Antibody repertoire development in fetal and neonatal piglets. XX. B cell lymphogenesis is absent in the ileal Peyer's patches, their repertoire development is antigen dependent, and they are not required for B cell maintenance

The continuous ileal Peyer's patches (IPP) of sheep are regarded as a type of mammalian bursal equivalent where B cells diversify their repertoire in an Ag-independent fashion. Anatomically and developmentally similar IPP occur in swine. Resection of ～90% of the IPP in piglets at birth did not alter Ig levels in serum and secretions or retard diversification of the Ab repertoire when animals were maintained in isolators and colonized with a defined gut flora. Resection or sham surgery elevated IgG and IgA in serum and in lavage fluid from the gut, lung, and in saliva. No changes in the frequency of IgG-, IgA-, and IgM-containing cells in the spleen and peripheral lymph node were observed. Using an index that quantifies diversification of the VDJ repertoire, no differences were seen in three secondary lymphoid tissues between piglets lacking IPP and colonized controls, whereas both groups displayed >10-fold greater diversification than did late-term fetal piglets or piglets maintained germ-free. Somatic hypermutation was very low in fetal IPP and the IPP of germ-free piglets but increased 3- to 5-fold after colonization. D-J signal joint circles were not recovered in IPP, and V-DJ signal joint circles were 5-fold lower than in bone marrow and similar to those in thymus and spleen. We conclude that the porcine IPP are not a site of B cell lymphogenesis, do not undergo Ag-independent repertoire diversification, and are not primary lymphoid tissue since they are not required for maintenance of Ig levels in serum and secretions.     

The inositol pyrophosphate pathway in health and diseases

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate‐containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet‐induced obesity (DIO), type‐2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio‐metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.     

Ileal Peyer's patches are not necessary for systemic B cell development and maintenance and do not contribute significantly to the overall B cell pool in swine

Based on studies of sheep, ileal Peyer's patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. Because bursectomy results in B cell deficiency, we wondered whether resection of the IPP of piglets would have a similar effect. Comparison of IPP-resected, surgical shams and untreated germ-free piglets, all of which were later colonized with a defined commensal flora, demonstrated that resection of the IPP did not alter the level and phenotype of B and T cells in lymphoid tissues and the blood 10 wk after surgery. Additionally, colonization of IPP caused a shift from the fetal type of lymphocyte distribution to the adult type that is characterized by prevalence of B cells, with many of them representing IgA(+) switched B cells or displaying a more mature CD2(-)CD21(+) and CD2(-)CD21(-) phenotype. Moreover, colonization leads to appearance of effector CD4(+)CD8(+) αβ T helper and CD2(+)CD8(-) γδ T cells. Comparison of germ-free with colonized pigs and experiments utilizing surgical transposition of jejunal Peyer's patch into terminal ileum or construction of isolated ileal loops indicated that lymphocyte development in IPP is dependent on colonization. Although our studies confirmed higher mitotic and apoptotic rates in IPP, they failed to identify any cell populations that resemble developing B lineage cells in the bone marrow. These results indicate that porcine IPP are not required for systemic B cell generation or maintenance, but they are secondary lymphoid tissue that appears important in immune responses to colonizing bacteria.     

Intestinal microflora and diversification of the rabbit antibody repertoire

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.     

Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.     

Antibody engineering for the analysis of affinity maturation of an anti-hapten response.

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-bearing anti-NP antibodies which by itself leads to an increase of hapten-binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.     

动物体细胞核移植技术的研究现状

动物体细胞核移植的成功表明动物体细胞的分化是可逆的,这是近年来人类在细胞生物学及发育生物学领域取得的最伟大的成就之一。１９９７年首例体细胞核移植绵羊在世界上诞生,之后不久出现了体细胞核移植小鼠及牛,并通过体细胞核移植技术制作了转基因动物。成就是不言而喻的,但其中也暴露出一些需克服和解决的问题。本文围绕世界上首例体细胞克隆羊诞生的背景、体细胞核移植技术目前的研究状况、体细胞核移植过程中的核质互作、体细胞核移植技术目前存在的问题、体细胞核移植技术在制作转基因动物上的应用等方面,扼要介绍了体细胞核移植技术在最近两年内取得的进展。     

A secreted salivary inositol polyphosphate 5-phosphatase from a blood-feeding insect: allosteric activation by soluble phosphoinositides and phosphatidylserine

Type II inositol polyphosphate 5-phosphatases (IPPs) act on both soluble inositol phosphate and phosphoinositide substrates. In many cases, these enzymes occur as multidomain proteins in which the IPP domain is linked to lipid-binding or additional catalytic domains. Rhodnius prolixus IPPRp exists as an isolated IPP domain which is secreted into the saliva of this blood-feeding insect. It shows selectivity for soluble and lipid substrates having a 1,4,5-trisphosphate substitution pattern while only poorly hydrolyzing substrates containing a D3 phosphate. With soluble diC8 PI(4,5)P(2) as a substrate, sigmoidal kinetics were observed, suggesting the presence of allosteric activation sites. Surprisingly, IPPRp-mediated hydrolysis of PI(4,5)P(2) and PI(3,4,5)P(3) was also stimulated up to 100-fold by diC8 PI(4)P and diC8 phosphatidylserine (PS). The activation kinetics were again sigmoidal, demonstrating that the allosteric sites recognize nonsubstrate phospholipids. Activation was positively cooperative, and analysis by the Hill equation suggests that at least three to four allosteric sites are present. In a vesicular system, hydrolysis of PI(4,5)P(2) followed a surface dilution kinetic model, and as expected, PS was found to be strongly stimulatory. If allosteric activation of type II IPPs by PI(4)P and PS is a widespread feature of the group, it may represent a novel regulatory mechanism for these important enzymes.     

A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset

The leukocyte common antigen (LCA, CD45) of humans and rodents is expressed exclusively by leukocytes, and has been implicated in a number of immune functions (1-4), although its precise function is still unknown. Three monoclonal antibodies (mAbs) were produced which identified different epitopes on the LCA of sheep. mAbs 1-11-32 and 38-42 reacted with determinants of LCA expressed on all leukocytes, but showed differential reactivity with thymocytes. Another antibody, 20-96, identified an epitope of LCA expressed mainly on B cells, but also on a unique lymphocyte subset contained mostly in peripheral blood, which was 20-96high, sIg-, CD4-, CD8-, SBU-T19-, but CD5+. These cells constituted only 5-6% of PBL. The cellular lineage of this latter subset is uncertain since these cells appeared to be unrelated to B cells, and were absent from the thymus. Unlike the other two mAbs to LCA, 20-96 was not reactive with macrophages and granulocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of material immunoprecipitated by the "pan" LCA-specific mAbs revealed lymphocyte forms with molecular weights (MW) of 220, 210, and 190K, whereas 20-96 immunoprecipitated only a 220K MW form. The expression and MW of LCA on thymocytes or ileal Peyer's patch (IPP) cells differed from those on peripheral lymphocytes. B cells in IPP, which constitute 98% of cells, expressed the 20-96 determinant at low density, in contrast to its high expression on peripheral B cells. LCA from IPP existed in two forms of 220 and 190K MW, whereas LCA from peripheral B cells was entirely 220K MW, and thymus 210 and 190K MW.     

A new model of sheep Ig diversification: shifting the emphasis toward combinatorial mechanisms and away from hypermutation

The current model of Ig repertoire development in sheep focuses on the rearrangement of a small number (approximately 20) of Vlambda gene segments. It is believed that this limited combinatorial repertoire is then further diversified through postrearrangement somatic hypermutation. This process has been reported to introduce as many as 110 mutations/1000 nucleotides. In contrast, our data have that indicated somatic hypermutation may diversify the preimmune repertoire to a much lesser extent. We have identified 64 new Vlambda gene segments within the rearranged Ig repertoire. As a result, many of the unique nucleotide patterns thought to be the product of somatic hypermutation are actually hard-coded within the germline. We suggest that combinatorial rearrangement makes a much larger contribution, and somatic hypermutation makes a much smaller contribution to the generation of diversity within the sheep Ig repertoire than is currently acknowledged.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.     

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.     

Sequence transfers between variable regions in a mouse antibody transgene can occur by gene conversion

Different vertebrate species show widely differing usage of somatic hyperconversion (SHC) as a mechanism for diversifying expressed Ab V genes. The basis for the differing levels of SHC in different species is not known. Although no clear evidence for SHC has been found in normal mouse B cells, transgenic mice carrying high-copy numbers of a gene construct designed to optimize detection of SHC have previously been shown to exhibit sequence transfers that resemble gene conversion events. However, these transgene sequence transfers could reflect multistep or reciprocal DNA recombination events rather than gene conversions. We now find in low-copy number transgenic mice that transgene sequence transfers can exhibit the unidirectional sequence information movement that is a hallmark of gene conversion. This indicates that gene conversion between V region sequences can occur in mouse B cells; we propose that the lack of efficient SHC contributions to Ab diversification in normal mice may be due, at least in part, to the particular pattern of V gene recombinational accessibility that occurs in differentiating mouse B cells.     

Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.     

SMUG1 is able to excise uracil from immunoglobulin genes: insight into mutation versus repair

Mammals harbour multiple enzymes capable of excising uracil from DNA, although their distinct physiological roles remain uncertain. One of them (UNG) plays a critical role in antibody gene diversification, as UNG deficiency alone is sufficient to perturb the process. Here, we show this unique requirement for UNG does not reflect the fact that other glycosylases are unable to access the U:G lesion. SMUG1, if overexpressed, can partially substitute for UNG to assist antibody diversification as judged by its effect on somatic hypermutation patterns (in both DT40 B cells and mice) as well as a restoration of isotype switching in SMUG-transgenic msh2-/- ung-/- mice. However, SMUG1 plays little natural role in antibody diversification because (i) it is diminishingly expressed during B-cell activation and (ii) even if overexpressed, SMUG1 more appears to favour conventional repair of the uracil lesion than assist diversification. The distinction between UNG and overexpressed SMUG1 regarding the balance between antibody diversification and non-mutagenic repair of the U:G lesion could reflect the association of UNG (but not SMUG1) with sites of DNA replication.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.