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CD1d-Independent NKT Cells in ₂-Microglobulin-Deficient Mice Have Hybrid Phenotype and Function of NK and T Cells ¹

Motoi Maeda^*, Ashleen Shadeo^*, Anna M. MacFadyen^* and Fumio Takei^2,*,

^* Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Unlike CD1d-restricted NK1.1⁺TCR⁺ (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in ₂-microglobulin (₂m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in ₂m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-indpendent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed ₂m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D^b killed RMA-S, but not RMA, cells. They also killed RMA (H-2^b) cells that were preincubated with the HY peptide. NKT cells from ₂m-deficient mice, upon CD3 cross-linking, secreted IFN- and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A population of murine T cells coexpresses the TCR and NK cell-associated molecules, including NK1.1 and the Ly49 family of receptors for MHC class I (1). This population, often termed NKT cells, is heterogeneous and can be divided into two major subpopulations, namely CD1d-restricted and CD1d-independent NKT cell subsets (2). The former recognizes lipid Ags presented by CD1d and has a highly skewed TCR repertoire, with the majority of cells expressing V14-J281 paired preferentially with V8.2 (and, to a lesser extent, V7 and V2) chain (V14i T cell) (3, 4, 5, 6, 7). V14i T cells are vigorously activated with -galactosylceramide (-GalCer), ³ originally isolated from marine sponge, presented by CD1d on APC (8, 9). Thus, most CD1d-restricted NKT cells can be identified by CD1d tetramers loaded with -GalCer (10, 11). NKT cells stained with the tetramer are either CD4⁺ or double negative. Upon activation, they rapidly secrete large amounts of cytokines, including IL-4 and IFN-. They appear to have unique immune regulatory functions in vivo. They seem to suppress autoimmune diseases in mice and man, involved in immunity to infectious agents such as Plasmodium, Toxoplasma gondii, Listeria, and prevent tumor metastasis in liver and lung (12, 13, 14, 15, 16, 17, 18).

In contrast to CD1d-restricted NKT cells, which have been intensively studied, very little is known about CD1d-independent NKT cells. They are unstained with CD1d-tetramers loaded with -GalCer and are found in mice lacking CD1d expression, such as CD1d^–/– and ₂-microglobulin (₂m)^–/– mice, as their development is independent of CD1d (19, 20, 21, 22). CD1d-independent NKT cells do not have skewed TCR repertoire (23, 24). Some CD1d-independent NKT cells are CD8⁺. The phenotype of CD8⁺ NKT cells, which are abundant in the liver and large intestine, has been characterized (25, 26, 27). However, the functions of CD1d-independent NKT cells are mostly unknown.

In the present report, we show that CD1d-independent NKT cells can be activated with an intermediate dose of IL-2, and IL-2-activated CD1d-independent NKT cells display both NK cell-like and CTL-like functions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 (B6) (Ly5.2), C57BL/6J: Pep 3b (Ly5.1), and ₂m-deficient mice of the B6 background were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility as previously described (28). The Abb/₂m double-knockout mice (MHC class I and II deficient) of B6 background and the recombinase-activating gene 2 (RAG2)-deficient transgenic mice with the male Ag HY-specific TCR of C57BL/6 background (29, 30) were purchased from Taconic Farms (Germantown, NY). Male mice (8–12 wk old) were used for this study unless stated otherwise.

Cells, mAbs, and flow cytometry

YAC-1 and the hybridoma 2.4G2 (anti-FcR) were obtained from American Type Culture Collection (Manassas, VA). The murine T cell lines RMA and RMA-S have been described previously (31). The mAbs YE1/48 (anti-Ly49A), 5E6 (anti-Ly49C/I), and 4D11 (anti-Ly49G2) have been described previously (32). These mAbs were biotinylated and used in this study. FITC-conjugated anti-Ly5.2 mAb AL14A2 and biotinylated anti-murine Mac-1 mAb M1/70 have been previously described (33). Biotinylated anti-murine NKG2D mAb C7 were purchased from BioLegend (San Diego, CA). Biotinylated anti-murine CD4 mAb GK1.5, biotinylated anti-murine CD8 mAb 53-6.7, biotinylated anti-murine CD44 mAb IM-7, biotinylated anti-murine 2B4 mAb 2B4, biotinylated anti-murine CD94 mAb 18d3, biotinylated anti-murine NKG2A/C/E mAb 20d5, biotinylated anti-murine CD132 (common -chain) mAb TUGm2, biotinylated and FITC-conjugated anti-TCR mAb H-57-597, purified and FITC-conjugated anti-CD3 mAb 145-2C11, FITC-conjugated anti-TCRV2 mAb B20.1, FITC-conjugated anti-TCRV3.2^b,c mAb RR3-16, FITC-conjugated anti-TCRV8.3 mAb B21.14, FITC-conjugated anti-TCRV11.1,11.2^b,d mAb RR8-1, FITC-conjugated V TCR screening panel mAbs, PE-conjugated anti-NK1.1 mAb PK136, PE-conjugated anti-CD25 (IL-2R -chain) mAb 3C7, PE-conjugated anti-CD122 (IL-2R -chain) mAb TM-1, isotype control mAbs, and streptavidin-allophycocyanin were purchased from BD Biosciences (Mississauga, Canada).

To detect V14i T cells, recombinant soluble dimeric mouse CD1d-Ig fusion protein (DimerX I; BD Biosciences) was used. After loading with -GalCer (provided by Dr. T. Yamamura, National Institute of Neuroscience, Tokyo, Japan), DimerX I was used for staining according to the manufacturer’s instruction. The NKT cell line described previously (34) was used as a positive control for CD1d dimer staining.

For flow cytometry and cell sorting, cells were first incubated with unlabeled 2.4G2 to block the FcR, then stained with mAbs. All incubations were performed for 30 min on ice. After washing, streptavidin-allophycocyanin was added. Dead cells were stained with propidium iodide and gated out for the analysis. A FACSCalibur (BD Biosciences, Mountain View, CA) equipped with CellQuest software (BD Biosciences) was used for analysis, and a FACSVantage (BD Biosciences) was used for cell sorting.

NKT-lymphokine activated killer (NKT-LAK) cell culture

Splenocytes (5 x 10⁷ cells) were cultured in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME in a 100-mm tissue culture plate overnight. Nonadherent cells were then harvested, transferred into a new plate, and cultured in the same medium containing 200 U/ml human rIL-2 (PeproTech, Rocky Hill, NJ). After 3 days, nonadherent cells were removed with gentle rinses, and the remaining adherent cells were cultured in the same medium with IL-2 for an additional 4 days. In some experiments NK1.1⁺TCR⁺ or NK1.1^–TCR⁺ cells were sorted from ₂m-deficient splenocytes (Ly5.2) after nylon-wool column purification and were cocultured with Ly5 congenic C57BL/6J:Pep 3b (Ly5.1) bulk splenocytes as described above. After 8 days of culture, Ly5.2-positive cells were analyzed by flow cytometry. IL-2-activated splenic NK (NK-LAK) cells were generated as previously described (31). Flow cytometric analysis of the cells showed that >95% of the cells were NK1.1⁺CD3^–.

Proliferation assay and cytokine ELISA

For cell proliferation assays, splenocytes from ₂m-deficient mice were passed through nylon-wool column and sorted into NK (NK1.1⁺CD3^–), NKT (NK1.1⁺TCR⁺), and T (NK1.1^–TCR⁺) cells. The three sorted fractions were cultured with 1000, 200, and 0 U/ml IL-2 in 96-well, round-bottom wells (2 x 10⁴ cells/well). At 3 or 7 days of culture, cells were [³H]thymidine-labeled for the last 8 h and harvested to count on a beta counter (LKB Wallac, Turku, Finland).

After nylon-wool column purification, NK1.1⁺TCR⁺ cells were sorted from ₂m-deficient splenocytes. Cells (5 x 10⁴ cells/well) were incubated in 96-well, flat-bottom plates coated with anti-CD3 mAb (BD Biosciences) for 48 h. The amounts of cytokines (IFN-, IL-2, and IL-4) in the supernatants were measured by Quantikine kits (R&D Systems, Minneapolis, MN).

In vivo stimulation

One microgram of anti-CD3 mAb or isotype control mAb was i.v. injected into mice. Spleens were removed 90 min later, and single-cell suspensions were prepared. The cells (10⁷) were incubated in 2 ml of medium in triplicate wells of 24-well plates at 37°C for 2 h as previously described (22, 35, 36, 37). IFN- in the supernatant was determined by ELISA as described above.

Cytotoxicity assay

For specific lysis of target cells, the standard ⁵¹Cr release assay was performed as previously described (31). In some experiments RMA cells were incubated with 10 µg/ml HY peptide (KCSRNRQYL) or a scrambled peptide (NYQRSLCKR) as a control before labeling with ⁵¹Cr (38).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Surface phenotype of CD1d-independent NKT cells

To characterize CD1d-independent NKT cells, we examined NK1.1⁺TCR⁺ cells in ₂m-deficient B6 mouse spleen, which lacks conventional CD8⁺ T cells and CD1d-restricted NKT cells. Approximately 0.4% of splenocytes from ₂m-deficient B6 mice were NK1.1⁺TCR⁺ (Fig. 1). This frequency was about one-half that in normal B6 spleen, which contains both CD1d-restricted and CD1d-independent NKT cells (10, 11, 23). Over 70% of NKT cells in ₂m-deficient B6 mice were double negative, but some expressed either CD4 (16%) or CD8 (13%). They were CD44^high, and some were IL-2R⁺, resembling memory T cells. They also expressed the Ly49 and CD94/NKG2 families of NK receptors. The expression patterns of these receptors on CD1d-independent NKT cells were very similar to those on NK cells, with the exception of Ly49A, which was expressed on CD1d-independent NKT cells at a significantly higher frequency (>50%) than that on NK cells (20%). They also expressed the activating NK receptor NKG2D as well as Mac-1 and 2B4. We also examined the TCR repertoire of CD1d-independent NKT cells. NK1.1⁺ TCR⁺ and NK1.1^– T cells in ₂m-deficient mice significantly differed in the expression of individual TCRV and TCRV. However, unlike CD1d-restricted NKT cells, which preferentially express V8.2, V7, and V2, no such skewed TCR repertoire was seen with NK1.1⁺TCR⁺ and NK1.1^– T cells in ₂m-deficient mice (Fig. 1B). These results show that CD1d-independent NKT cells in ₂m-deficient B6 mice have the phenotype of both memory T cells and NK cells.

View larger version (56K): [in this window] [in a new window]   FIGURE 1. Phenotype of CD1d-independent NKT (NK1.1+TCR+) cells. A, Splenocytes from 2m-deficient B6 mice were analyzed by three-color staining for TCR, NK1.1, and various cell surface molecules. The dot plot in the top left corner shows staining for TCR and NK1.1. The percentage on top of the dot plot shows the frequency of NKT cells in the spleen. Histograms show the expression of indicated cell surface molecules on NKT cells (gated as indicated by a box in the dot plot). Data represent the mean ± SD of at least three independent experiments. B, Splenocytes of 2m-deficient mice were stained for NK1.1, TCR, and various TCR V and V by three-color flow cytometry, and the percentages of NK1.1–TCR+ () and NK1.1+TCR+ () cells stained with individual anti-TCR mAb were determined. The mean values (±SD) obtained from three independent experiments are shown.

The developmental pathway of CD1d-independent NKT cells is currently unknown. As they are found in ₂m-deficient mice, their development is probably independent of classical MHC class I, CD1d, and Qa-1^b. They are also found in ₂m/MHC class II double-knockout mice (Fig. 2A), and their phenotypes are similar to those in ₂m single-knockout mice (Fig. 2B), indicating that their development is also independent of MHC class II expression in the host. To test whether CD1d-independent NKT cells undergo thymic selection, we analyzed splenocytes of HY Ag-specific, TCR-transgenic, RAG-deficient mice (30). NK1.1⁺TCR⁺ cells were detected in female transgenic mice of the H-2^b haplotype, in which the transgenic T cells are positively selected (Fig. 2C). They were also found in male transgenic H-2^b mice, in which T cells are negatively selected, as well as in transgenic H-2^d mice, in which T cells are not selected positively or negatively. Although the number of T (NK1.1^–TCR⁺) cells was significantly higher in the spleen of transgenic H-2^b mice than in H-2^d mice, the numbers of NK1.1⁺TCR⁺ cells were similar to each other (Fig. 2C). The phenotype of NK1.1⁺TCR⁺ cells in these mice was similar to that of NKT cells in ₂m-deficient mice. They expressed NK cell receptors and were dominantly double negative (Fig. 2D) and CD44^high (data not shown). However, the percentages of Ly49A⁺ cells among transgenic NKT cells were significantly lower than that of NKT cells in nontransgenic B6 or ₂m-deficient mice. It should be noted that the frequency of Ly49A⁺ NK cells in these transgenic mice was also lower than that in B6 mice (10.5 ± 2.6 vs 16.8 ± 1.3%). The frequencies of CD8⁺ NKT cells also varied among different transgenic NKT cells. The significance of these differences is unknown. NK1.1^–TCR⁺ cells in TCR-transgenic mice expressed no NK cell receptors, dominantly CD8⁺ and CD44^neg-high (data not shown). These results indicate that NKT cells in the TCR-transgenic mice are similar to normal CD1d-independent NKT cells, and they do not undergo conventional thymic selection process.

View larger version (51K): [in this window] [in a new window]   FIGURE 2. Development of CD1d-independent NKT cells. A, Spleen cells from wild-type, 2m-knockout (KO), and MHC class II/2m double-KO B6 mice were stained for TCR and NK1.1, and the number of NKT cells per spleen was calculated from the percentage of NKT cells and the total number of nucleated cells per spleen. The bars represent the mean ± SD of at least three independent experiments. B, NKT cells among splenocytes from wild-type B6 mice (), 2m KO mice (), and MHC class II/2m double-KO mice () were analyzed by flow cytometry for the expression of various cell surface molecules. The percentages of NKT cells expressing the indicated molecules were determined as described in Fig. 1. Data represent the mean ± SD of at least three independent experiments. C, Spleen cells from HY-specific, TCR-transgenic, RAG-deficient mice of the indicated sex and MHC haplotype were stained for NK1.1 and TCR, and the percentages and numbers of NKT cells per spleen were determined by flow cytometry as described in Fig. 1. Data represent the mean ± SD of at least three independent experiments. D, The percentages of NKT cells expressing the indicated molecules were determined by flow cytometry as described in Fig. 1. , H-2b male transgenic NKT cells; , H-2b female transgenic NKT cells; , H-2d male transgenic NKT cells. Data represent the mean ± SD of at least three independent experiments.

We established a simple culture method to expand the CD1d-independent NKT cell population. The method was adapted from classical NK-LAK cell culture by reducing the IL-2 concentration. Splenocytes were cultured overnight without IL-2 to remove adherent cells. Nonadherent cells were recovered and cultured in the presence of 200 U/ml IL-2. After 3 days of cultures, nonadherent cells were removed, and adherent cells were cultured with 200 U/ml IL-2 for an additional 4–5 days. The NK1.1⁺TCR⁺ cell population was preferentially expanded by this culture method, whereas NK cells were the dominant population when 1000 U/ml IL-2 was used (data not shown). Almost all cells recovered from the cultures after 7–8 days were CD3⁺ (Fig. 3A, left panel) and included NK1.1⁺TCR⁺ and NK1.1^–TCR⁺ cells (Fig. 3A, right panel) as well as TCR⁺ T cells (data not shown). The cultured NK1.1⁺TCR⁺ cells and NK1.1^–TCR⁺ cells generated from ₂m-deficient splenocytes expressed Ly49 and CD94/NKG2, but the frequencies of the cells expressing these NK receptors were significantly lower than those of freshly isolated splenic NKT cells (Fig. 3B). NK1.1^–TCR⁺ cells in particular were mostly negative for NK receptors. It seems that Ly49C/I, CD94, and NKG2A/C/E on NKT cells were down-regulated in the cultures.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Phenotype of NKT-LAK cells. A, Splenocytes from 2m-deficient mice were cultured for 8 days with 200 U/ml IL-2 as described in Materials and Methods. The cells harvested from the cultures were stained for NK1.1 and CD3 (left panel) or NK1.1 and TCR (right panel) and analyzed by flow cytometry as described in Fig. 1. Representative results from more than six independent experiments are shown. B, NKT-LAK cells generated from 2m-deficient mouse spleen as described above were stained for TCR, NK1.1 and various cell surface molecules, and the expression of the indicated molecules on NK1.1+TCR+ () and NK1.1–TCR+ () cells was determined by three-color flow cytometric analyses. The data represent the mean ± SD of at least three independent experiments. C, NKT-LAK cells derived from B6 splenocytes (right panel) were stained with -GalCer-loaded soluble dimeric mouse CD1d-Ig fusion protein. For a positive control, the CD1d-restricted NKT cell line (left panel) was used. Shaded histograms show the staining with -GalCer-loaded CD1d-Ig, and open histograms show control staining with CD1d-Ig incubated with DMSO. The results of one of two sets of independent experiments, both showing similar results, are shown.

Origin of NKT-LAK cells

To determine whether NK1.1⁺TCR⁺ and NK1.1^–TCR⁺ cells recovered from the cultures with 200 U/ml IL-2 derived from T cells or from NKT cells that acquired or lost NK receptors during the culture, NKT (NK1.1⁺TCR⁺) and T (NK1.1^–TCR⁺) cells from ₂m-deficient mice were purified by cell sorting. They were mixed with Ly5 (CD45) congenic Pep3b splenocytes (Ly5.1) and cultured with 200 U/ml IL-2. After 8 days, ₂m^–/– cells (Ly5.2) were analyzed by flow cytometry. More than 90% of NK1.1⁺TCR⁺ cells maintained the phenotype, and <10% lost NK1.1 expression (Fig. 4A). Similarly, almost 90% of NK1.1^–TCR⁺ cells remained NK1.1^–, whereas only 10% acquired NK1.1 during the cultures and became NK1.1⁺TCR⁺ cells. Therefore, most NK1.1⁺TCR⁺ cells recovered from the cultures seemed to derive from NKT cells, but some derived from T cells that acquired NK1.1 during the cultures. Similarly, some of the NK1.1^–TCR⁺ cells derived from NK1.1⁺TCR⁺ cells, but NK1.1^–TCR⁺ cells also gave rise to this population. It should be noted that CD8⁺ T cells in ₂m-deficient mice are not typical CD8 T cells, as the mice are deficient in MHC class I expression (39).

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Origin of NKT-LAK cells. A, NK1.1+TCR+ cells (upper panel) or NK1.1–TCR+ cells (lower panel) were sorted from 2m-deficient mouse spleen (Ly5.2) and cocultured with Pep3b splenocytes (Ly5.1) in NKT-LAK cultures. After 8 days of culture, cells were stained for Ly5.2, NK1.1, and TCR, and the phenotypes of Ly5.2+ cells were determined by three-color flow cytometry. The results from one of two independent experiments, both showing similar results, are shown. B, Spleen cells from 2m-deficient mice were stained for NK1.1, TCR, and the IL-2R, and the expression of the -, -, and -chains of the IL-2R on NK1.1+TCR+ cells (CD1d-independent NKT cells) and NK1.1+CD3– (NK cells) cells was determined by flow cytometry. The data represent the mean ± SD of at least three independent experiments. C, NK1.1+CD3– (NK), NK1.1+TCR+ (NKT), and NK1.1–TCR+ (T) cells from 2m-deficient mouse spleen were purified by cell sorting and cultured with various concentrations of IL-2, and their proliferation was measured by [3H]thymidine incorporation on days 3 and 7. The data represent the mean ± SD of triplicate samples. The results of one of two independent experiments, both showing similar results, are shown.

NK cell-like cytotoxicity of NKT-LAK cells

Splenocytes from B6 and ₂m^–/– mice were cultured with 200 or 1000 U/ml IL-2 as described above to generate NKT-LAK and NK-LAK cells, respectively, and were tested for cytotoxicity. IL-2-activated NKT and NK cells generated from B6 or ₂m^–/– mice efficiently killed the prototypic NK cell target YAC-1 and MHC class I-deficient RMA-S cells (Fig. 5A). NKT-LAK cells also killed RMA cells that express MHC class I, although the cytotoxicity was considerably lower than that against RMA-S cells. They killed ₂m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. Because the cultures contained both NK1.1⁺ and NK1.1^– cells, we purified NK1.1⁺TCR⁺ and NK1.1^–TCR⁺ cells from cultured ₂m^–/– splenocytes on day 5, and the sorted cells were cultured for 3 more days to remove the bound Abs. NK1.1⁺TCR⁺ cells recovered from the cultures efficiently killed RMA-S cells, but not RMA cells, indicating that they are inhibited by MHC class I (Fig. 5B). NK1.1^–TCR⁺ cells killed RMA-S cells as well as RMA cells, although the cytotoxicity was significantly lower than that against RMA-S cells, suggesting that the inhibition of cytotoxicity by MHC class I is incomplete. These results suggest that NK receptors on CD1d-independent NKT cells are functional and mediate NK-like cytotoxicity.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Cytotoxicity of NKT-LAK cells. A, Splenocytes from wild-type B6 or 2m-deficient B6 mice were cultured with 200 or 1000 U/ml IL-2 to generate NKT- and NK-LAK cells, respectively, and their cytotoxicities against indicated target cells were determined by 51Cr release assay (•, B6 NK-LAK cells; , B6 NKT-LAK cells; , 2m-deficient NK-LAK cells; , 2m-deficient NKT-LAK cells). Representative data from at least three independent experiments are shown. B, Splenocytes from 2m-deficient mice were cultured with 200 U/ml IL-2 for 5 days and stained for NK1.1 and TCR. NK1.1+TCR+ () and NK1.1–TCR+ () cells were sorted from the cultured cells and cultured for an additional 3 days with 200 U/ml IL-2. They were tested for cytotoxicity against RMA and RMA-S cells using an E:T cell ratio of 10. The results of one of two independent experiments, both showing similar results, are shown.

To test whether the TCR on CD1d-independent NKT cells also mediate CTL-like cytotoxicity, NK1.1⁺TCR⁺ cells in HY TCR-transgenic, RAG2-deificient H-2^b male mice were grown in NKT-LAK cultures. After 5 days, NK1.1⁺TCR⁺ cells in the cultures were sorted and cultured for 3 more days. IL-2-activated NKT cells thus generated were similar to NKT-LAK cells generated from nontransgenic mice and killed RMA-S cells, but not RMA cells (Fig. 6A). When the HY peptide was added to RMA cells, they efficiently killed RMA cells, indicating that they display MHC/peptide-specific cytotoxicity similar to that of CTL. These results indicate that both NK receptors and the TCR on CD1d-independent NKT cells are functional and mediate NK- and CTL-like cytotoxicity, respectively.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Functional TCR on NKT cells. A, Splenocytes from HY TCR-transgenic, RAG2-deficient H-2b male mice were cultured with 200 U/ml IL-2 for 5 days. TCR+NK1.1+ cells in the cultures were sorted and cultured for 3 more days, and their cytotoxicity against RMA cells preincubated with scramble peptide () or H-Y peptide () and RMA-S cells () was tested. Data are representative of three independent experiments. B, NK1.1+TCR+ cells were isolated from 2m-deficient mouse spleen and incubated in microwells coated with anti-CD3e mAb () or isotype control mAb () for 48 h, and cytokine production in the supernatants was determined by ELISA. Data represent the mean ± SD of triplicate cultures and are representative of three independent experiments. C, IFN- production by splenocytes from mice injected with anti-CD3 mAb. Wild-type B6 (), 2m-deficient (), and Abb/2m-deficient () mice were i.v. injected with 1 µg of anti-CD3 mAb. After 90 min, splenocytes were cultured in vitro in triplicate for 2 h without additional stimulation. Culture supernatants were harvested, and the amounts of IFN- secreted in the supernatants were determined. Control splenocytes from mice injected with 1 µg of isotype control mAb did not produce a detectable amount of IFN-. Data from two independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we have characterized a unique subset of NKT (NK1.1⁺ TCR⁺) cells. The NK1.1⁺TCR⁺ cell population in wild-type B6 mice can be divided into CD1d-restricted and CD1d-independent subsets (2, 23). The latter subset, which is also found in CD1d-deficient B6 mice, is heterogeneous and consists of multiple T cell populations. They include memory CD8⁺ T cells that have acquired NK1.1 after activation (40, 41, 42, 43), mucosa-associated T cells that express invariant TCR (V19-J33) and are restricted by the MHC class I-related molecule MR1 (44, 45), and CD8⁺ NKT cells that are prominent in the liver and large intestine (25, 26, 27). The NKT cells we have characterized in this study are different from these cell populations. They are present in ₂m-deficient mice, which are deficient in the expression of MHC class I and MR1 and lack conventional CD8⁺ T cells and MR1-restricted mucosa-associated T cells. The majority of splenic CD1d-independent NKT cells in ₂m-deficient mice are double negative, and this population has not been previously characterized. It is very likely that this subset of CD1d-indpendent NKT cells is also present in normal B6 mice. CD1d-independent NKT cells in this study have the phenotypes and functions of both NK and T cells. They express TCR and NK receptors, including Ly49, CD94/NKG2, and NKG2D. Moreover, upon activation with IL-2, they display NK-like cytotoxicity as well as CTL-like cytotoxicity. The former is inhibited by target cell MHC class I, whereas the latter is Ag⁺ MHC class I-specific. Upon cross-linking of the TCR, they also secrete IFN- and IL-2, but very little IL-4. These characteristics of CD1d-independent NKT cells are very different from those of V14i T cells. The majority of CD1d-restricted NKT cells do not express Ly49, and they have a highly skewed TCR repertoire (46). Upon stimulation with -GalCer or CD3 cross-linking, they secrete large amounts of IFN- and IL-4 (1). Although NK-like cytotoxicity of -GalCer-stimulated NKT cells has been reported, the cytotoxicity may not be directly mediated by V14i T cells, but may be mediated by NK cells that are stimulated with cytokines secreted by V14i T cells (9, 18, 47, 48, 49, 50, 51).

The origin of CD1d-independent NKT cells is still unclear. CD1d-independent NKT cells have a memory T cell-like phenotype (IL-2R⁺CD44^high), and some conventional CD8 T cells have been shown to acquire NK cell-associated molecules, including NK1.1 and Ly49, upon activation (40, 41, 42, 43), suggesting that some NK1.1⁺TCR⁺ cells may derive from activated CD8 T cells. However, CD1d-independent NKT cells in this study are unlikely to derive from conventional CD8 T cells, because the ₂m-deficient mice used in this study have very few CD8 T cells (39). It should also be noted that the TCRV usage of CD1d-independent NKT cells in this study significantly differs from that of memory CD8 T cell expressing Ly49 and NK1.1 reported previously (40). Approximately 20% of Ly49⁺CD8 T cell express V 5.1,5.2, whereas only 7% of CD1d-independent NKT cells in this study express V5.1, 5.2. Furthermore, the development of CD1d-independent NKT cells is independent of MHC class I or II, CD1d, or Qa-1^b, and they are not subjected to the same thymic selection that normal CD8 T cells undergo. Because they do not undergo thymic selection, they may be potentially self-reactive. The expression of Ly49 and CD94/NKG2 receptors may be important for the maintenance of self-tolerance of CD1d-independent NKT cells. CD1d-indendent NKT cells can be selectively grown in cultures in the presence of 200 U/ml IL-2. Why CD1d-independent NKT cells, but not V14i T cells or NK cells, grow in these cultures is still unknown. Both NKT and NK cells equally proliferate in the presence of 200 U/ml IL-2. Furthermore, early in the cultures (day 5) both NK and NKT cells are found, but in the following 3 days, most NK cells are lost, whereas NKT cells continue to grow. We tested whether this is due to killing of NK cells by NKT cells in the cultures, but purified IL-2 activated NKT cells did not display cytotoxicity against IL-2-activated NK cells as assessed by ⁵¹Cr release assay (data not shown). It is also unknown why CD1d-restricted NKT cells failed to grow in this culture. Nevertheless, it is a simple and convenient method to preferentially grow CD1d-independent NKT cells and may prove to be a useful tool to further analyze their functions.

IL-2-activated, CD1d-indpendent NKT (NKT-LAK) cells are very similar to IL-2-activated NK (NK-LAK) cells. Both kill YAC-1 and the TAP-deficient RMA-S, whereas RMA, which expresses MHC class I, is resistant. Furthermore, they kill ₂m-deficient Con A blasts, but not normal Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on targets. It should be noted that NKT-LAK cells consist of two populations, namely, NK1.1⁺ and NK1.1^– cells. Most known MHC class I-specific inhibitory receptors, Ly49 and CD94/NKG2, on NKT cells are down-regulated in cultured NKT-LAK cells. This is particularly prominent in NK1.1^–LAK cells generated from ₂m-deficient splenocytes. This may explain why they show low, but significant, cytotoxicity against RMA cells, suggesting an incomplete inhibition by MHC class I. Most NKT-LAK cells do not express Ly49A, C, I, or CD94/NKG2, which are the only known inhibitory receptors for H-2^b (52). Nevertheless, NKT-LAK cells are inhibited by MHC class I (H-2^b) on RMA cells and B6 Con A blasts. Therefore, it seems likely that NKT-LAK cells express unknown inhibitory receptor(s) that recognizes H-2^b. CD1d-independent NKT cells express NKG2D, which is known to mediate cytotoxicity to a wide range of NK-sensitive target cells (53). However, NKG2D ligands have not been detected on RMA-S cells, and activating receptors that mediate cytotoxicity against RMA-S cells have not been identified.

NKT-LAK cells generated from TCR-transgenic mice showed Ag/MHC-specific cytotoxicity, indicating that the TCR on CD1d-independent NKT cells is functional. Cross-linking of CD3 on resting CD1d-independent NKT cells from nontransgenic mice also activates them to secrete cytokines. Anti-CD3 mAb stimulates splenocytes of ₂m/MHC class II double-knockout mice in vivo. The stimulated splenocytes secrete IFN-. As these mice are deficient in conventional T cells and CD1d-restricted T cells, it seems likely that CD1d-independent NKT cells are stimulated with anti-CD3 in vivo and secrete IFN-. Therefore, the TCR on CD1d-independent NKT cells is potentially functional, although Ag/MHC specificity of the TCR on normal CD1d-independent NKT cells is unknown. It seems that the main function of CD1d-independent NKT cells is cytotoxicity, whereas CD1d-restricted NKT cells are an important source of cytokines. Thus, the NKT cell population is similar to conventional T cells and can be divided into two subsets, namely, cytotoxic CD1d-independent and cytokine-producing CD1d-restricted NKT cells. We are currently investigating whether CD1d-independent NKT cells are activated and expand during infections and whether they play a role in immune responses in vivo.

	Footnotes

 
¹ This work was supported by a grant from the National Cancer Institute of Canada. M.M. is a recipient of fellowships from the Leukemia Research Fund of Canada and the Michael Smith Foundation for Health Research.

² Address correspondence and reprint requests to Dr. Fumio Takei, Terry Fox Laboratory, British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. E-mail address: ftakei{at}bccrc.ca

³ Abbreviations used in this paper: -GalCer, -galactosylceramide; ₂m, ₂-microglobulin; LAK, lymphokine-activated killer; RAG, recombinase-activating gene.

Received for publication October 23, 2003. Accepted for publication March 15, 2004.

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