Profiling HLA motifs by large scale peptide sequencing 2009 Agilent Innovators Tour David K. Crockett ARUP Laboratories February 10, 2009
HLA Background The human leukocyte antigen system (HLA) is the name of the human major histocompatibility complex (MHC). MHC markers are present on the surface of almost all cells. HLA antigens are essential in immune HLA antigens are essential in immune function to determine self vs non-self.
HLA Class I On the cell surface, MHC class I proteins are bound to protein fragments (peptides) that have been exported from within the cell. MHC class I proteins display these cytosolic peptides to the immune system. (In contrast MHC class II molecules act to present (In contrast, MHC class II molecules act to present exogenous peptide fragments.)
HLA Function HLA Class I molecules bind protein fragments exported from within the cell. Class I proteins display these peptides to the immune system. If the immune system sees foreign peptides, it destroys the infected cell.
HLA Motifs Binding specificity conferred by amino acid frequency and position in the groove. MHC class I molecules bind peptides between 8-10 residues. Peptides presented by MHC class I molecules are anchored at two positions: P2 position and PΩ position.
HLA Allele l Diversity it HLA genes have many different normal variations, allowing each person's immune system to react to a wide range of foreign invaders. Hundreds d of versions (alleles) l of HLA A, B, and C are known, each given a particular number (ex. HLA-B27). MHC class I Locus # Locus # HLA A 649 HLA E 9 HLA B 1029 HLA F 21 HLA C 350 HLA G 31 (Major Antigens) (Minor Antigens) MHC class II HLA -A1 -B1 -B2 to B9* Potential Total Locus # # # Combinations Combinations DM 4 7 28 28 DO 12 9 108 3,024 DQ 34 91 3,094 9,356256 DP 26 128 3,328 3.11 x10 10 DR 3 640 84 161,280 5.02 x10 15 *DRB2 DRB9 have variable presence in humans IMGT-HLA database - May 2008
Objective HLA proteins play key roles in: Autoimmunity Graft rejection Infectious disease Cancer Classification of HLA allele motif binding specificity. Discovery of de novo ligands of disease state.
Experimental Rationale Analysis of natural MHC ligands by direct sequencing of eluted peptides (Rammensee 1991; Hunt & Engelhard 1992) Based on limited number of eluted peptides (10-15 peptides only) MHC motifs amino acid binding gives allele specificities at anchor positions Example: HLA-A*0201: xlxxxxxxv HLA-B*0702: xvxxxxxxr
Overview Analysis of HLA-B 3501 to 3508 subtypes by LC-MS/MS. Generate peptide catalogue for each subtype. Search for patterns of amino acid frequency/position. Summarize motif patterns by HLA B subtype.
Experimental Design Generation of cells expressing HLA-B35 alleles K562 (K) cell line - BEFORE MHC class I and II null Antigen-processing capable ) K562 (K) cell line - AFTER Retrovirus transfected Single HLA allele expressed Erp57 tapasin calreticulin K562 (K) K-3501 K-3502 K-3503 K-3504 K-3506 K-3508 TAP anti HLA-B
Experimental Design Step 1 Class I null cell line HLA B35 transfection cell culture cell lysis MHC I molecules Step 2 Y Y YY Y Y Y Y Y Y Y Y Y Y MHC I molecules purification elution 3 kda filtration HLA peptides Step 3 HLA peptides HPLC fractionation LC-MS/MS analysis peptide fragmentation peptide ID matching
Data Acquisition iti Parallel experiments for each HLA B35 allele (3501-08). Each experiment yielded some 40 HPLC fractions. All fractions analyzed by 6510 ChipCube QTOF. Approx. 1000 injections over 2months months.
R lt Results YTDNLVRVW VVYPWTQRF TTIPTKQTQTF TTLEHQKTF YTDNLVRVW LSFPTTKTY KPRQSNLPGNFT VVYPWTQRF VVYPWTQRF VVYPWTQRF TSQDVLHSW TSQDVLHSW TTLEHQKTF YTDNLVRVW TTIPTKQTQTF LTSLQTLKLSN RVIDVGSEW LEFRALLF ATFPDPNVKY TTLEHQKTF EEHVFTLY IAGGNPYGLF ATIPNARQF SPSSSMAQSKSQ Q ITSQDVLHSW LKTIPDLTF DPPPPESQ VVYPWTQRF VTSLAF ATFPDPNVKY TSVPDHVVW FYAPELLYYANK HTFQNDIHVY ITSVDKSYGGY LTSQGVDGVQDV VVYSFYGYVN KTIGGGDDSFNTF EPDSAPATSEG LTRGSSLAD Q Q TAGVSEEQHSH FQLVVLYLF EEIYLF TPYPDERSFQ SPGAYDRSF VTSLAF PTYDLLFLGTA LIFGCT ATAVPPQGSPT LSTPVTFPDG ITSVDKSYGGY VVYPWTQRF RVFPDKGYSF TALPLLKQF VVYPWTQRF LTRGSSLAD VSQLIQREF KTIGGGDDSFNTF YTTLRFPEMNIPRTG FALGF SPGAYDRSF EGPAISDGEEG EEIYLF FENHFQTRQ LKLSVPATF TALGGGKLEAT ATFPDPNVKY TEAMLVSVER KILAEGGGAKF KTIGGGDDSFNTF VTYVPVTTF TIQSTLGAEGKI VTYVPVTTF PVIPPPNQA VVYPWTQRF PDPPKTDL RVFPEKGYSF KFSLPPPLQLIPEVQKPL VLPPLRDV LSYQPNRGTK LYRFLP VVYCNTSKKW QGQLVAEFSSVFLRLEY VENVKSQTY MEPAPKPA KTIGGGDDSFNTF VGIATL VSGSTWCIST VVYPWTQRF ASLPLRVSF PTYFISSTVGK LSFPTTKTY LSYQPNRGTK GSRPCCRVF HTFQNDIHVY MEPAPKPA VVYPWTQRF
Results Over 1500 peptides total were found. 100 s of peptides were identified for each HLA-B35 experiment (K3501-K3508). Peptide data sets contain 20x to 50x the number of peptides compared to earlier studies. Motif patterns easier to find and confirm.
Peptide validation Known motif control included in each QTOF run. Spectrum Mill peptide score > 12 and ppm error < 5. In vitro peptide binding assays. Binding prediction algorithms: ann, smm, arb. (www.immuneepitome.org) Comparison to SYFPEITHI database. (www.syfpeithi.de)
Results HLA-B*3501: 372 unique peptides identified Percentage 100 90 80 70 60 50 40 30 20 10 0 K3501 Peptide Distribution 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Length K-3501. Amino acid in 1st Position K-3501. Amino acid in 3rd Position K-3501. Amino acid in 5th Position K-3501. Amino acid in 7th Position 100 100 K-3501. Amino acid in 2nd Position K-3501. Amino acid in 4th 100 K-3501. Amino acid in6th K-3501. Amino acid in LAST Position 100 90 90 90 80 100 80 100 90 100 80 100 70 90 70 90 80 90 70 90 70 60 80 60 80 80 60 80 60 50 70 64 50 70 70 50 70 50 40 60 40 60 60 40 60 40 50 30 50 30 50 50 30 50 30 20 16 40 20 40 16 40 2 6 9 9 5 4 8 6 2 4 3 4 9 10 10 20 40 8 10 30 5 1 2 4 6 6 9 7 7 6 9 3 1 0 2 4 3 6 12 20 10 30 3 2 4 30 10 9 2 1 10 5 5 5 4 5 5 4 5 2 20 1 0 12 0 20 10 11 7 P A V N L F E T S G10 Y R D M 2 I 3 W 1 H 2 Q 2 K 0 C 1 0 3 0 1 2 0 1 P 2 A 1 V N L F E T S G Y R 6 7 D M 4 I 6W H Q K C 0 2 3 7 12 1 1 3 4 6 5 9 10 5 10 14 4 7 7 30 10 5 4 2 4 10 20 0 0 10 1 3 1 5 1 7 9 8 11 14 1 2 3 1 2 5 5 3 22 P A V 1N L F E T S G Y R D M I W2 H 6 Q 8 K3 C 6 7 8 20 15 0 0 10 3 2 4 1 1 P2 A1 V 5 N 0 L F E T S G Y R D M10 I W H Q K C 0 3 2 1 1 0 0 1 1 1 1 2 0 0 0 1 0 AMINO ACID 0 AMINO ACID 0 AMINO ACID 0 AMINO ACID 0 P A V N L F E T S G Y R D M I W H Q K C P A V N L F E T S G Y R D M I W H Q K C P A V N L F E T S G Y R D M I W H Q K C P A V N L F E T S G Y R D M I W H Q K C AMINO ACID AMINO ACID AMINO ACID AMINO ACID
Length distribution ib ti Length distribution of peptides eluted from HLA-B35 molecules. J Immunol. 2008 Oct 1;181(7):4874-82.
Sequence logos Logos displaying the peptide binding motif of HLA-B35 molecules. J Immunol. 2008 Oct 1;181(7):4874-82.
HLA-B*35 Allele l Specificity it PyMol model of peptide binding groove of the HLA-B*3501 molecule. J Immunol. 2008 Oct 1;181(7):4874-82.
Disease Example HLA-B35 and susceptibility to AIDS progression. HLA-B*3501-like motif xpxxxxxy (slower progression) HLA-B*3502-like motif xpdxxxxl (rapid progression) Why?
Application Type 1 diabetes risk for human leukocyte antigen (HLA)-DR3 haplotypes depends on genotypic context: Association of DPB1 and HLA class I loci among DR3- and DR4-matched Italian patients and controls. Hum Immunol. 2008 Apr-May;69(4-5):291-300. Expression of HLA molecules on peripheral blood lymphocytes: a useful monitoring parameter in cardiac transplantation. Transplant Proc. 2007 Sep;39(7):2362-4. HLA-associated susceptibility to childhood B-cell precursor ALL: definition and role of HLA-DPB1 supertypes. Br J Cancer. 2008 Mar 25;98(6):1125-31. HLA-DRB1*04 and DRB1*10 are associated with resistance and susceptibility, respectively, in Brazilian and Vietnamese leprosy patients. Genes Immun. 2007 Jun;8(4):320-4. Cross-clade immune responses to Gag p24 in patients infected with different HIV- 1 subtypes and correlation with HLA class I and II alleles. Vaccine. 2008 Apr 22. [Epub ahead of print]
Application Discovery of autoimmune disease cell surface epitopes. Monitoring early markers of transplant rejection. Classification of HLA supertypes across ethnic groups. Survey disease resistance/susceptibility by ethnicity.
Conclusions The ChipCube QTOF is a superior technology for HLA peptide motif scanning and discovery. We have developed a sensitive and robust methodology for interrogating endogenous cell marker peptides. This technique has broad applications in research and patient treatment across many disease areas.
Publications Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs. Journal of Immunology. 2008 Oct 1;181(7):4874-82. Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction. Immunogenetics (in press). Three more manuscripts in preparation.
Future Directions Class II MHC surface peptide presentation. Viral cytotoxic T-lymphocyte (CTL) epitope discovery based on HLA motifs. Biomarker discovery in kidney transplant rejection.
Ak Acknowledgements ld Peter Jensen, MD Julio Delgado, MD Hernando Escobar, MS ARUP Institute for Clinical and Experimental Pathology