Mature Single-positive CD4 Research Articles

In Vivo Modeling of T-Cell Acute Lymphoblastic Leukemia Reveals Synergistic Oncogenic Pathways and Bcl11b Haploinsufficiency As a Potential Therapeutic Vulnerability

For decades, T-ALL models were based on retroviral overexpression of NOTCH1 in murine hematopoietic stem cells (HSCs). This induces aggressive T-ALL but does not reflect the genetic and clinical heterogeneity of the disease. Large-scale genomic analyses have mapped the detailed landscape of T-ALL driver events over recent years, but how these diverse oncogenic events interact in T-ALL pathogenesis and which vulnerabilities this creates remains incompletely explored. This is in part due to the lack of appropriate mouse models that recapitulate the complex disease biology and are suitable to identify and study potential therapeutic targets and interventions in a more realistic context. We modeled the genetic heterogeneity of T-ALL through in vivo multiplexing of gain-of-function and loss-of-function oncogenic events known to drive the disease. We developed a novel mouse strain that expresses Cre-recombinase and an inducible Cas9 only in the thymus ( LSL.Cas9 x Lck-cre) along with a lentiviral vector system into which T-ALL specific oncogenes are cloned in antisense flanked by LOX66/71 sites. Gain-of-function events, such as the overexpression of transcriptional regulators ( Tlx1, Tal1, or Lmo2) and signal transducers ( NRAS G12D, PIK3CD E1021K) were induced in developing thymocytes by Cre-mediated inversion together CRISPR-Cas9 editing of recurrent T-ALL loss-of-function drivers. This combinatorial approach allowed to probe more than 2000 possible oncogenic mutational combinations in vivo. Transplantation of HSCs from LSL.Cas9 x Lck-cre mice transduced with our vector system into sublethally irradiated mice led to T-ALL development with different morphology and disease phenotypes. Developing leukemia harbored reproducible phenotypes, covering very immature CD4-CD8-CD25+CD44+ T-ALL in the Tlx1_NRAS subgroup, in contrast to the classical cortical CD4+CD8+CD3- phenotype (Tlx1_PIK3CD subgroup), as well as mature single-positive CD4+CD3+ or CD8+CD3+ blasts (Tal1_Lmo2 subgroup). We identified Tlx1, Tal1, Lmo2, NRAS G12Dand PIK3CD E1021Kas the main driver of disease phenotype and gene expression profile, whereas the loss-of-function mutations contributed to and accelerated the onset of the disease. Tumor-derived cell lines could be easily established and kept their phenotypic characteristics and mutation patterns. The resulting T-ALL tumors contained up to eight different disease-relevant genetic alterations, thus recapitulating the genetic complexity in humans and overcoming the long latency for spontaneous mutations in less complex mouse models. Among these, co-evolution of insertions and deletions in Notch1, Cdkn2a, Bcl11b, and Pten were among the most frequently observed mutations in definitive T-ALL, whereas others, e.g., in Dnm2, Phf6, Etv6, and Lef1, occurred more randomly. While gene editing by CRISPR-Cas9 mostly resulted in frame-shift mutations in both alleles, Bcl11b exhibited a strong haploinsufficient phenotype with exactly one knock-out and one intact allele in each case examined. Following this observation, further suppression of Bcl11b by shRNAs in Bcl11b +/- cell lines derived from our tumor models demonstrated rapid induction of cell death, which was more intense in T-ALL cells compared to non-malignant thymocytes. In addition, re-analysis of CRISPR-based loss-of-function screens of human hematopoietic cell lines retrieved from the Cancer Dependency Map confirmed BCLL11B as a strong selective dependency in human T-ALL. In summary, we model subgroup-specific T-ALL transformation through conditional and multiplexed in vivo gene editing and oncogene overexpression, thus creating a resource of novel mouse models that recapitulate the genetic and biological heterogeneity of the disease. Using our resource, we identified Bcl11b as a strong and selective context-specific dependency in T-ALL. Although Bcl11b is a transcription factor, our results could serve as an starting point for novel therapies that interfere with Bcl11b function, such as molecular glue degraders or protein interaction inhibitors.

Thymic-specific regulation of TCR signaling by Tespa1.

Double-positive (DP) thymocytes undergo positive selection to become mature single-positive CD4+ and CD8+ T cells in response to T cell receptor (TCR) signaling. Unlike mature T cells, DP cells must respond to low-affinity self-peptide-MHC ligands before full upregulation of their surface TCR expression can occur. Thus, DP thymocytes must be more sensitive to ligands than mature T cells. A number of molecules have been found that are able to enhance the strength of the TCR signal to facilitate positive selection. However, almost all of these molecules are also active in mature T cells. Themis (thymocyte expressed molecule involved in selection) and Tespa1 (thymocyte expressed positive selection associated 1) are two recently discovered molecules essential for optimal TCR signaling and thymocyte development. A deficiency in both molecules leads to defects in positive selection. Here, we compared the relative contributions of Themis and Tespa1 to positive selection in thymocytes. We show that Tespa1 deficiency led to more limited and specific gene expression profile changes in cells undergoing positive selection. In mixed bone marrow transfer experiments, Tespa1-/- cells showed more severe defects in thymocyte development than Themis-/- cells. However, Tespa1-/- cells showed a substantial degree of homeostatic expansion and became predominant in the peripheral lymphoid organs, suggesting that Tespa1 is a thymic-specific TCR signaling regulator. This hypothesis is further supported by our observations in Tespa1 conditional knockout mice, as Tespa1 deletion in peripheral T cells did not affect TCR signaling or cell proliferation. The different regulatory effects of Tespa1 and Themis are in accordance with their nonredundant roles in thymocyte selection, during which Tespa1 and Themis double knockouts showed additive defects.

Sphingosine-1-Phosphate Receptor 1 Is Involved in Non-Obese Diabetic Mouse Thymocyte Migration Disorders.

NOD (non-obese diabetic) mice spontaneously develop type 1 diabetes following T cell-dependent destruction of pancreatic β cells. Several alterations are observed in the NOD thymus, including the presence of giant perivascular spaces (PVS) filled with single-positive (SP) CD4+ and CD8+ T cells that accumulate in the organ. These cells have a decreased expression of membrane CD49e (the α5 integrin chain of the fibronectin receptor VLA-5 (very late antigen-5). Herein, we observed lower sphingosine-1-phosphate receptor 1 (S1P1) expression in NOD mouse thymocytes when compared with controls, mainly in the mature SP CD4+CD62Lhi and CD8+CD62Lhi subpopulations bearing the CD49e− phenotype. In contrast, differences in S1P1 expression were not observed in mature CD49e+ thymocytes. Functionally, NOD CD49e− thymocytes had reduced S1P-driven migratory response, whereas CD49e+ cells were more responsive to S1P. We further noticed a decreased expression of the sphingosine-1-phosphate lyase (SGPL1) in NOD SP thymocytes, which can lead to a higher sphingosine-1-phosphate (S1P) expression around PVS and S1P1 internalization. In summary, our results indicate that the modulation of S1P1 expression and S1P/S1P1 interactions in NOD mouse thymocytes are part of the T-cell migratory disorder observed during the pathogenesis of type 1 diabetes.

Expression patterns of Ikaros family members during positive selection and lineage commitment of human thymocytes.

The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8+ thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8+ developmental stage, some thymocytes re-express CD4 and become CD3hi double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4+ thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4+ T-cell maturation. For thymocytes progressing toward the CD8+ T-cell lineage, TSP CD8+ thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8+ developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.

A quantitative study of the mechanisms behind thymic atrophy in Gαi2-deficient mice during colitis development.

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2−/− mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2−/− mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2−/− mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2−/− SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4+ and CD8+ thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC+ T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2−/− mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis.

Gads‐deficient thymocytes are blocked at the transitional single positive CD4+ stage

Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4(+)CD8(+) double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4(+) thymocytes. Then, cells are selected to become either mature single positive CD4(+) or mature single positive CD8(+) thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads(+/+) mice, most TSP CD4(+) thymocytes are TCR(hi)Bcl-2(hi)CD69(+), suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads(-/-) mice could readily generate TSP CD4(+) thymocytes, many Gads(-/-) TSP CD4(+) cells were TCR(lo)Bcl-2(lo)CD69(-), suggesting that Gads(-/-) cells proceeded to the TSP CD4(+) stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4(+) thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4(+) stage are separable events and Gads is only required for positive selection.

Development of CD4 T cells from Human Hematopoietic stem Cells in a Lentiviral-engineered Stromal Culture (46.15)

Abstract Hematopoietic precursor/stem cells (HPCs) are progenitors to all functional immune cell lineages. We established a novel stromal cell line expressing (Delta-like 1) DL1, interleukin-7 (IL-7), and FMS-like tyrosine kinase 3 Ligand (Flt3-L) and demonstrated efficient differentiation and expansion of early T cells from adult CD34+ HPCs. However, continuous IL-7 signaling led to the production of immature CD8+ T lymphocytes at the expense of CD4+ T lymphocytes. It is known that the developing double positive T cells shut down IL-7 mediated signaling and require TCR signaling for survival and positive selection. We hypothesize that IL-7 inhibits the TCR signaling event required for positive selection. We found that by removal of IL-7, followed with CD3/CD28 stimulation, the early T cells were able to differentiate into mature single positive CD4 T cells expressing CD3, CD28, and TCRαβ. This can also be demonstrated by bypassing TCR signaling through stimulation with PMA/ionomycin. When further stimulated, these in vitro derived CD4+ T cells differentiated into various T helper subtypes and produced effector cytokines including IL-2, IFN-γ, and IL-17. This novel culture system can greatly facilitate the study of late T cell development and has potential for immunotherapeutic applications. Supported by Vectorite Biomedica Inc. and Yongling Foundation.

Aberrant T‐cell ontogeny and defective thymocyte and colonic T‐cell chemotactic migration in colitis‐prone Gαi2‐deficient mice

Galphai2-deficient mice, which spontaneously develop colitis, have previously been reported to have an increased frequency of mature, single positive thymocytes compared to wild-type mice. In this study we further characterized the intrathymic changes in these mice before and during overt colitis. Even before the onset of colitis, Galphai2(-/-) thymi weighed less and contained fewer thymocytes, and this was exacerbated with colitis development. Whereas precolitic Galphai2(-/-) mice had unchanged thymocyte density compared to Galphai2(+/-) mice of the same age, this was significantly decreased in mice with colitis. Thymic atrophy in Galphai2(-/-) mice involved mainly the cortex. Using a five-stage phenotypic characterization of thymocyte maturation based on expression of CD4, CD8, TCRalphabeta, CD69 and CD62L, we found that both precolitic and colitic Galphai2(-/-) mice had significantly increased frequencies of mature single-positive CD4(+) and CD8(+) medullary thymocytes, and significantly reduced frequencies and total numbers of immature CD4(+) CD8(+) double-positive thymocytes compared to Galphai2(+/-) mice. Furthermore, cortical and transitional precolitic Galphai2(-/-) thymocytes showed significantly reduced chemotactic migration towards CXCL12, and a trend towards reduced migration to CCL25, compared to wild-type thymocytes, a feature even more pronounced in colitic mice. This impaired chemotactic migration of Galphai2(-/-) thymocytes could not be reversed by increased chemokine concentrations. Galphai2(-/-) thymocytes also showed reduced expression of the CCL25 receptor CCR9, but not CXCR4, the receptor, for CXCL12. Finally, wild-type colonic lamina propria lymphocytes migrated in response to CXCL12, but not CCL25 and, as with thymocytes, the chemokine responsiveness was significantly reduced in Galphai2(-/-) mucosal lymphocytes.

CD28 in thymocyte development and peripheral T cell activation in mice exposed to suspended particulate matter

The CD28:B7 signaling pathway is very important for the activity of mature peripheral T lymphocytes and thymocyte development. The proper development of thymocytes into mature single positive CD4+and CD8+ T cells is crucial for almost all immune functions. In naturally occurring conditions, T cells maturation in the thymus is influenced by environmental agents. The expression of CD28 and the distribution of CD28(low/high) thymocytes have been examined at various stages of thymocyte development in BALB/c mice exposed to air-suspended particulate matter (ASM). Acute exposure to ASM resulted in the decrease of CD28 expression in the total thymocyte population. The increase of the percentage of CD28(low) and the decrease of CD28(high) thymocytes were observed, which may account for the acceleration of thymocyte development under the conditions of elevated risk resulting from the exposure of animals to environmental xenobiotics. ASM exposure resulted in the increase of the level of proliferation of lymph node T cells induced by anti-CD3 and anti-CD28 monoclonal antibodies activation despite normal expression of CD28 molecule. In contrast, the level of proliferation of spleen T cells was lowered or normal dependently of the concentration of stimuli used for activation. Results of these studies demonstrate that acute exposure of mice to ASM can result in the progression of two contrasting processes in the immune system: upregulation of thymocyte development, which contributes to the maintenance of peripheral T cell pool, and over-activation of lymph node lymphocytes, which may lead to uncontrolled immunostimulation.

CD69 down‐modulation and inhibition of thymic egress by short‐ and long‐term selective chemical agonism of sphingosine 1‐phosphate receptors

Thymic development requires proliferation, selection, maturation and release of mature single-positive CD4 and CD8 T cells into the periphery. In mice, non-selective sphingosine-1 phosphate (S1P) receptor agonists, active on four of the five known S1P receptors, alter thymocyte phenotype and egress. Here, we show that down-modulation of CD69 occurs acutely and transiently at a discrete and late stage of medullary development after a single-dose administration of S1P(1) receptor-selective agonist, which induces long-term tonic receptor activation in the absence of receptor degradation. In addition, agonist acutely inhibited egress of mature thymocytes into peripheral lymphoid organs, suggesting that both the phenotype and migration of medullary thymocytes are regulated simultaneously and coordinately by agonism of S1P(1) alone. Long-term dosing shifted the early/late medullary thymocyte ratio with an expansion of the late medullary compartment, as mature CD69(-) thymocytes were retained within the thymus. Therefore, chemical agonism of S1P(1) accelerates medullary phenotypic maturation and inhibits egress, leading to the expansion and accumulation of the recent thymocyte emigrant population in the medulla. However, chemical agonism fails to replicate the S1P(1)-null CD69(hi) late medullary phenotype, suggesting that agonism and gene deletion operate by distinct mechanisms, and that functional receptor antagonism may not be required for lymphocyte sequestration.

A CXCR4-Dependent Chemorepellent Signal Contributes to the Emigration of Mature Single-Positive CD4 Cells from the Fetal Thymus

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62L(high) and CD69(low) recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.

CXCR4-Dependent Repulsion of Mature Single-Positive CD4 Cells Mediates Emigration from Thymus.

Developing thymocytes undergo maturation during their passage through the cortex to the medulla of the thymus before emigrating to peripheral lymphoid organs. Chemokines control T cell migration within and away from the thymus. We have previously shown that the chemokine SDF-1, produced by thymic stroma, repels mature thymocytes away from thymus via a CXCR4 mediated mechanism. We proposed that CXCR4 plays a specific role in thymic emigration. We evaluated the emigration of mature thymocytes away from thymus in a modified fetal thymic organ culture (FTOC) system in fetal thymi from CXCR4+/− (Control) and CXCR4 −/− (KO) mice at day 15.5. Fetal thymic emigrants were collected using a transwell system with a polycarbonate membrane (3 μm pore size). The immunophenotype of thymic emigrants was distinct from that of thymocytes resident in the thymus in both control and CXCR4-KO thymi. From day 9 of culture onwards, only mature SP cells were able to move away from thymus into the lower chamber of the transwell. Only SP CD4 thymocytes expressing CD62Lhigh were found among emigrants, whereas two distinct populations of SP CD8 cells expressing CD62Lhigh and CD62Llow were present in the emigrant fraction. Incubation of FTOC with pertussis toxin significantly inhibited emigration of mature SP thymocytes out of the thymus in control CXCR4 mice (p=0.002). The total number of emigrants collected from CXCR4-KO thymi was always significantly reduced compared to emigrants from control thymi. In addition, the total number of SP CD4 emigrants from CXCR4-KO thymi were strikingly reduced in comparison to control SP CD4 emigrants (p=0.035). There was no statistically significant difference in the maturation of intrathymic thymocytes from CXCR4-KO as compared to control FTOC. Analysis of CD62L expression in CXCR4-KO thymocytes showed retention of SP CD4 cells in the thymus with the immunophenotypic characteristic normally associated with recent thymic emigrants (p<0.05). Treatment of FTOC with the specific CXCR4 antagonist, AMD3100, significantly increased the total number of SP CD4 cells retained in the thymus compared to untreated FTOC (p=0.002). Treatment with AMD3100 significantly reduced the absolute number of emigrants compared to untreated FTOC (p=0.015) and again SP CD4 cells were most affected in their ability to migrate away from the thymus. Treatment of FTOC with sphingosine-1-phosphate (S1P) resulted in an increase in the emigration of SP CD4 cells from CXCR4-KO thymi (p=0.02). In conclusion these data support the view that CXCR4-mediated repulsion of mature thymocytes away from the medullary chemokine SDF-1, termed fugetaxis, is a key mechanism controlling T cell egress from the thymus. These data support the existence of a CXCR4 dependent mechanism governing thymic emigration which may itself have S1P dependent and/or independent components.

Generation of CD3+CD8low thymocytes in the HIV type 1-infected thymus.

Infection with the HIV type 1 (HIV-1) can result both in depletion of CD4(+) T cells and in the generation of dysfunctional CD8(+) T cells. In HIV-1-infected children, repopulation of the peripheral T cell pool is mediated by the thymus, which is itself susceptible to HIV-1 infection. Previous work has shown that MHC class I (MHC I) molecules are strongly up-regulated as result of IFN-alpha secretion in the HIV-1-infected thymus. We demonstrate in this study that increased MHC I up-regulation on thymic epithelial cells and double-positive CD3(-/int)CD4(+)CD8(+) thymocytes correlates with the generation of mature single-positive CD4(-)CD8(+) thymocytes that have low expression of CD8. Treatment of HIV-1-infected thymus with highly active antiretroviral therapy normalizes MHC I expression and surface CD8 expression on such CD4(-)CD8(+) thymocytes. In pediatric patients with possible HIV-1 infection of the thymus, a low CD3 percentage in the peripheral circulation is also associated with a CD8(low) phenotype on circulating CD3(+)CD8(+) T cells. Furthermore, CD8(low) peripheral T cells from these HIV-1(+) pediatric patients are less responsive to stimulation by Ags from CMV. These data indicate that IFN-alpha-mediated MHC I up-regulation on thymic epithelial cells may lead to high avidity interactions with developing double-positive thymocytes and drive the selection of dysfunctional CD3(+)CD8(low) T cells. We suggest that this HIV-1-initiated selection process may contribute to the generation of dysfunctional CD8(+) T cells in HIV-1-infected patients.

Interleukin-7 and infection itself by human immunodeficiency virus 1 favor virus persistence in mature CD4+CD8−CD3+ thymocytes through sustained induction of Bcl-2

The sequence of events and the mechanisms leading to the destruction of the thymus during human immunodeficiency virus (HIV) infection are still poorly characterized. Investigated here are the survival capacity on HIV-1 infection of the mature single-positive CD4(+)CD8(-)CD3(+) (SP CD4(+)) and the intermediate CD4(+) CD8(-)CD3(-) thymocytes previously shown to be able to replicate the virus in the thymic microenvironment. It is demonstrated that the mature SP CD4(+) thymocytes exhibit a high survival capacity despite the production of a high yield of viruses. Interleukin-7, reported to be a crucial cofactor of tumor necrosis factor (TNF) to promote HIV replication, is shown here to counteract the apoptotic activity of TNF. Resistance to apoptosis of SP CD4(+) cells is conferred by a high expression of the IL-7 receptor (IL-7R) associated with the capacity of IL-7 to permanently up-regulate Bcl-2. In addition, this high Bcl-2 level is further enhanced by infection itself. In contrast, intermediate thymocytes, which replicate the virus at a lower level, are more sensitive to apoptosis, and their differentiation into double-positive CD4(+)CD8(+)CD3(-) (DP CD3(-)) cells strongly increases their death rate on infection. This sensitivity is related to a lower expression of IL-7R and Bcl-2 in intermediate thymocytes, which further decreases at the DP CD3(-) stage. In addition, a decreased level of Bcl-2 is observed in this subset during infection. Altogether these data suggest that in vivo, HIV infection might create a persistent virus reservoir within the SP CD4(+) thymocytes, whereas the later infection of intermediate cells might lead to thymopoiesis failure.

Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.

Fas Is Expressed Early in Human Thymocyte Development But Does Not Transmit an Apoptotic Signal

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders.

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.