KLRB1 Human, Sf9

What is KLRB1 and what cellular distribution pattern does it exhibit?

KLRB1 (Killer Cell Lectin-Like Receptor B1), also known as CD161, is a C-type lectin-like receptor primarily expressed on natural killer (NK) cells, T cells, and NKT cells. It functions as an inhibitory receptor on NK cells when binding to its cognate ligand CLEC2D (also known as LLT1), inhibiting cytotoxic function and cytokine secretion . On T and NKT cells, it serves as a co-stimulatory receptor promoting IFNγ secretion . Most notably, KLRB1 is expressed on Th17 and Tc17 lymphocytes where it presumably promotes targeting into CLEC2D-expressing immunologically privileged niches .

Methodologically, KLRB1 expression can be quantified using flow cytometry, quantitative PCR, and single-cell RNA sequencing. Studies have shown that KLRB1 is only expressed by small subpopulations of human blood T cells, but its expression can be induced in specific environments such as tumor microenvironments .

How does KLRB1 function differ between NK cells and T cell subsets?

KLRB1 exhibits distinct functional profiles across different immune cell populations:

In NK cells:

• Functions as an inhibitory receptor

• Binding to CLEC2D inhibits cytotoxic function and cytokine secretion

• Expression decreases after interferon-β treatment in MS patients

In T cells:

• Serves as a co-stimulatory receptor promoting IFNγ secretion

• CD4+ KLRB1+ T cells exhibit greater IL-17A, IL-21, IL-22, and IFN-γ secretion upon stimulation

• Significantly higher proportions of CCR5+, CCR2+, CX3CR1+, CCR6+, and CXCR3+ cells are found among CD4+ KLRB1+ T cells compared to CD4+ KLRB1- T cells

• Marks activated T cells, with CD25 and Ki-67 expression significantly greater in CD4+ KLRB1+ T cells than in CD4+ KLRB1- T cells

These functional differences suggest context-specific roles in immune regulation and highlight the importance of studying KLRB1 function in isolated cell populations using techniques such as cell sorting followed by functional assays.

What is the structure of KLRB1 and how does it interact with its ligand?

Human KLRB1 is a C-type lectin-like receptor with an extracellular lectin-like domain connected to a transmembrane region via a stalk region. The natural form is expressed on cell surfaces, but soluble forms can be produced for research purposes .

Key structural features include:

• C-type lectin-like domain responsible for ligand binding

• N-terminal stalk region with variable length affecting expression efficiency

• Potential glycosylation sites that impact function and stability

KLRB1 interacts with its cognate protein ligand CLEC2D (LLT1), and this interaction has significant immunological consequences. Binding of CLEC2D to the KLRB1 receptor inhibits the cytotoxic function of NK cells and cytokine secretion . The interaction appears to have different effects in T cells, potentially serving a co-stimulatory function.

The detailed structural basis of this interaction can be studied using purified recombinant proteins. Research has shown that soluble KLRB1 can be produced that is "homogeneous, deglycosylatable, crystallizable and monomeric in solution," as confirmed by size-exclusion chromatography, multi-angle light scattering, and analytical ultracentrifugation .

What are the advantages and challenges of using Sf9 insect cells for KLRB1 expression?

Sf9 insect cells offer several advantages for KLRB1 expression:

Advantages:

• Can perform post-translational modifications including basic glycosylation

• Higher expression levels than many mammalian systems

• Better protein folding capacity than bacterial systems

• Cost-effective compared to mammalian cell culture

• Scalable in suspension culture

Challenges:

• Insect cell glycosylation differs from human patterns

• Requires optimization of construct design, particularly the length of the N-terminal stalk region

• May produce heterogeneous protein populations

• Variable expression levels between batches

Research has shown that expression of soluble KLRB1 in Sf9 cells requires careful optimization of constructs. Studies have tested libraries of expression constructs in pOPING vector containing the extracellular lectin-like domain with different lengths of the putative N-terminal stalk region . This approach helps identify the most productive variant for structural and functional studies.

How do different expression tags affect KLRB1 solubility and function?

Expression tags can significantly impact KLRB1 solubility, yield, and functionality when produced in heterologous systems:

Researchers have tested various protein tags (SUMO, TRX, GST, MsyB) on the expression of soluble KLRB1 in E. coli, but encountered challenges with protein folding and solubility . The choice of tag should consider:

• Impact on protein folding and native structure

• Ease of tag removal after purification

• Potential interference with ligand binding

• Effect on crystallization properties

For structural and functional studies, smaller tags with minimal impact on native structure are preferred, and tag removal may be necessary to ensure physiologically relevant results.

What is the optimal construct design for high-yield soluble KLRB1 expression?

Optimal construct design for soluble KLRB1 expression requires careful consideration of domain boundaries and expression systems:

The most successful approach involved:

• Using the extracellular lectin-like domain with optimized N-terminal stalk region length

• Testing multiple constructs with varying boundaries in expression vector libraries

• Evaluating expression in different systems (Sf9 and HEK293 cells)

The highest yield of soluble, functional KLRB1 was achieved by:

• Stable expression in suspension-adapted HEK293S GnTI- cells

• Utilizing pOPINGTTneo expression vector

• Including properly defined boundaries of the extracellular domain

This approach produced protein that was "homogeneous, deglycosylatable, crystallizable and monomeric in solution," as confirmed by size-exclusion chromatography, multi-angle light scattering, and analytical ultracentrifugation .

For researchers seeking to express KLRB1, a systematic approach testing multiple constructs with precisely defined domain boundaries is recommended, with particular attention to the length of the N-terminal stalk region.

How is KLRB1 expression altered in autoimmune diseases?

KLRB1 expression shows significant alterations in multiple autoimmune conditions:

In Multiple Sclerosis (MS):

• Blood from MS patients showed higher KLRB1 expression compared to healthy controls (P<0.001)

• KLRB1 expression decreased significantly (P<0.001) after interferon-β treatment

• Expression mainly decreased in NK cells of interferon-β treated patients

• A genome-wide association study identified a marginally significant association between the KLRB1 gene SNP rs4763655 and MS (P=0.046, odds ratio=1.06 (1.00-1.13))

In Primary Sjögren's Syndrome (pSS):

• KLRB1 expression in CD4+ T cells was markedly elevated compared to healthy controls

• Expression significantly correlated with clinical disease indicators

• KLRB1+ CD4+ T cells exhibited greater IL-17A, IL-21, IL-22, and IFN-γ secretion upon stimulation

• ROC curve analysis suggested potential utility as an auxiliary diagnostic marker for pSS

These disease-specific alterations in KLRB1 expression suggest it plays important roles in autoimmune pathogenesis, potentially by modulating T cell activation, cytokine production, or tissue targeting through chemokine receptor regulation.

What role does KLRB1 play in cancer immunology, particularly in glioblastoma?

KLRB1 has emerged as a significant factor in cancer immunology, with particular relevance to glioblastoma multiforme (GBM):

In GBM microenvironment:

• KLRB1 was identified as one of the top gene products expressed in the tumor

• Expression is induced in T cells within the GBM microenvironment

• Functions as an inhibitory receptor for human T cells by binding to the CLEC2D ligand on tumor cells

• Inactivation of the KLRB1 gene in primary human T cells greatly enhances their cytotoxic function within tumors in a humanized mouse model

Mechanistically, KLRB1-CLEC2D interaction appears to create an immunosuppressive axis in GBM:

• Binding of CLEC2D to the KLRB1 receptor inhibits cytotoxic function

• Suppresses cytokine secretion in tumor-infiltrating lymphocytes

• May contribute to immune evasion by GBM cells

These findings have led to therapeutic development strategies focusing on blocking KLRB1-CLEC2D interaction or reducing KLRB1 expression in tumor-infiltrating lymphocytes as potential approaches for enhancing anti-tumor immunity .

How does interferon-β treatment affect KLRB1 expression in multiple sclerosis?

Interferon-β (IFN-β) treatment in multiple sclerosis patients has a significant modulatory effect on KLRB1 expression:

Key findings:

• KLRB1 expression decreased significantly (P<0.001) after IFN-β treatment in MS patients

• The decrease in expression was predominantly observed in NK cells rather than T cells

• This alteration in expression correlates with treatment response

This differential regulation suggests a potential mechanism of action for IFN-β in MS therapy:

• Reduction of KLRB1 expression may alter NK cell function

• Changed receptor profile could affect immune cell trafficking or activation

• Modified cytokine production patterns in KLRB1-expressing cells

The cell-type specific effect (primarily in NK cells) highlights the complex immunomodulatory actions of IFN-β and suggests that monitoring KLRB1 expression could potentially serve as a biomarker for treatment response in MS patients.

What therapeutic strategies are being developed to target the KLRB1 pathway in cancer?

Several therapeutic approaches targeting KLRB1 are being developed for cancer treatment, particularly for glioblastoma multiforme (GBM):

• Blocking KLRB1-CLEC2D Interaction:

  • Antibodies or fragments that bind KLRB1

  • Antibodies or fragments that bind CLEC2D

  • Soluble CD161 protein or fragments that competitively bind CLEC2D ligands

• Reducing KLRB1 Expression:

  • Programmable nucleic acid modifying agents (CRISPR-Cas, zinc fingers, TALEs, or meganucleases)

  • CRISPR systems including Cas9, Cas12, Cas13, or Cas14 specifically targeting KLRB1

• Modified T Cell Approaches:

  • T cells modified to have decreased KLRB1 expression or activity

  • Combination with checkpoint blockade therapy (anti-PD-1, anti-CTLA4, anti-PD-L1, anti-TIM-3, anti-LAG3)

A patent application describes methods involving "administering an agent capable of blocking the interaction of KLRB1 with its ligand," which may include humanized or chimeric antibodies targeting either KLRB1 or its ligands .

Preliminary data from a humanized mouse model of GBM demonstrated that inactivation of the KLRB1 gene in primary human T cells greatly enhanced their cytotoxic function within tumors, providing proof-of-concept for this approach .

How might KLRB1 modulation be applied to autoimmune disease treatment?

In contrast to cancer therapy where KLRB1 inhibition is pursued, autoimmune disease treatment may benefit from KLRB1 pathway activation:

For chronic inflammatory and autoimmune diseases:

• Increasing expression of KLRB1

• Increasing expression of genes encoding CD161 ligands

• Activating or stimulating cell signaling through CD161

• Using agonistic antibodies of CD161

The divergent approaches for cancer versus autoimmune conditions reflect the complex, context-dependent roles of KLRB1 in immune regulation. While elevated KLRB1 expression is associated with diseases like multiple sclerosis and primary Sjögren's syndrome , its activation may potentially limit inflammatory responses in certain contexts.

Therapeutic development would require:

• Precise understanding of KLRB1 function in specific autoimmune conditions

• Cell-type targeted delivery of KLRB1-modulating agents

• Careful monitoring of immune responses to avoid unintended consequences

This bidirectional therapeutic potential highlights the importance of thoroughly characterizing KLRB1 pathway function in different disease contexts.

What are the advantages of genetic versus antibody-based approaches to targeting KLRB1?

Different approaches to KLRB1 modulation offer distinct advantages and limitations:

Genetic approaches may use CRISPR-Cas systems (Cas9, Cas12, Cas13, or Cas14) or other programmable nucleic acid modifying agents like zinc fingers, TALEs, or meganucleases . These techniques can be applied to modify T cells ex vivo before reinfusion, potentially in combination with other genetic modifications to enhance anti-tumor activity.

Antibody approaches can target either KLRB1 or its ligands (such as CLEC2D) and may include humanized or chimeric antibodies or antibody fragments . They offer the advantage of established development pathways but may face challenges in accessing certain anatomical locations like the central nervous system in GBM treatment.

How can single-cell technologies enhance our understanding of KLRB1 function?

Single-cell technologies offer powerful approaches to elucidate KLRB1 function in complex tissue environments:

Key methodological applications:

• Single-cell RNA sequencing (scRNA-seq):

  • Identifies specific cell populations expressing KLRB1

  • Reveals co-expression patterns with other receptors and effector molecules

  • Has been successfully used to analyze KLRB1 expression in T cells from primary Sjögren's syndrome patients

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) enables simultaneous detection of dozens of proteins

  • Multiparameter flow cytometry allows sorting and functional analysis of KLRB1+ subsets

• Spatial transcriptomics:

  • Maps KLRB1-expressing cells within tissue microenvironments

  • Provides context for receptor-ligand interactions in situ

• Single-cell multi-omics:

  • Combined analysis of transcriptome, proteome, and epigenome

  • Reveals regulatory mechanisms controlling KLRB1 expression

Research applications include:

• Characterizing KLRB1+ cell heterogeneity in disease tissues

• Tracking changes in KLRB1-expressing populations during disease progression

• Monitoring treatment effects on KLRB1+ cells

Studies have already utilized publicly available single-cell RNA-Seq data from primary Sjögren's syndrome patient PBMCs to analyze KLRB1 expression in T cells, demonstrating the value of these approaches .

What are the best experimental designs for studying KLRB1 signaling pathways?

Investigating KLRB1 signaling pathways requires sophisticated experimental designs:

• Receptor engagement approaches:

  • Plate-bound or soluble antibodies against KLRB1

  • Recombinant CLEC2D ligand stimulation

  • Co-culture with CLEC2D-expressing cells

• Signaling readouts:

  • Phosphoprotein analysis by flow cytometry

  • Western blotting for pathway components

  • Calcium flux measurement

  • Transcriptional reporter assays

• Genetic manipulation:

  • CRISPR-based knockout of KLRB1 or downstream components

  • Overexpression systems with wildtype or mutant receptors

  • Domain swapping to identify functional regions

• Advanced imaging:

  • Live-cell imaging to visualize receptor clustering

  • FRET-based approaches to detect molecular interactions

  • Super-resolution microscopy to study receptor organization

• Proteomics:

  • Immunoprecipitation coupled with mass spectrometry

  • Proximity labeling techniques to identify interacting partners

  • Phosphoproteomics to map signaling cascades

Comparative analysis between NK cells (where KLRB1 is inhibitory) and T cells (where it can be co-stimulatory) is particularly valuable for understanding context-dependent signaling mechanisms and identifying cell-specific signaling partners.

How can structural biology approaches advance KLRB1-targeted therapeutics?

Structural biology provides crucial insights for developing KLRB1-targeted therapeutics:

• Protein crystallography:

  • Determines atomic-resolution structure of KLRB1 alone or in complex with ligands

  • Identifies binding interfaces and key interaction residues

  • Requires homogeneous, crystallizable protein preparations as achieved with HEK293S GnTI- expression

• Cryo-electron microscopy:

  • Visualizes KLRB1-ligand complexes in different conformational states

  • Less dependent on crystallization, suitable for membrane-associated forms

• NMR spectroscopy:

  • Maps binding interfaces through chemical shift perturbations

  • Studies dynamics of receptor-ligand interactions

• Molecular dynamics simulations:

  • Models receptor-ligand interactions in silico

  • Predicts effects of mutations or small molecule binding

• Structure-guided drug design:

  • Rational design of antibodies targeting specific epitopes

  • Development of small molecule modulators of KLRB1-CLEC2D interaction

  • Protein engineering to create optimized soluble decoys

These approaches benefit from high-quality recombinant KLRB1 protein. Research has successfully produced soluble KLRB1 that is "homogeneous, deglycosylatable, crystallizable and monomeric in solution," providing a foundation for structural studies .

What are the emerging questions regarding KLRB1 tissue-specific functions?

Several critical questions remain regarding KLRB1's tissue-specific functions:

• Nervous system immunology:

  • How does KLRB1 expression in T cells contribute to GBM pathogenesis?

  • Is KLRB1 involved in other neurological disorders beyond MS?

  • What role might KLRB1 play in neuroinflammation more broadly?

• Exocrine gland immunity:

  • What mechanisms drive elevated KLRB1 expression in Sjögren's syndrome?

  • How do KLRB1+ T cells contribute to salivary and lacrimal gland pathology?

  • Could KLRB1 expression serve as a biomarker for disease progression?

• Tissue microenvironment regulation:

  • How does the tumor microenvironment induce KLRB1 expression in T cells?

  • What factors regulate CLEC2D expression in different tissues?

  • How does KLRB1-CLEC2D interaction influence immune cell trafficking?

• Therapeutic targeting considerations:

  • Which tissues would benefit most from KLRB1 pathway modulation?

  • How can tissue-specific delivery of KLRB1-targeting agents be achieved?

  • What biomarkers can predict response to KLRB1-targeted therapies?

Addressing these questions will require integrating advanced technologies like spatial transcriptomics, tissue-specific genetic models, and sophisticated organoid systems that recapitulate the complex cellular interactions in different tissue environments.

How might combination immunotherapies incorporate KLRB1 targeting?

KLRB1 targeting shows promise as a component of combination immunotherapy approaches:

• Checkpoint inhibitor combinations:

  • KLRB1 blockade could complement PD-1/PD-L1, CTLA-4, TIM-3, or LAG3 inhibition

  • Patent literature specifically mentions combining modified T cells with checkpoint blockade therapy

  • May overcome resistance to existing checkpoint inhibitors

• CAR-T cell therapy enhancement:

  • KLRB1 gene inactivation could improve CAR-T function in solid tumors

  • Potential for dual targeting of KLRB1 and tumor-specific antigens

  • May improve persistence and activity in immunosuppressive microenvironments

• Adoptive cell therapy optimization:

  • Ex vivo modification of TILs to reduce KLRB1 expression

  • Combined with cytokine treatment to enhance in vivo expansion

  • Potential delivery methods include infusion or injection into cerebrospinal fluid

• Bispecific antibody approaches:

  • One arm targeting KLRB1 or CLEC2D

  • Second arm engaging effector cells or additional tumor antigens

  • Could provide targeted modulation of the pathway

Research indicates that "inactivation of the KLRB1 gene in primary human T cells greatly enhances their cytotoxic function within tumors" in a humanized mouse model of GBM, suggesting significant potential for combining KLRB1 targeting with other immunotherapy approaches .

What role might artificial intelligence play in advancing KLRB1 research?

Artificial intelligence approaches offer transformative potential for KLRB1 research:

• Target identification and validation:

  • Mining multi-omics data to identify patient subsets likely to benefit from KLRB1 targeting

  • Predicting functional consequences of KLRB1 genetic variants

  • Identifying novel KLRB1-expressing cell populations in disease contexts

• Therapeutic design:

  • Structure-based AI models to design optimal antibodies against KLRB1 or CLEC2D

  • Predicting off-target effects of genetic modification approaches

  • Optimizing CRISPR guide RNA design for KLRB1 targeting

• Clinical translation:

  • Patient stratification algorithms incorporating KLRB1 expression data

  • Predicting response to KLRB1-targeted therapies

  • Monitoring treatment efficacy through image analysis of KLRB1+ cells

• Mechanistic understanding:

  • Network analysis to place KLRB1 in broader signaling contexts

  • Predicting dynamic changes in KLRB1 expression under different conditions

  • Modeling cell-cell interactions mediated by KLRB1-CLEC2D binding

As research progresses, integrating AI approaches with experimental validation will likely accelerate both fundamental discoveries about KLRB1 biology and therapeutic development targeting this pathway in cancer and autoimmune diseases.