CD8B Human, Sf9

What is CD8B Human, Sf9 and how is it produced?

CD8B Human, Sf9 is a recombinant form of the human CD8B protein produced in Sf9 Baculovirus cells. It is a single, glycosylated polypeptide chain containing 158 amino acids (residues 22-170 of the native protein) with a 6-amino acid His tag at the C-terminus . The production process involves:

• Cloning the human CD8B gene into a baculovirus expression vector

• Infecting Sf9 insect cells with the recombinant baculovirus

• Expressing the protein with post-translational modifications

• Purifying using proprietary chromatographic techniques that leverage the His tag

• Formulating in a buffer containing phosphate-buffered saline with 10% glycerol

This expression system offers advantages for producing complex proteins as it allows for proper folding and post-translational modifications while providing higher yields than mammalian expression systems.

What is the biological function of CD8B in T-cell immunity?

CD8B is a critical component of the CD8 coreceptor found on cytotoxic T lymphocytes. Its primary functions include:

• Acting as a coreceptor with the T-cell receptor (TCR) to recognize antigens presented by MHC class I molecules

• Facilitating efficient cell-cell interactions within the immune system

• Recruiting the Src kinase LCK to the vicinity of the TCR-CD3 complex through a palmitoylation site in its cytoplasmic tail

• Contributing to plasma membrane lipid raft partitioning where signaling proteins are enriched

• Initiating intracellular signaling pathways that lead to lymphokine production and cell motility

The CD8 coreceptor can exist either as a heterodimer composed of CD8A and CD8B chains or as a homodimer of two CD8A chains. Both the alpha and beta chains share significant homology to immunoglobulin variable light chains .

What are the structural characteristics of CD8B Human, Sf9?

The amino acid sequence is: ADPLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFC .

What storage conditions are recommended for CD8B Human, Sf9?

Optimal storage conditions for CD8B Human, Sf9 depend on the intended timeframe of use:

• Short-term (2-4 weeks): Store at 4°C

• Long-term: Store frozen at -20°C

• For extended stability: Add a carrier protein (0.1% HSA or BSA)

Important considerations include:

• Avoid multiple freeze-thaw cycles as they can lead to protein denaturation

• The typical formulation (10% glycerol in PBS, pH 7.4) provides some cryoprotection

• Aliquoting before freezing is recommended to avoid repeated freeze-thaw cycles

• Monitor protein stability through functional assays before experimental use

How does CD8B interact with other components of the immune system?

CD8B functions as part of a larger immunological network:

• Forms either a heterodimer with CD8A or contributes to CD8A homodimers on T cell surfaces

• Binds to MHC class I molecules presenting peptide antigens on antigen-presenting cells

• Cooperates with the T-cell receptor to recognize specific antigens

• Facilitates signal transduction through recruitment of LCK kinase

• Plays a role in T cell development in the thymus and peripheral T cell function

• Contributes to immunological memory formation and maintenance of virus-specific T cells

The importance of CD8B is evident in studies showing that CD8+ T cells specific for conserved epitopes correlate with milder disease outcomes in viral infections such as COVID-19 .

How can CD8B Human, Sf9 be utilized in virus-specific T-cell profiling experiments?

CD8B Human, Sf9 can be incorporated into sophisticated experimental designs for profiling virus-specific T cells:

• Enhanced MHC-multimer development:

  • Incorporate CD8B into tetramers or spheromers for increased detection sensitivity

  • Use for identifying low-frequency antigen-specific T cells in peripheral blood

  • Compare responses across different viral specificities (influenza, CMV, SARS-CoV-2)

• Cross-reactive T cell identification:

  • Identify T cells recognizing conserved epitopes across viral families

  • Analyze TCR motifs shared between different virus-specific T cell populations

  • Map the relationship between cross-reactivity and disease severity

• Methodological approach:

  • Isolate peripheral blood mononuclear cells from donor samples

  • Stain with CD8B-enhanced MHC-peptide multimers

  • Perform mass cytometry or single-cell RNA sequencing

  • Apply machine learning to extract phenotypic signatures

This approach has been validated through studies analyzing over 500 antigen-specific T cell responses across six different HLA alleles, revealing unique phenotypic patterns for T cells specific to different viral antigens .

What experimental considerations are important when comparing Sf9-expressed CD8B to native CD8B?

When utilizing Sf9-expressed CD8B in research, several important differences from native CD8B must be considered:

Key experimental considerations include:

How can CD8B Human, Sf9 contribute to understanding T-cell exhaustion in chronic infections and cancer?

CD8B Human, Sf9 can be utilized in sophisticated experimental systems to study T-cell exhaustion:

• Integrative single-cell mapping approach:

  • Combine CD8B-enhanced MHC multimer staining with single-cell RNA sequencing

  • Identify exhausted T cell subtypes based on transcriptional profiles

  • Establish connections between distinct cell subtypes through T cell receptor clonal analysis

  • Analyze phenotypic and functional transitions during exhaustion development

• Cross-disease comparative analysis:

  • Profile exhausted CD8+ T cells across multiple disease states

  • Identify common and disease-specific exhaustion signatures

  • Characterize the three distinct exhausted T cell subtypes recently discovered

  • Reveal diverse transcriptome and clonal sharing patterns in different inflammatory conditions

• Deep learning integration frameworks:

  • Apply computational tools like scAtlasVAE for integration of large-scale single-cell datasets

  • Enable automatic annotation of CD8+ T cell subtypes in query datasets

  • Facilitate unbiased and scalable analyses of exhaustion states

  • Establish comprehensive single-cell references for CD8+ T cell research

This methodology has been validated through the construction of an extensive human CD8+ T cell atlas comprising over 1.15 million cells from 961 samples across 68 studies and 42 disease conditions .

What protocols can maximize the sensitivity of CD8B-based detection systems for rare antigen-specific T cells?

To optimize detection of rare antigen-specific T cells using CD8B-based systems:

• Enhanced multimer design:

  • Incorporate CD8B into spheromer complexes rather than traditional tetramers

  • Optimize the ratio of CD8B to MHC molecules for maximum sensitivity

  • Include stabilizing components to improve multimer half-life

• Sampling and enrichment strategy:

  • Process samples within 8 hours of collection to preserve T cell viability

  • Perform magnetic pre-enrichment of multimer-positive cells

  • Implement a dual-staining approach with two differently labeled multimers

• Flow cytometry optimization:

  • Use high-sensitivity flow cytometers with appropriate compensation

  • Implement a dump channel to exclude dead cells and lineage markers

  • Apply Boolean gating strategies to identify true positive events

• Validation framework:

  • Confirm specificity using peptide-stimulated control samples

  • Perform functional validation through cytokine production assays

  • Compare frequencies across multiple donors and time points

Studies have demonstrated that CD8B-enhanced detection systems can identify significantly higher frequencies of antigen-specific T cells compared to conventional methods, particularly for influenza-M1 and HCMV-pp65 viral specificities .

How can researchers incorporate CD8B Human, Sf9 into studies of conserved coronavirus epitopes?

Researchers can leverage CD8B Human, Sf9 to study conserved coronavirus epitopes through the following methodological approach:

• Epitope conservation analysis:

  • Identify peptides with high sequence similarity across human coronaviruses

  • Compare responses to conserved versus unique SARS-CoV-2 epitopes

  • Correlate epitope conservation with disease severity

• T cell response profiling:

  • Develop CD8B-enhanced MHC multimers loaded with conserved coronavirus peptides

  • Isolate and characterize T cells specific for these conserved epitopes

  • Analyze their effector phenotypes indicating recent antigen activation

  • Compare frequencies in mild versus severe COVID-19 patients

• TCR repertoire analysis:

  • Identify TCR motifs shared between unexposed individuals and COVID-19 patients

  • Determine if TCR motifs against conserved epitopes are enriched in mild COVID-19

  • Assess whether TCRs specific for SARS-CoV-2-unique epitopes predominate in severe disease

This approach has revealed that CD8+ T cells specific for conserved coronavirus epitopes are more abundant in patients with mild COVID-19 compared to those with severe disease, suggesting that cross-reactive memory T cells from previous human coronavirus exposures may contribute to protective immunity .

What are the best practices for designing CD8B-based experimental systems to study baculovirus pseudotyping?

When designing experimental systems that incorporate both CD8B and baculovirus pseudotyping:

• Expression system optimization:

  • Develop stable Sf9 insect cell lines for consistent protein expression

  • Implement inducible promoter systems like the p XXL promoter for controlled expression

  • Select cell lines based on protein expression levels and functional properties

• Pseudotyping strategy:

  • Incorporate VSV-G protein for enhanced transduction efficiency

  • Optimize infection conditions for producing functional pseudotyped baculoviruses

  • Purify budded virions to assess protein incorporation

• Experimental validation:

  • Quantify transduction efficiency across diverse mammalian cell lines

  • Compare pseudotyped versus non-pseudotyped baculoviruses

  • Assess expression of the transgene using flow cytometry

• CD8B specific considerations:

  • Evaluate potential interactions between CD8B and the pseudotyping proteins

  • Assess whether pseudotyping affects CD8B functionality in downstream applications

  • Optimize the ratio of CD8B expression to viral production

Research has demonstrated that pseudotyped baculovirus consistently increases transduction efficiency across multiple mammalian cell lines, providing a valuable approach for enhancing gene delivery without inserting pseudotyping genes into baculoviral genomes .