Dependency of Ly49 Recognition on Anchor Residues of Peptide Bound to MHC-I by Elsa Aurora Marquez A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in IMMUNOLOGY Department of Medical Microbiology and Immunology University of Alberta © Elsa Aurora Marquez, 2015 ABSTRACT In rodents, Ly49s are prominent natural killer (NK) cell receptors that interact with peptide-bound class I major histocompatibility complex (MHC-I). This association generally induces inhibitory signals when MHC-I presents self-peptides, thus blocking NK cell activation against healthy cells. Inhibitory Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. NK cell inhibitory Ly49s associate with MHC-I at a region under the MHC-I peptide binding groove, without direct contact with the peptide bound; however, specific details of peptide dependent Ly49 recognition have not been fully explored. The mouse NK cell receptor Ly49C recognize its MHC-I ligand H-2Kb in a peptide selective and specific fashion. However, the molecular basis for Ly49C peptide specific recognition of H-2Kb has not been fully elucidated. Utilizing functional assays, we demonstrated that both the auxiliary anchor residue at position 3 (P3) and the adjacent residue at position 2 (P2), that also anchors into the peptide binding groove, influence Ly49C peptide selectivity in recognition of H-2Kb. In particular, we found that the Ly49C and H-2Kb interaction is supported by non-polar aliphatic residues at P3 and bulky non-polar aliphatic residues at P2. Previous studies in our laboratory also showed the importance of peptide anchor residues in determining recognition between a rat Ly49 receptor, Ly49i2, and its cognate ligand RT1-A1c. This suggests that peptide anchor residues bound to MHC-I may be modulators of Ly49 peptide specificity in rodent models of Ly49 and MHC-I interaction. ii In addition, we used functional assays to demonstrate peptide-dependent antagonism of mouse NK cell inhibition using as a model Ly49C and H-2Kb association. Partial NK cell antagonism, in other words partial depression of inhibition, was observed during the co-presentation of peptide-MHC-I (pMHC-I) complexes that support Ly49C- H-2Kb interaction and pMHC-I complexes that can bind Ly49C at an intermediate level. In contrast, a weaker antagonistic phenotype was observed by co-expression of pMHC-I complexes that confer H-2Kb and Ly49C interaction together with pMHC-I complexes that bind Ly49C at low to null levels. Collectively, our studies show that peptides bound to H-2Kb are fundamental in the integration of NK cell signaling events that determine NK activity. Rodent Ly49 receptors can play an important role in NK cell function for the clearance of tumorigenic cells as well as virally infected cells. Given that the peptide repertoire within a cell is subjected to modifications during cell stress, such as tumorigenesis or viral infection, it is of importance to understand how Ly49 directed NK cell function is affected by the nature of the peptides bound to MHC-I. iii ACKNOWLEDGEMENTS Firstly, I want to thank my supervisor Dr. Kevin Kane for his support through my years in graduate school and to my supervisory committee, Drs. Debby Burshtyn and John Elliott, for their feedback. The production of this thesis would not have been possible without a great team in both the Kane and Ostergaard labs, and I will always be grateful for their support inside and outside the lab. I also want to thank friends in the department that are not only kind but are also talented scientists that I hold in high regard. Lastly and more importantly I want to thank my family for their continuous encouragement, love and patience. iv TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION A. The Major Histocompatibility Complex. ................................................................. 1 B. Classical class I MHC. ............................................................................................. 2 C. The immunopeptidome ........................................................................................... 4 D. Role of natural killer cells ...................................................................................... 7 E. Natural killer cells in health and disease ................................................................ 9 F. Natural killer cell receptors ..................................................................................... 11 The Leukocyte Receptor Complex ....................................................................... 12 The Natural Killer Complex ................................................................................ 14 G. Ly49 receptor expression and role in NK cell biology ......................................... 16 H. A structural view of Ly49 and MHC-I. ................................................................ 17 I. MHC-I peptide dependent NK cell function. .......................................................... 19 J. Background and Rationale ...................................................................................... 21 II. MATERIALS AND METHODS Cell lines and peptides ........................................................................................ 33 Monoclonal antibodies and cell staining. ........................................................... 33 RMA/S stabilization assay and peptide titration assays ..................................... 36 Production of recombinant mouse β2m for cell culture ..................................... 36 Protein analysis .................................................................................................. 37 Structural analysis .............................................................................................. 38 Generation of RNK-16 cells expressing chimeric Ly49W/C. ............................. 38 Cytotoxicity assays ............................................................................................. 39 Statistical analysis .............................................................................................. 40 Prediction of peptide binding affinities to H-2Kb using NetMHCpan. ............... 40 v Sequential peptide loading. ................................................................................. 40 Animals ...................................................................................................................... 41 Mouse NK cell separation. .................................................................................. 41 III. PEPTIDE SEQUENCE REQUIREMENTS FOR EFFICIENT H-2Kb BINDING AT THE CELL SURFACE A. Introduction. ................................................................................................................. 43 B. Results .......................................................................................................................... 46 Detection of peptides bound to H-2Kb at the cell surface is dependent on peptide sequence ................................................................................................................... 46 Different H-2Kb specific antibodies have similar recognition of H-2Kb-peptide complexes ................................................................................................................. 48 Recombinant mouse β2m does not assist in overcoming poor stabilization of H-2Kb by peptides ................................................................................................ 50 Determining peptide concentrations yielding similar H-2Kb upregulation. ....... 51 C. Discussion. ........................................................................................................... 54 IV. AMINO ACIDS AT POSITIONS 2 AND 3 OF THE H-2Kb BOUND PEPTIDE DICTATE LY49C RECOGNITION OF H-2Kb A. Introduction. ............................................................................................................... 75 B. Results........................................................................................................................ 77 Ly49W/C chimeric receptors are peptide specific for recognition of H-2Kb ...... 77 Identity of the peptide auxiliary anchor residue at P3 correlates with Ly49C recognition of H-2Kb ........................................................................................... 79 Aliphatic residues at P3 are supportive of Ly49W/C recognition of H-2Kb ....... 80 P2 and P3 peptide residues combine to drive Ly49W/C recognition of H-2Kb...82 An LCMV immunodominant peptide and CTL escape mutants do not support Ly49C recognition of H-2Kb ............................................................................... 82 C. Discussion. ........................................................................................................... 84 vi V. TUNING MOUSE NK CELL FUNCTION BY THE MHC-I PEPTIDE A. Introduction. .............................................................................................................. 117 B. Results ........................................................................................................................ 120 Ex vivo NK cells for functional assays .............................................................. 120 Selection of H-2Kb peptides ............................................................................... 123 NK cell inhibition can be mediated by low SIINFEKL concentrations ............ 124 Peptide preventing H-2Kb-Ly49C interaction weakly antagonizes NK inhibition ................................................................................................................................ 127 Peptide supporting H-2Kb-Ly49C interaction at intermediate levels reduce NK inhibition by agonist H-2Kb complexes ............................................................. 130 Cooperative inhibition of NK cells by H-2Kb peptides that support H-2Kb-Ly49C interaction. ............................................................................................................. 133 C. Discussion. ........................................................................................................... 136 VI. DISCUSSION AND CONCLUSION A. Overview of thesis .............................................................................................. 159 Rationale: H-2Kb peptide binding studies and H-2Kb peptide dependent Ly49C recognition. ........................................................................................................ 159 Summary: H-2Kb peptide binding studies .......................................................... 160 Summary: H-2Kb peptide dependent Ly49C recognition .................................. 162 Rationale: MHC-I peptide dependent antagonism depresses NK cell inhibition ................................................................................................................................ 164 Summary: MHC-I peptide dependent antagonism depresses NK cell inhibition. ................................................................................................................................ 164 Overall contributions, remarks and research significance ................................ 166 B. Discussion and Future Studies ................................................................................ 166 MHC-I and peptide affinity ............................................................................... 166 Cell changes: role of PTMs in MHC-I-bound peptides ..................................... 168 Ly49 peptide specific recognition of MHC-I. .................................................... 169 vii Peptide antagonism and NK cell activity ........................................................... 170 C. Concluding remarks ............................................................................................... 171 REFERENCES ............................................................................................................ 173 viii LIST OF FIGURES FIGURE DESCRIPTION PAGE 1-1 The Major Histocompatibility Complex in humans and mice .................................. 24 1-2 General structure of classical MHC-I molecules ...................................................... 25 1-3 Peptide anchor residues stabilize peptide bound to MHC-I. ..................................... 26 1-4 Antigen processing and presentation pathway ..........................................................27 1-5 NK cell activating and inhibitory signaling. ............................................................. 28 1-6 NK cell education. ..................................................................................................... 30 1-7 Structural view of H-2Kb and Ly49C association ..................................................... 32 3-1 Differential peptide binding to H-2Kb on RMA/S cells using the Y3 antibody ....... 60 3-2 Mapping of determinants recognized by H-2Kb specific antibodies ........................ 62 3-3 Differential peptide binding to H-2Kb on RMA/S cells using the H-2Kb specific antibody AF6-88.5.5.3. ............................................................................................ 63 3-4 Differential peptide binding to H-2Kb on RMA/S cells using the H-2Kb specific antibody B8-24-3. ................................................................................................... 65 3-5 Differential peptide binding to H-2Kb on RMA/S cells using the M1/42, β2m dependent antibody.................................................................................................. 67 3-6 Purification of recombinant mouse β2m ................................................................... 69 3-7 Recombinant mouse β2m does not aid in the stabilization of peptide-H-2Kb complexes where the peptide has low “affinity” for H-2Kb .................................... 70 3-8 Peptide titration assays to determine similar H-2Kb expression on RMA/S cells using peptides with non-polar aliphatic P3 residues ......................................................... 72 3-9 Peptide titration assays to determine similar H-2Kb expression on RMA/S cells using peptides with aromatic P3 residues .................................................................73 3-10 Structural comparison of P8 anchor residue in the F-pocket of the H-2Kb peptide binding groove ............................................................................................ 74 ix 4-1 Expression of chimeric receptor Ly49W/C on RNK-16 cells .................................. 93 4-2 Chimeric receptor Ly49W/C recognizes H-2Kb in a peptide selective manner........ 94 4-3 Ly49C peptide dependent recognition of H-2Kb is retained in peptides with non- polar aliphatic auxiliary anchor residues ................................................................. 97 4-4 Ly49C peptide dependent recognition of H-2Kb decreases in peptides with non-polar aliphatic auxiliary anchor residues .......................................................................... 99 4-5 H-2Kb stabilization using Alanine SIINFEKL variants .......................................... 102 4-6 Solvent exposed residues of the H-2Kb bound peptide do not influence Ly49C recognition. ................................................................................................................. 103 4-7 Peptide amino acid substitution demonstrates that auxiliary anchor residue P3 is an important factor in determining Ly49C recognition ...............................................105 4-8 Peptide residues P2 and P3 determine Ly49C recognition of H-2Kb ..................... 107 4-9 H-2Kb stabilization using AAYAYAAL peptide variants to recapitulate SIINFEKL anchor residues ...................................................................................................... 109 4-10 LCMV immunodominant peptide and escape mutants are not recognized by Ly49W/C receptors when bound to H-2Kb ............................................................ 110 4-11 Structural analysis of peptide amino acid docking into the B-pocket of H-2Kb ... 112 4-12 Structural analysis of peptide amino acid docking into the D-pocket of H-2Kb ... 113 4-13 H-2Kb peptide dependent intramolecular interactions that potentially affect H-2Kb and Ly49C association. ...........................................................................................114 5-1 Analysis of ex vivo CD3-NK1.1+Ly49C&I+ NK cells ............................................ 145 5-2 Low concentrations of SIINFEKL on RMA/S cells yield inhibition of CD3- NK1.1+Ly49C&I+ NK cells .................................................................................... 147 5-3 Expression levels of H-2Kb on RMA/S cells utilizing sequential peptide loading of SIINFEKL and RGYVYQGL ................................................................................ 149 5-4 Peptide that does not support Ly49C and H-2Kb interaction can act to weakly antagonize the inhibitory effect of H-2Kb-SIINFEKL ........................................... 150 x