Isolation and Functional Use of Human NKT Cells

Mark A. Exley1, S. Brian Wilson2, Steven P. Balk3

1 Manchester Collaborative Centre for Inflammation Research, Manchester, 2 Diabetes Center of Excellence, University of Florida, Gainesville, 3 Cancer Biology Program, Hematology‐Oncology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.11
DOI:  10.1002/cpim.33
Online Posting Date:  November, 2017
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Abstract

This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term ‘iNKT’ derives from the fact that a large fraction of murine and some human NK marker+ T cells (‘NKT’) recognize the MHC class I–like CD1d protein and use an identical ‘invariant’ TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N‐region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, ‘iNKT’ cells). iNKT are mostly CD4+ or CD4–CD8– (‘double negative’), although a few CD8+ iNKT can be found in some humans. Basic Protocol 1 and Alternate Protocol 1 use multi‐color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high‐speed FACS sorting of iNKT cells. Basic Protocol 3 explains functional analysis of iNKT. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A support protocol for secondary stimulation and rapid expansion of iNKT cells is also included. © 2017 by John Wiley & Sons, Inc.

Keywords: NKT cells; human; CD1d; α‐galactosylceramide; invariant T cell receptor; iNKT cells

     
 
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Table of Contents

  • Introduction
  • Identification and Quantitation of Invariant NKT Cells
  • Basic Protocol 1: iNKT Cell Flow Cytometry with Anti‐Invariant TCR mAB 6B11 or CD1d Multimer
  • Alternate Protocol 1: Identification of Invariant NKT Cells by Flow Cytometry Using Vα24 and Vβ11 mAb
  • Isolation and Expansion of Invariant NKT Cells
  • Basic Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by Immunomagnetic Beads Followed by Selective Expansion with α−GalCer
  • Alternate Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by FACS Followed by Selective Expansion with α–GalCer
  • Alternate Protocol 3: Generation of Invariant NKT Cell Clones
  • Support Protocol 1: Secondary Stimulation of Invariant NKT Cell Lines and Clones
  • Basic Protocol 3: CD1d‐Specific Functional Assays for Invariant NKT Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: iNKT Cell Flow Cytometry with Anti‐Invariant TCR mAB 6B11 or CD1d Multimer

  Materials
  • Healthy human blood donor
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Flow cytometry buffer (FC buffer): PBS ( appendix 2A) with 1% human serum, 1% FBS, 0.1% sodium azide
  • Conjugated anti‐Vα24 mAb (clone C15B2, PE or FITC conjugate, Coulter Immunotech)
  • Conjugated 6B11 anti–invariant TCR mAb (Pharmingen, eBioSciences, Miltenyi Biotech) or stable α−GalCer analog, PBS‐57‐loaded CD1d tetramer (NIH Tetramer Core Facility; http://tetramer.yerkes.emory.edu/index.html), or other α−GalCer loaded CD1d multimer conjugated to appropriate chromophore such as APC
  • IgG1 isotype control for 6B11 or un‐loaded CD1d tetramer control conjugate (NIH Tetramer Core Facility)
  • Lineage markers for inclusion (e.g., CD3), exclusion (e.g., CD19), or sub‐typing (CD4, etc.; see below)
  • PBS with 4% (w/v) paraformaldehyde
  • Refrigerated centrifuge
  • Additional reagents and equipment for purifying peripheral blood mononuclear cells (Fuss, Kanof, Smith, & Zola, )
CAUTION: Paraformaldehyde is highly toxic! Heat only in a functional fume hood to 80°C with stirring bar to dissolve and then cool and aliquot in the hood into sealed tubes before removal for individual use; aliquots can be stored at −20°C. Paraformaldehyde is unstable at room temperature and 4°C. It can be stored at −20°C indefinitely and there are also stabilized commercial forms which can be stored at 4°C.

Alternate Protocol 1: Identification of Invariant NKT Cells by Flow Cytometry Using Vα24 and Vβ11 mAb

  Additional Materials (also see protocol 1)
  • Optional lineage markers for inclusion (e.g., CD3), exclusion (e.g., CD19), and sub‐typing (CD4 etc.; see below)
  • Conjugated anti‐Vα24 mAb (clone C15B2, PE or FITC conjugates, Coulter Immunotech)
  • Conjugated anti‐Vβ11 mAb (clone C21D2, Coulter FITC or PE conjugates: i.e., reciprocal chromophore)
  • Conjugated isotype matched control mAbs (Coulter, Pharmingen)
    • For further phenotypic characterization by multi‐color flow cytometry:Cy5 or other conjugated anti‐CD4, anti‐CD8α
    • Conjugated anti‐CD161 (the Coulter mAb has so far yielded the best shifts in our hands) or NKG2D
    • Compatibly conjugated Abs to other cell surface proteins of interest as available

Basic Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by Immunomagnetic Beads Followed by Selective Expansion with α−GalCer

  Materials
  • Healthy human blood donor
  • Growth medium (see recipe)
  • PBS/EDTA: phosphate‐buffered saline (PBS; appendix 2A) with 2 mM EDTA
  • FcR‐blocking reagent (Human IgG; Miltenyi Biotech, cat. no. 130‐059‐901)
  • Unconjugated or ‐PE‐conjugated anti‐Vα24 mAb (Coulter)
  • Unconjugated or ‐PE‐conjugated 6B11 anti‐invariant TCRα mAb (eBioSciences, Pharmingen)
  • Binding buffer: PBS ( appendix 2A) with 2 mM EDTA and 2% (v/v) human serum
  • Goat anti‐mouse IgG or anti‐PE microbeads (Miltenyi Biotech)
  • T cell medium (see recipe)
  • DMSO freezing mixture: 90% FBS/10% DMSO
  • α−GalCer (https://avantilipids.com; http://shop.diagnocine.com)
  • IL‐2 (optional)
  • Refrigerated centrifuge
  • MS columns (for up to 108 starting cells; Miltenyi Biotech; cat. no. 130‐042‐201)
  • LS columns (for up to 108 starting cells; Miltenyi Biotech, cat. no. 130‐042‐401)
  • Magnetic separation device (Miltenyi Biotec)
  • γ‐irradiator
  • Round‐bottomed 96‐well tissue culture plates
  • Flat‐bottom 96‐well tissue culture plates (optional)
  • 24‐well tissue culture plates (optional)
  • Additional reagents and equipment for purifying peripheral blood mononuclear cells (unit 7.1; Fuss et al., ) and counting cells ( appendix 3a; Strober, )
NOTE: Alternative to all reagents above is direct 6B11‐conjugated microbeads/iNKT kit (Miltenyi). Manufacturer's directions are similar to below.NOTE: Alternatives to α−GalCer (see below) are PHA‐P (Difco) or mitogenic CD3 mAb (such as OKT3 from ATCC or other CD3 clones).

Alternate Protocol 2: Isolation of Vα24+ or 6B11+ T Cells by FACS Followed by Selective Expansion with α–GalCer

  Additional Materials (also see Basic Protocols protocol 11 and protocol 32)
  • FACS buffer: PBS with 1% human serum and 1% FBS (no azide)
  • Fluorescence‐activated cell sorting (FACS) instrument (see Chapter 5)

Alternate Protocol 3: Generation of Invariant NKT Cell Clones

  Additional Materials (also see Basic Protocols protocol 11 and protocol 32)
  • Phosphate‐buffered serum (PBS; appendix 2A) with 10% human serum
  • T cell cloning medium: RPMI‐1640, 10% autologous human serum, 2 μg/ml PHA‐P, 20 U/ml IL‐2, and 20 U/ml IL‐7 (Genzyme)

Support Protocol 1: Secondary Stimulation of Invariant NKT Cell Lines and Clones

  Materials
  • Healthy human blood donor
  • B lymphoblastoid cell line (JY or C1R)
  • Expansion medium: RPMI‐1640 with 10% AB negative human serum, 10 mM HEPES, and gentamicin (FBS may be used in place of human serum, but lots of the human and bovine sera should be tested for their ability to stimulate these cells)
  • Mitogenic anti‐CD3 [clones UCHT1 (IgG1) from Ancell or OKT3 have been used with success]
  • Recombinant IL‐2 and IL‐7 (Genzyme)
  • γ‐irradiator
  • 25‐cm2 flasks
  • Microscope
  • Additional reagents and equipment for isolating human PBMC (unit 7.1; Fuss et al., )

Basic Protocol 3: CD1d‐Specific Functional Assays for Invariant NKT Cells

  Materials
  • Isolated iNKT cells (see protocol 3, protocol 4, or protocol 5)
  • T cell stimulation (TCS) medium (see recipe)
  • 42.1 human CD1d‐specific mAb (Pharmingen)
  • α‐GalCer‐pulsed APCs, optional
  • Cytokine ELISA kits (Endogen, Pharmingen, or other)
  • 96‐well flat‐bottom plates
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Figures

Videos

Literature Cited

  Altman, J. D., & Davis, M. M. (2016). MHC‐peptide tetramers to visualize antigen‐specific T cells. Current Protocols in Immunology, 115, 17.3.1‐17.3.44. doi: 10.1002/cpim.14.
  Behar, S. M., Podrebarac, T. A., Roy, C. J., Wang, C. R., & Brenner, M. B. (1999). Diverse TCRs recognize murine CD1. Journal of Immunology, 162(1), 161–167.
  Bendelac, A., Savage, P. B., & Teyton, L. (2007). The biology of NKT cells. Annual Review of Immunology, 25, 297–336. doi: 10.1146/annurev.immunol.25.022106.141711.
  Brigl, M., Bry, L., Kent, S. C., Gumperz, J. E., & Brenner, M. B. (2003). Mechanism of CD1d‐restricted natural killer T cell activation during microbial infection. Nature Immunology, 4(12), 1230–1237. doi: 10.1038/ni1002.
  Brigl, M., & Brenner, M. B. (2004). CD1: Antigen presentation and T cell function. Annual Review of Immunology, 22, 817–890. doi: 10.1146/annurev.immunol.22.012703.104608.
  Bricard, G., Cesson, V., Devevre, E., Bouzourene, H., Barbey, C., Rufer, N., … Speiser, D. E. (2009). Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors. Journal of Immunology, 182(8), 5140–5151. doi: 10.4049/jimmunol.0711086.
  Brossay, L., Chioda, M., Burdin, N., Koezuka, Y., Casorati, G., Dellabona, P., & Kronenberg, M. (1998). CD1d‐mediated recognition of an alpha‐galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. The Journal of Experimental Medicine, 188(8), 1521–1528. doi: 10.1084/jem.188.8.1521.
  Chiu, Y. H., Jayawardena, J., Weiss, A., Lee, D., Park, S. H., Dautry‐Varsat, A., & Bendelac, A. (1999). Distinct subsets of CD1d‐restricted T cells recognize self‐antigens loaded in different cellular compartments. The Journal of Experimental Medicine, 189(1), 103–110. doi: 10.1084/jem.189.1.103.
  Coico, R., & Lunn, G. (2005). Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Current Protocols in Immunology, 69, A.1V.1–A.1V.8. doi: 10.1002/0471142735.ima01vs69.
  Cui, J., Shin, T., Kawano, T., Sato, H., Kondo, E., Toura, I., … Taniguchi, M. (1997). Requirement for Valpha14 NKT cells in IL‐12‐mediated rejection of tumors. Science, 278(5343), 1623–1626.
  Dhodapkar, M. V., Geller, M. D., Chang, D. H., Shimizu, K., Fujii, S., Dhodapkar, K. M., & Krasovsky, J. (2003). A reversible defect in natural killer T cell function characterizes the progression of premalignant to malignant multiple myeloma. The Journal of Experimental Medicine, 197(12), 1667–1676. doi: 10.1084/jem.20021650.
  Durante‐Mangoni, E., Wang, R., Shaulov, A., He, Q., Nasser, I., Afdhal, N., … Exley, M. A. (2004). Hepatic CD1d expression in hepatitis C virus infection and recognition by resident proinflammatory CD1d‐reactive T cells. Journal of Immunology, 173(3), 2159–2166. doi: 10.4049/jimmunol.173.3.2159.
  Exley, M., Garcia, J., Balk, S. P., & Porcelli, S. (1997). Requirements for CD1d recognition by human invariant Valpha24+ CD4‐CD8‐ T cells. The Journal of Experimental Medicine, 186(1), 109–120. doi: 10.1084/jem.186.1.109.
  Exley, M. A., Tahir, S. M., Cheng, O., Shaulov, A., Joyce, R., Avigan, D., … Balk, S. P. (2001). Cutting edge: A major fraction of human bone marrow lymphocytes are Th2‐like CD1d‐reactive T cells that can suppress mixed lymphocyte responses. Journal of Immunology, 167(10), 5531–5534. doi: 10.4049/jimmunol.167.10.5531.
  Exley, M. A., He, Q., Cheng, O., Wang, R. J., Cheney, C. P., Balk, S. P., & Koziel, M. J. (2002). Cutting edge: Compartmentalization of Th1‐like noninvariant CD1d‐reactive T cells in hepatitis C virus‐infected liver. Journal of Immunology, 168(4), 1519–1523. doi: 10.4049/jimmunol.168.4.1519.
  Exley, M. A., Hou, R., Shaulov, A., Tonti, E., Dellabona, P., Casorati, G., … Wilson, S. B. (2008). Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR alpha‐chain CDR3 loop. European Journal of Immunology, 38(6), 1756–1766. doi: 10.1002/eji.200737389.
  Exley, M. A., Vriend, L., Alatrakchi, N., Yue, S., Clark, J., Friedlander, P., … Balk, S. (2017). Phase 1 clinical trial of highly purified invariant NKT cells for stage IV melanoma. Clinical Cancer Research, pii, clincanres.0600.2016. doi: 10.1158/1078‐0432.CCR‐16‐0600.
  Fujii, S., Shimizu, K., Steinman, R. M., & Dhodapkar, M. V. (2003). Detection and activation of human Valpha24+ natural killer T cells using alpha‐galactosyl ceramide‐pulsed dendritic cells. Journal of Immunological Methods, 272(1‐2), 147–159. PubMed PMID: 12505720. doi: 10.1016/S0022‐1759(02)00497‐0.
  Fuss, I. J., Kanof, M. E., Smith, P. D., & Zola, H. 2009. Isolation of whole mononuclear cells from peripheral blood and cord blood. Current Protocols in Immunology, 85, 7.1.1–7.1.8. doi: 10.1002/0471142735.im0701s85.
  Gadola, S. D., Dulphy, N., Salio, M., & Cerundolo, V. (2002). Valpha24‐JalphaQ‐independent, CD1d‐restricted recognition of alpha‐galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes. Journal of Immunology, 168(11), 5514–5520. doi: 10.4049/jimmunol.168.11.5514.
  Gansuvd, B., Hubbard, W. J., Hutchings, A., Thomas, F. T., Goodwin, J., Wilson, S. B., … Thomas, J. M. (2003). Phenotypic and functional characterization of long‐term cultured rhesus macaque spleen‐derived NKT cells. Journal of Immunology, 171(6), 2904–2911. PubMed PMID: 12960313. doi: 10.4049/jimmunol.171.6.2904.
  Gumperz, J. E. (2004). CD1d‐restricted “NKT” cells and myeloid IL‐12 production: An immunological crossroads leading to promotion or suppression of effective anti‐tumor immune responses? Journal of Leukocyte Biology, 76, 307–313. doi: 10.1189/jlb.0104038.
  Gumperz, J. E., Miyake, S., Yamamura, T., & Brenner, M. B. (2002). Functionally distinct subsets of CD1d‐restricted natural killer T cells revealed by CD1d tetramer staining. The Journal of Experimental Medicine, 195(5), 625–636. doi: 10.1084/jem.20011786.
  Kain, L., Webb, B., Anderson, B. L., Deng, S., Holt, M., Costanzo, A., … Teyton, L. (2014). The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α‐linked glycosylceramides. Immunity, 41(4), 543–554. doi: 10.1016/j.immuni.2014.08.017.
  Kawano, T., Cui, J., Koezuka, Y., Toura, I., Kaneko, Y., Motoki, K., … Taniguchi, M. (1997). CD1d‐restricted and TCR‐mediated activation of valpha14 NKT cells by glycosylceramides. Science, 278(5343), 1626–1629.
  Kenna, T., Golden‐Mason, L., Porcelli, S. A., Koezuka, Y., Hegarty, J. E., O'Farrelly, C., & Doherty, D. G. (2003). NKT cells from normal and tumor‐bearing human livers are phenotypically and functionally distinct from murine NKT cells. Journal of Immunology, 171(4), 1775–1779. doi: 10.4049/jimmunol.171.4.1775.
  Kim, C. H., Butcher, E. C., & Johnston, B. (2002). Distinct subsets of human Valpha24‐invariant NKT cells: Cytokine responses and chemokine receptor expression. Trends in Immunology, 23(11), 516–519. PubMed PMID: 12401396. doi: 10.1016/S1471‐4906(02)02323‐2.
  Kronenberg, M., & Kinjo, Y. (2009). Innate‐like recognition of microbes by invariant natural killer T cells. Current Opinion in Immunology, 21(4), 391–396. Epub 2009 Jul 29. doi: 10.1016/j.coi.2009.07.002.
  Kruisbeek, A. M., Shevach, E., & Thornton, A. M. (2004). Proliferative assays for T cell function. Current Protocols in Immunology, 60, 3.12.1–3.12.20. doi: 10.1002/0471142735.im0312s60.
  Kukreja, A., Cost, G., Marker, J., Zhang, C., Sun, Z., Lin‐Su, K., … Maclaren, N. (2002). Multiple immuno‐regulatory defects in type‐1 diabetes. The Journal of Clinical Investigation, 109(1), 131–140. doi: 10.1172/JCI0213605.
  Lee, P. T., Benlagha, K., Teyton, L., & Bendelac, A. (2002). Distinct functional lineages of human V(alpha)24 natural killer T cells. The Journal of Experimental Medicine, 195(5), 637–641. doi: 10.1084/jem.20011908.
  Mattner, J., Savage, P. B., Leung, P., Oertelt, S. S., Wang, V., Trivedi, O., … Bendelac, A. (2008). Liver autoimmunity triggered by microbial activation of natural killer T cells. Cell Host & Microbe, 3(5), 304–315. doi: 10.1016/j.chom.2008.03.009.
  Metelitsa, L. S., Naidenko, O. V., Kant, A., Wu, H. W., Loza, M. J., Perussia, B., … Seeger, R. C. (2001). Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL‐2 to activate NK cells. Journal of Immunology, 167(6), 3114–3122. doi: 10.4049/jimmunol.167.6.3114.
  Metelitsa, L. S. (2004). Flow cytometry for natural killer T cells: Multi‐parameter methods for multifunctional cells. Clinical Immunology, 110(3), 267–276. doi: 10.1016/j.clim.2003.11.005.
  Metelitsa, L. S., Wu, H. W., Wang, H., Yang, Y., Warsi, Z., Asgharzadeh, S., … Seeger, R. C. (2004). Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2. Journal of Experimental Medicine, 199(9), 1213–1221. doi: 10.1084/jem.20031462.
  Molling, J. W., K∂lgen, W., van der Vliet, H. J., Boomsma, M. F., Kruizenga, H., Smorenburg, C. H., … van den Eertwegh, A. J. (2005). Peripheral blood IFN‐gamma‐secreting Valpha24+Vbeta11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load. International Journal of Cancer, 116(1), 87–93. doi: 10.1002/ijc.20998.
  Motsinger, A., Azimzadeh, A., Stanic, A. K., Johnson, R. P., Van Kaer, L., Joyce, S., & Unutmaz, D. (2003). Identification and simian immunodeficiency virus infection of CD1d‐restricted macaque natural killer T cells. Journal of Virology, 77(14), 8153–8158. PubMed PMID: 12829854; doi: 10.1128/JVI.77.14.8153‐8158.2003.
  Nair, S., Archer, G. E., & Tedder, T. F. (2012). Isolation and generation of human dendritic cells. Current Protocols in Immunology, 99, 7.32.1–7.32.23. doi: 10.1002/0471142735.im0732s99.
  Porcelli, S., Yockey, C. E., Brenner, M. B., & Balk, S. P. (1993). Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4‐8‐ alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. The Journal of Experimental Medicine, 178(1), 1–16. doi: 10.1084/jem.178.1.1.
  Rogers, P. R., Matsumoto, A., Naidenko, O., Kronenberg, M., Mikayama, T., & Kato, S. (2004). Expansion of human Valpha24+ NKT cells by repeated stimulation with KRN7000. Journal of Immunological Methods, 285(2), 197–214. doi: 10.1016/j.jim.2003.12.003.
  Rout, N., Else, J. G., Yue, S., Connole, M., Exley, M. A., & Kaur, A. (2010). Paucity of CD4+ natural killer T (NKT) lymphocytes in sooty mangabeys is associated with lack of NKT cell depletion after SIV infection. PLoS One, 5(3), e9787. doi: 10.1371/journal.pone.0009787.
  Sandberg, J. K., Bhardwaj, N., & Nixon, D. F. (2003). Dominant effector memory characteristics, capacity for dynamic adaptive expansion, and sex bias in the innate Valpha24 NKT cell compartment. European Journal of Immunology, 33(3), 588–596. PubMed PMID: 12616479. doi: 10.1002/eji.200323707.
  Thomas, S. Y., Hou, R., Boyson, J. E., Means, T. K., Hess, C., Olson, D. P., … Luster, A. D. (2003). CD1d‐restricted NKT cells express a chemokine receptor profile indicative of Th1‐type inflammatory homing cells. Journal of Immunology, 171(5), 2571–2580. PubMed PMID: 12928408. doi: 10.4049/jimmunol.171.5.2571.
  Spada, F. M., Koezuka, Y., & Porcelli, S. A. (1998). CD1d‐restricted recognition of synthetic glycolipid antigens by human natural killer T cells. The Journal of Experimental Medicine, 188(8), 1529–1534. doi: 10.1084/jem.188.8.1529.
  Song, L., Asgharzadeh, S., Salo, J., Engell, K., Wu, H. W., Sposto, R., … Metelitsa, L. S. (2009). Valpha24‐invariant NKT cells mediate antitumor activity via killing of tumor‐associated macrophages. The Journal of Clinical Investigation, 119(6), 1524–1536. doi: 10.1172/JCI37869.
  Strober, W. (2001). Monitoring cell growth. Current Protocols in Immunology, 21, A.3A.1–A.3A.2. doi: 10.1002/0471142735.ima03as21.
  Tahir, S. M., Cheng, O., Shaulov, A., Koezuka, Y., Bubley, G. J., Wilson, S. B., … Exley, M. A. (2001). Loss of IFN‐gamma production by invariant NK T cells in advanced cancer. Journal of Immunology, 167, 4046‐4050.
  van der Vliet, H. J., Nishi, N., Koezuka, Y., von Blomberg, B. M., van den Eertwegh, A. J., Porcelli, S. A., … Giaccone, G. (2001). Potent expansion of human natural killer T cells using alpha‐galactosylceramide (KRN7000)‐loaded monocyte‐derived dendritic cells, cultured in the presence of IL‐7 and IL‐15. Journal of Immunological Methods, 247(1‐2), 61–72. doi: 10.1016/S0022‐1759(00)00272‐6.
  van der Vliet, H. J., Molling, J. W., Nishi, N., Masterson, A. J., K∂lgen, W., Porcelli, S. A., … Scheper, R. J. (2003). Polarization of Valpha24+ Vbeta11+ natural killer T cells of healthy volunteers and cancer patients using alpha‐galactosylceramide‐loaded and environmentally instructed dendritic cells. Cancer Research, 63(14), 4101–4106.
  Wilson, S. B., Kent, S. C., Patton, K. T., Orban, T., Jackson, R. A., Exley, M., … Hafler, D. A. (1998). Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature, 391(6663), 177–181. doi: 10.1038/34419.
  Wonderlich, J., Shearer, G., Livingstone, A., & Brooks, A. (2006). Induction and measurement of cytotoxic T lymphocyte activity. Current Protocols in Immunology, 72, 3.11.1–3.11.23. doi: 10.1002/0471142735.im0311s72.
Key References
  Bendelac, A., Savage, P. B., & Teyton, L. (2007). The biology of NKT cells. Annual Review of Immunology, 25, 297–336. Review. PubMed PMID: 17150027. doi: 10.1146/annurev.immunol.25.022106.141711.
  Authoritative classic review from the investigator who first demonstrated CD1d restriction of murine NKT cells and more recent colleagues.
  Brennan, P. J., Brigl, M., & Brenner, M. B. (2013). Invariant natural killer T cells: An innate activation scheme linked to diverse effector functions. Nature Reviews Immunology, 13(2), 101–117. doi: 10.1038/nri3369.
  Another authoritative NKT review from key investigators in the field.
  Crosby, C. M., & Kronenberg, M. (2016). Invariant natural killer T cells: Front line fighters in the war against pathogenic microbes. Immunogenetics, 68(8), 639–648. doi: 10.1007/s00251‐016‐0933‐y.
  Recent review on iNKT cell functions by leading group in the field.
  Exley, M., Garcia, J., Balk, S. P., & Porcelli, S. (1997). Requirements for CD1d recognition by human invariant Valpha24+ CD4‐CD8‐ T cells. The Journal of Experimental Medicine, 186(1), 109–120. doi: 10.1084/jem.186.1.109.
  First functional definition of human iNKT cells.
  Exley, M. A., Hou, R., Shaulov, A., Tonti, E., Dellabona, P., Casorati, G., … Wilson, S. B. (2008). Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR alpha‐chain CDR3 loop. European Journal of Immunology, 38(6), 1756–1766. doi: 10.1002/eji.200737389.
  Description and use of iNKTCR mAb 6B11 for analysis and purification of iNKT cells.
  Exley, M. A., Vriend, L., Alatrakchi, N., Yue, S., Clark, J., Friedlander, P., … Balk, S. (2017). Phase 1 clinical trial of highly purified invariant NKT cells for stage IV melanoma. Clinical Cancer Research, pii, clincanres.0600.2016. doi: 10.1158/1078‐0432.CCR‐16‐0600. [Epub] PMID: 28193627.
  Clinical trial of autologous iNKT cell adoptive transfer for cancer patients using iNKTCR mAb 6B11.
  Fujii, S., Shimizu, K., Steinman, R. M., & Dhodapkar, M. V. (2003). Detection and activation of human Valpha24+ natural killer T cells using alpha‐galactosyl ceramide‐pulsed dendritic cells. Journal of Immunological Methods, 272(1‐2), 147–159. doi: 10.1016/S0022‐1759(02)00497‐0.
  Activation of iNKT from the antigen‐presenting cell perspective by leading translational NKT investigators.
  Molling, J. W., K∂lgen, W., van der Vliet, H. J., Boomsma, M. F., Kruizenga, H., Smorenburg, C. H., … van den Eertwegh, A. J. (2005). Peripheral blood IFN‐gamma‐secreting Valpha24+Vbeta11+ NKT cell numbers are decreased in cancer patients independent of tumor type or tumor load. International Journal of Cancer, 116(1), 87–93. doi: 10.1002/ijc.20998.
  Early example of iNKT analysis from patients.
  Thomas, S. Y., Hou, R., Boyson, J. E., Means, T. K., Hess, C., Olson, D. P., … Luster, A. D. (2003). CD1d‐restricted NKT cells express a chemokine receptor profile indicative of Th1‐type inflammatory homing cells. Journal of Immunology, 171(5), 2571–2580. doi: 10.4049/jimmunol.171.5.2571.
  First description of human iNKT cell chemokine receptor expression.
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