Threshold of pre-T-cell-receptor surface expression is associated with alphabeta T-cell lineage commitment.

BACKGROUND: The development of immature thymocytes is regulated by the pre-T-cell receptor (pre-TCR). The pre-TCR is involved in several developmental processes including rescuing cells from programmed cell death, allelic exclusion and alphabeta versus gammadelta T-cell lineage commitment. A major issue is how the pre-TCR functions to integrate these processes in developing thymocytes. RESULTS: We have used a sensitive immunofluorescence technique to reveal the surface-expression profile of the pre-TCR on immature thymocyte subsets. We show that early pre-T cells (CD25(+)CD44(-)) can be subdivided on the basis of the level of surface pre-TCR expression. Detectable surface pre-TCR expression identified a rapidly cycling population of early pre-T cells which had successfully undergone beta-selection and been rescued from programmed cell death. Late pre-T cells (CD25(-)CD44(-)), which had traversed the beta-selection checkpoint, expressed surprisingly heterogeneous surface levels of the pre-TCR: high levels of surface pre-TCR expression were associated with commitment to the alphabeta T-cell lineage, whereas late pre-T cells with lower levels of surface pre-TCR could develop along both the alphabeta or gammadelta T-cell lineages. CONCLUSIONS: These data demonstrate that the surface expression of the pre-TCR can be used to reveal newly identified stages of T-cell development and to provide insights into alphabeta T-cell lineage commitment. They show that, although pre-TCR expression does not act as a developmental switch per se, its level of surface expression on late pre-T cells predicts their developmental potential.     

Differential requirement for RhoA GTPase depending on the cellular localization of protein kinase D

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

Biased V beta usage in immature thymocytes is independent of DJ beta proximity and pT alpha pairing

During thymus development, the TCR beta locus rearranges before the TCR alpha locus. Pairing of productively rearranged TCR beta-chains with an invariant pT alpha chain leads to the formation of a pre-TCR and subsequent expansion of immature pre-T cells. Essentially nothing is known about the TCR V beta repertoire in pre-T cells before or after the expression of a pre-TCR. Using intracellular staining, we show here that the TCR V beta repertoire is significantly biased at the earliest developmental stage in which VDJ beta rearrangement has occurred. Moreover (and in contrast to the V(H) repertoire in immature B cells), V beta repertoire biases in immature T cells do not reflect proximity of V beta gene segments to the DJ beta cluster, nor do they depend upon preferential V beta pairing with the pT alpha chain. We conclude that V gene repertoires in developing T and B cells are controlled by partially distinct mechanisms.     

Deconstructing Ras signaling in the thymus

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance.     

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.     

Integrin regulation by RhoA in thymocytes

The guanine nucleotide-binding protein Rho has essential functions in T cell development and is important for the survival and proliferation of T cell progenitors in the thymus. To explore the mechanisms used by RhoA to control thymocyte biology, the role of this GTPase in the regulation of integrin-mediated cell adhesion was examined. The data show that RhoA activation is sufficient to stimulate beta(1) and beta(2) integrin-mediated adhesion in murine thymocytes. RhoA is also needed for integrin activation in vivo as loss of Rho function impaired the ability of thymocytes to adhere to the extracellular matrix protein VCAM-1 and prevented integrin activation induced by the GTPases Rac-1 and Rap1A in vivo. The regulated activity of integrins is needed for cell motility and in the present study it was seen that RhoA activity is critical for integrin-mediated thymocyte migration to chemokines in vitro. Thus, RhoA has a critical role in regulating cell adhesion and migration during T cell development.     

A role for the cytoplasmic tail of the pre-T cell receptor (TCR) alpha chain in promoting constitutive internalization and degradation of the pre-TCR

Engagement of the alpha beta T cell receptor (TCR) by its ligand results in the down-modulation of TCR cell surface expression, which is thought to be a central event in T cell activation. On the other hand, pre-TCR signaling is a key process in alpha beta T cell development, which appears to proceed in a constitutive and ligand-independent manner. Here, comparative analyses on the dynamics of pre-TCR and TCR cell surface expression show that unligated pre-TCR complexes expressed on human pre-T cells behave as engaged TCR complexes, i.e. they are rapidly internalized and degraded in lysosomes and proteasomes but do not recycle back to the cell surface. Thus, pre-TCR down-regulation takes place constitutively without the need for extracellular ligation. By using TCR alpha/p Tau alpha chain chimeras, we demonstrate that prevention of recycling and induction of degradation are unique pre-TCR properties conferred by the cytoplasmic domain of the pT alpha chain. Finally, we show that pre-TCR internalization is a protein kinase C-independent process that involves the combination of src kinase-dependent and -independent pathways. These data suggest that constitutive pre-TCR down-modulation regulates pre-TCR surface expression levels and hence the extent of ligand-independent signaling through the pre-TCR.     

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

E2A proteins enforce a proliferation checkpoint in developing thymocytes

E2A proteins regulate multiple stages of thymocyte development and suppress T-cell lymphoma. The activity of E2A proteins throughout thymocyte development is modulated by signals emanating from the pre-TCR and TCR. Here we demonstrate that E2A is required for the complete arrest in both differentiation and proliferation observed in thymocytes with defects in proteins that mediate pre-TCR signaling, including LAT, Lck and Fyn. We show that E2A proteins are required to prevent the accumulation of TCRbeta negative cells beyond the pre-TCR checkpoint. E2A-deficient thymocytes also exhibit abnormal cell-cycle progression prior to pre-TCR expression. Furthermore, we demonstrate that E47 can act in concert with Bcl-2 to induce cell-cycle arrest in vitro. These observations indicate that E2A proteins function during early thymocyte development to block cell-cycle progression prior to the expression of TCRbeta. In addition, these data provide further insight into how deficiencies in E2A lead to T lymphoma.