Intrathymic Precursors Research Articles

Identification of a Common Developmental Pathway for Thymic Natural Killer Cells and Dendritic Cells

Current data support the notion that the thymus is seeded by a yet uncommitted progenitor cell able to generate T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). We assess in this report the developmental relationship of DCs and NK cells derived from a small subset of CD34(+) human postnatal thymocytes that, like the earliest precursors in the fetal thymus, display low CD33 surface expression. Culture of these isolated CD34(+) CD33(lo) thymic progenitors with a mixture of cytokines, including interleukin-7 (IL-7), IL-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, results in predominant generation of DCs. However, the addition of IL-2 to the cytokine mixture leads to the simultaneous development of DCs and NK cells. Both developmental pathways progress through a transient population of CD34(+)CD44(bright) CD5(lo/-)CD33(+) large-sized cells, distinct from small-sized T-lineage precursors, that contain bipotential NK/DC progenitors. These data provide evidence of linked pathways of NK cell and DC development from intrathymic precursors and suggest that NK cells and DCs branch off the T lineage through a common intermediate progenitor.

In vivo roles of receptor tyrosine kinases and cytokine receptors in early thymocyte development

The early phases of T-cell development require both cell—cell interactions and soluble factors provided by stromal cells within the thymic microenvironment. Still, the precise nature of the signals delivered in vivo by cytokines (resulting in survival, proliferation or differentiation) remains unclear. Recent studies using mice deficient in cytokines or in their receptors have helped to identify essential signaling pathways required for the development of intrathymic precursors to mature αβ and γβ T cells. In addition, cytokine requirements for the development of natural killer cells were revealed in such mutants. The results obtained demonstrate that the development of all classes of lymphocytes (natural killer, γδ T cells and αβ T cells) is cytokine dependent, but the specific requirements differ for each lineage.

Early Steps in T Cell Development Are Affected by Aging

Involution of the thymus accompanies aging, a process in which the organ diminishes in size and cellularity and becomes disorganized. The rate of T cell emigration from the thymus is markedly reduced with age, and phenotypic analyses have identified alterations in the relative proportions of the major thymocyte subpopulations. The present studies made use of the capacity of the thymus to regenerate following irradiation from an intrathymic radio-resistant precursor population. By analysis of the differentiation of this “wave” of thymocytes, it was determined that aging most severely affects the earliest developmental transitions. While the overall rate of differentiation does not appear to be affected in older mice, fewer thymic progenitors initiate differentiation. The reduced expansion of late pre-T cells in the middle-aged is due to the smaller pool size of these cells.

Adult Thymus Contains Dendritic Epidermal T Cell Precursors That Home to the Epidermis in Irradiated, but Not in Normal, Mice

It was generally assumed that Vγ5+fetal thymocytes are the only cells capable of homing to the epidermis. However, we have recently demonstrated that reconstitution of thymectomized irradiated mice with T-cell-depleted bone marrow (BM) cells results in the appearance of donor-type CD8+, TCR-αβ+dendritic epidermal T cells (DETC) phenotypically distinct from their normal γδ+counterpants. In the present study, we found that the epidermis of lethally irradiated, BM-reconstituted (XBM) mice was also repopulated by DETC of host origin as well as donor BM-derived DETC and that the host-derived DETC were rarely detected in the epidermis of athymic BM chimeras (ATXBM). This result indicates that the host-derived DETC are originated from intrathymic radioresistant cells that may share migratory capacities with the fetal thymocytes. We demonstrated that transfer of radioresistant intrathymic precursor cells into ATXBM mice resulted in the appearance of donor-type CD8+, TCR-αβ+DETC. Contrary to our initial expectation, however, transferred normal adult thymocytes also gave rise to donor-type DETC with frequency similar to radioresistant thymocyte-derived DETC. These thymocyte-derived DETC were phenotypically similar to those derived from BM cells but differed in some respects. Tissue tropism by the CD8+subset was observed only for the epidermis, but not for other lymphoid organs, such as lymph nodes. The epidermis of normal mice, unlike that of irradiated mice, was refractory to the appearance of the thymus-derived DETC. These results suggest that prior irradiation of the host may confer a susceptibility of the epidermis to repopulation of DETC precursors, which are otherwise incapable of migrating into the epidermis. This provides an explanation for why CD8+, TCR-αβ+DETC of adult thymus or BM origin are rarely detected in the normal epidermis, but abundantly observed in the irradiated mice.

CD4 and CD8 expression and T cell antigen receptor gene rearrangement in early intrathymic precursor cells.

The earliest T precursor population in the adult mouse thymus, considered to have the surface phenotype CD4lo8-3-44+25-Thy-1lo c-kit+ (termed the low CD4 precursor), has been shown to have the capacity to produce B cells and dendritic cells, as well as T cells, and to have the T cell antigen receptor (TCR) C beta gene region in germ-line configuration. Because of evidence that this precursor population may have low levels of CD8 as well as CD4 on the cell surface, it was isolated, stained for surface CD4 and CD8 and assayed for the expression of messenger RNA (mRNA) for CD4 and CD8 by the reverse transcriptase polymerase chain reaction (RT-PCR). The low CD4 precursors gave definite, moderate levels of staining for both CD8 and CD4, in contrast to downstream precursors which showed only marginal staining and so could be considered as genuine CD4-8-3- triple negatives. The low CD4 precursor expressed a significant level of mRNA for CD4, indicating that the surface CD4 was produced by these cells. However, the low CD4 precursor did not express a detectable level of mRNA for CD8, suggesting that the surface CD8 was acquired from other cells. Since the low CD4 precursor population was found already to express mRNA for enzymes involved in TCR gene rearrangement, including in this study terminal deoxynucleotidyl transferase (TdT), a PCR procedure was used to assay early precursors for D-J rearrangements at the TCR beta gene locus. However, the low CD4 precursor had the TCR beta D-J genes in germ-line configuration, D-J gene rearrangements being first detected several stages downstream in the CD3-4-8-44-25-+ precursor population. We conclude that a transient synthesis of CD4, but not of CD8, characterize these early thymus precursors. Although they have initiated synthesis of some recombination-associated enzymes, full commitment to the T lineage and TCR gene rearrangement is a later event.

Thymic stromal cells can support B cell differentiation from intrathymic precursors.

T lineage cells are the predominant intrathymic population, but low numbers of thymic B cells are also present in the thymus. Recent studies describing the contribution of thymic B cells to the shaping of the T cell repertoire have raised the question as to whether they are produced intrathymically and the potential role of the thymic stroma in this process. During the course of studies aimed at testing the B cell developmental potential of CD4-CD8-CD3/TCR- triple-negative thymocytes present in a newly developed, stromal cell-dependent, long term culture system, the ability of selected thymic stromal cells to support distinct stages of B cell development was revealed. Some thymic stromal cell lines maintained cells with B cell developmental potential but could not support their differentiation, while other selected lines efficiently supported the development of surface IgM-expressing cells from triple-negative precursors. Thymic surface IgM+ B cells were generated most efficiently from thymocytes harvested from relatively young mice. Taken together, the results of this study demonstrate that the thymic microenvironment exhibits a functional heterogeneity in its ability to support different stages of B cell development.

Herpesvirus saimiri immortalization of alpha beta and gamma delta human T-lineage cells derived from CD34+ intrathymic precursors in vitro.

Herpesvirus saimiri (HVS), an agent that can infect many human cell types, has been shown to immortalize selectively TCR alpha beta + CD3+ T lymphocytes. Human T cell precursors defined as CD34+CD3-CD4-CD8- were isolated from thymic samples and exposed to HVS in the presence of either IL-2 or IL-7. Cultures lacking the virus were non-viable by day 15. Test cultures, in contrast, showed a sustained proliferative activity lasting > 5 months, allowing the phenotypical and molecular analysis of the cellular progeny. In the presence of IL-7, TCR alpha beta + cells with three different phenotypes (mainly CD4+CD8-, but also CD4+CD8+ and CD4-CD8+) were immortalized, whereas no TCR gamma delta + cells were recovered. Kinetic studies showed that the expansion of immortalized TCR alpha beta + cells was preceded by a gradual loss of CD34+ cells followed by a transient accumulation of two distinct cell subsets: first CD1+CD4+CD3- cells and then CD4+CD8+ thymocytes. This resembles early phenotypic changes occurring during normal intrathymic T cell development. In the presence of IL-2, in contrast, only TCR gamma delta + cells were immortalized (mainly CD4-CD8+, but also CD4-CD8-). The results show that HVS can be used to read the CD3+ cellular outcome of T cell differentiation assays, including gamma delta + CD4-CD8+, gamma delta + CD4-CD8-, alpha beta + CD4+CD8-, alpha beta + CD4-CD8+ and alpha beta + CD4+CD8+ T cells. A clear role for different cytokines (IL-2 for gamma delta + cells, IL-7 for alpha beta + cells) in early T cell commitment was also apparent.

Pathways from hematopoietic stem cells to thymocytes

A long-standing debate has been whether commitment to the T cell lineage occurs exclusively following thymus colonization, or whether prethymic T lineage restricted progenitors exist. Recently, the analysis of murine fetal blood for the presence of hematopoietic progenitor cells has led to the identification of a T lineage committed precursor population (designated prothymocytes). Fetal blood prothymocytes lack multipotent progenitor potential as shown by the fact that they fail to reconstitute B lymphocyte, myeloid anderythroid lineages. In addition to prothymocytes, fetal blood also contains a phenotypically distinct, pluripotent progenitor population which can reconstitute both T and B lymphocytes as well as myeloid and erythroid lineages. The identification of a circulating, T lineage restricted precursor population, which is also found in the blood of fetal athymic mice, provides strong evidence that T lineage commitment can precede thymus colonization. The thymus is not, however, exclusively colonized by prothymocytes. Under appropriate developmental conditions, multipotent precursor activity for non-T lineages such as B lymphocytes and thymic dendritic cells can be revealed within the intrathymic precursor pool. Moreover, evidence has been accumulated for a common progenitor for T cells and natural killer cells whch may be distinct from multipotent intrathymic progenitors.

The development of T and non-T cell lineages from CD34+ human thymic precursors can be traced by the differential expression of CD44.

In addition to T-lineage cells, a small proportion of hematopoietic non-T cells are present in the human postnatal thymus. However, the origin of this minor non-T cell thymic compartment is presently unknown. In this study we have analyzed the developmental potential of the earliest human intrathymic precursors, characterized as CD34+ cells expressing intermediate levels of CD44. We show that these CD34+CD44int thymocytes cultured with interleukin 7 were able to develop simultaneously into both T- and non-T (monocytes and dendritic cells) -lineage cells. Both developmental pathways progress through a CD1+CD4+ intermediate stage, currently believed to be the immediate precursor of double positive thymocytes. However, separate progenitors for either T or non-T cells could be characterized within CD1+CD4+ thymocytes by their opposite expression of CD44. Downregulated levels of CD44 identified CD1+CD4+ T-lineage precursors, whereas CD44 upregulation occurred on CD1+CD4+ intermediates that later differentiated into non-T cells. Therefore, commitment of human early intrathymic precursors to either T or non-T cell lineages can be traced by the differential expression of the CD44 receptor.

Thymic stem cells in mouse bone marrow

There is still controversy concerning the nature of the stem cells from bone marrow that colonize the thymus during embryogenesis and continually throughout life. To identify the bone marrow stem cells that home to and populate the thymus, we screened murine bone marrow cells for the presence of a population of surface phenotype similar to the earliest known intrathymic precursor. We have identified a population characterized by expression of an intermediate level of heat stable antigen, a very low level of Thy-1, and high levels of CD44 and class I major histocompatibility complex antigens. It is negative for B- cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac- 1, and TER 119). All these markers are common to the intrathymic precursors and bone marrow stem cells. However, this new bone marrow population is Sca-2+, similar to the intrathymic precursor, which makes a clear distinction from the Sca-2- bone marrow hematopoietic stem cells previously characterized. The frequency of the new population in the normal mouse bone marrow is about 0.25%. When transferred intrathymically or intravenously to lethally irradiated mice, it has a higher expansion potential (2 x 10(5)) than has been found for the intrathymic precursors (10(3)), but less than was found for the Sca-2- multipotent stem cell (10(7)). These transfer studies also showed that it was pluripotent, in that its precursor activity was not restricted to the production of T or B lymphocytes. However, it gave a reduced spleen colony number and smaller colonies (day-12 colony-forming unit spleen) when compared with multipotent stem cells. Thus, the cell we have identified appears to be the latest pluripotent cells so far identified in bone marrow and is therefore a good candidate for a bone marrow prothymocyte, but it appears not to be T-cell-committed.

Thymic Stem Cells in Mouse Bone Marrow

There is still controversy concerning the nature of the stem cells from bone marrow that colonize the thymus during embryogenesis and continually throughout life. To identify the bone marrow stem cells that home to and populate the thymus, we screened murine bone marrow cells for the presence of a population of surface phenotype similar to the earliest known intrathymic precursor. We have identified a population characterized by expression of an intermediate level of heat stable antigen, a very low level of Thy-1, and high levels of CD44 and class I major histocompatibility complex antigens. It is negative for B-cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac-1, and TER 119). All these markers are common to the intrathymic precursors and bone marrow stem cells. However, this new bone marrow population is Sca-2+, similar to the intrathymic precursor, which makes a clear distinction from the Sca-2- bone marrow hematopoietic stem cells previously characterized. The frequency of the new population in the normal mouse bone marrow is about 0.25%. When transferred intrathymically or intravenously to lethally irradiated mice, it has a higher expansion potential (2 x 10(5)) than has been found for the intrathymic precursors (10(3)), but less than was found for the Sca-2- multipotent stem cell (10(7)). These transfer studies also showed that it was pluripotent, in that its precursor activity was not restricted to the production of T or B lymphocytes. However, it gave a reduced spleen colony number and smaller colonies (day-12 colony-forming unit spleen) when compared with multipotent stem cells. Thus, the cell we have identified appears to be the latest pluripotent cells so far identified in bone marrow and is therefore a good candidate for a bone marrow prothymocyte, but it appears not to be T-cell-committed.

Mouse stem cell antigen Sca-2 is a member of the Ly-6 family of cell surface proteins.

Mature T lymphocytes arise from intrathymic T-cell precursors, which in turn are derived from a multipotent stem cell in the bone marrow. Unlike bone marrow stem cells, the differentiation potential of the earliest intrathymic precursor cells is strongly biased toward the lymphoid lineage. The major difference in cell surface phenotype between early thymic precursor cells and bone marrow stem cells is that the former population expresses Sca-2. The progeny of the intrathymic precursor population continue to express Sca-2 until the transition from blast cells to small cells, at which stage expression of Sca-2 is down regulated. Mature thymocytes and peripheral T cells do not express detectable levels of Sca-2, whereas peripheral B cells are Sca-2-positive. We report herein the complete sequence of mouse Sca-2 deduced from a thymocyte cDNA clone. Sca-2 is a member of the Ly-6 family, a group of small cysteine-rich cell surface proteins that are anchored in the membrane by a glycosyl-phosphatidylinositol moiety.

Early thymic regeneration after irradiation

Whole body irradiation produces profound thymic atrophy. After sublethal irradiation, regeneration begins promptly and the earliest regeneration is from radioresistant intrathymic precursors. The progeny of these precursors expand rapidly and restore thymic cellularity to near normal within 2 weeks. We have used monoclonal antibodies specific for a variety of differentiation markers of the T lineage to analyze the early events in thymic regeneration. A three-color flow microfluorometric analysis revealed that the majority of the cells found early in the regenerative process have the phenotye of mature T cells. These include CD4 −/CD8 −; CD3 hi as well as CD4 4+/CD8 −; CD3 hi and Cd4 −/CD8 +; CD3 hi. The proportion of cells with mature phenotypes declines rapidly between day 6 and day 12. Not all of the early appearing cells have mature phenotypes. Among the early cells that do not express CD3 are both CD4 and CD8 single positive cells that express HSA and resemble the intrathymic precursors found in other systems. In these mice CD4 single positive predominate. There are other cells that are HSA positive but express low levels of CD4 and very low levels of Thy-1. These appear to include the earliest members of the T-lineage. In addition to relatively mature conventional T cells and early progenitors, the early developing population includes cells that express markers of the T-cell lineage including the T-cell receptor but do not express Thy-1. These Thy-1 negative T cells comprise a significant number of the earliest cells found after regeneration.

Intrathymic lymphoid precursor cells during fetal thymus development.

Our previous studies have demonstrated the presence, in the adult mouse thymus, of a population of early precursor cells able to give rise to T and B lymphocytes but not myeloid cells. This population of cells expresses low levels of CD4 and has been termed the "low CD4 precursors." All these precursors were found to be c-kit positive, and they precede the better known CD4-CD8- precursor stage. In this study, embryonic and neonatal thymuses were examined to see whether a similar low CD4 precursor was part of the pathway of T cell development during ontogeny. A population with the phenotypic characteristics of the adult low CD4 precursor was found from day 15 of embryonic development, although the expression of low levels of CD4 was apparent only from embryonic day 17. Functional tests of these putative precursors showed they had no thymus-reconstituting ability when isolated from thymuses at any time during embryonic life, and very low reconstituting ability even 24 days after birth. These results raise questions about the adult low CD4 precursor as an obligatory stage in the development of T cells in the thymus.

The CD44 expressed on the earliest intrathymic precursor population functions as a thymus homing molecule but does not bind to hyaluronate.

A minute population of cells representing the earliest lymphoid-restricted precursor cells in the adult mouse thymus has been isolated recently in our laboratory. This population expresses low levels of CD4, very high levels of CD44 and has a surface antigenic phenotype similar to that of bone marrow hemopoietic stem cells. To examine the role of CD44 on these cells, and to ascertain its ligand, we analysed the ability of the early thymic precursor cells to bind hyaluronate (HA) which functions as a cell adhesion molecule and a ligand for CD44. We also examined if anti-CD44 antibodies (clones IM7.8.1 and KM201) can block the thymic precursor activity. The majority of the thymic precursor cells did not show specific HA binding, indicating that HA is not the ligand for the CD44 molecules expressed on these precursor cells. HA and CD44 interactions are therefore unlikely to play a role in development of cells at this early intrathymic precursor stage. When the purified intrathymic precursor cells were incubated with anti-CD44 antibodies before intravenous transfer into recipients, very few progeny cells were detected in the thymus, although progeny were found in the spleen and lymph nodes. Coating the cells with other isotype-matched antibodies did not have this effect. However, when the same anti-CD44-coated cells were transferred directly into the recipient thymus, normal levels of progeny cells were detected in this organ. This suggests that the CD44 on the intrathymic precursor cells is a homing molecule, able to direct cells to the thymus but utilizing some ligand other than HA.

Lymphocyte lineage commitment: Instruction versus selection

In mammals, lymphocytes develop from hematopoietic stem cells throughout life. The exact branchpoint of irreversible commitment to the lymphocyte lineage is not known, and developmental stages committed to the T and B lymphocyte lineages are being defined. The T cell lineage branches further into cells with ~8 or a8 T cell receptors (TCRs) for antigen. The a8 TCR lineage is subdivided by surface markers and functional potential into CD4+8helper and CD4-8+ killer lineages, which are discussed here. CD4+ He/per and CDl Connolly et al., 1988): cross-linking of either a CD4 or CD8 coreceptor with the a8 TCR by the same MHC molecule is required for efficient activation of T cells (Emmrich et al., 1986; Gabert et al., 1987). For some time, it has not been clear whether CD4+ and CD8’ T cells can each express both class I and class II MHC-specific TCRs. If they can, those cells with a “mismatched” combination of coreceptor and a6 TCR (which would bind to different MHC molecules) would be wasted because these cells could not be efficiently activated. The alternative possibility is that the formation of the CD4+ helper and CD8+ killer lineages is crucially dependent on the specificity of the a8 TCR for class II and class I MHC molecules, respectively. To probe the latter idea, the contribution of a particular receptor (of defined antigen specificity) to the formation of CD4 and CD8 lineages needed to be examined. Because of the great TCR diversity, this could not be done in normal mice but required TCR transgenic mice. The results of the transgenic experiments showed, first, that binding of an up TCR to the polymorphic part of thymic MHC molecules in the absence of the specific peptide antigen is essential for the generation of functional CD4+ helper and CD8+ killer lineage cells from nonfunctional CD4+8+ intrathymic precursors. Second, a CISSS l-specific TCR exclusively supports the development of mature CD8+, while a class II-specific TCR exclusively supports the development of mature CD4+ lineage Cells (von Boehmer, 1990). These differentiation Steps are called positive selection. Minireview

Radioresistant cells of thymus--producers and targets of thymocyte growth factor and their possible role in postradiation restoration of thymus.

In the present study the association between the damaging and the stimulating action of radiation on the cells of thymus (in particular intrathymic T-lymphocyte precursors, TLP) is analyzed. The radioresistance and possibility to secrete humoral products, in particular, the thymocytes growth factor, THGF, is assessed. The possible role of THGF in the postradiation restoration of thymus is also discussed.

Lymphocyte development from stem cells.

Highly enriched pluripotent and multipotent hematopoietic stem cells (HSCs) are isolated from bone marrow and fetal liver as Thy-1loLin-Sca-1+ cells. Pluripotent HSCs express c-kit receptor on their surface, but the generation and proliferation of early fetal HSCs take place in the absence of steel factor. T precursor cells migrate into the fetal thymus by chemotactic mechanism. CD4lo precursors represent a newly defined phase of T-cell development in the thymus between the bone marrow-derived stem cells and the CD4-8- intrathymic precursors. Only fetal, but not adult, HSCs have the capacity to differentiate into V gamma 3+ and V gamma 4+ T cells under the fetal thymic microenvironment, and HSC themselves may lose some of their developmental potential during ontogeny. It is postulated that HSCs are the locus of a complicated but precise developmental clock that may determine both the time-dependent closure of some gene loci (e.g. V gamma 3 and V gamma 4 T cell receptor, and embryonic and fetal globin) and the activation of others (e.g. the N nucleotide insertion machinery).

Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.

This study was designed to resolve the long standing controversy as to whether hematogenous precursors or resident intrathymic precursors maintain thymocytopoiesis in postnatal life. The kinetics of thymic chimerism was examined over a 21-wk period in 105 pairs of nonirradiated, Thy-1-alloantigen-disparate, parabiotic B10.S mice. Thymic chimerism reached reached maximum levels of 25% in the Thy-1b and 16% in the Thy-1a partners (mean 20.5% and 9.8%, respectively) 6 to 7 wk after parabiotic union. Most of the donor-origin thymocytes were located in the thymus cortex and expressed high levels of Thy-1 Ag and terminal deoxynucleotidyl transferase. Thymic chimerism did not reach 50% because circulating prothymocytes, unlike peripheral T cells, do not distribute randomly in the blood of parabiotic partners. This was shown in irradiated pairs of Thy-1-alloantigen-identical parabiotic mice, one member of which was injected i.v. with Thy-1-alloantigen-disparate bone marrow cells. Only 10% of the total donor-origin prothymocyte activity was detected in the opposite parabiont 72 h later. Similarly, the level of thymic chimerism in nonirradiated, Thy-1-alloantigen-disparate parabionts closely corresponded to the donor: host ratio of prothymocytes in the blood (i.e., between 10 and 20%), as determined directly by an intrathymic adoptive transfer assay. The continued dependence of thymic chimerism on blood-borne precursors was formally demonstrated by surgically separating mice 9 wk after parabiosis. Twelve weeks later, thymic chimerism was 50 to 80% lower in the separated parabionts than in sham-operated controls. The possible role of "stress" in initiating or maintaining thymic chimerism during parabiosis appeared to be excluded by the existence of similar kinetics of thymocytopoiesis (including the onset of thymic involution) in parabiotic and nonparabiotic mice; and by the occurrence of similar levels of thymic chimerism in adrenalectomized and nonadrenalectomized parabiotic mice. Thus these experiments demonstrate that the maintenance of thymocytopoiesis in postnatal life, as in prenatal life, is primarily dependent upon blood-borne prothymocytes, and that intrathymic precursors are replaced at an average rate of 2 to 3%/day.

Developmental potential of the earliest precursor cells from the adult mouse thymus.

A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.