1. Understanding the immune system A Challenge or impossible dream

2. What do we (think we) understand Class I pathway Proteasomal cleavage TAP transport Binding to MHC Supertype clustering and epitope selection

3. http://www.nki.nl/nkidep/h4/neefjes/neefjes.htm Processing of intracellular proteins

4. 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 What do we NOT understand We don’t understand anything

5. 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

6. P9K ligands

7. 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.

8. Peptides from endogenous proteins are presented at the cell surface in complex with MHC class I TAP independent epitope presentation

9. Proteasome TAP exopeptidase MHC-I protein Hydrophobic peptides Sec6 1 Unknown entry route furin regurgitation ER How to gain access to MHC-I -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. -Unknown ER proteases cleave proteins in ER membrane or lumen -Furin cleave proteins in Post-ER compartment -Simple diffusion across membranes by hydrophobic peptides -Regurgitation -Unknown entry route

10. 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 MHC- I epitopes eluted from the surface of normal, TAP-containing human cells

11. A2 -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. -Unknown ER proteases cleave proteins in ER - membrane or lumen -Furin cleave proteins in Post-ER compartment -Simple diffusion across membranes by hydrophobic peptides -Regurgitation -Unknown entry route ? Epitope Ex for name of sourceprotein (acc. to sp or nr) Startpos epitope Signal- peptide SPase cleavage (NN/HMM) SPase cleavage site - Endpos.174/.4 5 LLSAEPVPACD79B_HUMAN20Yes280557 LLGPRLVLATMP21_HUMAN23Yes310253 ALSAYDLVLQ6LCB5_HUMAN29yes381186 SLWGQPAEACO4A5_HUMAN18Yes260124 VLAPRVLRARCN1_HUMAN21Yes290120 ALVVQVAEAHEXB_HUMAN34Yes28/420116 LLAAWTARAA4_HUMAN9Yes17092 VLLKARLVPAgb|AAY24258.119Yes32/28048 KMDASLGNLFAFAM3C_HUMAN30Yes24-1636 LLFSHVDHVIANAC1_HUMAN25Yes35028 FLGPWPAASAMRP_HUMAN22Yes28/32-2/224 SLYALHVKAVKOR1_HUMAN23Yes26/34-5/323 LLLSAEPVPACD79B_HUMAN19Yes28022 AMAPPSHLLLgb|AAC17709.1473Yes21-46118 FLLGPRLVLATMP21_HUMAN22Yes31018 LLLDVPTAAVGILT_HUMAN26Yes37218 LLLDVPTAAVQAGILT_HUMAN26Yes37015 LLDVPTAAVGILT_HUMAN27Yes37214 VLFRGGPRGLLAVASSRA_HUMAN19Yes20-1213 LLSAEPVPAACD79B_HUMAN20Yes2813 AVLALVLAPAGANRP1_HUMAN10Yes21013 LAPRVLRARCN1_HUMAN22Yes2904 AALLDVRSVPGDF5_MOUSE269Yes27-2514 LLATLAAAMLCLP24_HUMAN177Yes25-1610,05

12. ribosome Sec61 ER membrane CytosolER lumen Epitopes present in the N-terminal part of the SP N’ C’

13. A2, cont. B51 -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. -Unknown ER proteases cleave proteins in ER - membrane or lumen -Furin cleave proteins in Post-ER compartment -Simple diffusion across membranes by hydrophobic peptides -Regurgitation -Unknown entry route ? Protein with SP Protein without SP Epitope Ex for name of sourceprotein (acc. to sp or nr) Startos epitope Signal- Peptide (SignalP) SPase cleavage SPase cleavage site - Endpos.174/.4 5 ALLSSLNDFNIF3L_HUMAN5No na13 LLHPPPPPPPARANB9_HUMAN68No na13 QLQEGKNVIGLTAGL2_HUMAN165No na8 SLPKKLALLL10K_HUMAN72No na3 Epitope Ex for name of sourceprotein (acc. to sp or nr) Startpos epitope Signal- peptide (SignalP) SPase cleavage -site SPase cleavage site - Endpos.174/.45 HGVFLPLVK0247_HUMAN21Yes391192 MAPLALHLLFIG1_HUMAN1Yes211218 MASRWGPLIGCAB45_HUMAN8Yes36195 MAPRTLVL1A02_HUMAN4Yes24130,5 MAPRTLIL1C03_HUMAN4Yes24130,2 GSHSMRYF1A01_HUMAN25Yes24-80,2 ILAPAGSLPKIref|XP_514384.1|328No na6 KAPVTKVAAPDLI1_HUMAN240No na2 NPLPSKETITYB4_HUMAN26No na1 NPYDSVKKIFAT10_HUMAN25No na0,2 DALDVANKIGIIRL23A_HUMAN145No na0,07 YPFKPPKVUB2E3_HUMAN120No na0,04

14. Proteasome TAP exopeptidase MHC-I protein Hydrophobic peptides Sec6 1 Unknown entry route furin regurgitation ER How to gain access to MHC-I -Normal entry throug TAP -Peptides within SP gain entry through Sec61 translocon. -Unknown ER proteases cleave proteins in ER membrane or lumen -Furin cleave proteins in Post-ER compartment -Simple diffusion across membranes by hydrophobic peptides -Regurgitation -Unknown entry route

15. Presentation of alternatively spliced epitopes

16. Presentation of Noncontiguous peptides The conventional approach to epitope discovery is to use overlapping peptides What if splicing of noncontiguous peptides occure?

17. HLA-A3 Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006

18. HLA-A3 Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006 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 Identity -------------------------------------------------- 0 STPKRRHKK 0.4237 510 A3 1 TPKRRHKKK 0.1019 16598 A3 2 PKRRHKKKS 0.0071 46309 A3 3 KRRHKKKSL 0.0082 45761 A3 4 RRHKKKSLP 0.0137 43097 A3 5 RHKKKSLPR 0.1051 16035 A3 6 HKKKSLPRG 0.0085 45624 A3 7 KKKSLPRGT 0.0091 45326 A3 8 KKSLPRGTA 0.0109 44425 A3 9 KSLPRGTAS 0.0991 17110 A3 10 SLPRGTASS 0.0608 25887 A3 11 LPRGTASSR 0.0732 22656 A3 -------------------------------------------------- Number of high binders 0. Number of weak binders 0. Number of peptides 12

19. Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006

20. Antigen produced by splicing of Noncontiguous peptides Final peptide: SLPRGTSTPK A3 motif: P2:L, P9:K

21. HLA-A3 Antigen produced by splicing of Noncontiguous peptides Warren et al. Science, 313, p 1444, 2006 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 Identity -------------------------------------------------- 0 SLPRGSTPK 0.5029 216 WB SLPRGSTPK -------------------------------------------------- Number of high binders 0. Number of weak binders 1. Number of peptides 1

22. Supertypes. What are they good for? Alleles within supertypes present the same set of peptides!

23. Clustering of HLA alleles O Lund et al., Immunogenetics. 2004 55:797-810

24. 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!

25. The truth about supertypes! A2 A24 A26 A3 A1

26. 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

27. 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?

28. 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?

29. Information processing in the immune system How many different self peptides do we have? How much information is present in a 9- mer? Can you discriminate self from non-self based on the information in 9-mers? Burroughs, De Boer & Kesmir, Immunogenetics, 2004, 56(5):311-20

30. Size of self 10 7 << 20 9 : self is a small fraction of peptide space: Overlap with other “selfs” expected to be small.

31. Size of self - continued

32. Discriminating self 9-mers provide enough information to discriminate: Average overlap human and pathogens <1% (Overlap between unrelated bacteria also 1%)

33. Overlap with human depend on evolutionary distance

34. Conclusions Information in 9-mers sufficient to discriminate self from non-self The chance that an immunodominant pathogenic peptide resembles self < 0.5%

35. 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? x x

36. TCR recognition of MHC:peptide complex

37. TCR data processing Experimental data Normalization & frequency conversion Visualization (logo) or

38. G10, INFg, APL T5 INFg, APL G10 Cr, APL T5 CR, APL B7 Cr, APL 2G4 Cr, APL 161 cr, PSCL PBMC INFg, APL 3F4 Cr, PSCL, Amididated C-terminus

39. Shannon information

40. TCR and MHC specificity profile anti-correlates

41. HLA-A0201 HLA-A0201 restricted HIV Gag CTL clone

42. 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

43. Conclusions We might thinks we understand parts of the immune system, but nothing is ever always as we would like