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Cutting Edge: Analysis of Human V24⁺CD8⁺ NK T Cells Activated by -Galactosylceramide-Pulsed Monocyte-Derived Dendritic Cells

Tsuyoshi Takahashi^*,, Shigeru Chiba^*, Mie Nieda^¶, Takeshi Azuma, Soichiro Ishihara, Yoichi Shibata, Takeo Juji^¶ and Hisamaru Hirai^1,*

Departments of ^* Hematology and Oncology, Transfusion Medicine, Urology, and Surgery, Graduate School of Medicine, University of Tokyo, and ^¶ Department of Research, The Japanese Red Central Blood Center, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Human V24⁺ NKT cells constitute a counterpart of mouse V14⁺ NKT cells, both of which use an invariant TCR- chain. The human V24⁺ NKT cells as well as mouse V14⁺ NKT cells are activated by glycolipids in a CD1d-restricted manner and produce many immunomodulatory cytokines, possibly affecting the immune balance. In mice, it has been considered from extensive investigations that V14⁺CD8⁺ NKT cells that express invariant TCR do not exist. Here we introduce human V24⁺CD8⁺ NKT cells. These cells share important features of V24⁺ NKT cells in common, but in contrast to CD4^-CD8^- (double-negative) or CD4⁺ V24⁺ NKT cells, they do not produce IL-4. Our discovery may extend and deepen the research field of V24⁺ NKT cells as well as help to understand the mechanism of the immune balance-related diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Although mouse V14⁺ NKT cells that express TCR and NKR, NK1.1 (NKR-P1C), may play some important roles in the immune system (1, 2), the details are unknown. These cells use an invariant TCR- chain (V14-J281) that pairs preferentially with V8, V7, or V2 (3, 4, 5). The human homolog of mouse V14⁺ NKT cells appears to be V24⁺ NKT cells, because many features identified in the former are conserved in the latter. The V24⁺ NKT cells express CD161 (NKR-P1A) as an NKR and the TCR of the V24-JQ invariant TCR- chain that pairs preferentially with V11 (human V24 and V11 are homologs of mouse V14 and V8, respectively) (6, 7). Phenotypically, CD4^-CD8^- double-negative (DN)² and CD4⁺ NKT cells have been reported in both mice and humans (8, 9, 10). In mice, it has been considered from extensive investigations that V14⁺CD8⁺ NKT cells that express invariant TCR do not exist (5, 8, 11, 12). The function of human V24⁺ NKT cells is similar to that of mouse V14⁺ NKT cells in many respects. Both are activated by synthetic glycolipids such as -galactosylceramide (-GalCer) in a CD1d-restricted and an invariant TCR-mediated manner (13, 14, 15, 16). They exhibit cytotoxic activity against tumor cells (10, 17, 18), which may be important for natural anticancer immunity. On TCR stimulation, they produce a large amount of IFN- and IL-4, whereas the level of IL-4 production is much higher in CD4⁺ NKT cells than in DN NKT cells (9, 10, 19, 20, 21).

In this report, we describe a novel subpopulation of human V24⁺ NKT cells with a CD4^-CD8⁺ phenotype (CD8 single-positive, hereafter referred to as V24⁺CD8⁺ NKT), which have features similar to those of other V24⁺ NKT subpopulations such as the usage of invariant TCR, CD1d-restricted activation, and cytotoxicity against tumor cells. Interestingly, the cytokine production patterns are distinctly different among them. V24⁺CD8⁺ NKT cells do not produce IL-4, whereas DN and CD4⁺ V24⁺ NKT cells do. V24⁺CD4⁺ NKT cells produce more IL-10 and IL-13 than do DN and CD8⁺ V24⁺ NKT cells. These differences in cytokine production patterns potentially influence the immune balance such as the determination of the Th1-Th2 profile and activation or suppression of immune response. Relative and absolute numbers of V24⁺ NKT cells belonging to each subpopulation may be important in immune regulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Abs and reagents

The following Abs were purchased from Immunotech (Marseilles, France): IgG1 (679.1 Mc7); IgG2b (MOPC-195); anti-CD3 (UCHT-1); anti-CD4 (SFCI12T4D11(T4)); anti-CD8 (2ST8.5H7); anti-CD161 (191B8); anti-V24 (C15); and anti-V11 (C21). Anti-CD1d (55.3.1) (22) was a gift from Dr. S. Porcelli (Albert Einstein College of Medicine, Bronx, NY). -GalCer was obtained from Kirin (Gunma, Japan). rhGM-CSF and rhIL-4 were purchased from CellGenix (Freiburg, Germany), and rhIL-2 was obtained from Shionogi (Osaka, Japan).

Cell populations

V24⁺ NKT cells were established as follows. Monocytes from the healthy human donors were each cultured in AIM medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS with rhIL-4 (500 U/ml) and rhGM-CSF (500 U/ml) for 5 days, and used as monocyte-derived dendritic cells (Mo-DCs). Lymphocytes from the same donor were cultured with irradiated (50 Gy)-Mo-DCs which were pulsed for 12 h with -GalCer (100 ng/ml). After 7 days, V24⁺ cells were established by positive (V24) magnetic bead sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) and maintained in the presence of rhIL-2 (40 U/ml). The cells were restimulated every 7 days. When the cells were expanded, V24⁺V11⁺CD4^-CD8^-, V24⁺V11⁺CD4⁺, and V24⁺V11⁺CD8⁺ subpopulations were sorted using the FACSVantage apparatus (BD Biosciences, San Jose, CA) and maintained in the presence of rhIL-2 (40 u/ml) and by restimulation every 7–10 days.

Phenotypic assay

Cells were analyzed by flow cytometry using a FACSVantage apparatus. Immunofluorescence staining using specific mAb was performed according to standard procedures.

Molecular analysis of TCR- transcripts

Total RNA was extracted from 1 x 10⁶ NKT or T cells according to the manufacturer’s protocol (ISOGEN; Wako, Osaka, Japan) and reverse transcribed using the outer primer of the constant region of TCR-. Then the transcribed cDNA was subjected to the first-round PCR amplification using the outer primer of the variable region of TCR V24 in a thermal cycler (RoboCycler; Stratagene, La Jolla, CA). The second PCR was performed using the inner primer pair. The PCR products were electrophoresed, transferred to a nylon membrane, hybridized with ³²P-labeled JQ probe, and autoradiographed. The details of PCR primers and JQ probe sequences and PCR conditions were described elsewhere (13). For the detection of V-J sequence, the DNA band was excised from the gel, and DNA was extracted according to the manufacturer’s protocol (QIAquick Gel Extraction Kit; Qiagen, Hilden, Germany). Their nucleotide sequences were then determined (ABI PRISM 3100 Genetic Analyzer; PE Applied Biosystems, Foster City, CA) by a dideoxy chain termination method according to the manufacturer’s protocol, with a DNA sequencing kit (PE Applied Biosystems).

Proliferative response to -GalCer-pulsed Mo-DCs and their inhibition by anti-CD1d Ab

Proliferative response was assayed as follows. The bulk of 5 x 10⁴ NKT cells as the responders and 5 x 10⁴ irradiated-allogeneic Mo-DCs (all allogeneic monocytes were derived from the same donor) as the stimulators in 200 µl of culture medium were seeded onto flat-bottom microtiter wells with or without -GalCer (100 ng/ml). The culture was incubated for 48 h. For the final 6 h of incubation, 1 µCi [³H]TdR was added to each well, and the incorporation of [³H]TdR was determined by liquid scintillation counting. To determine whether the response is CD1d restricted, 55.3.1 (anti-CD1d mAb) or IgG2b as a control was added to the wells (5 µg/ml).

Cytotoxicity assays

⁵¹Cr release assay was performed as follows. ⁵¹Cr-labeled (Na₂⁵¹CrO₃) (Amersham, Arlington Heights, IL) U937 or K562 cell lines (5 x 10³) as the target cells and various numbers of effector cells in 200 µl of culture medium were seeded onto round-bottom microtiter wells. The culture was incubated for 4 h, and 100 µl of supernatant were collected from each well. The percentage of specific ⁵¹Cr release was calculated as [(cpm experimental release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release)] x 100.

Cytokine production

For the cytokine production assay, 5 x 10⁴ NKT and 5 x 10⁴ -GalCer-pulsed Mo-DCs (all allogeneic monocytes were derived from the same donor) were suspended in 200 µl of culture medium in 96-well plates. After 18 h, the supernatants were collected from each well and assayed for the concentrations of IFN-, IL-4, IL-10, and IL-13 by ELISA according to the manufacturer’s protocol (AN'ALYZA; Genzyme, Cambridge, MA).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Phenotypic and molecular characterization of lymphocytes activated by -GalCer-pulsed Mo-DCs

V24⁺ NKT cells have been reported to be activated by stimulation with -GalCer-pulsed Mo-DCs in a CD1d-restricted manner (14, 15, 16). We generated Mo-DCs from healthy donors using IL-4 and GM-CSF in vitro, which were then pulsed with -GalCer. The -GalCer-pulsed Mo-DCs were cocultured with autolymphocytes to generate V24⁺ NKT cells. On the basis of the responses of V24⁺-sorted lymphocytes to the stimulation, we identified CD8 single-positive V24⁺CD8⁺ NKT cells as well as DN and CD4⁺ V24⁺ NKT cells from two healthy donors (Fig. 1A). According to CD4/CD8 phenotype, each subpopulation was sorted. Each V24⁺ NKT subpopulation used V11 for its TCR V repertoire and expressed NK receptor, CD161 (NKR-P1A) (Fig. 1B). It did not express other NK receptors, CD16, CD56, or CD94 (data not shown). To confirm whether V24⁺CD8⁺ NKT cells use JQ as an invariant TCR- chain, we then performed RT-PCR with the V24 and C primers and analyzed the fragment encompassing the V-J junction, using >99% pure NKT subpopulations. A V24⁺V11^- T cell population was sorted and used as a control. When the PCR products were fractionated and hybridized with an oligonucleotide probe specific for JQ on a filter, the expression of JQ was detected in V24⁺CD8⁺ NKT cells as well as in DN and CD4⁺ V24⁺ NKT cells, but not in V24⁺V11^- T cells from the same donor (Fig. 2A). For further confirmation, we sequenced the PCR product of each subpopulation by a direct sequence method. V24⁺CD8⁺ NKT cells as well as DN and CD4⁺ V24⁺ NKT cells were confirmed to use the V24-JQ invariant chain without N region diversity (Fig. 2B). Next we examined the frequency of CD8⁺ V24⁺ NKT cells in fresh PBL from 10 healthy donors (age 32.8 ± 6.4 years (mean ± SD)) to identify their physiological counterpart in the peripheral blood. We performed four-color analysis using V11-FITC/CD8-PE/V24-biotin-avidin-PE-Texas Red (ECD)/CD4-allophycocyanin (APC) stain. The frequency of V24⁺V11⁺ cells in the CD3⁺ cells was 0.050 ± 0.030%. The percentage of CD4/CD8 phenotype in V24⁺V11⁺ cells was: CD4^-CD8^-, 71.2 ± 11.8; CD4⁺CD8^-, 24.8 ± 12.0; CD4^-CD8⁺, 2.7 ± 1.8; CD4⁺CD8⁺, 0.3 ± 0.6. Although the frequency of CD8⁺ V24⁺ NKT cells was <5%, this subpopulation clearly existed in fresh PBL (Fig. 1C).

View larger version (45K): [in this window] [in a new window]   FIGURE 1. Phenotypic analysis of V24+DN NKT, V24+CD4+ NKT, and V24+CD8+ NKT cells. The -GalCer-pulsed Mo-DCs were cocultured with autolymphocytes and selected for expression of V24. The cells were then restimulated with -GalCer-pulsed Mo-DCs and IL-2 for two cycles and then sorted into separate populations based on the expression of CD4 or CD8. The cells were stained by two-color flow cytometry using the indicated mAbs. Phenotypes of V24+ NKT cells were indicated before (A) and after (B) sorting based on CD4/CD8 expression. The numbers in the dot plots indicate the percentage of each population. Shown are the dot plots obtained from the first donor (HD1). Similar results were obtained from the second donor (HD2) (data not shown). Next the CD4/CD8 phenotype of V24+ NKT cells was examined in peripheral blood. Freshly isolated PBL from 10 healthy donors were analyzed by four-color flow cytometry analysis using V11-FITC/CD8-PE/V24-biotin-avidin-PE-Texas Red (ECD)/CD4-allophycocyanin (APC) stain. The cells were gated for lymphoid cells by light scatter characteristics, and expression of CD4/CD8 was examined within V24+V11+ cells. Representative dot plots are shown (C). The numbers in the dot plot indicate the percentage of each subpopulation among V24+V11+ cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Invariant TCR- chain expression of V24+ NKT cells. Samples were from the first donor (HD1). In all samples, PCR was performed using V24-specific 5' and C-specific 3' primers. The PCR products were electrophoresed on 2% agarose gel, and fractionated products were transferred to a nylon membrane and then hybridized with a 32P-labeled JQ probe. All NKT cells except V24+V11- T cells used the JQ sequence (A). For detection of the V-J sequence, the nucleotide sequences of the PCR products were determined (B). The V-J sequence of V24+V11- T cells could not be determined by this method.

A CD1d-restricted response of V24⁺CD8⁺ NKT cells to -GalCer-pulsed Mo-DCs

Next, we examined the response of V24⁺CD8⁺ NKT cells to -GalCer-pulsed Mo-DCs in a CD1d-restricted manner. The response of V24⁺CD8⁺ NKT cells as well as DN and CD4⁺ V24⁺ NKT cells to Mo-DCs was enhanced by -GalCer and inhibited by an anti-CD1d mAb, indicating that V24⁺CD8⁺ NKT cells recognize a glycolipid, -GalCer, in a CD1d-restricted manner similar to other V24⁺ NKT subpopulations (Fig. 3). It is still unknown whether there exists a murine homolog of human V24⁺CD8⁺ NKT cells, i.e., V14⁺CD8⁺ NKT cells in mice, although there have been reports that the forced expression of CD8 in transgenic mice resulted in deletion of this V14⁺ NKT population (5, 8) and that mouse CD8⁺NK1.1⁺ NKT cells do not use invariant V14 TCR nor are they restricted in CD1d (11, 12). Thus, CD8⁺ NKT subpopulations that express invariant TCR may exist in human V24⁺ NKT populations but not in mouse V14⁺ NKT populations.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. All subpopulations of human V24+ NKT cells respond to synthetic -GalCer in a CD1d-restricted manner. All subpopulations of V24+ NKT cells from healthy donor 1 (HD1) and healthy donor 2 (HD2) nonstimulated (), and stimulated with Mo-DC (), Mo-DC plus -GalCer (), Mo-DC plus -GalCer plus control Ig (), and Mo-DC plus -GalCer plus CD1d (). Cells were incubated under each condition for 48 h, and [3H]TdR was added to each well during the final 6 h of incubation. Results are the means ± SD of triplicate values. Representative results of three independent experiments.

Cytotoxic activity of V24⁺CD8⁺ NKT cells

It has been reported that human V24⁺ NKT cells as well as mouse V14⁺ NKT cells exhibit cytotoxicity against tumors. We thus examined the cytotoxic activity of human V24⁺CD8⁺ NKT cells. V24⁺CD8⁺ NKT cells as well as DN and CD4⁺ V24⁺ NKT cells exhibited cytotoxic activities against U937 cells (percent cytotoxicity, 20–40; E:T 10:1). However, none of them showed a cytotoxic activity against an erythroleukemic, NK-sensitive cell line, K562 (percent cytotoxicity, <10; E:T 10:1; data not shown).

Cytokine secretion of V24⁺ NKT cells

Because human and mouse DN and CD4⁺ NKT cells have been reported to produce a large amount of various cytokines such as IFN- and IL-4 when stimulated (9, 10, 19, 20, 21), we examined the cytokine production pattern of each subpopulation of V24⁺ NKT cells after stimulation with -GalCer-pulsed Mo-DCs. All subpopulations produced a large amount of IFN- (Fig. 4A). Regarding IL-4 production, both DN and CD4⁺ V24⁺ NKT cells produced a large amount of IL-4, and V24⁺CD4⁺ NKT cells tended to produce more IL-4 than did V24⁺DN NKT cells. Thus, in contrast to V24⁺DN NKT cells, V24⁺CD4⁺ NKT cells had more Th2 bias. However, V24⁺CD8⁺ NKT cells produced a small amount of IL-4 (Fig. 4B). Regarding IL-10 and IL-13 production, V24⁺CD4⁺ NKT cells produced a large amount ofthese cytokines on stimulation, but both DN and CD8⁺V24⁺ NKT cells produced a small amount of these cytokines (Fig. 4, C and D). These results indicated that the cytokine production pattern might be distinctly different among V24⁺ NKT subpopulations and thus that each subpopulation could have a different effect on immunological response, although it is possible that repeated GalCer/cytokine stimulation might influence the function such as cytokine production patterns. Recently, the immunoregulatory role of NKT cells has been well documented. NKT cell-derived IL-10 was essential for the differentiation of Ag-specific regulatory T cells in systemic tolerance (23). V24⁺CD4⁺ NKT cell-derived IL-13 prevented effective CTL-mediated tumor eradication (24). Furthermore, the relationship between autoimmune/allergy diseases and NKT cells has been recently reported in mouse and human (25, 26, 27, 28). Therefore, relative and absolute numbers of V24⁺ NKT cells belonging to each subpopulation may be important in physiological and pathological regulation of immune system. The relationship between changes in the ratio of each V24⁺ NKT subpopulation and the disease status such as autoimmune disease, allergy, malignancy, infection, and transplantation must be studied. In contrast, when considering V24⁺ NKT cells as a tool for cell therapy, we should choose the adequate subpopulation of NKT cells depending on its purpose in combination with selecting APC and/or ligands.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Assay for cytokine production. A total of 5 x 104 V24+ NKT cells and -GalCer-pulsed allogeneic Mo-DCs (all allogeneic Mo-DCs were derived from same donor) were cocultured for 48-h. The supernatant was collected from each well and assayed for the production of IFN- (A), IL-4 (B), IL-10 (C) or IL-13 (D) by ELISA. DN NKT (), CD4+ NKT (), CD8+ NKT (). Representative results of three independent experiments ± SD.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Hisamaru Hirai, Department of Hematology and Oncology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail address: hhirai-tky{at}umin.u-tokyo.ac.jp

² Abbreviations used in this paper: DN, double-negative; -GalCer, -galactosylceramide; rh, recombinant human; Mo-DC, monocyte-derived dendritic cell; HD, healthy donor.

Received for publication December 19, 2001. Accepted for publication February 7, 2002.

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Top Abstract Introduction Materials and Methods Results and Discussion References

 

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