Lambda light chain revision in the human intestinal IgA response - PubMed (original) (raw)

Lambda light chain revision in the human intestinal IgA response

Wen Su et al. J Immunol. 2008.

Abstract

Revision of Ab L chains by secondary rearrangement in mature B cells has the potential to change the specific target of the immune response. In this study, we show for the first time that L chain revision is normal and widespread in the largest Ab producing population in man: intestinal IgA plasma cells (PC). Biases in the productive and non-productive repertoire of lambda L chains, identification of the circular products of rearrangement that have the characteristic biases of revision, and identification of RAG genes and protein all reflect revision during normal intestinal IgA PC development. We saw no evidence of IgH revision, probably due to inappropriately orientated recombination signal sequences, and little evidence of kappa-chain revision, probably due to locus inactivation by the kappa-deleting element. We propose that the lambda L chain locus is available and a principal modifier and diversifier of Ab specificity in intestinal IgA PCs.

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Figures

Figure 1. Characteristics of Igk rearrangements from single IgA plasma cells.

Comparison of productive and non-productive rearrangements involving the Vκ families (A) and individual Jk segments (B). The only bias observed was a tendency not to observe Jk1 in the non-productive rearrangements, consistent with editing of these rearrangements as seen in another study (7,8). In most cells, the somatic hypermutation mechanism was only active on productive rearrangements of VκJκ (C), presumably due to the activity of the KDE in non-productive rearrangements. The activity of the KDE is illustrated in D, where a VκJκ rearrangement is identified by ‘a’. The KDE can inactivate this rearrangement if it is non-productive. The KDE can either recombine with an RSS in the intronic sequence between Jκ and Cκ identified by ‘b’, or with an upstream V segment identified by ‘c’. Evidence for revision or allelic inclusion was apparent in 3 different plasma cells where each had two mutated in-frame alleles. In one in-frame rearrangement involving the germline V segment V3-11, had acquired stops by somatic hypermutation, which may have been replaced by rearrangement at the second allele (E).

Figure 2. Biased ratio of productive: non-productive Igλ rearrangements in IgA plasma cells compared to mature naïve B cells.

Ratio of productive (open bars) to non-productive (black bars) rearrangements in the Vλ1 and 2 families and the Vλ5 family and related Vλ9 segment. Data from single B cells and from B cell lines in this study are shown alongside data from populations of IgA plasma cells and naïve B cells, including Peyer’s patch cells from other studies (10, 28). The actual number of sequences contributing is written inside the bars. In IgA plasma cells and immunoblasts, there is a bias towards productive rearrangements in the Vλ 1 and 2 families and towards non-productive rearrangements in the Vλ5 and 9 families. This skewing is not apparent in naïve B cells and is therefore a consequence of revision and not editing.

Figure 3. Illustration of generation and detection of λRECs.

When Vλ and Jλ segments rearrange, illustrated by ‘a’, a coding joint is formed between Vλ and Jλ. The intervening sequence forms a circular DNA, which we term the Igλ recombination excision circle (lREC), that includes a signal joint characterized by a pair of RSS in opposite orientations (illustrated by triangles). The binding sites of 5’- and 3’-primers used to amplify λRECs are shown as arrows.

Figure 4. Calculation of expected and observed frequency of λRECs.

The graph in (A) illustrates the decreasing frequency of λRECs in an exponentially expanding population (y axis) which starts with a frequency of λRECs of 1 in 3.5 cells, as observed in mature naïve IgD+ B cells, as cell cycles progress (x axis). The observed frequency of λRECs is the experimental observation of 1 λREC per 89 IgA immunoblasts, which would be derived from 4.7 cycles of proliferation if revision did not occur. Three expected frequencies of λRECs are illustrated. Two use the range of hypermutation frequencies of 0.07 to 0.11 mutations/ 100 bp/ cycle to generate the 4.9% observed mutations over 44.5 to 70 cycles of replication respectively (23). This would result in 1 λREC in between 1.2 x 1014 and 4.1 x 1021 cells. The third expected frequency of λRECs is derived from an estimate of 0.5 mutations/ V segment/ cycle, which would generate 4.9% mutation frequency over 27.5 cycles resulting in 1 λREC per 9.4 x 108 cells (22). (B) The frequencies of hypermutation in the IgH and IgL from the IgA immunoblast clones, IgH from the untransformed IgA immunoblasts from which clones were made, and IgH from gut plasma cells. The frequencies of hypermutation were on average 4.9%. The gels in (C) illustrate the frequency of λRECs when analysed by PCR. Cells for PCR were diluted by doubling dilutions from a starting cell number of 400 IgD+ lymphocytes or IgA immunoblasts per well. The top 4 rows are IgA immunoblasts, the bottom 4 rows are IgD+ lymphocytes from the same individual. The IgA immunoblasts are between 4 and 5 doubling dilutions (analogous to doubling during cell cycles) behind the IgD+ cells if it is assumed that revision does not occur, consistent with figures calculated from limiting dilution studies.

Figure 5. Analysis of J and V segment rearrangements in λRECs.

J segment involvement in λRECs from IgD+ B cells, IgA immunoblasts and IgA plasma cells from the gut (A). Jl1 is most common in the λREC from IgD+ cells, whereas there is a significant tendency to see Jl2/3 in the λRECs from IgA immunoblasts *(P<0.001) and plasma cells **(P<0.02). In contrast there is no strong bias in Vλ segments in λRECs, other than a tendency not to see the commonly rearranged Vλ2-14 segment in the λRECs from IgA immunoblasts (B). Similar profile of Vλ familes in productive rearrangements from naive B cells (28) and plasma cells (10) and in the λRECs from the IgA immunoblasts (C).

Figure 6. RAG-2 expression in Peyer’s patches.

A and B, double immunostain with goat anti-RAG-2 (brown) and CD20 (red) on paraffin sections of normal human Peyer’s patches. (A) Detection of RAG-2 positive B cells in the germinal center of a Peyer’s patch (arrowed with higher power detail in insert). (B) RAG-2 expressing cells infiltrating the epithelium adjacent to the Peyer’s patches (arrowheads). (C) Rabbit anti-RAG-2 also identified germinal center cells (arrows and in insert) and a subset of intraepithelial lymphocytes (not illustrated) consistent with the staining illustrated in B and C. D. Example of amplification of RAG-2 gene transcripts by RT-PCR from laser capture microdissected samples of germinal centers RNA. Molecular weight marker is identified by ‘M’, the cell line Namalwa (N) that rearranges light chain genes in vitro (unpublished) is a positive control. AID transcripts are the positive control for laser microcapture of germinal center cells.

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Lambda light chain revision in the human intestinal IgA response - PubMed (original) (raw)