Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells.

Abstract

SummaryThe splenic marginal zone (MGZ), which surrounds the mantle zone (MTZ) in human splenic white pulp, contains a phenotypically and morphologically distinct population of B cells. The origin of MGZ B cells is .uncertain. Whereas some experiments in rodents have suggested that they are a distinct cell lineage responsible for the immune response to T-independent type 2 antigens, others have suggested that they are memory B cells derived from a germinal center (GC) response. The progeny of a GC reaction is expected to have rearranged immunoglobulin (Ig) genes that are mutated. The distribution of mutations would be expected to reflect the selection of Ig by its affinity for antigen. We have analyzed rearranged Ig heavy chain variable region (VH) 6 and VH 4.21 genes in MGZ and MTZ B cells microdissected from frozen sections of human spleen to determine whether these genes have the properties of an affinity-selected memory B cell population. MTZ B cells contained germline Ig VH genes, confirming previous reports and providing an internal control for mutational analysis. MGZ B cells contained Ig VH genes that were mutated, showing that these cells had been subjected to a mutational mechanism characteristically active in the GC. The rearranged VH 6 genes showed patterns of mutation indicative of an antigen selection process, whereas the distribution of mutations in VH 4.21 genes was not characteristic of gene selection by conventional T-dependent antigen. Our studies provide the first evidence that the human splenic MGZ is a reservoir of memory B cells.T he marginal zone (MGZ) l of the spleen is a microanatomically defined lymphoid compartment at the border of the white and red pulp. The major cellular components of the MGZ are B cells and macrophages (1-3). In humans and rodents, MGZ B cells are characteristically IgM + , IgD-, and they strongly express CD21 (1, 4-6). They are not in cell cycle and do not express antigens associated with cellular activation (7,8).Two different functions have been ascribed to MGZ B cells in rodents: the antibody response to T-independent type 2 (TI-2) antigens and B cell memory. It has been well documented that an intact splenic MGZ is required for an efficient response to TI-2 antigen (9-11). A number of experiments have suggested that the B cells in the MGZ (12) and the B cells responding to TI-2 antigens (13) differ developmentally from other B cell populations, and they have prompted the suggestion that MGZ B ceUs are of a distinct 1 Abbreviations used in this paper: Fw, framework region; GC, germinal center; MGZ, marginal zone; MTZ, mantle zone; R, replacement; s, silent; TD, T dependent; TI-2, T independent type 2; Vs, heavy chain variable region.lineage. However, in vitro comparison of the ability of mufine MGZ B cells and follicle center B cells to respond to T-dependent (TD), TI-1, and TI-2 antigens have failed to show any major differences, suggesting that responsiveness to TI-2 antigens is due to factor(s) in the microenvironment of the MGZ (14). The role of MGZ macro...

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“…This datum coincides with others previously obtained in the analysis of the IgV H and bcl-6 genes, which corroborate that mantle cells in the spleen are naive B cells. 16 The data presented here seem to support the hypothesis that, in spite of initial morphological observations, a significant proportion of SMZL cases could derive from an unmutated naive precursor other than the marginal zone, and possibly situated in the mantle zone of the splenic lymphoid follicles. This is consistent with the high frequency of IgD expression by SMZL cells.…”

“…These findings confirm previous data on the frequency of IgV H mutations in the marginal zone cells of the spleen, thereby confirming that at least a significant proportion of splenic marginal zone B cells are composed of memory cells, which is consistent with their IGM + IgD − immunophenotype, suggesting that these cells are post-germinal center B cells (Table 3). 16,18 This study has also shown evidence of clonal expansion within the marginal zone. However, the mantle cells of the spleen that were analyzed, selected on the basis of their IgD expression, show a germinal configuration of the bcl-6 gene.…”

“…[11][12][13][14][15] Mutation analysis of rearranged IgV H genes in microdissected marginal zone B cells in the spleen, Peyer's patches and lymph nodes has shown that there are mutated IgV H genes in the majority of cells, as well as evidence of related B cell clones. [16][17][18][19] Although it was initially assumed that the process of somatic hypermutation in normal B lymphocytes was restricted to Ig loci, including heavy and light chain variable region genes, 20 analogous mutations in the bcl-6 gene were further described, showing promoter-dependent hypermutations in the noncoding first exon, paralleling and almost identical with the hypermutation of IgV H genes with respect to mutation frequency and the involvement of B cell subpopulations. [21][22][23] Bcl-6 is a gene located at chromosomal band 3q27, and encodes a POZ/zinc finger transcriptional repressor that is normally expressed in B cells within germinal centers, and which is required for germinal center formation and T celldirected immune response.…”

Immunoglobulin gene analysis as a tool for investigating human immune responses

“…Hence, when looking at the immunoglobulin gene rearrangements in a sample, the presence of mutations, in comparison to germline sequences, makes it evident that the cell has been activated by antigen. Thus, we could show for the first time that even though the B cells of the splenic marginal zone were not class switched, retaining IgM functionality, they were still antigen‐experienced cells as their Ig genes were mutated 20. In chronic lymphocytic leukemia (CLL) the extent of mutation was investigated to try and understand the etiology of the disease and it was found that there were two different classes of CLL with prognostic significance, those with mutated immunoglobulin genes and those carrying germline immunoglobulin genes 21.…”

“…It also potentially biased the measurements of J region usage and was open to the risk of V region bias due to faulty primers by virtue of the fact that the V region primers were a mix of family‐specific primers. While these early sequencing technologies were invaluable for the discovery of new cell populations, they often relied on expensive and time‐consuming cloning that did not capture the full repertoire; due to the single channel capabilities of Sanger Sequencing 20, 22, 29, 58…”