T Cell Factor-1 Controls the Lifetime of CD4+ CD8+ Thymocytes In Vivo and Distal T Cell Receptor α-Chain Rearrangement Required for NKT Cell Development

Natural killer T (NKT) cells are a component of innate and adaptive immune systems implicated in immune, autoimmune responses and in the control of obesity and cancer. NKT cells develop from common CD4+ CD8+ double positive (DP) thymocyte precursors after the rearrangement and expression of T cell receptor (TCR) Vα14-Jα18 gene. Temporal regulation and late appearance of Vα14-Jα18 rearrangement in immature DP thymocytes has been demonstrated. However, the precise control of lifetime of DP thymocytes in vivo that enables distal rearrangements remains incompletely defined. Here we demonstrate that T cell factor (TCF)-1, encoded by the Tcf7 gene, is critical for the extended lifetime of DP thymocytes. TCF-1-deficient DP thymocytes fail to undergo TCR Vα14-Jα18 rearrangement and produce significantly fewer NKT cells. Ectopic expression of Bcl-xL permits Vα14-Jα18 rearrangement and rescues NKT cell development. We report that TCF-1 regulates expression of RORγt, which regulates DP thymocyte survival by controlling expression of Bcl-xL. We posit that TCF-1 along with its cofactors controls the lifetime of DP thymocytes in vivo.


Introduction
T cell development in the thymus is characterized by the rearrangement and expression of T cell receptor (TCR) aand b-chains. TCRb chain rearranges first in CD4 2 CD8 2 double negative (DN) cells and is expressed on the cell surface with a pre-Ta chain and the components of CD3 complex. Thymocytes that express a pre-Ta/TCRb chain complex transition to the immature CD4 + CD8 + double positive (DP) stage and initiate the rearrangement of the TCRa chains. TCRa chain rearrangement persists until a productive interaction between the TCRab complex and the MHC complex is registered by the positive selection of the T cell. If the cell fails to be positively selected, a secondary Va-Ja rearrangement proceeds to replace the failed TCRa chain [1]. This temporal process is initiated at the 59 end of the Ja cluster and progresses through the 39 Jas during the lifetime of the DP thymocyte [2]. Natural killer T (NKT) cells develop from DP thymocytes with Va14-Ja18 TCRa chain rearrangement paired with Vb8, Vb7 or Vb2. The resulting limited TCR repertoire recognizes glycolipid antigens presented by the MHC class I-like molecule CD1d [3]. NKT cells produce cytokines when TCR is stimulated with their ligand a-galactosylceramide (a-GalCer) [4][5][6]. As Va14-Ja18 rearrangement is a temporally distal event, deletion of genes such as transcription factor RORct, that limit the lifetime of DP thymocytes, generally lead to impaired NKT cell development and immunity [2,3]. However, the transcriptional program that controls the lifetime of DP thymocytes in vivo remains to be fully defined.
TCF-1, encoded by the Tcf7 gene, and co-factor b-catenin are evolutionarily conserved transcription factors that work together and separately with other factors to regulate gene expression. In T cells, TCF-1 is induced by the Notch signaling pathway and participates in T cell commitment in the thymus [7,8]. b-Catenin is ubiquitously expressed and in T cells is augmented in response to TCR signals [9]. Cooperating together and functioning independently, these transcription factors regulate gene expression that control critical aspects of conventional T cell development and function [10][11][12][13]. In addition, we have demonstrated that TCF-1 and b-catenin regulate the generation of innate-like CD8 (iCD8) thymocytes [14]. Transcription factor RORct was shown to be a target of TCF-1 and shown to regulate thymocyte survival by controlling expression of Bcl-x L [15]. TCF-1 and b-catenin also regulate thymocyte survival in vitro [15,16]. However, their role in controlling the lifetime of DP thymocyte in vivo has not been defined. In particular, it remains to be demonstrated if TCF-1 and b-catenin regulate distal TCRa chain rearrangements and control NKT cell development.
In this study, we demonstrate that TCF-1 deletion results in significantly decreased NKT cells in the thymus. Enforced expression of Va14-Ja18 TCR (Va14) transgene resulted in the rescue of NKT cells, indicating that the reduction in the frequency of NKT cells was in part due to a failure to rearrange the Va14-Ja18 TCRa chain. Ectopic expression of Bcl-x L also rescued the frequency of Va14-Ja18 rearrangement and the NKT cell subset. Finally, we show that in vivo TCF-1 controls DP thymocyte lifetime by prompting expression of RORct as TCF-1-deficient DP thymocytes failed to express RORct. These studies demonstrate that the decrease in the frequency and number of NKT cells was due to a decrease in the lifetime of DP thymocytes in TCF-1-deficent mice. We posit that TCF-1 controls the lifetime of DP thymocytes in vivo.

TCF-1 is expressed in NKT cells in a developmentally relevant manner
To determine the role of TCF-1 in NKT cell generation, we first analyzed the expression of this protein by flow cytometry in NKT cells from control wild-type (WT) mice after staining target cells from lymphoid organs with anti-TCF-1, anti-TCRb and CD1d tetramers loaded with glycolipid a-GalCer analogue PBS-57 (CD1d-tet). We found that TCF-1 is expressed in NKT cells from thymus, liver ( Fig. 1A), spleen and lymph nodes (data not shown). To determine whether TCF-1 expression is regulated during NKT cell development, we also stained with CD44 and NK1.1 to be able to differentiate stage 1 (CD44 lo NK1.1 2 ), stage 2 (CD44 hi NK1.1 2 ) and stage 3 (CD44 hi NK1.1 + ) NKT cells [6,17]. The data showed that TCF-1 expression was highest in immature stage 1, followed by stage 2 and lowest in stage 3 thymic NKT cells (Fig. 1B, top). The decreased expression of TCF-1 in mature stage 3 NKT cells was also noticeable in liver (Fig. 1B, bottom). These data document changes in TCF-1 expression in NKT cells in a developmentally sensitive manner and suggest a functional role for TCF-1 during NKT cell development.
Recently, NKT cells were redefined on the basis of expression of transcription factors PLZF and T-bet [18]. NKT2 cells express higher PLZF compared to NKT1 cells and do not express T-bet (Fig. 1C). Interestingly, whereas all NKT cells express TCF-1, NKT2 cells express higher level of TCF-1 compared to NKT1 cells. We conclude that the expression pattern of TCF-1 in all NKT cells and during NKT cell development advocates a role for this transcription factor.

TCF-1-deficient mice have reduced proportion and number of NKT cells
To determine whether TCF-1 has a functional role in NKT cells, we analyzed thymocytes from TCF-1-KO mice. Surface staining with CD1d-tet and TCRb showed a significant decrease of NKT cell percentage and numbers in TCF-1deficient mice compared to control mice ( Fig. 2A). We conclude that transcription factor TCF-1 is required for the generation of all NKT cells.  Enforced expression of the Va14-Tg rescues NKT cells in TCF1deficient thymus One reason for the reduction in NKT cells could be failure to rearrange Va14-Ja18 TCRa chain to express the invariant TCR. To directly address this, we generated TCF-1-KO Va14-Tg mice by breeding TCF-1-KO mice with mice expressing a rearranged Va14-Ja18 TCRa transgene. Analysis of double-mutant (TCF-1-KO Va14-Tg) mice showed that enforced expression of Va14-Tg resulted in a 15-to-20-fold increase in the frequency of NKT cells compared to control mice (Fig. 2B). However, expression of Va14-Tg did not rescue the absolute number of thymocytes or total number of NKT cells found in wild type mice. Together, these results suggest that TCF-1-deficiency leads to decreased generation of NKT cells due to impaired rearrangement of Va14-Ja18 segments. As rearrangement of Va14-Ja18 segments is temporally a distal event, we hypothesized that a decrease in the lifetime of TCF-1-deficient DP thymocytes might prevent the rearrangement of distal 39 elements.
Ectopic expression of Bcl-x L in developing TCF-1-deficient thymocytes rescues Va14-Ja18 rearrangements and NKT cells TCF-1-deficient DP thymocytes have reduced survival when cultured in vitro, which was rescued by the expression of a proximal Lck promoter-driven Bcl-2 transgene [16]. This report showed that survival of DP thymocytes during culture in vitro was regulated by TCF-1 dependent expression of Bcl-family proteins. To determine if TCF-1 regulated the lifetime of DP cells in vivo that led to a reduction in NKT cells, we generated TCF-1-KOxBcl-x L transgenic mice (TCF-1-KO Bcl-x L -Tg). Representative data show that thymocyte numbers remained low in TCF-1-KO Bcl-x L -Tg mice (Fig. 3A). However, analysis of NKT cell populations in TCF-1-KO Bcl-x L -Tg mice demonstrated a rescue of the frequency of NKT cells (Fig. 3B). However, the number of NKT cells remained lower than observed in control mice. We conclude that expression of Bcl-x L from the proximal Lck promoter rescued the lifetime of TCF-1-deficient DP thymocytes in vivo and promoted development of NKT cells.
To further understand the role of TCF-1 in NKT cell generation, we tested the frequency of the Va14-Ja18 rearrangement in DP cells from TCF-1-KO, TCF-1-KO Bcl-x L -Tg or control mice. We noted that TCF-1-deficient DP thymocytes showed significant decreased representation of Va14-Ja18 rearrangements compared to control cells (Fig. 3C). The frequency of Va14-Ja18 rearrangements was rescued in TCF-1-KO Bcl-x L -Tg mice (Fig. 3D). These data demonstrate that TCF-1-deficient DP thymocytes do not rearrange distal TCRa chains and thus do not generate a complete TCR repertoire. Incidentally, transgenic overexpression of b-catenin did not enhance the frequency of Va14-Ja18 rearrangements (data not shown) indicating that b-catenin expression was not limiting in the definition of the lifetime of DP thymocytes. We conclude that TCF-1 is an essential component of the transcription factor profile required for proper T cell development and generation of NKT cells and T cell repertoire.

Discussion
In this report we demonstrate that TCF-1 controls the lifetime of DP thymocytes in vivo, temporally distal Va14-Ja18 rearrangements and NKT cell development. We deduce this from the observation that TCF-1-deficiency results in failure to rearrange distal Va14-Ja18 rearrangements that impacts NKT cell generation in vivo. We show that, in TCF-1-deficient mice, these cells are significantly reduced in frequency and enforced expression of the NKT cell specific Va14-Ja18 TCR transgene rescues the development of NKT cells in TCF-1-deficient mice. Finally, we demonstrate that ectopic expression of Bcl-x L transgene from the proximal Lck promoter in TCF-1-deficient DP thymocytes extends lifetime in vivo to rescue Va14-Ja18 rearrangements and NKT cells.
The transcriptional program that regulates thymic cellularity remains to be defined. TCF-1 was shown to regulate DP thymocyte survival and respond to dexamethasone challenge in a RORct-dependent manner [15]. However, reduced thymic cellularity TCF-1-deficient mice show was not rescued by expression of genes that confer cell survival from proximal Lck promoter. Held et al. expressed Bcl-2 transgene that rescued DP thymocyte survival in vitro but failed to salvage thymic cellularity in vivo [16]. Likewise, data presented in this report show that Lck-driven Bcl-x L transgene fails to rescue thymic cellularity. We posit that these data reflect requirement of TCF-1-dependent gene expression during early T cell development to generate thymic cellularity.
A requirement for the rearrangement of Va14-Ja18, a temporally distal event, makes the lifetime of DP thymocytes in vivo a major factor in NKT cell generation. Deletion of transcription factors that regulate DP thymocyte lifetime in vivo show decreased NKT cell numbers, which was rescued by enforced expression of Bcl-x L [19][20][21][22][23]. For example, Bcl-x L overexpression in c-Mybdeficient DP cells restored survival and Va14-Ja18 rearrangements but not NKT cell development suggesting that Va14-Ja18 rearrangement is not sufficient for NKT cell development. Id2-deficiency is damaging to DP survival and NKT cell numbers, and both of these defects were rescued by breeding with Bim-KO mice [24]. Other transcription factors, such as c-Myc [25], have been demonstrated to regulate proliferation but not survival of NKT cells. TCF-1 expression has been linked to decreased survival of DP thymocytes in vitro, which was rescued by Bcl-2 expression [16]. In this report we demonstrate that TCF-1-deficient DP thymocytes have significantly fewer Va14-Ja18 rearrangements and NKT cells. TCF-1 and LEF-1 have been shown to have redundant roles during T cell development [10][11][12][13]. We expect that similar overlap might be observed in the regulation of NKT cell generation in the thymus. However, in this report we show that transgenic expression of Bcl-x L in DP thymocytes rescues distal TCRa rearrangement but not NKT cell numbers. It is important to note that thymic cellularity was not rescued by transgenic expression of Bcl-x L suggesting that DP thymocyte lifetime is not an essential component of thymic cellularity during normal T cell development. We propose that TCF-1 controls specific gene expression required for the extended lifetime of DP thymocytes, in turn required to rearrange the distal Va14-Ja18 segments and thereby facilitates NKT cell development.
Va14-Ja18 rearrangement analysis RNA from DP cells was isolated using the Qiagen RNeasy Kit (Qiagen Inc.) following the manufacturer's instructions. cDNA was generated by RT-PCR with a SuperScript III kit (Invitrogen) and, serially diluted 1:2, it was amplified with a forward primer specific for Va14 (59-GTCCTCAGTCCCTGGTTGTC-39) paired with a downstream primer specific for the segment Ja18 (59-CAAAATGCAGCCTCCCTAAG-39). PCR products were separated by agarose gel electrophoresis and visualized by SYBR Safe DNA gel staining (Invitrogen). Results were normalized to Va14-Ca expression (downstream Ca primer 59-CAGTCAACGTGGCATCACA-39).

Statistics
Statistical significance was determined by the Student's t-test.