THEMIS2 Antibody

What is THEMIS2 and what are its primary functional roles in immune cells?

THEMIS2 (Thymocyte-expressed molecule involved in selection protein 2) is a scaffold protein predominantly expressed in B cells and macrophages. It functions as an adaptor protein that modulates B-cell selection in response to antigens by strengthening interactions between PLCγ2 and upstream kinases like Lyn . THEMIS2 has no known catalytic domain and determines the threshold for activation of B cells by low-affinity and low-avidity ligands via PLCγ2 activation and ERK1/2-dependent pathways . In macrophages, THEMIS2 constitutes a control point in inflammatory responses, promoting LPS-induced TNF production . Additionally, THEMIS2 inhibits activating NK receptor signaling by binding to GRB2 and phosphorylated SHP-1 and SHP-2 near activating NK receptors DNAM-1 and NKG2D .

What applications are most effective for THEMIS2 antibody detection in experimental settings?

THEMIS2 antibodies have proven effective in multiple applications with specific optimization parameters:

• Western Blot: Dilutions ranging from 1:500-1:5000 are recommended, with expected band sizes around 57 kDa, though multiple isoforms (73, 30, 57, 14, 58, 51 kDa) have been detected .

• Immunohistochemistry: Optimal dilutions of 1:200-1:500 for paraffin-embedded tissues. For human pancreatic cancer samples, a 1:400 dilution has shown good results using Leica Bond™ systems .

• Immunofluorescence: A dilution range of 1:50-1:200 is typically effective for cellular localization studies .

• ELISA: THEMIS2 antibodies can be employed in enzyme immunoassays for quantitative detection .

Subcellular localization studies indicate that THEMIS2 can be detected in both nucleoplasm and cytoplasm, requiring careful sample preparation and proper controls .

How should researchers design experiments to investigate contradictory findings about THEMIS2's role in B cell function?

Resolving contradictions regarding THEMIS2's role in B cell function requires careful experimental design:

• Antigen affinity consideration: One key contradiction in the literature stems from different antigen types used. While Themis2-deficiency showed no effect on B-cell development or antibody responses to high-valency forms of antigen (NP21-CGG), effects became evident only with lower valency antigens (NP3-CGG or soluble HEL) . Design experiments using both high and low-affinity antigens.

• B cell activation markers: Measure CD69 (required for retention and proliferation of activated B cells) and CD86 (required for T-cell help), which are differentially regulated by THEMIS2 .

• Developmental analysis: Use flow cytometry to assess all developmental stages from pro-B cells through mature B cells in bone marrow and peripheral lymphoid tissues.

• Transcriptional profiling: Employ RNA sequencing on resting and activated B cells, as minor changes may only be apparent after stimulation .

• In vivo models: Include multiple immunization protocols with T-dependent and T-independent antigens, and viral challenges (e.g., influenza virus) to detect subtle phenotypes .

This comprehensive approach will help delineate context-dependent effects of THEMIS2 in B cell function.

What methodological approaches should be used to investigate THEMIS2's interactions with signaling complexes?

To comprehensively characterize THEMIS2's interactions with signaling complexes:

• Chemical crosslinking: DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) is essential for detecting low-affinity interactions. Without crosslinking, THEMIS2 interactions with DAP12 and ZAP70 after receptor stimulation were undetectable in co-immunoprecipitation experiments .

• Tagged protein expression systems: Establish cell lines (e.g., NKL cells) expressing FLAG-tagged THEMIS2 and relevant receptors (e.g., Ly49H) to facilitate biochemical analysis .

• Proximity assays: Beyond traditional co-immunoprecipitation, proximity-based approaches can identify transient interactions in intact cells.

• Time-course analysis: Examine THEMIS2 complex formation at different timepoints after receptor stimulation to capture dynamic interactions .

• Domain mapping: Create deletion mutants to identify specific regions required for protein-protein interactions.

Research has shown that THEMIS2 forms complexes with signaling molecules including GRB2, VAV and LYN after BCR stimulation , and with GRB2, SHP-1 and SHP-2 near activating NK receptors , suggesting a scaffolding role in immune cell signaling.

How can researchers effectively investigate THEMIS2's role in NK cell-mediated antitumor immunity?

To investigate THEMIS2's role in NK cell-mediated antitumor immunity:

• Genetic manipulation approaches:

  • Generate conditional Themis2 knockout mice using NK-CreERT2 systems for temporal control

  • Employ CRISPR/Cas9 for THEMIS2 knockdown or overexpression in human NK cells

  • Compare Themis2-sufficient and Themis2-deficient NK cells in parallel experiments

• Functional assays:

  • Measure cytotoxicity against tumor targets

  • Assess production of effector cytokines (IFN-γ, TNF-α)

  • Analyze degranulation markers and activating receptor expression

• In vivo tumor models:

  • Challenge with viral pathogens (e.g., MCMV) that rely on NK-mediated clearance

  • Track expansion, contraction, and memory formation of Ly49H+ NK cells during viral infection

  • Perform adoptive transfer experiments to assess protective capacity of memory NK cells

• Signaling pathway analysis:

  • Analyze phosphorylation status of key signaling molecules after receptor stimulation

  • Examine the recruitment of phosphatases to inhibitory complexes

  • Investigate calcium flux and ERK activation in response to activating signals

Studies have demonstrated that Themis2-deficient NK cells show enhanced differentiation into memory NK cells and provide improved protection against MCMV infection, suggesting that targeting THEMIS2 could enhance NK cell-based cancer immunotherapies .

What methodological approaches are recommended for exploring THEMIS2 as a cancer biomarker?

To investigate THEMIS2 as a cancer biomarker:

• Expression analysis in multiple cancer types:

  • Compare THEMIS2 mRNA and protein expression between tumor and normal tissues using qRT-PCR, Western blot, and IHC

  • Analyze expression across cancer stages and histological subtypes

  • Correlate expression with patient age, gender, and other clinical parameters

• Bioinformatic approaches:

  • Perform weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with THEMIS2

  • Conduct LASSO regression analysis to establish THEMIS2 as a diagnostic biomarker (optimal cutoff: AUC > 0.65)

  • Develop nomogram models incorporating THEMIS2 expression with clinical parameters (age, stage) for prognostic prediction

• Pathway enrichment analysis:

  • Use gene set enrichment analysis (GSEA) to identify pathways associated with THEMIS2 expression

  • Focus on cancer-relevant pathways such as JAK-STAT, p53, and immune signaling pathways

• Immune infiltration correlation:

  • Apply CIBERSORT algorithm to determine levels of 22 immune cell types in relation to THEMIS2 expression

  • Analyze differences in immune cell infiltration between high and low THEMIS2 expression groups

  • Correlate THEMIS2 expression with immune checkpoint molecules

Research has identified THEMIS2 as a potential diagnostic and prognostic biomarker in thyroid cancer, with significant associations with JAK-STAT signaling, T and B cell receptor pathways, and p53 signaling .

How should researchers interpret differences in experimental outcomes when studying THEMIS2 function in different immune cell populations?

When interpreting differences in THEMIS2 function across immune cell types:

• Cell type-specific expression patterns:

  • THEMIS2 is predominantly expressed in B cells and macrophages, with differential regulation during activation

  • Expression levels vary across B cell developmental stages and after activation

  • In thyroid cancer, THEMIS2 expression shows age-dependent differences (lower in patients ≥55 years)

• Receptor context considerations:

  • THEMIS2 modulates different receptor systems (BCR in B cells, TLR in macrophages, activating receptors in NK cells)

  • Signal strength dependency - effects may only be apparent with low-affinity/avidity stimulation

  • Receptor-specific adaptor proteins may influence THEMIS2 function differently

• Functional interpretation framework:

  • For B cells: Focus on subtle effects on activation thresholds rather than complete developmental blocks

  • For NK cells: Examine both quantitative (cell numbers) and qualitative (functional capacity) parameters

  • For cancer cells: Consider both cell-intrinsic effects and influences on the tumor microenvironment

• Experimental validation across systems:

  • Verify key findings in both mouse and human systems

  • Confirm in vitro observations with in vivo models

  • Use both loss-of-function and gain-of-function approaches

This approach will help resolve apparent contradictions, such as why THEMIS2 appears dispensable for B cell development in some contexts while regulating B cell selection thresholds in others .

What analytical approaches should be used to correlate THEMIS2 expression with immune cell infiltration in cancer?

For analyzing THEMIS2 expression in relation to immune infiltration:

• Immune deconvolution methodologies:

  • Apply CIBERSORT algorithm to estimate proportions of 22 immune cell populations from bulk expression data

  • Use "limma" package in R to determine statistically significant differences between high and low THEMIS2 expression groups

  • Create visualization heatmaps showing differential immune cell infiltration patterns

• Correlation analysis with immune checkpoints:

  • Calculate Pearson or Spearman correlation coefficients between THEMIS2 and immune checkpoint genes

  • Analyze co-expression with key checkpoints including PD-L1/PD-1, CTLA4, LAG3, CD28/CD80, and CD40/CD40LG

  • Investigate whether THEMIS2 expression predicts immunotherapy response

• Multidimensional data integration:

  • Combine transcriptomic data with protein-level validation

  • Correlate immune infiltration patterns with clinical outcomes

  • Develop integrated prognostic models incorporating THEMIS2 and immune parameters

• Spatial analysis approaches:

  • Use multiplex immunohistochemistry to map THEMIS2-expressing cells relative to immune infiltrates

  • Analyze spatial relationships between THEMIS2+ cells and various immune cell types

  • Correlate patterns with tumor progression and patient outcomes

Research in thyroid cancer demonstrated that high THEMIS2 expression correlates with lower CD8+ T cells and activated NK cells but higher Tregs, suggesting an immunosuppressive role .

What are the critical quality control measures for validating THEMIS2 antibody specificity in research applications?

Essential quality control measures for THEMIS2 antibody validation:

• Genetic validation controls:

  • Test antibodies on Themis2-knockout or Themis2-knockdown samples as negative controls

  • Include overexpression systems as positive controls

  • Verify reactivity across species boundaries if working with both human and mouse samples

• Multi-technique confirmation:

  • Validate antibody performance across multiple applications (WB, IHC, IF)

  • Confirm consistent protein detection patterns across techniques

  • Verify predicted molecular weight detection (primary band at ~57 kDa)

• Epitope considerations:

  • Know the specific epitope region targeted by your antibody

  • Consider whether the antibody detects all six reported THEMIS2 isoforms or is isoform-specific

  • Use multiple antibodies targeting different epitopes for critical experiments

• Application-specific controls:

  • For IHC: Include isotype controls and blocking peptide competition controls

  • For WB: Run gradient gels to resolve multiple isoforms

  • For IF: Include secondary-only controls to rule out non-specific binding

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other THEMIS family members (e.g., THEMIS1, THEMIS3)

  • Perform peptide competition assays with the immunizing peptide

  • Validate in tissues known to express or lack THEMIS2

These validation steps ensure reliable results when studying THEMIS2 in complex biological systems.

How should researchers optimize protocols for detecting THEMIS2 protein complexes in primary immune cells?

For optimal detection of THEMIS2 protein complexes in primary immune cells:

• Cell isolation and preparation:

  • Use gentle isolation techniques to maintain native protein interactions

  • Minimize time between isolation and lysis to prevent complex dissociation

  • Consider crosslinking live cells before lysis to stabilize transient interactions

• Lysis and immunoprecipitation optimization:

  • Use mild detergents (e.g., 1% NP-40 or 1% Digitonin) to preserve protein complexes

  • Include phosphatase inhibitors to maintain phosphorylation-dependent interactions

  • Apply chemical crosslinkers (e.g., DTSSP) to capture low-affinity interactions

  • Pre-clear lysates thoroughly to reduce non-specific binding

• Stimulation considerations:

  • Compare resting cells with receptor-stimulated cells (e.g., BCR, NK activating receptors)

  • Use physiologically relevant stimuli at appropriate concentrations

  • Perform time-course experiments to capture dynamic interaction changes

• Detection strategies:

  • For Western blot detection: Use high-sensitivity ECL substrates

  • For mass spectrometry: Consider label-free quantification or SILAC approaches

  • For microscopy: Employ proximity ligation assays to visualize interactions in situ

Evidence shows that THEMIS2 forms distinct complexes in different cell types - with PLCγ2 and Lyn in B cells , and with GRB2, SHP-1, and SHP-2 in NK cells - requiring cell type-specific optimization approaches.