Effects of genetic polymorphism in MHC, KIR, and related loci on human disease

Division Of Basic Sciences - Nci

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Abstract

HLA-A02:01 is the most common allele in White and Black US populations, present at allelic frequencies 28% and 12%, respectively. This allele has been reported to associate with protection in human herpesvirus infection. We have conducted analysis in the UK Biobank dataset across various herpesvirus-associated diseases, and observed multiple protective associations, including herpes simplex infection, Zoster infection, multple sclerosis, and Hodgkin's lymphoma. We are interested in understanding the mechanism of HLA-A02:01 protection. Many herpesviruses affect MHC class I peptide presentation by inhibiting the function of TAP . HLA-A02:01 has been known for its relative resistance to TAP inhibition, partly due to its ability to present hydrophobic peptides derived from SPs. We hypothesized that HLA-A02:01 may confer protection by presenting viral peptides more efficiently than other alleles in the absence of TAP. This enhanced presentation could occur through (1) peptide exchange in endosomal compartments, where hydrophobic TAP-independent self-peptides are displaced by viral peptides; or (2) TAP-independent loading of viral peptides in the ER, such as SPs or transmembrane proteins fragments cleaved from the ER membrane into the lumen. To test the first hypothesis, we generated 721.221-TAP knockout cell line and transduced it with HLA-A02:01 and human cytomegalovirus (HCMV) protein UL83, which contains the immunodominant peptide NLVPMVATV (NV9). This peptide is known to elicit strong CD8 T cell responses in HLA-A02:01-positive individuals. However, we were unable to detect T cell activation in response to these cells, either using Jurkat reporter cells expressing a TCR specific for HLA-A02:01/NV9, or with primary CD8 T cells from NV9-responsive donors activated with the peptide in vitro. We next used computational prediction tools to identify HCMV-derived SP fragments that could potentially be cleaved into the ER lumen and presented by HLA-A02:01. These candidate peptides were assessed for HLA-A02:01 binding using a peptide-pulsing assay with 721.221 cells expressing HLA-A02:01. Peptides demonstrating stable binding will be further evaluated for their capacity to elicit T cell activation in CMV-seropositive individuals and individuals with CMV reactivation. We next identified HCMV SP peptide fragments that may potentially be cleaved into the ER lumen and loaded onto HLA-A02:01 using computational prediction tools. These peptides were tested for HLA-A02:01 binding in a peptide-pulsing assay using 721.221 cells expressing HLA-A02:01. Peptides that showed binding will be further tested for their ability to elicit T cell activation in CMV-seropositive individuals and individuals with CMV reactivation. These expriemnts aim to uncover a TAP-independent mechanism of viral peptide presentation that could explain the protective effect of HLA-A02:01 and inform future immunotherapeutic strategies. Nearly all nasopharyngeal carcinoma (NPC) are caused by infection with Epstein-Barr virus (EBV). EBV infection is nearly ubiquitous in humans, typically occurs early in life, and establishes lifelong, latent infections. Only a very small fraction of those infected with EBV develop NPC, however. Why some EBV-infected individuals develop NPC while most do not is not fully understood. Human leukocyte antigen (HLA) alleles and haplotypes have long been established as an important determinant of NPC risk among EBV-infected individuals. HLA alleles/haplotypes associated with increased risk include HLA A02:07, B46:01, A33 or B58. In contrast, HLA A11:01, a common HLA allele in Southeast Asian populations, where NPC incidence is high, has been consistently shown to be associated with a 2-fold reduction in risk of NPC. Most recently, studies that have evaluated another HLA allele, HLA A11:02, which differs from HLA A11:01 at a single amino acid site outside the HLA binding region, have suggested that while HLA A11:01 is associated with strong protection against NPC, HLA A11:02 is not associated with disease. The reason for this discrepancy, despite the fact that both alleles share the same binding motif, is unknown. To better understand why HLA A11:01 is a protective allele for NPC but HLA A11:02 is not, we submitted those allotypes to molecular modeling, peptide binding assays and characterized their peptidome through mass spectrometry analyses of mono allelic cell lines expressing each of them. Those analyses revealed that HLA-A11:02 is intrinsically less stable, presents a lesser number of peptides on the cell surface and that the bound peptides have a lower thermostability and a lower cell surface stability then when presented through HLA-A11:01.