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Understanding the immune system A Challenge or impossible dream What do we (think we) understand • Class I pathway • • • • Proteasomal cleavage TAP transport Binding to MHC Supertype clustering and epitope selection Processing of intracellular proteins http://www.nki.nl/nkidep/h4/neefjes/neefjes.htm What do we NOT understand • Class I pathway • • • • Proteasomal cleavage TAP transport Binding to MHC Supertype clustering and epitope selection • Exceptions • • • • K epitopes Alternative translocation to ER Alternative epitope splicing Supertypes do not binding identical set of peptides • Some alleles can not be supertype clustered P9K ligands • P9 of MHC ligands is generated by the proteasome!! • Frequency of amino acids at P9 in MHC ligands should reflect preference for proteasomal cleavage •This is not the case for all amino acids P9K ligands P9K ligands • P9 of MHC ligands is generated by the proteasome!! • Frequency of amino acids at P9 should reflect preference for proteasomal cleavage •This is not the case for all amino acids • Suggests a protease other than the proteasome is likely involved in the generation of the C-termini of P9K ligands. TAP independent epitope presentation Peptides from endogenous proteins are presented at the cell surface in complex with MHC class I How to gain access to MHC-I protein -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. MHC-I TAP Proteasome -Unknown ER proteases cleave proteins in ER membrane or lumen -Furin cleave proteins in Post-ER compartment exopeptidase Hydrophobic peptides Sec6 1 -Simple diffusion across membranes by hydrophobic peptides furin -Regurgitation -Unknown entry route regurgitation ER Unknown entry route Datasets From Andreas Weinzierl, Tübingen University: 40 MHC-I epitopes eluted from the surface the human .174 cell line that doesn’t express TAP For comparison: From SYFPEITHI (http://syfpeithi.bmi-heidelberg.com/): 308 MHCI epitopes eluted from the surface of normal, TAP-containing human cells A2 Epitope Ex for name of sourceprotein (acc. to sp or nr) Startpos epitope Signalpeptide SPase cleavage (NN/HMM) SPase cleavage site Endpos .174/.4 5 -Normal entry throug TAP LLSAEPVPA CD79B_HUMAN 20 Yes 28 0 LLGPRLVLA TMP21_HUMAN 23 Yes 31 0 ALSAYDLVL Q6LCB5_HUMAN 29 yes 38 1 -Peptides within SP gain 253 entry through Sec61 186 translocon. SLWGQPAEA CO4A5_HUMAN 18 Yes 26 0 124 VLAPRVLRA RCN1_HUMAN 21 Yes 29 0 120 ALVVQVAEA HEXB_HUMAN 34 Yes LLAAWTARA A4_HUMAN 9 Yes VLLKARLVPA gb|AAY24258.1 19 Yes KMDASLGNLFA FAM3C_HUMAN 30 Yes 24 -16 LLFSHVDHVIA NAC1_HUMAN 25 Yes 35 0 FLGPWPAAS AMRP_HUMAN 22 Yes 28/32 -2/2 SLYALHVKA VKOR1_HUMAN 23 Yes 26/34 -5/3 LLLSAEPVPA CD79B_HUMAN 19 Yes 28 0 AMAPPSHLLL gb|AAC17709.1 473 Yes 21 -461 18 FLLGPRLVLA TMP21_HUMAN 22 Yes 31 0 18 LLLDVPTAAV GILT_HUMAN 26 Yes 37 2 18 LLLDVPTAAVQA GILT_HUMAN 26 Yes 37 0 15 LLDVPTAAV GILT_HUMAN 27 Yes 37 2 14 VLFRGGPRGLLAVA SSRA_HUMAN 19 Yes 20 -12 13 LLSAEPVPAA CD79B_HUMAN 20 Yes 28 -1 13 AVLALVLAPAGA NRP1_HUMAN 10 Yes 21 0 13 LAPRVLRA RCN1_HUMAN 22 Yes 29 0 4 AALLDVRSVP GDF5_MOUSE 269 Yes 27 -251 4 LLATLAAAML CLP24_HUMAN 177 Yes 25 -161 0,05 28/42 17 32/28 0 0 0 557 -Unknown ER proteases cleave proteins in ER 116 membrane or lumen 92 48 -Furin cleave proteins in 36 Post-ER compartment 28 -Simple diffusion across 23 membranes by hydrophobic peptides 24 22 -Regurgitation -Unknown entry route ? Epitopes present in the N-terminal part of the SP ER membrane Cytosol ER lumen N’ C’ Sec61 ribosome -Normal entry throug TAP A2, cont. Epitope Ex for name of sourceprotein (acc. to sp or nr) SignalPeptide (SignalP) Startos epitope SPase cleavage SPase site cleavage Endpos -Peptides within SP gain entry through Sec61 translocon. .174/.4 5 ALLSSLNDF NIF3L_HUMAN 5 No na 13 LLHPPPPPPPA RANB9_HUMAN 68 No na 13 QLQEGKNVIGL TAGL2_HUMAN 165 No na 8 SLPKKLALL L10K_HUMAN 72 No na 3 B51 Epitope Ex for name of sourceprotein (acc. to sp or nr) HGVFLPLV K0247_HUMAN MAPLALHLL Startpos epitope Signalpeptide (SignalP) SPase SPase cleavage cleavage site -site Endpos -Unknown ER proteases cleave proteins in ER membrane or lumen -Furin cleave proteins in Post-ER compartment -Simple diffusion across membranes by hydrophobic peptides .174/.45 21 Yes 39 11 92 FIG1_HUMAN 1 Yes 21 12 18 MASRWGPLIG CAB45_HUMAN 8 Yes 36 19 5 MAPRTLVL 1A02_HUMAN 4 Yes 24 13 0,5 MAPRTLIL 1C03_HUMAN 4 Yes 24 13 0,2 GSHSMRYF 1A01_HUMAN 25 Yes 24 -8 0,2 ILAPAGSLPKI ref|XP_514384.1| 328 No na 6 KAPVTKVAA PDLI1_HUMAN 240 No na 2 NPLPSKETI TYB4_HUMAN 26 No na 1 NPYDSVKKI FAT10_HUMAN 25 No na 0,2 DALDVANKIGII RL23A_HUMAN 145 No na 0,07 YPFKPPKV UB2E3_HUMAN 120 No na 0,04 -Regurgitation -Unknown entry route Protein with SP Protein without SP ? How to gain access to MHC-I protein -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. MHC-I TAP Proteasome -Unknown ER proteases cleave proteins in ER membrane or lumen -Furin cleave proteins in Post-ER compartment exopeptidase Hydrophobic peptides Sec6 1 -Simple diffusion across membranes by hydrophobic peptides furin -Regurgitation -Unknown entry route regurgitation ER Unknown entry route Presentation of alternatively spliced epitopes Presentation of Noncontiguous peptides • The conventional approach to epitope discovery is to use overlapping peptides • What if splicing of noncontiguous peptides occure? HLA-A3 Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006 HLA-A3 Antigen produced by splicing of Noncontiguous peptides NetMHC version 3.0. Prediction using Neural Networks. Allele A0301. Strong binder threshold 50.00. Weak binder threshold 500.00. -------------------------------------------------pos peptide 1-log50k(aff) affinity(nM) Bind Level -------------------------------------------------0 STPKRRHKK 0.4237 510 1 TPKRRHKKK 0.1019 16598 2 PKRRHKKKS 0.0071 46309 3 KRRHKKKSL 0.0082 45761 4 RRHKKKSLP 0.0137 43097 5 RHKKKSLPR 0.1051 16035 6 HKKKSLPRG 0.0085 45624 7 KKKSLPRGT 0.0091 45326 8 KKSLPRGTA 0.0109 44425 9 KSLPRGTAS 0.0991 17110 10 SLPRGTASS 0.0608 25887 11 LPRGTASSR 0.0732 22656 -------------------------------------------------- Identity A3 A3 A3 A3 A3 A3 A3 A3 A3 A3 A3 A3 Number of high binders 0. Number of weak binders 0. Number of peptides 12 Warren et al. Science, 313, p 1444, 2006 Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006 Antigen produced by splicing of Noncontiguous peptides Final peptide: SLPRGTSTPK A3 motif: P2:L, P9:K Warren et al. Science, 313, p 1444, 2006 HLA-A3 Antigen produced by splicing of Noncontiguous peptides NetMHC version 3.0. Prediction using Neural Networks. Allele A0301. Strong binder threshold 50.00. Weak binder threshold 500.00. -------------------------------------------------pos peptide 1-log50k(aff) affinity(nM) Bind Level -------------------------------------------------0 SLPRGSTPK 0.5029 216 WB -------------------------------------------------- Identity SLPRGSTPK Number of high binders 0. Number of weak binders 1. Number of peptides 1 Warren et al. Science, 313, p 1444, 2006 Supertypes. What are they good for? • Alleles within supertypes present the same set of peptides! Clustering of HLA alleles O Lund et al., Immunogenetics. Supertypes. What are they good for? • Alleles with in supertypes present the same set of peptides! • Is this really so? • Less that 50% of A6802 binders will bind to A0201! • Less than 33% of A0201 binders will bind to A6802! The truth about supertypes! A3 A26 A24 A2 A1 Supertypes are good for getting funding, but.. • Need to define more refined method for identifying promiscuous epitopes • Need to develop method to predict binding across all HLA alleles • Supertypes is too simple a picture What more do we (think we) understand • Why are epitopes 9 amino acids long? • Why did nature not choose 15mers? • Which class I presented peptides can bind TCR? • Or can we estimate TCR cross reactivity? Why 9mers? • Why did the immune system settle on presentation of 9mer peptides? • The proteasome generates mostly fragments of 4-7 amino acids • TAP preference peptides of 8-18 amino acids • MHC preference peptides of 8-12 amino acids • So why 9? Information processing in the immune system • How many different self peptides do we have? • How much information is present in a 9mer? • Can you discriminate self from non-self based on the information in 9-mers? Burroughs, De Boer & Kesmir, Immunogenetics, 2004, 56(5):311-20 Size of self 107 << 209 : self is a small fraction of peptide space: Overlap with other “selfs” expected to be small. Size of self - continued Discriminating self 9-mers provide enough information to discriminate: Average overlap human and pathogens <1% (Overlap between unrelated bacteria also 1%) Overlap with human depend on evolutionary distance Conclusions • Information in 9-mers sufficient to discriminate self from non-self • The chance that an immunodominant pathogenic peptide resembles self < 0.5% TCR self-tolerance • What determines if a MHC ligand can elicit an immune responds? • Similarity to self peptide • What is similar? • What amino acids determine similarity? • How broad is the cross reactivity? xx TCR recognition of MHC:peptide complex TCR data processing Experimental data Normalization & frequency conversion or Visualization (logo) G10, INFg, APL G10 Cr, APL 161 cr, PSCL T5 INFg, APL 2G4 Cr, APL T5 CR, APL B7 Cr, APL PBMC INFg, APL 3F4 Cr, PSCL, Amididated C-terminus Shannon information 5 Shannon Information 4.5 4 3.5 3 data 2.5 median 2 1.5 1 0.5 0 0 1 2 3 4 5 6 Peptide position 7 8 9 10 TCR and MHC specificity profile anti-correlates HLA-A0201 restricted HIV Gag CTL clone HLA-A0201 Predict TRC cross reactivity • TCR cross-reactivity can (partly) be characterized from a specificity profile, and amino acid conservation. • Use cross reactivity model to predict cross reactivity • Explain lacking immunogenecity of predicted CTL epitopes • Overlap between cross reactivity space and host genome Conclusions • We might thinks we understand parts of the immune system, but nothing is ever always as we would like