An Introduction to NKT cells CD1d Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides Artificial ligand: a-galactosylceramide (a-GalCer) mu: Va14-Ja18 hu: Va24-Ja18 mu: Vb8.2/Vb7 hu: Vb11 CDR3b diverse Va14iNKT ( CD4) CD44high NK1.1 Autoimmune diseases Infectious diseases Tumors CD69high Ly49 ( DX5) IFN-g IL-4

CD1 molecules present glycolipids CD1 family represents 5 MHC class I like molecules: CD1a, b, c, d, e CD1 grooves provide “shoe-like” cavity serving to anchor the lipid antigens and to shield them from the aqueous environment In contrast to MHC class I and II genes, allelic variation of CD1 genes is extremely limited In mice only CD1d molecule is present *Moody DB, Zajonc DM, Wilson IA. Nat Rev Immunol. 2005;5:387-399

Developmental pathway of Va14i NKT cells CD4- CD8- TCR TCR Thymus NK1.1 CD4+ CD8+ CD1d NKT NKT MHC I MHC II T CD8+ T CD4+ TCR TCR NK1.1 T CD8+ T CD4+ NKT ? NKT Periphery MacDonald H.R., Science, 2002

DEVELOPMENT AND SELECTION OF Va14i NKT CELLS Cellular requirements for positive and negative selection Role of Vb domain in selection by endogenous glycolipids Role of c-myc in Va14i NKT cell development

Specific Identification of Va14i NKT cells Tetramers Dimers Streptavidin- fluorochrome Biotin muCD1d/ huCD1d muCD1d aGalCer aGalCer Va14-Ja18 Vb8.2/Vb7 Va14-Ja18 Vb8.2/Vb7 Va14iNKT Va14iNKT NK1.1 NK1.1

to Va14i NKT cells expressing Vb8.2 Human CD1d:aGalCer dimers bind preferentially to Va14i NKT cells expressing Vb8.2 Thymus Liver Thymus Liver 28 17 12 9 mouse dimers human dimers TCR-b TCR-b Counts 53 ± 4 58 ± 2 80 ± 7 84 ± 4 Counts Vb8.2 Vb8.2 Counts 16 ± 2 12 ± 2 6 ± 5 4 ± 1 Counts Vb7 Vb7

Targeted expression of human CD1d in transgenic mice CD1d expression in CD4+CD8+ (DP) thymocytes driven by lck proximal promoter CD1d expression in thymic dendritic cells driven by CD11c promoter Monitor human CD1d-reactive ( Vb8.2+) Va14i NKT cells on CD1d-/- background (positive selection) or CD1d+/- background (negative selection)

DP thymocytes but not DC expressing huCD1d positively select Vb8.2+ Va14i NKT cells muCD1d-/- % Vb8.2 % Vb7 pLck- huCD1d-tg muCD1d-/- CD11c- huCD1d-tg muCD1d-/- non-tg muCD1d+/-

Both DP thymocytes and DC expressing huCD1d negatively select Vb8.2+ Va14i NKT cells muCD1d+/- % Vb7 % Vb8.2 pLck- huCD1d-tg muCD1d+/- CD11c- huCD1d-tg muCD1d+/- non-tg muCD1d+/-

CONCLUSIONS FROM Human CD1d TRANSGENIC MICE Human CD1d bound to mouse endogenous glycolipid ligands selects preferentially Vb8.2 Va14i NKT cells (like human CD1d bound to aGalCer),implying that residues on Vb8.2 interact preferentially with human CD1d DP thymocytes expressing human CD1d are sufficient to induce both positive and negative selection of developing Va14i NKT cells Thymic DC expressing human CD1d are sufficient to induce negative but not positive selection of developing Va14i NKT cells Thymic DC induce negative selection of developing Va14i NKT cells more efficiently than DP thymocytes

Role of Vb domain in selection of Va14i NKT cells by CD1d-binding endogenous glycolipids a-Galactosylceramide (aGalCer) serves as a model CD1d antigen aGalCer is a glycosphingolipid found in marine sponge and has no known physiological function in mammalian immunity Isoglobotrihexosylceramide (iGb3) has been demonstrated as an endogenous agonist for CD1d restricted T cells

Higher avidity binding of mouse CD1d:aGalCer dimers by Va14i NKT cells expressing Vb8.2 Thymus Liver mouse dimers TCR-b % Vb8.2+ % Vb7+ Gate

Frequency of thymic Vb7+ and Vb8 Frequency of thymic Vb7+ and Vb8.2+ Va14i NKT cells reflects Vb rearrangement frequency in CD4+ CD8+ precursors Va14i NKT DP CD8a mouse dimer Vb8.2 (ic) Vb8.2 Vb7 (ic) Vb7 NK1.1 T % Vb8.2 or Vb7 (ic) CD4 TCR-b % Vb8.2 or Vb7 DP mature Va14i NKT 9.4 ± 0.5 50 ± 1 Vbb/b Ja18+/+ Vbb/b Ja18+/+ Vbb/b Ja18+/- Vba/b Ja18+/+ Vb8.2 (ic) Vb8.2 DP mature Va14i NKT DP thymocytes mature Va14i NKT cells 2.7 ± 0.3 14 ± 3 Vb7 (ic) Vb7

Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous ligand Concentration in vivo Vb8.2 (ic) Vb8.2 * Vb7 (ic) Vb7 % Vb8.2 Vbb/b % Vb7 DP CD8a * Vb7 (ic) Vb7 CD4 DP thymocytes CD1d+/- CD1d+/+ % Vb7 Vba/a MFI, 59 ± 7 MFI, 122 ± 15 CD1d CD1d+/+ CD1d+/- CD1d+/+ CD1d+/- DP thymocytes mature Va14i NKT cells

Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice expressing a low avidity invariant TCRa chain non-tg huVa24-tg Vb8.2 (ic) Vb8.2 NKT NKT NK1.1 Vb7 (ic) Vb7 non-NKT non-NKT % Vb8.2 or Vb7 TCR-b non- NKT TCR-b 88 ± 5 21 ± 10 non- NKT NKT NKT DP DN DP NKT NKT dimer+ dimer+ tetramer+ mouse tetramer non-tg huVa24-tg non-tg huVa24-tg 81 ± 7 7 ± 3 non- NKT non- NKT NKT NKT mouse dimer

Vb7+ NKT cells are preferentially selected by endogenous ligands or exogenous self-ligand iGb3 in thymic culture cell expansion (%) cell expansion (%)

Role of Vb domain in selection of Va14i NKT cells by CD1d-binding glycolipids Vb8.2 binds the artificial agonist ligand aGalCer better than Vb7 Vb7 binds endogenous ligands (including iGb3) better than Vb8.2 Vb DOMAIN CONTRIBUTES TO GLYCOLIPID BINDING

Diverse functions of the proto-oncogene c-Myc From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15:128-137

c-Myc deficiency in vivo Conventional c-Myc deficiency is embryonic lethal *Trumpp A, Refaeli Y, Oskarsson T, Gasser S, Murphy M, Martin GR, Bishop JM. 2001. Nature 2001; 414: 768-773 *Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN, Cleveland JL. Genes & Dev. 2002; 16:2530-2543 Conditional elimination of c-Myc in bone marrow (Mx cre;c-Myc flox/flox mice) results in failure to initiate normal stem cell differentiation *Wilson A, Murphy MJ, Oskarsson T, Kaloulis K, Bettess MD, Oser GM, Pasche AC, Knabenhans C, Macdonald HR, Trumpp A. Genes Dev. 2004;18:2747-2763 Haploinsufficiency of c-Myc leads to a significant decrease in the CD8 memory T cell population *Bianchi T, Gasser S, Trumpp A, Macdonald HR. Blood. 2006

Reduced numbers of Va14i NKT cells in c-myc haploinsufficient mice Dimer positive cell number: Thymus c-Myc +/+ c-Myc +/- x10e3 57% 28% Dimer TCRb Liver Thymus Dimer positive cell number: Liver 29% 15% x10e3 Dimer TCRb

c-Myc fl/fl c-Myc fl/wt c-Myc fl/fl 4myc +/+ 4myc +/- 4myc -/- T-cell specific conditional deletion of c-myc leads to a dramatic and selective reduction in thymic Va14i NKT cells CD4cre- CD4cre+ CD4cre+ c-Myc fl/fl c-Myc fl/wt c-Myc fl/fl 4myc +/+ 4myc +/- 4myc -/- 44% 38% 6% Dimer TCRb Dimer positive T cell number: Thymus Dimer negative T cell number: Thymus Thymus x10e3 x10e5

Requirement for IL-15 in Va14i NKT cell development Dimer positive cell number: Thymus IL15+/+ IL15+/- IL15-/- x10e3 51% 30% 5% Dimer TCRb Liver Thymus Dimer positive cell number: Liver x10e3 33% 18% 5% Dimer TCRb

Synergistic reduction in Va14i NKT cells in combined c-myc and IL-15 haploinsufficiency c-Myc+/+ c-Myc+/- c-Myc+/+ c-Myc+/- IL15+/+ IL15+/+ IL15+/- IL15+/- Thymus 56% 41% 43% 7% Dimer TCRb Dimer positive cell number: Thymus x10e3

Preliminary conclusions (c-myc) C-myc plays a crucial cell-autonomous role in Va14i NKT cell development C-myc may be involved in IL-15 responsiveness of developing Va14i NKT cells Va14i NKT cells share properties with CD8 memory T cells