TIMD4 Antibody

What is the optimal application for detecting TIMD4 in tissue samples?

Immunofluorescence (IF-P) is highly effective for detecting TIMD4 in tissue samples, particularly in human tonsillitis tissue. For optimal results, use a dilution range of 1:50-1:500, with the specific dilution requiring optimization for each experimental system . When performing IF-P detection, maintain consistent fixation protocols as TIMD4 expression can be affected by fixation methods. Storage of antibodies at -20°C with 50% glycerol is recommended to maintain stability, and exposure to light should be minimized due to the fluorescent conjugate properties .

Which tissues typically express high levels of TIMD4?

TIMD4 demonstrates distinct expression patterns across various tissues. High expression is observed in peripheral lymphoid tissues including tonsils, thymus, spleen, and lymph nodes . In contrast, lung, liver, and kidney tissues show consistently low TIMD4 expression levels . Positron emission tomography (PET) imaging with radioactively-labeled anti-murine Tim-4 antibody confirms strong signal in liver, spleen, bones, and lymph nodes, validating these expression patterns . When designing experiments to detect TIMD4, prioritize these high-expression tissues as positive controls to ensure proper antibody functionality.

What cell types express TIMD4 that should be targeted in immunological research?

TIMD4 was initially considered to be primarily expressed by antigen-presenting cells (APCs) such as macrophages and mature dendritic cells (DCs), but research has established a broader expression profile . The following table summarizes key TIMD4-expressing cell types:

When designing experiments to study TIMD4 function, consider that tissue-resident macrophages, particularly those in peritoneal and pleural cavities, show especially high expression levels compared to monocyte-derived macrophages .

How should I validate the specificity of a TIMD4 antibody?

Proper validation of TIMD4 antibody specificity requires multiple complementary approaches. Begin with Western blotting against tissues known to express TIMD4 at high levels (lymphoid tissues) and low levels (lung tissue) as positive and negative controls respectively . Importantly, include TIM4-deficient samples as definitive negative controls, as demonstrated in studies with TIM4-deficient mice .

For immunofluorescence applications, perform blocking experiments by pre-incubating with excess unlabeled TIMD4 antibody to confirm signal reduction in high-expressing tissues . PET imaging studies have demonstrated that specific uptake of radiolabeled anti-TIMD4 antibody is reduced with co-infusion of excess unlabeled antibody in tissues with known TIMD4 expression . Additionally, verify antibody reactivity across species if conducting translational research, as epitope conservation may vary between human and murine TIMD4.

What controls are essential when using TIMD4 antibodies in tumor immunology studies?

When investigating TIMD4 in tumor immunology, several controls are critical for accurate interpretation:

• Cellular compartment controls: Discriminate between resident macrophages and infiltrating monocytes/macrophages, as TIMD4 expression is significantly higher in tissue-resident populations .

• Tumor type specificity: Different tumor types show variable patterns of TIMD4 expression and function. For instance, studies show non-specific blood pooling in well-vascularized tumors like B16F10 melanoma and MC38 colon carcinoma .

• Patient-specific variability: In human samples (particularly cancer patients), there is substantial interpatient variability in TIMD4 expression on macrophages. Nearly half of lung cancer patient samples show high levels of Tim-4 expression, necessitating proper stratification .

• Technical controls: When studying TIMD4's role in apoptotic cell clearance, use both TIMD4-blocking antibodies and TIMD4-deficient models to distinguish antibody effects from genetic deficiency .

• T cell subset analysis: Always analyze multiple T cell populations (CD8+, CD39+, PD-1+) when studying TIMD4's immunomodulatory effects, as TIMD4 may differentially affect specific subsets rather than total T cell populations .

What is the optimal methodology for detecting soluble TIMD4 (sTIMD4) in serum samples?

For detecting soluble TIMD4 (sTIMD4) in serum samples, enzyme-linked immunosorbent assay (ELISA) methodology has been effectively employed. When establishing this assay, consider the following parameters:

• Sensitivity requirements: The optimal cut-off value for sTIMD4 has been determined to be 0.34 ng/mL for distinguishing healthy controls from coronary heart disease patients, with 74.9% sensitivity and 66.7% specificity .

• Sample handling: Process serum samples consistently to minimize variability, with standardized collection, processing times, and storage temperatures.

• Validation: Validate assay performance using samples with known sTIMD4 levels, including positive controls from patients with conditions associated with elevated sTIMD4 (such as CHD patients) .

• Cross-reactivity testing: Ensure the antibody pairs used in the ELISA do not cross-react with other TIM family members or related proteins.

• Clinical correlation: When interpreting results, note that sTIMD4 levels correlate positively with coronary heart disease events, with an area under the curve (AUC) of 0.787 in ROC curve analysis .

How does TIMD4 expression differ between tissue-resident macrophages and infiltrating monocytes in pathological conditions?

TIMD4 expression demonstrates distinct patterns between tissue-resident macrophages and infiltrating monocytes, particularly in pathological conditions. Tissue-resident macrophages, especially cavity-resident macrophages in the peritoneum and pleura, constitutively express high levels of TIMD4 . In contrast, infiltrating monocytes and monocyte-derived macrophages show minimal TIMD4 expression .

In tumor microenvironments, this differential expression becomes particularly relevant. Human studies of pleural effusions from lung cancer patients and peritoneal ascites from ovarian cancer patients demonstrate that resident macrophages maintain high TIMD4 expression, serving as a distinctive marker to differentiate them from newly recruited monocytes . This expression pattern creates functional heterogeneity within the tumor-associated macrophage population.

The resident macrophage-specific expression of TIMD4 is regulated through distinct transcriptional programs. In murine peritoneal macrophages, GATA-6 regulates TIMD4 expression, though interestingly, human pleural and peritoneal macrophages do not appear to express GATA-6 despite high TIMD4 levels, suggesting species-specific regulatory mechanisms . When studying macrophage populations, researchers should use TIMD4 in conjunction with other markers like VSIG4, which has been used to identify human peritoneal resident macrophages .

How can researchers resolve contradictory findings regarding TIMD4's role in tumor immunity?

The literature reveals apparently contradictory roles for TIMD4 in tumor immunity, presenting an important research challenge. To resolve these contradictions, consider the following methodological approaches:

• Context-specific analysis: TIMD4 functions differently depending on its cellular context. In antigen-presenting cells, it may promote T cell differentiation toward Th2 cells through interaction with TIM-1 , while in cavity-resident macrophages, it appears to suppress anti-tumor CD8+ T cell responses .

• Distinct forms analysis: Differentiate between membrane-bound TIMD4 (mTIMD4) and soluble TIMD4 (sTIMD4), which have distinct biological activities. Under inflammatory conditions like exposure to oxidized low-density lipoprotein (ox-LDL), mTIMD4 decreases while sTIMD4 increases through ADAM17-mediated cleavage . This process is accompanied by increased expression of proinflammatory factors (IL-6, IL-1β) and decreased anti-inflammatory factors (IL-10) .

• Mechanistic experiments: Use ADAM17 inhibitors like TAPI-1 to block mTIMD4 cleavage and observe the effects on downstream signaling pathways including NF-κB and TLR-4 . This approach helps elucidate whether contradictory findings stem from differential regulation of membrane-bound versus soluble forms.

• Compartment-specific investigation: Recognize that TIMD4 function varies by anatomical compartment. TIM4-deficient mice show defective apoptotic cell clearance specifically in the peritoneum but not in the spleen, yet this compartment-specific defect is sufficient to break tolerance to nuclear antigens and promote autoimmunity .

• T cell subset analysis: Analyze specific T cell populations beyond total CD8+ counts. High TIMD4+ macrophages are associated with reduced percentages of CD39+ among CD8+ T cells (which are enriched for tumor antigen-reactive cytotoxic T cells) despite no differences in total CD8+ T cell counts .

What experimental approaches can determine if changes in TIMD4 expression are causal or consequential in disease progression?

To establish whether TIMD4 expression changes are causal or consequential in disease progression, researchers should implement multiple complementary approaches:

• Temporal expression analysis: Monitor TIMD4 expression throughout disease progression using longitudinal sampling. In atherosclerosis models, TIMD4 regulation can be tracked by treating RAW264.7 cells with ox-LDL at different times (0, 3, 6, 12, 24h) and concentrations (0, 10, 20, 40, 80 μg/mL) to establish temporal relationships between TIMD4 changes and disease markers .

• Genetic manipulation strategies: Utilize TIM4-deficient mouse models to determine how TIMD4 absence affects disease onset and progression. TIM4-deficient mice develop autoantibodies against double-stranded DNA and display hyperactive immune responses, demonstrating that even compartment-specific TIMD4 deficiency can have systemic consequences .

• Pharmacological intervention: Apply specific inhibitors to modulate TIMD4 function at different disease stages. ADAM17 inhibitors like TAPI-1 prevent the cleavage of membrane TIMD4 to soluble TIMD4, thereby altering inflammatory signaling cascades through NF-κB, TLR-4, and IL-6 pathways .

• Correlative clinical studies: Analyze TIMD4 expression in patient cohorts with varying disease severity and outcomes. In coronary heart disease, serum sTIMD4 levels correlate with disease events and provide diagnostic value (AUC 0.787) for distinguishing patients from healthy controls .

• Mechanistic pathway analysis: Investigate molecular mechanisms linking TIMD4 to disease phenotypes. In inflammatory conditions, ADAM17 activation leads to TIMD4 shedding from the membrane, which occurs alongside activation of TLR-4/NF-κB signaling and altered cytokine production. Blocking this pathway with TAPI-1 abolishes the expression of phosphorylated NF-κB, TLR-4, and IL-6 that are upregulated by ox-LDL .

What factors might contribute to inconsistent TIMD4 antibody staining in human clinical samples?

Inconsistent TIMD4 antibody staining in human clinical samples stems from several factors that researchers should systematically address:

• Biological variability: Notable interpatient variability in TIMD4 staining intensity on CD3-CD14+ macrophages has been documented, particularly in lung cancer cohorts where approximately half of the samples show high levels of TIMD4 expression on macrophages . This biological heterogeneity is intrinsic and should be accounted for in study design through adequate sample sizes.

• Sample preservation methods: Fixation protocols significantly impact TIMD4 epitope accessibility. Standardize fixation times, concentrations, and buffer compositions to minimize technical variability.

• Antibody clone selection: Different antibody clones may recognize distinct epitopes on TIMD4, which can be differentially affected by sample processing. For immunofluorescence applications of TIMD4, polyclonal antibodies like CL488-12008 have demonstrated reliable detection in human samples .

• Signal amplification requirements: TIMD4 expression may require signal amplification techniques in tissues with low expression levels. A recommended starting dilution of 1:50-1:500 for immunofluorescence should be optimized for each tissue type and experimental system .

• Disease and treatment effects: Prior chemotherapy, radiation therapy, or immunotherapy may alter TIMD4 expression patterns, though univariate analysis has not found significant associations between these factors and TIMD4 expression levels on macrophages in lung cancer patients .

How can researchers distinguish between membrane-bound TIMD4 (mTIMD4) and soluble TIMD4 (sTIMD4) in experimental systems?

Distinguishing between membrane-bound TIMD4 (mTIMD4) and soluble TIMD4 (sTIMD4) requires specific methodological approaches:

• Form-specific detection methods:

  • For mTIMD4: Use flow cytometry with non-permeabilizing conditions and antibodies targeting the extracellular domain

  • For sTIMD4: Implement ELISAs optimized for serum/plasma samples with an established cut-off value of 0.34 ng/mL for clinical significance

• Differential regulation analysis: Track the relationship between mTIMD4 and sTIMD4 levels under experimental conditions. When RAW264.7 cells are treated with ox-LDL, mTIMD4 levels decrease while sTIMD4 levels increase in a gradient manner, accompanied by changes in inflammatory markers .

• ADAM17 inhibition experiments: ADAM17 mediates the cleavage of mTIMD4 to generate sTIMD4. Treating cells with TAPI-1 (an ADAM17 inhibitor) increases mTIMD4 expression while decreasing sTIMD4 levels in ox-LDL-stimulated conditions . This manipulation provides a controlled system to study form-specific functions.

• Transcriptional versus post-translational regulation: Ox-LDL increases ADAM17 mRNA expression but does not affect mTIMD4 mRNA expression, suggesting that changes in protein levels occur through post-translational mechanisms . Monitoring both mRNA and protein levels helps distinguish between transcriptional regulation and protein processing.

• Functional consequence assessment: Membrane and soluble forms may have distinct biological activities. Changes in NF-κB phosphorylation, TLR-4 expression, and IL-6 production correlate with alterations in the mTIMD4/sTIMD4 balance and can be modulated by ADAM17 inhibition .

What are the most critical methodological considerations when using TIMD4 antibodies to analyze apoptotic cell clearance?

When using TIMD4 antibodies to study apoptotic cell clearance, researchers should consider these methodological factors:

How can TIMD4 expression be leveraged to distinguish resident vs. infiltrating macrophages in tumor microenvironments?

TIMD4 expression provides a valuable marker to distinguish tissue-resident macrophages from infiltrating monocyte-derived macrophages in tumor microenvironments, offering important research applications:

• Dual-marker strategies: Combine TIMD4 staining with monocyte markers like Ly6C (mouse) or CD14high/CD16low (human) to definitively separate resident from infiltrating populations. In human studies, TIMD4 works effectively in combination with VSIG4 as markers for resident macrophages in peritoneal environments .

• Multi-compartment analysis: When studying cancer with metastatic spread to serous cavities (like pleural effusions or peritoneal ascites), analyze TIMD4 expression in both primary tumor-associated macrophages and cavity-resident macrophages to understand compartment-specific immunity. Human studies of pleural effusions from lung cancer patients demonstrate high TIMD4 expression specifically in resident macrophages .

• Functional correlation: Correlate TIMD4+ resident macrophage abundance with T cell functionality metrics. High TIMD4+ resident macrophage presence is associated with reduced percentages of CD39+ (tumor-reactive) CD8+ T cells without affecting total CD8+ T cell numbers . This correlation provides insight into the immunosuppressive mechanisms operating within the tumor microenvironment.

• Therapeutic targeting assessment: Evaluate how therapies differentially affect TIMD4+ resident versus TIMD4- infiltrating macrophages. This distinction is crucial for immunotherapies that may depend on reprogramming specific macrophage populations.

• Ontogeny studies: In lineage tracing experiments, TIMD4 expression can help confirm the embryonic origin of tissue-resident macrophages versus adult bone marrow-derived infiltrating cells in tumor contexts.

What are the experimental challenges in studying TIMD4's dual roles in autoimmunity and tumor immunity?

Investigating TIMD4's seemingly contradictory roles in autoimmunity and tumor immunity presents several experimental challenges that require sophisticated methodological approaches:

• Tissue-specific knockout models: Develop conditional knockout systems that target TIMD4 in specific tissue compartments or cell types rather than global knockouts. This approach helps delineate compartment-specific functions, as TIMD4 deficiency in peritoneal macrophages produces systemic autoimmune effects despite normal apoptotic cell clearance in the spleen .

• Temporal regulation systems: Implement inducible knockout or overexpression systems to study TIMD4's role at different disease stages. This strategy distinguishes between TIMD4's effects during disease initiation versus progression.

• Context-dependent signaling analysis: TIMD4 interacts with multiple partners that influence downstream signaling. While TIMD4 on dendritic cells interacts with TIM-1 to promote Th2 differentiation , its phosphatidylserine-binding capability facilitates apoptotic cell clearance . Comprehensive co-immunoprecipitation studies and proximity ligation assays can map context-specific binding partners.

• Parallel pathway investigation: Study how TIMD4 signaling intersects with other immunoregulatory pathways. TIMD4 cleavage by ADAM17 affects NF-κB and TLR-4 signaling , while TIMD4's role in efferocytosis influences self-tolerance . Pathway inhibition studies with readouts for multiple signaling cascades help establish these connections.

• Translational validation: Validate findings across species barriers. While murine TIMD4 studies provide mechanistic insights, expression patterns may differ in humans. For instance, human pleural and peritoneal macrophages express high TIMD4 levels without apparent GATA-6 expression, unlike their murine counterparts .

How does TIMD4 contribute to the regulation of tumor-infiltrating lymphocytes at the molecular level?

TIMD4's regulation of tumor-infiltrating lymphocytes involves several molecular mechanisms that can be investigated through specific experimental approaches:

• T cell subset characterization: TIMD4+ cavity-resident macrophages are associated with selective reduction in CD39+ among CD8+ T cells, which represent tumor antigen-reactive cytotoxic T cells . Comprehensive phenotyping of tumor-infiltrating lymphocytes should include markers for:

  • Antigen specificity (CD39)

  • Activation status (PD-1)

  • Exhaustion profile (TIM-3, LAG-3)

  • Effector function (IFN-γ, TNF-α, Granzyme B)

• Direct vs. indirect effects distinction: Determine whether TIMD4+ macrophages directly interact with T cells or act through soluble mediators. Co-culture experiments with TIMD4+ macrophages and T cells, with or without transwell separation, can establish the requirement for direct contact.

• Efferocytosis-dependent mechanisms: Investigate whether TIMD4+ macrophages regulate T cells through enhanced efferocytosis of antigen-specific T cells expressing phosphatidylserine. Previous studies have shown that F4/80+TIM-4+ macrophages engulf antigen-specific T cells expressing PS, decreasing antigen-specific T cells entering the periphery and inducing immune tolerance .

• Cytokine profile analysis: Measure the cytokine production profile of TIMD4+ macrophages compared to TIMD4- macrophages in tumor contexts. TIMD4 has been shown to inhibit the production of cytokines like TNF-α and IL-6 from macrophages in other inflammatory contexts .

• Checkpoint pathway interaction: Investigate how TIMD4 expression on macrophages influences checkpoint receptor expression on T cells. While CD8+PD-1+ T cells are associated with tumor reactivity and improved responses to immunotherapy in lung cancer, their relationship with TIMD4+ macrophages remains to be fully characterized .