NKT Cell-TCR Expression Activates Conventional T Cells in Vivo, but Is Largely Dispensable for Mature NKT Cell Biology

Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.


Introduction
Natural Killer T (NKT) cells represent a subset of T cells in mice and humans that express NK cell markers and recognize a small class of glycolipid (auto-) antigens [1,2]. Most mouse NKT cells express an invariant Va14-Ja18 (Va14i) TCRa rearrangement (Va24-Ja18 in humans). In principle, all TCRb-chains are able to pair with this Va14i-TCR chain [3]. However, the selection of NKT cells by endogenous glycolipids presented by the monomorphic MHC class I-like CD1d induces a strong bias towards TCRs containing Vb8, Vb7, or Vb2 [1,3], which is abrogated in the absence of selection [3,4]. Recently, crystallographic analysis demonstrated a conserved binding mode of the NKT cell TCR to various glycolipids, where only germlineencoded residues were in direct antigen contact, reminiscent of innate pattern-recognition receptors [5]. Moreover, several observations suggest that this receptor is inherently auto-reactive [1,2] and thereby determines NKT cell identity and influences their function. The expression of several inhibitory NK cell receptors on NKT cells was suggested to control their selfreactivity and avoid autoimmune activation [6,7].
During development in the thymus, the few T cells expressing a Va14i-TCR are selected upon recognition of self-lipids on double-positive thymocytes. Although several good candidates have been put forward [8][9][10], the exact nature of the selecting glycolipids remains controversial. Homotypic interactions involving the SLAM family (SLAMf) receptors 1 and 6 are additionally required for NKT cell differentiation [11]. Auto-reactive activation during thymic selection is thought to induce a substantially stronger TCR stimulus in comparison to that during the development of conventional T cells [12,13]. As a consequence, expression of the transcription factors Egr1 and Egr2 is strongly increased [13], which in turn directly induce PLZF, the key transcription factor controlling NKT cell differentiation, migration, and functions [13].
Interestingly, the homeostatic proliferation of NKT cells after adoptive transfer was similar in CD1d-deficient and wild-type mice, indicating that this process is mostly cytokine-driven and does not depend on continued TCR-mediated self-lipid-recognition [14,15]. However, as the transferred cells contained CD1d, a role for antigen could not be completely excluded. In addition, tonic antigen-independent TCR signals might contribute to NKT cell maintenance and phenotype. During immune responses, NKT cell activation depends mostly on two parameters: engagement of the TCR and the presence of proinflammatory cytokines released from antigen-presenting cells activated by innate immune path-ways such as toll-like receptor (TLR) signals. Lipids derived from different bacteria [16][17][18][19] were shown to directly activate mouse and human NKT cells in a TLR-and IL-12-independent manner, and NKT cells are required for productive immune responses against these pathogens. NKT cells can also be activated indirectly through cytokines such as IL-12, IL-18, or type I interferons (IFNs) [20]. However, it remains controversial whether, depending on the strength of the cytokine signal, weak responses to self-antigens presented by CD1d are an additional obligate requirement. In one study, CD1d-dependent signals were found to be necessary for full NKT cell activation in response to all tested pathogens [20]. In contrast, others reported that IL-12-dependent NKT cell activation after LPS injection [21] or MCMV infection [22] is independent of either foreign or self-glycolipid antigen presentation by CD1d.
Upon activation, the most distinguishing feature of NKT cells is their ability to rapidly produce and secrete large amounts of cytokines (Th1 and Th2 cytokines, among others). Their fast, effector-like response could be based on steady-state expression of cytokine mRNA in mice [23,24] that was suggested to be a consequence of tonic self-reactive activation [2]. Recently, it was reported that human NKT cells do not constitutively express cytokine mRNAs. Instead, rapid cytokine-induced innate IFNc production by NKT cells was suggested to rely on obligate continuous recognition of self-lipids, which retains histone acetylation patterns at the IFNG locus that favor transcription [25]. Another characteristic feature of NKT cells, their surface marker expression reminiscent of memory or recently activated T cells, was also connected to their inherent autoreactivity [2].
To thoroughly address the open questions regarding the nature and importance of TCR signaling for NKT cells, we generated a novel mouse model that allowed us to study the extent of Va14i-TCR-mediated auto-antigen recognition in the periphery and its relevance for NKT cell identity. Furthermore, we monitored the fate of NKT cells after TCR ablation. Our results prove the inherent self-reactivity of the NKT cell TCR and demonstrate that although essential for positive selection, tonic TCR signaling is not required for NKT cell homeostasis, lineage identity, and rapid cytokine secretion.

Correct Timing and Endogenous Control of Va14i-TCR Expression Produces Large Numbers of Bona Fide NKT Cells
In order to produce large numbers of NKT cells in a physiological manner and to manipulate the expression of the semi-invariant Va14i-TCR in a conditional fashion, we generated Va14i StopF knock-in mice. To this end we cloned a productive Va14-Ja18 rearrangement, including the Va14 leader exon, intron and 1.8 kb of upstream regulatory sequence, and 0.2 kb intronic sequence downstream of Ja18. These elements were inserted by homologous recombination 39 of Ja1 upstream of the Ca constant region of the Tcra locus ( Figure 1A). Expression of putative upstream rearrangements is aborted by four SV40 polyA sites at the 59 end of the construct, and expression of Va14i is rendered conditional through a loxP-flanked STOP cassette. We obtained over 80% (271 of 325) homologous recombinant ES cell clones during gene targeting, indicating an unusually high targeting efficiency of our construct ( Figure S1A). The development of conventional T and NKT cells, identified by staining with mouse CD1d-PBS57-tetramers (tetramer+), occurs unperturbed in Va14i StopF /wt heterozygous mice. In homozygous Va14i StopF/F mice, T cell development is abolished due to transcriptional termination of TCRa expression before the Ca exons ( Figure 1B). We bred Va14i StopF to CD4-Cre mice, in order to express the inserted Va14i-chain in double-positive thymocytes, mimicking the physiological timing of TCRa-chain rearrangement and expression [26,27]. On average 23 times more thymic and 43 times more splenic NKT cells were generated in these, compared to wild-type mice ( Figures 1B and 2A-E). Around 9% of the tetramer+ T cells in CD4-Cre Va14i StopF /wt mice expressed the CD8 co-receptor (over 80% as CD8ab heterodimer; Figures 1C and S1B,C), which is also expressed by some human NKT cells, but normally not in mice [28]. The proportions of CD42 CD82 double negative (DN) and CD4+ cells were comparable between transgenic (tg) and wild-type NKT cells ( Figure 1C). Furthermore, the tgNKT cells were largely comparable to wild-type NKT cells with respect to Vb-chain bias ( Figure 1D) and surface phenotype ( Figure 1E). Finally, we found that NKT cells from CD4-Cre Va14i StopF /wt animals expressed the critical transcription factors promyelocytic leukemia zinc finger (PLZF), GATA binding protein 3 (GATA-3), and T-helper-inducing POZ/Krüppel-like factor (Th-POK) ( Figure 1F) [28,29]. Interestingly, we also detected a substantial proportion of the recently described NKT17 subset in the transgenic animals. These DN NK1.1 2 NKT cells express the transcription factor ROR-ct and were shown to produce the cytokine IL-17 upon activation ( Figure 1F) [29,30].

Timing of Transgenic Va14i-TCR Expression Is Critical for Normal NKT Cell Development
Premature TCRa expression leads to aberrant T cell development in transgenic mouse models [26,27]. To directly compare the consequence of premature to CD4-Cre-mediated timely Va14i-TCRa-chain expression in our knock-in approach, we bred our mice to a germline Cre-deleter strain (Nestin-Cre) [31]. Compared to CD4-Cre-induced Va14i-TCRa-chain expression, premature expression in Cre-deleter Va14i StopF /wt led to significantly reduced numbers of NKT cells in thymus and spleen, especially of CD4+ NKT cells (Figure 2A-C). In addition, we

Author Summary
Immune system natural killer T (NKT) cells help to protect against certain strains of bacteria and viruses, and suppress the development of autoimmune diseases and cancer. However, NKT cells are also central mediators of allergic responses. The recognition of one's own glycolipid antigens (self-glycolipids) in the thymus via the unique Va14i T cell receptor, Va14i-TCR, triggers the NKT cell developmental program, which differs considerably from that of conventional T cells. We generated a mouse model to investigate whether the Va14i-TCR on mature NKT cells constantly recognizes self-glycolipids and to assess whether this TCR is required for survival and continued NKT cell identity. Switching the peptide-recognizing TCR of a mature conventional T cell to a glycolipid-recognizing Va14i-TCR led to activation of the T cells, indicating that this TCR is also autoreactive on peripheral T cells or can signal autonomously. But TCR ablation did not affect the half-life, characteristic gene expression or innate functions of mature NKT cells. Therefore, the inherently autoreactive Va14i-TCR is dispensable for the functions of mature peripheral NKT cells after instructing thymic NKT cell development. Thus the Va14i-TCR serves a similar function to pattern-recognition receptors, in mediating immune recognition of foreign invasion or diseased cells.

NKT Cell Maturation in CD4-Cre Va14i StopF /wt Animals
To test the functionality of our transgenic NKT cells, we injected CD4-Cre Va14i StopF /wt mice with the NKT cell ligand a-Galactosylceramide (a-GalCer) and determined their cytokine production directly ex vivo. The transgenic NKT cells were able to mount a rapid and robust cytokine response. Although a reduced proportion of transgenic NKT cells responded, in absolute cell numbers there was a 6-10-fold increase compared to wild-type NKT cells ( Figure 3A). We did not observe significant steady-state cytokine production by transgenic or control NKT cells, and we detected only minor increases in cytokine levels in the serum of some of these mice ( Figure S1D). Since cytokine production also varies with NKT cell maturation, we analyzed NKT cell development in CD4-Cre Va14i StopF /wt mice in more detail. This revealed a strong bias toward immature fractions in the thymus, due to the dramatic increase in NKT cell progenitors. In the periphery, 20% of NKT cells fully matured, as judged by the expression of NK1.1 and other NK cell markers ( Figure 3B,C). This view is further supported by the reduced proportion of CD69 and T-bet-expressing NKT cells in CD4-Cre Va14i StopF /wt compared to wild-type mice ( Figure 3D). The expression of both CD69 and T-bet strongly correlated with NK1.1 surface levels ( Figure  S1E,F). This also explains the higher intracellular PLZF expression in CD4+ and DN NKT cells of CD4-Cre Va14i StopF /wt animals in comparison to control animals ( Figure 1F), as it was shown that PLZF expression is downregulated during NKT cell development [36]. Reduced maturation seems to be a common feature in mice with overabundance of NKT cells ( Figure S1G and Table S1) [33]. Indeed, a comparison of different Va14i-tg mice suggests that independently of the total number of NKT cells generated, the size of the homeostatic niche for mature NKT cells appears to be around two million cells (Table S1).
IL-15 is critical for the final maturation of NKT cells [37] and together with IL-7 required for their peripheral maintenance [14,38]. NKT cells compete with NK cells for these resources [38]. The halved number of NK cells in CD4-Cre Va14i StopF /wt mice ( Figure 3E) suggests that the availability of these and maybe other cytokines might be insufficient due to the dramatically increased NKT cell numbers. The fact that a similar effect was observed in Va11p-Va14itg mice ( Figure 3E) underscores this notion. These results let us conclude that while large amounts of NKT cells can Investigating NKT Cell-TCR Signals PLOS Biology | www.plosbiology.org be produced in mice, depending on the mode of Va14i expression, the number of fully mature NKT cells is restricted by homeostatic constraints, some of which are shared with NK cells.

The Exchange of the Endogenous TCR Repertoire for a Va14i-Restricted One on Mature Conventional T Cells Leads to a Significant Population of Tetramer+ T Cells
The strong self-lipid-induced TCR stimulus that early NKT cell progenitors receive in the thymus can be visualized through high GFP expression under the control of the Nur77 gene locus, reporting TCR signal strength [12]. However, the subsequent loss of GFP in mature NKT cells suggests that these cells are either not exposed to or not responsive to self-antigens. In order to answer this question and to study NKT cell TCR-autoreactivity in the periphery, we investigated the consequences of Va14i-TCR signals for conventional naïve T cells. We wondered whether Va14i-TCR expression on naïve T cells, lacking inhibitory receptors and generally a NKT cell ''identity'', would lead to activation upon (self-)lipid recognition and what cellular fate(s) are elicited by such activation.
To this end, we generated mice enabling us to exchange the endogenous TCR-repertoire present on naïve peripheral T cells for a Va14i-restricted TCR repertoire. The induction of Cre expression in Mx-Cre Ca F /Va14i StopF mice inactivates the Ca F allele and simultaneously turns on the Va14i StopF allele, leading to substitution of endogenous TCRa-chains with the Va14i TCRa- chain ( Figure 4A). As mentioned above, the Va14i-chain can pair with all TCRb-chains [3], although only Vb2-, Vb7-, and Vb8containing Va14i-TCRs can recognize endogenous lipids such as iGb3 [3,4]. Since TCRs containing one of these Vb-chains constitute approximately 30% of the CD4+ and CD8+ peripheral T cell pool ( Figure 1D and unpublished data), we predicted that our genetic switch experiment should generate sufficient numbers of T cells able to recognize self-lipids.
In Mx-Cre transgenic mice, Cre expression can be induced through injection of dsRNA, such as poly(I:C) [39]. However, lowlevel ''leaky'' recombination occurs also in absence of an inducer [39,40], leading to increased numbers of tetramer+ T cells in naive Mx-Cre Ca F /Va14i StopF mice ( Figure S2A). Therefore, splenocytes were depleted of tetramer+ T cells by magnetic cell separation (MACS, Figure S2A), and 20610 6 purified cells were injected intravenously (i.v.) into recipient animals lacking conventional ab T cells and NKT cells (Ca 2/2 or Va14i StopF/F ). After cells were allowed to engraft for 2 wk, the TCR switch was induced by poly(I:C) injection. Importantly, except for a short-term activation of the immune system, poly(I:C) injection in Mx-Cre mice per se has no significant long-lasting effect on peripheral conventional T cells [40,41] or on the number and phenotype of NKT cells (unpublished data). To definitely exclude any effect of poly(I:C) injection on our results, we waited 2-4 mo before analyzing the animals after the induced TCR switch.
We found significant numbers of tetramer+ CD4+ and CD8+ T cells as a result of this switch experiment ( Figure 4B-E). ''Unloaded'' tetramers did not stain these cells, demonstrating that they were not reactive against CD1d itself ( Figure S2B). The TCR-switched tetramer+ T cells were predominantly enriched in cells expressing Vb-chains that are associated with high avidity auto-antigen binding: Vb2, Vb8.1/8.2, and Vb7 ( Figure 4D,E) [3,4,42]. The exceptions were CD8+ TCR-switched tetramer+ T cells, in which Vb7-expressing cells were not enriched. The bias toward tetramer+ CD8+ T cells ( Figure 4C) is most likely due to more efficient Mx-Cre-mediated recombination in these cells [40].

Sterile Inflammation in Mice Containing TCR-Switched T Cells
Animals containing TCR-switched tetramer+ T cells, but not controls, displayed splenomegaly ( Figure 5A,B), characterized by increased numbers of macrophages/monocytes, neutrophils, and Ter119+ erythroid progenitor cells, suggesting an inflammatory state ( Figure 5C-E). In line with these findings, we could detect elevated serum TNF in more than half of these mice ( Figure 5F). Elevated levels of other cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, and IFN-c, were not found in the sera of these mice (unpublished data). Interestingly, we found that 6 (highlighted in red throughout the figure) of 17 spleens containing TCR-switched T cells were almost completely devoid of B cells ( Figure 5G) as well as dendritic cells (DCs, Figure 5H), which present lipid antigens to NKT cells via CD1d [1]. Furthermore, tetramer-''conventional'' T cells were also strongly reduced in these animals (unpublished data). Together, these results suggest that induced expression of the Va14i-TCR on conventional naïve T cells causes sterile inflammation, possibly due to autoimmune activation.

Va14i-TCR Signaling Induces Cellular Activation, But Not NKT Cell Differentiation of TCR-Switched Tetramer+ T Cells
The appearance of tetramer+ cells displaying a Vb bias similar to antigen-selected NKT cells, together with signs of inflammation upon TCR switch and the absence of CD1d-expressing B cell and DCs in some cases, suggested auto-antigen-mediated activation of TCR-switched cells. To verify that the newly assembled Va14i-TCR on conventional T cells is functional, we injected recipients of Mx-Cre Ca F /Va14i StopF and control cells with a-GalCer or PBS 2 mo after switch induction. Ninety minutes after a-GalCer, but not PBS, injection, CD4+ and CD8+ tetramer+ T cells produced IFN-c and TNF ( Figure 6A), demonstrating the functionality of the newly assembled Va14i-TCR. In comparison to NKT cells from wild-type or CD4-Cre Va14i StopF /wt animals, a smaller proportion of tetramer+ T cells produced cytokines (Figures 6A  and S2C). Tetramer+ TCR-switched T cells could also be activated in vitro through a-GalCer-pulsed A20 cells overexpressing CD1d (unpublished data) [43].
To study the consequences of Va14i-TCR expression on tetramer+ TCR-switched T cells in more detail, we analyzed their surface phenotype and transcription factor expression. Absence of NK cell markers (Figures 6B and S2D) and PLZF expression ( Figure 6C) indicated that the Va14i-TCR signals are not sufficient to induce NKT cell differentiation of mature conventional T cells. However, the TCR-switched tetramer+ T cells expressed signifi- cantly higher levels of Egr2 in comparison to tetramer2 T cells in the same animals ( Figure 6D), suggesting that the switched cells receive stronger TCR signals [13]. TCR-switched T cells showed further signs of cellular activation, as they expressed elevated levels of CD69 ( Figure 6E). Interestingly, these T cells displayed also significantly increased surface levels of PD-1, LAG-3, and less frequently, BTLA and TIM-3, which is typical of exhausted/ anergic cells ( Figure 6F-H and unpublished data) [44,45].
To test whether exhaustion/anergy of tetramer+ TCR-switched T cells prevented a more dramatic form of autoimmune inflammation, we injected mice with PD-L1 and PD-L2 blocking or control antibodies twice a week for 4 consecutive weeks, starting 2 d before switch induction. The administration of these blocking antibodies has previously been shown to efficiently prevent anergy induction of conventional T as well as NKT cells, and to partially reverse the exhaustion of CD8+ T cells [44,45]. However, we did not observe any dramatic differences in spleen weight or cellularity, or signs of increased inflammation, between animals receiving PD-L blocking or control antibodies (unpublished data). In response to PD-1 blockade, other inhibitory receptors such as LAG-3, BTLA, or TIM-3 might control the TCR-switched T cells.
Taken together, our results showed that expression of the Va14i-TCR on mature conventional T cells is not sufficient to induce a NKT cell differentiation program. Still, it is likely that Va14i-TCR signals induce auto-antigen-mediated activation, possibly to the point of exhaustion. We therefore present strong evidence that the Va14i-TCR can constitutively recognize selflipids in the naïve steady state situation in vivo.

Maintenance of Mature NKT Cells Is TCR-Independent
The evidence for autoreactivity of the Va14i-TCR on mature peripheral T cells raised the old but still not completely resolved question whether and to what extent interactions with self-lipidpresenting APCs are required for NKT cell maintenance, cellular identity, and function. In order to evaluate the importance of constitutive TCR expression and signaling for NKT cells directly in vivo and for long periods of time, we ablated the TCR on mature T cells using poly(I:C) injection of Mx-Cre Ca F/F mice [40].
Two weeks after induced Cre-mediated recombination, around 30% of CD4 and 65% of CD8 T cells had lost functional TCR expression in these mice ( Figure 7A and [40]). To unambiguously identify TCR-deficient NKT cells, we developed a robust staining strategy based on CD4, NK1.1, CD5, and CD62L expression ( Figure S3A). This limited us to CD4+ NKT cells, but our staining identified over 50% of the total NKT cell populations in thymus and spleen ( Figure S3B). Around 65% of the thus identified NKT cells had lost TCR surface expression 2 wk after Cre induction ( Figure 7A,B).
Due to complete Cre-mediated recombination in lymphoid progenitors, T cell development is blocked at the double positive stage in Mx-Cre Ca F/F mice after induction of Cre [40]. This allowed us to study the T cell decay in the absence of cellular efflux from the thymus. In agreement with previous studies [40,46], we found that loss of the TCR leads to decay of naïve CD4+ CD44 low and memory/effector-like CD4+ CD44 high T cells with a half-life of 40 d and 297 d, respectively ( Figure 7C,D). Interestingly, we observed essentially no decay of receptor-less NKT cells, with a calculated half-life of 322 d ( Figure 7E), and could find significant numbers of TCR-deficient NKT cells even 45 wk after TCR deletion (unpublished data). To evaluate the role of TCR signals during in situ homeostatic proliferation, we administered BrdU for 4 wk via the drinking water, starting 2 wk after induced TCR ablation. Naïve CD4+ CD44 low as well as CD4+ CD44 high memory/effector-like T cells showed significantly decreased BrdU incorporation in TCR-deficient compared to TCR-expressing cells ( Figure 7F,G). In contrast, TCR ablation did not affect NKT cell proliferation ( Figure 7F,G). Interestingly, the BrdU incorporation was identical in TCR-deficient CD4+ CD44 high T and NKT cells, indicating that in the absence of TCR signals the cytokine-driven expansion of CD4+ CD44 high memory/effectorlike T and NKT cells is similar ( Figure 7F,G). Our results therefore indicate that long-term in situ NKT cell homeostasis is completely independent of TCR-induced signals.

The TCR Is Dispensable for the Identity and Cytokine-Secretion Ability of Mature NKT Cells
In absence of de novo T cell generation, we found elevated Egr2 expression in mature thymic, but not splenic, NKT cells compared to DP thymocytes and CD4+ T cells, respectively ( Figure 8A). This indicates that NKT cells receive stronger TCR signals in the thymus, which is supported by the decreased Egr2 expression of mature thymic TCR-deficient NKT cells ( Figure 8A). Surprisingly, in mature NKT cells in thymus and spleen, expression of the TCR-signal-induced key transcription factor PLZF is completely unaffected by TCR ablation ( Figure 8B).
In order to more generally evaluate to what extent NKT cell TCR-expression is required for the maintenance of characteristic lineage-specific gene expression (resembling recently activated T cells), we extensively analyzed the cell-surface phenotype of NKT cells 6 wk after TCR ablation. Of all the analyzed markers, the only significant changes that we observed on splenic NKT cells upon TCR ablation were downregulation of NK1.1, CD4, CD5, and ICOS ( Figures 8C,D and S3C-E). NK1.1 expression was also reduced in thymic TCR-deficient NKT cells, in addition to CXCR6 expression (unpublished data). CD5 and ICOS expression were also reduced in TCR-deficient splenic naïve as well as CD62L low CD4+ T cells ( Figure S3C,D). CD4 was upregulated on TCR-deficient CD4+ naïve, but downregulated on NKT and CD4+ CD44 high T cells ( Figure S3E). Strikingly, all other cell surface markers characteristic for the NKT cell lineage, among them the transcription factors PLZF, GATA-3, T-bet, and Th-POK, as well as many cell surface markers whose expression is also induced upon TCR engagement, remained largely unaffected by loss of the NKT cell TCR ( Figure 8D).
Treatment of mice with LPS, a cell wall component of gramnegative bacteria, leads to release of IFN-c by NKT cells via stimulation with IL-12 and IL-18 produced by innate immune cells. This does not require acute TCR engagement [21]. However, it has been proposed that the ability of NKT cells to rapidly release IFN-c in this context critically requires continuous weak TCR activation in the steady state [25]. We therefore analyzed IFN-c release of TCR+ and TCR-NKT cells after in vivo injection of LPS, a-GalCer, and PBS ( Figure 9A,B). As expected, Egr2 expression could only be detected in NKT cells that were activated through their TCR ( Figure 9A). Accordingly, 90 min after a-GalCer injection, the majority of TCR+ NKT cells, but virtually none of the TCR-NKT cells or the CD4+ conventional T cells, produced IFN-c protein ( Figure 9B). Interestingly, NKT cell activation through LPS injection in vivo was able to induce similar IFN-c production by TCR-NKT cells in comparison to their TCR+ counterparts ( Figure 9B). Our results thus clearly demonstrate that homeostasis and key features defining the nature of NKT cells, namely the unique activated cell-surface phenotype and the innate capacity for instant production of IFN-c, do not require continuous auto-antigen recognition in the mouse.

Discussion
The elucidation of NKT cell function and their intriguing semi-invariant TCR benefited enormously from Va14i-TCR transgenic mouse models [11,32,47,48]. Over the last years, it became increasingly clear that premature expression of transgenic TCRa chains, including Va14i [11,32], leads to various unwanted side-effects such as impaired b-selection and the generation of large numbers of DN T cells both in the periphery and in the thymus [26,27]. This drawback affects even TCR alleles generated through nuclear transfer of mature NKT cells [33]. For that reason, Baldwin et al. developed a system in which a transgenic CAGGS-promoter-driven TCRa-chain is expressed upon CD4-Cre-mediated excision of a loxP-flanked STOP cassette, mimicking the physiologic expression time point [26]. Likewise, Griewank and colleagues expressed the Va14i-TCR under direct control of CD4 promoter and enhancer sequences [11]. These are clear improvements, but carry the inbuilt caveats of the respective heterologous expression construct. For example, it has been shown that a large proportion of activated mature T cells loses expression from such transgenic CD4 promoter enhancer constructs [49].
Here, we present a novel approach, in which the expression of the transgenic Va14i-TCRa-chain, and in the future any other TCRa-chain of interest, can be initiated via CD4-Cre at the DP stage in the thymus, and is under endogenous control of the Tcra locus throughout the lifespan of the cell. In these mice, large numbers of bona fide CD4+ and DN NKT cells were generated. The reduced proportions of fully mature stage 3 NKT cells (NK1.1+, CD69 high , T-bet+), as well as the reduced numbers of NK cells, are most likely a consequence of limiting amounts of common differentiation and maintenance factors, such as IL-15 [14,37,50]. In addition, attenuated TCR-signaling due to increased competition for self-antigen/ CD1d-complexes might delay the full maturation of NKT cells in the transgenic animals. TCR signals have been proposed to Moreover, we observed the generation of tetramer+ CD8+ T cells. CD8+ NKT cells are found in the human, but not in wildtype mice. CD8 expression on Va14i NKT cells does not interfere with negative selection, avidity for antigen presented by CD1d, or NKT cell function [28]. Instead, it was proposed that the absence of CD8+ NKT cells in the mouse is due to the constitutive expression of the transcription factor Th-Pok in all CD4+ as well as DN NKT cells [28]. Th-Pok has been shown to be crucial for the maturation and function of NKT cells, and directly represses CD8 expression [28]. This scenario fits well with the fact that the CD8+ tetramer+ T cells in the CD4-Cre Va14i StopF /wt (as well as in the Va11p-Va14itg animals) did not express Th-Pok. These cells also lack many other characteristic features of NKT cells, including PLZF expression. Therefore, we refer to them as tetramer+ CD8+ T cells.
Given the faithful recapitulation of endogenous TCRa-chain expression timing and strength in our knock-in mice, combined with the extremely high homologous recombination efficiency, we believe that our strategy should prove useful for the generation of further novel TCR-transgenic mouse models. By replacing RAGmediated Va14 to Ja18 recombination with Cre-mediated activation of Va14i expression in CD4-Cre Va14i StopF /wt mice, we can directly couple conditional gain or loss of gene function with Va14i-TCR expression in NKT cells. NKT cell-specific gene targeting in mice with physiological NKT cell numbers could be achieved through the generation of mixed bone marrow chimeras with Ja18 2/2 bone marrow, which cannot give rise to Va14i-NKT cells.
Our studies were designed to elucidate whether or to what extent the expression of the autoreactive semi-invariant TCR would activate a peripheral mature naïve conventional T cell, convert it into an NKT cell, or induce gene expression typical of NKT cells. We took advantage of the conditional nature of the Va14i-TCR knock-in transgene for a TCR switch experiment on conventional peripheral T cells. Naïve CD4+ T cells inherit a high plasticity [51]. Depending on TCR signaling strength and cytokine environment, they can differentiate in various subsets in periphery. This differentiation includes the induction of specific transcription factors, namely T-bet (Th1), GATA-3 (Th2), ROR-ct (Th17), and FoxP3 (peripherally derived regulatory T cells). For NKT cells, it is believed that strong TCR signaling, together with homotypic interactions involving the SLAM family (SLAMf) receptors 1 and 6, ultimately leads to PLZF induction during thymic development [11,13]. DP thymocytes, presenting auto-antigen via CD1d and also expressing SLAMf members, are crucial for thymic NKT cell selection [11]. These SLAMf receptors are expressed on peripheral lymphocytes in comparable levels to double positive thymocytes (www.immgen.org). Therefore, lymphocytes, especially marginal zone B cells, which express CD1d to a similar level as DP thymocytes, should be able to present antigen and SLAMfmediated co-stimulation, to naïve conventional T cells with a newly expressed Va14i-TCR on their surface. The elevated levels of the TCR-induced transcription factor Egr2 in switched tetramer+ T cells suggest that they receive an (auto-)antigenic signal. This finding is in principle in agreement with our finding that tetramer+ TCR-switched T cells are enriched in cells that express Vb2-and Vb8.1-/8.2-containing Va14i-TCRs. These TCRs were shown to have the highest avidity for NKT cell antigens [3]. Furthermore, Vb7-containing Va14i-TCRs were shown to be favored when endogenous ligand concentration are suboptimal in CD1d +/2 mice [42]. In fact, in CD4+ tetramer+ TCR-switched T cells the relative enrichment for Vb7-expressing cells was slightly higher than for Vb2-and Vb8.1-/8.2-expressing cells (unpublished data). However, the interpretation that this advantage is due to antigenic selection is at odds with the fact that Vb7-expressing cells are not enriched in tetramer+ TCR-switched CD8+ T cells. We currently have no satisfactory explanation for this discrepancy. Both CD4+ and CD8+ Va14i-TCR-expressing conventional T cells show features of activation and exhaustion/ anergy, but do not develop into NKT cells, judged by absent PLZF and NK cell marker expression. This indicates that either mature T cells have lost the ability to enter the NKT cell lineage, the peripheral Va14i-TCR signal is not strong enough, or as yet unidentified components of the thymic microenvironment are required to induce an NKT cell fate. Indeed, the high Egr2 expression of mature NKT cells that matured in the periphery and migrated back to the thymus ( Figure 8A) suggests that stronger self-antigens are presented at this location. Interestingly, unlike TCR-switched tetramer+ T cells, Egr2 expression in mature splenic NKT cells was similar to that of conventional mature CD4+ T cells. Our data therefore suggest that in the periphery, the Va14i-TCR can recognize self-lipids, but maturing NKT cells undergo a developmental program that prevents an auto-reactive inflammatory response. At this point, we cannot exclude the possibility that the observed cellular activation was antigenindependent. The fact that the internal control cells, the cotransferred tetramer2 T cells, show no or significantly less signs of activation strongly argues for an involvement of antigen recognition or tonic signaling by the Va14i-TCR. It also remains possible that the transient immune activation caused by the poly(I:C) administration contributes to the observed phenotypes. In all likelihood, this contribution is small, as we never observed any significant immune activation, not to mention loss of CD1dexpressing antigen-presenting B cells and dendritic cells, in Mx-Cre Ca F /wt control mice that received poly(I:C). Despite these caveats, our results clearly show that under our experimental conditions, Va14i-TCR expression on conventional naïve T cells leads to their activation and general immune deregulation.
These findings seemed to support notions that NKT cell maintenance [52], their activated surface phenotype, and especially their rapid cytokine expression abilities might depend on constant antigen recognition [25]. However, by ablating the TCR on mature NKT cells in situ, we unequivocally demonstrated that long-term mouse NKT cell homeostasis and gene expression are nearly completely independent of TCR signals. In this regard, they are similar to memory T and B cells, which can maintain their numbers, identity, and functional capabilities in the absence of antigen [53,54]. Our results are hard to reconcile with a recent report suggesting that NKT cell maintenance requires lipid presentation by B cells [52]. While there might be some differences between mouse and man, a more likely scenario is that the observations of Bosma et al. reflect rather acute local activation than true homeostatic requirements. Most of the known functions of NKT cells critically depend on their ability to rapidly secrete large amounts of many different immune-modulatory cytokines shortly after their activation. Still, it is not fully understood how NKT cell activation is triggered in different disease settings, and especially to what extent signaling in response to TCR-mediated recognition of antigens versus activation by proinflammatory cytokines contributes to this. Various studies reported that CD1ddependent signals were required for full NKT activation in vitro [19,20,55], although most of them contained the caveat of potentially incomplete blockade of CD1d function by blocking antibodies. Our experiments, in line with a recent report [21], show that even in the complete absence of TCR signaling for 4 wk, NKT cells can be robustly activated in vivo to produce IFNc upon LPS injection in similar amounts as their TCR+ counterparts. Thus, we demonstrate that in mouse NKT cells continuous steady-state TCR-signaling is not required to maintain the Ifng locus in a transcriptionally active state, as recently proposed for human NKT cells [25]. Therefore, our results clearly demonstrate that cellular identity and critical functional abilities of mature NKT cells, such as steady-state proliferation and innate cytokine secretion ability, although initially instructed by strong TCR signals, do not require further antigen recognition through their TCR.
Collectively, our data strongly support the view that Va14i-TCR expression on developing NKT cells triggers a program that makes them unresponsive to peripheral self-antigens, which can continuously be recognized by their auto-reactive TCR. NKT cells are extremely potent immune-modulatory cells that upon activation can instantly secrete a large array of cytokines. Although they are selected by high affinity to auto-antigens, similar to regulatory T cells, they are not mainly suppressive cells. Therefore, it seems plausible that NKT cells are rendered ''blind'' to peripheral auto-antigens, rather than depend on continuous stimulation by self-lipids to maintain their cellular identity and innate functions. By keeping their activated state independent of self-antigen recognition, NKT cells can stay poised to secrete immune-activating cytokines while minimizing the risk of causing damage to self during normal physiology. On the other hand, the presence of the auto-reactive Va14i-TCR serves to detect pathogenic states when a stronger signal is generated by the enhanced presentation of potentially more potent self-antigens or foreign lipids.

Genetically Modified Mice
To generate Va14i StopF mice, B6 ES cells (Artemis) were transfected, cultured, and selected as previously described for Bruce 4 ES cells [56]. Mx-Cre [39], Ca F [40], CD4-Cre [57], Nestin-Cre [31], Va11p-Va14i-tg [32], and Va14i StopF mice were kept on a C57BL/6 genetic background. As we did not observe any differences between CD4-Cre and Va14i StopF /wt mice in NKT cell biology, they were sometimes grouped together as controls. Mice were housed in the specific pathogen-free animal facility of the MPIB. All animal procedures were approved by the Regierung of Oberbayern.

Cre Induction in Mx-Cre Animals
At the age of 6-8 wk (or 2 wk after cell transfer for the TCR switch experiment), animals were given a single i.p. injection (400 mg) of poly(I:C) (Amersham). All mice were analyzed 6-8 wk after injection, unless otherwise indicated. . PLZF antibody and the CXCL16-Fc fusion were generous gifts from Derek Sant'Angelo and Mehrdad Matloubian, respectively. mCD1d-tetramers were provided by the NIH tetramer core facility. For intracellular transcription factor stainings, cells were fixed and permeabilized with the FoxP3 staining kit (eBioscience). For intracellular cytokine stainings, mice were injected i.v. in the tail vein with 40 mg of LPS (Sigma) or 2 mg aGalCer (Funakoshi) in a total volume of 200 ml PBS. Afterwards, cells were treated according to manufacturer's instructions with the Cytofix/Cytoperm kit (BD). For multiplex measurement of cytokines in the serum, we used the mouse Th1/ Th2 10plex Cytomix kit according to manufacturer's instructions (eBioscience). Samples were acquired on a FACSCanto2 (BD) machine, and analyzed with FlowJo software (Treestar). The heat map was generated using perseus (part of the MaxQuant software [58]).

BrdU Incorporation
Mice were fed with 0.5 mg/ml BrdU (Sigma) in the drinking water for 4 consecutive weeks. Directly afterwards, BrdU incorporation was analyzed with a BrdU Flow Kit (BD).

ELISA
Serum TNF levels were determined by ELISA as recommended by the manufacturer (BD).

Quantitative RT-PCR
RNA was isolated (QIAGEN RNeasy Micro Kit) and reverse transcribed (Promega) for quantitative real-time polymerase chain reaction (PCR) using probes and primers from the Universal Probe Library (Roche Diagnostics) according to the manufacturer's instructions.

Statistics
Statistical analysis of the results was performed by one-way ANOVA followed by Tukey's test, or by student t test, in Prism software (GraphPad). The p values are presented in figure legends where a statistically significant difference was found.