B-like Cells Research Articles

Discovery of an unconventional lamprey lymphocyte lineage highlights divergent features in vertebrate adaptive immune system evolution

Lymphocyte receptors independently evolved in both jawed and jawless vertebrates with similar adaptive immune responses. However, the diversity of functional subtypes and molecular architecture in jawless vertebrate lymphocytes, comparable to jawed species, is not well defined. Here, we profile the gills, intestines, and blood of the lamprey, Lampetra morii, with single-cell RNA sequencing, using a full-length transcriptome as a reference. Our findings reveal higher tissue-specific heterogeneity among T-like cells in contrast to B-like cells. Notably, we identify a unique T-like cell subtype expressing a homolog of the nonlymphoid hematopoietic growth factor receptor, MPL-like (MPL-L). These MPL-L+ T-like cells exhibit features distinct from T cells of jawed vertebrates, particularly in their elevated expression of hematopoietic genes. We further discovered that MPL-L+ VLRA+ T-like cells are widely present in the typhlosole, gill, liver, kidney, and skin of lamprey and they proliferate in response to both a T cell mitogen and recombinant human thrombopoietin. These findings provide new insights into the adaptive immune response in jawless vertebrates, shedding new light on the evolution of adaptive immunity.

Isolation of CD35+ follicular dendritic cells and its role in the differentiation from B cells to IgA+GL7+ cells

Follicular dendritic cells (FDCs) are non-hematopoietic cells that are localized in the germinal centers (GCs) of lymph nodes (LNs) and are involved in humoral immunity. FDCs are a rare population that are sensitive to mechanical and chemical stimuli, making their isolation for analysis difficult. In Peyer's Patches, which are the main IgA-inductive sites, FDCs have been reported to be activated by retinoic acid receptor (RAR) and toll-like receptor (TLR) signals to induce IgA production. However, little is known about FDCs in mesenteric LNs (MLNs), although MLNs are also an IgA-inductive site. In this study, we efficiently isolated FDCs as CD35+ cells using anti-CD35 antibodies (Abs) and magnetic bead sorting. We found that CD35+ FDCs facilitated differentiation from B220+ B cells into IgA+GL7+ GC B-like cells but not IgA+CD138+ plasma cells. Furthermore, using CD35+ FDCs from LPS-resistant C3H/HeJ mice, the generation of IgA+GL7+ GC B-like cells was not altered significantly between wild-type and LPS-resistant mice. Moreover, the addition of RAR antagonists and agonists revealed that differentiation into IgA+GL7+ GC B-like cells required the activation of RAR, especially RAR-β, in FDCs. The differentiation of IgA+GL7+ cells was promoted by FDCs in peripheral LNs as well as MLNs in our in vitro assay. Taken together, these results indicate that magnetic bead sorting with anti-CD35 Abs enable the efficient isolation of FDCs. Our data suggested that CD35+ FDCs can support differentiation of B cells into IgA+GL7+ GC B-like cells in environments that are not limited to MLNs, which can be stimulated by retinoic acid.

Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

Evolution of variable lymphocyte receptor B antibody loci in jawless vertebrates

SignificanceJawless vertebrates, lampreys and hagfish, use three types of variable leucine-rich repeat proteins as antigen receptors: VLRB on B-like cells and VLRA or VLRC on T-like lymphocytes. In their incomplete germline status, theVLRBgenes in different lamprey species uniquely possess long noncoding intervening regions, which contain different sets of transposable elements and potential donor cassettes. Phylogenetic analysis of the germlineVLRgenes reveals ongoing evolution over the last 250 million y. Comparative assessment of genomic sequences in two lamprey species yields a detailed map of theirVLRBloci. The use of this map, together with an analysis of cassette usage in a large repertoire of expressedVLRBs, supports a fragmented Lego-like model ofVLRBassembly.

Pattern of Tumor-Infiltrating Lymphocytes in Mixed Epithelial and Stromal Tumor of the Kidney: A Review of Five Cases.

Mixed epithelial and stromal tumor of the kidney (MESTK), a benign rare tumor with malignant transformation potential, is thought to be derived from fetal or immature cells originating from the mesonephric and Müllerian ducts. However, due to its rarity, little is known about the anti-tumor immune responses in MESTK. Herein, we present five cases of MESTK and evaluate the population of tumor-infiltrating lymphocytes (TILs) using a freshly obtained MESTK sample. Microscopically, TILs were scattered or clustered in large aggregates in the stroma in all five cases; furthermore, three cases exhibited heavy, large lymphocytic aggregates with no well-organized tertiary lymphoid structures with germinal centers. Flow cytometric analysis of TILs in one freshly obtained MESTK sample revealed that >40% of CD3+ T cells were effector memory Fas+CD28− γδ T cells expressing high levels of programmed cell death protein 1 and inducible T-cell co-stimulator, but low levels of CD44 and CD27. Most αß T cells exhibited a naïve phenotype. Additionally, we detected many activated class-switched CD21+CD27+ B cells as well as CD11chighIgMhigh marginal zone B-like and CD27−CD21−CD23− immunoglobulin (Ig)DhighIgMlow age-associated B-like cells. Collectively, for the first time, we report the immune microenvironment pattern of MESTK to oncogenic stress.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Co-evolution of mutagenic genome editors and vertebrate adaptive immunity.

The adaptive immune systems of all vertebrates rely on self-DNA mutating enzymes to assemble their antigen receptors in lymphocytes of their two principal lineages. In jawed vertebrates, the RAG1/2 recombinase directs V(D)J recombination of B cell and T cell receptor genes, whereas the activation-induced cytidine deaminase AID engages in their secondary modification. The recombination activating genes (RAG) 1 and 2 evolved from an ancient transposon-encoded genome modifier into a self-DNA mutator serving adaptive immunity; this was possible as a result of domestication, involving several changes in RAG1 and RAG2 proteins suppressing transposition and instead facilitating-coupled cleavage and recombination. By contrast, recent evidence supports the notion that the antigen receptors of T-like and B-like cells of jawless vertebrates, designated variable lymphocyte receptors (VLRs), are somatically assembled through a process akin to gene conversion that is believed to be dependent on the activities of distant relatives of AID, the cytidine deaminases CDA1 and CDA2, respectively. It appears, therefore, that the precursors of AID and CDAs underwent a domestication process that changed their target range from foreign nucleic acids to self-DNA; this multi-step evolutionary process ensured that the threat to host genome integrity was minimized. Here, we review recent findings illuminating the evolutionary steps associated with the domestication of the two groups of genome editors, RAG1/2 and cytidine deaminases, indicating how they became the driving forces underlying the emergence of vertebrate adaptive immune systems.

Abstract 2348: BRD4 regulates the expression of Livin and thereby confers resistance to Fas-mediated immune cytotoxicity in aggressive B-cell lymphoma

Abstract Aggressive types of non-Hodgkin’s lymphoma (NHL) include Burkitt’s lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), which are germinal center (GC) B-derived lymphomas. The death receptor Fas is expressed in GC B-lymphocytes and contributes to the elimination of nonspecific and autoreactive B cells. Fas has been implicated in lymphomagenesis on the basis of its pro-apoptotic function and in autoimmune lymphoproliferative syndrome, with germline mutations in the Fas gene having been found to predispose individuals to the development of lymphoma. However, the possible mechanisms underlying the impairment of Fas-mediated apoptosis induction during human lymphomagenesis have remained unknown. In the present study, we established a mouse model of mature B-cell lymphoma based on transplantation into recipients of ex vivo-induced GC B-like cells derived from Ink4a/Arf-/- mice and transduced with MYC. Overexpression of MYC and deletion or silence of INK4A/ARF (also known as CDKN2A) are frequently found in both BL and DLBCL. The downregulation of Fas was necessary for both lymphomagenesis and lymphoma cell survival in vivo, suggesting that GC-derived lymphomas require persistent Fas downregulation to evade antitumor immunity by Fas ligand (FasL)-expressing cells. CD40 signal activation in lymphoma cells significantly restored the expression of Fas and thereby induced apoptosis following FasL treatment. Similarly, the majority of human BL and DLBCL cell lines exhibited downregulated Fas, and its restoration by CD40 signal activation. While half of the lymphoma cell lines exhibited high sensitivity to Fas-mediated apoptosis, the remaining cell lines exhibited resistance to it. We found that an inhibitor of the apoptotic protein Livin was responsible for the resistance to Fas-mediated apoptosis and that a high expression of Livin is a poor prognostic factor for patients with BL and DLBCL. We further demonstrated that a bromodomain and extra-terminal (BET) protein BRD4 (bromodomain 4) drives the expression of Livin through its direct binding to the proximal region of the Livin gene promoter, suggesting that the expression of Livin is epigenetically regulated in refractory human lymphoma cells to protect them from immune cytotoxity. Accordingly, the combination of CD40-mediated Fas expression with the targeting of Livin or BET inhibitors is a potential immunotherapeutic strategy for treating aggressive B-cell NHLs. Citation Format: Sugihara Eiji, Norisato Hashimoto, Satoru Osuka, Sayaka Ueno, Takatsune Shimizu, Shogo Okazaki, Taka-aki Sato, Shinichiro Okamoto, Hideyuki Saya. BRD4 regulates the expression of Livin and thereby confers resistance to Fas-mediated immune cytotoxicity in aggressive B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2348.

Evolution of Alternative Adaptive Immune Systems in Vertebrates.

Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.

Abstract P5-04-10: Phosphodiesterase type 5 promotes the invasive potential of breast cancer cells through Rho GTPase activation

Abstract The impairment of cyclic guanosine monophosphate (cGMP) signaling by overexpression of PDE5 isoform has been recently described in multiple human carcinomas. In addition, accumulating evidences indicate that PDE5 inhibitors could have direct anti-cancer activities as well as they may enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs. However, despite these studies, neither the expression of PDE5 in breast cancer subtypes nor the underlying regulatory molecular mechanisms by which PDE5 expression may contribute to breast cancer progression have been deeply studied. We demonstrated that PDE5 was expressed in different subtypes of breast cancer cell lines at higher levels than in non tumorogenic human epithelial breast cell lines. Increased levels were detected in more aggressive endocrine non responsive basal-like breast cancer cells. Interestingly, PDE5 was expressed at very low levels in luminal A-type breast cancer cell lines, which display low ki67 expression, weak invasive behavior and endocrine responsiveness (MCF-7 and T47D cells) compared to luminal B-like cells (such as ZR-75 cells). These results well correlated with data obtained in immunohistochemistry analyses of human breast cancer tissues, showing PDE5 expression in 30 of 35 tumor entities analyzed, with the highest intensity staining in high-grade tumors. Concomitantly, no cytoplasmic PDE5 staining was observed in non neoplastic tissues examined (n=5). In addition, retrospective analyses (n=1959, median follow-up time: 25 years) showed that high PDE5 expression in breast cancer patients was correlated with a statistically significant poorer survival compared to low PDE5-expressing patients. A more relevant discrimination is achieved in lymphnode-negative patients, suggesting a role of PDE5 for identifying early patients at high risk of rapid progression. In order to better ascertain the role of PDE5 in breast tumorogenesis, we selected a breast tumor cell line that express low levels of this enzyme, MCF-7 and engineered stable clones for overexpression studies. Both vector- and PDE5-stable MCF-7 clones demonstrated comparable proliferation rates; whereas, cell motility and invasion were dramatically increased in PDE5-overexpressing cells. RNA sequencing to compare the transcriptomes of vector- and PDE5-overexpressing MCF-7 cells identified differential expression of genes involved in cell migration and invasion. Particularly, based on pathway analysis we found marked changes in the expression of Rho GTPase family members, proteins involved in cell cytoskeleton organization, migration, and metastasis dissemination (Rho A, cdc42 and Rac signaling, activation score= 1.9, 1.342, and 0.302, respectively). Indeed, Rho and cdc42 pull-down assays revealed increased Rho GTPase activity in cells overexpressing PDE5. Moreover, the selective ROCK inhibitor Y-27632 as well as the PDE5 inhibitor sildenafil were able to significantly reduce both migration and invasion of PDE5 clones. Our data reveal that PDE5 expression enhances motility and invasiveness of breast cancer cells through the activation of the Rho family of GTPases, and highlight, for the first time, a novel role for PDE5 as a marker of poor outcome in breast cancer patients. Citation Format: Barone I, Campana A, Giordano C, Tarallo R, Rinaldi A, Bruno G, Gyorffy B, Lanzino M, Bonofiglio D, Catalano S, Ando' S. Phosphodiesterase type 5 promotes the invasive potential of breast cancer cells through Rho GTPase activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-10.

Characterization of Lamprey IL-17 Family Members and Their Receptors.

IL-17 is an ancient cytokine implicated in a variety of immune defense reactions. We identified five members of the sea lamprey IL-17 family (IL-17D.1, IL-17D.2, IL-17E, IL-17B, and IL-17C) and six IL-17R genes (IL-17RA.1, IL-17RA.2, IL-17RA.3, IL-17RF, IL-17RE/RC, and IL-17RD), determined their relationship with mammalian orthologs, and examined their expression patterns and potential interactions to explore their roles in innate and adaptive immunity. The most highly expressed IL-17 family member is IL-17D.1 (mammalian IL-17D like), which was found to be preferentially expressed by epithelial cells of skin, intestine, and gills and by the two types of lamprey T-like cells. IL-17D.1 binding to rIL-17RA.1 and to the surface of IL-17RA.1-expressing B-like cells and monocytes of lamprey larvae was demonstrated, and treatment of lamprey blood cells with rIL-17D.1 protein enhanced transcription of genes expressed by the B-like cells. These findings suggest a potential role for IL-17 in coordinating the interactions between T-like cells and other cells of the adaptive and innate immune systems in jawless vertebrates.

A TRUE “CANDIDATE IG KAPPA GENE” IN THE SEA-STAR: <i>ASTERIAS RUBENS</i> (ECHINODERMA)

The axial organ of the sea-star Asterias rubens is a primitive immune organ. The B-like cells, when stimulated by various antigens, produce antibody substances correlated with Ig Kappa gene. A candidate Ig kappa gene (IgK chain V-IV region S107B precursor) more convincing in term of genome was shown.

Evidence of complement genes in the sea-star Asterias rubens. Comparisons with the sea urchin

The axial organ of the sea star Asterias rubens is a primitive immune organ. The B-like cells, when stimulated by various antigens, produce antibody substances correlating with Ig kappa genes, .On the other hand,component complement genes were found. For each component, one or several contigs were analyzed.It is said that Asterias forbesi, another sea-star, in earlier results, showed complement-like activity.A brief comparison with the complement system in sea urchin was performed, especially about the C3 component.

COMPLEMENT GENES IN THE ASTERIDS COMPARISONS WITH THE ECHINIDS (ECHINODERMA)

The axial organ of the sea star Asterias rubens (Asterid) is a primitive immune organ. The B-like cells, when stimulated by various antigens, produce antibody substances correlating with ig kappa genes. On the other hand, component complement genes were found. For each component, one or several contigs were analysed. It is said that Asterias forbesi, another sea-star, in earlier results, showed complement-like activity. A brief comparison with the complement system in sea urchin (Echinid) was performed, especially about the C3 component.

INNATE AND ADAPTATIVE IMMUNITY IN THE SEA-STAR ASTERIAS RUBENS

The axial organ of the sea star Asterias rubens is a primitive immune organ. The B-like cells, when stimulated by various antigens, produce antibody substances correlating with Mouse Ig Kappa genes. On the other hand elements of the innate immunity are present in Asterias rubens such as Toll-like receptors, interleukin receptors, complement components which contribute to initiate the «antibody factor».

A thymus candidate in lampreys

Immunologists and evolutionary biologists have been debating the nature of the immune system of jawless vertebrates--lampreys and hagfish--since the nineteenth century. In the past 50 years, these fish were shown to have antibody-like responses and the capacity to reject allografts but were found to lack the immunoglobulin-based adaptive immune system of jawed vertebrates. Recent work has shown that lampreys have lymphocytes that instead express somatically diversified antigen receptors that contain leucine-rich-repeats, termed variable lymphocyte receptors (VLRs), and that the type of VLR expressed is specific to the lymphocyte lineage: T-like lymphocytes express type A VLR (VLRA) genes, and B-like lymphocytes express VLRB genes. These clonally diverse anticipatory antigen receptors are assembled from incomplete genomic fragments by gene conversion, which is thought to be initiated by either of two genes encoding cytosine deaminase, cytosine deaminase 1 (CDA1) in T-like cells and CDA2 in B-like cells. It is unknown whether jawless fish, like jawed vertebrates, have dedicated primary lymphoid organs, such as the thymus, where the development and selection of lymphocytes takes place. Here we identify discrete thymus-like lympho-epithelial structures, termed thymoids, in the tips of the gill filaments and the neighbouring secondary lamellae (both within the gill basket) of lamprey larvae. Only in the thymoids was expression of the orthologue of the gene encoding forkhead box N1 (FOXN1), a marker of the thymopoietic microenvironment in jawed vertebrates, accompanied by expression of CDA1 and VLRA. This expression pattern was unaffected by immunization of lampreys or by stimulation with a T-cell mitogen. Non-functional VLRA gene assemblies were found frequently in the thymoids but not elsewhere, further implicating the thymoid as the site of development of T-like cells in lampreys. These findings suggest that the similarities underlying the dual nature of the adaptive immune systems in the two sister groups of vertebrates extend to primary lymphoid organs.

NKX6.1 combined with PDX1 induces mesenchymal stem cell differentiation into B-like cells

Objective To study the synergistic effect of NKX6.1 and PDX1 in inducing differentiation of fetal liver-derived mesenchymal stem cells(FL-MSCs) into the pancreatic B cells and to explore the underlying mechanisms,so as to obtain enough islet-like body for transplantation.Methods Recombinant adenovirus vector harboring both PDX1 and NKX6.1 genes was constructed,and the vector was used to infect FL-MSCs.Then a series of cytokines were used to induce the differentiation of infected FL-MSCs into pancreatic B cells.The expressions of PDX1,NKX6.1 gene,transcription factors NGN3,NeuroD1/Beta2,MafA as well as C-peptide were examined.Results PDX1 and NKX6.1 were detected in FL-MSCs cells 24 h after infection;cells began to express NGN3,NeuroD1,and MafA and stably expressed pancreatic B cell related factors including insulin after induction.The expression of these molecules was in a certain order.Conclusion PDX1,NKX6.1 combined with a series of cytokines can effectively induce FL-MSCs to differentiate into pancreatic islet B cells in vitro,which might be through activation of transcription factors NGN3,NeuroD1,and MafA in turn,inducing FL-MSCs to differentiate towards endocrine precursor cells,B endocrine precursor cells and B cells in turn.

A Life of Adventure in Immunobiology

This article outlines my early start in medicine, a late start in immunology research, and my efforts to integrate the two activities. I first describe some of the background information, excitement, and implications of the recognition of T and B cells as separate but functionally intertwined arms of the adaptive immune system. The article continues with a brief account of my colleagues' and my efforts to use the model of hematopoietic stem cell differentiation along T and B cell lines to gain a better understanding of immunodeficiency diseases and lymphoid malignancies. It concludes with the discovery of a more ancient adaptive immune system in which T-like and B-like cells in jawless vertebrates use variable lymphocyte receptors constructed with leucine-rich-repeat sequences to recognize antigens.

Antibody repertoire development in swine

Swine belong to the Order Artiodactyla and like mice and humans, express IgM, IgD, IgG, IgE and IgA antibodies but a larger number of IgG subclasses. Like rabbits and chickens, expressed V H genes belong to the ancestral V H3 family and only 5 comprise >80% of the pre-immune repertoire. Since they use primarily two D H segments and have a single J H like chickens, junctional diversity plays a relatively greater role in repertoire formation than in humans and mice. Proportional light chain usage surprisingly resembles that in humans and is therefore distinctly different from the predominant kappa chain usage (>90%) of lab rodents and predominant lambda chain usage in other ungulates (>90%). The pre-immune V κ repertoire also appears restricted since >95% of V κJ κ rearrangements use only a few members of the IGKV2 family and only J κ2. Two V λ families (IGLV3 and IGLV8) are used in forming the pre-immune repertoire. Antibodies that do not utilize light chains as in camelids, or the lengthy CDR3 regions seen in cattle that use V H4 family genes, have not been reported in swine. B cell lymphogenesis first occurs in the yolk sac but early VDJ rearrangements differ from mice and humans in that nearly 100% are in-frame and N-region additions are already present. Swine possess ileal Peyers patches like sheep which may be important for antigen-independent B cell repertoire diversification. The presence of pro B-like cells in interlobular areas of thymus and mature B cells in the thymic medulla that have switched to especially IgA in early gestation, is so far unique among mammals. The offspring of swine are believed to receive no passive immunity in utero and are precosial. Thus, they are a useful model for studies on fetal–neonatal immunological development. The model has already shown that: (a) colonization of the gut is required for responsiveness to TD and TI-2 antigens, (b) responsiveness due to colonization depends on bacterial PAMPs and (c) some viral pathogens can interfere with the establishment of immune homeostasis in neonates. Studies on swine reinforce concerns that caution be used when paradigms arising from studies in one mammal are extrapolated to other mammals, even when similarities are predicted by taxonomy and phylogeny. Swine exemplify a situation in which evolutionary diversification of the immune system is not characteristic of an entire order or even of other related systems in the same species.

Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin.

Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system.