SummaryTo gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7 b fetal liver cells into nonirradiated congenic RT7 a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonaUy. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating.YhProliferation and differentiation from pluripotent emopoietic stem cells to lymphocytes, cell populations with highly diverse antigen-receptor specificities are generated and maintained. To understand this generation of the antigen-receptor repertoire, a better insight into the donal organization of the lymphoid system is required. Particularly relevant issues are the numbers orb and T lymphoid cell clones (a clone is the progeny of a single ancestor cell) that simultaneously contribute to the generation of lymphocytes, the size and antigen receptor composition of individual clones, and the nature of the interactions of the progeny cells with the environment. To study the number of clones simultaneously engaged in production of hemo/lymphopoietic cells, chimeric animals are used since genotypic fluctuations in these animals are an indication for progenitor cell numbers.

T and B lymphocytes are descended from a common ancestor, a muhipotent stem cell (1, 2). The question arises as to the stage at which these two lineages diverge. Do multipotent stem cells directly generate B and T cells according to their local microenvironment, or do they first give rise to descendant stem cells that are restricted in their potentiality? There would be prima facie evidence for such committed stem cells, able to yield only one or other type of lymphocyte, if procedures were found that differentially replenish the mature B and T compartments of repopulated irradiated hosts. Abramson et al. (2) induced chromosome markers by irradiation and followed the progeny of uniquely marked individual stem cells. They found signs of a committed T stem cell in bone marrow. However, their protocol did not permit the characterization or isolation of committed stem cells. The formal objection has been raised (3) that the induction of the marker may itself have caused the apparent commitment. In the studies reported here, we have capitalized on naturally occurring B and T cell markers in the rat. We set out to confirm the existence of committed T stem cells by studying their surface antigenic properties and their radiosensitivity in vivo.In rats, a 95,000 mol we, heavily glycosylated glycoprotein defined by the mouse monoclonal antibody W3/13 (4, 5) is found on some bone marrow cells, on thymocytes, mature T lymphocytes, and immunoglobulin-secreting cells, but not B lymphocytes. This peculiar distribution prompted us to study this antigen on stem cells assayed by their ability to form T and B lymphocyte chimeras (6). Marrow from donors simultaneously carrying T and B alloantigenic genetic markers was incubated with W3/13 and sorted into bright or dim or dull fractions before injection into syngeneic irradiated hosts along with a fixed dose of competing host-type marrow. We found differential T and B lymphocyte chimerism in the recipients of W3/13 dim marrow.There are several reports (7-11) that some T cells are more radioresistant than B cells. We therefore studied whether B and T lymphoid stem cells could also be distinguished by their radiation sensitivities in vivo by competing the endogenous stem cells left immediately after irradiation with exogenous B and T genetically marked marrow. PVG rats were subjected to various doses of gamma irradiation and injected with a fixed dose of doubly marked marrow. We then looked for split chimerism in peripheral T and B lymphocytes.

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To investigate early stages of B lymphocytopoiesis in rat bone marrow (BM) before the expression of surface IgM (s mu), the populations of cytoplasmic mu-chain-positive (c mu+) pre-B cells and terminal deoxynucleotidyl transferase-positive (TdT+) cells were studied by double immunofluorescence microscopy. B lymphocytes that were s mu+ constituted 5%, c mu+s mu- pre-B cells 23%, and TdT+ cells 4% of nucleated cells in the BM of juvenile rats. TdT+ and pre-B cells ranged between 7 and 17 microns in diameter. TdT+ cells were slightly larger, with a modal diameter of 10.5 microns against 9 microns for pre-B cells. mu-Chains were absent from nearly all TdT+ cells. Their surface antigenic phenotype was studied by using a panel of mouse monoclonal antibodies (MAb) to rat B lymphocyte-associated antigens (Ig, Ia, and others) and T lymphocyte-associated antigens. Both pre-B cells and TdT+ lacked surface Ig and Ia but carried most of the other B lymphocyte-associated antigens analyzed. TdT+ and pre-B cells lacked those antigens found only on the T lineage. By using MAb HIS24 (detecting a non-Ig/Ia B lymphocyte-associated antigen) and fluorescence-activated cell sorting, TdT+ and pre-B cells were highly enriched. The results show that most TdT+ cells in rat BM are mu- but demonstrate strong similarity with pre-B cells in surface antigenic phenotype. Therefore, as suggested for man, a major proportion of rat BM TdT+ cells may be B lineage-cells before mu heavy chain gene expression.

In rat bone marrow (BM), the B lineage surface antigen HIS24 is expressed by all surface mu chain-bearing (s mu+) B cells, by cytoplasmic mu chain-containing (c mu+s mu-) pre-B cells and TdT+ cells, and by lymphoid cells lacking both mu and TdT. Because TdT+ and HIS24+TdT-mu- cells may represent stages in B lymphocytopoiesis before mu chain expression, we investigated their kinetics. The metaphase arrest method was combined with immunofluorescence staining to detect proliferation and to quantitate cell production in the BM pre-B, TdT+, and HIS24+TdT-mu- compartments. Their apparent cell cycle times (tC(a)) were 38, 36, and 19 hr, and the number of cells produced per hour per femur were 58, 9, and 41 X 10(4), respectively. The HIS24+ compartments showed further phenotypic heterogeneity. Six percent of TdT+ cells expressed mu chains and were therefore pre-B cells. Twenty percent of HIS24+TdT-mu- cells expressed Ig other than mu chains, with size distribution and kinetics similar to HIS24+TdT-Ig- cells. Thus, the rate of production in the truly Ig-HIS24+ compartment was about 40 X 10(4)/hr/femur (8.5 by TdT+mu- and 33 by TdT-Ig-). In each phenotypic compartment, mitoses were confined to subsets of large (greater than 11 to 12 micron) cells with tC(a) of 13 to 15 hr. Surface mu+ B cells were essentially non-cycling. To quantitate whole body BM cell production, the recovery of marrow from bone and the distribution of BM were measured in 59Fe distribution experiments. The number of cells produced by whole body BM was estimated as follows: for pre-B cells, 4.5 X 10(8)/day; for TdT+mu-, 0.7 X 10(8)/day; and for HIS24+TdT-Ig- 2.6 X 10(8)/day. From the derived cell flux in these compartments we suggest that 1) many more pre-B cells are produced than needed by the peripheral B cell pool; 2) if TdT is an obligatory stage in B cell genesis, there must be at least two cell cycles in the pre-B cell compartment; 3) if it is not, the TdT+ stage may be bypassed, with HIS24+TdT-Ig- cells perhaps feeding directly into the pre-B cell compartment.