Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8alphaalpha+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.     

Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors

Interactions between T cell precursors and thymic stromal cells are essential during thymocyte development. However, the role of the thymus in initial commitment of lymphoid progenitors to the T lineage remains controversial, with data providing evidence for both extra- and intrathymic commitment mechanisms. In this context, it is clear that Notch1 is an important mediator during initiation of T cell development. Here we have analyzed the mechanisms regulating Notch activation in lymphoid precursors at extrathymic sites and in the thymus, including stages representing the first wave of embryonic thymus colonization on embryonic day 12 of gestation. We show that Notch activation in migrant lymphoid precursors requires entry into the thymic microenvironment where they are exposed to Notch ligands expressed by immature thymic epithelial cells. Moreover, continued Notch signaling in such precursors requires sustained interactions with Notch ligands. Collectively, these findings suggest a role for Notch in an intrathymic mechanism of T cell lineage commitment involving sustained interactions with Notch ligand bearing thymic epithelium.     

Impaired development of T lymphoid precursors from pluripotent hematopoietic stem cells in New Zealand Black mice.

Bone marrow cells from autoimmune-prone New Zealand Black (NZB) mice are less efficient at colonizing fetal thymic lobes than cells from normal strains. This study demonstrates that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Rather, the T lymphopoietic defect of NZB mice is due to an impaired ability of pluripotent hematopoietic stem cells (PHSCs) to generate more committed lymphoid progeny, which could include common lymphoid precursors and/or other T cell-committed progenitors. Although PHSCs from NZB mice were not as efficient at thymic repopulation as comparable numbers of PHSCs from control strains, the ability of common lymphoid precursors from NZB mice to repopulate the thymus was not defective. Similarly, more differentiated NZB T cell precursors included in the intrathymic pool of CD4(-)CD8(-) cells also exhibited normal T lymphopoietic potential. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that generation of lymphoid progeny from the PHSCs is a regulated event.     

Developmental regulation of the intrathymic T cell precursor population

The maturation potential of CD4-8- thymocytes purified from mice of different developmental ages was examined in vivo after intrathymic injection. As previously reported, 14-day fetal CD4-8- thymocytes produced fewer CD4+ than CD8+ progeny in peripheral lymphoid tissues, resulting in a CD4+:CD8+ ratio of less than or equal to 1.0. In contrast, adult CD4-8- thymocytes generated CD4+ or CD8+ peripheral progeny in the proportions found in the normal adult animal (CD4+:CD8+ = 2 to 3). Here we have shown that CD4-8- precursor cells from the 17-day fetal thymus also produced peripheral lymphocytes with low CD4+:CD8+ ratios. Precursors from full term fetuses produced slightly higher CD4+:CD8+ ratios (1.1-1.6) and precursors from animals three to 4 days post-birth achieved CD4+:CD8+ ratios intermediate between those produced by fetal and adult CD4-8- thymocytes. Parallel changes in the production of alpha beta TCR+ peripheral progeny were observed. Fetal CD4-8- thymocytes generated fewer alpha beta TCR+ progeny than did adult CD4-8- thymocytes. However, peripheral lymphocytes arising from either fetal or adult thymic precursors showed similar proportions of gamma delta TCR+ cells. The same pattern of progeny was observed when fetal CD4-8- thymocytes matured in an adult or in a fetal thymic stromal environment. In contrast to fetal thymic precursors, fetal liver T cell precursors resembled adult CD4-8- thymocytes by all parameters measured. These results suggest that fetal thymic precursors are intrinsically different from both adult CD4-8- thymocytes and fetal liver T cell precursors. Moreover, they lead to the hypothesis that the composition of the peripheral T cell compartment is developmentally regulated by the types of precursors found in the thymus. A model is proposed in which migration of adult-like precursors from the fetal liver to the thymus approximately at birth triggers a transition from the fetal to the adult stages of intrathymic T cell differentiation.     

Thymic microenvironment induces HIV expression. Physiologic secretion of IL-6 by thymic epithelial cells up-regulates virus expression in chronically infected cells

The hallmark of infection with HIV-1 is progressive depletion and qualitative dysfunction of the CD4+ Th cell population in infected individuals. Clinical trials of antiretroviral agents have shown that, despite suppression of virus replication, regeneration of the T cell pool does not occur. One proposed explanation for the defective regenerative capacity of the CD4+ T cell pool is infection of early T lymphocyte progenitors or stem cells. An additional explanation could be failure of cells of the intrathymic microenvironment (thymic epithelial (TE) cells) to carry out critical nurturing functions for developing thymocytes, i.e., secretion of thymocyte-trophic cytokines and expression of adhesion molecules. This study examines the effect of HIV on cultured TE cells and determines the role of TE cells in the regulation of viral expression in chronically HIV-infected cells. We found no evidence of infection of TE cells after exposure to HIV-1. However, normal human serum induced secretion of IL-6 by TE cells; induction of TE IL-6 was partially blocked by anti-IFN-gamma antibodies. Moreover, supernatants from TE cells maintained in normal human serum up-regulated HIV replication in chronically HIV-1-infected cells. Because intrathymic T cell precursors can be infected with HIV and T cell precursors come into close contact with TE cells in the thymus, IL-6 secreted by TE cells during normal intrathymic development may induce HIV expression in infected thymocytes in vivo and promote the intrathymic spread of HIV.     

Sonic hedgehog regulates early human thymocyte differentiation by counteracting the IL-7-induced development of CD34+ precursor cells

The Hedgehog (Hh) family of signaling molecules normally functions in the development of numerous tissues by regulating cellular differentiation and proliferation. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate the potential role of Sonic hedgehog (Shh) in human intrathymic T cell maturation. Results show that the expression of the two components of the Hh receptor, Patched and Smoothened, is mostly restricted to CD34+ precursor cells that are committing to the T cell lineage. Shh significantly increased the viability of CD34+ T cell precursors modulating bcl-2 and bax protein expression, and also inhibited their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with Shh resulted in an arrested thymocyte differentiation and an accumulation of CD34+ progenitor cells. This effect was mainly attributed to the ability of Shh to counteract the IL-7-induced proliferation and differentiation of CD34+ cells. Shh down-regulated in the precursor cell population the expression of IL-7R as well as stromal-derived factor-1 chemokine receptor, CXCR4, and inhibited IL-7-dependent STAT5 phosphorylation. Therefore, Shh may function as a maintenance factor for intrathymic CD34+ precursor cells.     

Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes.     

The Expansion of Thymopoiesis in Neonatal Mice Is Dependent on Expression of High Mobility Group A 2 Protein (Hmga2)

Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.     

The functional capabilities of cells leaving the thymus

There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.     

Extra-thymic physiological T lineage progenitor activity is exclusively confined to cells expressing either CD127, CD90, or high levels of CD117

T cell development depends on continuous recruitment of progenitors from bone marrow (BM) to the thymus via peripheral blood. However, both phenotype and functional characteristics of physiological T cell precursors remain ill-defined. Here, we characterized a putative CD135(+)CD27(+) T cell progenitor population, which lacked expression of CD127, CD90, and high levels of CD117 and was therefore termed triple negative precursor (TNP). TNPs were present in both BM and blood and displayed robust T lineage potential, but virtually no myeloid or B lineage potential, in vitro. However, TNPs did not efficiently generate T lineage progeny after intravenous or intrathymic transfer, suggesting that a physiological thymic microenvironment does not optimally support T cell differentiation from TNPs. Thus, we propose that physiological T cell precursors are confined to populations expressing either CD127, CD90, or high levels of CD117 in addition to CD135 and CD27 and that TNPs may have other physiological functions.     

Gated importation of prothymocytes by adult mouse thymus is coordinated with their periodic mobilization from bone marrow

The wavelike pattern of fetal T cell neogenesis is largely determined by the intermittent generation and exportation of waves of prothymocytes by the hemopoietic tissues in coordination with their gated importation by the thymus. Having previously shown that the importation of prothymocytes by the adult mouse thymus is also gated and that thymocytopoiesis proceeds in discrete (albeit overlapping) waves, we now demonstrate that prothymocytes are periodically exported in saturating numbers from the adult mouse bone marrow. Experiments in normal, radioablated, and parabiotic mice document the cyclical accumulation (3-5 wk) of prothymocytes in both the steady state and regenerating bone marrow, followed by their release into the blood approximately 1 wk before intrathymic gate opening. The results also show that circulating donor-origin thymocyte precursors can transiently ( approximately 1 wk) establish high level chimerism in the bone marrow after the mobilization of endogenous prothymocytes, presumably by occupying vacated microenvironmental niches. Hence, by analogy with the fetal state, we posit the existence of a feedback loop whereby diffusible chemokines of thymic origin regulate the production and/or release of bone marrow prothymocytes during each period of thymic receptivity. Because each resulting wave of thymocytopoiesis is accompanied by a wave of intrathymic dendritic cell formation, these coordinated events may help to optimize thymocyte selection as well as production.     

Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes

A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.     

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.     

Recombinant interleukin 4/BSF-1 promotes growth and differentiation of intrathymic T cell precursors from fetal mice in vitro.

Recombinant mouse interleukin 4/BSF-1 (rIL4/BSF-1) together with phorbol myristate acetate (PMA) promotes growth of one out of approximately four intrathymic T cell precursors from fetal mice (14-15 days gestation). This response is not inhibited by even high concentrations of monoclonal antibody against the receptor for interleukin 2. Fetal thymocytes activated by rIL4/BSF-1 plus PMA give rise to cytolytic T cells after 7-21 days of culture. All the proliferating cells are Thy1+, some of them express Lyt2 but none has detectable L3T4 T cell differentiation antigens nor T cell antigen receptor (F23.1) on the cell membrane as assessed by immunofluorescence staining and flow fluorocytometry analysis. It is concluded that rIL4/BSF-1 exerts both growth and differentiation activities on normal intrathymic T cell precursors. The results provide evidence for an alternative growth factor to interleukin 2 involved in proliferation of T cell precursors. These findings open new and direct ways of studying cellular and molecular events during the differentiation of normal intrathymic T cell precursors in vitro and extend the spectrum of target cells for IL4/BSF-1.     

Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR(+) progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.     

Differentiation of human T lymphocytes. I. Acquisition of a novel human cell surface protein (p80) during normal intrathymic T cell maturation

The thymus is thought to be the primary central lymphoid organ in which T cells mature. Although thymic cortical and medullary compartments are distinct histologically, few antigens have been described that are absolutely acquired during the presumed intrathymic maturation pathway from cortical to medullary thymocytes. In this paper, we describe the acquisition during human intrathymic T cell maturation of a novel protein (p80) defined by a monoclonal antibody (A1G3). Although the p80-A1G3 antigen is distributed throughout the body and is not T cell specific, our study demonstrates that expression of p80-A1G3 antigen in normal human thymus is associated with thymocyte functional maturity and location in the thymus medulla. Moreover, in contrast to other markers of mature human T cells, the p80-A1G3 cell surface protein is not expressed on T6+ cortical thymocytes, and, therefore, is absolutely acquired by medullary thymocytes during T cell maturation. Thus, the p80-A1G3 antigen and the A1G3 antibody provide a heretofore unavailable system for the study of molecular events that transpire during the maturation of thymocytes.     

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.