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Impact of the Major Histocompatibility Complex Class I Peptide Repertoire on Natural Killer Cell Function

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UNIVERSITY OF SOUTHAMPTON FACULTY OF MEDICINE Clinical and Experimental Sciences Impact of the Major Histocompatibility Complex class I peptide repertoire on Natural Killer cell function by Berenice Mbiribindi Nvunabandi Thesis for the degree of Doctor of Philosophy December 2016 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE Clinical and Experimental Sciences Transfer Thesis for the degree of Doctor of Philosophy IMPACT OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I PEPTIDE REPERTOIRE ON NATURAL KILLER CELL FUNCTION Berenice Mbiribindi Nvunabandi Background: Natural Killer (NK) cell activation requires the integration of inhibitory and activating signalling. Inhibitory signals are determined by members of the KIR family, which have MHC class I ligands. Peptide antagonism of MHC class I provides an alternative mechanism for loss of inhibition of KIR2DL2/3-positive NK cells. This occurs when a weak KIR binding peptide disrupts the inhibitory signalling of a strong binding peptide. Peptide antagonism has been defined only for HLA-C*0102, endogenously expressed in TAP-deficient cells, using a peptide variant of VAPWNSLSL (VAPWNSDAL and VAPWNSDYL), therefore it may be a unique property of HLA-C*0102 or more general finding. Hypothesis: We hypothesize that small changes in MHC-I peptide repertoire can impact on NK cell activation and that additional peptides can antagonise inhibitory KIR can be discovered. Results: 721.221 cells were transfected with HLA-C*0304 and ICP47 used for peptide loading and as target cells in CD107a assays. We studied previously described position 8 (P8) derivatives and also generated novel P7 derivatives of the endogenously processed peptide GAVDPLLAL. In contrast to our observations for HLA-C*0102, arginine at P7 triggered stronger inhibition than phenylalanine. L7D did not inhibit KIR2DL2/3-positive NK cells. However it did antagonise inhibition by L7R in CD107a assays. I used peptide elution and HPLC analysis to demonstrate that peptide antagonism was not related to displacement of L7R or L7F by LD7. In a study of the immunopeptidome of Hepatitis C virus expressing cells expressing HLA- C*0102 were generated to study its peptide repertoire and to identify potential NK activating or inhibitory peptides. I identified that the peptide repertoire of HLA-C was preferentially altered in comparison to HLA-A. To improve our methods for studying the influence of peptides on NK cell, I developed a Granzyme B assay based on HPLC technology. This showed that it is possible to perform a functional assay using NK lines with low backgrounds, however it was not robust enough to detect small differences in peptides. Conlcusions: Peptide antagonism is a generalizable phenomenon and it appears that HLA-C alleles has become specialised to NK cells. Contents Contents ................................................................................................................................... i List of tables ...................................................................................................................... vii List of figures ......................................................................................................................ix List of accompanying materials ............................................................................. xv DECLARATION OF AUTHORSHIP ........................................................................... xvii Acknowledgements .................................................................................................... xviii Definitions and Abbreviations ............................................................................... xix 1. Introduction................................................................................................................. 1 1.1 Natural Killer cells ....................................................................... 1 1.1.1 General description ..................................................................... 1 1.1.2 NK cells development: transcription factors and lineage commitment 1 1.1.3 NK cells development: the journey from the bone marrow to the periphery .................................................................................................. 3 1.2 NK cell education ........................................................................ 4 1.2.1 Chronic exposure of NK cells to activating ligands make them tolerant. 4 1.2.2 Inhibitory receptor repertoire selectivity of NK cells receptors ..... 6 1.2.3 Self-MHC Class I and Inhibitory receptors recognition is involved in NK Cell Education ........................................................................................... 8 1.2.4 Mechanisms leading to hyporesponsive NK cells: licensing or disarming? 9 1.3 NK cells in health and disease ................................................... 11 1.3.1 NK cells in cancer ..................................................................... 11 1.3.2 NK cells in pregnancy ............................................................... 12 1.3.3 NK cells in infection .................................................................. 13 1.3.4 NK cells in autoimmune diseases .............................................. 15 1.4 Therapeutic applications of NK cells ......................................... 17 1.4.1 NK cells in Liver disease ............................................................ 19 1.4.2 NK cells as therapeutic targets for the treatment of liver disease20 1.5 The molecular specificity of NK cell receptors ........................... 21 1.5.1 Receptor-mediated NK cell responses ....................................... 22 1.5.2 NK cell recognition and response receptors .............................. 23 1.5.3 Activating receptors .................................................................. 24 1.5.4 Inhibitory receptors .................................................................. 27 i 1.6 Killer Immunoglobulin like Receptors: KIR ................................. 28 1.6.1 Structural and diversity of KIR receptors ................................... 28 1.6.2 Structural basis of KIR-HLA recognition ..................................... 30 1.6.3 HLA-C Recognition by inhibitory HLA-C specific KIR ................... 32 1.6.4 The role of the peptide: peptide presentation ........................... 33 1.6.5 The role of the peptide: KIR recognition .................................... 36 1.6.6 NK cells inhibitory receptors and peptide selectivity .................. 37 1.6.7 Antagonism of NK cells inhibition through inhibitory receptors . 38 1.6.8 Considerations for viral infections............................................. 40 1.7 Specificity of Activating KIR2D receptors ................................... 41 1.8 NK cell activation ...................................................................... 42 1.9 Hypothesis, aims and Experimental plan ................................... 43 1.9.1 Aim 1: Demonstrate that peptide antagonism is not restricted to only one HLA-C allele ...................................................................................... 44 1.9.2 Aim 2: Design of a novel method for peptide high throughput screen …………………………………………………………………………………44 1.9.3 Aim 3: Identifying viral induced changes in peptide repertoire and assessing how this may affect NK cell reactivity ....................................... 45 2. Materials and Methods ...................................................................................... 46 2.1 Peptides ................................................................................... 46 2.2 Cells lines, PBMC and cell culture .............................................. 46 2.3 ICP47 gene transfection in 721.221:C*0304 cells ..................... 47 2.4 Peptide stabilization assay ........................................................ 48 2.5 Measurement of the decay of cell surface HLA-C molecules ....... 49 2.6 Degranulation Assays ............................................................... 49 2.7 Peptide elution from MHC class I and HPLC analysis .................. 50 2.8 PBMC DNA extraction ................................................................ 51 2.9 KIR genotyping ......................................................................... 52 2.10 Confocal microscopy ................................................................ 53 2.11 KIR binding assay ..................................................................... 53 2.11.1 Affinity of P7 peptide variants for KIR ....................................... 53 2.11.2 KIR binding and peptide titration .............................................. 54 2.11.3 KIR binding, HLA-C and Degranulation Assay ............................ 54 2.12 Minigenes for endogenous peptide presentation ....................... 55 2.12.1 Minigene preparation ................................................................ 55 2.12.2 Minigenes transformation into competent cells ......................... 56 2.12.3 Extraction of plasmids containing minigenes ............................ 56 ii 2.13 Transfection of minigenes constructs into 721.221:C*0304 cells by electroporation ......................................................................... 57 2.14 Cytotoxicity Assay using Propidium iodide (PI) .......................... 58 2.15 Granzyme B Assay optimisation ................................................ 58 2.15.1 Determining the optimal wavelength for Caspase 3 Peptide (C3P) detection 58 2.15.2 Determining the optimal conditions for
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