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The Importance of “Seeing” Self Thymic selection Lymphocyte survival: A red queen hypothesis Enhancing foreign antigen recognition MCC: moth cyt. C 88-103 ADLLALYKQATK 99 Ehrich et al. (1993) J Exp Med 178: 713. TCR CDR 3 Loops Exhibit High Conformational Mobility and Heterogeneity Willcox, et al. ‘99, Garcia et al. ‘98 Comparison of Peptide-MHC Epitope Contribution to TCR Stability and Association Stability Association TCR may dock on the MHC first, and the CDR3s can adjust to different peptide. A proof of principal: Altered peptide ligand and differential signaling Thymic selection Lymphocyte survival: A red queen hypothesis Necessities of life. . Running to stay in place. The presence of the transcription factor LKLF is essential for the survival of naïve T cells, the cells that respond to foreign antigens. Initiation of TCR Signaling Multivalent engagement of peptide/MHC ligand The need for membrane reorganization and the formation of specialized junction (SMAC or immunological synapse) Ligand KD kon koff t1/2 (s) (mM) (M-1s-1) (s-1) -----------------------------------------------------------------------MCC/2B4 40 900 0.06 12.1 Ld/2C 3.3 8300 0.027 26 IgM 0.011 110,000 0.0012 580 IgG 0.001 270.000 0.00025 2800 CD80/CD28Ig 2.5 >600,000 >1.6 <1.6 Davis, SJ and van der Merwe, A. (1996) Imm Today 4:177. Talin-green, PKC q-red Monk et al. Nature 395:82 ‘98 Bilayers as artificial APCs • Defined, uniform surface for imaging • Mobile lipids and lipid-linked glycoproteins • Control molecular composition and MHC + p protein density • Molecules can be fluorescently labeled prior to incorporation • Extent of membrane interaction determined glass ICAM-1 can be PC 2B4 T cells with IE/MCC IE-green, ICAM-1-red Grakoui et al. Science 285: 221 Ligand Relative Classification KA kon t1/2 (s) Density Total number activity (mM-1) (M-1s-1 ) (molec./µm2) of molecules -----------------------------------------------------------------------MCC 100 T102S 1 Agonist 16.6 900 12.1 352 770 Weak agonist 4.2 1,500 1.92 193 310 T102G <0.001 Antagonist 0.66 3,400 0.14 53 50 K99A 0 Null ND ND ND <10 <10 MHC-peptide density in cSMAC MHC-peptide density is determined by off-rate when MHCpeptide dose is fixed 250 250 250 200 200 200 150 150 150 100 100 100 50 50 50 0 0 50 100 KA (mM-1 ) 0 0 10000 20000 kon (M-1 s-1 ) 0 0 5 10 15 Half-life (s) Clustering of CD3 z Krummel et al. Science 289: 1349 ‘00 Wulfing et al. (2002) Nat Immunol 3:42-47. “Null” peptide MCC 99A can form dense clusters in the presence of trace amounts of MCC/ I-Ek complexes Inhibitory peptide (MCC 99E)/IE complexes do not Synapse Formation • Synapse formation appears to be driven by costimulation and involves the active transport of membrane/cytoskelatal associated molecules/rafts to the interface (Wuelfing et al. 1998, Viola et al. 1999, Wuelfing, Irvine et al., unpublished). It is sensitive to PI3Kinase and myosin motor inhibition. Costimulation dependant bead movement. Wuelfing et al.Science 1998 Stage 1 2 3a 3b 4 5 Morphology T Synapse Kummel and Davis, Current Opinion of Immunology ‘02 Integrin TCR/CD3 CD4 APC TCR engagement TCR micro clustering CD4 co-cluster Centralization CD4, CD45 excluded Internalization of TCR Polarizes T cell secretory apparatus Sustained TCR signaling gd T Cells are important but their functions are unclear gd k/o mice fare worse or differently in several infection models. Function-the importance of location abIEL vs. gd IEL Antigen recognition Targets Innate vs. Adaptive gdIELs may be able to deal with a broad range of pathological situation quickly, despite the diversity of TCRs gdIELs are constitutively transcribing cytolytic, NK activating and inhibitory genes. Activation of cytotoxic function, however, is likely to occur through the overcoming of inhibitory receptor signals. Thus, gd IEL are ready to act, with no requirement for new gene synthesis. gdIELs could be "fired" either through general activatory surface molecules, or through the T cell receptor, with potentially different outcomes in each case. Fahrer AM et al. P.N.A.S. 98:10261 (2001). CDR3 Length Distribution of Immune Receptor Chains Rock et al. J. Exp. Med. 179:323 ‘94 gd TCR recognizes antigens directly, with no antigen processing and presentation requirement. ImplicationCells and pathogens can be recognized directly, responses can be initiated. ChallengeTo identify such ligands and to find a normal population of gd T cells recognizing them. T10/T22 has been found twice! G8- Balb/c nude immunized with B10.BR spleen cells KN6- Double negative thymocyte from C57B/6 . A population of gdT cells recognize T22 QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. T22 HLA-A2 QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. T22 tetramer 1:100-1:500 peripheral gd T cells recognize T22 Anti-gd TCR Crowley et al. Science 287: 314, 00 T cell Status of immune system Frequency1 T10/T22- specific gd T cells ~ 1/250 MHC/peptide specific ab T cells (not primed) ~ 1/1.000,000 MHC/peptide specific ab T cells (Immunized; effector phase) ~1/2-1/100 Comparison of ligand affinities of ab and gdTCR TCR Type Ligand Kd (mM) kon (M-1s-1) koff (s-1) t1/2 G8 gd T10 /T22 0.1 65,000 0.008 88 2C ab p2Ca/Ld 3.3 8,300 0.027 26 2B4 ab MCC/IEk 40.0 1,600 0.060 12 T102S/IEk 240.0 1,500 0.360 2 > 240.0 N.D. LBK5 gd IEk N.D. (N.D. = not determined) T10/T22 on cell surface are likely to have a shorter half-life than classical MHC molecules Tm(oC) DG(kcal/mole) T10/mb2m 43 1.5 peptide/MHC 65-72 >5 FcRn (pH 6) (pH 8) 62 51 Immunoregulatory role for T10/T22 specific gd T cells? lymphocyte gd Tcell activation T22 T10 gd TCR cytotoxicity? cytokine release? Others? Crowley et al. Science 287:314 ‘00 Expression of multiple class I MHC subclasses in distinct regions of the mature CNS. Huh et al. Science 290:2155, 00