Stages Of Thymic Development Research Articles

Fetal and adult progenitors give rise to unique populations of CD8+ T cells

Abstract Neonates often generate incomplete immunity against intracellular pathogens. As CD8+ T cells are essential in clearing intracellular infections, it is crucial to understand how neonatal CD8+ T cells respond to infection. We showed that neonatal CD8+ T cells fail to form memory. However, the underlying basis for this is unclear. Here, we investigated whether neonatal CD8+ T cells fail to form memory cells because (i) they have undergone more extensive homeostatic proliferation (HP) prior to infection (proliferation model) or (ii) they are created from a distinct hematopoietic stem cell (HSC) population (origin model). We first compared the behavior and gene expression profiles of different aged CD8+ T cells that had undergone similar amounts of HP. Interestingly, neonatal CD8+ T cells still possessed unique gene expression profiles and failed to form memory post-infection. Hence, the proliferation model cannot fully explain the differences. We tested the origin model by comparing neonatal and adult CD8+ T cells at the same stage of thymic development. Again, we observed distinct gene expression profiles, indicating that CD8+ T cells are created differently in neonates. To examine whether different HSCs underlie these differences, we adoptively transferred fetal and adult progenitors into adult thymii and found that fetal progenitor derived CD8+ T cells exhibited a memory phenotype and selectively formed short-lived effectors after infection. The adult progenitor derived cells exhibited a more naïve phenotype and preferentially formed memory precursors after infection. Collectively, our data extends the layered immune hypothesis to CD8+ T cells and suggests that neonatal and adult CD8+ T cells are derived from different HSCs.

T-cell selection in the thymus: a spatial and temporal perspective.

The ability of T cells to respond to a wide array of foreign antigens while avoiding reactivity to self is largely determined by cellular selection of developing T cells in the thymus. While a great deal is known about the cell types and molecules involved in T-cell selection in the thymus, our understanding of the spatial and temporal aspects of this process remain relatively poorly understood. Thymocytes are highly motile within the thymus and travel between specialized microenvironments at different phases of their development while interacting with distinct sets of self-peptides and peptide presenting cells. A knowledge of when, where, and how thymocytes encounter self-peptide MHC ligands at different stages of thymic development is key to understanding T-cell selection. In the past several years, our laboratory has investigated this topic using two-photon time-lapse microscopy to directly visualize thymocyte migration and signaling events, together with a living thymic slice preparation to provide a synchronized experimental model of T-cell selection insitu. Here, we discuss recent advances in our understanding of the temporal and spatial aspects of T-cell selection, highlighting our own work, and placing them in the context of work from other groups.

Targeting IRAK1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) represents expansion of cells arrested at specific stages of thymic development with the underlying genetic abnormality often determining the stage of maturation arrest. Although their outcome has been improved with current therapy, survival rates remain only around 50% at 5 years and patients may therefore benefit from specific targeted therapy. Interleukin receptor associated kinase 1 (IRAK1) is a ubiquitously expressed serine/threonine kinase that mediates signaling downstream to Toll-like (TLR) and Interleukin-1 Receptors (IL1R). Our data demonstrated that IRAK1 is overexpressed in all subtypes of T-ALL, compared to normal human thymic subpopulations, and is functional in T-ALL cell lines. Genetic knock-down of IRAK1 led to apoptosis, cell cycle disruption, diminished proliferation and reversal of corticosteroid resistance in T-ALL cell lines. However, pharmacological inhibition of IRAK1 using a small molecule inhibitor (IRAK1/4-Inh) only partially reproduced the results of the genetic knock-down. Altogether, our data suggest that IRAK1 is a candidate therapeutic target in T-ALL and highlight the requirement of next generation IRAK1 inhibitors.

Expression of Helios, an Ikaros Transcription Factor Family Member, Differentiates Thymic-Derived from Peripherally Induced Foxp3+ T Regulatory Cells

Helios, a member of the Ikaros transcription factor family, is preferentially expressed at the mRNA level by regulatory T cells (Treg cells). We evaluated Helios protein expression using a newly generated mAb and demonstrated that it is expressed in all thymocytes at the double negative 2 stage of thymic development. Although Helios was expressed by 100% of CD4(+)CD8(-)Foxp3(+) thymocytes, its expression in peripheral lymphoid tissues was restricted to a subpopulation ( approximately 70%) of Foxp3(+) T cells in mice and humans. Neither mouse nor human naive T cells induced to express Foxp3 in vitro by TCR stimulation in the presence of TGF-beta expressed Helios. Ag-specific Foxp3(+) T cells induced in vivo by Ag feeding also failed to express Helios. Collectively, these results demonstrate that Helios is potentially a specific marker of thymic-derived Treg cells and raises the possibility that a significant percentage of Foxp3(+) Treg cells are generated extrathymically.

Rare Development of Foxp3+ Thymocytes in the CD4+CD8+ Subset

The CD4(+)CD8(+) (double positive, DP) stage of thymic development is thought to be the earliest period that generates natural Foxp3(+) regulatory T (Treg) cells important for the prevention of autoimmunity. However, we found that most Foxp3(+) DP cells identified by routine flow cytometry represent doublets comprised of Foxp3(-) DP and Foxp3(+) CD4(+)CD8(-) (CD4SP) cells. This was determined using analysis of flow cytometric height and width parameters, postsort contaminants, and thymocyte mixing studies. Temporal analysis of Treg cell development arising from bone marrow precursors in neonatal bone marrow chimeras suggested that Foxp3(+) DP cells are not a major percentage of Foxp3(+) thymocytes, and it supported the notion that most Treg cell development occurred at the immature HSA(high) CD4SP stage. Thus, these data demonstrate that the frequency of Foxp3(+) cells generated at the DP stage is much smaller than previously recognized, suggesting that additional thymocyte maturation may be required to facilitate efficient induction of Foxp3.

The Transmembrane Adapter Protein SIT Regulates Thymic Development and Peripheral T-Cell Functions

SIT is a transmembrane adapter protein that modulates signals emanating from the T-cell receptor (TCR). Here, we have used gene-targeted mice to assess the role of SIT for T-cell development and peripheral T-cell functions. SIT(-/-) double-positive thymocytes show an upregulation of the activation markers CD5 and CD69, suggesting that SIT negatively regulates TCR-mediated signals at the CD4(+) CD8(+) stage of thymic development. This assumption is further supported by the observation that in female H-Y TCR transgenic mice, positive selection is enhanced and even converted to negative selection. Similarly, mature peripheral T cells are hyperresponsive towards TCR-mediated stimuli and produce larger amounts of T-helper 1 (TH1) cytokines, and SIT-deficient mice show an increased susceptibility to develop experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. These results demonstrate that SIT is a critical negative regulator of TCR-mediated signaling and finely tunes the signals required for thymic selection and peripheral T-cell activation.

Critical Roles for Transcription Factor GATA-3 in Thymocyte Development

The transcription factor GATA-3 is expressed at every stage of thymic development, but its role in thymocyte differentiation is unknown. The fact that RAG chimeric animals lacking GATA-3 cannot generate early thymocytes from common lymphoid progenitors has thus far precluded investigation of the function of GATA-3 in the thymus. To address this, we generated mice deficient in GATA-3 at early and late stages of thymic differentiation. Our studies revealed that GATA-3 is involved in β selection and is indispensable for single-positive CD4 thymocyte development. Thus, our data demonstrate that the coordinated and regulated expression of GATA-3 at each stage of thymic development is critical for the generation of mature T cells.

Antigen recognition and differentiation of T lymphocytes

During their intrathymic development, T lymphocytes acquire the ability to recognize myriads of antigens bound to self major histocompatibility molecules. This property is linked to the activation at early stages of thymic development of the biosynthesis of the antigen recognition receptors, the T cell receptors. It is also the consequence of intrathymic selective processes, that will favor maturation of thymocytes interacting with self histocompatibility products while preventing production of self-reactive lymphocytes. Here will be detailed the main intrathymic developmental stages and the mechanisms leading to the generation and intrathymic selection of the T cell repertoire.

Defective depletion of CD45-null thymocytes by the Staphylococcus aureus Enterotoxin B superantigen

The development of a normal T-cell repertoire is critically dependent on the negative and positive selection events which occur at the CD4 +CD8 + (double positive, DP) stage of thymic development. Depending on the avidity of the T-cell antigen receptor (TCR) for peptides presented within the thymus, DP thymocytes are either positively selected for maturation to CD4 +CD8 + single positive cells or are depleted by apoptosis. The addition of superantigen to thymocytes within foetal thymic organ culture (FTOC) mimics the negative selection signal of potentially autoreactive thymocytes and induces the responding population of thymocytes to apoptose. Here we present evidence that the transmembrane phosphotyrosine phosphatase CD45 critically regulates TCR-induced signals in thymic differentiation and present data to show defective depletion of CD45-null transgenic TCR-Vβ8 DP thymocytes in FTOC by the Staphylococcus aureus Enterotoxin B (SEB) superantigen.

Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells.

The ability of thymocytes to express cytokine genes changes during the different stages of thymic development. Although CD4- CD8- thymocytes are able to produce a wide spectrum of cytokines in response to a T-cell receptor (TcR)-independent stimulus, as they approach the double-positive (DP) CD4+ CD8+ stage, they lose the ability to produce cytokine. After the DP stage, thymocytes become single-positive CD4+ or CD8+ thymocytes which reacquire the ability to secrete cytokines. In an attempt to understand the molecular basis of this specific regulatin, we use AP-1-luciferase and newly generated NFAT-luciferase transgenic mice to analyze the transcriptional and DNA-binding activities of these two transcription factors that are involved in the regulation of cytokine gene expression. Here, we show that both AP-1 and NFAT transcriptional activities are not inducible in the majority of DP cells but that during the differentiation of DP cells to the mature single-positive stage, thymocytes regain this inducibility. Subpopulation analysis demonstrates that this inducibility is reacquired at the DP stage before the down-modulation of one of the coreceptors. Indeed AP-1 inducibility, just like the ability to express the interleukin-2 gene, is reacquired during the differentiation of DP TcRlow CD69low heat-stable antigen (HSA)high thymocytes to DP TcRhigh CD69high HSAhigh cells, which is considered to be the consequence of the first signal that initiates positive selection. We therefore propose that the inability of DP thymocytes to induce AP-1 and NFAT activities is one of the causes for the lack of cytokine gene expression at this stage and that this inducibility is reacquired at the latest stage of DP differentiation as a consequence of positive selection. This could be a mechanism to prevent the activation of DP thymocytes before selection has taken place.

Sublethal gamma-radiation induces differentiation of CD4-/CD8- into CD4+/CD8+ thymocytes without T cell receptor beta rearrangement in recombinase activation gene 2-/- mice.

DNA recombination of the immunoglobulin (Ig) or T cell receptor (TCR) gene loci is an essential step in the production of lymphocytes bearing antigen-specific receptors. Mice that lack the ability to rearrange their Ig and TCR gene loci are devoid of mature B and T cells. Complete rearrangement and expression of the TCR-beta chain has been suggested to allow immature thymocytes to switch from the CD4-/CD8- to the CD4+/CD8+ stage of thymic development. Thus, thymocytes from severe combined immune deficient (SCID) mice or mice deficient in recombinase activation genes (RAG), which do not undergo proper DNA rearrangement, are arrested at the early CD4-/CD8- stage of development. B cell precursors in SCID or RAG mice do not progress from the B220+/sIgM-/heat stable antigen (HSA)+/CD43+ to the B220+/sIgM-/HSA+/CD43- stage. In an attempt to reconstitute RAG-2-/- mice with bone marrow- or fetal liver-derived progenitor cells, we subjected these mice to sublethal doses of gamma-radiation. It is surprising that in the absence of donor cells, irradiated RAG-2-/- mice revealed a dramatic change in their lymphoid phenotype. 14 d after irradiation, the majority of thymocytes had advanced to the CD4+/CD8+ stage of T cell development and a small number of bone marrow precursors had progressed to the CD43-, HSAhi stage of B cell development. Analysis of the resulting CD4+/CD8+ thymocytes revealed no surface expression of the TCR/CD3 complex and no V-D-J rearrangement of the TCR-beta gene locus. Our findings provide evidence for a novel pathway that allows the transition of thymocytes from the CD4-/CD8- to the CD4+/CD8+ stage and that does not appear to require TCR-beta chain rearrangement.

Clonal deletion of V beta 5+ T cells by transgenic I-E restricted to thymic medullary epithelium.

A variety of cell types expressing MHC class II molecules is known to function as APC in vitro. We employed the Ig kappa gene enhancer and promoter to target the class II E alpha gene, and thereby I-E, exclusively to B cells to address their APC function in vivo. Although transgenic I-E was expressed on B lymphocytes, we unexpectedly obtained I-E on thymic medullary epithelium but not macrophages and at low frequency on dendritic cells. Using these transgenic mice, we constructed bone marrow irradiation chimeras with I-E expressed only on medullary epithelium, in order to determine the role of this cell type in tolerance by clonal deletion in the thymus. Although it is accepted that bm-derived cells play a primary role in deletion, and thymic epithelium can delete clones to a lesser degree, the role of cortical vs medullary thymic epithelium has not been directly dissected. We demonstrate that medullary epithelium alone can tolerize by partial deletion of I-E-reactive V beta 5+ T cells. These results indicate a role for medullary epithelium in deletion during the later stages of thymic development, and support the notion that positive and negative selection of developing T cells can occur in distinct temporal and anatomic compartments.

T Cell receptor rearrangements in various S49 lymphoma sublines

The S49 cell lines are a unique series of tumor sublines isolated from a single BALB/c thymoma. Several different sublines were previously isolated from non-mutagenized cells using pharmacologie agents that would select for different stages of thymic development. In this report we show that all seven of the sublines studied express TL class I Ag confirming their derivation from immature thymocytes. This uniform TL expression is in contrast to the previously characterized locus-specific shut off of K d,D d, and/or L dAg by various S49 sublines. Furthermore, S49 sublines were found to display disparate CD4/CD8 expression. Whereas the unselected subline is a CD4 +/CD8 + double positive, each of the selected sublines is singly positive for either CD4 or CD8. All seven sublines were found to be CD3 + and express αβ TCR heterodimers. To establish whether the S49 sublines have a monoclonal or polyclonal origin, their TCR rearrangements were compared. Based on the detection of identical but unusual TCR γ rearrangements and similarity of the a and β rearrangements, we propose that the S49 sublines probably had a monoclonal origin. However, significant differences between the TCR a and β gene rearrangement were observed, suggesting that these sublines have undergone further differentiation at TCR loci in addition to CD4/CD8 and MHC loci. Evidence is presented that much of this phenotypic diversity preceded their in vitro selection.

LYMPHOCYTE FORMATION IN THE THYMUS OF THE EMBRYONIC CHICK.

AbstractEmbryonic thymus provides a distinct advantage over the definitive thymus because the earliest formation of lymphocyte and lymphocytic precursors can be observed in a relatively simple and uncomplicated situation. Special cytological techniques combined with light, phase, and electron microscopy have been performed in this investigation involving over 150 chick embryos between 5 and 18 days of embryonic development.Sequential cytological changes in the development of the thymus into an active lymphocytopoietic organ indicate that lymphoblasts develop by the gradual proliferation and transformation of “undifferentiated” epithelial cells comprising the primordial thymus. Lymphoblastic transformation begins on the seventh day of embryonic development in the chick and is characterized by increased cytoplasmic and nucleolar basophilia and chromatin condensation. “Undifferentiated” epithelial cells undergo two distinct lines of differentiation between the seventh and tenth days: into lymphoblasts and into stellate reticular‐epithelial cells which constitute the organ parenchyma. All stages of lymphocytic maturation may be observed by 10–11 days as the thymus assumes a predominately lymphocytic character. Absence of lymphocytes or lymphocytic precursors in the connective tissue surrounding the embryonic thymus before and during the period of initial lymphoblastic transformation; presence of a continuous basement membrane surrounding the developing thymus; and absence of cells passing through this membrane during this phase of development indicate that the lymphocytic elements appearing in the embryonic thymus parenchyma are of epithelial rather than mesenchymal derivation. Although a contribution of mesenchymal elements to the lymphocytic population via vascular invasion and lobular formation in later stages of thymic development is unlikely, this question cannot be answered at this time. In spite of functional immunological distinctions between the lymphocytes of the chick thymus and bursa of Fabricius, the similarity of origin of the lymphocytic elements of these lympho‐epithelial organs is apparent.