Reaggregate Thymic Organ Cultures Research Articles

Comprehensive phenotypic analysis of diverse FOXN1 variants

Thymus hypoplasia due to stromal cell problems has been linked to mutations in several transcription factors, including Forkhead box N1 (FOXN1). FOXN1 supports T-cell development by regulating the formation and expansion of thymic epithelial cells (TECs). While autosomal recessive FOXN1 mutations result in a nude and severe combined immunodeficiency phenotype, the impact of single-allelic or compound heterozygous FOXN1 mutations is less well-defined. With more than 400 FOXN1 mutations reported, their impact on protein function and thymopoiesis remains unclear for most variants. We developed a systematic approach to delineate the functional impact of diverse FOXN1 variants. Selected FOXN1 variants were tested with transcriptional reporter assays and imaging studies. Thymopoiesis was assessed in mouse lines genocopying several human FOXN1 variants. Reaggregate thymus organ cultures were used to compare the thymopoietic potential of the FOXN1 variants. FOXN1 variants were categorized into benign, loss- or gain-of-function, and/or dominant-negatives. Dominant negative activities mapped to frameshift variants impacting the transactivation domain. A nuclear localization signal was mapped within the DNA binding domain. Thymopoiesis analyses with mouse models and reaggregate thymus organ cultures revealed distinct consequences of particular Foxn1 variants on T-cell development. The potential effect of a FOXN1 variant on T-cell output from the thymus may relate to its effects on transcriptional activity, nuclear localization, and/or dominant negative functions. A combination of functional assays and thymopoiesis comparisons enabled a categorization of diverse FOXN1 variants and their potential impact on T-cell output from the thymus.

Defining the impact of FOXN1 variants with functional assays and reaggregate thymus organ cultures reveals those with loss- and gain- of function and dominant negative consequences

Thymus hypoplasia can occur in individuals with autosomal recessive, compound heterozygous and single allelic Forkhead Box N1 (FOXN1) mutations. FOXN1 regulates thymus epithelial cell (TEC) development and function, which is essential for T cell development. The impact of an ever-increasing number of FOXN1 mutations, identified in patients, on TECs and consequently, thymopoiesis, remains unclear for most variants. We developed a systematic approach to determine the consequence of different FOXN1 variants on protein activity (luciferase reporter assays), localization (cell imaging), and dominant negative capabilities (co-expression studies). With these assays, we mapped the nuclear localization signal in FOXN1, identified de novo sequences in human FOXN1 that mediate dominant negative functions and uncovered two domains that modulate transcriptional activities. Such strategies reveal both loss- and gain- of function variants. To determine the impact of the variants on thymopoiesis, we modified reaggregate thymus organ cultures (RTOC) by incorporating flow sorting to isolate and recombine different cell subsets. In this system, only FOXN1 sufficient TECs can support thymopoiesis when combined with hematopoietic, mesenchymal, and endothelial cell subsets. Murine TECs from “nude” embryos (lacking FOXN1) were unable to support RTOC growth. The flow sorted “nude” TECs were reconstituted with purified TAT-Foxn1 fusion proteins prior to reaggregation. The transduction of wildtype TAT-Foxn1 re-established RTOC growth, thymopoiesis and Tcell development within a 10-day culture period. This RTOC strategy can enable one to define the FOXN1 variants of unknown significance (VUS) as benign, partially, or fully attenuated in their capacity to support thymopoiesis. Taken together, our findings establish FOXN1 genotype-phenotype relationships and reveal a thymopoietic screening strategy that obviates the need for generating mouse knock-in lines.

Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing Methodologies.

Thymic epithelial cells (TECs) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Cortical (c)TECs, present in the cortex, are essential for early thymocyte development including selection of thymocytes expressing functional TCRs (positive selection). Medullary (m)TECs, located in the medulla, play a key role in late thymocyte development, including depletion of self-reactive T cells (negative selection) and selection of regulatory T cells. In recent years, transcriptomic analysis by single-cell (sc)RNA sequencing (Seq) has revealed TEC heterogeneity previously masked by population-level RNA-Seq or phenotypic studies. We summarize the discoveries made possible by scRNA-Seq, including the identification of novel mTEC subsets, advances in understanding mTEC promiscuous gene expression, and TEC alterations from embryonic to adult stages. Whereas pseudotime analyses of scRNA-Seq data can suggest relationships between TEC subsets, experimental methods such as lineage tracing and reaggregate thymic organ culture (RTOC) are required to test these hypotheses. Lineage tracing - namely, of β5t or Aire expressing cells - has exposed progenitor and parent-daughter cellular relationships within TEC.

Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs.

In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, further enhanced by cancer therapies. Identifying thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution. We identified a new population of progenitor cells, present in both the thymus and bone marrow (BM) of mice, that coexpress the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintain the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed coexpression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+ EpCAM+ GFP-expressing cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing thymic stroma. Sorted BM-derived CD45+ EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1-expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm. BM-derived CD45+ EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+ EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may help counteract thymic involution in cancer patients.

Bone marrow-derived cells contribute to the maintenance of thymic stroma including thymic epithelial cells

Abstract In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, which are further enhanced by cancer therapies. Identification of thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution. We identified a new population of progenitor cells, present in both thymus and bone marrow (BM), that co-express the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintains the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed co-expression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+EpCAM+GFP+ cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing stromal cell. Sorted BM-derived CD45+EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1 expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm. BM-derived CD45+EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may prove useful for counteracting thymic involution in cancer patients.

Disassembling and Reaggregating the Thymus: The Pros and Cons of Current Assays.

This review briefly describes the last decades of experimental work on the thymus. Given the histological complexity of this organ, the multiple embryological origins of its cellular components and its role in carefully regulating T lymphocyte maturation and function, methods to dissect and understand this complexity have been developed through the years. The possibility to study ex vivo the thymus organ function has been achieved by developing Fetal Thymus Organ Cultures (FTOC). Subsequently, the combination of organ disaggregation and reaggregation in vitro represented by Reaggregate Thymus Organ cultures (RTOC) allowed mixing cellular components from different genetic backgrounds. Moreover, RTOC allowed dissecting the different stromal and hematological components to study the interactions between Major Histocompatibility Complex (MHC) molecules and the T-cell receptors during thymocytes selection. In more recent years, prospective isolation of stromal cells and thymocytes at different stages of development made it possible to explore and elucidate the molecular and cellular players in both the developing and adult thymus. Finally, the appearance of novel cell sources such as embryonic stem (ES) cells and more recently induced pluripotent stem (iPS) cells has opened new scenarios in modelling thymus development and regeneration strategies. Most of the work described was carried out in rodents and the current challenge is to develop equivalent or even more informative assays and tools in entirely human model systems.

Dissecting and modeling the emergent murine TEC compartment during ontogeny.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45- ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII- CD80- → MHCIIlo CD80- → MHCIIhi CD80- → MHCIIhi CD80hi TECs, whereby MHCIIhi CD80- and MHCIIhi CD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhi CD80- cTECs directly generate mature MHCIIhi CD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

Reconstituted Thymus Organ Culture.

Reconstituted thymus organ culture is based on fetal thymus organ culture (FTOC). Purified thymocyte populations, from genetically modified mice or even from other species, are cultured in vitro with thymic lobes depleted of their endogenous thymocytes (by 2'-deoxyguanosine treatment) to form a new thymus. This potent and timesaving method is distinct from FTOC, which assesses development of unmodified thymic lobes, and reaggregate thymic organ culture, in which epithelial cells are separately purified before being aggregated with thymocytes.

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures [corrected].

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire

Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire+ and Aire− MEC subsets through flow cytometric analysis of 5-bromo-2′deoxyuridine (BrdU) incorporation. The Aire+ MEC subset was almost entirely postmitotic and derived from cycling Aire− precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire− MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire+ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a “terminal differentiation” model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire+ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.

EphrinB1‐EphB signaling regulates thymocyte‐epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2-Fc or ephrinB1-Fc fusion proteins to fetal thymus organ cultures established from 17-day-old fetal mice decreases the numbers of both double-positive (CD4(+)CD8(+)) and single-positive (both CD4(+)CD8(-) and CD4(-)CD8(+)) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4(+)CD8(+) thymocytes, we have also demonstrated that ephrinB1-Fc proteins are able to disorganize the three-dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB-Fc treatment also decreases the formation of cell conjugates by CD4(+)CD8(+) thymocytes and TEC as well as the TCR-dependent signaling between both cell types. Finally, immobilized EphB2-Fc and ephrinB1-Fc modulate the anti-CD3 antibody-induced apoptosis of CD4(+)CD8(+) thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three-dimensional organization of TEC.

Fetal Thymus Organ Culture: Figure 1.

INTRODUCTIONThe generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic stromal cell function. This system remains the only in vitro system that supports a complete program of T-cell development, including positive and negative selection of the developing T-cell receptor repertoire. Modifications of the basic fetal thymus organ culture system, such as hanging drop cultures and reaggregate thymus organ cultures, provide useful systems to analyze thymus colonization and thymic stromal cell function, respectively.

Metalloproteinase‐Dependent Control of Thymocyte Differentiation and Proliferation

The development of T cells in the thymus is dependent on interactions between thymocytes and thymic stromal cells, on stimulation by growth factors, and on the binding to and migration along extracellular matrix (ECM) components. As metalloproteinases (MP) are involved in processes such as growth factor release and ECM modelling, we assessed the effect of MP inhibitors on T-cell development using fetal thymic organ culture systems. MP inhibitors significantly reduced the numbers of CD4/CD8 double-positive (DP) and mature single-positive thymocytes generated, correlated with a reduced number of cell cycles between the double-negative (DN)3 and DP stages. The progression of early thymocyte progenitors through the DN1-4 stages of development was also severely affected, including incomplete upregulation of CD25, decreased DN3 cell numbers, reduced rearrangement of the T-cell receptor (TCR)-beta locus and expression of intracellular TCR-beta by fewer DN3 cells. When purified DN1 cells were utilized as donor cells in reaggregate thymic organ cultures, essentially no DP thymocytes were produced in the presence of MP inhibitors. The results suggest that MP inhibitors affect the differentiation of developing thymocytes before, and reduce proliferation after, pre-TCR-mediated selection.

In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9‐DL1 Cell Coculture

Most T cell development occurs within the thymus and includes a series of selection processes that are, in large part, still poorly understood. Studies of T cell development have been greatly advanced by the description of multiple phenotypic subsets of T cells and their maturational relationships. This unit describes a system for observing and modulating T cell development in vitro via the culture of entire mouse fetal thymic lobes. Methods are included for the isolation of fetal thymi and culture to allow for normal T cell development on either transwell plates or Gelfoam sponges. A method for depleting hematopoietic cells from thymic lobes using 2'-deoxyguanosine and subsequent reconstitution with precursor cells is also described. This protocol is valuable for the study of tolerance and T cell selection. A support protocol describing methods of altering and monitoring T cell development are outlined. In addition, methods for culturing fetal thymic lobes under high oxygen submersion conditions and for the preparation of reaggregate thymus organ cultures are provided. Finally, a simple and practical method that allows for the thymus-independent generation of T cells from defined sources of stem/progenitor cells by OP9-DL1 coculture is described.

The TCRβ Enhancer Is Dispensable for the Expression of Rearranged TCRβ Genes in Thymic DN2/DN3 Populations but Not at Later Stages

The Ebeta enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRbeta segments but appears to be required for TCRbeta V to DJ rearrangement. Ebeta dependency of the subsequent expression of VDJ-rearranged TCRbeta genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vbeta8.2Dbeta1Jbeta1.3-rearranged TCRbeta bacterial artificial chromosome, which lacked Ebeta, and monitored transgene expression by flow cytometry using Vbeta-specific mAbs and an IRES-eGFP reporter. Transgene expression was found in double negative (DN)2 and DN3 but not at later stages of thymopoesis. There was no toxicity associated with the transgene given that apoptosis in DN3, DN4 was not increased, and the number of DN4 cells generated from DN3 cells in reaggregate thymic organ cultures was not diminished. The transgenic TCRbeta gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRbeta rearrangement, to facilitate beta-selection on a TCRbeta-deficient background and to inhibit gammadelta T cell lineage development. The results suggest that the Vbeta8.2 promoter is sufficient to drive expression of rearranged TCRbeta VDJ genes Ebeta independently in DN2/DN3 but not at later stages.

Phenotypic Characterization of CD3−7+ Cells in Developing Human Intestine and an Analysis of Their Ability to Differentiate into T Cells

We have identified a large population of CD3(-)7(+) cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8alphaalpha homodimer. In contrast about half of CD3(+) cells expressed CD4 and half expressed CD8alpha. A large proportion of CD3(-)7(+) cells expressed CD56, CD94, and CD161, and whereas CD3(+) T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3(-)7(+) cells have the potential to differentiate into CD3(+) cells. About half of CD3(-)7(+) cells contain intracellular CD3epsilon. Rearranged TCR gamma-chains were detected in highly purified CD3(-)7(+) cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5' Jgamma segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3(-)7(+) cells also gave rise to CD3(+) T cells. Thus, we demonstrate that the CD3(-)7(+) cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3(+) T cells.

Interleukin-7 Over-Expression Regulates T Cell Versus B Cell Lineage Development in the Thymus.

We have shown that high IL-7 transgene (Tg) over-expression (39-fold at day 1 in thymic tissue) under the T cell specific, proximal lck promoter had a dose effect on TCRαβ that was accompanied by active B cell development in the thymus. To further characterize these affects in the thymus of IL-7 transgenic mice, we analyzed thymi from day 18 embryos and newborn Tg mice, as well as fetal thymic organ culture (FTOC) derived from using day 16 embryos. We show that arrested T-cell and increased B-cell thymic development is initiated during fetal development. Using mixed bone marrow chimeras and anti-IL-7 monoclonal antibody injection, we further demonstrate that abnormalities in thymic T and B cell development are non-cell autonomous and are due to IL-7 over-expression. Recently, it was shown that only the early thymocyte progenitor (ETP, c-kit+IL-7R−/lo) fraction within the DN1 subpopulation had a T-cell proliferative potential in contrast to the c-kit−IL-7R+ DN1 subset. Here we show that in Tg mice the ETPs were decreased, while the c-kit−IL-7R+ cells are increased in both percentage and absolute count when compared to normal controls. In order to explore the T vs. B ETP potential, we seeded re-aggregate thymic organ cultures with sorted lin−CD44+CD25−c-kit+IL-7R+ cells. While ETPs derived from normal controls were able to proliferate and produce 83% of DP thymocytes, ETPs sorted from Tg mice developed poorly (10-fold less) into DP cells (30%) and produced 14% of B220+ cells vs. 6% in controls. Moreover, sorted Pro/Pre B derived thymic B cells from Tg mice, but not BM-derived Pro/Pre B cells had the TCRβD-J rearrangement, suggesting a T-specific origin. Since the B-cell differentiation pathway in normal mice is selectively inhibited by thymic presentation of Notch ligands, we hypothesized that IL-7 down-regulates Notch signaling. To test this hypothesis, we analyzed thymocyte progenitors (DN1-DN4) in normal and Tg mice for the intra-cytoplasmic part of Notch, that is cleaved upon Notch/Notch-ligand activation. Notch staining was decreased in the lin−CD44+CD25inter representing the only DN2 population present in these Tg mice. These data favour a decrease of Notch signalling in mice with high IL-7 Tg over-expression, inducing a block in TCRαβ development, and skewing of thymic B cell development by T vs. B lineage subversion. These conclusions may have implications for IL-7 in the clinical setting.

Delayed and restricted expression limits putative instructional opportunities of Vgamma1.1/Vgamma2 gammadelta TCR in alphabeta/gammadelta lineage choice in the thymus.

Development of alphabeta and gammadelta T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRbeta and TCRgammadelta proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRgammadelta proteins or to a more rapid progression to DN4 of thymocytes expressing TCRgammadelta. The answer to this question bears on the distinction between instructive and stochastic models of alphabeta/gammadelta lineage decision. To study this question, we first monitored initial TCR protein expression in wild-type and TCR transgenic mice in reaggregate thymic organ cultures. A TCRbeta transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRgammadelta proteins. In contrast, TCRgammadelta transgenes were expressed only in a fraction of DN4 cells, did not accelerate DN3 to DN4 transition, and did not reduce the number of DN4 cells expressing TCRbeta proteins. The TCRbeta transgene partially inhibited endogenous TCRgamma rearrangements, whereas the TCRgammadelta transgenes did not inhibit endogenous TCRbeta rearrangements. Second, we analyzed frequencies of productive TCRbeta and TCRgammadelta V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRgammadelta rearrangements (Vdelta5, Vgamma1.1, and Vgamma2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding gammadeltaTCR, severely limiting their putative instructional opportunities in alphabeta/gammadelta divergence.

In vitro generation of T lymphocytes from embryonic stem cell–derived prehematopoietic progenitors

AbstractEmbryonic stem (ES) cells can differentiate into most blood cells in vitro, providing a powerful model system to study hematopoiesis. However, ES cell–derived T lymphocytes have not been generated in vitro, and it was unresolved whether such potential is absent or merely difficult to isolate. Because the latter case might result from rapid commitment to non–T-cell fates, we isolated ES cell–derived prehematopoietic precursors for reconstitution of fetal thymic organ cultures. We found a transient Flk1+CD45– subset of these precursors generated T lymphocytes in vitro, and the use of reaggregate thymic organ cultures greatly enhanced reconstitution frequency. These findings reveal that ES cells can exhibit in vitro T-cell potential, but this is restricted to early stages of ES cell differentiation. Moreover, the results support the notion that the thymic microenvironment can induce T-cell differentiation from a subset of prehematopoietic progenitors and suggest deficient migration into intact thymi hindered previous attempts to generate T cells in vitro from ES cell–derived progenitors. These findings demonstrate that a defined subset of ES cells has the potential to generate T cells in vitro and could contribute to greater understanding of the molecular events of hematopoietic induction and T-cell lineage commitment.