1. Class I pathway Prediction of proteasomal cleavage and TAP binidng Morten Nielsen, CBS, BioCentrum, DTU

2. Outline MHC class I epitopes –Antigen processing Proteasome –Specificity and Polymorphism –Prediction methods TAP –Binding motif Evolution Immune escape

3. Peptide generation in the class I pathway

4. Proteasomal cleavage ~20% of all peptide bonds are cleaved Average peptide length 8-9 amino acids Not all peptide bonds are equally likely cleaved Cleavage more likely after hydrophobic than after hydrophilic amino acids

5. Proteasome specificity Low polymorphism –Constitutive & Immuno- proteasome Evolutionary conserved Stochastic and low specificity –Only 70-80% of the cleavage sites are reproduced in repeated experiments

6. Proteasome evolution (  1 unit) Constitutive Immuno Human (Hs) - Human Drosophila (Dm) - Fly Bos Taurus (Bota) - Cow Oncorhynchus mykiss (Om) - Fish …

7. Immuno- and Constitutive proteasome specificity... LVGPTPVNIIGRNMLTQL.. P1P1’ Immuno Constitutive

8. NetChop –Neural network based method PaProc –Weight matrix based method FragPredict –Based on a statistical analysis of cleavage- determining amino acid motifs present around the scissile bond i.e. also weight matrix like Predicting proteasomal cleavage

9. NetChop 3.0 Cterm (MHC ligands) LDFVRFMGVMSSCNNPA LVQEKYLEYRQVPDSDP RTQDENPVVHFFKNIVT TPLIPLTIFVGENTGVP LVPVEPDKVEEATEGEN YMLDLQPETTDLYCYEQ PVESMETTMRSPVFTDN ISEYRHYCYSLYGTTLE AAVDAGMAMAGQSPVLR QPKKVKRRLFETRELTD LGEFYNQMMVKAGLNDD GYGGRASDYKSAHKGLK KTKDIVNGLRSVQTFAD LVGFLLLKYRAREPVTK SVDPKNYPKKKMEKRFV SSSSTPLLYPSLALPAP FLYGALLLAEGFYTTGA NetChop-3.0 C-term –Trained on class I epitopes –Most epitopes are generated by the immuno proteasome –Predicts the immuno proteasome specificity

10. NetChop20S-3.0 In vitro digest data from the constitutive proteasome Toes et al., J.exp.med. 2001

11. Prediction performance TP FP AP AN A roc =0.5 A roc =0.8 1 - spec Sens

12. Predicting proteasomal cleavage NetChop20S-3.0 NetChop-3.0 Relative poor predictive performance –For MHC prediction CC~0.92 and AUC~0.95

13. Proteasome specificity NetChop is the best available cleavage method –www.cbs.dtu.dk/services/NetChop-3.0

14. What does TAP do?

15. TAP affinity prediction Transporter Associated with antigen Processing Binds peptides 9-18 long Binding determined mostly by N1-3 and C terminal amino acids

16. TAP binding motif matrix Peters et el., 2003. JI, 171: 1741. A low matrix entry corresponds to an amino acid well suited for TAP binding

17. TAP affinity prediction

18. Predicting TAP affinity 9 meric peptides>9 meric Peters et el., 2003. JI, 171: 1741. ILRGTSFVYV -0.11 + 0.09 - 0.42 - 0.3 = -0.74

19. Proteasome, TAP and MHC co-evolution Antigen processing and presentation is highly ineffective Only 1 in 200 peptides will bind a given MHC complex If proteasome and TAP do not effectively produce MHC restricted peptides, antigen processing would be a severe bottleneck for antigen recognition

20. Co-evolution of Proteasome, TAP and MHC CP-P1: Constitutive proteasome specificity at P1 position TAP-9: TAP motif at P9 position MHC-9: Average MHC motif at P9

21. Co-evolution of Proteasome, TAP and MHC IP-P1: Immuno proteasome specificity at P1 position CP-P1: Constitutive proteasome specificity at P1 position TAP-9: TAP motif at P9 position MHC-9: Average MHC motif at P9

22. Co-evolution (continued) Kesmir et al. Immunogenetics, 2003, 55:437

23. More evolution Constitutive proteasome!!!

24. What is going on at the N terminal?

25. S T R K F L D G N E M T L... Epitope identification TAP precursor A2 Epitope FLDGNEMTL FLDGNEMTL 2.0100 KFLDGNEMTL -2.5300 RKFLDGNEMTL -3.7400 TRKFLDGNEMTL -2.4400 0.0101 0.6483 0.9955 0.9984 0.4299 0.2261 0.0103 0.0265 0.0099 0.0099 0.9590 0.4670 0.9989 Proteasomal cleavage

26. N terminal trimming >50% need 2-3 amino acids N terminal trimming

27. TAP and proteasome independent presentation CTL epitopes are presented at the cell surface on TAP deficient cell lines Some CTL epitopes have very poor TAP binding affinity Dominate CTL epitopes can have very poor C terminal cleavage signal Many CTL epitope have strong internal cleavage sites Other important players in the class I pathway –Signal peptides –Sec61 –Diffusion –Proteases Mette will tell you more

28. Immune escape Pathogens evolve under strong selection pressure to avoid CTL recognition Generate point mutations or insertions/deletions to disturb –Peptide binding to MHC –CTL recognition Only involve the antigentic peptide region –Antigen processing Can involve peptide flanking region

29. Immune escape via antigen processing 189 S N 0.38 SSWDFITV 190 S S 0.59 SSWDFITV 191 W S 0.92 SSWDFITV 192 D N 0.23 SSWDFITV 193 F S 0.87 SSWDFITV 194 I S 0.84 SSWDFITV 195 T N 0.27 SSWDFITV 196 V S 0.96 SSWDFITV 197 N S 0.82 SSWDFITV 189 S N 0.38 SSWDFITV 190 S S 0.59 SSWDFITV 191 W S 0.92 SSWDFITV 192 D N 0.23 SSWDFITV 193 F S 0.87 SSWDFITV 194 I S 0.83 SSWDFITV 195 T N 0.13 SSWDFITV 196 V S 0.92 SSWDFITV 197 D S 0.97 SSWDFITV Moloney murine leukemia virus (MuLV) epitope SSWDFITV Processed right and recognized by CTL Processed as SSWDFITVD and has wrong C terminal for MHC binding, not recognized by CTL

30. Immune escape Proteasome-mediated digestion analysis of a synthetic 26- mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules.. Beekmanet al., JI 2000

31. Summary The most important players (MHC, TAP and proteasome) in the MHC class I pathway have co evolved to a share a common C terminal pathway specificity Proteasomal cleavage prediction tools exist –NetChop3.0 and NetChop20S-3.0 are among the best TAP binding motif characterized in a weight matrix –Binding mostly determined by the N1-3 and C terminal amino acids Proteasome produces and TAP transports precursor T cell epitopes of length 9-13 amino acids Epitope trimming in the ER by several amino peptidases (ERAP) We still do not understand every thing –Many more important players are involved in the class I path way