Subtle defects in pre-TCR signaling in the absence of the Tec kinase Itk

alphabeta T cell development in the thymus is dependent on signaling through the TCR. The first of these signals is mediated by the pre-TCR, which is responsible for promoting pre-T cell proliferation and the differentiation of CD4(-)8(-)3(-) (DN) thymocytes into CD4(+)8(+)3(+) (DP) cells. In many cases, T cell signaling proteins known to be essential for TCR signaling in mature T cells are also required for pre-TCR signaling in DN thymocytes. Therefore, it came as a surprise to discover that mice lacking the Tec kinases Itk and Rlk, enzymes required for efficient activation of phospholipase C-gamma1 in mature T cells, showed no obvious defects in pre-TCR-dependent selection events in the thymus. In this report, we demonstrate that DN thymocytes lacking Itk, or Itk and Rlk, are impaired in their ability to generate normal numbers of DP thymocytes, especially when placed in direct competition with WT DN thymocytes. We also show that Itk is required for maximal pre-TCR signaling in DN thymocytes. These data demonstrate that the Tec kinases Itk and Rlk are involved in, but are not essential for, pre-TCR signaling in the thymus, suggesting that there is an alternative mechanism for activating phospholipase C-gamma1 in DN thymocytes that is not operating in DP thymocytes and mature T cells.     

Early ontogeny of thymocytes in pigs: sequential colonization of the thymus by T cell progenitors

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3epsilon-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3epsilonhigh and belonged to the gammadelta T cell lineage. Mature CD3epsilonhigh alphabeta thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3epsilonlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Ralpha (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRgammadelta+ thymocytes that were cycling late during their development in the thymus.     

Early TCR alpha beta expression promotes maturation of T cells expressing Fc epsilon RI gamma containing TCR/CD3 complexes

In a previous study we presented data indicating that the expanded population of CD4(-)CD8(-) (DN) alphabeta T cells in TCRalpha-chain-transgenic mice was partially if not entirely derived from gammadelta T cell lineage cells. The development of both gammadelta T cells and DN alphabeta T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN alphabeta T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRalpha-chain is expressed by precursor thymocytes already at the CD3(-)CD4(-)CD8(-) (triple negative, TN) CD44(+)CD25(-) stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRbeta-chains matured into DN alphabeta T cells at several stages of development. The genes encoding the gamma-chain of the high affinity receptor for IgE (FcepsilonRIgamma) and the CD3zeta protein were found to be reciprocally expressed in TN thymocytes such that during development the FcepsilonRIgamma expression decreased whereas CD3zeta expression increased. Furthermore, in a fraction of the transgene-induced DN alphabeta T cells the FcepsilonRIgamma protein colocalized with the TCR/CD3 complex. These data suggest that similarly to gammadelta T cells and NKT cells, precursors expressing the TCR early in the common alphabetagammadelta developmental pathway may use the FcepsilonRIgamma protein as a signaling component of the TCR/CD3 complex.     

The TCR C beta FG loop regulates alpha beta T cell development

The TCRbeta chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an alphabeta TCR cavity against which CD3epsilongamma may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15alphabeta TCR transfectants expressing a FG loop-deleted chain (betaDeltaFG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type beta-chain (betawt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3epsilongamma heterodimer and the beta-chain in TCR complexes containing the betaDeltaFG variant. To further investigate the biologic role of the Cbeta FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2-/- mice using bone marrow cells from betawt or betaDeltaFG transgenic C57BL/6 (B6) mice. Both betawt and betaDeltaFG precursor cells generate thymocytes representative of all maturational stages. However, betaDeltaFG-expressing thymocytes dominate during subsequent development, resulting in an excess of betaDeltaFG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique Cbeta FG loop appendage primarily controls alphabeta T cell development through selection processes.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

A NK1.1+ thymocyte-derived TCR beta-chain transgene promotes positive selection of thymic NK1.1+ alpha beta T cells

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR beta-chain derived from conventional alphabeta T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion alpha-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a beta-chain derived from nonclassical, NK1.1+ alphabeta T cells, the thymus-derived, CD1. 1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 beta Tg thymocytes revealed that the frequency of the parental alpha-chain sequence, in this instance the Valpha14-Jalpha281 canonical alpha-chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1+ alphabeta T cells in DN32H6 beta Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR beta-chain from a thymus-derived NK1.1+ Valpha14+ T cell hybridoma promotes positive selection of thymic NK1.1+ alphabeta T cells. These observations indicate that the strong influence of productive beta-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical alphabeta T cells and therefore represents a general phenomenon in intrathymic alphabeta T lymphocyte development.     

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.     

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.     

Transgenic expression of the p16(INK4a) cyclin-dependent kinase inhibitor leads to enhanced apoptosis and differentiation arrest of CD4-CD8- immature thymocytes

In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16(INK4a) is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16(Ink4a) controls thymocyte development by analyzing transgenic mice expressing the human p16(INK4a) into the T cell lineage. We show that forced expression of p16(INK4a) in thymocytes blocked T cell differentiation at the early CD4-CD8-CD3-CD25+ stage without significantly affecting the development of gammadelta T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2(-/-)). Upon anti-CD3epsilon treatment in vivo, p16(INK4a)-expressing RAG-2(-/-) thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16(INK4a) prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.     

Phosphoinositide-dependent kinase l (PDK1) haplo-insufficiency inhibits production of alpha/beta (alpha/beta) but not gamma delta (gamma/delta) T lymphocytes

In the present study, we have explored the impact of deleting a single allele of PDK1 in T cell progenitors on alpha/beta and gamma/delta T cell development. The data show that deleting a single allele of PDK1 allows differentiation of alpha/beta T cells but prevents their proliferative expansion in the thymus. Accordingly, mice with T cells that are haplo-insufficient for PDK1 have reduced numbers of thymocytes and alpha/beta peripheral T cells. T cell progenitors also give rise to gamma/delta T cells but in contrast to the loss of alpha/beta T cells in T-PDK1 null and haplo-insufficient mice, there were increased numbers of gamma/delta T cells. The production of alpha/beta T cells is dependent on the proliferative expansion of thymocytes and is determined by a balance between the frequency with which cells enter the proliferative phase of the cell cycle and rates of cell death. Herein, we show that PDK1 haplo-insufficient thymocytes have no defects in their ability to enter the cell cycle but show increased apoptosis. PDK1 thus plays a determining role in the development of alpha/beta T lymphocytes but does not limit gamma/delta T cell development.     

Signal-transducing adaptor molecules STAM1 and STAM2 are required for T-cell development and survival

We previously reported that the STAM family members STAM1 and STAM2 are phosphorylated on tyrosine upon stimulation with cytokines through the gammac-Jak3 signaling pathway, which is essential for T-cell development. Mice with targeted mutations in either STAM1 or STAM2 show no abnormality in T-cell development, and mice with double mutations for STAM1 and STAM2 are embryonically lethal; therefore, here we generated mice with T-cell-specific double mutations for STAM1 and STAM2 using the Cre/loxP system. These STAM1(-/-) STAM2(-/-) mice showed a significant reduction in thymocytes and a profound reduction in peripheral mature T cells. In proliferation assays, thymocytes derived from the double mutant mice showed a defective response to T-cell-receptor (TCR) stimulation by antibodies and/or cytokines, interleukin-2 (IL-2) and IL-7. However, signaling events downstream of receptors for IL-2 and IL-7, such as activations of STAT5, extracellular signal-regulated kinase (ERK), and protein kinase B (PKB)/Akt, and c-myc induction, were normal in the double mutant thymocytes. Upon TCR-mediated stimulation, prolonged activations of p38 mitogen-activated protein kinase and Jun N-terminal protein kinase were seen, but activations of ERK, PKB/Akt, and intracellular calcium flux were normal in the double mutant thymocytes. When the cell viability of cultured thymocytes was assessed, the double mutant thymocytes died more quickly than controls. These results demonstrate that the STAMs are indispensably involved in T-cell development and survival in the thymus through the prevention of apoptosis but are dispensable for the proximal signaling of TCR and cytokine receptors.     

In vivo overexpression of Dad1, the defender against apoptotic death-1, enhances T cell proliferation but does not protect against apoptosis.

The Dad1 protein has been shown to play a role in prevention of apoptosis in certain cell types. Dad1 is also a subunit of the oligosaccharyltransferase enzyme complex that initiates N-linked glycosylation. It is encoded by a gene located adjacent to the TCR alpha and delta genes on mouse chromosome 14. We have investigated the role of Dad1 during T cell development and activation. We observe that endogenous Dad1 levels are modulated during T cell development to reach maximal expression in mature thymocytes. Transgenic mice that overexpress Dad1 in both the thymus and peripheral immune system have been generated. Apoptosis of thymocytes from such mice is largely unaffected, but peripheral T cells display hyperproliferation in response to stimuli. Therefore, the linkage between the TCR and Dad1 genes may have important consequences for T cell function.     

Absence of programmed death receptor 1 alters thymic development and enhances generation of CD4/CD8 double-negative TCR-transgenic T cells

Programmed death receptor 1 (PD-1) is expressed on thymocytes in addition to activated lymphocyte cells. Its ligation is thought to negatively regulate T cell activation, and PD-1(-/-) mice develop autoimmunity. To study the role of PD-1 on the development and function of a monoclonal CD8(+) T cell population, 2C TCR-transgenic/recombination-activating gene 2(-/-)/PD-1(-/-) mice were generated. Unexpectedly, approximately 30% of peripheral T cells in these mice were CD4/CD8 double negative (DN). Although the DN cells were not activated by Ag-expressing APCs, they functioned normally in response to anti-CD3/anti-CD28. These cells had a naive surface phenotype and lacked expression of NK1.1, B220, and gammadelta TCR; and the majority did not up-regulate CD8alphaalpha expression upon activation, arguing that they are not predominantly diverted gammadelta-lineage cells. The thymus was studied in detail to infer the mechanism of generation of DN peripheral T cells. Total thymus cellularity was reduced in 2C TCR-transgenic/recombination-activating gene 2(-/-)/PD-1(-/-) mice, and a relative increase in DN cells and decrease in double-positive (DP) cells were observed. Increased annexin V(+) cells among the DP population argued for augmented negative selection in PD-1(-/-) mice. In addition, an increased fraction of the DN thymocytes was HSA negative, suggesting that they had undergone positive selection. This possibility was supported by decreased emergence of DN PD-1(-/-) 2C cells in H-2(k) bone marrow chimera recipients. Our results are consistent with a model in which absence of PD-1 leads to greater negative selection of strongly interacting DP cells as well as increased emergence of DN alphabeta peripheral T cells.     

WDR55 is a nucleolar modulator of ribosomal RNA synthesis, cell cycle progression, and teleost organ development

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.