SummaryWe have resolved B220+IgM -B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen) . Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system . Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7 -pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFLlb.l, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D -J rearrangement, but no V-D-J . Finally, functional analysis demonstrates that the proliferative response to ID7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A .

B cell development is a highly regulated process whereby functional peripheral subsets are produced from hematopoietic stem cells, in the fetal liver before birth and in the bone marrow afterward. Here we review progress in understanding some aspects of this process in the mouse bone marrow, focusing on delineation of the earliest stages of commitment, on pre-B cell receptor selection, and B cell tolerance during the immature-to-mature B cell transition. Then we note some of the distinctions in hematopoiesis and pre-B selection between fetal liver and adult bone marrow, drawing a connection from fetal development to B-1/CD5(+) B cells. Finally, focusing on CD5(+) cells, we consider the forces that influence the generation and maintenance of this distinctive peripheral B cell population, enriched for natural autoreactive specificities that are encoded by particular germline V(H)-V(L) combinations.

Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene-encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.

Since the distinction was made between immunoglobulin-bearing (B) lymphocytes that give rise to antibody forming cells and thymus derived, Thy-l-bearing (T) lymphocytes responsible for a host of other immune functions, substantial effort has been directed toward finding individual cell surface markers that subdivide these populations. In the mid-1970s, the Lyt-1, Lyt-2, and Lyt-3 antigens were shown (with the assays then available) to be represented exclusively on T cells (1, 2) and to identify functionally distinct T cell subpopulations. Lyt-1 appeared to be restricted to the helper-amplifier subset, and Lyt-2 and Lyt-3 defined the suppressor-cytotoxic subset (3-6).The development of monoclonal anti-Lyt reagents increased the sensitivity with which these antigens could be measured and significantly changed the status of the Lyt-1 (Ly-1) 1 marker: quantitative immunofluorescence studies with the fluorescenceactivated cell sorter (FACS) z revealed that all Thy-l-bearing cells have some Ly-1 (8); that lower levels of Ly-1 on cytotoxic-suppressor cells explains the previous failure to detect this antigen on the Lyt-2,3 subset with cytotoxic depletion assays; and that the frequency of Ly-1 + cells in normal spleen, in fact, is usually slightly greater than the frequency of Thy-1 + cells in the same organ (9).Recent studies (10, 11) demonstrating the existence of Ly-l-bearing B cells (Ly-1 B) indicate that such cells account for at least part of this excess. These findings, documented by FACS analyses on cell populations from normal animals, are consistent with a variety of previous observations: small numbers of Ly-1 + cells are present in B cell areas of stained tissue sections in normal spleen (8); certain mouse B cell lymphomas synthesize and coexpress Ly-1 and IgM (7); old NZB mice tend to have increased numbers of Ly-1 bearing cells (12) that we have now shown to be IgMpositive and Thy-1-negative (unpublished observations); human B cell chronic lymphocytic leukemias tend to carry IgM and Leu-1 (13), a human cell surface molecule whose properties are homologous to mouse Ly-1 (14). Furthermore, a subpopulation * This work was supported in part by grants GM-17367, HD-01287, and CA-04681 from the National Institutes of Health.:~ Fellow of the American Cancer Society. t Several years after its initial description as the first in a series of lymphocyte differentiation antigens, Ly-1 was renamed Lyt-1 to reflect its apparently exclusive expression on T cells. We return here to the original usage since this antigen was recently demonstrated on B cell tumors (7) and we now report its presence on a subpopulation of normal B ceils. No other cell surface antigen is presently called Ly-l.2 Abbreviations used m this paper: Bi, biotin; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; Fl, fluorescein; Ly-1 B, Ly-l-bearing B cell; PFC, plaque-forming cells; PI, propidium iodide; R1A, radioimmunoassay; TR, Texas red. 202J. Exp. MEn.

The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.