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Distinct Functional Lineages of Human Vα24 Natural Killer T Cells

Distinct Functional Lineages of Human Vα24 Natural Killer T Cells CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy sub- jects express 1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents -galactosyl- ceramide (GalCer) loaded CD1d-tetramers and anti-V24 monoclonal antibody, to specifi- cally identify and characterize these rare cells in fresh PBLs. We report here that CD4 and CD4 CD8 (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4 NKT cells were the exclusive pro- ducers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases. Key words: CD1 • NKT cells • cytokine • IDDM • T cell development Introduction NKT cells are a conserved subpopulation of  T cells, ple, they can suppress type I diabetes in NOD mouse which are restricted by the antigen-presenting molecule through the secretion of IL-4 and IL-10 (5, 6) and their de- CD1d, and appear to regulate several disease processes fects in both NOD mice and humans with IDDM may ranging from tumor rejection to autoimmune diseases (for contribute to pathogenesis (7, 8). Conversely, they naturally a review, see references 1 and 2). They express a con- suppress methylcholantrene-induced carcinogenesis through served canonical TCR (V14J18-V8 in mouse and IFN- (9). In another report, secretion of the Th2 cytokine V24J18-V11 in human) that is thought to recognize a IL-13 was found to inhibit the immune rejection of a tu- self-antigen mimicked by the glycolipid GalCer. Since mor graft (10). Collectively, these findings suggest that reg- NKT cells are present at high frequency in various mouse ulated expression of Th1 or Th2 cytokines by NKT cells, tissues and in human liver, they seem to participate in the rather than mere changes in its frequency (7, 11, 12), might innate, rather than the adaptive arm of the immune re- control the outcome of some disease conditions. sponse and resemble other innate lymphocytes such as B-1 How could the Th1- versus Th2-promoting functions of B cells and  T cells which express canonical antigen re- NKT cells be selectively recruited? It has been suggested that ceptors responding to cell stress and tissue damage (3, 4). altered GalCer ligands with shorter sphingosine chain could Importantly, CD1d is mainly expressed on dendritic cells selectively activate Th2 functions (13), whereas NK1.1 sig- (DCs), macrophage, and B cells, implying that NKT cells naling could favor Th1 response (14). However, the possibil- primarily interact with APCs rather than tissue cells. ity that subsets of NKT cells might specialize in Th1 versus The secretion of Th1 and Th2 cytokines by NKT cells is Th2 functions has not been thoroughly investigated. thought to underlie their regulatory properties. For exam- One obstacle to the identification of NKT cell subsets has been that, until recently, NKT cells could not be unambigu- ously identified. The generation of CD1d-GalCer tetramers Address correspondence to Albert Bendelac, Dept. of Molecular Biology, specific for both mouse and human canonical TCR makes it Princeton University, Washington Rd., Princeton, NJ 08544. Phone: 609- 258-5454; Fax: 609-258-2205; E-mail: [email protected] possible to identify NKT cells based on their specificity rather 637 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/03/637/05 $5.00 Volume 195, Number 5, March 4, 2002 637–641 http://www.jem.org/cgi/content/full/195/5/637 than their phenotype (15–17). CD1d tetramers have already cells above background staining levels, we combined the revealed several important findings, including a subset of use of two TCR-specific reagents, a mAb to V24 and CD1d-restricted murine NKT cells that do not express the CD1d-GalCer tetramers. We found that, although NK1.1 marker and differ from the NK1.1 cells with respect CD1d-GalCer tetramer staining is completely inhibited to their pattern of integrins (15). However, detailed examina- by prior incubation with the anti-V24 mAb (15), the re- tion of human NKT cells has not been performed. verse reaction order allowed significant binding of anti- An additional challenge to the study of fresh human V24, presumably because the tetramers require contigu- PBLs is the very low frequency of canonical NKT cells, of- ous clusters of TCR to bind, leaving a significant amount ten well below common background level staining of of unbound TCR available for bright V24 staining. Fig. 1 0.1%. Here, we have used a combination of CD1d-Gal- A shows that the frequency of V24/CD1d-GalCer dou- Cer tetramers and anti-V24 mAb, which specifically ble positive canonical NKT cells in the fresh PBLs of identifies the canonical NKT cells even at the very low fre- healthy volunteers is between 0.01 and 0.1%. Importantly, quencies found in human PBLs, to investigate human tetramer staining alone invariably included 0.01–0.05% NKT cell subsets. We have dissected the phenotype of nonV24 cells, which are noncanonical cells that presum- these cells into CD4 and double negative (DN) popula- ably reflect background staining (Fig. 1 B, top left quad- tions, and found that they systematically differed in many rants in left dot plots). Thus, whereas the population de- functionally relevant ways with respect to Th cytokine fined by conventional tetramer staining alone included a profile, pattern of chemokine receptors, and integrin ex- significant proportion of nonNKT cells in most healthy in- pression, and array of NK receptors displayed on the cell dividuals, especially in those with low NKT cell frequency, surface. These findings suggest that human CD4 and DN the double staining combination allowed complete speci- V24 NKT cells represent functionally separate lineages ficity. This is shown by the 100% expression of V11 that may promote different Th responses. among the V24/CD1d-GalCer double-positive cells (Fig. 1 B, right dot plots). In the sample expressing 0.01% NKT cells, 100 out of 100 gated cells were V11 , indi- Materials and Methods cating that this technique specifically identified all of the 100 canonical NKT cells present among 1 million PBL. Antibodies. Fluorochrome or biotin conjugates of antibodies against V24, V11, CD4, CD25, CD28, CD56, CD94, NKG2A CD4 and DN Subsets Have Different Cytokine Secretion (Beckman Coulter); CCR1, CCR2, CXCR6 (R&D Systems); Profiles. A fraction of V24 NKT cells expresses CD4 CCR4, CCR5, CCR6, CCR7, CXCR3, CXCR4, CD45RA, while the remaining is CD8-negative and is called DN. CD45RB, CD45RO, CD49a, CD49b, CD49d, CD49e, CD49f, While the proportion of the CD4 and DN varied consider- CD69, CD152, CD154, CD158a, CD158b, CD161, IL-4, IL-13, ably between individuals, on average, they were roughly TNF-, and IFN- (BD Biosciences) were used. equal (50%) in a group of 10 healthy individuals (Fig. 1 B, Flow Cytometric Analysis. PBLs were obtained from whole and data not shown). Surprisingly, despite reports that the blood of healthy donors by centrifugation over Ficoll (Amersham corresponding mouse subsets exhibit similar cytokine secre- Pharmacia Biotech) gradient. Cells were then washed three times tion properties (18), systematic differences were found in with PBS before surface staining. Staining with CD1d-GalCer tet- 10/10 individual human subjects examined. Thus, upon ex ramers was as follows. Cells were incubated with 1 g/ml unlabeled vivo stimulation with the combination of ionomycin and streptavidin (Pierce Chemical Co.) for 15 min at room temperature, followed by incubation with CD1d-GC tetramers (15) for 1 h at room temperature. Other mAbs such as CD4 and V24 were then added for a further 30 min incubation on ice. Cells were then washed with staining buffer (PBS, 0.1% BSA; Sigma-Aldrich) and 0.01% sodium azide (Sigma-Aldrich) and analyzed by flow cytome- try using FACSort™ and CELLQuest™ software (Becton Dickin- son). For studies involving CD152 or intracellular cytokines, cells were first stained with tetramers, then permeabilized with Cytofix/ Cytoperm™ (BD Biosciences), and washed with Perm/Wash™ buffer (BD Biosciences). Appropriate mAbs were then added for 30 min before two further washes with Perm/Wash™ buffer. Stimulation of Cytokine Production. PBLs were cultured for 12 h at a concentration of 5 10 cells per milliliter in RPMI 1640 supplemented with 10% FCS (Biofluids) in the presence of 1 ng/ ml of phorbol-myristate-acetate (PMA), 1 M of ionomycin, and Figure 1. Identification of human NKT cells. (A) NKT cells present 5 g/ml of Brefeldin A (all from Sigma-Aldrich). among the PBL of 10 healthy adults were enumerated after double stain- ing with CD1d-GC tetramers followed by anti-V24. Each dot repre- sents an individual sample. (B) PBL from individuals expressing high (top Results panels) or low (bottom panels) NKT cell numbers were stained with CD1d-GC tetramers followed by anti-V24, -V11, and CD4 mAb. Specific Identification of Human NKT Cells Using CD1d- Two-color combinations are displayed to illustrate correlations between GalCer Tetramers and Anti-V24 mAbs. To accurately iden- different subsets. Right panels are gated on V24/CD1d-GC double- tify potential subsets of the rare circulating human NKT positive cells. 638 Functional Lineages of Human V24 NKT Cells Figure 2. Different cytokine and cytokine receptor profiles of CD4 and DN NKT cells. Fresh PBLs were rested or stimulated with PMA/ionomycin for 12 h before stain- ing with CD1d-GalCer tetramers followed by permeabilization and staining with mAbs to V24, CD4, and cytokines or cytokine receptors. (A) FACS dot plots from a rep- resentative individual are shown, after gating on CD1d-GalCer/V24 double-positive cells. Numbers in the quadrants indicate percentages of cells within the CD4 or DN subsets (top and bottom quadrants, respec- tively). (B) Summary of cytokine expression from 10 individual subjects. White circles represent the CD4 subset while black circles represent the DN subset. Significant differ- ences (Student’s paired t test) were found for IL-4 (P  0.01) and IL-13 (P  0.01). Staining of CD25 (IL-2R) was performed without membrane permeabilization. PMA, nearly all the IL-4 and IL-13 stained by intracellular tegrins were similarly expressed in both subsets, includ- FACS were present in CD4 cells (Fig. 2). In contrast, ing CCR1 and CCR2 (2%), CXCR3 and CXCR4 both the CD4 and DN subsets produced abundant Th1 cy- (2–100%), CCR4 (100%), and CCR7 (2%) (data not tokines such as TNF- and IFN-. Interestingly, the IL- shown). Likewise, both CD4 and DN NKT cells expressed 2R chain (CD25) was exclusively expressed by CD4 cells abundant CD49d, CD49e, and CD49f but limited CD49b (10–80% positive), indicating that CD4 NKT cells repre- (data not shown). sent a fraction of human CD4 CD25 regulatory cells. NK lineage receptors are mainly expressed by the DN CD4 and DN Subsets Have Different Patterns of Chemokine subset. One of the hallmarks of mouse NKT cells is the ex- Receptors and Integrins. When we examined a broad range pression of receptors of the NK lineage, which regulate of chemokine receptors and integrins (Fig. 3), significant cellular activation by fine tuning TCR signaling. Using a differences were systematically found between CD4 and battery of NK receptor-specific reagents, we have found DN cells with respect to the expression of CCR5, CCR6, systematic differences between CD4 and DN NKT cells CXCR6, and CD49a. Other chemokine receptors and in- (Fig. 4). Thus, CD161, which costimulates TCR activation Figure 3. Different chemokine receptor and integrin pattern of CD4 Figure 4. Different patterns of NK receptor expression by CD4 and and DN NKT subsets. The CD1d-GalCer/V24 double-positive PBLs DN NKT cells. The CD1d-GalCer/V24 double-positive PBLs were were gated for analysis of CD4 and chemokine receptors or CD49a. (A) gated for analysis of CD4 and 2B4, CD94, NKG2A, or CD161. (A) ® ® FACS dot plots of a representative PBL sample. Numbers in the quad- FACS dot plots of a representative PBL sample. Numbers in the quad- rants indicate percentages of cells within the CD4 or DN subsets (top and rants indicate percentages of cells within the CD4 or DN subsets (top and bottom quadrants, respectively). (B) Summary of chemokine receptor and bottom quadrants, respectively). (B) Summary of NK receptor expression CD49a expression from 10 individual subjects. White circles represent from 10 individual subjects. White circles represent the CD4 subset, while black circles represent the DN subset. Significant differences (Stu- the CD4 subset, while black circles represent the DN subset. Significant differences (Student’s paired t test) were found for CCR5 (P  0.05), dent’s paired t test) were found for 2B4 (P  0.001), CD94 (P  0.05), CCR6 (P  0.01), CXCR6 (P  0.05), and CD49a (P  0.05). NKG2A (P  0.05), and CD161 (P  0.01). 639 Lee et al. Brief Definitive Report (14), was expressed at a higher density and at greater fre- cretion or NK receptor expression (18). Further studies are quency by DN NKT cells. Likewise, 2B4, CD94, and required to elucidate the mechanisms underlying Th2 ver- NKG2A were nearly exclusively expressed by DN NKT sus Th1 regulation in mouse. It is possible that other subsets cells. On the other hand, CD56 was highly expressed by or alternative mechanisms, such as altered glycolipid ligand both subsets, whereas the KIR CD158a and CD158b were (13) or NK receptor signaling (14), could account for the generally not expressed (data not shown). control of Th function in these conditions. Other Surface Receptors Expressed by Fresh NKT Cell Sub- In conclusion, our study of fresh human V24 NKT re- sets. There were no systematic differences for costimula- vealed the existence of two functionally distinct lineages of tory receptors such as CD28, which was generally ex- CD4 and DN cells. These lineages might be differentially pressed by most CD4 and DN NKT cells. In contrast, altered or recruited in various disease conditions, providing cytotoxic T lymphocyte antigen 4 and CD40L were ex- a potential mechanism explaining how NKT cells might pressed by very small fractions of NKT cells (data not promote opposite Th1 or Th2 responses. A detailed under- shown). Most CD4 and DN NKT cells expressed standing of this regulation will be critical to design future CD45RO and CD45RB, but not CD45RA (data not strategies to manipulate the immune response through shown). CD69 was equally expressed by 50% of both NKT cell activation. CD4 and DN NKT cells. We thank Kirin Pharmaceutical Laboratories for the gift of GalCer. This research is supported by grants from the National Institutes of Health (to A. Bendelac and L. Teyton) and the Juvenile Diabetes Discussion Research Foundation (to A. Bendelac), and fellowships from the Juvenile Diabetes Research Foundation (to P.T. Lee) and the Leu- By combining the use of TCR-specific reagents such as kemia and Lymphoma Society of America (to K. Benlagha). CD1d-GalCer tetramer and anti-V24 mAb, we could specifically identify all canonical V24 NKT cells, even at Submitted: 14 November 2001 Revised: 13 December 2001 the very low frequencies found among the PBLs of most Accepted: 8 January 2002 healthy individuals. Indeed, we demonstrated that the dou- ble staining method allowed correct and specific identification of every NKT cell among 1 million PBLs, a 100–1,000- References fold improvement over conventional tetramer tracking 1. Park, S.H., and A. Bendelac. 2000. CD1-restricted T-cell re- methods, which is likely to apply to other T cell subsets. sponses and microbial infection. Nature. 406:788–792. Using this methodology, we were able to characterize 2. Godfrey, D.I., K.J.L. Hammond, L.D. Poulton, and A.G. functionally distinct subsets and show that they segregate Baxter. 2000. NKT cell facts, functions and fallacies. Immu- with the CD4 and DN subsets of NKT cells. nol. Today. 21:573–583. While both CD4 and DN NKT cells could produce 3. Janeway, C.A., Jr. 1990.  T cells: research on the frontlines Th1 cytokines, the release of Th2 cytokines such as IL-4 of defence. Res. Immunol. 141:688–695. and IL-13 was the exclusive property of CD4 T cells. CD4 4. Benlagha, K., and A. Bendelac. 2000. CD1d-restricted and DN NKT cells also exhibited systematic differences in mouse V14 and human V24 T cells: lymphocytes of in- their pattern of chemokine receptors and integrins, suggest- nate immunity. Semin. Immunol. 12:537–542. 5. Hammond, K.J.L., L.D. Poulton, L.J. Palmisano, P.A. Sil- ing different migratory properties. Finally, the expression of veira, D.I. Godfrey, and A.G. Baxter. 1998. /-T cell re- several NK receptors was restricted to the DN lineage. Al- ceptor (TCR) CD4 CD8 (NKT) thymocytes prevent together, these findings reveal that the CD4 and DN sub- insulin-dependent diabetes mellitus in nonobese diabetic sets represent distinct lineages with markedly different (NOD)/Lt mice by the influence of interleukin (IL)-4 and/ functional properties. or IL-10. J. Exp. Med. 187:1047–1056. The restricted production of Th2 cytokines by fresh 6. Lehuen, A., O. Lantz, L. Beaudoin, V. Laloux, C. Carnaud, CD4 NKT cells apparently conflicts with a prior study A. Bendelac, J.F. Bach, and R.C. Monteiro. 1998. Overex- showing that DN NKT cell clones derived from healthy pression of natural killer T cells protects V14-J281 trans- individuals produced Th2 cytokines, whereas those of genic nonobese diabetic mice against diabetes. J. Exp. Med. IDDM patients were Th1 (7). It is possible that the culture 188:1831–1839. system modified their primary cytokine profile, as repeated 7. Wilson, S.B., S.C. Kemt, K.T. Patton, T. Orban, R.A. Jack- son, M. Exley, S. Porcelli, D.A. Schatz, M.A. Atkinson, S.P. stimulation was reported to downmodulate Th1 cytokines Balk, et al. 1998. Extreme Th1 bias of invariant V24JQ T and upregulate Th2 (19). Our study suggests that a reexam- cells in type I diabetes. Nature. 391:177–181. ination of the functional status of NKT cell subsets in 8. Shi, F.D., M. Flodstrom, B. Balasa, S.H. Kim, K. Van Gunst, IDDM patients is warranted to verify whether the conclu- J.L. Strominger, S.B. Wilson, and N. Sarvetnick. 2001. Germ sions derived from the in vitro studies will apply to fresh line deletion of the CD1 locus exacerbates diabetes in the NKT cell subsets. NOD mouse. Proc. Natl. Acad. Sci. USA. 98:6777–6782. This study of human V24 NKT cells also points to a 9. Smyth, M.J., K.Y. Thia, S.E. Street, E. Cretney, J.A. Tra- surprising difference between the mouse and the human pani, M. Taniguchi, T. Kawano, S.B. Pelikan, N.Y. Crowe, system. Indeed, the mouse CD4 and DN NKT cells do not and D.I. Godfrey. 2000. Differential tumor surveillance by clearly differ with respect to Th1 versus Th2 cytokine se- natural killer (NK) and NKT cells. J. Exp. Med. 191:661– 640 Functional Lineages of Human V24 NKT Cells 668. V24JQ T cell receptor  chains. J. Exp. Med. 188:867– 10. Terabe, M., S. Matsui, N. Noben-Trauth, H. Chen, C. Wat- 876. son, D.D. Donaldson, D.P. Carbone, W.E. Paul, and J.A. 15. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, and A. Ben- Berzofsky. 2000. NKT cell-mediated repression of tumor delac. 2000. In vivo identification of glycolipid antigen spe- immunosurveillance by IL-13 and the IL-4R-STAT6 path- cific T cells using fluorescent CD1d tetramers. J. Exp. Med. way. Nat. Immunol. 1:515–520. 191:1895–1903. 11. Sumida, T., A. Sakamoto, H. Murata, Y. Makino, H. Taka- 16. Karadimitris, A., S. Gadola, M. Altamirano, D. Brown, A. hashi, S. Yoshida, K. Nishioka, I. Iwamoto, and M. Tanigu- Woolfson, P. Klenerman, J.L. Chen, Y. Koezuka, I.A. Rob- chi. 1995. Selective reduction of T cells bearing invariant erts, D.A. Price, et al. 2001. From the Cover: human CD1d- V24-JQ antigen receptor in patients with systemic sclero- glycolipid tetramers generated by in vitro oxidative refolding sis. J. Exp. Med. 182:1163–1168. chromatography. Proc. Natl. Acad. Sci. USA. 98:3294–3298. 12. Illes, Z., T. Kondo, J. Newcombe, N. Oka, T. Tabira, and 17. Matsuda, J.L., O.V. Naidenko, L. Gapin, T. Nakayama, M. T. Yamamura. 2000. Differential expression of NK T cell Taniguchi, C.R. Wang, Y. Koezuka, and M. Kronenberg. V24J Q invariant TCR chain in the lesions of multiple 2000. Tracking the response of natural killer T cells to a gly- sclerosis and chronic inflammatory demyelinating polyneur- colipid antigen using CD1d tetramers. J. Exp. Med. 192:741– opathy. J. Immunol. 164:4375–4381. 754. 13. Miyamoto, K., S. Miyake, and T. Yamamura. 2001. A syn- 18. Bendelac, A., M.N. Rivera, S.-H. Park, and J.H. Roark. thetic glycolipid prevents autoimmune encephalomyelitis by 1997. Mouse CD1-specific NK1 T cells: development, spec- inducing TH2 bias of natural killer T cells. Nature. 413:531– ificity, and function. Annu. Rev. Immunol. 15:535–562. 534. 19. Burdin, N., L. Brossay, and M. Kronenberg. 1999. Immuni- 14. Exley, M., S. Porcelli, M. Furman, J. Garcia, and S. Balk. zation with -galactosylceramide polarizes CD1-reactive NK 1998. CD161 (NKR-P1A) costimulation of CD1d-depen- T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29: dent activation of human T cells expressing invariant 2014–2025. 641 Lee et al. Brief Definitive Report http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Distinct Functional Lineages of Human Vα24 Natural Killer T Cells

Lee, Peter T.; Benlagha, Kamel; Teyton, Luc; Bendelac, Albert
The Journal of Experimental Medicine , Volume 195 (5) – Mar 4, 2002
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Copyright © 2002, The Rockefeller University Press
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0022-1007
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10.1084/jem.20011908
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Abstract

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy sub- jects express 1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents -galactosyl- ceramide (GalCer) loaded CD1d-tetramers and anti-V24 monoclonal antibody, to specifi- cally identify and characterize these rare cells in fresh PBLs. We report here that CD4 and CD4 CD8 (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4 NKT cells were the exclusive pro- ducers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases. Key words: CD1 • NKT cells • cytokine • IDDM • T cell development Introduction NKT cells are a conserved subpopulation of  T cells, ple, they can suppress type I diabetes in NOD mouse which are restricted by the antigen-presenting molecule through the secretion of IL-4 and IL-10 (5, 6) and their de- CD1d, and appear to regulate several disease processes fects in both NOD mice and humans with IDDM may ranging from tumor rejection to autoimmune diseases (for contribute to pathogenesis (7, 8). Conversely, they naturally a review, see references 1 and 2). They express a con- suppress methylcholantrene-induced carcinogenesis through served canonical TCR (V14J18-V8 in mouse and IFN- (9). In another report, secretion of the Th2 cytokine V24J18-V11 in human) that is thought to recognize a IL-13 was found to inhibit the immune rejection of a tu- self-antigen mimicked by the glycolipid GalCer. Since mor graft (10). Collectively, these findings suggest that reg- NKT cells are present at high frequency in various mouse ulated expression of Th1 or Th2 cytokines by NKT cells, tissues and in human liver, they seem to participate in the rather than mere changes in its frequency (7, 11, 12), might innate, rather than the adaptive arm of the immune re- control the outcome of some disease conditions. sponse and resemble other innate lymphocytes such as B-1 How could the Th1- versus Th2-promoting functions of B cells and  T cells which express canonical antigen re- NKT cells be selectively recruited? It has been suggested that ceptors responding to cell stress and tissue damage (3, 4). altered GalCer ligands with shorter sphingosine chain could Importantly, CD1d is mainly expressed on dendritic cells selectively activate Th2 functions (13), whereas NK1.1 sig- (DCs), macrophage, and B cells, implying that NKT cells naling could favor Th1 response (14). However, the possibil- primarily interact with APCs rather than tissue cells. ity that subsets of NKT cells might specialize in Th1 versus The secretion of Th1 and Th2 cytokines by NKT cells is Th2 functions has not been thoroughly investigated. thought to underlie their regulatory properties. For exam- One obstacle to the identification of NKT cell subsets has been that, until recently, NKT cells could not be unambigu- ously identified. The generation of CD1d-GalCer tetramers Address correspondence to Albert Bendelac, Dept. of Molecular Biology, specific for both mouse and human canonical TCR makes it Princeton University, Washington Rd., Princeton, NJ 08544. Phone: 609- 258-5454; Fax: 609-258-2205; E-mail: [email protected] possible to identify NKT cells based on their specificity rather 637 J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/03/637/05 $5.00 Volume 195, Number 5, March 4, 2002 637–641 http://www.jem.org/cgi/content/full/195/5/637 than their phenotype (15–17). CD1d tetramers have already cells above background staining levels, we combined the revealed several important findings, including a subset of use of two TCR-specific reagents, a mAb to V24 and CD1d-restricted murine NKT cells that do not express the CD1d-GalCer tetramers. We found that, although NK1.1 marker and differ from the NK1.1 cells with respect CD1d-GalCer tetramer staining is completely inhibited to their pattern of integrins (15). However, detailed examina- by prior incubation with the anti-V24 mAb (15), the re- tion of human NKT cells has not been performed. verse reaction order allowed significant binding of anti- An additional challenge to the study of fresh human V24, presumably because the tetramers require contigu- PBLs is the very low frequency of canonical NKT cells, of- ous clusters of TCR to bind, leaving a significant amount ten well below common background level staining of of unbound TCR available for bright V24 staining. Fig. 1 0.1%. Here, we have used a combination of CD1d-Gal- A shows that the frequency of V24/CD1d-GalCer dou- Cer tetramers and anti-V24 mAb, which specifically ble positive canonical NKT cells in the fresh PBLs of identifies the canonical NKT cells even at the very low fre- healthy volunteers is between 0.01 and 0.1%. Importantly, quencies found in human PBLs, to investigate human tetramer staining alone invariably included 0.01–0.05% NKT cell subsets. We have dissected the phenotype of nonV24 cells, which are noncanonical cells that presum- these cells into CD4 and double negative (DN) popula- ably reflect background staining (Fig. 1 B, top left quad- tions, and found that they systematically differed in many rants in left dot plots). Thus, whereas the population de- functionally relevant ways with respect to Th cytokine fined by conventional tetramer staining alone included a profile, pattern of chemokine receptors, and integrin ex- significant proportion of nonNKT cells in most healthy in- pression, and array of NK receptors displayed on the cell dividuals, especially in those with low NKT cell frequency, surface. These findings suggest that human CD4 and DN the double staining combination allowed complete speci- V24 NKT cells represent functionally separate lineages ficity. This is shown by the 100% expression of V11 that may promote different Th responses. among the V24/CD1d-GalCer double-positive cells (Fig. 1 B, right dot plots). In the sample expressing 0.01% NKT cells, 100 out of 100 gated cells were V11 , indi- Materials and Methods cating that this technique specifically identified all of the 100 canonical NKT cells present among 1 million PBL. Antibodies. Fluorochrome or biotin conjugates of antibodies against V24, V11, CD4, CD25, CD28, CD56, CD94, NKG2A CD4 and DN Subsets Have Different Cytokine Secretion (Beckman Coulter); CCR1, CCR2, CXCR6 (R&D Systems); Profiles. A fraction of V24 NKT cells expresses CD4 CCR4, CCR5, CCR6, CCR7, CXCR3, CXCR4, CD45RA, while the remaining is CD8-negative and is called DN. CD45RB, CD45RO, CD49a, CD49b, CD49d, CD49e, CD49f, While the proportion of the CD4 and DN varied consider- CD69, CD152, CD154, CD158a, CD158b, CD161, IL-4, IL-13, ably between individuals, on average, they were roughly TNF-, and IFN- (BD Biosciences) were used. equal (50%) in a group of 10 healthy individuals (Fig. 1 B, Flow Cytometric Analysis. PBLs were obtained from whole and data not shown). Surprisingly, despite reports that the blood of healthy donors by centrifugation over Ficoll (Amersham corresponding mouse subsets exhibit similar cytokine secre- Pharmacia Biotech) gradient. Cells were then washed three times tion properties (18), systematic differences were found in with PBS before surface staining. Staining with CD1d-GalCer tet- 10/10 individual human subjects examined. Thus, upon ex ramers was as follows. Cells were incubated with 1 g/ml unlabeled vivo stimulation with the combination of ionomycin and streptavidin (Pierce Chemical Co.) for 15 min at room temperature, followed by incubation with CD1d-GC tetramers (15) for 1 h at room temperature. Other mAbs such as CD4 and V24 were then added for a further 30 min incubation on ice. Cells were then washed with staining buffer (PBS, 0.1% BSA; Sigma-Aldrich) and 0.01% sodium azide (Sigma-Aldrich) and analyzed by flow cytome- try using FACSort™ and CELLQuest™ software (Becton Dickin- son). For studies involving CD152 or intracellular cytokines, cells were first stained with tetramers, then permeabilized with Cytofix/ Cytoperm™ (BD Biosciences), and washed with Perm/Wash™ buffer (BD Biosciences). Appropriate mAbs were then added for 30 min before two further washes with Perm/Wash™ buffer. Stimulation of Cytokine Production. PBLs were cultured for 12 h at a concentration of 5 10 cells per milliliter in RPMI 1640 supplemented with 10% FCS (Biofluids) in the presence of 1 ng/ ml of phorbol-myristate-acetate (PMA), 1 M of ionomycin, and Figure 1. Identification of human NKT cells. (A) NKT cells present 5 g/ml of Brefeldin A (all from Sigma-Aldrich). among the PBL of 10 healthy adults were enumerated after double stain- ing with CD1d-GC tetramers followed by anti-V24. Each dot repre- sents an individual sample. (B) PBL from individuals expressing high (top Results panels) or low (bottom panels) NKT cell numbers were stained with CD1d-GC tetramers followed by anti-V24, -V11, and CD4 mAb. Specific Identification of Human NKT Cells Using CD1d- Two-color combinations are displayed to illustrate correlations between GalCer Tetramers and Anti-V24 mAbs. To accurately iden- different subsets. Right panels are gated on V24/CD1d-GC double- tify potential subsets of the rare circulating human NKT positive cells. 638 Functional Lineages of Human V24 NKT Cells Figure 2. Different cytokine and cytokine receptor profiles of CD4 and DN NKT cells. Fresh PBLs were rested or stimulated with PMA/ionomycin for 12 h before stain- ing with CD1d-GalCer tetramers followed by permeabilization and staining with mAbs to V24, CD4, and cytokines or cytokine receptors. (A) FACS dot plots from a rep- resentative individual are shown, after gating on CD1d-GalCer/V24 double-positive cells. Numbers in the quadrants indicate percentages of cells within the CD4 or DN subsets (top and bottom quadrants, respec- tively). (B) Summary of cytokine expression from 10 individual subjects. White circles represent the CD4 subset while black circles represent the DN subset. Significant differ- ences (Student’s paired t test) were found for IL-4 (P  0.01) and IL-13 (P  0.01). Staining of CD25 (IL-2R) was performed without membrane permeabilization. PMA, nearly all the IL-4 and IL-13 stained by intracellular tegrins were similarly expressed in both subsets, includ- FACS were present in CD4 cells (Fig. 2). In contrast, ing CCR1 and CCR2 (2%), CXCR3 and CXCR4 both the CD4 and DN subsets produced abundant Th1 cy- (2–100%), CCR4 (100%), and CCR7 (2%) (data not tokines such as TNF- and IFN-. Interestingly, the IL- shown). Likewise, both CD4 and DN NKT cells expressed 2R chain (CD25) was exclusively expressed by CD4 cells abundant CD49d, CD49e, and CD49f but limited CD49b (10–80% positive), indicating that CD4 NKT cells repre- (data not shown). sent a fraction of human CD4 CD25 regulatory cells. NK lineage receptors are mainly expressed by the DN CD4 and DN Subsets Have Different Patterns of Chemokine subset. One of the hallmarks of mouse NKT cells is the ex- Receptors and Integrins. When we examined a broad range pression of receptors of the NK lineage, which regulate of chemokine receptors and integrins (Fig. 3), significant cellular activation by fine tuning TCR signaling. Using a differences were systematically found between CD4 and battery of NK receptor-specific reagents, we have found DN cells with respect to the expression of CCR5, CCR6, systematic differences between CD4 and DN NKT cells CXCR6, and CD49a. Other chemokine receptors and in- (Fig. 4). Thus, CD161, which costimulates TCR activation Figure 3. Different chemokine receptor and integrin pattern of CD4 Figure 4. Different patterns of NK receptor expression by CD4 and and DN NKT subsets. The CD1d-GalCer/V24 double-positive PBLs DN NKT cells. The CD1d-GalCer/V24 double-positive PBLs were were gated for analysis of CD4 and chemokine receptors or CD49a. (A) gated for analysis of CD4 and 2B4, CD94, NKG2A, or CD161. (A) ® ® FACS dot plots of a representative PBL sample. Numbers in the quad- FACS dot plots of a representative PBL sample. Numbers in the quad- rants indicate percentages of cells within the CD4 or DN subsets (top and rants indicate percentages of cells within the CD4 or DN subsets (top and bottom quadrants, respectively). (B) Summary of chemokine receptor and bottom quadrants, respectively). (B) Summary of NK receptor expression CD49a expression from 10 individual subjects. White circles represent from 10 individual subjects. White circles represent the CD4 subset, while black circles represent the DN subset. Significant differences (Stu- the CD4 subset, while black circles represent the DN subset. Significant differences (Student’s paired t test) were found for CCR5 (P  0.05), dent’s paired t test) were found for 2B4 (P  0.001), CD94 (P  0.05), CCR6 (P  0.01), CXCR6 (P  0.05), and CD49a (P  0.05). NKG2A (P  0.05), and CD161 (P  0.01). 639 Lee et al. Brief Definitive Report (14), was expressed at a higher density and at greater fre- cretion or NK receptor expression (18). Further studies are quency by DN NKT cells. Likewise, 2B4, CD94, and required to elucidate the mechanisms underlying Th2 ver- NKG2A were nearly exclusively expressed by DN NKT sus Th1 regulation in mouse. It is possible that other subsets cells. On the other hand, CD56 was highly expressed by or alternative mechanisms, such as altered glycolipid ligand both subsets, whereas the KIR CD158a and CD158b were (13) or NK receptor signaling (14), could account for the generally not expressed (data not shown). control of Th function in these conditions. Other Surface Receptors Expressed by Fresh NKT Cell Sub- In conclusion, our study of fresh human V24 NKT re- sets. There were no systematic differences for costimula- vealed the existence of two functionally distinct lineages of tory receptors such as CD28, which was generally ex- CD4 and DN cells. These lineages might be differentially pressed by most CD4 and DN NKT cells. In contrast, altered or recruited in various disease conditions, providing cytotoxic T lymphocyte antigen 4 and CD40L were ex- a potential mechanism explaining how NKT cells might pressed by very small fractions of NKT cells (data not promote opposite Th1 or Th2 responses. A detailed under- shown). Most CD4 and DN NKT cells expressed standing of this regulation will be critical to design future CD45RO and CD45RB, but not CD45RA (data not strategies to manipulate the immune response through shown). CD69 was equally expressed by 50% of both NKT cell activation. CD4 and DN NKT cells. We thank Kirin Pharmaceutical Laboratories for the gift of GalCer. This research is supported by grants from the National Institutes of Health (to A. Bendelac and L. Teyton) and the Juvenile Diabetes Discussion Research Foundation (to A. Bendelac), and fellowships from the Juvenile Diabetes Research Foundation (to P.T. Lee) and the Leu- By combining the use of TCR-specific reagents such as kemia and Lymphoma Society of America (to K. Benlagha). CD1d-GalCer tetramer and anti-V24 mAb, we could specifically identify all canonical V24 NKT cells, even at Submitted: 14 November 2001 Revised: 13 December 2001 the very low frequencies found among the PBLs of most Accepted: 8 January 2002 healthy individuals. Indeed, we demonstrated that the dou- ble staining method allowed correct and specific identification of every NKT cell among 1 million PBLs, a 100–1,000- References fold improvement over conventional tetramer tracking 1. Park, S.H., and A. Bendelac. 2000. CD1-restricted T-cell re- methods, which is likely to apply to other T cell subsets. sponses and microbial infection. Nature. 406:788–792. Using this methodology, we were able to characterize 2. Godfrey, D.I., K.J.L. Hammond, L.D. Poulton, and A.G. functionally distinct subsets and show that they segregate Baxter. 2000. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Mar 4, 2002

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