A genomic association study revealing subphenotypes of childhood steroid-sensitive nephrotic syndrome in a larger genomic sequencing cohort.

Dissecting the genetic components that contribute to the two main subphenotypes of steroid-sensitive nephrotic syndrome (SSNS) using genome-wide association studies (GWAS) strategy is important for understanding the disease. We conducted a multicenter cohort study (360 patients and 1835 controls) combined with a GWAS strategy to identify susceptibility variants associated with the following two subphenotypes of SSNS: steroid-sensitive nephrotic syndrome without relapse (SSNSWR, 181 patients) and steroid-dependent/frequent relapse nephrotic syndrome (SDNS/FRNS, 179 patients). The distribution of two single-nucleotide polymorphisms (SNPs) in ANKRD36 and ALPG was significant between SSNSWR and healthy controls, and that of two SNPs in GAD1 and HLA-DQA1 was significant between SDNS/FRNS and healthy controls. Interestingly, rs1047989 in HLA-DQA1 was a candidate locus for SDNS/FRNS but not for SSNSWR. No significant SNPs were observed between SSNSWR and SDNS/FRNS. Meanwhile, chromosome 2:171713702 in GAD1 was associated with a greater steroid dose (>0.75 mg/kg/d) upon relapse to first remission in patients with SDNS/FRNS (odds ratio = 3.14; 95% confidence interval, 0.97-9.87; P = 0.034). rs117014418 in APOL4 was significantly associated with a decrease in eGFR of greater than 20% compared with the baseline in SDNS/FRNS patients (P = 0.0001). Protein-protein intersection network construction suggested that HLA-DQA1 and HLA-DQB1 function together through GSDMA. Thus, SSNSWR belongs to non-HLA region-dependent nephropathy, and the HLA-DQA/DQB region is likely strongly associated with disease relapse, especially in SDNS/FRNS. The study provides a novel approach for the GWAS strategy of SSNS and contributes to our understanding of the pathological mechanisms of SSNSWR and SDNS/FRNS.

Biomarker role of maternal soluble human leukocyte antigen G in pre-eclampsia: A meta-analysis.

INTRODUCTION: Human leukocyte antigen-G (HLA-G) is a non-classical class I HLA molecule shown to regulate the immunomodulation of maternal immune cells to prevent fetal tissue destruction. Low levels of freely circulating maternal soluble HLA-G (sHLA-G) have been observed in pre-eclampsia, however, no pooled evidence exists. This meta-analysis aimed to generate pooled findings on the association of sHLA-G levels with pre-eclampsia and is the first study to perform a trimester-wise comparison of the levels of sHLA-G in preeclamptic cases and normal pregnant controls. METHODS: The databases PubMed, Emba, Web of Science, and Google Scholar through May 31, 2023. Preeclamptic women were defined as cases and normal pregnancies as controls. Data on the level of sHLA-G in cases and controls was extracted and subjected to a meta-analysis using a random-effects model. The pooled effect was expressed in terms of standardized mean difference (SMD). Sensitivity analysis was performed to investigate the effect of the exclusion of each study on the pooled results. Publication bias was assessed statistically. RESULTS: Nine studies with altogether 567 PE cases and 1132 normal pregnancy controls were included in the meta-analysis. The first and third trimester levels of sHLA-G in PE cases were significantly lower than that of normal pregnant controls: (SMD: -0.84 [-1.29; -0.38]; p = .003; I2 = 54%) and (SMD: -0.39 [-0.71; -0.06]; p = .02; I2 = 79%) respectively. Sensitivity analysis revealed significant fluctuations in the pooled findings when few studies were excluded, raising questions on the consistency of results among studies. CONCLUSION: Although we found that first and third-trimester sHLA-G levels in pre-eclampsia are significantly lower, taking into consideration the inconsistent results from the sensitivity analysis, our findings advocate the demand for more studies with larger sample sizes to generate solid ground pooled evidence on the predictive role of sHLA-G in pre-eclampsia.

HLA-DRB1 and HLA-DQB1 genes in patients diagnosed with systemic lupus erythematosus in Guatemala.

Systemic lupus erythematosus (SLE) is an autoimmune disorder that impacts connective tissue and can affect various organs and systems within the body. One important aspect of this disease is the role of the human leukocyte antigen (HLA) system, a protein complex that plays a role in the immune response. Specifically, the HLA-DRB1 and HLA-DQB1 genes have been implicated in the development of SLE. In order to better understand this relationship in the Guatemalan population, a study was conducted with the objective of characterizing the allelic and haplotype profiles of the HLA-DQB1 and HLA-DRB1 loci in 50 patients diagnosed with SLE who were receiving treatment at a hospital in Guatemala. Allele and haplotype frequencies were determined and compared to 127 healthy Guatemalan subjects as a control group. The results of the analysis showed a reduction in the frequencies of HLA-DQB1*03 and HLA-DRB1*14 in SLE patients, which could suggest a protective effect on the development of the disease. In contrast, a risk association was found between HLA-DRB1*07, HLA-DRB1*08, HLA-DQB1*02 and HLA-DQB1*06 in SLE patients. Finally, we observed an additional protective associated of haplotype HLA-DRB1*04∼DQB1*03 with SLE patients, while haplotypes HLA-DRB1*07∼DQB1*02 and DRB1*08-DQB1*06 showed a risk association.

Exploring the immunological relevance of pre-transplant donor-specific antibody in intestinal transplantation, with special consideration to the liver.

Despite recent advances that have improved outcomes following intestinal transplantation (ITx), achieving long-term patient survival and rejection-free survival is still challenging. Understanding the relevance of pre-transplant human leukocyte antigen (HLA) donor-specific antibody (DSA) in ITx and the immunomodulatory potential of the liver within the allograft is crucial to providing an accurate assessment of pre-transplant immunological risk, which could influence and improve post-transplant outcomes further. This was the primary objective of this retrospective study of 95 adult ITx transplants which took place at Cambridge University Hospitals (United Kingdom) between 2007 and 2019. Two novel programs were developed and validated to identify DSA (tested by Luminex single antigen beads) in this dataset. Data analysis utilised Kaplan-Meier survival methods, and statistical analysis was performed using log-rank tests and adjusted Cox models. Fifty-four (57%) ITx cases contained a liver, and 36 (38%) were sensitised to HLA. Pre-transplant DSA > 500 mean fluorescent intensity appeared to negatively affect post-ITx patient survival and rejection outcomes. Additionally, liver-inclusive allografts seemed to show particular resistance to HLA class I DSA. Our data hints towards consistency with other ITx studies where deleterious effects of DSA have been demonstrated, and where liver inclusion is protective from HLA class I DSA. This is in line with current national guidelines for immunological risk. Our publicly available research programs could support future large or multicentre studies where statistically relevant data might be gained.

Comparison of human leukocyte antigen in patients with paroxysmal nocturnal hemoglobinuria of different clone sizes.

Glycosylphosphatidylinositol-anchored protein-deficient hematopoietic stem and progenitor cell development caused by PIGA mutations cannot fully explain the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Herein, patients newly diagnosed with PNH at our hospital between April 2019 and April 2021 were recruited. The human leukocyte antigen (HLA) class I and II loci were analyzed, and patients were stratified by PNH clone sizes: small (< 50%) and large (≥ 50%). In 40 patients (29 males; 72.5%), the median PNH clone size was 72%. Thirteen (32.5%) and twenty-seven (67.5%) patients harbored small and large PNH clones, respectively. DRB1*15:01 and DQB1*06:02 had higher frequencies in patients with PNH than in healthy controls (adjusted P-value = 4.10 × 10-4 and 4.10 × 10-4, respectively). Whole HLA class I and II allele contributions differed (P = 0.046 and 0.065, not significant difference) when comparing patients with small and large PNH clones. B*13:01 and C*04:01 allelic frequencies were significantly higher in patients with small clones (P = 0.032 and P = 0.032, respectively). Patients with small clones had higher class II HLA evolutionary divergence (HED) (P = 0.041) and global class I and II HED (P = 0.019). In the entire cohort, 17 HLA aberrations were found in 11 (27.5%) patients. No significant differences in HLA aberrations were found between patients with small or large clones. In conclusion, patients with small clones tended to have a higher frequency of immune attack-associated alleles. A higher HED in patients with small clones may reflect a propensity for T cell-mediated autoimmunity. HLA aberrations were similar between patients with small and large clones.

Haploidentical and matched unrelated donor allogeneic hematopoietic stem cell transplantation offer similar survival outcomes for acute leukemia.

BACKGROUND: Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has emerged as an effective approach for acute leukemia, primarily due to the inherent difficulty in finding human leukocyte antigen-matched unrelated donors (MUD). Nevertheless, it remains uncertain whether haplo-HSCT and MUD-HSCT can provide comparable outcomes in patients with acute leukemia. AIMS: This study aimed to assess the overall survival (OS) and leukemia-free survival (LFS) outcomes between the MUD-HSCT and haplo-HSCT groups. METHODS AND RESULTS: This retrospective analysis encompassed adult patients with acute leukemia undergoing the initial allo-HSCT. Among these 85 patients, we stratified 33 patients into the MUD-HSCT group and 52 to the haplo-HSCT group. The primary outcomes were OS and LFS. The median OS was not reached in the haplo-HSCT group, while it reached 29.8 months in patients undergoing MUD-HSCT (p = .211). Likewise, the median LFS periods were 52.6 months in the haplo-HSCT group and 12.7 months in the MUD-HSCT group (p = .212). Importantly, neither the OS nor LFS showed substantial differences between the MUD-HSCT and haplo-HSCT groups. Furthermore, univariate analyses revealed that haplo-HSCT did not demonstrate a significantly higher risk of worse LFS (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.38-1.25; p = .216) or OS (HR, 0.67; 95% CI, 0.36-1.26; p = .214) than MUD-HSCT. Notably, a high European Group for Blood and Marrow Transplantation risk score (HR, 1.44; 95% CI, 1.10-1.87; p = .007) and non-complete remission (HR, 2.48; 95% CI, 1.17-5.23; p = .017) were significantly correlated with worse OS. CONCLUSION: Haplo-HSCT may serve as an alternative to MUD-HSCT for the treatment of acute leukemia, offering similar survival outcomes.

LILRB2 promotes immune escape in breast cancer cells via enhanced HLA-A degradation.

PURPOSE: Increased expression of leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is associated with immune evasion in breast cancer (BC). The aim of this study to elucidate the role of LILRB2 in BC progression. METHODS: LILRB2 expression in tumor tissues was detected by immunohistochemical staining. Human leukocyte antigen A (HLA-A) expression in BC cells was detected by Western blotting, and HLA-A ubiquitination was detected by immunoprecipitation and histidine pulldown assay. An in-situ tumor model was established in nude BALB/c mice to verify the role of LILRB2 in immune escape. Finally, the functions and potential mechanisms of LILRB2 in BC progression were explored using in silico data. RESULTS: LILRB2 was upregulated in BC tissues and cells, and correlated positively with poor prognosis. LILRB2 promoted BC progression by downregulating HLA-A expression. Mechanistically, LILRB2 facilitates the ubiquitination and subsequent degradation of HLA-A by promoting the interaction between the ubiquitin ligase membrane-associated ring finger protein 9 (MARCH9) and HLA-A. In syngeneic graft mouse models, LILRB2-expressing BC cells evaded CD8 + T cells and inhibited the secretion of cytokines by the cytotoxic CD8 + T cells. CONCLUSION: LILRB2 downregulates HLA-A to promote immune evasion in BC cells and is a promising new target for BC treatment.

A novel framework for human leukocyte antigen (HLA) genotyping using probe capture-based targeted next-generation sequencing and computational analysis.

Human leukocyte antigen (HLA) genes play pivotal roles in numerous immunological applications. Given the immense number of polymorphisms, achieving accurate high-throughput HLA typing remains challenging. This study aimed to harness the human pan-genome reference consortium (HPRC) resources as a potential benchmark for HLA reference materials. We meticulously annotated specific four field-resolution alleles for 11 HLA genes (HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5) from 44 high-quality HPRC personal genome assemblies. For sequencing, we crafted HLA-specific probes and conducted capture-based targeted sequencing of the genomic DNA of the HPRC cohort, ensuring focused and comprehensive coverage of the HLA region of interest. We used publicly available short-read whole-genome sequencing (WGS) data from identical samples to offer a comparative perspective. To decipher the vast amount of sequencing data, we employed seven distinct software tools: OptiType, HLA-VBseq, HISAT genotype, SpecHLA, T1K, QzType, and DRAGEN. Each tool offers unique capabilities and algorithms for HLA genotyping, allowing comprehensive analysis and validation of the results. We then compared these results with benchmarks derived from personal genome assemblies. Our findings present a comprehensive four-field-resolution HLA allele annotation for 44 HPRC samples. Significantly, our innovative targeted next-generation sequencing (NGS) approach for HLA genes showed superior accuracy compared with conventional short-read WGS. An integrated analysis involving QzType, T1K, and DRAGEN was developed, achieving 100% accuracy for all 11 HLA genes. In conclusion, our study highlighted the combination of targeted short-read sequencing and astute computational analysis as a robust approach for HLA genotyping. Furthermore, the HPRC cohort has emerged as a valuable assembly-based reference in this realm.

Clinical conundrums in pediatric kidney transplantation: What we know about the role of angiotensin II type I receptor antibodies in pediatric kidney transplantation and the path forward.

Antibodies to angiotensin II type 1 receptor (AT1R-Abs) are among the most well-studied non-HLA antibodies in renal transplantation. These antibodies have been shown to be common in pediatric kidney transplantation and associated with antibody-mediated rejection (AMR), vascular inflammation, development of human leukocyte donor-specific antibodies (HLA DSA), and allograft loss. As AT1R-Ab testing becomes more readily accessible, evidence to guide clinical practice for testing and treating AT1R-Ab positivity in pediatric kidney transplant recipients remains limited. This review discusses the clinical complexities of evaluating AT1R-Abs given the current available evidence.

Immune response stability to the SARS-CoV-2 mRNA vaccine booster is influenced by differential splicing of HLA genes.

Many molecular mechanisms that lead to the host antibody response to COVID-19 vaccines remain largely unknown. In this study, we used serum antibody detection combined with whole blood RNA-based transcriptome analysis to investigate variability in vaccine response in healthy recipients of a booster (third) dose schedule of the mRNA BNT162b2 vaccine against COVID-19. The cohort was divided into two groups: (1) low-stable individuals, with antibody concentration anti-SARS-CoV IgG S1 below 0.4 percentile at 180 days after boosting vaccination; and (2) high-stable individuals, with antibody values greater than 0.6 percentile of the range in the same period (median 9525 [185-80,000] AU/mL). Differential gene expression, expressed single nucleotide variants and insertions/deletions, differential splicing events, and allelic imbalance were explored to broaden our understanding of the immune response sustenance. Our analysis revealed a differential expression of genes with immunological functions in individuals with low antibody titers, compared to those with higher antibody titers, underscoring the fundamental importance of the innate immune response for boosting immunity. Our findings also provide new insights into the determinants of the immune response variability to the SARS-CoV-2 mRNA vaccine booster, highlighting the significance of differential splicing regulatory mechanisms, mainly concerning HLA alleles, in delineating vaccine immunogenicity.

[The Polymorphism Analysis of HLA Class II Alleles Based on Next-Generation Sequencing and Prevention Strategy for Allele Dropout].

OBJECTIVE: To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system. METHODS: NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods. RESULTS: A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System. CONCLUSION: The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

Tumor beta2-microglobulin and HLA-A expression is increased by immunotherapy and can predict response to CIT in association with other biomarkers.

Background: Downregulation of MHC class I expression and/or defects in the antigen presentation pathways are commonly reported in human cancers. Numerous studies previously have explored extensively the molecular mechanisms that underlie HLA-class I and Beta2-Microglobulin (B2M) downregulation. However, the techniques presently available to detect expression of MHC class I proteins lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. Furthermore, the dynamics of HLA-class I and B2M expression have not been comprehensively studied as a potential biomarker for immunotherapy. Methods: Using novel, validated, immunohistochemistry (IHC)-based methods for quantifying B2M and HLA-A in tumor samples from diverse cancer types, we have determined loss of B2M and HLA-A proteins in 336 archived, primary specimens and 329 biopsies from metastatic patients collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapy candidates as monotherapy or in combination with CPI. Results: Up to 56% of cases with B2M or HLA-A loss were noted in the investigated tumor types. The frequency of loss was dependent on indication and stage of disease and revealed heterogeneous expression patterns across patients. B2M and HLA-A loss was increased in metastatic lesions compared to primary tumors, indicating selection of MHC class I low clones in metastatic and refractory tumor cells. High on-treatment B2M expression correlated with successful clinical outcome (RECIST), while high baseline B2M did not. A treatment-induced increase of B2M expression was noted in most of the patients with low B2M levels at baseline. The triple biomarker combination of B2M, CD8 and PDL1 strongly improved response prediction to cancer immunotherapy. Conclusion: Our results indicate that B2M and HLA-A loss occurs frequently in tumors and is reversed in most instances following immunotherapy which supports the conclusion that MHC class I loss is not the dominant resistance mechanism to CPI treatment. This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by indication, metastatic status, immunophenotype and immunotherapy treatment. Baseline expression levels of B2M on tumors may be of utility as a constituent of a biomarker panel used for selecting patients for immunotherapy clinical trials.

Frequency distribution of HLA class I and II alleles in Greek population and their significance in orchestrating the National Donor Registry Program.

Human leukocyte antigens (HLA) represent one of the most polymorphic systems in humans, responsible for the identification of foreign antigens and the presentation of immune responses. Therefore, HLA is considered to play a major role in human disorders, donor-recipient matching and transplantation outcomes. This study aimed to determine the HLA class I and II alleles and haplotypes in the Greek population. Moreover, a comparative analysis of HLA alleles and haplotype frequencies found in Greek and pooled European populations was also performed to acquire a better knowledge about the HLA alleles distribution. A total number of 1896 healthy individuals were typed for their HLA alleles in the National Tissue Typing Center of Greece. High-resolution HLA typing for the HLA-A, -B, -C and -DR, -DQ, -DP with the use of the next-generation sequencing analysis was performed, followed by data analysis for establishing the HLA allele and haplotype differences. The results of this study showed that the most frequent alleles for the HLA-A were the A*02:01:01 (27.1%), *24:02:01 (14.4%), *01:01:01 (9.3%), for the HLA-B were the B*51:01:01 (15.3%), *18:01:01 (9.7%), *35:01:01 (6.8%) and for the HLA-C were the C*04:01:01 (15.4%), *07:01:01 (13.1%), *12:03:01 (9.6%). For the HLA class II, the most frequent alleles for the HLA-DRB1 were the DRB1*11:04:01 (16.4%), *16:01:01 (11.3%), *11:01:01 (9.5%), for the HLA-DQB1 were the DQB1*03:01:01 (30.5%), *05:02:01 (15.1%), *05:01:01 (10.6%) and for the HLA-DPB1 were the DPB1*04:01:01 (34.8%), *02:01:01 (11.6%), *04:02:01 (7.3%). Additionally, the most frequent haplotypes were the A*02:01:01∼C*07:01:01-B*18:01:01∼DRB1*11:04:01 (2.3%), followed by the A*01:01:01∼C*07:01:01∼B*08:01:01∼DRB1*03:01:01 (2.2%), A*24:02:01∼C*04:01:01∼B*35:02:01∼DRB1*11:04:01 (1.4%) and A*02:01:01∼C*04:01:01∼B*35:01:01-DRB1*14:01:01 (1.2%). The results herein were comparable to those obtained from the pooled European populations. Moreover, these results can be used for the improvement of the donor-recipient matching procedure and to understand better the disease association in Greece.

Human Leukocyte Antigen Markers for Distinguishing Pustular Psoriasis and Adult-Onset Immunodeficiency with Pustular Reaction.

Pustular skin diseases, with pustular psoriasis (PP) being the prototype, are immune-mediated diseases characterized by the presence of multiple pustules, resulting from neutrophil accumulation in the layer of epidermis. Sterile skin pustular eruption, like PP, is also observed in 20-30% of patients with adult-onset immunodeficiency syndrome (AOID) and anti-interferon γ autoantibodies (IFN-γ), leading to challenges in classification and diagnosis. While the mechanism underlying this similar phenotype remains unknown, genetic factors in relation to the immune system are suspected of playing an important role. Here, the association between human leukocyte antigen (HLA) genes, which play essential roles in antigen presentation, contributing to immune response, and the presence of skin pustules in AOID and PP was revealed. HLA genotyping of 41 patients from multiple centers in Thailand who presented with multiple sterile skin pustules (17 AOID patients and 24 PP patients) was conducted using a next-generation-sequencing-based approach. In comparison to healthy controls, HLA-B*13:01 (OR = 3.825, 95%CI: 2.08-7.035), C*03:04 (OR = 3.665, 95%CI: 2.102-6.39), and DQB1*05:02 (OR = 2.134, 95%CI: 1.326-3.434) were significantly associated with the group of aforementioned conditions having sterile cutaneous pustules, suggesting a common genetic-related mechanism. We found that DPB1*05:01 (OR = 3.851, p = 0.008) and DRB1*15:02 (OR = 3.195, p = 0.033) have a significant association with pustular reaction in AOID patients, with PP patients used as a control. A variant in the DRB1 gene, rs17885482 (OR = 9.073, p = 0.005), was observed to be a risk factor for PP when using AOID patients who had pustular reactions as a control group. DPB1*05:01 and DRB1*15:02 alleles, as well as the rs17885482 variant in the DRB1 gene, were proposed as novel biomarkers to differentiate PP and AOID patients who first present with multiple sterile skin pustules without known documented underlying conditions.

Mass Spectrometry-Based Immunopeptidomics of Peptides Presented on Human Leukocyte Antigen Proteins.

Human leukocyte antigen (HLA) proteins are a group of glycoproteins that are expressed at the cell surface, where they present peptides to T cells through physical interactions with T-cell receptors (TCRs). Hence, characterizing the set of peptides presented by HLA proteins, referred to hereafter as the immunopeptidome, is fundamental for neoantigen identification, immunotherapy, and vaccine development. As a result, different methods have been used over the years to identify peptides presented by HLA proteins, including competition assays, peptide microarrays, and yeast display systems. Nonetheless, over the last decade, mass spectrometry-based immunopeptidomics (MS-immunopeptidomics) has emerged as the gold-standard method for identifying peptides presented by HLA proteins. MS-immunopeptidomics enables the direct identification of the immunopeptidome in different tissues and cell types in different physiological and pathological states, for example, solid tumors or virally infected cells. Despite its advantages, it is still an experimentally and computationally challenging technique with different aspects that need to be considered before planning an MS-immunopeptidomics experiment, while conducting the experiment and with analyzing and interpreting the results. Hence, we aim in this chapter to provide an overview of this method and discuss different practical considerations at different stages starting from sample collection until data analysis. These points should aid different groups aiming at utilizing MS-immunopeptidomics, as well as, identifying future research directions to improve the method.

Helicobacter pylori enhances HLA-C expression in the human gastric adenocarcinoma cells AGS and can protect them from the cytotoxicity of natural killer cells.

Helicobacter pylori (H. pylori) seems to play causative roles in gastric cancers. H. pylori has also been detected in established gastric cancers. How the presence of H. pylori modulates immune response to the cancer is unclear. The cytotoxicity of natural killer (NK) cells, toward infected or malignant cells, is controlled by the repertoire of activating and inhibitory receptors expressed on their surface. Here, we studied H. pylori-induced changes in the expression of ligands, of activating and inhibitory receptors of NK cells, in the gastric adenocarcinoma AGS cells, and their impacts on NK cell responses. AGS cells lacked or had low surface expression of the class I major histocompatibility complex (MHC-I) molecules HLA-E and HLA-C-ligands of the major NK cell inhibitory receptors NKG2A and killer-cell Ig-like receptor (KIR), respectively. However, AGS cells had high surface expression of ligands of activating receptors DNAM-1 and CD2, and of the adhesion molecules LFA-1. Consistently, AGS cells were sensitive to killing by NK cells despite the expression of inhibitory KIR on NK cells. Furthermore, H. pylori enhanced HLA-C surface expression on AGS cells. H. pylori infection enhanced HLA-C protein synthesis, which could explain H. pylori-induced HLA-C surface expression. H. pylori infection enhanced HLA-C surface expression also in the hepatoma Huh7 and HepG2 cells. Furthermore, H. pylori-induced HLA-C surface expression on AGS cells promoted inhibition of NK cells by KIR, and thereby protected AGS cells from NK cell cytotoxicity. These results suggest that H. pylori enhances HLA-C expression in host cells and protects them from the cytotoxic attack of NK cells expressing HLA-C-specific inhibitory receptors.

Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells.

Pancreatic cancer is a lethal disease, harboring a five-year overall survival rate of only 13%. Current treatment approaches thus require modulation, with attention shifting towards liberating the stalled efficacy of immunotherapies. Select chemotherapy drugs which possess inherent immune-modifying behaviors could revitalize immune activity against pancreatic tumors and potentiate immunotherapeutic success. In this study, we characterized the influence of gemcitabine, a chemotherapy drug approved for the treatment of pancreatic cancer, on tumor antigen presentation by human leukocyte antigen class I (HLA-I). Gemcitabine increased pancreatic cancer cells' HLA-I mRNA transcripts, total protein, surface expression, and surface stability. Temperature-dependent assay results indicated that the increased HLA-I stability may be due to reduced binding of low affinity peptides. Mass spectrometry analysis confirmed changes in the HLA-I-presented peptide pool post-treatment, and computational predictions suggested improved affinity and immunogenicity of peptides displayed solely by gemcitabine-treated cells. Most of the gemcitabine-exclusive peptides were derived from unique source proteins, with a notable overrepresentation of translation-related proteins. Gemcitabine also increased expression of select immunoproteasome subunits, providing a plausible mechanism for its modulation of the HLA-I-bound peptidome. Our work supports continued investigation of immunotherapies, including peptide-based vaccines, to be used with gemcitabine as new combination treatment modalities for pancreatic cancer.

HLA Genetics for the Human Diseases.

Highly polymorphic human leukocyte antigen (HLA) molecules (alleles) expressed by different classical HLA class I and class II genes have crucial roles in the regulation of innate and adaptive immune responses, transplant rejection and in the pathogenesis of numerous infectious and autoimmune diseases. To date, over 35,000 HLA alleles have been published from the IPD-IMGT/HLA database, and specific HLA alleles and HLA haplotypes have been reported to be associated with more than 100 different diseases and phenotypes. Next generation sequencing (NGS) technology developed in recent years has provided breakthroughs in various HLA genomic/gene studies and transplant medicine. In this chapter, we review the current information on the HLA genomic structure and polymorphisms, as well as the genetic context in which numerous disease associations have been identified in this region.

Aortitis after switching short-acting granulocyte colony-stimulating factors in a lymphoma patient with HLA-B52.

Aortitis is a rare adverse event of granulocyte colony-stimulating factor (G-CSF) treatment. Several previous studies have described recurrent aortitis caused by re-administration of the same G-CSF. However, no previous studies have examined the safety of switching between short-acting G-CSFs in patients who develop aortitis. We report the case of a 55-year-old man with refractory diffuse large B-cell lymphoma, who developed G-CSF-associated aortitis. The aortitis was triggered by filgrastim and recurred after treatment with lenograstim. The patient possessed human leukocyte antigen B52, which has been implicated in Takayasu arteritis. In addition, a drug-induced lymphocyte stimulation test for lenograstim performed upon detection of recurrent G-CSF-associated aortitis produced a positive result. Our case suggests that switching from one short-acting G-CSF to another does not prevent recurrence of G-CSF-associated aortitis. Although the etiology of G-CSF-associated aortitis has not been fully elucidated, our case also suggests that some patients may be genetically predisposed to aortitis.