DRB5 Antibody

What is HLA-DRB5 and what is its structural composition?

HLA-DRB5 belongs to the HLA class II beta chain paralogues, forming a heterodimer with an alpha (DRA) chain, both anchored in the cell membrane. The beta chain weighs approximately 26-28 kDa and its gene contains 6 exons with specific functions: exon one encodes the leader peptide, exons 2 and 3 encode the two extracellular domains, exon 4 encodes the transmembrane domain, and exon 5 encodes the cytoplasmic tail. Within the DR molecule, the beta chain contains all the polymorphisms that specify peptide binding specificities, making it critical for immune function .

The expression of HLA-DRB5 is linked with specific allelic variants of DRB1; when these variants are absent, HLA-DRB5 is not expressed. Notably, DRB1 is expressed at levels approximately five times higher than its paralogues DRB3, DRB4, and DRB5. There are also four related pseudogenes: DRB2, DRB6, DRB7, DRB8, and DRB9, which contribute to the genetic complexity of this region .

How do HLA-DRB5 molecules function in the immune system?

HLA-DRB5 plays a central role in the immune system by presenting peptides derived from extracellular proteins to immune cells. This MHC class II molecule is primarily expressed in antigen-presenting cells (APCs), including B lymphocytes, dendritic cells, and macrophages . The antigen presentation process involves capturing extracellular proteins, processing them into peptides, and displaying these peptides on the cell surface via HLA-DRB5 and other MHC class II molecules.

Research has demonstrated that HLA-DRB5 participates in critical immune processes including T cell activation. In immune thrombocytopenia (ITP) studies, elevated expressions of HLA-DRB5, MHC-II, and co-stimulatory molecules (CD80, CD86) were observed, along with increased percentages of CD8+ T cells in the peripheral blood of affected mice. Knockdown of HLA-DRB5 mitigated ITP by increasing platelet levels, downregulating expressions of MHC-II and co-stimulatory molecules, and inactivating CD8+ T cells .

What methodological approaches are most effective for studying HLA-DRB5 expression?

When investigating HLA-DRB5 expression, researchers typically employ multiple complementary techniques to ensure robust and reliable results:

• Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR): This method allows precise quantification of HLA-DRB5 mRNA expression levels. Primers should be designed to specifically target the unique regions of DRB5 to avoid cross-reactivity with other HLA-DRB genes .

• Western blot analysis: For protein-level detection, western blotting using specific anti-HLA-DRB5 antibodies allows researchers to quantify expression levels across different experimental conditions or disease states .

• Immunofluorescence assays: This technique enables visualization of HLA-DRB5 localization within cells and tissues, providing spatial information about expression patterns .

• Flow cytometry: Particularly useful for analyzing HLA-DRB5 expression on specific cell populations, flow cytometry allows for quantification of expression levels on individual cells within heterogeneous samples .

For optimal results, researchers should validate antibody specificity using positive and negative controls, as well as knockdown or knockout models where possible.

What are the known disease associations with HLA-DRB5?

HLA-DRB5 has been associated with several diseases and immune-related conditions:

Research indicates that HLA-DRB5 may influence disease pathogenesis through its role in antigen presentation and subsequent T cell activation. The mechanisms underlying these associations involve complex interactions between genetic factors and immune system components .

How does HLA-DRB5 influence COVID-19 vaccine-induced antibody responses?

Recent population-based research involving 357,806 vaccinated participants with high-resolution HLA genotyping data has revealed significant associations between specific HLA alleles and antibody responses to COVID-19 vaccines. HLA-DRB501:01 was identified as part of a cluster (Cluster 1: DQA101:02; DRB501:01; DRB115:01) associated with antibody response .

The study methodology involved:

• Cross-sectional cohort analysis of 175,000 participants with antibody serology test results who received COVID-19 vaccines.

• Stratification of participants based on vaccination status (one dose vs. two doses).

• Comprehensive HLA genotyping and antibody response measurement.

• Statistical analysis to identify associations between HLA alleles and antibody positivity.

Results demonstrated that while HLA-DQB106 alleles showed the strongest association with enhanced antibody response (particularly DQB106:04), several other HLA alleles, including those in linkage disequilibrium with HLA-DRB5*01:01, independently influenced antibody responses to COVID-19 vaccination. These findings suggest that HLA-DRB5 participates in a network of HLA genes that collectively modulate vaccine-induced immunity .

What experimental approaches can effectively elucidate the role of HLA-DRB5 in antigen presentation?

Investigating HLA-DRB5's role in antigen presentation requires sophisticated experimental approaches:

• Peptide binding assays: Researchers can use purified HLA-DRB5 molecules to assess binding affinity for various peptides. This can be accomplished through competitive binding assays using labeled reference peptides or through direct binding measurements using techniques such as surface plasmon resonance .

• Structural biology approaches: X-ray crystallography and cryo-electron microscopy can reveal the three-dimensional structure of HLA-DRB5 in complex with peptides, providing insights into binding mechanisms and specificities.

• Immunopeptidomics: This approach involves identifying peptides naturally presented by HLA-DRB5 molecules using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This technique allows for comprehensive characterization of the peptide repertoire presented by HLA-DRB5 in various cellular contexts .

• T cell activation assays: Functional studies using antigen-presenting cells expressing HLA-DRB5 and T cells with appropriate T cell receptors can demonstrate the ability of HLA-DRB5 to present specific peptides and activate T cells.

• CRISPR-Cas9 gene editing: Creating cellular models with specific modifications to HLA-DRB5 can help determine structure-function relationships in antigen presentation.

These methodological approaches provide complementary information about HLA-DRB5's role in antigen presentation and can be combined to obtain a comprehensive understanding of its function.

How can researchers investigate the combined effects of multiple HLA alleles on immune responses?

Investigating the combined effects of multiple HLA alleles represents a significant challenge due to the complex linkage disequilibrium patterns and functional interactions between these genes. Recent research has demonstrated effective methodological approaches:

• Genetic scoring systems: In the COVID-19 vaccine response study, researchers developed a genetic score (GS) based on the combined effects of multiple independent HLA alleles (including seven validated HLA alleles: DQB106:04, DQA101:02, DRB301:01, C16:01, DPB110:01, A03:01, DQA1*01:01). This scoring system allowed for the identification of individuals with significantly increased or decreased risk of breakthrough COVID-19 based on their HLA genotype .

• Cluster analysis: Researchers identified several haplotypes or clusters of HLA alleles among validated alleles affecting antibody responses to vaccines:

  • Cluster 1: DQA101:02; DRB501:01; DRB1*15:01

  • Cluster 2: DRB301:01; DRB103:01; DQB1*02:01

  • Cluster 3: DRB101:01; DQB105:01; DQA1*01:01

  • Cluster 4: DQB106:04; DRB303:01; DRB1*13:02

• Removal of highly correlated alleles: After identifying clusters, researchers removed highly correlated alleles from each cluster to identify the independent effects of specific HLA alleles on the outcome of interest .

• Dose-dependent correlation analysis: This approach examines the relationship between antibody response and clinical outcomes predicted by each HLA allele, revealing dose-dependent effects across multiple HLA genes .

These methodological approaches allow for a more comprehensive understanding of how multiple HLA alleles, including HLA-DRB5, collectively influence immune responses and disease susceptibility.

What are the methodological considerations when studying HLA-DRB5's role in autoimmune disorders?

Research into HLA-DRB5's role in autoimmune disorders requires careful methodological considerations:

• Disease model selection: For immune thrombocytopenia (ITP) research, investigators established murine models through injection of guinea pig anti-mouse platelet serum (GP-APS). This approach allowed for monitoring of platelet counts in peripheral blood during disease progression .

• Comprehensive phenotyping: Researchers quantified expressions of HLA-DRB5, MHC-II, and co-stimulatory molecules (CD80, CD86) using multiple techniques (qRT-PCR, western blot, immunofluorescence) to comprehensively characterize the molecular changes associated with disease .

• Immune cell analysis: Flow cytometry analysis of CD8+ T cell percentages provided insights into the cellular immune response mechanisms influenced by HLA-DRB5 .

• Gene knockdown validation: To establish causality, researchers utilized HLA-DRB5 knockdown approaches to demonstrate that downregulation of HLA-DRB5 restored peripheral platelet counts in ITP mice by reducing MHC-II-mediated antigen presentation of macrophages and inhibiting the activation of CD8+ T cells .

• Mechanistic pathway investigation: Studies revealed that HLA-DRB5 affects autoimmune conditions through specific mechanisms, such as MHC-II-mediated antigen presentation by macrophages to CD8+ T cells, providing valuable insights into potential therapeutic targets .

These methodological approaches enable researchers to elucidate the complex roles of HLA-DRB5 in autoimmune disorders, from molecular mechanisms to cellular interactions and disease manifestations.

How can researchers assess the clinical significance of HLA-DRB5 genetic variations?

Assessing the clinical significance of HLA-DRB5 genetic variations requires robust methodological approaches:

• Population-based studies with large cohorts: The COVID-19 vaccine response study included 357,806 vaccinated participants with high-resolution HLA genotyping data, providing sufficient statistical power to detect significant associations .

• Clinical outcome correlation: Researchers examined the relationship between HLA alleles (including those in linkage disequilibrium with HLA-DRB5) and clinical outcomes, such as breakthrough COVID-19 infection and disease severity, demonstrating that while individual alleles might not show significant associations, their combined effects were clinically relevant .

• Mendelian randomization analysis: This approach was used to estimate the portion of vaccine effectiveness against SARS-CoV-2 infection and against severe COVID-19 attributable to vaccine-induced antibodies, thereby establishing causal relationships between genetic factors, immune responses, and clinical outcomes .

• Genetic scoring systems: A genetic score based on validated HLA alleles identified 5% of participants who had a 26% (HR 1.26 95% CI 1.01 to 1.58) or a 34% (HR 1.34 95% CI 1.08 to 1.67) increased risk of severe COVID-19 based on first-dose or second-dose genetic scores, respectively .

• Ethnic group stratification: Researchers validated that the associations between HLA alleles and immune responses were consistent across different ethnic groups, enhancing the generalizability of their findings .

These methodological approaches provide a framework for assessing the clinical significance of HLA-DRB5 genetic variations in various disease contexts and population groups.