CD127 Expression Confers Enhanced Survival on Human B-Lineage Cells; Implications for the Role of IL-7 Signaling in Human B Lymphopoiesis.

The IL-7Rα chain (CD127) is expressed on some, but not all, CD19+/surface μ-B-lineage cells isolated from fetal and adult marrow, and umbilical cord blood, but the functional consequences of CD127 expression in early human B cell development during all stages of human life are unresolved. We have described a xenogeneic culture model for generating CD19+ B-lineage cells from CD34+ cord blood stem cells following a 4 wk co-culture on murine MS-5 stromal cells (Johnson SE et al., 2005 J Immunol.). The majority of these cells express a pro-B cell phenotype i.e., CD19+/CD10+/CD20lo/CD21−/CD22+/CD24+/pre-BCR-. Moreover, 1–4% express surface μ heavy chains (HC), and some express κ or λ light chains. We have also reported that 20–40% of CD19+ cells emerging in the xenogeneic culture express CD127, CD127+ cells are more blastic than CD127- cells, and development of CD19+ cells is substantially blocked in xenogeneic cultures treated with anti-murine IL-7 (Johnson SE, ibid). The goal of the present study was to further characterize the developmental potential and patterns of gene expression in CD19+/CD127+ and CD19+/CD127− cells that emerge in xenogeneic cultures. We hypothesized that CD127 expression defines a subset of CD19+ B cell precursors with heightened potential to become mature B-lymphocytes. CD19+/CD127+ and CD19+CD127− populations that emerged in 4 wk xenogeneic cultures were sorted to > 95% purity by FACS. Immunofluorescent staining showed that surface and intracellular μ HC positive cells were present in both sorted populations at frequencies of 3–4%. Using an RT-PCR-based sequencing approach, we analyzed approximately 100 IgM V region sequences from both CD19+/CD127+ and CD19+/CD127− FACS-purified populations. The results indicated that the sequences from both populations were:

1. non-mutated and used VH4, D and J gene segments that were similar to what would be expected in a cord blood B cell repertoire, and

2. exhibited no statistically significant difference in N segment length, CDR3 length, or CDR3 charges.

These results suggest that the amplified sequences were likely derived from the 3–4% cytoplasmic μ HC+ pre-B cells present in both sorted populations. The immunofluorescent staining and VDJH rearrangement sequence results suggest that large CD19+/CD127+/cytoplasmic μ HC+ pre-B cells may differentiate into small CD19+/CD127−/cytoplasmic μ HC+ cells in the xenogeneic culture. This was further supported by showing that intra-tibial injection of FACS-purified CD19+/CD127+ and CD19+/CD127− cells into sub-lethally irradiated NOD-SCID mice led to the appearance of surface μ HC+ cells from both sorted populations. Western blotting of sorted CD19+/CD127+ and CD19+/CD127− cells showed the presence of Bcl-2, p-GSK3β (specifically p-Ser9, the residue phosphorylated by AKT) and pERK1/2 in CD19+/CD127+ cells, while these proteins/phosphoproteins were undetectable in CD19+/CD127− cells. By contrast, expression of the cell cycle inhibitor p27KIP1 was elevated in CD19+/CD127− cells compared to CD19+/CD127+ cells. Furthermore, FACS-purified CD19+/CD127+ cells survived longer than CD19+/CD127− cells when cultured in the absence of MS-5, and the survival of the former was enhanced by stimulation with IL-7. The collective results indicate that CD127 expression identifies a population of normal human CD19+ B cell precursors with a pattern of gene expression and survival/proliferation attributes consistent with a crucial role in the development of the B-lymphocyte arm of the human immune system.