Mikael Niku

Bos taurus genome sequence reveals the assortment of immunoglobulin and surrogate light chain genes in domestic cattle.

BackgroundThe assortment of cattle immunoglobulin and surrogate light chain genes has been extracted from the version 3.1 of Bos taurus genome sequence as a part of an international effort to sequence and annotate the bovine genome.Results63 variable lambda chain and 22 variable kappa chain genes were identified and phylogenetically assigned to 8 and 4 subgroups, respectively. The specified phylogenetic relationships are compatible with the established ruminant light chain variable gene families or subgroups. Because of gaps and uncertainties in the assembled genome sequence, the number of genes might change in the future versions of the genome sequence. In addition, three bovine surrogate light chain genes were identified. The corresponding cDNAs were cloned and the expression of the surrogate light chain genes was demonstrated from fetal material.ConclusionThe bovine kappa gene locus is compact and simple which may reflect the preferential use of the lambda chain in cattle. The relative orientation of variable and joining genes in both loci are consistent with a deletion mechanism in VJ joining. The orientation of some variable genes cannot be determined from the data available. The number of functional variable genes is moderate when compared to man or mouse. Thus, post-recombinatorial mechanisms might contribute to the generation of the bovine pre-immune antibody repertoire. The heavy chains probably contribute more to recombinational immunoglobulin repertoire diversity than the light chains but the heavy chain locus could not be annotated from the version 3.1 of Bos taurus genome.

The bovine genomic DNA sequence data reveal three IGHV subgroups, only one of which is functionally expressed

A comprehensive analysis of cattle shotgun sequencing data reveals 36 immunoglobulin heavy chain variable genes. The previously described bovine subgroup IGHV1 contains 10 functional genes with a conserved promoter including the consensus octamer and several other transcription factor binding sites, intact exons and matching cDNA sequences. Subgroups IGHV2 and IGHV3 consist entirely of pseudogenes. Thus, the bovine germline IGHV repertoire is very limited. The IGHV genes are distributed in mammalian clans I and II, while no clan III genes were detected. Clan-specific PCR of genomic DNA from cattle, sheep, Eurasian elk, white-tailed deer, pig and dolphin indicates highly dynamic evolution of IGHV gene usage within Cetartiodactyla. The bovine germline IGHV repertoire was probably generated by recent duplications of an IGHV1-IGHV2 homology unit. Immunoglobulin heavy chain genes are largely incorrectly assembled in the current cattle genome versions Btau_4.2 and UMD_3.1. FISH experiments confirm an IGHV locus close to terminus of BTA21.

Persistent differences in the level of chimerism in B versus T cells of freemartin cattle

Parabiosis during pregnancy regularly results in an exchange of hematopoietic stem cells between cattle twins. We have exploited this phenomenon and show differences in the levels of chimerism between the descendant cell types. Female recipients were screened for the levels of male donor contribution in surface IgM-bearing B lymphocytes versus CD3(+) T lymphocytes using immunomagnetic fractionation and Y-chromosome specific in situ hybridization. Two calves of 15 were discovered to have less than 10% of B cells but over 70% of T cells and other blood leukocytes of male origin. The donor cell ratios remained stable for 9 months. Analysis of lymphoid tissues revealed a similar cell type specific pattern of male cell ratios in both female calves and one twin brother. These findings are in agreement with the existence of an essentially self-sufficient population of developing B cells that gives rise to the peripheral pool of B lymphocytes in young cattle.

Limited contribution of circulating cells to the development and maintenance of nonhematopoietic bovine tissues

Bone marrow‐derived stem cells appear surprisingly multipotent in experimental settings, but the physiological significance of such plasticity is unclear. We have used sex‐mismatched cattle twins with stably chimeric hematopoietic systems to investigate the general extent of integration of circulating cells to the nonhematopoietic cell lineages in an unmanipulated large mammal. The donor‐derived (Y+) nonhematopoietic cells in female recipient tissues were visualized by Y‐chromosome specific in situ hybridization combined with pan‐leukocyte labeling. Y+ leukocytes were frequent in all tissues, but in 11 of 12 animals, average contribution to nonhematopoietic lineages was in any tissue below 1% (in brain <0.001%). Significantly higher integration rate was detected in regenerating granulation tissue. Also, one animal showed a high frequency of nonhematopoietic Y+ cells in several tissues, including intestinal epithelium and mammary gland stroma. In conclusion, circulating cells do not appear significant in the development and maintenance of nonhematopoietic bovine tissues, but may be important in regeneration and other special conditions.

Activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer’s patch and spleen and is associated with expansion of the primary antibody repertoire in the absence of exogenous antigens

Due to a limited range of immunoglobulin (Ig) genes, cattle and several other domestic animals rely on postrecombinatorial amplification of the primary repertoire. We report that activation-induced cytidine deaminase (AID) is strongly expressed in the fetal bovine ileal Peyer’s patch and spleen but not in fetal bone marrow. The numbers of IGHV (immunoglobulin heavy chain variable) mutations correlate with AID expression. The mutational profile in the fetuses is similar to postnatal and immunized calves, with targeting of complementarity-determining region (CDR) over framework region (FR), preference of replacement over silent mutations in CDRs but not in FRs, and targeting of the AID hotspot motif RGYW/WRCY. Statistical analysis indicates negative selection on FRs and positive selection on CDRs. Our results suggest that AID-mediated somatic hypermutation and selection take place in bovine fetuses, implying a role for AID in the diversification of the primary antibody repertoire in the absence of exogenous antigens.

Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

BackgroundCattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization.ResultsBull-derived CD34+ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34+ cells was lower in the freemartin than in its male siblings. Bull (Y+) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y+CD34+ cells, Y+CD34- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation.ConclusionThe fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.

B-cell development in bovine fetuses proceeds via a pre-B like cell in bone marrow and lymph nodes

The production of B cells and the primary antibody repertoire in mammalian species other than rodents or man appears to depend on gut-associated lymphoid tissue. Bovine B cells are generated in ileal Peyers patch from late gestational to juvenile age. However, little is known about where and when the bona fide B lymphopoiesis takes place. We analyzed bovine fetuses for signs of ongoing B lymphopoiesis using a combination of immunohistochemistry, flow cytometry, real-time quantitative PCR and RNA in situ hybridization. In fetal bone marrow and lymph node, we could demonstrate pre-B like cells positive for intracellular Ig mu but negative for membrane IgM. Strong expression of immunoglobulin lambda-like polypeptide 1 and recombination activating genes was also detected in the same tissues. Similar analyses did not reveal pre-B like cells in the corresponding adult tissues. These results suggest that bovine fetal bone marrow and lymph node support B lymphopoiesis via a pre-B cell like stage before and in parallel to the development of the ileal Peyers patch.

Oligoclonal Peyer's patch follicles in the terminal small intestine of cattle.

In small ruminants, the development of B cells differs from that in mice or in man. The anti-body repertoire is expanded in the Peyers patches of the terminal ileum where each B-cell follicle is found by a few cells. To investigate the amount of founder clones in bovine ileal follicles, we have used sex mismatched cattle twins. These animals are chimeric due to placental anastomoses. Y-chromosome targeted in situ hybridization was used to trace donor-derived cells of the male genotype in a female recipient (called a freemartin). A strong clustering of lymphoid cells originating from either twin was seen in the ileal Peyers patches (IPPs). Furthermore, the follicles displayed a low amount of immunoglobulin heavy chain gene configurations in comparison with the splenic or jejunal follicles. These findings strongly suggest that as in sheep, the B-cell follicles in cattle IPPs develop oligoclonally.

Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period.

Identification of major cell types in paraffin sections of bovine tissues

BackgroundIdentification of cell types in bovine tissue sections is complicated by the limited availability of anti-bovine antibodies, and by antigen retrieval treatments required for formalin-fixed tissue samples. We have evaluated an antibody and lectin panel for identifying major cell types in paraffin-embedded bovine tissue sections, and report optimized pretreatments for these markers.ResultsWe selected 31 useful antibodies and lectins which can be used to identify cell types of epithelia, connective tissue, muscle, and nervous tissue, as well as cell proliferation and apoptosis.ConclusionThe panel of markers allows the identification of all major cell types in paraffin-embedded cattle tissue sections by immunohistochemistry or lectin histochemistry. Heat-induced epitope retrieval methods are required for most antibodies.