The kappa immunoglobulin light chain repertoire of peripheral blood B cells in patients with juvenile rheumatoid arthritis.

The frequent appearance of antinuclear antibodies in patients with juvenile rheumatoid arthritis (JRA) indicates a loss of tolerance in B cell differentiation and/or activation. In this analysis, we were interested whether particular changes in the immunoglobulin light chain repertoire might exist in early-onset pauciarticular arthritis (EOPA) patients thereby potentially revealing distinct molecular patterns, which characterize defects in central tolerance mechanisms as well as an autoreactive peripheral B cell repertoire. Using single cell sorting and single cell PCR the distribution of Vkappa Jkappa rearrangements has been analyzed in individual naïve B cells of patients with EOPA-JRA and healthy individuals. The immunoglobulin kappa light chain repertoire of peripheral blood B cells in EOPA patients seems to be skewed to a decreased use of downstream Vkappa gene segments indicating increased events of secondary V(D)J-recombination. Another prominent molecular pattern in JRA B cells seem to be a restricted combination of Vkappa Jkappa rearrangements based on the predominant utilization of the Jkappa 1 and 2 gene segment. The current study indicates disturbances in the peripheral B cell pool in juvenile rheumatoid arthritis. The peripheral blood B cell pool of JRA patients did show molecular changes in the kappa light chain repertoire which, in part, could be a sequel of secondary V(D)J-recombination and of a molecular bias during immunoglobulin rearrangement in the bone marrow. Thus, B cell tolerance might be broken by more than one pathogenic mechanism.

The porcine Ig delta gene: unique chimeric splicing of the first constant region domain in its heavy chain transcripts.

The pig delta gene is located approximately 3.4 kb downstream of the second transmembrane exon of the micro gene and shows a similar genomic structure to its counterpart in cow with three exons encoding the CH1, CH2, and CH3 domains. The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately upstream of the delta gene. The deltaCH1 exhibits a 98.7% similarity (314 of 318 bp) to the micro CH1 at the DNA level, whereas the homologies between the deltaCH2 and micro CH3, and the deltaCH3 and micro CH4 are only 33.3 and 35.8%, respectively. Either of the two CH1 exons ( micro and delta) could be observed in the expressed porcine IgD H chain cDNA sequences VDJ- micro CH1-H-deltaCH2-deltaCH3 or VDJ-deltaCH1-H-deltaCH2-deltaCH3, showing a pattern that has not been observed previously in vertebrates. In addition, transfection of a human B cell line, using artificial constructs resembling the porcine C micro -Cdelta locus, also generated both VDJ- micro CH1-deltaCH1-H1-deltaCH2 and VDJ -deltaCH1-H1-deltaCH2 transcripts. An examination of the pig delta genomic sequence shows a putative, second hinge region-encoding exon. Due to the lack of a normal branchpoint sequence for RNA splicing, this exon is not present in the normal pig delta cDNA. However, the exon could be spliced into most of the expressed transcripts in vitro in cell transfection experiments after introduction of a single T nucleotide to restore the branchpoint sequence upstream of the putative H2 exon.

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.

Rhabdovirus infection induces public and private T cell responses in teleost fish.

Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian alphabeta TCRs have been recently characterized in rainbow trout and in several teleost species, but the alphabeta T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCRbeta chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VbetaJbeta combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of Vbeta4Jbeta1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.

Sequence changes at the V-D junction of the VH1 heavy chain of anti-phosphocholine antibodies alter binding to and protection against Streptococcus pneumoniae.

X-linked immune deficient (Xid) mice fail to produce anti-phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC-specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H-Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.