lymphocytes to extrinsic antigen.

Infectious bursal disease virus (IBDV) is a lymphotropic virus with cytocidal effect on B lymphocytes of the bursa of Fabricius. We investigated the susceptibility of clonal populations of reticuloendotheliosis virus-transformed chicken B lymphocytes of both spleen and bursal origin to IBDV infection. The infected cells were metabolically-labelled and the viral polypeptides were analyzed by immunoprecipitation using monoclonal antibodies (MAbs). Virus adsorption and the effects of neutralizing convalescent antisera and MAbs on virus attachment were studied using flow cytometry. The results of the study indicate firstly that the transformed B cells support virus replication and provide an efficient system for studying IBDV-lymphocyte interactions. Secondly, results obtained also showed that the most potent neutralizing antibodies may not be those involved in preventing the receptor-mediated viral attachment but rather those involved in the inhibition of downstream events such as virus penetration or uncoating.

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (VH1 and VL1, respectively). The distribution and nature of nucleotide variation among these and previously identified VH1 and VL1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-VH and pseudo-VL gene families, which lie 5' to VH1 and VL1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH1 and VL1 genes. These results demonstrate that the chicken VH1 and VL1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of B cell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.