The role of DNA methylation in the regulation of HLA expression.

Variation within the non-coding genome may influence the regulation and expression of important genes involved in immune control such as the human leukocyte antigen (HLA) system. Class I and Class II HLA molecules are essential for peptide presentation which is required for T lymphocyte activation. Single nucleotide polymorphisms within non-coding regions of HLA Class I and Class II genes may influence the expression of these genes by affecting the binding of transcription factors and chromatin modeling molecules. Furthermore, an interplay between genetic and epigenetic factors may also influence HLA expression. Epigenetic factors such as DNA methylation and non-coding RNA, regulate gene expression without changing the DNA sequence. However, genetic variation may promote or allow genes to escape regulation by epigenetic factors, resulting in altered expression. The HLA system is central to most diseases, therefore, understanding the role of genetics and epigenetics on HLA regulation will tremendously impact healthcare. The knowledge gained from these studies may lead to novel and cost-effective diagnostic approaches and therapeutic interventions. This review discusses the role of non-coding variants on HLA regulation. Furthermore, we discuss the interplay between genetic and epigenetic factors on the regulation of HLA by evaluating literature based on polymorphisms within DNA methylation and miRNA regulatory sites within class I and Class II HLA genes. We also provide insight into the importance of the HLA non-coding genome on disease, discuss ethnic-specific differences across the HLA region and provide guidelines for future HLA studies.

Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Chapter 9 - Human leukocyte antigen-typing: a significant immunogenetic application in clinical medicine

Background: Pancreatic adenocarcinoma is one of the deadliest type of cancers with a 5-year overall survival rate of 10%. A standard chemotherapy approach for pancreatic cancer is treatment with gemcitabine. Gemcitabine is also considered a potential immunomodulatory agent, capable of increasing human leukocyte antigen (HLA) class I. HLA class I is a heterodimer of a highly polymorphic alpha heavy chain (HLA-A, -B, or -C) and the light chain beta-2-microglobulin (β2m) that is loaded with an endogenous antigen in the endoplasmic reticulum. The pool of antigens available for HLA class I loading is selected by the transporter associated with antigen processing protein (TAP). However, HLA class I peptide loading can occur independently of TAP, causing the HLA class I molecules to present a different antigen pool to cytotoxic T cells. Because many cancers downregulate HLA class I and prevent intracellular tumor antigens from being presented to cytotoxic T cells, understanding more about gemcitabine's capability to increase HLA class I expression is of great importance. Methods: Alterations in HLA class I and TAP protein levels were monitored via western blot analysis in a pancreatic cancer cell line (S2-013) in both a time and dose-dependent manner. The time points and dosages which solicited the largest increase in HLA class I expression were also examined by flow cytometry. Results: Administration of gemcitabine to S2-013 pancreatic cancer cells increased total HLA class I α heavy chain protein, with maximal increases of 141% (HLA-A) and 323% HLA-B/C. Flow cytometry revealed a 3-fold increase in HLA-A2 surface expression post-gemcitabine treatment and a maximal 2-fold increase in HLA-B/C expression at the corresponding gemcitabine concentration, but differing time point. Both western blot and flow cytometry analyses indicate HLA-B/C generally has its largest increase in expression at an earlier time point than HLA-A. In addition, high concentrations of gemcitabine that promoted HLA-A,B,C surface expression were associated with a large decrease in TAP total protein. Conclusion: In summary, gemcitabine demonstrates an ability to stimulate expression of HLA class I in pancreatic cancer cells, with the rate dependent on the HLA class I allele. Because gemcitabine treatment increases HLA class I surface expression with a simultaneous decrease in TAP expression, our findings indicate that gemcitabine may induce TAP-independent peptide loading of HLA class I. These results suggest the potential for gemcitabine treatment as a priming mechanism to enhance immunotherapy efficacy and potentially alter vaccination strategies in pancreatic cancer. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine impacts expression of antigen presentation proteins by pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1658.

Relationship Between HLA Class I and II Polymorphisms with Susceptibility and Clinical Outcome in Diffuse Large B-Cell Lymphoma.

Background: Pancreatic adenocarcinoma, or PDAC, is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine, a nucleoside analog, is a primary standard of care in pancreatic cancer. In addition to its normative cytotoxic function, evidence suggests that this chemotherapy drug also harnesses immunomodulatory capabilities in the form of increasing human leukocyte antigen (HLA) class I expression in lung, breast, colon, and cholangiocarcinoma cells. HLA class I is a complex (alpha heavy chain and beta 2-microglobulin light chain) located at the surface of nearly all cells where it presents peptides, including cancer-associated peptides, to cytotoxic T cells. Recognition of these peptides as atypical leads to T-cell mediated lysis of the presenting cell. Subsequently, understanding the ability of gemcitabine and the mechanisms by which it influences the HLA class I complex are of great importance. Methods: To investigate the effect of gemcitabine treatment on HLA class I expression in pancreatic cancer, alterations in HLA class I protein levels were monitored via western blot analysis, flow cytometry, and quantitative polymerase chain reaction (qPCR) in three pancreatic cancer cell lines. Changes in surface stability of the HLA class I complex were evaluated through brefeldin A (BFA) assays at the 72-hour time point. For western blot and flow cytometry experiments, impacts on the alpha heavy chain were further assessed for all three types of alpha heavy chains (HLA-A, HLA-B/C) at 72 and 96 hours. In qPCR experiments, alterations were analyzed for the HLA-A, HLA-B, and beta 2-microglobulin transcripts after a 48-hour gemcitabine exposure period. Results: Administration of gemcitabine to pancreatic cancer cell lines (S2-013, Capan-1, PANC-1) increased total protein levels of both HLA class I constituents (alpha heavy chain and beta-2-microglobulin light chain). All PDAC cell lines evaluated demonstrated enhanced surface expression of HLA-A2 and HLA-B/C with maximal increases of 3 and 2.5-fold respectively, as indicated by flow cytometry. Our qPCR analysis of the Capan-1 and S2-013 cell lines revealed increases in the levels of HLA-A, HLA-B, and beta 2-microglobulin transcripts (3-12-fold). BFA assays suggested that gemcitabine treatment also enhances stability of the surface HLA class I in the S2-013 and PANC-1 cell lines. Conclusion: In summary, gemcitabine exhibits an immunomodulatory ability to stimulate expression of HLA class I in pancreatic cancer cells. We have demonstrated that this increase in HLA class I is seen at the mRNA, total protein, surface, and stability level. Characterizing the ability of gemcitabine to influence the HLA class I complex and defining the mechanisms by which it does so will increase the potential for identification of suitable immunotherapies, including novel peptide-based cancer vaccines with enhanced treatment efficacy for PDAC patients. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P071.

Drug-induced liver injury: Is it time for genetics to change our clinical practice?

Suspicion that host genetic factors determine variability in the course of natural HIV-1 infection deepened throughout the 1980s and early 1990s [1,2], but only relatively recently have we recognized the impact of human genetic variation on HIV/AIDS as a complex phenomenon [3–11]. As the control and prevention of HIV-1 infection with highly active antiretroviral therapy (HAART) became more widely available, the growing opportunity for pharmacogenetic studies [12– 14] was also accompanied by uncertainty about the residual host genetic influences on HIV/AIDS.

Detection of genome DNA methylation change in spinach induced by 5-azaC

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

The human leukocyte antigen (HLA) system is a major factor controlling cancer immunosurveillance and response to immunotherapy, yet its status in pediatric cancers remains fragmentary. We determined high-confidence HLA genotypes in 576 children, adolescents and young adults with recurrent/refractory solid tumors from the MOSCATO-01 and MAPPYACTS trials, using normal and tumor whole exome and RNA sequencing data and benchmarked algorithms. There was no evidence for narrowed HLA allelic diversity but discordant homozygosity and allele frequencies across tumor types and subtypes, such as in embryonal and alveolar rhabdomyosarcoma, neuroblastoma MYCN and 11q subtypes, and high-grade glioma, and several alleles may represent protective or susceptibility factors to specific pediatric solid cancers. There was a paucity of somatic mutations in HLA and antigen processing and presentation (APP) genes in most tumors, except in cases with mismatch repair deficiency or genetic instability. The prevalence of loss-of-heterozygosity (LOH) ranged from 5.9 to 7.7% in HLA class I and 8.0 to 16.7% in HLA class II genes, but was widely increased in osteosarcoma and glioblastoma (~15-25%), and for DRB1-DQA1-DQB1 in Ewing sarcoma (~23-28%) and low-grade glioma (~33-50%). HLA class I and HLA-DR antigen expression was assessed in 194 tumors and 44 patient-derived xenografts (PDXs) by immunochemistry, and class I and APP transcript levels quantified in PDXs by RT-qPCR. We confirmed that HLA class I antigen expression is heterogeneous in advanced pediatric solid tumors, with class I loss commonly associated with the transcriptional downregulation of HLA-B and transporter associated with antigen processing (TAP) genes, whereas class II antigen expression is scarce on tumor cells and occurs on immune infiltrating cells. Patients with tumors expressing sufficient HLA class I and TAP levels such as some glioma, osteosarcoma, Ewing sarcoma and non-rhabdomyosarcoma soft-tissue sarcoma cases may more likely benefit from T cell-based approaches, whereas strategies to upregulate HLA expression, to expand the immunopeptidome, and to target TAP-independent epitopes or possibly LOH might provide novel therapeutic opportunities in others. The consequences of HLA class II expression by immune cells remain to be established. Immunogenetic profiling should be implemented in routine to inform immunotherapy trials for precision medicine of pediatric cancers.

Background: Endomyocardial fibrosis (EMF) is the most common form of restrictive cardiomyopathy worldwide. It has been linked to poverty and various environmental factors, but—for unknown reasons—only some people who live in similar conditions develop the disease. EMF cases cluster within both families and ethnic groups, suggesting a role for a genetic factor in host susceptibility. The human leukocyte antigen (HLA) system is associated with predisposition to various diseases. This two-center study was designed to investigate variation in the HLA system between EMF patients and unaffected controls. We provide the first genetic investigation of patients with EMF, as well as a comprehensive review of the literature. Methods: HLA class I (HLA-A, -B, -C) and class II (DRB1, DQB1) types were determined in 71 patients with severe EMF and 137 controls from Uganda and Mozambique. Chi Square analysis was used to identify any significant difference in frequency of class I and class II HLA types between cases and controls. Results: Compared to ethnically matched controls, HLA-B*58 occurred more frequently in Mozambique patients with EMF and HLA-A*02:02 occurred more frequently in Ugandan patients with EMF. Conclusions: Ample subjective evidence in the historical literature suggests the importance of a genetically susceptible host in EMF development. In this first formal genetic study, we found HLA alleles associated with cases of EMF in two populations from sub-Saharan Africa, with EMF patients being more likely than controls to have the HLA-B*58 allele in Mozambique (p-0.03) and the HLA-A*02:02 in Uganda (p = 0.005). Further investigations are needed to more fully understand the role of genetics in EMF development.

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

Human Leukocyte Antigen Contributes to Childhood Endemic Burkitt Lymphoma in Eastern Africa: A Case-Control Association Study