Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo.

Abstract

Cell lines and clones were established from PB76‐positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum‐substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL‐7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP‐1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP‐1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ‐line configuration. Either one or both of their H‐chain‐gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg‐positive surface, MHC class II negative, LPS‐reactive B cells within 2–3 days, in the absence of contacts with stromal cells and/or IL‐7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig‐, MHC class II‐positive LPS‐reactive cells for greater than 25 weeks. The long‐term in vitro proliferative capacity of these DH‐JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.

Analysis of Gene Expression and Ig Transcription in PU.1/Spi-B-Deficient Progenitor B Cell Lines

“…3A, upper panels). After IL-7 withdrawal, an increased fraction (2.5 Ϯ 0.8%, n ϭ 3) of wild-type pro-B cells expressed Ig on their surface, similar to what has previously been reported (49). In contrast, a larger proportion of PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ cells rearranged and expressed Ig on their surface (10.9 Ϯ 1.5%, n ϭ 3) compared with wild-type cell lines (Fig.…”

“…Culture in IL-7 arrests developing B cells at the late pro-B cell stage (49). When IL-7-dependent cell lines are cultured on stromal cells in the absence of IL-7, they initiate apoptotic cell death but also initiate differentiation, by rearrangement of L chain genes, (49,50).…”

“…When IL-7-dependent cell lines are cultured on stromal cells in the absence of IL-7, they initiate apoptotic cell death but also initiate differentiation, by rearrangement of L chain genes, (49,50). Therefore, we withdrew IL-7 from culture media of wild-type and PU.1 Ϫ/Ϫ Spi-B Ϫ/Ϫ pro-B cells for 72 h to test the ability of these cells to successfully rearrange and express Ig L chain on their surface relative to wild-type pro-B cells.…”

The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B cells

“…Removal of IL-7 allows their further maturation as reflected by completion of Ig rearrangement and surface marker expression [36,37]. We have classed the cells generated in this system as immature, defined by the expression of the Tg IgM, but it was considered possible that in certain aspects they may still be capable of further maturation upon removal of IL-7.…”

Both the pre-BCR and the IL-7Rα are essential for expansion at the pre-BII cell stagein vivo

“…After subsequent Ig light (L)-chain recombination, although not followed by an expansion phase, the end result is the production of large numbers of immature B cells, each carrying a unique BCR (IgM) on the cell surface. Immature B cells migrate to the spleen, where they mature and co-express IgD [9].Expansion at the pre-BI cell stage has been shown, both in vitro [10][11][12][13] and in vivo [14][15][16][17][18][19][20][21], to depend on the IL-7Ra [22]. Mice deficient in the IL-7Ra chain show a block in B cell development at the pre-BI stage.…”