VSIG4 (V-set and immunoglobulin domain containing 4), also known as CRIg or Z39IG, is a B7 family-related protein that functions as both a complement receptor involved in pathogen clearance and a negative regulator of T cell activation. VSIG4 is a type I transmembrane glycoprotein structurally related to the B7 family of immune regulatory proteins, containing one complete V-type Ig domain and one truncated C-type Ig domain .
The protein is exclusively expressed on tissue-resident and tumor-associated macrophages (TAMs), making it a unique target for immunotherapy research . VSIG4's significance in research stems from its role in suppressing T cell activation, proliferation, and IL-2 production, contributing to peripheral T cell tolerance and suppression of established inflammation . Importantly, increased VSIG4 expression correlates with worse survival outcomes in multiple cancer indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma .
VSIG4 exhibits highly specific expression patterns that differ between normal and pathological states. In normal tissues, VSIG4 is predominantly expressed in liver Kupffer cells, as demonstrated by immunohistochemical staining . Quantitative PCR studies have revealed high VSIG4 mRNA expression in the liver, dendritic cells, neutrophils, and macrophages, with lower expression levels in the lung, heart, spleen, and lymph nodes .
In pathological conditions, particularly cancer, VSIG4 expression is elevated in tumor-associated macrophages. Single-cell RNA-seq analysis of non-small cell lung cancer (NSCLC) patients has shown that VSIG4 is localized to tumor-associated myeloid populations within the tumor microenvironment and is absent in other cellular populations in both the TME and periphery . Analysis of approximately 10,000 human tumors spanning 33 cancer types from The Cancer Genome Atlas (TCGA) dataset found consistent VSIG4 expression across diverse tumor types, with the highest expression in glioblastoma, mesothelioma, NSCLC, and pancreatic adenocarcinoma .
VSIG4 is a 44 kilodalton protein with specific structural domains that determine its function. It is synthesized as a 280 amino acid precursor containing a signal sequence, an IgV-type immunological domain (amino acids 36-115), one potential N-linked glycosylation site, and a single transmembrane domain .
Two main isoforms of human VSIG4 have been identified and are referred to as "long" and "short" isoforms. Both isoforms share a conserved IgV domain . Cancer-specific patterns of VSIG4 isoform distribution have been observed, suggesting altered functional regulation in cancer . The IgV domain of mouse VSIG4 shares 86% amino acid sequence identity with rat VSIG4 and 80% with human VSIG4 , which is important to consider when developing cross-species reactive antibodies.
Validation of VSIG4 antibody specificity requires multiple complementary approaches to ensure reliable experimental results. Based on the literature, researchers should:
Confirm target recognition using recombinant proteins: Test antibody binding to both isoforms of recombinant VSIG4 protein using techniques such as ELISA or biolayer interferometry (BLI). For example, the 12A12c antibody was validated by confirming binding to both the long and short isoforms of recombinant human VSIG4 .
Verify cellular expression patterns: Use flow cytometry to confirm antibody binding to endogenous VSIG4 on appropriate cell types (macrophages) while showing absence of binding to cells known to lack VSIG4 expression (T and B cells) .
Employ genetic controls: Use VSIG4-deficient (VSIG4-/-) cells or tissues as negative controls, as demonstrated in several studies where antibody responses were absent in VSIG4-/- samples .
Cross-validate with multiple detection methods: Combine immunohistochemistry, immunofluorescence, and flow cytometry data to confirm consistent staining patterns. In particular, immunofluorescent double staining has shown that VSIG4 is present on CD68+ macrophages but absent from CD3+ T cells, CD31+ endothelial cells, and CK-18+ epithelial cells .
Assess cross-reactivity across species: Test the antibody against VSIG4 from multiple species if cross-species reactivity is claimed. For instance, the NLA14 antibody has been specifically tested and does not cross-react with rat or human VSIG4 .
Detection protocols should be tailored to specific sample types and research questions:
For tissue sections:
For frozen tissue sections: Use perfusion-fixed samples when possible. The R&D Systems AF4674 antibody has been successfully used at 1.7 µg/mL with overnight incubation at 4°C, followed by HRP-DAB staining and hematoxylin counterstaining .
Some antibodies, like NLA14, require formaldehyde fixation and permeabilization for successful detection of VSIG4 .
For flow cytometry:
For mouse peritoneal exudate cells: The NLA14 monoclonal antibody can be used at ≤0.5 µg per test (defined as the amount of antibody that will stain a cell sample in a final volume of 100 µL) .
Note that peritoneal macrophages in BALB/c mice express significantly higher levels of VSIG4 than those in C57BL/6 or Swiss Webster mice, which may affect detection sensitivity .
For in vitro differentiated macrophages:
When working with M-CSF plus IL-10-driven monocyte-derived M2c macrophages, it's important to note that pro-inflammatory stimuli such as TNF and LPS have been reported to down-regulate VSIG4 expression, which may affect antibody detection .
The epitope accessibility of VSIG4 can be significantly affected by fixation and permeabilization methods. Based on available information:
Formaldehyde fixation: Some antibodies, such as the NLA14 clone, specifically require formaldehyde fixation and permeabilization to recognize VSIG4 . This suggests that the epitope recognized by this antibody may be affected by cross-linking of proteins during fixation.
Perfusion fixation for tissues: For optimal detection in tissue sections, perfusion fixation prior to freezing has been used successfully, as demonstrated in mouse liver sections stained with the AF4674 antibody .
Live cell detection: Not all antibodies can recognize the native conformation of VSIG4 on live cells. Researchers should specifically verify if their antibody of interest has been validated for live cell applications.
Epitope masking considerations: Given that VSIG4 interacts with complement components C3b and iC3b , researchers should be aware that these interactions might mask certain epitopes in samples where complement activation has occurred, potentially affecting antibody binding.
Anti-VSIG4 antibodies have been shown to repolarize tumor-associated macrophages from an immunosuppressive M2 phenotype to a pro-inflammatory M1 phenotype, initiating a cascade of immunological events that ultimately lead to enhanced anti-tumor immunity.
Mechanism of macrophage repolarization:
Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages .
Treatment with anti-VSIG4 antibodies results in significant upregulation of cytokines involved in TAM repolarization and T cell activation, as well as chemokines involved in immune cell recruitment across patient-derived tumor samples from multiple tumor types .
Downstream effects:
Cytokine production: Anti-VSIG4 treatment induces secretion of pro-inflammatory cytokines and chemokines from repolarized macrophages .
T cell activation: The repolarized macrophages subsequently activate T cells, particularly CD8+ T cells, which are critical for the anti-tumor effect .
Enhanced anti-tumor response: In syngeneic mouse models, anti-VSIG4 treatment inhibits tumor growth either as monotherapy or in combination with anti-PD-1, with the effect being dependent on the systemic availability of CD8+ T cells .
A key finding is that the anti-tumor effect of VSIG4 blockade is CD8+ T cell-dependent, indicating that the primary mechanism involves activating the adaptive immune response through initial modulation of innate immunity .
VSIG4 functions within a complex network of immune regulatory molecules in the tumor microenvironment:
Co-expression with other B7 family members: Immunofluorescent double staining has shown that VSIG4 is co-expressed on B7-H1+ (PD-L1) and B7-H3+ cells in tumor specimens . This co-expression pattern suggests potential functional cooperation between these inhibitory molecules.
Complementary mechanisms with PD-1/PD-L1: While PD-1/PD-L1 primarily acts directly on T cells, VSIG4 appears to function through macrophage-mediated immunosuppression. This distinction provides the rationale for combination therapies targeting both pathways, as demonstrated by enhanced efficacy when anti-VSIG4 is combined with anti-PD-1 in preclinical models .
Unique expression pattern: Unlike many other immune checkpoints, VSIG4 expression is largely restricted to macrophages and is not found on T cells, B cells, endothelial cells, or epithelial cells . This unique expression pattern makes it a complementary target to other checkpoint molecules with different cellular distributions.
Role in inflammasome regulation: VSIG4 has been shown to mediate transcriptional inhibition of Nlrp3 and Il-1β in macrophages . VSIG4-deficient macrophages show enhanced NLRP3 inflammasome activation, suggesting that VSIG4 may regulate inflammation through multiple mechanisms beyond direct T cell suppression.
Several experimental models have been developed to evaluate anti-VSIG4 antibody efficacy:
In vitro models:
M2c macrophage differentiation system: Human monocytes are differentiated with M-CSF plus IL-10 to generate M2c macrophages with high VSIG4 expression, providing a system to test antibody-mediated repolarization .
Co-culture systems: VSIG4-expressing cells (either transfected or primary macrophages) are co-cultured with T cells to assess the ability of anti-VSIG4 antibodies to relieve T cell suppression and enhance proliferation and cytokine production .
Ex vivo models:
Patient-derived tumor explants: Fresh patient-derived tumor samples treated ex vivo with anti-VSIG4 antibodies have been used to assess cytokine production and immune cell activation in a more physiologically relevant context .
Ascites-derived macrophages: In ovarian cancer research, macrophages isolated from patient ascites express high levels of VSIG4 and display an immunosuppressive M2 phenotype, providing a relevant model for testing antibody effects .
In vivo models:
Syngeneic mouse models: Lewis lung carcinoma models in VSIG4-deficient versus wild-type mice have demonstrated reduced tumor growth in the absence of VSIG4 . Additionally, anti-mouse VSIG4 antibodies have shown efficacy in syngeneic models, both as monotherapy and in combination with anti-PD-1 .
Humanized mouse models: CD34+ cell and PBMC-humanized mouse models have been used to verify the therapeutic efficacy of antibodies like EU103 in ovarian cancer, particularly for demonstrating M2-to-M1 conversion in a more human-relevant system .
Distinguishing direct from indirect effects requires careful experimental design:
Use of selective cellular depletion: Researchers can deplete specific cell populations (e.g., CD8+ T cells) to determine which effects of anti-VSIG4 antibodies are dependent on these cells. This approach has demonstrated that the anti-tumor effects of VSIG4 blockade are dependent on CD8+ T cells, indicating an indirect mechanism through T cell activation .
Time-course experiments: Analyzing early versus late events after anti-VSIG4 treatment can help establish causality. Early changes in macrophage phenotype followed by later T cell activation would support a direct effect on macrophages with indirect effects on T cells.
In vitro versus in vivo comparison: Differences between antibody effects in purified macrophage cultures versus complex in vivo systems can help identify which effects require additional cell types or factors.
Genetic confirmation: Comparing antibody effects to genetic VSIG4 deficiency can help confirm target specificity. For example, VSIG4-deficient macrophages show enhanced NLRP3 inflammasome activation, suggesting this pathway might be directly regulated by VSIG4 .
Mechanistic blockade experiments: Blocking specific downstream pathways (e.g., with cytokine-neutralizing antibodies) can help determine which effects are dependent on specific mediators induced by VSIG4 targeting.
Researchers should anticipate several technical challenges:
Isoform selectivity: VSIG4 exists in multiple isoforms, particularly "long" and "short" variants. Therapeutic antibodies must recognize both isoforms if complete blockade is desired . The development of 12A12 antibody specifically addressed this by targeting the conserved IgV domain common to both isoforms .
Species cross-reactivity: Many VSIG4 antibodies are species-specific. The NLA14 antibody, for example, does not cross-react with rat or human VSIG4 . This limits the translation of findings between preclinical models and human applications, potentially requiring development of species-specific antibodies for different research stages.
Functional testing complexity: Since VSIG4 operates through complex cellular interactions, functional assays require carefully designed co-culture systems that recapitulate relevant aspects of the tumor microenvironment. Simple binding assays are insufficient to predict therapeutic efficacy.
Strain-dependent expression variations: Studies have shown that peritoneal macrophages in BALB/c mice express significantly higher levels of VSIG4 than such macrophages in C57BL/6 or Swiss Webster mice . This strain-dependent variation must be considered when designing and interpreting experiments.
Dynamic regulation: VSIG4 expression is dynamically regulated, with pro-inflammatory stimuli such as TNF and LPS reported to down-regulate its expression . This dynamic regulation may affect timing and dosing requirements for therapeutic applications.
Genetic and phenotypic variations in TAMs significantly impact anti-VSIG4 antibody efficacy:
Heterogeneity in VSIG4 expression: While VSIG4 is generally expressed on TAMs, the level of expression can vary between cancer types and even within the same tumor. Single-cell RNA-seq data from non-small cell lung cancer patients has shown VSIG4 localized to tumor-associated myeloid populations, but expression levels may vary .
Macrophage polarization state: The baseline polarization state of TAMs may affect their responsiveness to anti-VSIG4 therapy. Studies have shown that inhibitory TAMs expressing high levels of CD163 also express high levels of VSIG4, while pro-inflammatory M1 macrophages do not express VSIG4 . This suggests that tumors with predominantly M2-polarized TAMs may respond better to anti-VSIG4 therapy.
Tumor type-specific responses: Analyses of approximately 10,000 human tumors spanning 33 cancer types have shown varying levels of VSIG4 expression, with the highest in glioblastoma, mesothelioma, NSCLC, and pancreatic adenocarcinoma . This variation may predict differential responsiveness to anti-VSIG4 therapy across cancer types.
Inflammatory context: The inflammatory state of the tumor microenvironment may affect VSIG4 expression and function. Pro-inflammatory stimuli like TNF and LPS have been shown to down-regulate VSIG4 expression , potentially reducing the efficacy of anti-VSIG4 antibodies in highly inflamed tumors.
Interaction with complement system: VSIG4 binds complement components C3b and iC3b , and variations in complement activation within tumors may affect the accessibility of VSIG4 to therapeutic antibodies or alter its functional role in TAMs.
The discovery that VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1β in macrophages has important implications for cancer immunotherapy:
Enhanced inflammasome activation: VSIG4-deficient (VSIG4-/-) peritoneal macrophages show significantly enhanced expression of Nlrp3 and Il-1β mRNA compared to wild-type controls . This suggests that targeting VSIG4 could potentially enhance inflammasome activation as part of its immunostimulatory mechanism.
Increased IL-1β production: VSIG4-/- macrophages treated with NLRP3 inflammasome stimuli (ATP, nigericin, or crystalline silica) show enhanced caspase-1 activation and IL-1β secretion compared to wild-type controls . This increased IL-1β production may contribute to the anti-tumor effects of VSIG4 blockade by promoting inflammatory responses.
Enhanced pyroptosis: NLRP3 inflammasome stimuli significantly enhance caspase-1-dependent pyroptosis in VSIG4-/- macrophages compared to wild-type controls . This form of inflammatory cell death could potentially enhance tumor immunogenicity.
Therapeutic implications: The connection between VSIG4 and inflammasome regulation suggests that combining anti-VSIG4 antibodies with other immunotherapies that benefit from inflammasome activation might be particularly effective. It also implies that monitoring inflammasome-related biomarkers might help predict or assess response to anti-VSIG4 therapy.
Potential adverse effects: Enhanced inflammasome activation could potentially lead to increased inflammatory side effects with anti-VSIG4 therapy, requiring careful monitoring in clinical applications.