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Control of NKT Cell Differentiation by Tissue-Specific Microenvironments ¹

Yang Yang^2,*, Aito Ueno^*, Min Bao, Zhongying Wang^*, Jin Seon Im, Steven Porcelli and Ji-Won Yoon^2,3,

Departments of ^* Biochemistry and Molecular Biology and Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, NY 10461

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD1d-restricted V14 NKT cells play an important role in both Th1- and Th2-type immune responses. To determine whether NKT cells develop two functionally distinct subsets that provoke different types of responses, we examined the phenotypes and cellular functions of NK1.1⁺ and DX5⁺ T cells. We found that both NK1.1⁺ and DX5⁺ T cells are CD1d-restricted V14 T cells with identical Ag specificities, phenotypes, tissue locations, and functions. Similar to the NK1.1 marker, the DX5 marker (CD49b) is expressed on mature NKT cells in both NK1.1 allele-positive and allele-negative strains. However, when NK1.1⁺ and DX5⁺ NKT cells isolated from different tissues were compared, we found that thymic and splenic NKT cells differed not only in their cytokine profiles, but also in their phenotype and requirements for costimulatory signals. Thymic NKT cells displayed the phenotype of activated T cells and could be fully activated by TCR ligation. In contrast, splenic NKT cells displayed the phenotype of memory T cells and required a costimulatory signal for activation. Furthermore, the function and phenotype of thymic and splenic NKT cells were modulated by APCs from various tissues that expressed different levels of costimulatory molecules. Modulation of NKT cell function and differentiation may be mediated by synergic effects of costimulatory molecules on the surface of APCs. The results of the present study suggest that the costimulatory signals of tissue-specific APCs are key factors for NKT cell differentiation, and these signals cannot be replaced by anti-CD28 or anti-CD40 ligand Abs.

	Introduction

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Natural killer T cells have characteristics of both T and NK cells. NKT cells have TCRs like T cells, which are restricted by CD1 rather than by class I or class II MHC molecules. Like NK cells, NKT cells possess CD16 and other typical receptors and killer activity. Although NKT cells are a small subset of T cells, they can produce large amounts of both Th1 and Th2 types of cytokines as an immediate response to TCR ligation (1, 2, 3). Some studies showed that NKT cells contribute to the development of Th2 immune responses (4, 5, 6), whereas others showed that NKT cells suppress the Th2 immune response (7) and promote the IL-12-elicited Th1 immune responses involved in tumor surveillance (8, 9, 10). It is unclear whether these contradictory observations are due to the presence of two distinct subsets of NKT cells that have different functions or to a single population that functions differently within different microenvironments.

Compared with major T cell populations, NKT cells express an intermediate level of TCR (TCR^int) with a restricted TCR repertoire. In mice, most NKT cells use the invariant V14-J281 chain paired preferentially with V8.2, V7, or V2 chains (11) and recognize the glycolipid -galactosylceramide (GalCer) ³ in the context of CD1d molecules (12, 13, 14). NKT cells were initially called NK1.1⁺ T cells (3) because in C57BL/6 mice, NKT cells express NK1.1 Ag, a pan-NK cell marker on their surface. In NK1.1 allele-negative strains of mice, NKT cells express another pan-NK cell marker, the DX5 Ag (CD49b) (15). DX5⁺ NKT cells were found to have regulatory functions similar to those of NK1.1⁺ NKT cells in some circumstances (16, 17, 18, 19). However, a number of studies suggested that unlike NK1.1⁺ NKT cells, most DX5⁺ NKT cells may not be CD1d-restricted V14 NKT cells and may not exist in the thymus (20, 21, 22, 23).

This investigation was initiated to determine whether NK1.1⁺ NKT cells and DX5⁺ NKT cells are different subsets of NKT cells with respect to their tissue location, phenotype, and function using NK1.1 allele-positive and -negative strains of mice. We found that depletion of DX5⁺ T cells or NK1.1⁺ T cells from thymocytes or splenocytes eliminates the response to GalCer, indicating that most CD1d-restricted, GalCer-responding cells express DX5 Ag. GalCer/CD1d tetramer staining further revealed that V14 NKT cells in the thymus and spleen express both NK1.1 and DX5 markers at different levels. Furthermore, the DX5 marker is only expressed in CD24^-CD44⁺ mature NKT cells. We also found that thymic and splenic NK1.1⁺ and DX5⁺ T cells display substantial differences in their phenotypes, requirements for costimulatory signals, and cytokine profiles. However, the cytokine profile and phenotype of purified NKT cells can be modulated by APCs from different tissues. These results suggest that differentiation and functions of the mature NKT cells can be largely controlled by the microenvironment of specific tissues.

	Materials and Methods

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Mice and Abs

All mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained in pathogen-free units of the Animal Resources Center at University of Calgary. CD1 gene-deficient 129 mice were crossed to the C57BL/6 background for seven generations. Deficiency of the CD1 gene was screened by PCR. The PCR primers used for the CD1.1 and CD1.2 genes were 5'-GGTCTGGGGACAATCTGAAG-3' and 5'-CTGGATCTCAATGGGATCTG-3'. Defective CD1 expression in thymocytes was also examined by FACS. All Abs were purchased from BD PharMingen Canada (Mississauga, Canada).

FACS analysis of NKT cells

Thymocytes, lymph node cells, bone marrow cells, and splenocytes were isolated from 6- to 8-wk-old mice and preincubated with Ab against CD16/32 (2.4G2) in PBS containing 2% FCS. The cells were then incubated with allophycocyanin-labeled GalCer-CD1d tetramers at room temperature for 40 min, washed, and further incubated with a mixture of anti-TCR-FITC (H57-597), anti-NK1.1-PE (PK136), or DX5-PE Abs. The cells were analyzed by FACScan. FITC-labeled anti-CD1d, CD24, CD44, CD62L, and CD69 Abs and allophycocyanin-labeled GalCer-CD1d tetramers were also used as indicated.

Isolation and purification of NK1.1⁺TCR⁺, DX5⁺TCR⁺, and DX5^-TCR⁺ cells.

Thymocytes and splenocytes from C57BL/6 and AKR mice were incubated with PE-conjugated anti-NK1.1 or DX5 Ab and FITC-conjugated anti-TCR-chain Ab. NK1.1⁺ or DX5⁺ cells were first enriched by magnetic cell sorting (Miltenyi Biotec, Auburn, CA). NK1.1⁺TCR⁺, DX5⁺TCR⁺, and DX5^-TCR⁺ cells were further purified by FACS cell sorting. Magnetic cell sorting was also used to deplete NK1.1⁺ or DX5⁺ cells from thymocytes or splenocytes.

Assay for cytokine production

For ELISAs, purified (>95%) DX5⁺TCR⁺, NK1.1⁺TCR⁺, and DX5^-TCR⁺ cells were incubated in 96-well plates (1 x 10⁵ cells/well) with immobilized Ab to CD3 (10 µg/ml) for 48 h. Ab to CD28 (2 µg/ml) was added as indicated. Cells were also activated by PMA (20 ng/ml) and ionomycin (0.5 µg/ml). For Ag activation, DX5⁺TCR⁺, NK1.1⁺TCR⁺, and DX5^-TCR⁺ cells (1 x 10⁵ cells/well) were incubated in the presence of GalCer (Kirin Brewery Co., Tokyo, Japan) with or without DX5⁺ cell-depleted thymocytes or splenocytes as APCs (2 x 10⁵ cells/well) for 48 h. Culture supernatant was collected for ELISA (R&D Systems, Minneapolis, MN) to determine the concentrations of IFN- and IL-4. Thymocytes, splenocytes, or NK1.1⁺ cell-depleted or DX5⁺ cell-depleted thymocytes or splenocytes (1 x 10⁶ cell/well) were also activated with GalCer (100 ng/ml) or immobilized anti-CD3 Ab for 48 h for ELISA.

Detection of the restricted TCR V-chain usage of NKT cells

To detect TCR V14-J281 expression in DX5⁺ NKT cells, DX5⁺TCR⁺, NK1.1⁺TCR⁺, and DX5^-TCR⁺ cells were isolated and purified from thymocytes and splenocytes from C57BL/6 and AKR mice. Total RNA was extracted from identical numbers of purified DX5⁺TCR⁺, NK1.1⁺TCR⁺, and DX5^-TCR⁺ cells, and 2.5 µg RNA from each cell sample was used for cDNA synthesis. TCR V14-J281 rearrangements were amplified by RT-PCR (V14 primer, TAAGCACAGCACGCTGCACAT-3'; C primer, 5'-GTCAAAGTCGGTGAACAGGC-AGAG-3'), and PCR products were cloned using Topo-cloning kit (Invitrogen, Carlsbad, CA) and sequenced. RT-PCR reactions specific for V14-J281 rearrangements were also performed using serially diluted cDNA samples of NKT and T cells isolated from AKR mice. The J281 primer sequence and the conditions of RT-PCR reactions were previously described (12). As a control, RT-PCR for -actin expression was also conducted. The primers for -actin cDNA amplification were: forward, 5'-GTTACCAACTGGGACGACA-3'; and reverse, 5'-TGGCCATCTCCTGCTCGAA-3'.

Coculture experiments

Purified NK1.1⁺TCR⁺ thymocytes or splenocytes were incubated in 96-well plates (2 x 10⁵ cells/well) with DX5⁺ cell-depleted thymocytes or splenocytes (2 x 10⁶cells/well) in the presence or the absence of GalCer (100 ng/ml) for 2 days. The culture medium was collected to measure cytokine production and was replaced with fresh medium containing IL-2 (20 µg/ml; Takeda Chemical Industries, Osaka, Japan). Cells were cultured for an additional 5 days and then harvested for phenotypic analysis. In control experiments, splenocytes of CD1d-gene deficient mice were cocultured with purified NK1.1⁺TCR⁺ thymocytes. For the experiments on signal blockage, purified NK1.1⁺TCR⁺ thymocytes (5 x 10⁴/well) were cocultured with splenic APCs in the presence of GalCer (100 ng/ml) and in the presence or the absence of anti-CD40, anti-CD80, or anti-CD86 Ab (40 µg/ml) or a combination of anti-CD80 and anti-CD86 Abs (20 µg/ml). Purified NK1.1⁺TCR⁺ thymocytes were also activated by anti-CD3 (5 mg/ml) or a combination of anti-CD3 and anti-CD28 Abs for 2 days. The proliferation and cytokine production of thymic NKT cells activated by Ab or GalCer were determined and compared.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Both DX5⁺ and NK1.1⁺ T cells are responsive to GalCer

CD1d-restricted NKT cells are characterized by their dominant usage of the V14-J281 invariant TCR chain (11, 24, 25) and by specificity for the glycolipid, GalCer (13, 14). As several studies suggested that most mature V14 NKT cells do not express DX5 Ag (20, 21, 22, 23), we first tested whether the depletion of DX5⁺ T cells affects the response to GalCer. We isolated splenocytes from young C57BL/6 mice (8 wk old) and activated the splenocytes with anti-CD3 Ab or GalCer for 48 h. In a parallel experiment, DX5⁺ or NK1.1⁺ cells were removed from the splenocytes using Ab-conjugated microbeads before activation. As shown in Table I, splenocytes of C57BL/6 mice activated with anti-CD3 Ab produced a significant amount of IFN-, and IFN- production was slightly decreased when DX5⁺ or NK1.1⁺ cells were depleted. Splenocytes isolated from CD1d gene-deficient C57BL/6 (CD1d-KO) mice also produced a significant amount of IFN-, similar to DX5⁺ or NK1.1⁺ cell-depleted splenocytes. In contrast, DX5⁺ cell-depleted or NK1.1⁺ cell-depleted splenocytes as well as splenocytes from CD1d-KO mice produced significantly less IL-4 than C57BL/6 splenocytes when activated by anti-CD3 Ab. C57BL/6 splenocytes also produced significant amounts of IFN- and IL-4 when activated by GalCer. The cytokine production induced by GalCer is a CD1d-dependent response, as splenocytes of CD1d-KO mice produced neither IFN- nor IL-4. As well, DX5⁺ or NK1.1⁺ cell-depleted splenocytes produced undetectable levels of IFN- and very small amounts of IL-4 in response to GalCer (Table I), indicating that depletion of DX5⁺ or NK1.1⁺ cells eliminated the GalCer-responsive population. These results also showed that IL-4 was almost entirely produced by GalCer-responsive cells during the first 48 h of activation.

Mature V14 NKT cells coexpress NK1.1 and DX5 Ags in thymus and spleen of NK1.1⁺ mice (C57BL/6)

The results of the depletion experiments indicated that splenic DX5⁺ cells include GalCer-responding cells; however, it was suggested that DX5⁺TCR⁺ cells were less frequent than NK1.1⁺TCR⁺ cells in the thymus (20, 21, 22). To determine whether NK1.1⁺TCR⁺ cells and DX5⁺TCR⁺ cells are different populations in the thymus, we examined the expression of NK1.1 and DX5 markers in V14 thymic NKT cells from young C57BL/6 mice. A similar proportion of thymocytes expressed a high level of NK1.1 Ag (0.87%) and a low level of DX5 Ag (0.78%) or were stained by GalCer/CD1d tetramers (0.90%), and all these thymocytes expressed an intermediate level of TCR (Fig. 1a). DX5 expression was very low, and clear labeling was only achieved with PE-labeled anti-DX5 Ab, whereas fewer cells were detected with FITC-labeled anti-DX5 Ab (data not shown); therefore, PE-labeled anti-DX5 Ab was used for the remainder of this study.

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Coexpression of NK1.1 and DX5 Ags in mature Va14 NKT cells in C57BL/6 mice. a, Flow cytometric analysis of C57BL/6 thymocytes showed that similar portions of TCRint thymocytes expressed NK1.1 or DX5, and TCRint thymocytes were GalCer/CD1d tetramer+. b, In gated GalCer/CD1d tetramer+TCR+ thymocytes, most cells expressed both NK1.1 and DX5 Ags, and most NK1.1+ thymic T cells expressed variable levels of DX5 Ag. c, In DX5+ cell-depleted thymocytes, both GalCer/CD1d tetramer+TCR+ and NK1.1+TCR+ cells were removed. d, In GalCer/CD1d tetramer+TCR+ thymocytes, DX5+ cells were CD24- CD44+. e, In splenocytes from C57BL/6 mice, GalCer/CD1d tetramer+TCR+ cells expressed DX5 marker, and NK1.1+ T cells coexpressed DX5. Data represent the results from at least six young (5- to 8-wk-old) mice per group.

Thymic and splenic NKT cells differ in their requirements for costimulatory signals and cytokine production

To determine whether DX5⁺ and NK1.1⁺ T cells have different functions, we purified NK1.1⁺TCR⁺ and DX5⁺TCR⁺ cells from spleens of C57BL/6 mice and activated these cells with PMA and ionomycin or immobilized anti-CD3 Ab in the presence or the absence of anti-CD28 Ab as a costimulatory signal. DX5^-TCR⁺ cells were also isolated and activated under identical conditions. We found that splenic NK1.1⁺TCR⁺ and DX5⁺TCR⁺ cells produced equal amounts of IFN- and IL-4 under all conditions (Fig. 2a). Both splenic NK1.1⁺ and DX5⁺ T cells produced 2-fold more IFN- and at least 10-fold more IL-4 than DX5^- T cells. In addition, both splenic NK1.1⁺ and DX5⁺ T cells produced less IFN- and IL-4 in the absence of anti-CD28 Ab, indicating that costimulatory signals are required for the full activation of these splenic NK1.1⁺ and DX5⁺ T cells. Thus, NK1.1⁺TCR⁺ and DX5⁺TCR⁺ cells are identical with respect to activation requirements and cytokine production (Fig. 2a). However, when the purified thymic NK1.1⁺TCR⁺ cells were activated, substantial functional differences were seen between splenic and thymic NKT cells. Thymic NKT cells produced 2- to 3-fold more IFN- and 5-fold more IL-4 than splenic NKT cells (Fig. 2a). Therefore, thymic NKT cells not only have a higher capacity for cytokine production, but have a different profile than splenic NKT cells. Furthermore, thymic NKT cells produced similar amounts of cytokines with or without costimulatory signals from anti-CD28 Ab (Fig. 2a), indicating that ligation of TCR is sufficient for the activation of thymic NKT cells.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Different requirements for costimulatory signals, cytokine production, and surface markers between thymic and splenic NKT cells. a, Purified thymic NK1.1+ T cells and DX5- T cells (T) and splenic NK.1.1+ T cells, DX5+ T cells, and DX5-TCR+ cells (T) of C57BL/6 (B6) mice were activated by PMA/ionomycin (P+I), anti-CD3 Ab (anti-CD3), or anti-CD3 plus anti-CD28 Abs (anti-CD3/CD28). Splenic NK1.1+ T and DX5+ T cells produced identical IFN- and IL-4 under various activation conditions, and both NK1.1+ T and DX5+ T produced much more cytokines than DX5- T cells. However, thymic NK1.1+ T cells produced far more cytokines, especially IL-4, than splenic NKT cells. Thymic NKT cells produced the same amount of cytokines when activated by anti-CD3 Ab with or without anti-CD28 Ab. In contrast, cytokine production, particularly IFN- production, by peripheral NK1.1+ T and DX5+ T cells was enhanced when they were activated by anti-CD3 Ab in the presence of anti-CD28 Ab. b, Thymic and splenic NK1.1+ T cells of C57BL/6 mice and DX5+ T cells of AKR mice were gated, and the expressions of CD1d, CD44, CD62L, and CD69 Ags were compared. Most thymic and splenic NKT cells expressed identical levels of CD1d, but a small number of splenic NKT cells expressed a higher level of CD1d. Thymic and splenic NKT cells expressed identical high levels of CD44. However, in both C57BL/6 and AKR mice, thymic NKT cells showed an activated phenotype, CD62LlowCD69high. In contrast, splenic NKT cells showed a naive phenotype of CD62LhighCD69low. Data represent similar results from four mice per group.

DX5⁺ T cells in NK1.1^- strains of mice are CD1d-restricted V14 NKT cells

As most inbred strains do not express the NK1.1 Ag, we asked whether DX5⁺ T cells in NK1.1^- strains are CD1d-restricted V14 NKT cells. GalCer/CD1d tetramer staining revealed the presence of V14 NKT cells in the thymocytes of AKR, DA/2, BALB/c, and nonobese diabetic (NOD) mice. Different percentages of the thymocytes were GalCer/CD1d tetramer⁺, ranging from 1.3% in AKR mice to 03% in NOD mice. Regardless of the different portions of GalCer/CD1d tetramer⁺ NKT cells among thymocytes, most GalCer/CD1d tetramer⁺ V14 NKT cells expressed various levels of DX5 Ag (Fig. 3a). NOD mice have been known to have a deficient NKT cell population (27, 28); this deficiency can be clearly revealed by DX5 Ab staining, as significantly fewer DX5⁺ cells were found in NOD than in other strains of mice (Fig. 3a).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. V14 NKT cells in NK1.1 allele-negative strains express various levels of DX5 Ag, whereas thymic and splenic NKT cells are different in costimulatory signal and cytokine production. a, In the thymus of NK1.1 allele-negative mice, different portions of TCRint thymocytes were GalCer/CD1d tetramer+, and most GalCer/CD1d tetramer+ thymocytes expressed various levels of DX5 Ag (lower panel). b, Thymic and peripheral DX5+ T cells showed high IFN- production under all activation conditions compared with DX5-TCR+ cells (T). The presence of anti-CD28 Ab enhanced cytokine production by peripheral DX5+TCR+ cells and DX5-TCR+ cells activated by anti-CD3 Ab. Cytokine production by thymic DX5+TCR+ cells was not affected by anti-CD28 Ab. Data represent similar results from four independent experiments.

Splenic APCs can modify the phenotype and functional properties of thymic NKT cells

As thymic and splenic NKT cells develop from the same thymic precursor (29, 30, 31), but display different phenotypes and cellular functions, we hypothesized that thymic NKT cells may migrate to the periphery and interact with more diversified APCs, resulting in a change in the phenotype and functional properties of NKT cells. To test this hypothesis, we purified thymic and splenic NK1.1⁺ T cells from C57BL/6 mice and cocultured these cells with DX5⁺ cell-depleted thymocytes or splenocytes as APCs in the presence or the absence of GalCer. After 48 h, the culture supernatant was collected to determine the cytokine profile. Thymic NKT cells produced a large amount of IL-4, but a relatively low level of IFN-, when they were cocultured with thymic APCs in the presence of GalCer. However, coculture with splenic APCs significantly increased IFN- production and only slightly affected IL-4 production (Fig. 4a). Similar results were observed when splenic NKT cells were activated with different APCs, although they produced much less IL-4 and slightly less IFN- than thymic NKT cells (Fig. 4a). As thymic and splenic NKT cells differ not only in their cytokine production, but also in surface marker expression, we then examined the expression of CD62L in thymic and splenic NKT cells when they were cocultured with different APCs. Thymic NKT were CD62L^low; however, these cells expressed increased levels of CD62L when cocultured with splenic APCs for 5 days with or without GalCer. In contrast, no change in the expression levels of CD62L were detected in thymic NKT cells cocultured with thymic APCs. In addition, the increased CD62L expression in thymic NKT cells was CD1d dependent, because CD62L expression of thymic NKT cells did not increase when they were cocultured with splenocytes from CD1d-KO mice (Fig. 4b). The expression of CD62L in splenic NKT cells was not significantly changed during activation with different APCs (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Modification of cytokine profiles and CD62L expression in NKT cells by splenic and thymic APCs. a, Purified splenic and thymic NKT cells (1 x 105/well) were cocultured with splenic (columns 1 and 3) or thymic (columns 2 and 4) APCs in the absence (columns 1 and 2) or the presence of GalCer (columns 3 and 4; 100 ng/ml). In the absence of GalCer, NKT cells produced almost no cytokines. In the presence of GalCer, thymic and splenic NKT cells produced a significant amount of IFN-, which was significantly reduced when these cells were cocultured with thymic APCs. Similar amounts of IL-4 were produced when NKT cells were activated with splenic or thymic APCs, although splenic NKT cells produced much less IL-4 than thymic NKT cells (*, p = 0.02, by Student’s t test). b, Thymic NKT cells cocultured with splenic APCs for 5 days with (+) or without (-) GalCer expressed increased levels of CD62L compared with NKT cells cocultured with thymic APCs. In contrast, thymic NKT cells cocultured with splenic APCs from CD1d-KO mice did not show increased expression levels of CD62L compared with NKT cells cocultured with thymic APCs.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Modification of NKT cell function by multiple signaling molecules on the surface of APCs. a, Splenic APCs expressed significantly higher levels of CD1d and CD86 than thymic APCs, and both APCs expressed similar levels of CD80. b, Thymic NKT cells (5 x 104/well) purified from C57BL/6 mice were activated by GalCer presented by splenic APCs (columns 1, 4, 5, 6, and 7), anti-CD3 Ab (column 2), or a combination of anti-CD3 and anti-CD28 Abs (column 3). Ab-activated NKT cells proliferated more vigorously, but produced much less IFN-, than NKT cells activated by GalCer, although they produced similar amounts of IL-4 and IL-2 in different activation reactions. The presence of anti-CD80 (column 4; 40 µg/ml), anti-CD86 (column 5; 40 µg/ml), or anti-CD40 (column 7; 40 mg/ml) significantly decreased cytokine production, but did not affect the proliferation of NKT cells. However, the presence of both anti-CD80 and anti-CD86 (column 6; 20 µg/ml) in the activation reaction almost blocked the activation of NKT cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We first tested whether depletion of DX5⁺ cells has any effect on the GalCer-specific responses, as in both human and mouse, GalCer is only recognized by CD1d-restricted T cells. We found that DX5⁺ cells in both thymocytes and splenocytes from either NK1.1⁺ and NK1.1^- strains of mice contain GalCer-responsive populations. The results of GalCer/CD1d tetramer staining also clearly revealed that most of NK1.1⁺ and DX5⁺ NKT cells are V14 NKT cells, and the DX5 Ab-mediated depletion removed most GalCer/CD1d tetramer⁺ and NK1.1⁺ NKT cells. In fact, we have cloned and sequenced TCR cDNA of DX5⁺TCR⁺ cells purified from either C57BL/6 and AKR mice and found the predominant usage of V14-J281 invariant chain in these cells (data not shown). When purified NK1.1⁺TCR⁺ and DX5⁺TCR⁺ cells were activated under various conditions, these two populations displayed identical cytokine capacities and profiles. From the results of the functional and phenotype characterizations, we conclude that most NK1.1⁺ and DX5⁺ T cells do not represent different subsets of NKT cells. In addition, we found that similar to NK1.1 Ag, DX5 Ag is only expressed in CD24^-CD44⁺ mature NKT cells.

In the present study we found that thymic NKT cells in many strains express DX5 Ag at low levels that can be clearly detected by PE-conjugated Ab and only weakly by FITC-conjugated Ab. FITC-conjugated Ab was used in previous studies that reported negative results of DX5 staining in V14 NKT cells (20, 21, 22). As DX5⁺TCR⁺ cells demonstrate the functions and phenotypes of V14 NKT cells, DX5 Ag can be used as marker to identify and even enrich V14 NKT cells in NK1.1^- strains without affecting the functions of NKT cells. In fact, we are able to readily isolate GalCer/CD1 tetramer⁺ cells from thymocytes of NK1 allele-negative strains, such as BALB/c and NOD mice, using the DX5 marker. However, a small number of T cells in the spleens of young mice express DX5 Ag, but not NK1.1 Ag (32). Therefore, it is difficult to exclude the possibility that a small number of T cells expressing DX5 Ag in the spleen may be different from CD1d-dependent NKT cells. The nature of these cells remains to be determined.

Although NK1.1⁺ and DX5⁺ NKT cells are not distinct subsets of NKT cells, substantial differences in function between thymic and splenic NKT cells attracted our attention. Under various conditions, thymic NKT cells produced a higher amount of cytokines, especially IL-4, than splenic NKT cells. Apparently thymic NKT cells are able to produce more cytokines and have a cytokine profiles biased to Th2 responses. The results of the present study further revealed that thymic and splenic NKT cells from both NK1.1⁺ and NK1.1^- strains of mice differ not only in their cytokine production, but also in the expression of activation markers and requirements for costimulatory signals. Splenic NKT cells required both TCR- and CD28-mediated signals to be fully activated, whereas engagement of CD28 had no detectable effect on the activation of thymic NKT cells. We also found that the engagement of CD40 ligand (CD40L) on the surface of thymic NKT cells did not affect their cytokine production (data not shown), although the CD40 signal was shown to be important for the cytokine profile of splenic NKT cells (34). The effects of costimulatory signals on the cytokine profiles of NKT cells have been studied using peripheral NKT cells, including splenic (34) and hepatic (35) NKT cells. The present study provides the first evidence for different requirements for costimulatory signals between thymic and splenic NKT cells. Importantly, the different costimulatory signal requirement is consistent with different expression patterns of the activation markers between thymic and splenic NKT cells. As we showed in this study, splenic NKT cells are mostly CD44⁺CD62L⁺CD69^-, indicating that splenic NKT cells are memory T cells. The production of a large quantity of cytokines from thymic NKT cells in vitro by TCR ligation is consistent with other studies (21, 26). However, cytokine production from thymic NKT cells in vivo is not readily detected after anti-CD3 Ab or GalCer injection. Whether this difference between in vitro and in vivo conditions is due to thymus-specific factors that control the production and secretion of cytokines from NKT cells is presently under investigation.

To understand the mechanisms of NKT cell differentiation, we cocultured thymic and splenic NKT cells with APCs from different tissues. It is known that subsets of APCs, such as dendritic cells, enhance IFN- production by peripheral NKT cells (36, 37, 38). In the present study we found that IFN- production, but not IL-4 production, by either thymic or splenic NKT cells can be regulated by different APCs, suggesting that costimulatory signals conferred by splenic APCs enhance IFN- production. Interestingly, splenic APCs also enhance CD62L expression in thymic NKT cells after 5 days of culture, even in the absence of GalCer. Because the increased CD62L expression was CD1d dependent, it was likely that engagement of thymic NKT cells with endogenous Ags presented by splenic APCs increases CD62 expression when thymic NKT cells migrate into spleen. CD62L is required for the homing of memory-type T cells to secondary lymphoid organs (39); thus, the increase in CD62L expression may be an important step for NKT cell differentiation when they migrate from thymus to periphery. Diversified functions among NKT cells from different organs have been reported, and it is believed that differential distribution of CD4⁺ and CD4^-CD8^- NKT cells may be responsible for the functional diversities in the periphery (40, 41, 42), because CD4⁺ and CD4^-CD8^- NKT cells display different cytokine profiles (43, 44). Most NKT cells develop from the thymus, and most thymic NKT cells are CD4⁺ (29, 30, 31), thus CD4^-CD8^- NKT cells may differentiate from CD4⁺ NKT cells, but the mechanisms by which NKT cells differentiate remain unclear. A low, but consistent, level of activation by endogenous Ag on the surface of peripheral APCs may also affect CD4^-CD8^- double-negative NKT cell differentiation in secondary lymphoid organs, because it has been shown that a large fraction of splenic CD4⁺ NKT cells become double-negative NKT cells after repeated activation (45). Further, it has been found that NKT cells in the circulation express different chemokine receptors with distinct cytokine-producing capacities (46), suggesting that migration destinations are associated with the functions of NKT cells. All these data support the idea that the tissue-specific microenvironment determines the differentiation of thymic NKT cells into subsets of peripheral cells that are functionally and phenotypically distinct.

It is unclear how splenic APCs enhance IFN- production by thymic NKT cells. In our study we found different expression levels of CD1d and CD86 between thymic and splenic APCs. To determine which signal might be critical for the functional modification of NKT cells, we blocked candidate signaling molecules, CD80 and CD86, individually or together using their specific Abs. Blockage of CD80 or CD86 significantly hampered cytokine production, but not the proliferation of thymic NKT cells in the presence of GalCer presented by splenic APCs. A similar inhibitory effect was seen when anti-CD40 Ab was added to the culture. In addition, blockage of both CD80 and CD86 resulted in the almost complete elimination of cytokine production and proliferation of thymic NKT cells. In contrast, we found that engagement with anti-CD28 and anti-CD40L Abs had no direct effect on the function of thymic NKT cells. These results suggest that the signal mediated by anti-CD28 or anti-CD40L Ab could not replace the signal mediated by CD80/CD86 or CD40 expressed on splenic APCs. It is possible that multiple signals from APCs may synergistically enhance the function of NKT cells. Another possibility is that the Abs against the molecules on the surface of APCs disrupt structure formation, such as the lipid raft (47), which is required for Ag presentation or other unidentified costimulatory signals. Further studies are needed to clarify the role of complex signals in NKT cell function. However, these results have provided clear evidence that the roles of signals in different tissue-specific microenvironments, consisting of various APC subpopulations, are critical for the development of Th1- or Th2-oriented responses by NKT cells.

In conclusion, both NK1.1⁺ and DX5⁺ T cells are CD1d-restricted V14NKT cells, and thymic NKT cells differentiate into functionally different NKT cells under tissue-specific microenvironments. In mice, CD1d-restricted NKT cells preferentially evoke a Th1-oriented immune response in the spleen and a Th2-oriented immune response in the thymus.

	Acknowledgments

 
We are grateful to Lori Bryant for assistance with flow cytometry, and Dr. Ann Kyle for editorial assistance.

	Footnotes

 
¹ Y.Y. is a recipient of the Career Development Award from the Juvenile Diabetes Research Foundation International. J.W.Y. holds a Canada Research Chair in Diabetes.

² Address correspondence and reprint requests to Dr. Ji-Won Yoon, Department of Microbiology and Infectious Diseases, or Dr. Yang Yang, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail addresses: yoon{at}ucalgary.ca or yyang{at}ucalgary.ca

³ Current address: The Chicago Medical School, FUHS, 3333 Green Bay Road, North Chicago, IL 60064

⁴ Abbreviations used in this paper: GalCer, -galactosylceramide; CD1d-KO, CD1d gene-deficient C57BL/6 mice; CD40L, CD40 ligand; I-A-KO, MHC II 1-A gene-deficient C57BL/6 mice; NOD, nonobese diabetic.

Received for publication February 4, 2003. Accepted for publication September 26, 2003.

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