HLA-DRB5 Antibody

What is HLA-DRB5 and what is its role in the immune system?

HLA-DRB5 belongs to the HLA class II beta chain paralogues. It forms a heterodimeric protein with an alpha (DRA) chain, both anchored in the cell membrane. This molecule plays a central role in immune system function by presenting peptides derived from extracellular proteins to T cells. HLA-DRB5 is primarily expressed on antigen-presenting cells (APCs) including B lymphocytes, dendritic cells, and macrophages .

The HLA-DRB5 gene contains 6 exons with specific functions:

• Exon 1: Encodes the leader peptide

• Exons 2-3: Encode the two extracellular domains

• Exon 4: Encodes the transmembrane domain

• 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 antigen presentation and immune recognition .

How does HLA-DRB5 expression compare to other HLA-DRB paralogues?

HLA-DRB1 is expressed at levels approximately five times higher than its paralogues, including HLA-DRB3, HLA-DRB4, and HLA-DRB5. The presence of DRB5 is linked with specific allelic variants of DRB1; when these variants are absent, DRB5 is not expressed. This linkage disequilibrium has significant implications for both research methodology and clinical applications .

There are also four related pseudogenes identified: DRB2, DRB6, DRB7, DRB8, and DRB9, which add complexity to research in this area . Understanding this expression pattern is essential when designing experimental protocols to study HLA-DRB5.

What are the standard methods for detecting HLA-DRB5 in research samples?

For HLA-DRB5 detection, several validated techniques are employed:

• TaqMan gene-specific assay: This PCR-based approach uses specific primers (5′-ACGTTTCCTGTGGCAGCCTAA-3′ and 5′-TGCACTGTGAAGCTCTCCACAA-3′) along with VIC-labeled DRB5-specific probes. The reaction includes internal controls (β-globin) to confirm successful amplification .

• Western blot analysis: Typically performed with antibody dilutions of 1:1000-1:3000, this technique can detect the HLA-DRB5 protein (approximately 26-28 kDa) in cell lysates. Secondary antibody detection commonly uses HRP Goat Anti-Rabbit IgG at 1:10000 dilution, with ECL-based visualization .

• Next-Generation Sequencing (NGS): This approach uses PCR-based amplification of HLA followed by Illumina MiSeq technology for comprehensive genotyping. For HLA-DRB5, exons 2 and 3 are typically sequenced to determine allelic variants .

How does HLA-DRB5 contribute to the pathogenesis of immune thrombocytopenia (ITP)?

Recent research indicates that HLA-DRB5 plays a significant role in ITP pathogenesis through several mechanisms:

• Elevation in ITP: Expression of HLA-DRB5 is markedly increased in peripheral blood of ITP models compared to control groups. In murine models, HLA-DRB5 mRNA expression increases significantly (p < 0.001) within 2-3 days following ITP induction .

• MHC-II-mediated antigen presentation: HLA-DRB5 upregulates MHC-II and co-stimulatory molecules (CD80, CD86) on antigen-presenting cells, enhancing antigen presentation to CD8+ T cells, which subsequently contribute to platelet destruction .

• Impact on CD8+ T cell activation: Flow cytometry analyses have shown that HLA-DRB5 increases the percentage of CD8+ T cells while decreasing CD4+ T cells in peripheral blood of ITP mice. When HLA-DRB5 is knocked down, this imbalance is reversed .

Experimental data from ITP mouse models demonstrates that HLA-DRB5 knockdown mitigates ITP by:

• Increasing peripheral platelet levels

• Downregulating expression of MHC-II and co-stimulatory molecules

• Inhibiting activation of CD8+ T cells

What experimental approaches are used to study HLA-DRB5 in ITP mouse models?

Researchers employ several methodological approaches to investigate HLA-DRB5 in ITP:

• ITP mouse model induction: Models are established using guinea pig anti-mouse platelet serum (GP-APS) injections every other day for 15 days. Platelet counts are monitored in tail venous blood using automated blood cell counters .

• Gene expression analysis:

  • qRT-PCR: For quantifying HLA-DRB5, MHC-II, CD80, and CD86 mRNA expressions

  • Western blot: Using antibodies against HLA-DRB5 (30 kDa, Proteintech), MHC-II (29 kDa, Abcam) and GAPDH (36 kDa, Abcam)

  • Immunofluorescence assay: For detecting positive expressions of MHC-II, CD80, and CD86 in peripheral blood mononuclear cells (PBMCs)

• Genetic manipulation approaches:

  • HLA-DRB5 knockdown using shRNA vectors

  • HLA-DRB5 overexpression using adenoviral vectors

  • Co-administration of immune receptor expressed on myeloid cells-1 (IREM-1) monoclonal antibody to assess reversal effects

• Flow cytometry analysis: To determine CD8+ and CD4+ T cell percentages in peripheral blood following ITP induction and genetic manipulations .

What is the relationship between HLA-DRB5 and multiple sclerosis (MS)?

The relationship between HLA-DRB5 and multiple sclerosis is complex:

This research highlights the importance of studying populations with diverse HLA haplotypes to distinguish between primary susceptibility genes and disease modifiers.

Why is HLA-DRB5 typing important in transplantation medicine?

HLA-DRB5 typing is critical in transplantation medicine for several reasons:

• Transplant outcome prediction: Complete HLA typing, including HLA-DRB5, is essential for accurately predicting graft compatibility and transplant outcomes. The beta chain contains polymorphisms that specify peptide binding specificities, which influence immune responses to transplanted tissues .

• Virtual crossmatching reliability: The International Antibody Consensus Group supported by the Transplantation Society recommends donor typing for multiple HLA loci, including HLA-DRB3, DRB4, and DRB5, particularly for recipients enrolled in paired exchange registries. DSA should be consistently negative against these antigens to rely on virtual crossmatching alone .

• Unexpected positive crossmatches: Incomplete HLA typing that omits DRB5 can lead to unexpected positive crossmatches, especially in highly sensitized transplant candidates. This can result in transplant delays or cancellations .

The Histocompatibility Committee regularly reviews and updates the equivalency tables in transplantation policy to reflect advancements in HLA testing. These updates provide transplant professionals with current resources for performing and interpreting final crossmatches and considering organ offers .

What methodologies are recommended for comprehensive HLA-DRB5 typing in transplantation research?

For comprehensive HLA-DRB5 typing in transplantation research, the following methodologies are recommended:

• Next-Generation Sequencing (NGS):

  • PCR-based amplification of HLA followed by Illumina MiSeq technology

  • Includes bar coding for individual sample tracking

  • Covers exons 2 and 3 for all DR alleles, including DRB5

  • Analytical tools to define haplotypes and genotypes (developed in collaboration with specialized genetics companies)

  • Demonstrated 100% accuracy on >2,000 control samples

• Risk stratification approaches: Combination of Single Antigen Beads (SAB) and Flow Cytometric Crossmatch (Flow XM) testing is recommended. The following table outlines risk stratification guidelines:

AMR = Antibody Mediated Rejection

• Complete HLA typing panel: The International Antibody Consensus Group recommends typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA, DQB, DPA, and DPB for all potential recipients in paired exchange registries .

How can researchers differentiate between antibodies to native versus denatured HLA-DRB5?

Differentiating between antibodies to native versus denatured HLA-DRB5 is crucial for accurate interpretation of crossmatch results. Methodological approaches include:

• Acid treatment of Single Antigen Beads (SAB): When SAB results are positive but Flow Cytometric Crossmatch results are negative, researchers should perform acid treatment of SAB. If antibody reactivity persists after acid treatment, it suggests the alloantibody is directed toward denatured HLA rather than native conformations .

• Laboratory consultation: In cases where discrepancies exist between SAB and crossmatch results, consultation with HLA laboratory specialists is recommended to rule out technical factors that may affect antibody detection .

• Evaluation of donor/recipient HLA typing completeness: Unexpected positive crossmatches may occur if donor and/or recipient are not completely typed for HLA, especially for highly sensitized transplant candidates. Comprehensive typing including HLA-DRB5 is essential .

This methodological approach helps distinguish clinically relevant antibodies from those that may not impact transplant outcomes.

What are the challenges in studying HLA-DRB5 polymorphisms and how can they be addressed?

Studying HLA-DRB5 polymorphisms presents several challenges that researchers must address:

• Strong linkage disequilibrium: The identification of true predisposing genes within susceptibility haplotypes is handicapped by strong linkage disequilibrium across the locus. This makes it difficult to isolate the effects of HLA-DRB5 from other genes .

  Solution: Study populations with greater MHC haplotypic diversity and distinct patterns of linkage disequilibrium, such as African Americans, who are particularly informative for fine mapping efforts .

• Conditional dependencies: The presence of HLA-DRB5 is linked with allelic variants of HLA-DRB1, and it may be omitted in certain haplotypes .

  Solution: Use conditional haplotype analysis to reveal independent susceptibility signals. This approach has been successful in identifying signals at class III loci independent of DRB1 .

• DRB5-null individuals: Rare DRB5-null individuals have been described in African populations, complicating standardized detection approaches .

  Solution: Implement validated gene-specific TaqMan assays with internal positive controls (β-globin) to confirm reaction success and accurate detection of DRB5 presence or absence .

• Expression level variations: DRB1 is expressed at a level five times higher than its paralogues DRB3, DRB4, and DRB5, which can affect detection sensitivity .

  Solution: Adjust assay sensitivity parameters and use standardized quantification approaches when comparing expression levels across these paralogues.

How can researchers effectively analyze the impact of HLA-DRB5 on specific autoantibody associations?

To effectively analyze the impact of HLA-DRB5 on specific autoantibody associations, researchers should consider the following methodological approaches:

• Z-score analysis: When assessing associations between HLA-DRB haplotypes and autoantibodies, researchers can employ z-score analysis to indicate positive associations, negative associations, or lack of statistical association. This approach helps in interpreting complex patterns between various haplotypes and multiple autoantibodies .

• Reference haplotype comparison: Establish a common reference haplotype (e.g., DRB104:01:01-DRB401:03:01) against which other haplotypes can be compared. This controls for background genetic effects and isolates the impact of specific HLA-DRB5 alleles .

• Autoantibody-specific analysis: Rather than looking at general autoantibody presence, analyze associations with specific autoantibodies. Research has shown that different HLA-DRB3, DRB4, and DRB5 alleles may regulate autoantibody associations through specific autoantibodies (e.g., ZnT8QA and ZnT8RA) .

• Uncoupling genetic effects: In populations where HLA-DRB5 can be present or absent independent of other loci, analyze disease associations in both contexts to determine if HLA-DRB5 is acting as a primary susceptibility factor or as a modifier of other genetic effects .

What are emerging applications of HLA-DRB5 antibodies in immunopeptidomics?

HLA and MHC antibodies, including HLA-DRB5 antibodies, are increasingly important in the emerging field of immunopeptidomics. Key methodological approaches include:

• Peptide identification workflow:

  • Immunoprecipitation of HLA-peptide complexes using HLA-DRB5 antibodies

  • High-performance liquid chromatography coupled to tandem Mass Spectrometry for peptide identification

  • Bioinformatic analysis to characterize neoantigens

• Applications in neoantigen discovery: HLA-DRB5 antibodies facilitate the identification and characterization of neoantigens, which are critical for:

  • Understanding autoimmune disease mechanisms

  • Developing targeted immunotherapies

  • Creating personalized cancer vaccines

• Methodological considerations: Researchers must carefully select antibodies with appropriate specificity and affinity for HLA-DRB5, as cross-reactivity with other HLA-DRB molecules can confound results due to the high degree of sequence homology among these paralogues .

This growing field represents a promising frontier in understanding the role of HLA-DRB5 in antigen presentation and immune response modulation.

How might HLA-DRB5 research contribute to developing new therapeutic approaches for autoimmune diseases?

Based on current research findings, HLA-DRB5-targeted therapeutic approaches hold significant potential for autoimmune disease treatment:

• For Immune Thrombocytopenia (ITP):

  • Targeting HLA-DRB5 expression: Research has shown that HLA-DRB5 knockdown mitigates ITP by increasing peripheral platelet levels, suggesting RNA interference or CRISPR-based approaches to modulate HLA-DRB5 expression might be therapeutic .

  • Co-stimulatory molecule inhibition: Since HLA-DRB5 upregulates co-stimulatory molecules (CD80, CD86), therapeutic approaches targeting these interactions may disrupt the pathogenic pathway .

  • Combined approach with IREM-1 mAb: Studies have shown that IREM-1 monoclonal antibody can reverse the effects of HLA-DRB5 overexpression, suggesting potential combination therapies .

• For Multiple Sclerosis (MS):

  • Protective role of HLA-DRB5: Since HLA-DRB5-null subjects appear to be at increased risk for developing secondary progressive MS, therapeutic approaches that enhance HLA-DRB5 function might help attenuate disease severity .

  • Differential targeting based on genetic profile: Treatment strategies could be personalized based on patient HLA-DRB5 status, with different approaches for those with and without functional HLA-DRB5 .

• Methodological considerations for therapeutic development:

  • Model systems must account for species-specific differences in HLA expression

  • Targeted therapies should consider linkage disequilibrium with other HLA loci

  • Safety monitoring must evaluate potential impacts on beneficial immune functions

These approaches represent promising avenues for translating HLA-DRB5 research into clinical applications for autoimmune disease management.

What are the optimal storage and handling conditions for HLA-DRB5 antibodies in research settings?

For optimal results in HLA-DRB5 antibody research, the following storage and handling protocols are recommended:

• Storage temperature: Store antibodies at -20°C to maintain stability and activity. This is particularly important for polyclonal antibodies against HLA-DRB5 .

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles can degrade antibody quality and reduce specificity. Aliquot antibodies upon receipt to minimize the number of freeze-thaw cycles .

• Formulation considerations:

  • Optimal formulation: PBS (pH 7.3) with 50% glycerol provides stability during storage

  • Preservative: Low concentrations (e.g., 0.01% Thimerosal) help prevent microbial contamination without affecting antibody function

• Shipping conditions: Antibodies should be shipped with polar packs to maintain appropriate temperature during transport. Upon receipt, immediate storage at the recommended temperature is crucial .

• Working dilution preparation: When preparing working dilutions for specific applications like Western blot (1:1000-1:3000), use fresh dilutions rather than storing diluted antibody for extended periods .

Adherence to these technical considerations ensures reliable and reproducible results in HLA-DRB5 antibody-based research protocols.

What are the most sensitive and specific methods for detecting low-abundance HLA-DRB5 in complex biological samples?

Detecting low-abundance HLA-DRB5 in complex biological samples requires highly sensitive and specific methodologies:

• TaqMan gene-specific PCR assay:

  • Sensitivity: Can detect even single copies of the HLA-DRB5 gene

  • Specificity: Uses validated gene-specific primers and probes

  • Internal controls: Incorporates β-globin as a positive control to confirm successful amplification

  • Technical parameters: 40 cycles of 95°C for 15s and 60°C for 1 min using specialized detection systems

• Enhanced Western blot techniques:

  • Sample preparation: Homogenize samples in RIPA Buffer according to manufacturers' protocols

  • Protein quantification: Use BCA Protein Assay Kit for precise loading

  • Signal amplification: ECL-based detection systems enhance sensitivity

  • Loading controls: GAPDH (36 kDa) serves as an appropriate loading control

• Next-Generation Sequencing approaches:

  • Depth of coverage: Higher coverage depth enhances detection of rare alleles

  • Targeted sequencing: Focus on exons 2 and 3 for enhanced sensitivity

  • Bioinformatic analysis: Specialized analytical tools for accurate haplotype determination even in complex samples

• Immunoprecipitation followed by mass spectrometry:

  • Enhanced sensitivity: Can detect low-abundance proteins in complex mixtures

  • Specificity: Can distinguish between highly similar HLA-DR molecules based on unique peptide signatures

  • Application: Particularly valuable for immunopeptidomic studies