Early Human B-Lineage Cells Can Make Functional Igκ Gene Rearrangements and Express Igκ Protein in the Absence of Functional VDJH Rearrangements.

One of the tools available to study human B cell development are xenogeneic murine stromal cells that serve as a microenvironment to support development of B-lineage cells from human hematopoietic stem cells. Efforts to clarify the role of IL-7 signaling in human B cell development have been rejuvenated by the observation that cord blood CD34+ stem cells cultured on murine MS-5 stromal cells develop into CD19+ B-lineage cells expressing the IL-7R (i.e., IL-7Rα chain, CD127 and gamma common chain, CD132), and that signaling through IL-7R is essential for development of CD19+ B-lineage cells in this model. CD19+/CD127+ cells have survival attributes and patterns of Bcl-2 and p27KIP1 expression that are distinct from CD19+/CD127− cells. CD127+ cells are Bcl-2hi, p27KIP1lo and CD127− cells are Bcl-2lo, p27KIP1hi. However, the relationship between IL-7R expression/signaling and the onset/progression of IgH and IgL rearrangement patterns in early human B-lineage cells is unknown. We hypothesized that IL-7R expression would signify the potential for IgH gene rearrangement and eventually IgL gene rearrangement, whereas absence of expression would signify failure to make in-frame rearrangements. To test this hypothesis, CD19+ cells from three independent 4-wk cord blood/MS-5 xenogeneic cultures were FACS-purified into CD19+/CD127+ and CD19+/CD127− fractions, and a quantitative real time PCR assay was employed to characterize IgH and IgL rearrangements at the level of the sorted populations. The majority of cells in both fractions had incomplete DJH rearrangements. However, < 10% of CD127+ and CD127− cells had complete VDJH rearrangements and those that did primarily used VH1, 2 and 3 families. This data was in agreement with immunofluorescent staining which showed that ~ 5% of CD127+ and CD127− cells expressed cytoplasmic Igμ heavy chains. Neither elimination of IL-7 signaling nor addition of exogenous IGF-1 promoted the appearance of cells expressing Igμ heavy chains. Thus, many of the early B-lineage cells that developed in this model had an IgH rearrangement profile that resembled pre-B-I cells isolated from pediatric marrow. Unexpectedly, analysis of IgL gene rearrangements in the CD19+/CD127+ and CD19+/CD127− fractions showed profound differences. CD127+ cells harbored very few rearrangements at the Igκ and Igλ light chain loci (estimated to be < 5% of cells). However, CD127− cells showed extensive VJκ and VJλ rearrangements, as well as evidence that the kappa deleting element had undergone rearrangement with Vκ genes and the intron recombination signal sequence. Thus, despite having only a small percentage of cells with complete VDJH rearrangements, the CD127− fraction showed a high level of recombinatorial activity in the light chain loci. Many of the VJκ rearrangements were in-frame, since Igκ protein was detected in the majority of CD127− cells by flow cytometry, immunohistochemistry, and Western blotting. These results show that in-frame rearrangement at the IgH locus is not a requirement for developing human B-lineage cells to initiate rearrangement at the IgL loci. They further suggest that the low frequency of cells with complete VDJH rearrangements reflects epigenetic silencing of VH gene clusters, or the absence of a stimulus in the xenogeneic culture that induces VDJH rearrangements. The existence of cells expressing Igκ light chain protein in the absence of Igμ heavy chain protein may represent a heretofore unrecognized population of human B-lineage cells with heightened recombinatorial flexibility and/or a population destined to die in the marrow.