Evidence that gammadelta versus alphabeta T cell fate determination is initiated independently of T cell receptor signaling

Two types of T cells, alphabeta and gammadelta, develop in vertebrates. How these two T cell lineages arise from a common thymic T progenitor is poorly understood. Differentiation of alphabeta lineage T cells requires the surrogate alpha chain (pTalpha), which associates with the T cell receptor (TCR) beta chain to form the pre-TCR. gammadelta lineage development does not appear to involve an obligatory surrogate chain, but instead requires productive rearrangement and expression of both TCR gamma and delta genes. It has been proposed that the quality of signals transmitted by the pre-TCR and gammadelta TCR are distinct and that these "instructive" signals determine the lineage fate of an uncommitted progenitor cell. Here we show that the thymic T progenitor cells (CD25(+)CD44(+)c-kit(+)CD3(-)CD4(-)CD8(-) thymocytes, termed pro-T cells) from young adult mice that have yet to express TCRs can be subdivided based on interleukin 7 receptor (IL-7R) expression. These subsets exhibit differential potential to develop into gammadelta versus alphabeta lineage (CD4+CD8+ cells) in the thymus. Upon intrathymic injection, IL-7R(neg-lo) pro-T cells generated a 13-fold higher ratio of alphabeta lineage to gammadelta lineage cells than did IL-7R(+) pro-T cells. Much of this difference was due to a fivefold greater potential of IL-7R(+) pro-T cells to develop into TCR-gammadelta T cells. Evidence indicates that this biased developmental potential is not a result of enhanced TCR-gamma gene rearrangement/expression in IL-7R(+) pro-T cells. These results indicate that the pro-T cells are heterogeneous in developmental potential before TCR gene rearrangement and suggest that in some precursor cells the initial lineage commitment is independent of TCR-mediated signals.     

Defined alphabeta T cell receptors with distinct ligand specificities do not require those ligands to signal double negative thymocyte differentiation

During T cell development in the thymus, pre-T cell receptor (TCR) complexes signal CD4(-) CD8(-) (double negative [DN]) thymocytes to differentiate into CD4(+) CD8(+) (double positive [DP]) thymocytes, and they generate such signals without apparent ligand engagements. Although ligand-independent signaling is unusual and might be unique to the pre-TCR, it is possible that other TCR complexes such as alphabeta TCR or alphagamma TCR might also be able to signal the DN to DP transition in the absence of ligand engagement if they were expressed on DN thymocytes. Although alphagamma TCR complexes efficiently signal DN thymocyte differentiation, it is not yet certain if alphabeta TCR complexes are also capable of signaling DN thymocyte differentiation, nor is it certain if such signaling is dependent upon ligand engagement. This study has addressed these questions by expressing defined alphabeta TCR transgenes in recombination activating gene 2(-/-) pre-Talpha(-/-) double deficient mice. In such double deficient mice, the only antigen receptors that can be expressed are those encoded by the alphabeta TCR transgenes. In this way, this study definitively demonstrates that alphabeta TCR can in fact signal the DN to DP transition. In addition, this study demonstrates that transgenic alphabeta TCRs signal the DN to DP transition even in the absence of their specific MHC-peptide ligands.     

Premature expression of T cell receptor (TCR)alphabeta suppresses TCRgammadelta gene rearrangement but permits development of gammadelta lineage T cells

The T cell receptor (TCR)gammadelta and the pre-TCR promote survival and maturation of early thymocyte precursors. Whether these receptors also influence gammadelta versus alphabeta lineage determination is less clear. We show here that TCRgammadelta gene rearrangements are suppressed in TCRalphabeta transgenic mice when the TCRalphabeta is expressed early in T cell development. This situation offers the opportunity to examine the outcome of gammadelta versus alphabeta T lineage commitment when only the TCRalphabeta is expressed. We find that precursor thymocytes expressing TCRalphabeta not only mature in the alphabeta pathway as expected, but also as CD4(-)CD8(-) T cells with properties of gammadelta lineage cells. In TCRalphabeta transgenic mice, in which the transgenic receptor is expressed relatively late, TCRgammadelta rearrangements occur normally such that TCRalphabeta(+)CD4(-)CD8(-) cells co-express TCRgammadelta. The results support the notion that TCRalphabeta can substitute for TCRgammadelta to permit a gammadelta lineage choice and maturation in the gammadelta lineage. The findings could fit a model in which lineage commitment is determined before or independent of TCR gene rearrangement. However, these results could be compatible with a model in which distinct signals bias lineage choice and these signaling differences are not absolute or intrinsic to the specific TCR structure.     

T cell receptor-instructed alphabeta versus gammadelta lineage commitment revealed by single-cell analysis

alphabeta and gammadelta T cell lineages develop in the thymus from a common precursor. It is unclear at which stage of development commitment to these lineages takes place and in which way T cell receptor signaling contributes to the process. Recently, it was demonstrated that strong TCR signals favor gammadelta lineage development, whereas weaker TCR signals promote alphabeta lineage fate. Two models have been proposed to explain these results. The first model suggests that commitment occurs after TCR expression and TCR signaling directly instructs lymphocytes to adopt one or the other lineage fate. The second model suggests that commitment occurs before TCR expression and that TCR signaling merely confirms the lineage choice. By tracing the fate of single T cell precursors, this study shows that there is no commitment to either the alphabeta or gammadelta lineage before TCR expression and that modulation of TCR signaling in progeny of a single TCR-expressing cell changes lineage commitment.     

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.     

Synergy between the pre-T cell receptor and Notch: cementing the alphabeta lineage choice

Notch1 signaling suppresses B cell development and promotes T lineage commitment in thymus-seeding hematopoietic progenitors. Notch1 is also activated in early T cell progenitors, but the functions of these later Notch signals have not been clearly defined. Recent studies reveal that Notch signaling is not essential for pre-T cell receptor (TCR) expression or gammadelta lineage choice. Rather, pre-TCR signaling enhances progenitor competitiveness for limiting Notch ligands, leading to preferential expansion of TCRbeta-bearing progenitors.     

Pre-TCRalpha and TCRalpha are not interchangeable partners of TCRbeta during T lymphocyte development

In contrast with the alphabeta T cell receptor (TCR), the pre-TCR spontaneously segregates to membrane rafts from where it signals in a cell-autonomous fashion. The disparate behaviors of these two receptors may stem either from differences inherent to the distinct developmental stages during which they are expressed, or from features intrinsic and unique to the receptor components themselves. Here, we express TCRalpha precisely at the pre-TCR checkpoint, at levels resembling those of endogenous pre-TCRalpha (pTalpha), and in the absence of endogenous pTalpha. Both in isolation and more dramatically when in competition with pTalpha, TCRalpha induced defective proliferation, survival, and differentiation of alphabeta T lymphocyte precursors, as well as impaired commitment to the alphabeta T lymphocyte lineage. Substitution of TCRalpha transmembrane and cytoplasmic domains with those of pTalpha generated a hybrid molecule possessing enhanced competitive abilities. We conclude that features intrinsic to the pre-TCR, which are absent in TCRalpha, are essential for its unique function.     

Presenilins regulate alphabeta T cell development by modulating TCR signaling

TCRalphabeta signaling is crucial for the maturation of CD4 and CD8 T cells, but the role of the Notch signaling pathway in this process is poorly understood. Genes encoding Presenilin (PS) 1/2 were deleted to prevent activation of the multiple Notch receptors expressed by developing thymocytes. PS1/2 knockout thymocyte precursors inefficiently generate CD4 T cells, a phenotype that is most pronounced when thymocytes bear a single major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR). Diminished T cell production correlated with evidence of impaired TCR signaling, and could be rescued by manipulations that enhance MHC recognition. Although Notch appears to directly regulate binary fate decisions in many systems, these findings suggest a model in which PS-dependent Notch signaling influences positive selection and the development of alphabeta T cells by modifying TCR signal transduction.     

The E delta enhancer controls the generation of CD4- CD8- alphabetaTCR-expressing T cells that can give rise to different lineages of alphabeta T cells

It is well established that the pre-T cell receptor for antigen (TCR) is responsible for efficient expansion and differentiation of thymocytes with productive TCRbeta rearrangements. However, Ptcra- as well as Tcra-targeting experiments have suggested that the early expression of Tcra in CD4- CD8- cells can partially rescue the development of alphabeta CD4+ CD8+ cells in Ptcra-deficient mice. In this study, we show that the TCR E delta but not E alpha enhancer function is required for the cell surface expression of alphabetaTCR on immature CD4- CD8- T cell precursors, which play a crucial role in promoting alphabeta T cell development in the absence of pre-TCR. Thus, alphabetaTCR expression by CD4- CD8- thymocytes not only represents a transgenic artifact but occurs under physiological conditions.     

Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection

The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.     

CD3+CD16+NK1.1+B220+ large granular lymphocytes arise from both alpha- beta TCR+CD4-CD8- and gamma-delta TCR+CD4-CD8- cells

Cultivation of CD4-CD8- double negative (DN) mouse thymocytes and splenocytes with recombinant interleukin 2 (IL2) in the absence of other stimulation results in the generation of DN- CD3/TCR+CD16+NK1.1+B220+ large granular lymphocytes (LGL). Purified DN alpha-beta TCR+ thymocytes and splenocytes are CD16+IL2R alpha-IL2R beta+NK1.1+B220-CD5high. These cells are unique in that they express both CD16 and T cell receptor (TCR) which are usually mutually exclusive. In addition, they express the natural killer (NK) marker, NK1.1. Cultivation of these cells with IL2 for several days results in the generation of DN alpha-beta TCR+CD16+NK1.1+B220+CD5- LGL, suggesting that DN alpha-beta TCR+ cells in thymus and spleen are the precursors of the DN LGL reported previously. DN gamma-delta TCR+CD16- NK1.1-B220-CD5high thymocytes and splenocytes also give rise to DN gamma-delta TCR+CD16+NK1.1+B220+CD5- LGL which, as shown previously with DN alpha-beta TCR+ LGL cells, are cytotoxic against NK-sensitive YAC-1 cells. Cytotoxic activity is also induced through either CD16 or the gamma-delta TCR. DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL cells are thus similar in phenotype to TCR- NK cells. DN alpha-beta TCR+ thymocytes express low levels of the gamma subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI gamma) molecule, an essential component of CD16 expression. Fc epsilon RI gamma expression is greatly enhanced after cultivation with IL2, resulting in a higher surface expression of CD16. In contrast to DN alpha-beta TCR+ thymocytes, DN gamma-delta TCR+ thymocytes do not express detectable CD16 or Fc epsilon RI gamma mRNA but expression of both is induced by cultivation with IL2, leading to the expression of CD16 on the surface. Whereas CD16 molecules on both DN alpha-beta TCR+ and DN gamma-delta TCR+ LGL are associated with only Fc epsilon RI gamma homodimers, the TCR on these cells are associated with an Fc epsilon RI gamma homodimer and/or CD3 zeta-Fc epsilon RI gamma heterodimers. These results demonstrate that the Fc epsilon RI gamma subunit is a component of the TCR in a fraction of T lineage cells.     

Involvement of the TCR Cbeta FG loop in thymic selection and T cell function

The asymmetric disposition of T cell receptor (TCR) Cbeta and Calpha ectodomains creates a cavity with a side-wall formed by the rigid Cbeta FG loop. To investigate the significance of this conserved structure, we generated loop deletion (betaDeltaFG) and betawt transgenic (tg) mice using the TCR beta subunit of the N15 CTL. N15betawt and N15betaDeltaFG H-2(b) animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4(-)CD8(-) double-negative (DN) compartment. N15betaDeltaFG facilitates transition from DN to CD4(+)8(+) double-positive (DP) thymocytes in recombinase activating gene (RAG)-2(-/-) mice, showing that pre-TCR function remains. N15betaDeltaFG animals possess approximately twofold more CD8(+) single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Valpha repertoire observed in N15betaDeltaFG mice may confound the deletion's effect, we crossed N15alphabeta TCR tg RAG-2(-/-) with N15betaDeltaFG tg RAG-2(-/-) H-2(b) mice to generate N15alphabeta RAG-2(-/-) and N15alphabeta.betaDeltaFG RAG-2(-/-) littermates. N15alphabeta.betaDeltaFG RAG-2(-/-) mice show an 8-10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15alphabeta, N15alphabeta.betaDeltaFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cbeta FG loop facilitates negative selection of thymocytes and activation of T cells.     

Pre-T cell receptor (TCR) and TCR-controlled checkpoints in T cell differentiation are set by Ikaros.

T cell differentiation relies on pre-T cell receptor (TCR) and TCR signaling events that take place at successive steps of the pathway. Here, we show that two of these T cell differentiation checkpoints are regulated by Ikaros. In the absence of Ikaros, double negative thymocytes can differentiate to the double positive stage without expression of a pre-TCR complex. Subsequent events in T cell development mediated by TCR involving transition from the double positive to the single positive stage are also regulated by Ikaros. Nonetheless, in Ikaros-deficient thymocytes, the requirement of pre-TCR expression for expansion of immature thymocytes as they progress to the double positive stage is still maintained, and the T cell malignancies that invariably arise in the thymus of Ikaros-deficient mice are dependent on either pre-TCR or TCR signaling. We conclude that Ikaros regulates T cell differentiation, selection, and homeostasis by providing signaling thresholds for pre-TCR and TCR.     

Density of the Notch ligand Delta1 determines generation of B and T cell precursors from hematopoietic stem cells

Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. To address whether different amounts of Notch ligand influence lineage choices, we cultured murine bone marrow lin(-)Sca-1(+)c-kit+ cells with increasing densities of immobilized Delta1(ext-IgG) consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1. We found that relatively lower densities of Delta1(ext-IgG) enhanced the generation of Sca-1(+)c-kit+ cells, Thy1(+)CD25+ early T cell precursors, and B220(+)CD43(-/lo) cells that, when cocultured with OP9 stroma cells, differentiated into CD19+ early B cell precursors. Higher densities of Delta1(ext-IgG) also enhanced the generation of Sca-1(+)c-kit+ precursor cells and promoted the development of Thy1(+)CD25+ cells, but inhibited the development of B220(+)CD43(-/lo) cells. Analyses of further isolated precursor populations suggested that the enhanced generation of T and B cell precursors resulted from the effects on multipotent rather than lymphoid-committed precursors. The results demonstrate the density-dependent effects of Delta1 on fate decisions of hematopoietic precursors at multiple maturational stages and substantiate the previously unrecognized ability of Delta1 to enhance the development of both early B and T precursor cells.     

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.     

Abnormal thymic development, impaired immune function and gamma delta T cell lymphomas in a TL transgenic mouse strain

During derivation of transgenic mouse strains with various TL and TL/H-2 chimeric genes, one strain, Tg.Tlaa-3-1, introduced with a TL gene (Tlaa-3), was found to have an abnormal thymic T cell population and to develop a high incidence of T cell lymphomas. To investigate the etiology of the thymic abnormalities and of the lymphomas, the development of lymphoid organs in transgenic mice was studied. The thymus of these mice goes through three unusual successive events: perturbation of thymic development during embryogenesis, disappearance of thymocytes between day 14 and day 21 after birth, and subsequent proliferation of large blast-like cells. These events are associated with the abolishment of T cell receptor (TCR) alpha beta lineage of the T cell differentiation, leading to preponderance of cells belonging to the TCR gamma delta L3T4-Lyt-2- double negative (DN) lineage. Bone marrow transplantation and thymic graft experiments demonstrate that the abnormality resides in the bone marrow stem cells rather than in the thymic environment. The expression of TL antigen in the transgenic mice is greatly increased and TL is expressed in a wide range of T cells, including normally TL- DN cells and L3T4+ Lyt-2- and L3T4-Lyt-2+ single positive cells. These quantitative and qualitative abnormalities in TL expression most likely cause the abnormal T cell differentiation. The gamma delta DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, as T cell function is defective in antibody production to sheep red blood cells, in mixed lymphocyte culture reaction to allogenic spleen cells and also in stimulation with concanavalin A. These results indicate that the gamma delta cells are incapable of participating in these reactions. Molecular and serological analysis of T cell lymphomas reveal that they belong to the gamma delta lineage, suggesting that the gamma delta DN cells in this strain are susceptible to leukemic transformation. Based on cell surface phenotype and TCR expression of the DN thymocytes and T cell lymphomas, a map of the sequential steps involved in the differentiation of gamma delta DN cells is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death

Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4- CD8- double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre-T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.