Impact of the Major Histocompatibility Complex class I peptide repertoire on Natural Killer cell function

Abstract

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 HLAC*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.

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Identifiers

ORCID for Salim Khakoo: orcid.org/0000-0002-4057-9091

ORCID for Christopher Mccormick: orcid.org/0000-0002-6155-9161

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