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Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.

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)(8)(9)(10)(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.

Materials and Methods
with an argon laser at 488 nm was used at the following settings: laser, 100 mW; photomultiplier, 680 V; fluorescence gain 8/1 for bone marrow, 16/1 for fetal liver. Low-angle light scatter thresholds were set to exclude erythrocytes, dead cells, and aggregates. 5,000 cells were sorted per second for 6-8 h. Cell recovery was 30-40% of the input, the loss being due to a combination of cells aborted through coincidence in one drop, cells falling outside the chosen fluorescence windows, adherence to the feed tube, disintegration on passage through the nozzle, and handling losses in the collecting tubes. Suspension of the input cells in isopycnic medium (70% Percoll [Pharmacia Fine Chemicals, Piseataway, N.J.] in Dulbecco's A plus B solution containing 10 mM NaNa) helped to prevent aggregation in the sample and feed tubes. Purities, checked by re-analysis of sorted fractions, averaged 91% for bright cells, 81% for dim cells, and 93% for negative cells.

Results
W3/13 Labeling of Bone Marrow. A typical fluorescence intensity profile of W3/13labeled cells among marrow nucleated cells (Fig. 1 a) reveals a bimodal distribution with a trough demarcating W3/13 bright cells and a more weakly labeled shoulder of a W3/13 dim population merging into but distinguishable from background labeling. The proportions of W3/13 bright and W3/13 dim cells stabilized at ~40 and 25%, respectively, at about 6 wk after birth. The dot plot ( Fig. 1 b) of low-angle light scatter vs. fluorescence intensity showed the bright cells to be mostly large cells (granulocytes and blasts) and the dim cells to be mostly lymphoid. A differential count in smears of marrow sorted for these populations is shown in Table I, confirming this interpretation.
Functional Studies on W3/13-sorted Bone Marrow Cells: B and T Lymphocyte Stem Cell Assay. Initial experiments used PVG-la congenic rats as donors, carrying only the kappa chain marker, so only B and not T lymphocyte genesis was studied. PVG-la bone marrow cells were competed in various doses against a fixed inoculum of 107 PVG marrow cells in irradiated PVG hosts. Four different fractions of PVG-Ia bone marrow were used: unlabeled, labeled but unsorted, and the two sorted fractions, W3/13 bright and W3/13 dim-and-negative. Recipients were analyzed for chimerism of the B lymphocytes bearing the kappa allotype at various times after irradiation, usually 4-6 wk. We checked first the linearity of the stem cell assay to confirm previous observations (6). Competition between unlabeled PVG-la and PVG marrow was equal. Ideally, 107 PVG-la bone marrow cells should produce a chimerism of nearly 50%; in fact, four different animals were found to have 45, 46, 48, and 50% ( Fig. 2). Labeling cells with antibody caused only a marginal reduction in stem cell activity (Fig. 2).
The data in Fig. 2 show that by sorting labeled bone marrow.into W3/13 bright and W3/13 dim-and-negative fractions, B lymphocyte stem cell activity was contained entirely in the W3/13 dim-plus-negative fraction. The slight activity (3.6% chimerism) generated by the high dose of 107 W3/13 dim-plus-negative cells could be attributed to contamination with W3/13 bright cells. W3/13 bright cells, on the other hand, were able to generate more B cell progeny on a cell for cell basis than labeled but unsorted cells.
Similar experiments were then performed using PVG-(PtaDa + la) donor rats, which allow analysis of both T and B lymphocyte genesis simultaneously within one recipient. The use of these doubly congenic animals prevented artefacts due to variation in the thresholds used for sorting that otherwise might have occurred from one experiment to the next. Results from two experiments are summarized in Fig. 3. Both labeled and labeled but unsorted fractions of congenic marrow competed equally with PVG marrow. The lesser chimerism among T lymphocytes than B lymphocytes is the predicted pattern because T cells (defined as sIg-) include ~25% Pta-negative cells (6): fully donor-type Pta-bearing cells would therefore register as only 75% chimeric among all T cells. The results from the sorted fractions show that, as before, B stem cell activity was confined to the W3/13 bright population, which was roughly twice as potent per cell as unseparated marrow, as would be anticipated from their   "Early" analysis was at 5-7 wk after reconstitution; "late" analysis was at 24-44 wk. Note stability of B cell chimerism, whereas T cell chimerism in W3/13 bright recipients increased and W3/13 dim-plus-negative decreased.
reconstitution (Table II). In the interval between the analyses, B cell chimerism remained unchanged both in the sorted and unsorted fractions. For example, in the W3/13 bright fraction, the drive to proliferate was sufficiently vigorously maintained to continue competing equally with the whole PVG reference marrow inoculum. For T cells, however, although the chimerism generated by W3/13 bright cells almost doubled, that from W3/13 dim-plus-negative was halved though still detectable above background. The division between the T stem cell activities of bright and dim-plusnegative, initially equal, had shifted to about 3:1. These corresponding changes in T lymphocyte chimerism suggest that the more primitive T lymphopoietic stem cells are W3/13 bright.
In one further experiment, the thresholds for sorting were altered so as to separate W3/13 negative cells from W3/13 dim cells, discarding the bright fraction. Table III confirms the absence of B stem cell activity from dim or negative cells (Figs. 2 and 3) and shows that the T cell repopulating activity is in the dim not the negative population.

B and T Stem Cell Assays of Early Fetal
Liver. The preceding studies with bone marrow suggested that it contained a mixture of different kinds of stem cell, both multipotential and committed. To characterize the surface phenotype of the most primitive stem cells, we studied cells from the developmentally earliest lymphopoietic organ. We first tried yolk sac cells. 106 viable yolk sac cells at day 10, dissociated with trypsin, yielded no detectable cell chimerism in the standard competition assay 7 wk after reconstitution. Turning then to fetal liver, the earliest practicable day for analysis of function was day 14, when in the B and T competition assays there was potent activity. Chimerism from 105 cells was readily measurable within 2 mo after reconstitution (Table IV). Activity per cell fluctuated from one experiment to the next (perhaps at this early time the exact stage of gestation is more crucial than we were able to control), but was usually more vigorous even than the day I7 to day 20 fetal liver previously examined (6). Table IV shows how the titration of unlabeled cells confirms the much greater potency of day 14 liver than marrow (17), by a factor of at least 20 on a per cell basis. The more cells, the greater was the chimerism, but unlike marrow (Fig. 2), the relationship was nonlinear and tended to saturate at high doses. However, at lower doses (<106), the assay could be used with confidence to monitor the enrichment or depletion of activity among sorted cells.
Day 14 fetal liver was labeled with W3/13 antibody. Fig. 1 c shows clear labeling above the background seen with a control incubation with an irrelevant monoclonal supernate, W6/32. There was a continuous gradation of intensity of labeling, and the thresholds for defining a cell as negative, dim, or bright were taken to correspond to Chimeras were analyzed 8 wk after irradiation and reconstitution in the normal competition assay. In the dim fraction, the chimerisms show T stem cell activity to be approximately eightfold greater per injected cell than the unsorted cells. Note the absence of B stem cell activity from the sorted fractions.

Day-14 fetal liver cells were labeled with W3/13 antibody and fluorescent rabbit anti-mouse lg (experiments 1-5). Thresholds for sorting were set cor~sponding to those used for bone marrow. In experiments 1 and 2, donors were PVG-la and chimeras were analyzed at 5 and 8 wk after reconstitution. In experiments 3 and 4, heterozygous (PVG × PVG-Ptaoa) donors were used and analysis was at 8 and 12 wk; allowance was
made for the lower intensity of anti-Pta staining of heterozygotes during analysis. In experiment 5, donors were (PVG-(PtaDa + la) × PVGla)F1 hybrids, beterozygous for Pta and homozygous for Ig-la; analysis was at 10 wk. Table IV, experiments 1,

Radiosensitivity of Endogenous Stem Cells. Three experiments were performed in which
groups of PVG rats were exposed for various periods to the 137Cs source and injected intravenously 6 h later with 10 7 PVG-(PtaDA + la) young adult bone marrow cells.
Radiation doses ranged from 4.5 to 10 Gy (450-1,000 rad). Chimeras were analyzed at different times up to 9 mo after reconstitution, in some cases by cannulating the thoracic duct twice, before day 50 (early) and after day 100 (late). Even at early times the hourly output of lymphocytes from the thoracic duct was restored nearly to normal; the first night outputs were -2 × 107/h at the lower radiation doses, falling to about 107/h at the highest. If this output can be taken as a guide to the total mobilizable pool (e.g., 60 times the initial hourly output [18]), then the total pool of lymphocytes in recipients of an intermediate dose, 850 rad, can be estimated at ~8 × 108, a very substantial expansion from the 107 inoculated. The proportion of B cells among all TDL was independent of radiation dose, -60% at early times and 40-50% (comparable with normal PVG rats) at later times. The general properties of these chimeras were thus very similar to the fully exogenous chimeras described earlier (6). In addition to the usual sera detecting donor components, antibodies recognizing host Ig allotype (RI-lb) and host Pta (Ptapvo) were used to determine directly cells descended from endogenous sources. Fig. 4 shows the percentage of host-type Igbearing cells among host plus donor allotype bearers (B chimerism) and correspondingly for Pta-bearing T cells, which were ~70% of surface Ig-cells, irrespective of radiation dose. A crude but convenient index of radiosensitivity is the dose required to produce 50% chimerism, i.e., when the endogenous equivalent of 107 injected cells survive. For B lymphocytes, this was ~6.0-6.5 Gy, estimated within 50 d of reconsti-100. tution, and it hardly changed when examined much later, past 100 d. The late analyses included some chimeras analyzed at >8 too, but still the B cell chimerism remained steady. However, for T cells, 8.5 Gy were needed to produce 50% chimerism upon early analysis (before day 50) dropping to the same dose as for B cells, 6 Gy, at later times. In other words, rats given 8.5 Gy when analyzed soon after reconstitution contained substantial numbers of host T cells in the virtual absence of host B lymphocytes, another example of split chimerism. Longer-term repopulation revealed that the stem cells yielding T lymphocytes were equally as radiosensitive as those producing B lymphocytes. For permanent chimerism they might thus be one and the same.

Discussion
The monoclonal mouse antibody, W3/13, originally raised against rat thymocytes and present also on granulocytes, was found to label brightly the cells that can permanently repopulate the T and B lymphoid compartments of irradiated hosts. Both fetal liver and young adult bone marrow gave comparable results except that with marrow a W3/13 dim population was found that generated mainly T cells and not B cells. This exception is further considered below. W3/13 antigen, a glycophorinlike molecule (5), is therefore a marker that extends back from the thymocyte and granulocyte lineages to a stem cell, just as Thy-1, a variable region-like molecule (19), extends back from the lymphoid lineage (20)(21)(22). The combination of these two markers narrows the surface antigenic specification of primitive stem cells in rats and suggests a method for their enrichment that may improve still further on the 100-to 300-fold purification achieved by Goldschneider et al. (23).
Two circumstances are reported here in which substantial T cell chimerism without B cell chimerism was observed in irradiated reconstituted rats. First, W3/13 dim bone marrow generated predominantly T cells and not B cells. Second, host lymphocytes recovering in rats given 8.5 Gy were mainly T for the first 50 d after irradiation. In both cases, the surplus T chimerism waned later on. We take this to suggest the existence of committed T lymphocyte stem cells. Ideally, a committed stem cell must be shown to have three properties: self-replication, production of mature lymphocytes, and preferential generation of one rather than another lineage, in this case a greater number of T rather than B cell progeny. The first point is not formally demonstrated here, but the very substantial and continued expansion in cell numbers during reconstitution would be hard to achieve without self-replication. The second point is met by confining our analysis to peripheral lymphocytes carrying the surface antigens, Ig and Pta, characteristic of mature cells. The third criterion depends on the interpretation of the percentage chimerisms. Both sets of experiments here were performed as competition experiments, with the test cells racing against a reference standard of 10 7 normal untreated cells. Provided the way in which these competing cells proliferate is unaffected by the test cells the percentage chimerisms reflect the numerical expansion of the test cells. Normal untreated test bone marrow competes in irradiated hosts equally with normal untreated reference marrow (6); furthermore, in the present radiation dose experiments there was no great systematic variation with dose in the numbers or T:B ratio of the chimeric lymphocytes. To this extent, the presumption of undisturbed proliferation of reference marrow is justified. However, there may remain some minor fraction of marrow exhibiting proliferative inequality between test and reference inocula whose activity is amplified in the present experiments. Such disparity might result for example from the residual alloantigenic differences between the congenic lines, leading to artefactual imbalance in the relative production of T and B cells. However, these differences must be very small if they exist at all, because test skin allografts between the PVG and double-congenic strains are permanently accepted (our unpublished data). Unless the alloantigens used here as markers can be shown to be interfering, we interpret split chimerism as a manifestation of committed stem cells.
The differential radiosensitivity of T and B stem cells was an incidental finding from experiments whose first purpose was to ascertain the minimum dose at which the host component could be regarded as negligible in the chimerism assay. Fig. 4 shows this to be 9.5 Gy for chimeras assayed after day 50; earlier than this, 10.0 Gy should be used. The effect of residual host T cells in the chimerism assay would be to lower its sensitivity by reducing the percentage of cells of donor origin to below the expected value. The higher radiation doses needed for the eradication of host T stem cells than for B stem cells emphasize the need for careful monitoring of residual T cell activity when it is often presumed to be absent, for example in the preparation of B rats or of bone marrow chimeras for H-2-restriction experiments. Unwanted survival of host T cells could explain some of the discrepancies between findings on nude mice and B mice (24). The present results fit the observation that in semiallogeneic chimeras there is a detectable radioresistant host vs. graft component (10).
The experiments reported here do not establish whether the W3/13 dim committed stem cell sorted from marrow suspensions is the same as the more radioresistant component in the radiation dose experiments. At present, the only circumstantial evidence suggesting that they may be is that the surplus T cell chimerism was detected in analysis up to 2 mo after reconstitution, but in both cases, it diminished after that to approach the B cell chimerism, which stayed steady all the time. Thus the longterm proliferative capacity of the committed stem cell was weaker than the stem cell, presumably more primitive, that maintained the permanent chimerism. Further circumstantial evidence could be sought from a study of the subsets of T cells generated from committed stem cells that might distinguish them from progeny of more primitive stem cells. For more formal proof of identity it would be necessary to discover whether in vitro radiosensitivity corresponded to that in vivo and then to expose W3/13-sorted bone marrow to graded doses of radiation. The repopulation assay is not sufficiently sensitive to permit this kind of analysis.
In bone marrow there are a few recirculating T cells that can be removed by thoracic duct drainage (25). In addition, peripheral lymphocytes contain some T but not B repopulating activity when tested in chimeras (S. V. Hunt, unpublished data). This raises the question whether the W3/13 dim restricted T stem cell is pre-or postthymic. At first sight, the latter seems unlikely, because peripheral T cells carry large amounts of W3/13 and so correspond to W3/13 bright, not dim, cells in marrow. Also, peripheral T cells lack the thymocyte and early erythroid marker W3/15 (4), and in unpublished experiments we have found no surplus T chimerism in recipients of W3/15-negative marrow. However, the conclusion that the committed T stem cell is pre-thymic would be fortified by examining other peripheral T cell antigens such as MRC OX 8, W3/25 (26), and Pta itself. The question would be properly resolved by comparing the T repopulating activity of the marrow of thymus-deprived rats with that of the thymocyte-reconstituted B rats. Whether the committed T stem cell turns out to be pre-or post-thymic will not alter the conclusion that W3/13 is a prime example of a patchy or "jumping" antigen (27). It switches on, off, and on again during differentiation from the multipotent stem cell through both B and T lineages.

Summary
The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor ceils. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled -40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.