CD21 Human

What is the basic structure of human CD21?

Human CD21 (CR2) is a 145 kDa glycoprotein that contains 15-16 short consensus repeats (SCRs), with each domain consisting of approximately 60 amino acid residues . The N-terminal two repeats (SCR1-SCR2) form a critical functional region that serves as the binding site for multiple ligands including complement fragments, EBV, and CD23 . Crystal structure analysis reveals that CD21 SCR1-SCR2 assumes a compact V-shaped conformation, but with surprising flexibility at the SCR1-SCR2 interface . This structural flexibility may facilitate interactions with diverse ligands while maintaining specificity for each binding partner .

How is CD21 expression analyzed in research settings?

Researchers typically employ multiple complementary techniques to analyze CD21 expression:

• Flow cytometry: Using fluorescently-labeled anti-CD21 monoclonal antibodies (such as OKB7, HB5, and B2) to quantify surface expression on different cell populations .

• Semi-quantitative PCR: For measuring CD21 mRNA levels in purified cell populations or cell lines .

• Immunoblotting: To detect CD21 protein expression and assess glycosylation patterns .

• Surface plasmon resonance: For studying binding interactions between CD21 and its ligands .

When analyzing CD21-/low B cell populations specifically, researchers often use additional markers including CD27, CD11c, and T-bet to properly characterize these cells in different disease states .

What are the primary ligands of human CD21?

Human CD21 serves as a receptor for four distinct classes of ligands:

• Complement fragments: iC3b, C3dg, and C3d, which are covalently attached to target antigens .

• Viral proteins: The gp350/220 glycoprotein of Epstein-Barr virus (EBV) .

• Immunoregulatory proteins: CD23, the low-affinity receptor for IgE .

• Cytokines: IFN-α, a multifunctional cytokine important in innate immunity .

All four classes of ligands interact with the SCR1-SCR2 domains of CD21, although they may bind to distinct but overlapping regions within these domains . The affinity of these interactions is in a similar range across all four ligand types, as determined by surface plasmon resonance and ELISA approaches .

How does glycosylation affect CD21 function?

CD21 is heavily glycosylated, but the functional significance of this glycosylation varies by context. Experimental evidence indicates that the ability of CD21 to bind EBV is independent of glycosylation . Both glycosylated and deglycosylated SCR1-SCR2 fragments can prevent EBV attachment to CD21-expressing B cells in a dose-dependent manner . This contrasts with related complement regulatory proteins like CD46, which requires glycosylation for interaction with measles virus . In CD46, glycosylation is thought to help maintain the receptor's conformation, while CD21 appears to maintain its functional structure independently of its glycan modifications .

What methods are used to study CD21-ligand interactions?

Researchers employ several approaches to characterize CD21-ligand interactions:

• Virus binding inhibition assays: Using soluble CD21 fragments to competitively inhibit viral attachment to CD21+ cells .

• Erythrocyte rosetting assays: Sheep erythrocytes coated with C3d (EAC1-C3d) are used to assess C3d-binding capacity of CD21-expressing cells .

• Site-directed mutagenesis: To identify specific amino acids involved in ligand binding, such as the mutation of residues 66-NKYS to 66-NKTI in CD21 .

• Molecular modeling: Computational approaches to predict interactions between CD21 and its ligands, incorporating structural data from crystallography studies .

• Flow cytometry with fluorescently-labeled ligands: Such as FITC-labeled EBV to quantify binding to receptor-bearing cells .

What characterizes CD21-/low B cell populations in humans?

CD21-/low B cells represent a heterogeneous population with several distinct phenotypic and functional characteristics:

• Surface markers: Typically CD21-/lowCD27-CD11c+T-bet+ phenotype, often with expression of inhibitory receptors like FcRL4 and FcRL5 .

• Functional properties: Compared to conventional memory B cells, CD21-/low B cells generally show reduced proliferation in response to BCR stimulation but may respond similarly to TLR or cytokine stimulation .

• Tissue distribution: Found primarily in peripheral blood during disease states, but also present in lymphoid tissues like tonsils in healthy individuals .

• Antigen specificity: In infection contexts, CD21-/low B cells are often enriched for pathogen-specific responses, such as HIV-specific antibodies in HIV infection .

The table below summarizes functional characteristics of CD21-/low B cells in different conditions:

How are CD21-/low B cells implicated in viral infections?

CD21-/low B cells expand in various viral infections and show distinct characteristics depending on the pathogen:

• COVID-19: Critically ill COVID-19 patients exhibit expanded CD21-/lowCD27- B cells compared to healthy individuals . These cells are typically CD21-/lowCD27-IgD-CD38-CD11c+T-bet+/hi and their persistence correlates with poor clinical outcomes . Notably, infection-induced memory B cells generate more CD21-CD27-CD11c+ cells than vaccination-induced memory B cells .

• HIV: During HIV infection, peripheral blood CD21-/lowCD27-CD11c+FcRL4+ B cells appear in correlation with viremia . These cells produce HIV-specific antibodies and decrease with antiretroviral treatment . In lymph nodes of HIV patients, HIV-specific B cells are enriched among CD19hiT-bethi memory B cells, with a CD21-/low phenotype that is not detected in healthy lymph nodes .

• EBV: CD21 serves as the primary receptor for EBV, with the virus glycoprotein gp350/220 binding to SCR1-SCR2 domains . This interaction is distinct from the binding site for C3d .

What are the experimental approaches to studying CD21-/low B cells?

Researchers employ several complementary methods to characterize CD21-/low B cells:

• Flow cytometry panels: Comprehensive phenotyping using markers including CD21, CD27, CD11c, T-bet, FcRL4, and other lineage and activation markers .

• Functional assays: Comparative analysis of proliferation and differentiation potential upon stimulation with various stimuli (BCR crosslinking, TLR ligands, cytokines, CD40L) .

• Antigen-specific assays: Using labeled antigens to identify pathogen-specific B cells within the CD21-/low compartment .

• Transcriptional profiling: RNA sequencing to characterize gene expression patterns and potential regulatory mechanisms .

• Longitudinal studies: Tracking changes in CD21-/low B cell populations over the course of disease progression or treatment .

How do the structural features of CD21 impact its multiple ligand interactions?

The crystal structure of CD21 SCR1-SCR2 reveals a compact V-shaped conformation with flexibility at the domain interface . This structural characteristic likely facilitates interaction with diverse ligands while maintaining specificity. The binding sites for different ligands appear to be overlapping but distinct. For example, site-directed mutagenesis studies identified that residues 66-NKYS in CD21 are critical for EBV binding but not for C3d interaction . Research approaches to address this question include:

• Comparative crystallography: Solving structures of CD21 in complex with different ligands to identify shared and unique interaction surfaces.

• Hydrogen-deuterium exchange mass spectrometry: To map ligand-binding interfaces and conformational changes upon ligand binding.

• Molecular dynamics simulations: To model flexibility at the SCR1-SCR2 interface and its impact on ligand recognition.

What explains the discrepancy between CD21 mRNA expression and surface protein levels in T cells?

Human B and T lymphocytes express similar amounts of CD21 mRNA, yet surface expression of CD21 glycoprotein is detectable on B cells but not on CD4+ or CD8+ T cells . This suggests post-transcriptional regulation mechanisms that might include:

• Alternative splicing: Both B and T cell populations express CD21 mRNA with and without exon 11, contradicting earlier reports that exon 11 expression was restricted to follicular dendritic cells .

• Translational control: Potential differences in translation efficiency between cell types.

• Protein trafficking: Differences in transport of CD21 to the cell surface.

• Protein stability: Differential degradation of CD21 protein in B versus T cells.

Experimental approaches to investigate this discrepancy include ribosome profiling, pulse-chase experiments to track protein synthesis and degradation, and subcellular fractionation to locate intracellular CD21 protein in T cells.

How does CD21 function as an IFN-α receptor and what are the implications for autoimmunity?

CD21 has been identified as a receptor for IFN-α, binding this cytokine with similar affinity as its other well-characterized ligands . The interaction occurs in the same region of SCR1-SCR2 that serves as the binding site for C3d/iC3b, EBV-gp350, and CD23 . This finding has significant implications for understanding autoimmunity, particularly in systemic lupus erythematosus (SLE) where IFN-α plays a major pathogenic role . Research approaches to explore this relationship include:

• Comparative signaling studies: Investigating potential differences between signaling through CD21 versus the canonical type 1 IFN receptor (IFNAR1/2).

• Blocking antibody experiments: Using inhibitory anti-CR2 monoclonal antibodies (such as mAb 171) to assess the specific contribution of CD21 to IFN-α responses in B cells .

• Gene expression analysis: Studying IFN-α-responsive gene induction with and without CD21 inhibition .

• In vivo models: Using CD21-deficient or conditional knockout animals to assess the role of CD21 in IFN-α-mediated autoimmunity.

What are the optimal methods for isolating and characterizing CD21-expressing cells?

When designing experiments to study CD21-positive cells, researchers should consider:

• Cell isolation techniques:

  • Magnetic separation using anti-CD21 antibodies for positive selection

  • Flow cytometry-based cell sorting for high purity and additional marker characterization

  • Density gradient centrifugation for initial enrichment of B cells before CD21-specific isolation

• Antibody selection:

  • Use of well-characterized anti-CD21 clones (OKB7, HB5, B2) for consistency across experiments

  • Validation of antibody binding to the epitope of interest, especially when studying CD21 variants

• Functional validation:

  • Complement binding assays using C3d-coated erythrocytes (EAC1-C3d) to confirm CD21 activity

  • EBV binding assays using FITC-labeled virus to verify receptor functionality

• Controls:

  • Include CD21-negative cell lines (like CEM) as negative controls

  • Use CD21-high cell lines (like Raji) as positive controls

How should researchers approach the study of CD21-/low B cells in different disease contexts?

When investigating CD21-/low B cells in various diseases, consider these methodological approaches:

• Comprehensive phenotyping:

  • Include multiple markers beyond CD21, such as CD27, CD11c, T-bet, and FcRL4/5 to properly characterize subpopulations

  • Use consistent gating strategies across studies to facilitate comparison

• Appropriate controls:

  • Compare with age-matched healthy controls, as CD21-/low B cells expand with age even in healthy individuals

  • Include disease controls to distinguish disease-specific from general inflammatory changes

• Functional assessment:

  • Test multiple stimulation conditions (BCR, TLR, cytokine, CD40L) as CD21-/low B cells respond differently to various stimuli

  • Compare to conventional B cell populations from the same donor

• Longitudinal analysis:

  • Track changes in CD21-/low populations during disease progression or treatment

  • Correlate with clinical parameters to establish relevance

What technical challenges exist in CD21 research and how can they be addressed?

Several technical challenges complicate CD21 research, but methodological solutions exist:

• Challenge: Heterogeneity within CD21-/low populations
  Solution: Use multiparameter flow cytometry or mass cytometry (CyTOF) with clustering algorithms to identify distinct subpopulations

• Challenge: Low frequency of CD21-/low cells in healthy individuals
  Solution: Implement enrichment strategies before analysis or use high-dimensional techniques that require fewer cells

• Challenge: Distinguishing functionally relevant from artifactual loss of CD21 expression
  Solution: Include viability dyes and examine multiple functional parameters alongside CD21 expression

• Challenge: Variability in glycosylation affecting antibody binding
  Solution: Use multiple antibody clones recognizing different epitopes or enzymatic deglycosylation to standardize detection

• Challenge: Translating in vitro findings to in vivo relevance
  Solution: Correlate ex vivo properties with clinical parameters and use appropriate animal models where possible

How does CD21 expression correlate with clinical outcomes in different diseases?

Altered CD21 expression, particularly expansion of CD21-/low B cells, has important clinical implications:

• COVID-19: Expanded CD21-/lowCD27- B cells correlate with disease severity, persisting in patients who succumb to the disease while decreasing in those who recover .

• HIV: CD21-/lowCD27-CD11c+FcRL4+ B cells correlate with viremia and decrease with effective antiretroviral treatment .

• Autoimmune diseases: In systemic lupus erythematosus, CD21-/low B cells expressing high levels of T-bet expand and may contribute to disease pathogenesis through interaction with IFN-α .

• Common Variable Immunodeficiency (CVID): CD21-/low B cells are expanded and show altered functional properties including reduced proliferation to BCR stimulation but increased antibody secretion with cytokine and CD40L stimulation .

Understanding these correlations can inform both diagnostic approaches and therapeutic strategies targeting CD21 or CD21-/low B cells in various clinical contexts.

What are the potential therapeutic implications of targeting CD21 or CD21-dependent pathways?

CD21's involvement in multiple immune processes suggests several therapeutic approaches:

• Blocking viral entry: Soluble forms of CD21 containing only SCR1 and SCR2 can prevent EBV infection by competing for viral binding . This suggests potential therapeutic strategies for preventing or managing EBV-associated diseases.

• Modulating B cell activation: CD21 plays a role in lowering the threshold for B cell activation . Targeting this function could help manage B cell-mediated autoimmune conditions.

• Interfering with IFN-α signaling: The inhibitory anti-CR2 mAb 171 diminishes induction of IFN-α-responsive genes in human peripheral blood B cells . This approach might offer therapeutic benefits in conditions like SLE where IFN-α plays a pathogenic role.

• Targeting CD21-/low B cells: Given their expansion in various diseases, specific depletion or functional modulation of CD21-/low B cells might represent a more targeted approach than general B cell depletion in certain conditions.

These potential therapeutic strategies require further investigation in preclinical models before clinical translation, with careful consideration of the multifunctional nature of CD21 to avoid unintended consequences.