Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.

Abstract

Epstein-Barr virus transformation of Bcell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin K or A light chain without heavy chains. Four K-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had K-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity andjoining gene segments). All clones contained K mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced K protein. No ,L mRNA or immunoglobulin heavy-chain molecules were detected in any of the K+ clones. The results suggest that the ,L heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements.Variable (V) regions of immunoglobulin molecules are encoded by separate clusters of germ-line gene segments: VH, diversity (D), and joining (JH) segments for immunoglobulin heavy (H) chains and VL and JL segments for immunoglobulin light (L) chains. Stem-cell progeny entering the B-cell pathway must undergo rearrangement of these gene segments in order to express H and L chains (1). These rearrangements have been thought to occur in an ordered fashion so that u H-chain expression precedes L-chain expression during the conversion of pre-B cells to B cells (2-6). A model for the regulation of this ordered sequence of gene rearrangements has been proposed based primarily on studies of pre-B-cell lines transformed by the Abelson murine leukemia virus (Ab-MuLV) (7). According to this model, the ix polypeptide that is produced following a functional V-D-J rearrangement would play a dual role: (i) termination offurther H-chain gene assembly to guarantee allelic exclusion and (ii) induction of L-chain gene assembly (7,8).We have used Epstein-Barr virus (EBV) transformation to analyze human B-cell precursors at different developmental stages. EBV has long been known to immortalize B cells, but its ability to transform B-cell precursors, which requires prior depletion of surface IgM-positive (sIgM+) B cells, has been appreciated only more recently (9-11). Unlike many AbMuLV-transformed cell lines, which can continue to rearrange immunoglobulin genes in culture, EBV transformants stably maintain their immunoglobulin genes during in vitro propagation (10-13). In addition, EBV can transform a broad spectrum of B-lineage cells, including Ig-"null" cells with germ-line H-and L-chain genes (11,14,15 (100 ttCi each; 1 ttCi = 37 kBq) for 4 hr at 370C. Solid-phase immunoisolation from the cells solubilized in 1% Nonidet P-40 and from the culture supernatant was performed in microtitier wells precoated with rat mAb to mouse K chains (187.1) and then with mouse mAbs to human isotypes as described (17). The isolated, radiolabeled proteins were analyzed by NaDodSO4/PAGE under bot...

Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development

“…Our experiments show that human control and Igµ-deficient pro-B cells are similar to their mouse counterparts in that they undergo Igκ and Igλ gene rearrangements. These results are in agreement with the finding of rare IgL chain gene recombination in normal human pro-B cells and in Epstein-Barr virus-transformed fetal B cell precursors (67,72). Thus, IgL rearrangement in the human is similar to the mouse in that it is not strictly dependent on Igµ expression or pre-B cell development.…”

IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells

“…Surprisingly, analysis of Ig light-chain locus recombination revealed extensive rearrangements in CD127 Ϫ cells that greatly exceeded CD127 ϩ cells ( Figure 2C). There are several possible explanations for the existence of CD19 ϩ CD127 Ϫ cells with extensive Ig light-chain rearrangements but few VDJ H rearrangements, including heightened recombinatorial flexibility 41,42 and Ig H chain deficiency. 43 However, Ig light-chain rearrangement before Ig heavy chain rearrangement still appears to be a rare event.…”

The status of Ig loci rearrangements in single cells from different stages of B cell development

“…This shows that the vast majority of precursor B cells rearrange the DH-J H and VH-D|-|JH segment on the H chain loci before they rearrange the L chain loci. It makes the possible alternate pathway of B cell development improbable, or at least infrequent (21). While the large u.H/SL + and u.H/SL~ pre-B II cells are both candidates for precursor of small pre-B II cells, it remains to be investigated whether both of them, or only one of them, play(s) this role in B cell differentiation.…”