Working with NKT cells — pitfalls and practicalities

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  • ls d Melbourne, Royal Parade, Parkville, Victoria, 3010, Australia the moniker of the ‘double-edged sword’ of the immune system [1–3]. NKT cells apparently regulate fundamental The stringency of tetramer staining is very high, but NKT cell numbers can be overestimated through nonspecific aspects of immunity because their dysfunction, or defi- ciency, predisposes mice, and possibly humans, to an array of autoimmune diseases and cancers [2,4–7]. Sig- nificantly, many of these diseases have been prevented or cured in mice by replenishing [8,9] or stimulating [10,11] the residual NKT cell pool. tetramer binding and/or autofluorescence (SP Berzins et al., unpublished; [20,21]). Co-staining with anti-ab- TCR monoclonal antibodies (mAbs) enables non-T cells prone to nonspecific binding to be excluded from analysis [22], but further precautions are helpful for human stu- dies, in which NKT cell levels vary 100-fold between Current Opinion in Immunology 2005, 17:448–454 2 Cancer Immunology Program, Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria 3002, Australia Corresponding author: Berzins, Stuart P ( Current Opinion in Immunology 2005, 17:448–454 This review comes from a themed issue on Immunological techniques Edited by Daniel Speiser 0952-7915/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2005.05.012 Introduction NKT cells are emerging as a T cell subset whose activity could one day be harnessed in the treatment of auto- immune diseases and cancer. Their clinical potential lies in the rapid release of cytokines that promote or suppress different immune responses, which has earned NKT cells Working with NKT cells — pitfal Stuart P Berzins1, Mark J Smyth2 an Our understanding of NKT cells has been rapidly advancing over recent years, with many research groups studying how these cells behave and how they can be manipulated to prevent disease. Although good progress has been made, a difficulty is the lack of a clear consensus about how to assay, or even identify, NKT cells. The different approaches have been an ongoing source of uncertainty about the biological behaviour and function of NKT cells and have complicated efforts to define their role in immunity. An important step towards reaching agreement on the behaviour of NKT cells is to have a clear appreciation of the advantages and disadvantages of the different approaches that are employed in this field of study. This should help determine the most appropriate ways to investigate NKT cell function, thus bringing us closer to successfully exploiting these cells in the treatment of human diseases. Addresses 1 Department of Microbiology and Immunology, University of and practicalities Dale I Godfrey1 In this review, we discuss the challenges of working with NKT cells and suggest approaches that might resolve controversies that stem, at least in part, from the different techniques used to identify and manipulate these cells. Identifying NKT cells What are NKT cells? NKT cells are CD1d-restricted T cells: Type-1, or ‘clas- sical’, NKT cells express a semi-invariant TCR (incor- porating Va14Ja18 in mice and the homologous Va24Ja18 in humans), which distinguishes them from CD1d-dependent T cells that do not express this semi- invariant TCR (Type-2 NKT cells) [12��]. Although the functional relationship between Type-1 ‘classical’ NKT cells and Type-2 NKT cells is unclear, there is some evidence that Type-2 cells are a functionally important T cell subset, albeit distinct in phenotype and function from Type-1 NKT cells [12��]. This review will primarily focus on the more widely studied Type-1 NKT cells because their functional significance is far better characterized than other NKT like cells, and the term ‘NKT cells’ hereafter refers to Type-1 NKT cells unless otherwise specified. The distinctive TCR of NKT cells facilitates their spe- cific identification using CD1d-tetramers (or dimers) loaded with the glycosphingolipid antigen a-galactosyl- ceramide (aGC) [13–15] (Figure 1). The tetramer binds NKT cells in mice and humans, but human NKT cells can also be identified with anti-Va24 (or 6B11, an anti- body specific for the CDR3 region of the TCRa chain used by human NKT cells) and anti-Vb11 [16,17]. A reliable Va14-specific antibody is not available for mice. NK1.1 and/or DX5 expression by T cells is sometimes used to identify NKT cells, but many NKT cells do not express NK1.1 and not all NK1.1+ T cells are CD1d- dependent [12��]. Likewise, the DX5 antigen isn’t uni- formly expressed by [18], nor restricted to [19], NKT cells, and its effectiveness as a marker is further compro- mised by the weak reactivity and staining variability of anti-DX5 reagents (DG Pellicci et al. personal commu- nication).
  • Working with NKT cells Berzins, Smyth and Godfrey 449 s ge T en en st Figure 1 Mouse only Reagent(s) ProTargetSpecies Human and mouse αGC-loaded CD1d tetramer (or dimer) Anti-NK1.1/αβTCR Anti-DX5/αβTCR High strin Good NK enrichm Fair NKT cell Works in mo healthy individuals, and thymus levels can be below 1 in 100 000 (SP Berzins et al. unpublished; [21,23]). In such instances, the NKT cell region on a fluorescence-acti- vated cell sorting (FACS) plot can be ‘swamped’ by nonspecific events, and a FACS channel dedicated to a nonspecific reagent (e.g. unloaded CD1d tetramer) may help to exclude autofluorescent and nonspecifically labelled cells (SP Berzins et al., unpublished; see Update; [21]). Alternatively, an additional antibody such as anti- Va24 can further improve separation between CD1d tetramer+ NKT cells and background [21]. Technical challenges Because NKT cell identification typically relies on multi- ple reagents binding to the TCR, steric interference between reagents may reduce staining intensities. In our experience, this can occur when anti-Va24 is used in combination with aGC-loaded CD1d tetramer. Although both labels remain detectable, care must be taken to avoid underestimating NKT cell numbers. Another significant issue is fluorescence resonance emis- sion transfer (FRET), which can occur when different Human only Anti-Vα24/Vβ11 6B11 mAb High stringe High stringe Adoptive transfer modelling of NKT cell function. Careful selection of donor valuable information about the role of NKT cells in different disease models recipient strains are shown. Type 1 NKT cells are the CD1d-restricted ‘class are also CD1d-dependent but show a more diverse TCR gene usage and th products that may impact upon NKT cell function. For example, cytokines, Cons Comments Engages antigen- binding region of TCR. Might stimulate or blockTCR signaling ncy cell t richment strains Not entirely NKT specific Only works in some strains (eg. C57BL/6) Many/most DX5+ T cells are not NKT cells Best combined with anti-TCR or Vα24 to enhance specificity Highest stringency from thymus or liver. Poor from other tissues Not recommended if high NKT cell purity is required antibodies are in close proximity. If the emission spec- trum of one fluorochrome overlaps with the absorption spectrum of the other, unpredictable nonspecific emis- sions can complicate FACS compensation and analysis [24]. For example, by staining NKT cells with aGC- loaded CD1d tetramer–phycoerythrin (PE) and anti-ab- TCR–allophycocyanin (APC), we and others [25] have observed nonspecific emissions in the PE-Cy5 channel. In such instances, it is unwise to stain for low intensity antigens in the PE-Cy5 channel because NKT cells might appear falsely positive. A sample stained only with TCR-targeted reagents (with the FL3 channel unused) can control for FRET, or changing fluorochromes can avoid unwanted FRET interactions. Finding and isolating NKT cells Where to find NKT cells NKT cells are found wherever mainstream T cells reside. In mice, >30% of liver lymphocytes are NKT cells, but levels elsewhere are usually 0.1–1.0% [26]. Human NKT cell frequencies are more variable between individuals, and typically lower than they are in mice. This is most ncy ncy Might include some non-NKT cells Engages antigen- binding region of TCR. Might stimulate or block TCR signaling Suitable for most applications Best combined with anti-TCR or Vα24 to enhance specificity Current Opinion in Immunology and recipient strains in adoptive transfer experiments can provide . Advantages and disadvantages of commonly used donor and ical’ NKT cells that form the basis of this review. Type 2 NKT cells eir function is less clearly defined. ‘Factor X’ encompasses gene or other effector molecules. Current Opinion in Immunology 2005, 17:448–454
  • 450 Immunological techniques obvious in the thymus, where levels are at least 100-fold lower in humans [23], and liver, where only �1% of lymphocytes are NKT cells [27]. In situ localization Only a few studies have clearly identified NKT cells in situ [28,29], partly because of their very low frequency and the lack of a reliable mouse NKT cell-specific antibody. The CD1d tetramer has not been successfully used to detect NKT cells in situ, and less stringent markers such as NK1.1 require complicated staining protocols that are not widely applied [29]. One recent study examined mice in which green fluor- escent protein (GFP) was expressed under the transcrip- tional regulation of the CXCR6 chemokine receptor [30��]. Although CXCR6 is not exclusively an NKT cell marker, most CXCR6high cells in the liver are NKT cells, and this enabled intravital fluorescence microscopy to visualize NKT cell trafficking and interactions in a live mouse. Enrichment and purification of NKT cells Although analysis of NKT cells by flow cytometry can usually be achieved by the selective gating of unfractio- nated lymphocytes, functional assays often require prior enrichment and/or purification. For mice, NKT cell fre- quencies can be greatly increased by depleting non-NKT cells using complement-mediated killing or magnetic- bead separation. Alternatively, positive enrichment using anti-PE conjugated magnetic cell sorting (MACS) beads can isolate NKT cells labelled with reagents such as CD1d tetramer–PE [18,20,31]. FACS sorting is the best option for purifying NKT cells because the requirement to use at least two markers (e.g. CD1d tetramer and abTCR or CD3; or Va24 or 6B11 and Vb11), makes single-parameter techniques, such as magnetic beads, less effective. An important caveat to purification with TCR-specific reagents, particularly tet- ramers, is that signalling might be induced or blocked. In our experience, NKT cells isolated using tetramers can sometimes be partially activated and spontaneously pro- duce cytokines in culture (SP Berzins et al., unpublished data). For this reason, some researchers favour enrich- ment by NK1.1 [9] or DX5 [32]. We find that NK1.1 versus ab-TCR sorting isolates a greatly enriched NKT cell population that is not activated (SP Berzins et al., unpublished data). As NK1.1 and DX5 are not definitive NKT cell markers, however, these approaches carry the uncertainty that results may reflect the activity of non- NKT cell contaminants. Unfortunately, a choice often needs to be made between high purity enrichment that requires TCR ligation, and partial enrichment in which varying levels of contaminant cells remain. Activation might not always occur when using TCR- specific reagents (and may be irrelevant when sorted cells Current Opinion in Immunology 2005, 17:448–454 are to be stimulated [33]) but, for transfer studies, parti- cularly where a possible NKT cell response to endogen- ous antigens is being tested [9,34], the potential for flow cytometry reagents to alter NKT cell function should be considered. Perhaps the most cautious approach is to employ two methods. For example, if unfractionated liver (a rich source of NKT cells), provides similar results to FACS-sorted NK1.1+ abTCR+ cells, it is strong evidence that NKT cells are involved, independently of stimula- tion caused during their isolation [9]. Working with NKT cells NKT cell subsets NKT cells are often regarded as functionally singular, but distinct subsets probably exist. Functional differences have been reported for NKT cells that differentially express CD4 [33,35], NK1.1 [20,22,36] and other surface molecules [37] and may also exist between NKT cells from different organs [38–40]. There is no clear consensus on this issue, but it is important to recognize that the composition and origin of NKT cells may affect their overall response. For that reason, it is highly recom- mended that functional assays be assessed in context of the origin and phenotype of NKT cells at the begin- ning and end of experiments. The diversity of the NKT cell pool presents additional problems for human studies. As blood is often the only source of NKT cells, findings are often extrapolated to NKT cells in general. This is a risky assumption because the frequency and function of NKT cells from different organs might be unrelated [39,41,42]. In non-obese dia- betic (NOD) mice, for example, systemic NKT cell deficiencies are evident in all locations except blood [42]. It is not clear whether a similar phenomenon exists for humans, but it cannot be assumed that blood NKT cells are representative of NKT cells in other organs — even from the same donor. NKT cell expansion in vitro Expansion cultures are sometimes used to generate enough human NKT cells for multiple functional tests on cells from one donor; they might also become impor- tant for treating NKT cell deficiencies by adoptive trans- fer of autologous NKT cells. Human NKT cells can be cultured for many months and, with some exceptions [43�], appear to retain their functional activity and phe- notypic diversity [44�]. Although methods can differ between groups, the expansion of NKT cells is usually achieved by culturing peripheral blood mononuclear cells (PBMCs) with aGC and IL-2. Human serum is recom- mended, but not essential, and IL-15 or IL-7 (less effec- tive) can enhance, or substitute for, the actions of IL-2 in most instances [43�,44�,45]. Cultures need to be supple- mented with antigen-presenting cells (APCs) after the primary expansion and, although purified DCs are prob- ably most effective, irradiated PBMCs should suffice.
  • Working with NKT cells Berzins, Smyth and Godfrey 451 Functional assays Cytokine or cytotoxicity assays for mouse or human NKT cells employ similar conditions to those for mainstream T cells, with the exception of culture times. Strong cytokine production occurs within two hours, although longer culture times increase the sensitivity of ELISA assays. NKT cells respond rapidly to stimulation by plate-bound anti-CD3 (� anti-CD28), phorbol 12-myristate 13-acetate (PMA) and ionomycin (administered together) and Con- A, but other cells will also be stimulated and may com- plicate analysis unless NKT cells are purified before culture [46,47]. An alternative approach is to use specific glycolipid agonists, such as aGC or related compounds. Such antigens require presentation by CD1d+ APCs, or on CD1d-coated plates. If CD1d-coated plates are used, chaperone proteins such as saposins might be needed for efficient glycolipid loading — as was the case for the recently identified natural NKT cell agonist iGb3 [48�]. Cytokines released by activated NKT cells can have bystander effects on surrounding cells (including other NKT cells) [43�,49], but these can be minimized when sorted NKT cells are stimulated using irradiated agonist- pulsed APCs, or CD1d-coated plates. Developmental assays Fetal thymic organ cultures (FTOCs) provide the most physiological conditions for in vitro NKT cell develop- ment. They have been successfully used to establish the maturational hierarchy of thymic subsets in mice and are easily manipulated to investigate the effect of blocking or stimulating different receptor–ligand interactions [22,50]. Similar cultures of adult thymus fragments may be useful in human studies, but are hampered by tissue availability and the very low frequency of NKT cells [51]. Whether de novo NKT cells can be generated from suspension cul- tures of progenitor cells remains unclear. A problem is that NKT cells branch from mainstream T cell develop- ment at the intrathymic CD4+CD8+ stage and also rely on these cells for positive selection signalling. Hence, as for mainstream T cells, NKT cell development appears to be critically dependent on the thymic microenvironment. In vivo analysis of NKT cell functions Adoptive transfer Many functional interactions between NKT cells and other immune cells have been dissected by adoptive transfer experiments involving recipients that lack NKT cells (Figure 2). NKT cell-deficient mice are typi- cally Ja18�/� mice, which lack the genetic elements to produce the semi-invariant TCRa chain, or CD1d�/� mice, which cannot provide the CD1d-dependent signal- ling for NKT cell development. Both models provide an NKT cell-deficient immune environment that is other- wise largely normal, but the CD1d+ environment of Ja18�/� mice enables transferred NKT cells to respond to TCR-mediated stimuli, whereas NKT cells transferred into CD1d�/� mice can only respond to TCR-indepen- dent signals (such as cytokines). CD1d�/� mice also have the disadvantage that they lack other CD1d-dependent cells (Type-2 NKT cells), which can further complicate interpretation of the results. One example of a series of successfully applied adoptive transfer experiments was the characterization of NKT cell-mediated rejection of methylcholanthrene-induced sarcomas [9]. Tumours spontaneously rejected by wild- type mice grew vigorously in NKT cell-deficient Ja18�/� mice unless NKT cells were transferred. Comparing the protective effect of NKT cells from donor mice lacking different effector molecules revealed that NKT cell- derived IFN-g production, but not perforin or TNF, was essential for tumour rejection, whereas the failure to similarly protect CD1d�/� mice indicated that trans- ferred NKT cells required a TCR-mediated stimulus. Similar experiments have been performed in other mod- els of cancer [52,53], allergy and hypersensitivity [34,54] and autoimmunity [8,11]. Tracking NKT cells Carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling is a powerful tool for tracking ‘mainstream’ T cells (the most commonly used T cells, including MHC- restricted CD4+ and CD8+ T cells) in mice, and adoptively transferred NKT cells can be similarly tracked, albeit with some complicating factors [55,56]. First and foremost is a low recovery rate, and secondly, activation can rapidly dilute CFSE levels and cause the surface receptors used in NKT cell identification to be downregulated [56]. NKT cell tracking beyond 2–3 weeks is further complicated by the basal level of proliferation that sees NKT cells under- going 1–2 rounds of division per week even without exogenous stimulation (S Berzins, personal communica- tion; [55]). Congenically marked donor NKT cells are a practical solution for studies that do not require a measure of proliferation (e.g. NKT cell survival, homing or home- ostasis), and can be used in conjunction with CFSE to include proliferation as part of the analysis. In vivo activation Peripheral NKT cells are rapidly activated in vivo by intravenous or intraperitoneal administration of aGC. The cytokine response is clearly evident within two hours and continues at high levels for three days, accompanied by several rounds of proliferation. Numbers peak around day three, before returning to normal by day six [56,57]. NK1.1 and TCR downregulation by activated NKT cells can make them difficult to identify directly in the days following activation [56,57]. Receptor downregulation may also explain the similar, transient disappearance of NKT cells reported in humans within 1–2 days of aGC therapy [58]. Although aGC typically induces a Th0-like response, analogues, such as OCH (a truncated derivative of Current Opinion in Immunology 2005, 17:448–454
  • 452 Immunological techniques T Im d Figure 2 Recipient model Treatment Ja18–/– Deficiency of host Host immune function Type-1 NKT cell deficient Impaired Type-1 NKT cell dependent immune response aGC) [59] and a-c-glycoside [60], appear to produce Th2 or Th1 responses, respectively. Thus, it may become possible to tailor the type of NKT cell stimulation to promote a suppressive or proinflammatory response. This has exciting implications for the future potential of NKT cell-based therapies for various clinical conditions [61,62]. Conclusions Increasing agreement on the defining characteristics of NKT cells has resolved many of the conflicts that initially dogged the field. The semi-invariant TCR provides a reliable target to identify NKT cells and to directly assay NKT cells at the exclusion of contaminant cells. The recent identification of a natural NKT cell ligand is potentially a major advance, but important questions remain about how NKT cells ‘decide’ to promote Th1 or Th2 effects. The answers probably lie with the dif- ferent agonist activities of glycolipid antigens and their analogues and the characterization of functionally distinct NKT cell subsets. Adoptive transfer of wild-type Type -1 NKT cells Adoptive transfer of Factor -X-deficient NKT cells Restore Type-1 NKT cell function Outcome: Fully functional immune system Problem addressed: Is NKT cell derived Factor-X critical for NKT cell function? Res Typ Outco f d P Facto -in Strategies for identifying NKT cells by flow cytometry. While reagents that b CD1d tetramer) provide the highest stringency, alternative approaches may used approaches and comment on the pros and cons of each. Current Opinion in Immunology 2005, 17:448–454 CD1d–/– RAG-1–/– ype-1 and Type-2 NKT cell deficient paired Type -1 and Type-2 NKT cell ependent immune response T cell, B cell, Type-1 and Type-2 NKT cell deficient Impaired T cell, B cell, Type-1 and Type-2 NKT cell dependent immune response With the emerging consensus of how best to identify and assay NKT cells in human and mice, the potential for these cells to become part of the therapeutic arsenal to treat and prevent diseases, such as cancer and type 1 diabetes, appears increasingly attainable. Update The work referred to in the text as (SP Berzins et al., unpublished) has now been published [63]. Acknowledgements We are especially grateful to Jonathan Coquet from DIG’s laboratory for discussions concerning NKT cell activation and to Rohan Berzins for assistance with artwork. Other members of our laboratories are also thanked for their contributions and valuable scientific discussions. Research undertaken in our laboratories is supported by grants from the National Health and Medical Research Council (NHMRC) of Australia, National Institutes of Health (NIH), and Association of International Cancer Research (AICR). SPB is a long-term fellow of the Human Frontier Science Program (HFSP). MJS and DIG are each supported by a Research Fellowships and a Program Grant from the NHMRC. We apologize to those colleagues whose work has only been referenced indirectly through reviews as a result of space limitations. tore TCR-independent e-1 NKT cell function. me: No Type-2 NKT cell unction – No CD1d ependent signaling roblem addressed: Is NKT cell derived r-X critical for their TCR dependent function? Restore Type-1 NKT cell function Outcome: Normal Type-1 NKT cell funtion in absence of other lymphocytes (T cell, B cell and Type-2 NKT cells) Problem addressed: Is NKT cell-derived Factor-X necessary in absence of other lymphocytes? Current Opinion in Immunology ind the CDR3 region of the NKT cell TCR (such as aGC-loaded sometimes be necessary. Here we compare the most commonly
  • Working with NKT cells Berzins, Smyth and Godfrey 453 References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: � of special interest �� of outstanding interest 1. Smyth MJ, Godfrey DI: NKT cells and tumour immunity–a double-edged sword. Nat Immunol 2000, 1:459-460. 2. Godfrey DI, Kronenberg M: Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest 2004, 114:1379-1388. 3. van der Vliet HJ, Molling JW, von Blomberg BM, Nishi N, Kolgen W, van den Eertwegh AJ, Pinedo HM, Giaccone G, Scheper RJ: The immunoregulatory role of CD1d-restricted natural killer T cells in disease. Clin Immunol 2004, 112:8-23. 4. Hammond KJ, Godfrey DI: NKT cells: potential targets for autoimmune disease therapy? Tissue Antigens 2002, 59:353-363. 5. Smyth MJ, Crowe NY, Hayakawa Y, Takeda K, Yagita H, Godfrey DI: NKT cells - conductors of tumour immunity? Curr Opin Immunol 2002, 14:165-171. 6. 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Parekh VV, Singh AK, Wilson MT, Olivares-Villagomez D, Bezbradica JS, Inazawa H, Ehara H, Sakai T, Serizawa I, Wu L et al.: Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids. J Immunol 2004, 173:3693-3706. 63. Berzins SP, Cochrane AD, Pellicci DG, Smyth MJ, Godfrey DI: Limited correlation between human thymus and blood NKT cell content revealed by an ontogeny study of paired tissue samples. Eur J Immunol 2005, 35:1399-1407. Working with NKT cells - pitfalls and practicalities Introduction Identifying NKT cells What are NKT cells? Technical challenges Finding and isolating NKT cells Where to find NKT cells In situ localization Enrichment and purification of NKT cells Working with NKT cells NKT cell subsets NKT cell expansion in vitro Functional assays Developmental assays In vivo analysis of NKT cell functions Adoptive transfer Tracking NKT cells In vivo activation Conclusions Update Acknowledgements References and recommended reading