Autoreactivity by design: innate B and T lymphocytes.

Innate B and T lymphocytes are a subset of lymphocytes that express a restricted set of semi-invariant, germ-line-encoded, autoreactive antigen receptors. Although they have long been set apart from mainstream immunological thought, they now seem to represent a distinct immune-recognition strategy that targets conserved stress-induced self-structures, rather than variable foreign antigens. Innate lymphocytes regulate a range of infectious, tumour and autoimmune conditions. New studies have shed light on the principles and mechanisms that drive their unique development and function, and show their resemblance to another subset of innate lymphocytes, the natural killer cells.

The human fetal omentum: a site of B cell generation.

The fetal mouse omentum has been shown to be a source of precursors that exclusively reconstitutes Ly1+ B cells and the closely related Ly1- sister population, but not conventional B cells or T cells. We have extended these studies to compare B cell development in the human fetal omentum, liver, and spleen, and to demonstrate that the pro/pre-B cell compartment (CD24+, sIgM-) is detected in the omentum and liver but not spleen as early as 8 wk of gestation. From 8 to 12 wk of gestation, the proportions of IgM+ cells that were pre-B cells (cIgM+/sIgM-) in the omentum and liver were 53 +/- 15% and 45 +/- 13%, respectively, and IgM+ cells were not detectable in the spleen. After 12 wk, the percentage of pre-B cells was unchanged in the fetal liver (41 +/- 10%) but decreased significantly in the omentum (25 +/- 14%); pre-B cells were now detected in the spleen but at much lower percentages (2 +/- 3%) than either the omentum or liver. The nuclear enzyme, Tdt, was detected in approximately 25% of the CD24+ cells in the omentum and liver during the 8-12-wk time period, however, Tdt+ cells were not detected in the spleen. Approximately 40% of the mature B cells found in the omentum and spleen were CD5+ compared with only 20% in the liver. These results demonstrate that the fetal omentum, like the fetal liver and bone marrow, is a primary site of B cell development.

Fine idiotype analysis of B cell precursors in the T-dependent and T-independent responses to alpha 1-3 dextran in BALB/c mice.

In this study BALB/c B cell precursors responsive to the T-independent (TI) type 2 (TI-2) antigen, dextran B1355S (DEX), and the T-dependent (TD) derivative, dextran-Limulus hemocyanin (DEX-Hy) were examined for isotype and idiotope expression using the splenic focus assay. The predominant isotype detected in the TI assay was IgM, while IgA was the predominant isotype expressed in the TD assay. There was also a fourfold increase in the number of foci secreting more than one isotype in the TD assay vs. the TI assay without an overall change in anti-DEX precursor frequency, suggesting that carrier-primed T cells enhance the expression of non-IgM isotypes possibly by increasing the frequency of isotype switching by individual B cell precursors. A panel of distinct monoclonal antiidiotype antibodies (MAIDs) was then used to examine idiotope expression by antibodies secreted in splenic foci responding to DEX and DEX-Hy. This analysis revealed considerable diversity in the idiotope profiles expressed by all isotypes tested. There appeared to be no differences in idiotope diversity among the various isotypes. A similar diversity of idiotope profiles was obtained from both TI and TD splenic foci, indicating that a comparable degree of diversity was associated with the antibodies generated by TI and TD precursors. Idiotype analysis of IgM-IgA-secreting foci with a panel of monoclonal antiidiotope antibodies revealed slight idiotypic differences between the two isotypes secreted in the same focus in about half the cases. These results suggest that somatic variation occurs during the antigen-driven maturation of B cell precursors, within the 15-d time frame of the splenic focus assay, and may be associated with isotype switching.

Idiotypic network connectivity and a possible cause of myasthenia gravis.

Extensive idiotypic connectivity has been discovered between the antibodies composing the immune responses against the acetylcholine receptor (AChR) and alpha-1,3-dextran. The idiotypic connections form an elaborate network linking these disparate antigen systems, and there is an hierarchical organization of the antibodies in this network. The key anti-Ids that interconnect these two responses are more crossreactive, lower-affinity antibodies. Interestingly, 15% of patients with MG, which is caused by autoantibodies against the AChR, have serum antibodies against DEX. Control sera are negative for anti-DEX antibodies. Certain anti-DEX antibodies also bind to anti-AChR antibodies via idiotypic interactions. These findings suggest a model for the initiation of autoimmunity in MG. Antibodies made in response to DEX epitopes on the surface of certain bacteria would elicit the production of anti-Ids. However, some of these anti-Ids would also be autoantibodies against the AChR. Thus, is some circumstances, autoimmunity may develop as a consequence of the normal operation of regulatory idiotypic networks.

The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform.

During variable/diversity/joining (V[D]J) recombination, the enzyme terminal deoxynucleotidyl transferase (Tdt) adds random nucleotides at the junctions of the rearranging gene segments, increasing diversity of the antibody (Ab) and T cell receptor repertoires. Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro. We have expressed each splice variant of Tdt in transgenic (Tg) mice and found that the TdtS transgene catalyzes N addition on the endogenous Tdt(-/)- background and in fetal liver, but that the long isoform of Tdt (TdtL) transgene does neither. In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model. Furthermore, TdtL/TdtS double Tg mice exhibit less N addition in fetal liver than do TdtS Tg mice. Whereas the TdtS transgene was shown to have functional consequences on the antiphosphorylcholine (PC) B cell repertoire, TdtL Tg mice exhibit a normal PC response, and Tdt(-/)- mice actually exhibit an increase in the PC response and in TEPC 15 idiotype(+) Ab production. We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.

Cross-reactivity between C-reactive protein and idiotypic determinants on A phosphocholine-binding murine myeloma protein.

Binding of human 125I-C-reactive protein (CRP) to sheep erythrocytes sensitized with pneumococcal C polysaccharide (E-PnC) was found to be Ca++ dependent and inhibitable by phosphocholine, CRP, and HOPC 8. Binding of 125I-HOPC 8 to EPnC was Ca++ -independent but could also be inhibited by phosphocholine, CRP, and HOPC 8. Thus, CRP and HOPC 8, despite a differential Ca++ requirement, share a common binding specificity for phosphocholine. A monoclonal anti-idiotypic antibody (MAB), GB4-10, prepared in A/J mice immunized with BALB/c HOPC 8 inhibited the binding of both 125I-CRP and 125I-HOPC 8 to E-PnC. In addition, both proteins bound to GB4-10 immobilized on polysterene tubes. Interestingly, binding of 125I-CRP to GB4-10 required Ca++. Similar results were also obtained with another MAB (AB1-2) prepared similarly to GB4-10, whereas neither protein bound to a control MAB (EB3-7) against an alpha1 leads to 3 dextran-binding myeloma protein, J558. Binding of 125I-HOPC 8 to GB4-10 could be inhibited by HOPC 8, keyhole limpet hemocyanin-phosphocholine but not phosphocholine but not phosphocholine, and in the presence of Ca++ by CRP. These data indicate that CRP bears antigenic determinants cross-reacting with certain idiotypic determinants on HOPC 8. They also suggest that Ca++ acts as an allosteric effector, perhaps stabilizing the phosphocholine-binding site of CRP.

Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

Immunoglobulin VH determinants defined by monoclonal antibodies.

Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

Ontogeny of Ia and IgD on IgM-bearing B lymphocytes in mice.

We used immunofluorescence to examine the developmental relationship of Ia and IgD on B cells. Pre-B cells in fetal liver did not express Ia. Only very few surface IgM-positive (sIgM+) B cells in fetal spleen were found to be Ia+ and were weakly stained for Ia. After birth there was a linear increase in the proportion of sIgM+ spleen cells which expressed Ia, reaching 95% by 9 days. Adult bone marrow also contains a sizeable proportion of sIgM+ Ia- cells. Unstimulated cells from fetal or newborn liver and spleen expressed Ia at the same rate in culture. Anti-Ia antisera suppressed the LPS-induced differentiation of IgM and IgG plasma cells in cultures of neonatal lymphocytes. Ia was also detected on IgM and IgG plasma cells in vitro suggesting that lipopolysaccharide (LPS)-stimulated B cells by may express Ia antigens, induced by LPS, or appearing as part of normal differentiation. IgD did not appear on sIgM+ cells until 3 days of age and then rose slowly to reach adult levels later than Ia antigens.

Murine Peyer's patch T cell clones. Characterization of antigen-specific helper T cells for immunoglobulin A responses.

We successfully cloned antigen-specific T cells from murine gut-associated lymphoreticular tissue, i.e., Peyer's patches, which are dependent upon T cell growth factor and independent of antigen for continuous growth. These clones exhibit helper activity for IgA responses to sheep erythrocytes (SRBC) and have been designated T helper (Th) A. Two broad categories of Th A clones have been maintained in continuous culture. The first group supports IgM and largely IgA anti-SRBC plaque-forming cell (PFC) responses in both normal and SRBC-primed splenic B cell cultures, whereas the second group supports low IgM, IgG1, and IgG2 and high IgA PFC responses. Subclones derived from single cells maintain the parent helper properties when propagated in culture for long periods (greater than 7 mo). Cloned Th A cells are antigen specific and do not support polyclonal or immune responses to other thymus dependent antigens in normal B cell cultures. Th A cells require full histocompatibility for helper functions because addition of cloned Th A cells to B cell cultures from other H-2 types does not result in IgA responses. Cloned Th A cells are Thy-1.2+ and Lyt-1+ and Lyt-2-, Ig-, and I-A-. Th A cells bear Fc receptors for IgA and do not possess receptors for IgM or IgG isotypes. Thus, T cells that primarily promote IgA isotype responses have been isolated in high frequency from murine PP, an anatomical site of major importance for induction and regulation of the IgA response.

Isotype specificity of helper T cell clones. Peyer's patch Th cells preferentially collaborate with mature IgA B cells for IgA responses.

The nature of the IgA B cell precursors that receive preferential help from selected clones of T helper cells from mouse Peyer's patches (PP Th A) were studied. Activation of the PP Th A clones required the presence of antigen, sheep erythrocytes (SRBC), in a culture system supporting development of antibody-secreting plasma cells. Two types of PP Th A cells were used. Both gave vigorous IgA responses; the first also supported low IgM, and the second low IgM and IgG subclass antibody responses. Removal of sIgA+ B cells from either splenic or PP B cell cultures selectively depleted precursors of IgA antibody producers. Cultures of purified sIgA+ B cells, cloned PP Th A cells and SRBC, selectively yielded IgA antibody producers. Finally, PP Th A cells did not support IgA responses in B cell cultures derived from spleens of young mice (days 1-25), and full IgA responses were not seen until the donor mice were 6-7 wk of age. These results suggest that cloned T helper cells can recognize and collaborate with mature, IgA committed B cells.

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein.

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.

Developmentally controlled expression of immunoglobulin VH genes.

Although antibody diversity arises mainly from apparently random combinatorial and somatic mutational mechanisms acting upon a limited number of germline antibody genes, the antibody repertoire develops in an ordered fashion during mammalian ontogeny. A series of early pre-B and B-lymphocyte cell lines were examined to determine whether an ordered rearrangement of gene families of the variable region of immunoglobulin heavy chains (VH) may be the basis for the programmed development of the antibody response. The results indicated that the VH repertoire of fetal B-lineage cells is largely restricted to the VH 7183 gene family and that subsequent recruitment of additional VH gene families occurs during neonatal development. These results have important implications in understanding the ontogeny of immune function.

Short communication: genetic relationships between the Holstein cow populations of three European dairy countries.

The degree of relatedness was studied in 3 dairy cow populations from Great Britain (GBR), Italy (ITA), and Ireland (IRL) by using cows born from 2003 to 2006. Effective population size, inbreeding coefficient (F), and average relationship in the top and bottom 4,000 cows ranked on a profit index value (PIV) or milk yield evaluations were studied. Average inbreeding was approximately 2% in GBR and ITA, was 1% in IRL, but was slightly more than 2% when the joint pedigree was used. The average F for the joint population was 10 to 15% higher than estimates averaged across the 3 populations, reflecting the increased completeness of pedigree information in the joint pedigree. Effective population size in the joint pedigree was approximately 12% lower than estimates within the individual countries. The average genetic relationships for the top 4,000 PIV cows were not markedly different from those based on milk evaluation in GBR and ITA, but were approximately 2% lower in IRL. This was due to the use of an index with less weight on production traits in IRL compared with GBR and ITA. However, selection of the top 4,000 cows on PIV reduced the degree of relatedness across the 3 countries. The use of common sires accounted for most of the relatedness across the 3 countries, more than did the use of related sires or common foreign dams.

CD5+ B-cell networks.

The relationship between precursors for B1 and B2 cells remains controversial. On the basis of recent experimental results, it is highly probable that B1 cells can be generated from the same sources as B2 cells. However, the precursor cell types involved, the microenvironment and the age of donor and recipient may all determine whether B1 cells are generated under the variety of experimental conditions that have been described in the literature. Definition of these influences will be important in understanding the role of B1 cells in autoimmune disease. Further differences in antigen responsiveness, localization and signaling between B1 and B2 cells will also be informative with respect to their roles in antibody production.

B1 cells: similarities and differences with other B cell subsets.

Since their discovery, B1 B cells' origins and developmental pathways have eluded characterization. In the past year, focus on B1 B cells has shifted dramatically from developmental to functional aspects of these cells. Most advances have been made in describing the physiological activities of B1 cells, including their migration, activation by antigen and role in both autoimmunity and malignancy.

Inbreeding effects on milk production, calving performance, fertility, and conformation in Irish Holstein-Friesians.

The objective of this study was to determine the effect of inbreeding on milk production, somatic cell count, fertility, survival, calving performance, and cow conformation in Irish Holstein-Friesian pluriparous dairy cows. Inbreeding was included in a linear mixed model as either a class variable or a continuous variable, where higher order polynomials of the latter were also tested in the model as an indicator of nonlinear inbreeding depression. The effects of dam inbreeding and calf inbreeding on calving-related traits were analyzed separately. Inbreeding had a deleterious effect on most of the traits analyzed, although inbreeding depression was sometimes nonlinear or differed significantly across parities. A primiparous animal, 12.5% inbred (i.e., following the mating of noninbred half-sibs), had milk, fat, and protein yields reduced by 61.8, 5.3, and 1.2 kg, respectively; fat and protein concentrations reduced by 0.05 and 0.01%, respectively; and somatic cell scores (i.e., natural log of somatic cell count divided by 1,000) increased by 0.03. The 12.5% inbred animal was also expected to have a 2% greater incidence of dystocia, a 1% greater incidence of stillbirth, a 0.7% greater incidence of male calves, an increase in calving interval of 8.8 d, an increase in age at first calving of 2.5 d, and a reduced survival to second lactation of 4 percentage units. Inbred animals were also taller, narrower, and more angular. Although the effects of inbreeding were statistically significant, they were small and are unlikely to cause great financial loss on Irish dairy farms.

Inference of population structure of purebred dairy and beef cattle using high-density genotype data.

Information on the genetic diversity and population structure of cattle breeds is useful when deciding the most optimal, for example, crossbreeding strategies to improve phenotypic performance by exploiting heterosis. The present study investigated the genetic diversity and population structure of the most prominent dairy and beef breeds used in Ireland. Illumina high-density genotypes (777 962 single nucleotide polymorphisms; SNPs) were available on 4623 purebred bulls from nine breeds; Angus (n=430), Belgian Blue (n=298), Charolais (n=893), Hereford (n=327), Holstein-Friesian (n=1261), Jersey (n=75), Limousin (n=943), Montbéliarde (n=33) and Simmental (n=363). Principal component analysis revealed that Angus, Hereford, and Jersey formed non-overlapping clusters, representing distinct populations. In contrast, overlapping clusters suggested geographical proximity of origin and genetic similarity between Limousin, Simmental and Montbéliarde and to a lesser extent between Holstein, Friesian and Belgian Blue. The observed SNP heterozygosity averaged across all loci was 0.379. The Belgian Blue had the greatest mean observed heterozygosity (HO=0.389) among individuals within breed while the Holstein-Friesian and Jersey populations had the lowest mean heterozygosity (HO=0.370 and 0.376, respectively). The correlation between the genomic-based and pedigree-based inbreeding coefficients was weak (r=0.171; P<0.001). Mean genomic inbreeding estimates were greatest for Jersey (0.173) and least for Hereford (0.051). The pair-wise breed fixation index (F st) ranged from 0.049 (Limousin and Charolais) to 0.165 (Hereford and Jersey). In conclusion, substantial genetic variation exists among breeds commercially used in Ireland. Thus custom-mating strategies would be successful in maximising the exploitation of heterosis in crossbreeding strategies.

Ultra-low-density genotype panels for breed assignment of Angus and Hereford cattle.

Angus and Hereford beef is marketed internationally for apparent superior meat quality attributes; DNA-based breed authenticity could be a useful instrument to ensure consumer confidence on premium meat products. The objective of this study was to develop an ultra-low-density genotype panel to accurately quantify the Angus and Hereford breed proportion in biological samples. Medium-density genotypes (13 306 single nucleotide polymorphisms (SNPs)) were available on 54 703 commercial and 4042 purebred animals. The breed proportion of the commercial animals was generated from the medium-density genotypes and this estimate was regarded as the gold-standard breed composition. Ten genotype panels (100 to 1000 SNPs) were developed from the medium-density genotypes; five methods were used to identify the most informative SNPs and these included the Delta statistic, the fixation (F st) statistic and an index of both. Breed assignment analyses were undertaken for each breed, panel density and SNP selection method separately with a programme to infer population structure using the entire 13 306 SNP panel (representing the gold-standard measure). Breed assignment was undertaken for all commercial animals (n=54 703), animals deemed to contain some proportion of Angus based on pedigree (n=5740) and animals deemed to contain some proportion of Hereford based on pedigree (n=5187). The predicted breed proportion of all animals from the lower density panels was then compared with the gold-standard breed prediction. Panel density, SNP selection method and breed all had a significant effect on the correlation of predicted and actual breed proportion. Regardless of breed, the Index method of SNP selection numerically (but not significantly) outperformed all other selection methods in accuracy (i.e. correlation and root mean square of prediction) when panel density was ⩾300 SNPs. The correlation between actual and predicted breed proportion increased as panel density increased. Using 300 SNPs (selected using the global index method), the correlation between predicted and actual breed proportion was 0.993 and 0.995 in the Angus and Hereford validation populations, respectively. When SNP panels optimised for breed prediction in one population were used to predict the breed proportion of a separate population, the correlation between predicted and actual breed proportion was 0.034 and 0.044 weaker in the Hereford and Angus populations, respectively (using the 300 SNP panel). It is necessary to include at least 300 to 400 SNPs (per breed) on genotype panels to accurately predict breed proportion from biological samples.