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V 14iNKT mu: V 14-J 18 hu: V 24-J 18 mu: V 8.2/V 7 hu: V 11 NK1.1 CD1d Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides.

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Presentation on theme: "V 14iNKT mu: V 14-J 18 hu: V 24-J 18 mu: V 8.2/V 7 hu: V 11 NK1.1 CD1d Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides."— Presentation transcript:

1 V 14iNKT mu: V 14-J 18 hu: V 24-J 18 mu: V 8.2/V 7 hu: V 11 NK1.1 CD1d Endogenous ligand: Isoglobotrihexosylceramide (iGb3) Foreign ligand: Microbial a-glycuranosylceramides Artificial ligand: a-galactosylceramide ( -GalCer) IFN- IL-4 Autoimmune diseases Infectious diseases Tumors An Introduction to NKT cells CD44 high CD69 high Ly49 ( DX5) ( CD4) CDR3 diverse

2 CD1 molecules present glycolipids *Moody DB, Zajonc DM, Wilson IA. Nat Rev Immunol. 2005;5: 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

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

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 V 14iNKT V 14-J 18V 8.2/V 7 NK1.1 GalCer muCD1d Biotin Streptavidin- fluorochrome V 14iNKT V 14-J 18V 8.2/V 7 NK1.1 GalCer muCD1d/ huCD1d Specific Identification of Va14i NKT cells Tetramers Dimers

6 53 ± 458 ± 2 16 ± 212 ± 2 Counts 28 Thymus mouse dimers 17 Liver TCR ± 784 ± 4 6 ± 54 ± 1 V 8.2 V 7 Counts Thymus human dimers Liver TCR- V 8.2 V 7 Human CD1d: GalCer dimers bind preferentially to V 14i NKT cells expressing V

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 muCD1d -/- % V 8.2 % V 7 non-tg muCD1d +/- pLck- huCD1d-tg muCD1d -/- CD11c- huCD1d-tg muCD1d -/- DP thymocytes but not DC expressing huCD1d positively select V V 14i NKT cells

9 muCD1d +/- non-tg muCD1d +/- pLck- huCD1d-tg muCD1d +/- CD11c- huCD1d-tg muCD1d +/- % V 8.2 % V 7 Both DP thymocytes and DC expressing huCD1d negatively select V V 14i NKT cells

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 -Galactosylceramide ( GalCer) serves as a model CD1d antigen GalCer 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 Role of Vb domain in selection of Va14i NKT cells by CD1d-binding endogenous glycolipids

12 mouse dimers ThymusLiver % V % V 7 + Gate Higher avidity binding of mouse CD1d: GalCer dimers by V 14i NKT cells expressing V TCR-

13 T V 14i NKT TCR- mouse dimer V 8.2 mature V 14i NKT 50 ± 1 V 7 14 ± 3 mature V 14i NKT DP CD4 CD8 V 8.2 (ic) 9.4 ± 0.5 DP 2.7 ± 0.3 DP NK1.1 % V 8.2 or V 7 (ic) % V 8.2 or V 7 DP thymocytes mature V 14i NKT cells V 8.2 V 7 V b/b J 18 +/- V b/b J 18 +/+ V a/b J 18 +/+ V b/b J 18 +/+ V 8.2 (ic) V 7 (ic) Frequency of thymic V 7 + and V V 14i NKT cells reflects V rearrangement frequency in CD4 + CD8 + precursors

14 CD1d CD4 CD8 DP thymocytes DP MFI, 122 ± 15 MFI, 59 ± 7 CD1d +/- CD1d +/+ * % V 7 % V 8.2 V b/b V 8.2 V 7 V 8.2 (ic) V 7 (ic) % V 7 * CD1d +/+ DP thymocytes CD1d +/- CD1d +/+ mature V 14i NKT cells CD1d +/- V 7 (ic) V 7 V a/a Preferential Selection of Vb7 NKT Cells at Limiting CD1d:endogenous ligand Concentration in vivo

15 TCR- mouse dimer mouse tetramer TCR- NK1.1 non-tg huV 24-tg NKT non-NKT NKT non-NKT NKT non- NKT 88 ± 5 non- NKT 21 ± 10 NKT non- NKT 81 ± 7 NKT non- NKT 7 ± 3 % V 8.2 or V 7 tetramer + non-tg dimer + DP DN DP huV 24-tg non-tg huV 24-tg V 8.2 V 7 V 8.2 (ic) V 7 (ic) NKT Preferential Selection of Vb7 NKT Cells in Va24 Transgenic Mice expressing a low avidity invariant TCRa chain

16 Vb7+ NKT cells are preferentially selected by endogenous ligands or exogenous self-ligand iGb3 in thymic culture 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 From: Murphy MJ, Wilson A, Trumpp A. Trends Cell Biol 2005;15: Diverse functions of the proto-oncogene c-Myc

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 Nature 2001; 414: *Baudino TA, McKay C, Pendeville-Samain H, Nilsson JA, Maclean KH, White EL, Davis AC, Ihle JN, Cleveland JL. Genes & Dev. 2002; 16: 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: 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 c-Myc +/+c-Myc +/- Liver Thymus 57%28% 15%29% x10e3 Dimer positive cell number: Thymus Dimer positive cell number: Liver x10e3 TCRb Dimer TCRb Dimer Reduced numbers of Va14i NKT cells in c-myc haploinsufficient mice

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

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

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

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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