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The Journal of Immunology, 2004, 172: 6093-6100.
Copyright © 2004 by The American Association of Immunologists

Restoration of NK T Cell Development in fyn-Mutant Mice by a TCR Reveals a Requirement for Fyn During Early NK T Cell Ontogeny 1

Paul Gadue2,3,*, Liqun Yin3,{dagger}, Sumesh Jain{ddagger} and Paul L. Stein4,{dagger}

* Graduate Group in Immunology and {dagger} Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104; and {ddagger} Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1– NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn–/– mice exhibit a similar turnover rate as wild-type cells. The fyn–/– NK T cells respond to {alpha}-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR {alpha}-chain V{alpha}14J{alpha}18 leads to a complete restoration of NK T cell numbers in fyn–/– mice. Together, these results suggest that Fyn may have a role before {alpha}-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the V{alpha}14J{alpha}18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn–/– lymphoid populations to respond.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer T cells are a lymphocyte population with characteristics of both NK cells and T lymphocytes. The majority express a TCR with a specific TCR {alpha}-chain rearrangement, V{alpha}14J{alpha}18 (V{alpha}14) 5 (1, 2). They also express markers specific to NK cells such as NK1.1 and members of the Ly-49 family (3). NK T cells can recognize glycolipid Ags bound to the MHC-related molecule CD1d (4). These cells have an activated phenotype and can be induced to rapidly secrete multiple cytokines of both the Th1 and Th2 types, such as IFN-{gamma} and IL-4, respectively (5, 6). This rapid activation has led to the hypothesis that NK T cells are a more innate cell type, which may then influence the ensuing immune response (7). In addition, multiple studies have demonstrated that NK T cells can have important roles in the prevention of autoimmune disease, immune tolerance, as well as the regulation of certain cancers (3, 8, 9).

NK T cells exhibit similarities to both NK and T lymphocytes in their developmental requirements. Like NK cells, NK T cells require IL-15, lymphotoxin {alpha}{beta}, and the Ets-1 transcription factor for proper development (10, 11, 12). As with conventional T cells, NK T cell development requires the pre-TCR and the tyrosine kinase Lck (13, 14, 15). In addition, NK T cells are thymically derived, although a minor population may develop extrathymically (3, 8).

The study of genes uniquely required for NK T cell development, but not involved in conventional T cell or NK cell ontogeny, may help elucidate specific developmental requirements for NK T cells. This lymphoid population is selected by CD1d, presumably on double-positive thymocytes (16, 17), and mice that lack CD1d expression are found to have a selective deficiency in NK T cell numbers (18, 19, 20). To date, the fyn–/– mouse represents the only mutation to selectively affect NK T cells while leaving other lymphocyte subsets intact. The fyn-mutant mice exhibit a 5- to 10-fold reduction in the numbers of CD1d-restricted NK T cells (14, 15) due to a cell intrinsic defect (14).

The paucity of NK T cells in fyn–/– mice posed a number of questions regarding the function of this tyrosine kinase in NK T cells. First, is Fyn required during ontogeny, in peripheral survival and maintenance, or both? Second, what is the role of Fyn in mature NK T cell activation? In this study, it is reported that a transgene encoding the {alpha}-chain of the NK T cell-specific V{alpha}14 TCR is capable of completely restoring NK T cell development in fyn mutants. This suggests Fyn is required early in NK T cell development, before TCR {alpha}-chain rearrangement. This finding is also supported by directly examining NK T cell progenitors in nontransgenic mice using {alpha}-galactosylceramide ({alpha}GalCer)-loaded CD1d tetramers. There is a decrease of both immature NK1.1– and mature NK1.1+ CD1d-restricted thymocytes in fyn–/– mice. In addition, both wild-type (wt) and fyn–/– NK T cells exhibit similar 5-bromo-2'-deoxyuridine (BrdU)-labeling kinetics. These studies indicate that Fyn is not required to maintain NK T cell numbers in the periphery. The few NK T cells present in fyn–/– animals are capable of secreting cytokines at almost wt levels in response to TCR stimulation in vivo. However, proliferation induced by the NK T cell Ag {alpha}GalCer is decreased in the fyn mutants. Finally, the secondary activation of other lymphocytes by NK T cells is abrogated in Fyn-deficient mice because of decreased NK T cell numbers, and not due to intrinsic defects in the other lymphoid populations. Thus, the primary role for Fyn appears to be in regulating the early events underlying NK T cell ontogeny.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The generation of fyn–/–, lck–/–, and V{alpha}14 transgenic mice has been described previously (21, 22, 23). The lck–/–, V{alpha}14 transgenic, and lck–/– V{alpha}14 were maintained on a C57BL/6 background. Most fyn–/– mice on the C57BL/6 background die a few weeks after birth due to hydroencephaly (P. Gadue and P. L. Stein, unpublished observation). To obtain mice with matched genetic backgrounds, the following cross was performed. Fyn-null mice on a mixed background (129/Sv x C57BL/6) were mated to fyn+/– V{alpha}14 transgenic mice on the C57BL/6 background. The resulting offspring of various genotypes were then used in all experiments unless noted otherwise. To ensure that genetic background differences were not influencing results, a limited number of fyn–/– mice on the C57BL/6 background were also examined and found to have similar phenotypes as those on the mixed genetic background. All mice used were between 4 and 12 wk of age unless otherwise noted and maintained under American Association of Laboratory Animal Care and institutional guidelines.

Abs and reagents

The following Abs were used for flow cytometry: anti-TCR{beta}-FITC (H57-597), anti-NK1.1-PE (PK136), anti-NK1.1-biotin (PK136), anti-CD45.1-FITC (A20), Fc Block (2.4G4), anti-Thy1.2-FITC (53-2.1), anti-TCR V{alpha}2-FITC (B20.1), anti-TCR V{alpha}8.3-FITC (B21.4), anti-TCR V{alpha}11.1/11.2-FITC (RR8-1), anti-heat-stable Ag (HSA)-biotin (M1/69), anti-IL-4-PE (BVD4-1D11), anti-IFN-{gamma}-PE (XMG1.2), anti-CD69-FITC (H1.2F3), anti-B220-PE (RA3-6B2), and streptavidin-CyChrome were from BD PharMingen (San Diego, CA). The following Abs were obtained from hybridoma supernatants: anti-macrophage (F4/80), anti-Mac1 (M1/70.15.11.5.HL), anti-MHC II (M5/114.15.2), and anti-CD8 (2.43). The {alpha}GalCer was obtained from Kirin Brewery (Gunma, Japan) as a 220 µg/ml stock in 0.5% polysorbate-20 and 0.9% NaCl. The conditioned medium from J558 cells was used as a source of IL-7. PE-labeled CD1d tetramer loaded with {alpha}GalCer was a gift from Dr. M. Kronenberg (La Jolla Institute for Allergy and Immunology, La Jolla, CA) (24).

BrdU labeling

The 2.5-wk-old fyn–/– and C57BL/6 mice were injected i.p. with 100 µl of a 10 mg/ml solution of BrdU (BD PharMingen), and then sacrificed 3 days later. Lymphocytes from thymus, spleen, and liver were first stained with {alpha}GalCer-CD1d tetramer-PE, and then positively selected using anti-PE MicroBeads (Miltenyi Biotec, Auburn, CA) to enrich for NK T cells. This was particularly important for analyzing fyn–/– mice, because so few NK T cells are present. The cells were then stained with TCR{beta}-CyChrome, NK1.1-allophycocyanin, and BrdU-FITC (BrdU flow kit; BD PharMingen), following the manufacturer’s instructions, and analyzed by flow cytometry.

Construction of competitive chimeric mice

To distinguish between wt and fyn–/– cells, V{alpha}14 TCR transgenic mice were mated to CD45.1-congenic mice (The Jackson Laboratory, Bar Harbor, ME). Bone marrow (BM) cells from the first generation V{alpha}14/CD45.1 mice were mixed with fyn–/– V{alpha}14 BM cells at different ratios. A total of 6 x 106 cells were injected into irradiated C57BL/6 mice. Lymphocytes from thymus, spleen, and liver were isolated after 1 mo, and then stained with {alpha}GalCer-CD1d tetramer-PE, anti-TCR{beta}-CyChrome, and anti-CD45.1-FITC, and analyzed by flow cytometry.

Flow cytometry

Single-cell suspensions of thymocytes and splenocytes were obtained by mashing tissue between two frosted microscope slides. Splenocytes were also depleted of RBC by 0.14 M ammonium chloride treatment. Liver lymphocytes were isolated as described elsewhere (25). All cells were treated in Fc Block for 15 min at 4°C. Intracellular staining was performed using the Cytofix/Cytoperm kit (BD PharMingen) per the manufacturer’s protocol. Three-color immunofluorescence analysis was performed using a FACScan flow cytometer and analyzed using CellQuest software (BD Biosciences, San Jose, CA).

{alpha}GalCer stimulations

The {alpha}GalCer stock was diluted in PBS to a final concentration of 53 µg/ml. Mice were injected with 150 µl (8 µg) of {alpha}GalCer or vehicle i.v. via the retro-orbital sinus. The mice were then sacrificed either 2 or 6 h later, as indicated in the individual experiments. Organs were harvested and stained for flow-cytometric analysis. For the in vitro proliferation assay, splenocytes were enriched for NK T cells by immunodepletion as follows. Cell suspensions were incubated with F4/80, anti-Mac1, anti-MHC II, and anti-CD8 for 30 min and washed; then BioMag goat anti-rat IgG (PolySciences, Warrington, PA) was added, and cells positive for the above Abs were magnetically removed. The enriched cells were labeled with CFSE (Molecular Probes, Eugene, OR). In brief, cells were incubated with 10 µM CFSE in DMEM at 37°C for 10 min, and then washed with PBS. The enriched CFSE-labeled splenocytes were then stimulated with IL-7 or IL-7 plus {alpha}GalCer on irradiated fyn–/– splenocytes as a source of APCs as indicated in Results. Without the addition of IL-7, significant numbers of NK T cells did not survive the culture period in the absence {alpha}GalCer (data not shown). After 4 days, live cells were harvested by centrifugation through Lympholyte-M (Accurate Chemical and Scientific, Westbury, NY), and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fyn-mutant mice have decreased numbers of NK1.1– immature NK T cell progenitors

Several studies indicate that NK T cells go through a stage in development where they express the V{alpha}14 TCR, but lack NK cell markers such as NK1.1 and members of the Ly-49 family. First, mice that lack the common cytokine receptor {gamma}-chain have cells in the thymus that use the NK T cell invariant TCR but do not express NK cell markers (26). Second, most CD4–CD8– (double-negative) TCR+ thymocytes present in adult mice express NK1.1 and use the V{alpha}14J{alpha}18 rearrangement (3). However, in 4-wk-old mice, a large percentage of double-negative TCR+ cells do not express NK1.1 (27), and other studies suggest that a burst of NK T cell development occurs at this age (17, 28). Finally, more recent studies using CD1d tetramers also find that immature NK T cells are negative for NK markers (29, 30, 31, 32).

The majority of NK T cells will bind tetramers of CD1d loaded with a specific ligand, {alpha}GalCer (24, 33). Young fyn–/– mice were examined to determine whether the immature NK1.1-negative, NK T cell progenitors, were also affected by the fyn mutation. Fyn–/– and fyn+/– littermates aged 4 or 16 wk were stained with CD1d tetramer and NK1.1, and then analyzed by flow cytometry. Fig. 1A shows expression of NK1.1 on tetramer-positive cells. The vast majority of tetramer-positive cells from adult fyn+/– or fyn–/– mice express NK1.1. A similar distribution was noted in wt mice (24, 33). However, at 4 wk of age, only half of the tetramer-positive cells express NK1.1 in both fyn+/– and fyn–/– mice. Significantly, both the NK1.1+ and NK1.1– populations are decreased to a comparable extent in the fyn–/– mice (Fig. 1B). Similar results were obtained with C57BL/6 congenic fyn mutants (data not shown), indicating that the results were not due to strain differences. Mice heterozygous for Fyn expression were found to be identical with wt for all experiments in this report (data not shown). These data suggest that Fyn is required relatively early in NK T cell ontogeny, before NK1.1 marker up-regulation.



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  		FIGURE 1. Both NK1.1+ and NK1.1– CD1d tetramer-binding cells are decreased in Fyn-deficient mice. Thymocytes from 4- or 16-wk-old fyn+/– and fyn–/– were isolated and depleted of CD8 cells. The remaining cells were then stained for NK1.1 and {alpha}GalCer-loaded CD1d tetramer and analyzed by flow cytometry, collecting 5000 tetramer-positive events per sample. A, NK1.1 expression on tetramer+-gated cells from the indicated mice. The percentage of tetramer+ cells that express NK1.1 is shown. The data presented are representative of five independent experiments. B, The absolute number of NK1.1+tetramer+ and NK1.1–tetramer+ cells is shown as a percentage relative to wt mice (n = 5).

 
Previous studies showed that NK T cell expansion occurs predominantly when mice are 2–3 wk of age. At that time, ~20% of the thymic NK T cells label with BrdU, whereas <10% of the cells in the adult will label with BrdU (17). To evaluate whether decreased NK T cell numbers in the fyn mutant represented altered turnover of NK T cell progenitors, 2.5-wk-old mice were injected with BrdU, and then sacrificed 3 days later. The NK T cells isolated from thymus, spleen, and liver of both wt and fyn–/– mice had similar BrdU-labeling profiles (Fig. 2). Furthermore, no differences were noted between wt and fyn–/– mice when the BrdU+ population was analyzed for the distribution of immature (NK1.1–) and mature (NK1.1+) NK T cells (data not shown). Thus, although few NK T cells develop in Fyn-deficient mice, their homeostasis appears normal. These results suggest that generation of NK T cells from an early progenitor might be impaired in fyn mutants.



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  		FIGURE 2. Incorporation of BrdU into NK T cells from fyn–/– and C57BL/6 mice. Percentage of BrdU incorporation by {alpha}GalCer-CD1d tetramer+TCR{beta}+ cells in the thymus, spleen, and liver of fyn–/– and C57BL/6 mice. Representative data from one of three experiments are shown.

 
A V{alpha}14J{alpha}18 transgene completely restores the development of NK T cells in Fyn-deficient mice

Transgenic expression of the invariant NK T cell V{alpha}14 TCR {alpha}-chain leads to a 5- to 10-fold increase in the number of NK T cells (23). This transgene was used to help elucidate the function of Fyn in NK T cells. If Fyn is required for peripheral expansion or survival, then it would be anticipated that expression of the transgene would have a minimal effect on NK T cell numbers in fyn-mutant mice. However, if Fyn is required early in NK T cell development, before {alpha}-chain rearrangement, then forced expression of the {alpha}-chain may bypass the necessity for Fyn, leading to normal NK T cell numbers.

Transgenic and littermate controls from fyn+/– and fyn–/– mice were analyzed by flow cytometry to determine the number of NK T cells in the thymus, spleen, and liver. Analysis of liver lymphocytes indicated that both the fyn+/– and fyn–/– mice with the V{alpha}14 transgene have equivalent numbers of NK T cells (Fig. 3A). In contrast, nontransgenic fyn–/– mice have a >5-fold reduction in NK T cells relative to fyn+/– or wt mice. Absolute numbers of NK T cells in the thymus and spleen were similar in fyn+/– or fyn–/– V{alpha}14 mice (Fig. 3B). In both cases, NK T cell numbers were increased ~5-fold over nontransgenic wt mice. Conventional T cell numbers were similar in wt and fyn-mutant V{alpha}14 transgenic mice. To determine whether the NK T cells identified in the transgenic mice were CD1d reactive, NK1.1+TCR{beta}+ thymocytes were analyzed using {alpha}GalCer-loaded CD1d tetramers. Approximately 98% of the NK T cells isolated from transgenic fyn-mutant as well as transgenic wt mice bound tetramer (Fig. 3C). Therefore, expression of the V{alpha}14 transgene can support a complete rescue of NK T cell numbers in mice lacking Fyn. The cell surface phenotype of the NK T cells from fyn+/– and fyn–/– V{alpha}14 transgenic mice was also examined. Mature NK T cells are known to express high levels of CD44, have a proportion of Ly-6Chigh cells, are CD4+CD8– or CD4–CD8–, and skew their V{beta} repertoire toward V{beta}8.2 (3). Consequently, NK T cells from fyn+/– and fyn–/– V{alpha}14 transgenic mice were analyzed for the expression of CD44, Ly-6C, CD4, CD8, and TCR V{beta}8.2, and found to have similar expression patterns (data not shown). Thus, the NK T cells from fyn–/– V{alpha}14 transgenic mice express the appropriate markers and exhibit a mature phenotype.



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  		FIGURE 3. The V{alpha}14J{alpha}18 transgene rescues NK T cell development in fyn–/– mice. Thymocytes, splenocytes, and liver lymphocytes were isolated from mice with the indicated genotypes and stained for TCR{beta}, NK1.1, and HSA. A, Liver lymphocytes from mice were then gated on HSA-low cells, and dot plots of TCR vs NK1.1 are displayed. The percentage of NK1.1+TCR{beta}+ cells are indicated in the upper right quadrant of each plot. The data presented are representative of four independent experiments. B, The absolute number of NK T cells in the spleen and thymus from the indicated genotypes is shown. This was calculated by multiplying the percentage of TCR{beta}+NK1.1+ cells by the total number of cells obtained from each organ (n = 5). C, Thymocytes were gated on NK1.1+TCR{beta}+ cells, and the percentage of {alpha}GalCer-CD1d tetramer+ cells is indicated in histogram plots. The data presented are representative of four independent experiments.

 
Restoration of NK T cell numbers in the transgenic mice could be due to a complex interplay between endogenous TCR {alpha}-chains and the V{alpha}14 transgene. The endogenous TCR {alpha}-chains may help select for NK T cells, and then final maturation and function may be imparted by the V{alpha}14 transgene. This scenario implies that the NK T cells found in the transgenic mice would express endogenous TCR {alpha}-chains. Therefore, NK T cells from wt, V{alpha}14, and Fyn V{alpha}14 transgenic mice were stained with a mixture of Abs that recognize TCR V{alpha}2, V{alpha}8.3, and V{alpha}11.1/11.2. Together, these Abs bind 15–20% of the conventional T cell pool. In contrast, there was virtually no staining on the NK T cells from any of the mice examined (Table I). This indicates that, in all likelihood, there are no endogenous TCR {alpha}-chains expressed on the V{alpha}14 transgenic NK T cells, and that development/survival of NK T cells in the transgenic background is probably conferred by the transgenic V{alpha}14 TCR chain, rather than by endogenous TCR {alpha}-chains. These observations are consistent with a previously published report demonstrating that the V{alpha}14 transgene is capable of supporting NK T cell development in TCR C{alpha} mutants that cannot express endogenous {alpha}-chains (23).


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  		Table I. Percentage of mature thymocytes and NK T cells expressing the V{alpha}2, 8.3, 11.1/11.2 TCR{alpha} chainsa

 
The large pool of NK T cell progenitors created by the V{alpha}14 transgene may mask an important role for Fyn during their development. This might be reflected as differences in survival/development between wt and mutant cells. To test this, a series of competitive chimeric mice were constructed. BM cells from V{alpha}14/CD45.1-congenic mice were mixed with fyn–/– V{alpha}14 BM cells at different ratios, and then injected into irradiated hosts. After 1 mo, the relative contribution of wt NK T cells was assessed by determining the percentage of NK T cells that were positive for the CD45.1 allele. Lymphocytes were isolated from thymus, spleen, and liver, and then the percentage of CD45.1+tetramer+TCR{beta}+ cells was determined. At all ratios examined, the percentage of wt cells approximated the expected value (Table II). This indicates that, under the conditions studied here, the transgenic wt and fyn–/– NK T cell progenitors are capable of contributing equivalently. These data are consistent with a role for Fyn early in NK T cell development, but once this block is circumvented by the V{alpha}14 transgene, further development and peripheral homeostasis appear to be normal.


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  		Table II. Contribution of NK T cells in competitive chimeric micea

 
The V{alpha}14J{alpha}18 transgene leads to a minor increase in NK T cells in Lck mice

Lck, the other Src family member expressed in NK and T lymphocytes, is also required for NK T cell development (14, 15). To determine whether the V{alpha}14 transgene could restore NK T cell development in lck–/– mice as it had in the fyn mutant, lymphocytes from wt, lck–/–, and lck–/– V{alpha}14 transgenic mice were isolated, and the presence of NK T cells was determined by flow cytometry. As shown in Fig. 4, expression of the transgene results in an increased percentage of NK T cells in both the thymus and periphery of Lck-deficient mice. Unlike the fyn mutant, this rescue is only partial. The substantial difference in NK T cell numbers found in fyn and lck mutants with the V{alpha}14 transgene suggests that these molecules have very different functions during NK T cell development.



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  		FIGURE 4. The V{alpha}14J{alpha}18 transgene leads to a partial rescue in NK T cell numbers in lck–/– mice. Thymocytes and liver lymphocytes from wt, lck–/–, and lck–/– V{alpha}14J{alpha}18 mice were isolated and analyzed as in Fig. 1. The percentage of NK1.1+TCR{beta}+ cells are indicated in the upper right quadrant of each plot. The data presented are representative of four independent experiments.

 
Fyn is important for optimal proliferation of NK T cells to {alpha}GalCer but has only a minor role in cytokine secretion

To determine whether Fyn is important for the function of NK T cells in addition to its developmental role, the activation of mature fyn–/– NK T cells was examined. The glycolipid {alpha}GalCer was used to stimulate NK T cells from fyn+/– and fyn–/– mice, and then proliferation as well as cytokine production were analyzed. Flow-cytometric assays were used to allow analysis on a per-cell basis, because there are so few NK T cells in fyn–/– animals. Furthermore, these assays can discriminate between NK T cells and other lymphoid cells that are capable of secreting cytokine and proliferating in response to NK T cell activation (34, 35). Splenic NK T cells were enriched from fyn+/– and fyn–/– mice, labeled with the vital dye CFSE, and then stimulated with {alpha}GalCer plus APCs. The proliferation of NK T cells was analyzed by flow cytometry, examining the CFSE content of cells expressing TCR{beta} and NK1.1 (Fig. 5, A and B). These stimulations also contained IL-7 as a survival factor, because cultures with medium alone yielded too few NK T cells to analyze CFSE content (data not shown). Stimulations with {alpha}GalCer plus IL-7 or {alpha}GalCer alone yielded similar results in all experiments (data not shown). In the presence of IL-7 alone, the majority of fyn+/– or fyn–/– NK T cells did not divide appreciably (Fig. 5B). Gating on total NK T cells, the cells divided an average of 1.7 and 1.6 times, respectively. Because NK T cells are thought to recognize an unknown endogenous ligand, the low level of proliferation observed may result from this interaction. Addition of {alpha}GalCer to cultures of fyn+/– cells leads to a dramatic increase in proliferation, with an average of 4.6 divisions for the gated NK T cells. The Fyn-deficient NK T cells also proliferate in response to {alpha}GalCer, but to a lesser extent, with an average of 3.1 divisions per gated cell. There are also consistently more cells that do not divide in the fyn-null compared with the fyn+/– mice, suggesting that Fyn may be important to reach a threshold of stimulus necessary to initiate cell division. Similar results were also obtained when comparing fyn+/– V{alpha}14 transgenic to Fyn-deficient V{alpha}14 transgenic mice (data not shown).



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  		FIGURE 5. Proliferation and cytokine production in fyn–/– NK T cells. A and B, NK T cell-enriched splenocytes from fyn+/– and fyn–/– mice were labeled with CFSE and stimulated on irradiated APCs with 1% supernatant of the IL-7-producing cell line J558 or IL-7 plus 10 ng/ml {alpha}GalCer. IL-7 was included to promote survival of unstimulated cells. After 4 days of stimulation, live cells were isolated and stained with TCR{beta} and NK1.1. A, Gates used to identify NK T cells. B, Histograms of CFSE profiles gated on NK T cells are shown. C and D, Mice were injected with 8 µg of {alpha}GalCer i.v., and liver lymphocytes were isolated 2 h later. The cells were stained for Thy-1 and NK1.1, fixed, permeabilized, and stained for intracellular IFN-{gamma} or IL-4. C, Gating of NK T cells in fyn+/– and fyn–/– liver lymphocytes. D, Histograms for intracellular IFN-{gamma} and IL-4 on gated NK T cells. The dotted line shows staining from unstimulated mice, whereas the bold solid line represents staining from {alpha}GalCer-stimulated mice. The data shown are representative of four independent experiments.

 
Stimulation with {alpha}GalCer leads to rapid cytokine production from NK T cells (4). To determine whether induction was impaired in fyn mutants, both fyn+/– and fyn–/– mice were injected with {alpha}GalCer, and the liver lymphocytes were isolated 2 h later. Intracellular staining followed by flow-cytometric analysis was used to directly examine cytokine secretion by NK T cells. Because the {alpha}GalCer stimulation leads to significant TCR down-regulation (data not shown), liver NK T cells were identified as Thy1highNK1.1int (Fig. 5C). This population was found to contain the majority of tetramer+NK1.1+ cells. In contrast, only 2–4% of the Thy1highNK1.1high cells stained with tetramer (data not shown). Stimulation with {alpha}GalCer induced 85 and 53% of fyn+/– NK T cells to synthesize IFN-{gamma} and IL-4, respectively (Fig. 5D). Although cytokines were produced in the fyn mutants, the percentage of positive NK T cells was slightly decreased relative to wt (Fig. 5D). Fyn+/– and fyn–/– mice expressing the V{alpha}14 transgene also had similar cytokine production profiles after {alpha}GalCer stimulation (data not shown). These data indicate that Fyn is required for optimal proliferation, but has only a modest effect on cytokine production by NK T cells stimulated with Ag.

Secondary B cell and NK cell activation by {alpha}GalCer requires sufficient numbers of NK T cells

Activation of NK T cells by {alpha}GalCer leads to up-regulation of CD69 on B cells and IFN-{gamma} production by NK cells (34, 36). These changes have been shown to require NK T cells, because they fail to occur in CD1d-deficient mice, which lack NK T cells (34). To determine the role of Fyn in these secondary activation events, mice were injected with {alpha}GalCer i.v., and then spleens were harvested and analyzed 6 h later. Eighty-four percent of B cells from fyn+/– mice up-regulated CD69 following treatment with {alpha}GalCer (Fig. 6A). In contrast, the fyn–/– B cells did not respond. Similarly, IFN-{gamma} production by NK cells was also impaired in fyn mutants. Approximately 30% of NK cells in fyn+/– mice treated with {alpha}GalCer are IFN-{gamma} positive, whereas {alpha}GalCer-stimulated fyn–/– mice have levels similar to those of untreated animals (Fig. 6B).



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  		FIGURE 6. Secondary activation events in fyn+/–, fyn–/–, and fyn–/– V{alpha}14J{alpha}18 transgenic mice. Mice were injected i.v. with 8 µg of {alpha}GalCer. Six hours later, the mice were sacrificed and splenocytes were isolated. A, Splenocytes were stained for B220 and CD69 and analyzed by flow cytometry. CD69 expression on B220-gated B cells is shown for unstimulated (dotted line) and {alpha}GalCer-stimulated (bold solid line) mice. B, Splenocytes were stained for NK1.1 and TCR{beta}, and then fixed and permeabilized. They were then stained for intracellular IFN-{gamma} and analyzed by flow cytometry. Histograms for IFN-{gamma} expression are shown for NK cells gated as NK1.1+TCR{beta}– cells. The dotted line indicates unstimulated mice, whereas the bold solid line is from {alpha}GalCer-stimulated mice. The data shown are representative of three separate experiments.

 
There are several possible explanations for the lack of secondary lymphocyte activation in the Fyn-null mice. First, because NK T cells in fyn-mutant animals can still produce IFN-{gamma} and IL-4, there may be a requirement for Fyn to produce some other cytokine(s) responsible for this secondary activation. Second, because B cells and NK cells both express Fyn, the impairment in activation may result from an intrinsic defect in these cells due to a lack of Fyn. Finally, the low numbers of NK T cells present in the fyn mutants may be insufficient to secrete amounts of cytokines necessary to induce these secondary effects. To address these issues, similar experiments were also performed with fyn–/– V{alpha}14 mice, because they have robust numbers of NK T cells. If either of the first two possibilities are correct, then the fyn–/– V{alpha}14 mice should still exhibit defects in activation of B and/or NK cells. The fyn–/– V{alpha}14 animals display completely restored activation of both the B cells and NK cells compared with fyn–/– mice (Fig. 6). This indicates that the lack of secondary responses in Fyn-deficient mice is due to the paucity of NK T cells, rather than an intrinsic defect in the B and NK cells. The data obtained with the V{alpha}14 transgenic animals suggest that, if sufficient NK T cell-derived cytokine can be supplied, then these secondary activation events can occur normally without Fyn.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study presented here addresses some of the unanswered questions about the role of the tyrosine kinase Fyn in NK T cell development and peripheral activation. It had been previously shown that loss of Fyn results in a 5- to 10-fold decrease in the numbers of CD1d-restricted NK T cells in both the thymus and the periphery (14, 15). This decrease in thymic NK T cell number does not distinguish whether Fyn is important for development or for homeostasis of mature cells. The majority of NK T cells found in the adult thymus have a very long half-life and are probably not newly generated (17). The NK T cells in thymus and periphery of fyn mutants exhibit similar BrdU-labeling profiles as wt mice, arguing that Fyn may have a minimal role in homeostasis. Moreover, the V{alpha}14 transgene restores NK T cell development in fyn mutants, such that wt, fyn+/–, and fyn–/– mice harboring the V{alpha}14 transgene all have identical NK T cell numbers in the thymus and periphery. This suggests that Fyn is dispensable for the upkeep of NK T cell numbers. It is still formally possible that Fyn plays an undescribed role in homeostasis. For example, fyn–/– NK T cells may undergo decreased cell division in the periphery but have lower levels of cell death, leading to similar steady-state levels as wt. Although unlikely, further experimentation is needed to test this hypothesis.

Fyn also may be required before TCR {alpha}-chain rearrangement, because the V{alpha}14 transgene fully rescues NK T cell development on the fyn–/– background. The fact that the V{alpha}14 transgenic BM from both wt and fyn mutants can successfully compete with each other suggests that both BM populations produce similar numbers of NK T cell progenitors. Transgenic expression of the semi-invariant NK T cell {alpha}-chain in fyn mutants may circumvent the developmental block by allowing TCR-driven expansion to occur. These observations also exclude the possibility that Fyn is required for positive selection of NK T cells.

The reduction of NK T cell numbers in Fyn-deficient mice coupled with the fact that Fyn is not required for the upkeep of NK T cells indicates that Fyn must be required for development. These studies suggest that Fyn plays an important role at an early stage in NK T cell development. First, analysis of NK T cell progenitors in fyn mutants demonstrate that both mature NK1.1+ as well as immature NK1.1– populations are decreased to a similar extent in the fyn mutants. This indicates that Fyn is required relatively early in NK T cell ontogeny, before NK1.1 up-regulation. In addition, the few NK T cells developing in fyn–/– mice have similar BrdU-labeling kinetics as wt mice, suggesting that Fyn may allow for efficient generation of NK T cells from precursors.

It is also possible that expression of the V{alpha}14 transgene may alter normal NK T cell development. In some cases, early expression of TCR transgenes can occur during thymic development in transgenic mice (37, 38). In addition, TCR {alpha}-chain transgenes can outcompete the pre-TCR {alpha}-chain for pairing with the {beta}-chain (39). Thus, the V{alpha}14 transgene may express early or in a progenitor that is distinct from the one normally giving rise to NK T cells. In any case, ectopic expression of the V{alpha}14 {alpha}-chain can bypass the requirement for Fyn and result in precocious development of NK T cells.

Regardless of the mechanism by which the V{alpha}14 transgene promotes NK T cell development, several conclusions about NK T cell homeostasis can be drawn from this work. First, if Fyn were required to maintain viability of NK T cells, then fewer NK T cells should be present in the V{alpha}14 transgenic fyn mutant. The peripheral NK T cells present in the transgenic mice appear to be phenotypically normal as measured by expression of various NK T cell specific markers as well as functionally normal by cytokine production upon TCR stimulation (data not shown). These data suggest that Fyn is not required for maintaining peripheral NK T cell numbers. Second, the V{alpha}14 transgene does not rescue NK T cell development in lck–/– mice to the same degree as in fyn–/– mice. However, it does skew the few T cells that do develop toward the NK T cell lineage. Lck is required for pre-TCR signaling, and this process is important for expansion and developmental progression of T cell progenitors (22, 40). These data are consistent with Lck having a similar role in NK T cell development. The phenotypic differences between V{alpha}14 transgenic fyn- and lck-mutant mice suggest that each kinase has distinct roles in NK T cell development.

In contrast to the important role for Fyn during NK T cell development, peripheral responses from fyn–/– NK T cells were only mildly affected. The most prominent defect observed was decreased proliferation induced by the NK T cell ligand {alpha}GalCer. The fyn-mutant NK T cells divided less in response to activation and contained a higher percentage of undivided cells compared with fyn+/– NK T cells. It is also formally possible that the fyn-deficient NK T cells may undergo increased cell death, specifically in the populations which have undergone cell division, leading to a decreased proportion of this population. Further experimentation is necessary to determine which interpretation is correct.

Ligand-induced stimulation of the few NK T cells present in fyn-null mice results in almost normal IFN-{gamma} or IL-4 secretion on a per-cell basis, although absolute cytokine production is decreased due to the paucity of NK T cells in the mutant. Although the secondary activation responses of B cells and NK cells were absent in fyn–/– mice, rescuing NK T cell numbers with the V{alpha}14 transgene restored these responses. This suggests that the reduced numbers of NK T in fyn mutants caused the defect, rather than their ability to respond to {alpha}GalCer.

Two competing models have been developed to account for the origin of NK T cells (reviewed in Ref.41). In the instructive model, NK T cells are thought to arise from CD4+8+ thymocytes bearing the semi-invariant V{alpha}14 TCR. This is supported in part by the observation that early double-positive (TCR–CD4+8+) thymocytes from CD1d–/– mice can develop into NK T cells following intrathymic injection into wt recipients (29). In contrast, the precommitment model argues that a distinct parallel lineage exists in the thymus, and this population develops into NK T cells if they assemble the semi-invariant TCR, permitting selection by CD1d. Evidence in favor of this is less direct. However, conventional and NK T cell ontogeny exhibit a differential requirement for a variety of gene products and signaling pathways, an observation consistent with the possibility that the two lymphocyte populations arise from discrete progenitors. For example, unlike mainstream T cells, NK T cell development does not rely on an intact Ras/mitogen-activated protein kinase pathway (42). In contrast, disruption of Ets-1 (12) or AP-1 activity (43) has a more profound effect on NK T cell ontogeny than on conventional T cells. Similarly, NK T cell development is highly compromised in fyn mutants (14, 15), whereas mainstream T cell development and TCR repertoire are normal (21, 44). The data presented here are consistent with the prospect that NK T cells may be derived from a separate lineage with a specific requirement for Fyn.

In summary, the studies presented in this paper have demonstrated that Fyn plays an obligatory role in early NK T cell development, whereas it is dispensable for upkeep of peripheral NK T cell numbers. In peripheral activation, Fyn is important for optimal proliferation to ligand, but has only a minor role in regulating cytokine production. These data indicate that Fyn has a similar role in mature conventional T cells and NK T cells. Both cell types show minor deficiencies in activation in the absence of Fyn, as manifested by modest proliferative defects (21, 44, 45). In contrast, conventional T cells and NK T cells diverge in their requirement for Fyn during development. Future study into how Fyn functions in NK T cell ontogeny may give further insights into how these two lymphocyte subsets are related developmentally.


   Acknowledgments
 
We thank Paul Menard-Katcher for help with maintaining the animal colony and Kirin Brewery for providing the {alpha}GalCer. We are indebted to Dr. M. Kronenberg for providing tetramers and Dr. A. Bendelac for providing the V{alpha}14 transgenic mice.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health and the Juvenile Diabetes Research Foundation, International. P.L.S. was also supported, in part, by an Arthritis Foundation Investigator Award. Back

2 Current address: Department of Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, NY 10029. Back

3 P.G. and L.Y. contributed equally to this work. Back

4 Address correspondence and reprint requests to Dr. Paul L. Stein at the current address: Departments of Dermatology and Microbiology/Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: p-stein2{at}northwestern.edu Back

5 Abbreviations used in this paper: V{alpha}14, V{alpha}14J{alpha}18; {alpha}GalCer, {alpha}-galactosylceramide; wt, wild type; BrdU, 5-bromo-2'-deoxyuridine; BM, bone marrow; int, intermediate; HSA, heat-stable Ag. Back

Received for publication November 17, 2003. Accepted for publication March 12, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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