NKT Cells in Mice Originate from Cytoplasmic CD3-Positive, CD4−CD8− Double-Negative Thymocytes that Express CD44 and IL-7Rα

Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4−CD8− (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25− (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44−CD25− (DN3) and CD44− CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3− and surface CD3+TCR-β− cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.

T lymphocytes expressing NK cell lineage markers (NK1.1, CD56) are referred to as NKT lymphocytes and have characteristics of both T and NK cells 1 . NKT cells are a unique and small subset of regulatory T cells. NKT cells recognize glycolipid antigens, such as α-galactosylceramide (αGalCer), bridge innate and adaptive immunity and modulate immune responses in autoimmunity, malignancies and infection [2][3][4] . NKT cells can produce large amounts of both Th1 and Th2 cytokines as an immediate response to TCR ligation 5,6 . However, NKT cells have also been shown to display cytotoxic activity, in a mechanism similar to that of NK cells 7 . In adult mice, subsets of immature double-negative thymocytes, termed DN 1 and DN 2 , have NK-cell potential 8,9 . Previous studies demonstrated that T and NK cells were derived from a common precursor. Although NK1.1 + T cells may have a developmental pathway similar to that of T and NK cells, it has not been clear where NK1.1 + T cells branch off from this common pathway 10,11 . A previous study showed that NKT cells likely develop from DP cells 12    CD3 chains play essential roles in intracellular assembly, transport of TCR-CD3 complex to cell surface, receptor internalization and differential signal transduction. The pre-TCR and TCR contain four distinct signal-transducing subunits-CD3γ, CD3δ, CD3ε, and ζ_α [14][15][16] . CD3 chain specific transcripts are expressed as early as DN 1 stage and almost all the DN 3 thymocytes express high amounts of clonotype-independent CD3 complexes (CIC) in pro-thymocytes before the expression of pTα and TCRβ 17 . The intracytoplasmic (IC) domain of CD3 is essential for early thymocyte maturation and deletion of the CD3ε intracellular chains, but not the CD3γ or CD3δ intracellular domains results in a block in early thymocyte development 18 . While some previous studies show that intracellular CD3 are expressed as early as DN 1 stage, and all CD25 + cells express intracellular CD3 19 , many other reports say that intracellular CD3 was first detected in CD25CD44 lo CD117 lo thymocytes 20 . The TCR lacks intracellular signaling domains, but interacts with CD3 subunits, which possess intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation 21,22 .
Interleukin (IL)-7 is a cytokine essential for lymphocyte development and survival, and stimulates the proliferation of thymocytes, and mature T cells through an interaction with its high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily 23 . IL-7R consists of a common cytokine receptor γ-chain (γ c ) and a unique IL-7R α-chain (IL-7Rα). It is reported that γ c -deficient mice lack thymic NKT cells 24 . In contrast, the proportion of NKT cells is not reduced in the IL-7-or IL-7Rα-deficient thymus, although absolute numbers of thymic NKT cells are drastically reduced. Matsuda et al. observed that IL-7/IL-15 doubly deficient mice exhibited more severe reduction in NKT cells than IL-15-deficient mice 25 . Nonetheless, the roles of the IL-7R in development of NKT cells in the thymus remain unclear, and little is known about the roles of IL-7Rα in different stages of T-cell development.
CD44 is a widely distributed cell surface marker and cell adhesion molecule in normal adult and fetal tissues. CD44 has the potential to participate in several processes associated with the induction of cell motility, activation of cell survival responses, and promotion of cell adhesion 26 . Various reports have implicated CD44 in the development and function of the immune system and its expression during a particular stage or in a subset of thymocyte progenitors suggests its functional involvement in the homing and thymic colonization of precursor cells 27 . CD44 and lymphocyte activation and its relatively high level of cell surface expression on NKT cells have been observed 28 . Our previous study demonstrated that CD44 expression positively correlated with Foxp3 expression and suppressive function of CD4 + T regs 29 . In this study, we examined NK1.1, CD44, surface CD3, and cytoplasmic CD3 expression in thymocytes, and found that NK1.1 expression was correlated with CD44 and IL-7Rα expression in NKT cells. T and NKT cells developed from CD4 − CD8 − T progenitor cells through different pathway.

Materials and Methods
Experimental animals. C57BL/6 mice were supplied by the Laboratory Animal Center of Xi'an's Jiaotong University. Female mice were studied at 5 weeks old, and maintained under specific pathogen-free conditions. All studies were approved under the Institutional Animal Care and Use Committee at Xi'an Jiaotong University protocol (approval number: xjtu2018-005), and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Detection DN cells by improved flow cytometry. TCR regulate T-cell proliferation and differentiation
in the thymus and the periphery 30,31 . While flow cytometry is one of the most important research methods for examining T cell development, traditional flow cytometry cannot accurately predict percentages for cells that are expressed below a certain percentage or a certain amount. CD4 − CD8 − DN cells comprise only 3-5% of the total thymocytes within the thymus 32 . We here used an advanced flow cytometry method to explore DN-T and T cell development in the thymus 33 . Furthermore, the expression of many genes, such as the TCR-β gene is very low in DN thymocytes; therefore, accurate detection of protein molecules in various stages of DN thymocytes by flow cytometry is challenging. As shown in Fig. S1. Therefore, using this improved the flow cytometry detection method (5 × 10 6 thymocytes were collected for each sample). Moreover, lower expression protein molecules in each subpopulation of DN cells could be detected to reveal previously uncharacterized data on subsets of DN cells.

Flow cytometric method for elimination of contaminating cells within DN thymocytes.
Traditionally, contaminated cells (non-T-cell lineages) must be removed by specific blocking antibodies before detection of DN cells. We found cytoplasmic CD3 was expressed in the majority of DN thymocytes, and removed contaminating cells by the cytoplasmic CD3 gated (a detection software technology of flow cytometry) and then analyzed protein molecules in DN thymocytes (Fig. S2). The methods can be used to detect the DN thymocytes and remove contaminating cells (such as CD11b, B220).

Statistical analysis.
Results are presented as the mean and standard deviation. The software of GraphPad Prism was used in all analysis. More than three independent experiments were performed. The Tukey ' test was used to compare 3 or more means and a two-tailed unpaired t test was used to compare 2 groups. P ≤ 0.05 was considered to indicate a statistically significant difference between values. Statistically significant values are given in all figures.

Results
Surface CD3 and NK1.1 expression in thymocytes is higher within DN than DP thymocytes.
Surface CD3 and NK 1.1 expression is highest within DN 1 and DN 2 populations. Natural killer T (NKT) cells are a unique subset of T cells that express both T cell receptor (TCR) and NKR-P1B/C (NK1.1; CD161) on their surfaces but at lower density than conventional T cells and NK cells, respectively 1 . The αβ-TCR is part of a multi-chain complex that is composed of the peptide-MHC-binding TCRα and β chains that are  (Fig. 2B). The number of NKT cells was much higher in DN 1 (16%) than DN 2 , DN 3 , and DN 4 cells (0%) (Fig. 2C). These data suggest NKT cell development may initiate from DN 1 and DN 2 without passing through DN 3 or DN 4 stages.
Maturational stages of NKT cells can be followed by differential expression of cytoplasmic and surface CD3 in thymocytes. Cells from the murine thymus were stained with CD25 (PE-Cy7), CD4 (FITC), CD8 (PerCP), CD44 (APC-Cy7), NK1.1 (APC), CD3 (PE), and CD3 (PE-Texas Red) and then they were analyzed by flow cytometry. NK1.1 and surface CD3 expression were examined first (Fig. 4). The fluorescence intensity mean of surface CD3 in DN-T cells was significant lower (645) than in mature T cells (CD4-CD8+ cells in thymus: 8234 and CD8+ cells in spleen: 9039) (Fig. 4A). The fluorescence mean of surface CD3 in DN-NKT cells was significant lower (1634) than in mature NKT cells (CD4-CD8+ cells in thymus: 4902 and CD8+ cells in spleen: 9039) (Fig. 4B). The flow cytometry data from more than three independent experiments performed in duplicate is shown (Fig. 4C,D). Cytoplasmic CD3 expression was also examined for confirmation of our findings (Fig. 5). The fluorescence intensity mean of cytoplasmic CD3 in DN-T cells was significant lower (1845) than in mature T cells (CD4 − CD8 + cells in thymus: 13,268 and CD8 + cells in spleen: 14,328) (Fig. 5A). The fluorescence intensity mean of cytoplasmic CD3 in DN-NKT cells was also significant lower (854) than in mature NKT cells (CD4-CD8+ cells in thymus: 14373 and CD8 + cells in spleen: 15108) (Fig. 5B). The flow cytometry data from more than three independent experiments is shown (Fig. 5C,D). These data suggest that the maturation states of T cells could be compared with the fluorescence intensity mean of cytoplasmic and surface CD3. The NKT cells in DN thymocytes showed the least mean fluorescence intensity of cytoplasmic and surface CD3 within thymocytes.

Discussion
In this study, we used an advanced flow cytometry method to explore NKT cell development in the thymus. We examined changes in the expression of CD25, CD44, CD3, and NK1.1 in thymocytes. The use of specific mAbs allowed us to analyze NKT cells in the thymus. The level of NK1.1 expression was highest in CD4 − CD8 − (DN) cells, after that CD4 + CD8 − and CD4 + CD8 + cells (Fig. 1). The level of NK1.1expression was highest in DN 1 cells, after that DN 2 , while DN 3 and DN 4 cells did not express NK1.1 ( Fig. 2A). Similarly, the number of NKT cells was highest in DN 1 cells, and could not be identified in DN 2 , DN 3 , and DN 4 cells (Fig. 2B). Cytoplasmic CD3 was expressed in various stages of DN thymocytes and in various stages of thymocytes (Fig. 3). This result suggests that cytoplasmic CD3 could be used as a marker for early stage of T cells. From the above research, we found that  (Figs 4 and 5), suggests that the maturation states of T cells could be compared with the fluorescence mean of cytoplasmic CD3 and surface CD3.NKT cells in DN thymocytes showed the least fluorescence intensity mean of cytoplasmic and surface CD3 within thymocytes. T cells expressing CD3 did not show that it must be mature cells, such as CD3 + DN-T cells and CD3 + DN-NKT cells, which express weak strength of the fluorescence mean CD3 are immature cells. The number of NKT cells in DN-cytoplasmic CD3 was higher than in DN-surface CD3 cells (Fig. 6), this result suggest at least part of NKT cells in DN thymocytes are immature cells. CD3 expression was higher than TCR-β in DN 1 thymocytes. The number of CD3-NK1.1 cells was higher than TCR-β-NK1.1 cells in DN 1 thymocytes (Fig. 7). These results suggest that there are not only mature T cells but also immature T cells in surface CD3 positive DN 1 thymocytes, some of them do not express TCR-β and are immature NKT cells. NKT cells development may from DN thymocytes. NK1.1 expression and the number of NKT cells were correlated with CD44 expression in the murine thymus (Fig. 8). The surface IL-7Rα expression correlates with NK1.1, and CD44 expression (Fig. 9). Additionally, IL-7Rα was expressed at much higher levels in NKT cells than in T cells (Fig. 10). The discovery that there was more IL-7Ra expression in NKT cells than in conventional T cells is important with potential clinical implication. This would imply that IL-7Ra deficient patients would suffer from a more profound NKT deficiency than originally thought. We recently studied T reg cells and examined Foxp3 expression in Thymus by flow cytometry and found T reg cell development does not accord with the theory of DN T cell development (DN 1 , DN 2 , DN 3 , DN 4 ), nor the theory of T-cell development (DN, DP, SP). In the present study, we found that NKT cells like T reg cells develop in the thymus. NKT cell development may involve transition from DN-T progenitor cells directly to SP cells. Surprisingly, both NKT cells and T regs are the phenotype of T cell; yet do not share the T cell development pathway. Although NKT cells are present in the thymus, the developmental origin of these cells are controversial. Previous studies showed that NKT cell development was probably from CD8 cells 36 , DP cells 12,37 , CD4 cells 38,39 , and BM cells 40 . However in the present study, we found that NKT cells within DN thymocytes, much like DN T cells, expressed lower levels of fluorescence mean of CD3. DN 2 and DN 3 did not express surface CD3, but expressed higher level of cytoplasmic CD3. There is a gradual expression of CD3 from weak to strong and from intracellular to surface in T cell development, especially in the early stage, although some cells weakly express CD3 but are immature cells, such as in CD3 (30%), TCRβ (17%) in DN 1 thymocytes (Fig. 7C). If TCR-β is the marker for mature T cells, then half of CD3 T cells could be classified as immature in DN 1 thymocytes; therefore, it is unlikely that all CD3-expressing cells are mature cells.
There are not only mature T cells but also immature among cells in surface CD3 positive DN 1 thymocytes. Some of them do not express TCR-β and are immature surface CD3 + TCR-β − NKT cells (Fig. 7D). Furthermore, the number of cytoplasmic CD3-NKT cells was significantly higher than the surface of CD3-NKT cells in DN 1 thymocytes, which also suggest that NKT cells within DN thymocytes are immature cells, at least some of them are immature (cytoplasmic CD3 + surface CD3 − ) NKT cells. Although previous studies have indicated that DN-NKT cells are mature cells, this conjecture is only derived from indirect evidence, strong direct evidence is still needed to confirm this observation. If you want to prove that NKT cells derived from mature cells, you must to prove that no immature cells in the DN thymocytes. Our study has proved very clear that the development of NKT can be derived from the immature cytoplasmic CD3 + surface CD3 − and surface CD3 + TCR-β − DN 1 thymocytes. In this study, we found a higher population of NKT cells are immature cells in the DN 1 stage. NKT cells may develop from DN progenitor, involved the cytoplasmic CD3 + DN 1 , DN 2 stage, yet without the involvement of the DN 3 , DN 4 , and CD4 + CD8 + (DP) stages. We propose that NKT cells in mice may development originate from cytoplasmic CD3 positive, CD44 and IL-7Rα expressing DN thymocytes.