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V a 14iNKT

An Introduction to NKT cells. CD1d. Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides Artificial ligand: a-galactosylceramide ( a -GalCer). mu: V a 14-J a 18 hu: V a 24-J a 18. mu: V b 8.2/V b 7 hu: V b 11. CDR3 b diverse. ( CD4).

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V a 14iNKT

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  1. 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 ( CD4) Va14iNKT CD44high NK1.1 Autoimmune diseases Infectious diseases Tumors CD69high Ly49 ( DX5) IFN-g IL-4

  2. 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

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

  4. 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

  5. Specific Identification of Va14i NKT cellsTetramers 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

  6. 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 53 ± 4 58 ± 2 80 ± 7 84 ± 4 Counts Counts Vb8.2 Vb8.2 Counts 16 ± 2 12 ± 2 6 ± 5 4 ± 1 Counts Vb7 Vb7

  7. 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)

  8. 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+/-

  9. 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+/-

  10. 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

  11. 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

  12. 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

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

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

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

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

  17. 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

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

  19. 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

  20. 57% 28% 29% 15% Reduced numbers of Va14i NKT cells in c-myc haploinsufficient mice Dimer positive cell number: Thymus c-Myc +/+c-Myc +/- x10e3 Dimer TCRb Liver Thymus Dimer positive cell number: Liver x10e3 Dimer TCRb

  21. 44% 6% 38% 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 -/- Dimer TCRb Dimer positive T cell number: Thymus Dimer negative T cell number: Thymus x10e3 x10e5 Thymus

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

  23. 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

  24. 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

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