Bovine B cells : Antibody repertoire diversification in fetal cattle

The majority of research studies on immunological mechanisms have been conducted in human or rodent models and the results are generalized in respect of other vertebrates. However, the generation of B cells varies considerably between species. B cells are produced in the bone marrow of rodents and humans throughout life. The initial antibody diversity is produced by the random assembly of a great variety of antibody encoding gene segments. The repertoire of these gene segments is limited in many domestic species such as cattle, sheep, chicken, rabbit and pig and de novo B lymphopoiesis takes place only during fetal and/ or neonatal life. This results in a basal B cell population but one that is capable of being further expanded by other mechanisms. In this thesis, the specific diversification mechanisms in the bovine are explored. Knowledge of immunoglobulin genes is essential in order to distinguish various biological mechanisms, which are involved in antibody repertoire diversification. After the bovine genome was sequenced in 2009, the characterization of the immunoglobulin loci became possible. Previously, the bovine immunoglobulin light chain loci were characterized by only a small number of functional gene segments that had been found. Subsequently, we were able to analyse the heavy chain locus and found a total number of 62 heavy chain variable gene segments, of which 10-20 were verified as functional genes. Many livestock species and chicken rely on gut-associated lymphoid tissue for further expanding their fetal/neonatal antibody repertoire to compensate for the limited effective recombinatorial diversity. In cattle, the ileal Peyer s patch (IPP) is considered to be the major organ for B cell proliferation. In this research, the fingerprints of somatic hypermutation (SHM) in the IPP were analysed. SHM is dependent on activation-induced cytidine deaminase (AID), which contributes to modifying the immunoglobulin variable regions. SHM is conventionally considered to be a secondary diversification mechanism, which is activated with external antigens. AID-mediated SHM was shown to diversify the antibody repertoire in the fetal IPP, before the exposure of exogenous antigen encounters. Junctional diversity is produced by the random additions and excisions of nucleotides between immunoglobulin gene segments that occur during the somatic recombination. Non-templated nucleotides are added by terminal deoxynucleotidyl transferase (TdT). Sequence investigation in this study indicated that TdT-mediated junctional diversity contributes to the diversification of the antibody repertoire in bovine fetuses. Extensive junctional diversity mainly in the heavy chain sequences in bone marrow, ileum and spleen but also to a lesser extent in the light chains was detected in this study. The following model for bovine preimmune repertoire diversification was suggested in this study. First, the restricted immunoglobulin germline repertoire is diversified by junctional diversity in fetal bone marrow. Second, a small population of the B cell clones migrates to the fetal/neonatal IPP. In the IPP, further diversification by AID-mediated SHM takes place, which is associated with extensive proliferation. Third, these clones migrate to other peripheral organs where they are subjected to secondary, antigen-induced modifications. The IPP starts to involute in young animals and the animal survives for the rest of its life by proliferating and differentiating its B cell clones from the peripheral repertoire.Laaja vasta-ainekirjo on elintärkeä osa immuunipuolustusta, jotta elimistö pystyy tunnistamaan ja torjumaan taudinaiheuttajat. Eri eläinlajit käyttävät erilaisia strategioita vasta-ainekirjon luomiseen. Tietämyksemme immunologisista mekanismeista perustuu suurimmaksi osaksi tutkimuksiin, jotka on tehty ihmisillä tai hiirillä. Näitä tietoja ei kuitenkaan suoraan voida yleistää kaikkiin selkärankaisiin, sillä esimerkiksi B-solujen tuotanto eroaa huomattavasti eri lajien välillä. Ihmisellä ja hiirellä uusia B-soluja tuotetaan luuytimessä koko elinajan. Näillä lajeilla vasta-aineiden monimuotoisuus perustuu pitkälti siihen, että vasta-ainegeenit muodostuvat lukuisista vaihtoehtoisista palasista. Naudalla B-soluja on osoitettu syntyvän sikiöaikana mutta ei enää aikuisena. Myös naudan vasta-ainegeenivalikoima on ollut huonosti tunnettu ja samoin kun geenien monimuotoistumismekanismit. Tämän väitöskirjan tavoitteena on tutkia miten nauta muodostaa monipuolisen vasta-ainekirjon sikiöaikana. Erilaistuneet B-solut tuottavat miljoonia erilaisia vasta-aineita. Vasta-aineet rakentuvat kahdesta identtisestä kevyt- ja raskasketjusta, jotka edelleen koostuvat variaabeleista- ja vakioalueista. Monimuotoistumiseen vaikuttavia mekanismeja on lähes mahdoton tutkia jos vasta-ainegeenien alkuperäinen sekvenssi ei ole selvillä. Vuonna 2009 naudasta (Bos taurus) tuli kanan jälkeen toinen kotieläinlaji, jonka genomi on kokonaan sekvensoitu. Tämä mahdollisti naudan vasta-ainegeenien tarkemman karakterisoinnin. Pian tämän jälkeen osoitettiin, että naudalla on vain kohtalainen määrä erilaisia kevyenketjun rakentumiseen tarvittavia geenejä. Tämän väitöskirjan ensimmäisessä osatyössä tutkittiin monimuotoisuutta raskasketjun osalta. Raskasketjun variaabeleita geenejä löytyi 62, joista 10-20 ovat toimivia geenejä tai niiden alleeleja. Kevyt- ja raskasketjun yhdistelmät eivät siis yksinään pysty tuottamaan tarpeeksi monimuotoista vasta-ainekirjoa sikiöaikana. Somaattinen hypermutaatio on tunnettu vasta-aineiden monimuotoisuuteen vaikuttava mekanismi, jota välittää aktivaatio-indusoitu sytidiini deaminaasi (AID) entsyymi. AID-entsyymin toiminta on aikaisemmin osoitettu käynnistyvän syntymän jälkeen immuunivasteen yhteydessä. Väitöskirjan toisessa osatyössä osoitettiin AID-välitteisen somaattisen hypermutaation tapahtuvan naudalla jo sikiöaikana ileumin Peyerin levyissä. Tässä tutkimuksessa saatiin uutta tietoa AID-entsyymin toiminnasta ennen ulkoista antigeenialtistusta. Kolmannessa osatyössä tutkittiin vasta-ainegeenien liitoskohtien monimuotoisuutta naudan sikiön luuytimessä. Somaattisen rekombinaation aikana vasta-ainegeenit muodostuvat useasta palasesta. Geenien järjestelyn yhteydessä liitoskohtiin voi liittyä tai niistä voi poistua nukleotideja terminaalisen deoksinukleotidyylitransferaasi (TdT) entsyymin vaikutuksesta. Väitöskirjassa tämän entsyymin osoitettiin toimivan naudalla aktiivisesti jo sikiöaikana, kasvattaen vasta-ainekirjon monimuotoisuutta. Nukleotidilisäyksiä löytyi sekä kevyt- että raskasketjuista mutta erityisin pitkiä lisäyksiä löytyi osasta raskasketjuja. Näiden tulosten perusteella naudalla on kohtuullisen pieni alkuperäinen vasta-ainegeenivalikoima, jota sikiöaikana monimuotoistetaan muokkaamalla vasta-ainegeenien liitoskohtia sekä kohdistetuilla mutaatioilla. Tässä väitöskirjassa tehdyt löydökset viittaavat huomattaviin lajityypillisiin eroihin immuunijärjestelmän kehittymisessä ja toiminnassa. Immunologista tutkimusta ei tule rajoittaa vain muutamaan lajiin, vaan vertailevan immunogian tutkimus on ensiarvoisen tärkeää

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

<p>Abstract</p> <p>Background</p> <p>The assortment of cattle immunoglobulin and surrogate light chain genes has been extracted from the version 3.1 of <it>Bos taurus </it>genome sequence as a part of an international effort to sequence and annotate the bovine genome.</p> <p>Results</p> <p>63 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.</p> <p>Conclusion</p> <p>The 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 <it>Bos taurus </it>genome.</p

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

Peer reviewe

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

Analysis of nucleotide additions in bovine fetal IGH (bone marrow), IGL and IGK (bone marrow and ileum).

<p>Analysis of nucleotide additions in bovine fetal IGH (bone marrow), IGL and IGK (bone marrow and ileum).</p

The effect of removal of 63 cDNAs linked to high exonuclease activity to the frequency of <i>IGHD</i> segments in bovine fetal immunoglobulin cDNAs.

<p>Exonuclease activity was deduced from the alignments with the best matching <i>IGHD</i> segment and quantified by the number of apparently excised nucleotides. High exonuclease activity: excision of over 29 nucleotides from either end of the <i>IGHD</i> segment.</p

TdT mRNA expression level in adults and fetuses.

<p>The expression level was measured with RT-qPCR. Material consisted of 3 fetuses and 2 adults. Thymus, bone marrow and lymph node had elevated expression levels compared to liver, ileum and spleen. Fetuses did not differ from adults. 18S normalized cycle threshold values (ΔC_t) are shown. Tissues not differing statistically (α = 0.05) from each other are indicated by a horizontal bar. White points indicate fetuses and black points indicate adults.</p

PCR primers and probes.

<p>*Nucleotides in square brackets refer to locked nucleic acids.</p

Junctional diversity in bovine fetuses.

<p>N nucleotide additions in IGH (VD and DJ junctions, n = 645) IGL (λVJ junction, n = 65) and IGK (κVJ junction, n = 83). Dark line in the middle represents the median and 50% of the cases lie within the box. Whiskers extend to 1.5 times the height of the box. Circles represent outliers. We did not detect statistically significant differences between tissues or individuals. Heavy chain data is pooled from bone marrow, ileum and spleen (two fetuses). Light chain data is pooled from bone marrow and ileum (one fetus).</p

Immunofluorescence staining of fetal bovine bone marrow (A) and lymph node (B).

<p>Red: B lymphocyte marker CD79α. Green: T lymphocyte marker CD3. White: TdT. Blue: DAPI. White arrows: TdT positive B cells. Black arrows: TdT positive T cells. Scale bar: 50 µm.</p