FETAL AND ADULT B LYMPHOCYTE DIFFERENTIATION

RICHARD R. HARDY, Ph.D., Senior Member

B lymphocytes are generated throughout life from hematopoietic stem cells by a complex program of cell differentiation, in the liver before birth and in the bone marrow afterward. B lineage cells in the bone marrow can be recognized by expression of B220, the high molecular weight form of the common leukocyte antigen, CD45. This population is very heterogeneous, consisting of cells at various stages of differentiation, from newly committed progenitors to mature B cells. We have employed multiparameter flow cytometry to resolve these stages into seven fractions (A, B, C, C', D, E, and F) based on differential expression of several cell surface molecules. We have found that these phenotypic subsets show important molecular and functional distinctions; early fractions possess no or only incomplete heavy chain rearrangements (D-J_H) and are critically dependent on cell contact for survival; later fractions, possessing complete rearrangements (V_H-D-J_H), no longer have this requirement. Furthermore, cells that have just completed heavy chain rearrangement are in rapid cell cycle, as shown by analysis of the DNA content per cell of sorted fractions (fraction C' in Figure 1).

In our current work, we use this fractionation scheme as a framework for investigating several important issues in lymphocyte development, including 1) the relationship between B cell development and Ig rearrangement, 2) the differences between fetal and adult B cell development, particularly for generation of autoreactive malignant-prone CD5⁺ B cells, and 3) the extent of B lineage commitment during the earliest recognizable stages of B cell development. In the first area, we have devised a model system to investigate the role of heavy chain expression in the progression of B cell differentiation. In the second area, we have continued investigation of V_H11, a fetal-biased V_H gene that is frequently rearranged in CD5⁺ B cells. We have also begun characterization of V_H3609, another CD5⁺ B cell derived V_H gene that is associated with anti-thymocyte autoreactivity. In the third area, we have continued our study of the earliest stages of B lineage development, assessing the capacity of cells in these fractions to generate other hematopoietic lineages.

Assembly of newly formed immunoglobulin (Ig) M heavy chains with pre-existing peptide chains (l5, VpreB, Iga, and Igb) early in B cell development generates the pre-B cell receptor (BCR) complex. This complex is analogous to the BCR that functions in antigen-dependent activation of mature B cells in immune responses to foreign antigen, but contains K (k) or light (l) chains instead of l5 and VpreB. Previously, we found that B lineage cells from sever combined immuno-deficient (SCID, Rag^-) mice are blocked in their development at a stage just prior to pre-BCR expression and that this block can be complemented by Ig transgenes. We are studying the mechanism of pre-BCR signaling by introducing rearranged heavy chains into Ig^- early B lineage cell lines and investigating the changes in gene expression. Using an in vitro cell line model that mimics in vivo events, expression of a transfected heavy chain in the ret/02 pro-B cell line results in the sharp down-regulation of terminal deoxynucleotidyl transferase (TdT), an enzyme important in the rearrangement process. We hypothesize that upon expression of Ig heavy chain, the pre-B receptor is assembled to generate a signaling complex (Figure 1B, Figure 2A). Thus, understanding how the pre-BCR signals down-regulation of TdT (and by extension, normal B cell development) is a major goal of the laboratory. We are now testing whether mutations in the heavy chain, known to abolish important interactions within the pre-BCR complex, also alter the downregulation of TdT. In this work, we have shifted to a retroviral transduction system for introducing heavy chains into the ret/02 pro-B cell line. This approach has the advantage of providing a wider variety of target cell types and may allow extending this work to experiments with normal cells. Using a bicistronic eGFP retroviral vector, we have introduced mutated heavy chain genes (Figure 2B) lacking critical amino acids necessary for interaction with Ig-a /Ig-b (YS:VV) or lacking the V domain that normally binds VpreB (DV). Consistent with our model, both of these mutant genes, when expressed in the pro-B line, induce accumulation of heavy chain protein (not shown), but fail to induce down-regulation of TdT (Figure 2C).

STRUCTURE OF V_H11 HEAVY CHAIN IN PRE-B CELLS. HARDY, WEI, in collaboration with SEEHOLZER^§

The V_H11 heavy chain isolated from pre-B cells migrates in SDS-PAGE gels as two distinct species, a minor component at approximately the normal size for heavy chain and a major component that is 15-20 kDa larger. The "aberrant" size species disappears in V_H11⁺ B cells. We can replicate these analyses, originally done using normal cells from transgenic mice, by using a pro-B cell line transfected with either the heavy chain alone (where the larger species predominates) or together with a kappa light chain (where the normal size species predominates). Previously, we showed that the apparent size difference is not due to differences in glycosylation or ubiquitination. We are currently isolating large amounts of both species for peptide analysis in the Mass Spectrometry Facility.

LIGHT CHAIN REPERTOIRE OF ANTI-THYMOCYTE V_H3609 TRANSGENIC B CELLS. HARDY, ASANO, in collaboration with HAYAKAWA^§

We have generated immunoglobulin transgenic mice using a heavy chain isolated from an autoreactive CD5⁺ B cell-derived hybridoma, which secretes antibodies that bind to thymocytes. A population of CD5⁺ B cells expressing the transgene develops in the peritoneal cavity of these mice. These cells secrete antibody, at least some of which binds to thymocytes. We have investigated the diversity of light chains expressed in this cell fraction by generating hybridomas from sorted cells, then making RNA. Reverse transcribed cDNA was amplified with a consensus V_k/C_k primer pair using PCR and the amplified material cloned into the "TA" vector. Sequence analysis (Table 1) revealed repetitive (7/12) usage of the particular V_k21c gene found in the original V_H3609⁺ autoreactive hybridoma. Two other hybridomas used a different V_k21 family member. Thus, it appears that there is strong preference for association of particular light chains with the V_H3609 TG in the CD5⁺ B cell population, resulting in anti-thymocyte self-reactivity.

In adult mice, B lymphocytes arise from pluripotent stem cells in the bone marrow (BM). Current models predict that the differentiation of multipotential BM progenitor cells into B lineage committed precursors, incapable of giving rise to alternative hematopoietic lineages, requires the stage-specific activity of key gene products. However, despite intensive interest in early B cell development, elucidation of the molecular mechanisms underlying B lineage commitment has been hampered by the inability to clearly define the earliest B lineage restricted progenitor cells and their immediate unrestricted precursors. Recently, we described three novel cell populations in adult BM termed fractions A0, A1 and A2 (Figure 3A). Among these, fractions A1 and A2 exhibit numerous characteristics consistent with their designation as precursors for pro-B cells or pre-pro-B cells. We have now investigated the developmental potential of these pre-pro-B cells and the role that immunoglobulin heavy chain recombination plays in the restriction of multipotential progenitor cells to the B lineage. We find that, while the precursors of pre-pro-B cells in fraction A0 exhibit progenitor activity for multiple hematopoietic lineages, pre-pro-B cells in fractions A1 and A2 are B lineage restricted (Figure 3B) while still lacking D_HJ_H rearrangements. This finding demonstrates that B lineage commitment occurs very early in B cell development, before initiation of D_HJ_H recombination and coincident with surface B220 expression.

PUBLICATIONS

ALLMAN, D., LI, J., HARDY, R.R. Commitment to the B-lymphoid lineage occurs prior to D_H-J_H recombination. J. Exp. Med. (in press).

HARDY, R.R., MALISSEN, B. Lymphocyte development. The (knock-) ins and outs of lymphoid development. Curr. Opin. Immunol. 10:155-157, 1998.

TUMANG, J.R., OWYANG, A., ANDJELIC, S., JIN, Z., HARDY, R.R., LIOU, M.L., LIOU, H.C. c-Rel is essential for B lymphocyte survival and cell cycle progression. Eur. J. Immunol. 28:4299-4312, 1998.

ZENG, X.X., ZHANG, H., HARDY, R.R., WASSERMAN, R. The fetal origin of B-precursor leukemia in the E-mu-ret mouse. Blood 92:3529-3536, 1998.

Papers in press at time of previous report:

DAVÉ, V.P., ALLMAN, D., KEEFE, R., HARDY, R.R., KAPPES, D.J. HD mice: a novel mouse mutant with a specific defect in the generation of CD4+ T cells. Proc. Natl. Acad. Sci. USA 95:8187-8192, 1998.

WASSERMAN, R., ZENG, X.-X., HARDY, R.R. The evolution of B precursor leukemia in the Eu-Ret mouse. Blood 92: 273-282, 1998.

^§   Fox Chase researcher

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998