C10ORF54 Mouse

What is C10ORF54 and what are its alternative names in the scientific literature?

C10ORF54, located on chromosome 10 (10q22.1), is a gene that encodes the V-set immunoregulatory receptor protein also known as VISTA. This protein belongs to the immunoglobulin superfamily and functions as an important immunoregulatory molecule. In scientific literature, you may encounter C10ORF54 under several alternative designations including VISTA, GI24, B7-H5, B7H5, DD1alpha, Dies1, PD-1H, PP2135, and SISP1 . When conducting literature searches, it's advisable to include these alternative names to ensure comprehensive coverage of relevant research.

How conserved is VISTA (C10ORF54) across species and what does this suggest about its function?

VISTA demonstrates remarkable evolutionary conservation, suggesting its fundamental biological importance. Among B7 family members, VISTA exhibits the highest conservation level with 76% sequence identity between mouse and human orthologs. Even more striking is the 31% sequence identity between mouse and zebrafish counterparts, with the cytoplasmic tail showing an impressive 59.4% identity . The cytoplasmic domain is particularly well-preserved, sharing 90.6% identity between mouse and human versions, strongly indicating a tightly conserved functional role . For comparison, the cytoplasmic tails of human and mouse PD-1 share only 59% identity. This exceptional conservation across distant species suggests that VISTA likely plays a critical and evolutionarily ancient role in immune regulation that predates the divergence of mammalian lineages.

What is the cellular distribution pattern of VISTA in mouse immune cells?

VISTA expression in mice follows a distinct pattern primarily centered in the hematopoietic compartment. It is abundantly expressed in dendritic cells (DCs), particularly CD11c+ DCs, making these antigen-presenting cells important subjects for VISTA-focused research . VISTA is also present, though at lower levels, in T cells and is notably upregulated in activated regulatory T cells (Tregs) . This expression pattern is significant for experimental design as it suggests potential roles in both the antigen-presenting and responding arms of the immune response. When designing flow cytometry panels to detect VISTA, optimal results typically require approximately 10 μg of antibody per test when examining cell populations like human platelets, though titration for specific mouse cell populations is advisable .

How does VISTA's molecular structure differ from other B7 family members and what functional implications does this have?

VISTA exhibits distinct structural characteristics that differentiate it from other B7 family members and impact its functional properties. Unlike classic B7 molecules, VISTA lacks the conventional Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) or Immunoreceptor Tyrosine-based Activation Motif (ITAM) in its cytoplasmic domain . Instead, VISTA possesses a conserved Src homology 2 (SH2)-binding domain (YxxQ), which resembles features found in CD28 and CTLA-4 .

This unique structural arrangement suggests that VISTA likely employs signaling mechanisms distinct from other inhibitory receptors. The absence of conventional signaling motifs combined with the highly conserved cytoplasmic tail indicates that VISTA may utilize alternative molecular partnerships or signaling pathways to exert its immunoregulatory functions. When designing experiments to investigate VISTA signaling, researchers should consider techniques that can identify non-canonical protein-protein interactions, such as proximity ligation assays or immunoprecipitation coupled with mass spectrometry, rather than limiting investigations to known ITIM/ITAM-dependent pathways.

What is known about the synergistic effects between VISTA and other immune checkpoint molecules in mouse cancer models?

Research has demonstrated significant synergistic effects when targeting VISTA in combination with other immune checkpoint molecules in mouse cancer models. Particularly notable is the combination of anti-VISTA and anti-PD-L1 antibodies in the CT26 colon cancer mouse model . This dual checkpoint blockade results in substantially reduced tumor growth and markedly increased long-term survival compared to either monotherapy alone .

This synergy suggests that VISTA and PD-L1 operate through complementary but distinct immunosuppressive mechanisms. From an experimental perspective, researchers investigating combination therapies should:

• Include appropriate single-agent control groups alongside combination treatment

• Monitor survival beyond tumor volume metrics to capture durable response effects

• Examine changes in the tumor immune microenvironment, particularly focusing on T cell infiltration, activation status, and functional capacity

• Consider potential compensatory upregulation of alternative checkpoint molecules following blockade

The observed synergy provides compelling evidence for exploring VISTA-targeted approaches in combination immunotherapy strategies, particularly in contexts where single-agent PD-1/PD-L1 blockade shows limited efficacy.

How does genomic location and conservation of VISTA influence our understanding of its evolutionary and functional significance?

VISTA's genomic context provides fascinating insights into its evolutionary history and potential functional constraints. Unlike other B7 family members that typically cluster together in the genome, VISTA is isolated on chromosome 10 (10q22.1) with no neighboring immunoglobulin superfamily members . Intriguingly, VISTA is consistently located within a large intron of the CDH23 gene across all genomes starting from the most primitive predicted ortholog in ray-finned fish .

This unusual genomic arrangement has several implications:

• The consistent intronic location within CDH23 across diverse species suggests potential co-regulation or functional relationships that have been maintained throughout vertebrate evolution

• The isolation from other immunoglobulin superfamily members indicates VISTA likely evolved independently rather than through tandem gene duplication events typical of other immune receptors

• The high sequence conservation, particularly in the cytoplasmic tail, points to strong selective pressure maintaining VISTA's function

Researchers investigating VISTA should consider that this molecule likely represents an ancient and distinct branch of immune regulation separate from conventional B7 family evolution. Comparative studies examining VISTA function across species may provide valuable insights into fundamental immunoregulatory mechanisms that have been conserved for hundreds of millions of years.

What are the optimal approaches for detecting VISTA expression in mouse tissues and isolated immune cells?

Detection of VISTA in mouse tissues and cells requires careful consideration of methodology, reagent selection, and protocol optimization. Based on available research tools, the following approaches are recommended:

Immunohistochemistry (IHC):

• Mouse monoclonal antibodies such as clone UMAB271 have been validated for IHC applications

• Recommended dilution for IHC is typically 1:50, but optimization may be required for specific tissue types

• Appropriate antigen retrieval methods are essential, particularly for formalin-fixed tissues

• Positive control tissues should include spleen and lymph nodes where VISTA expression is abundant

Flow Cytometry:

• Clone ORF.4 purified from hybridoma cell culture has been validated for flow cytometry

• Standard protocol recommends approximately 10 μg antibody per test for human samples; mouse samples may require titration

• Include appropriate isotype controls (IgG1 for UMAB271 and ORF.4 clones)

• A comprehensive panel should include markers to identify specific immune subsets (e.g., CD11c for dendritic cells, Foxp3 for regulatory T cells) where VISTA expression is expected

Western Blotting:

• When analyzing protein lysates, compare results across multiple antibody clones to confirm specificity

• Expected molecular weight is approximately 33.7 kDa, though post-translational modifications may result in higher observed weights

• Reducing conditions are typically required for optimal detection

For all applications, verification of specificity using VISTA knockout tissues or cells as negative controls is strongly recommended when available.

What considerations should researchers take into account when designing in vivo experiments using anti-VISTA antibodies in mouse models?

When designing in vivo experiments with anti-VISTA antibodies in mouse models, several important considerations should guide experimental planning:

Antibody Selection and Dosing:

• Choose antibodies with demonstrated in vivo efficacy and appropriate isotype controls

• Dosing regimens typically range from 100-250 μg per injection every 3-4 days, but should be optimized for specific experimental contexts

• Consider the half-life of the antibody and pharmacodynamic effects when determining dosing frequency

Experimental Design:

• Include appropriate control groups: isotype control antibody, single-agent controls when testing combinations

• Power analysis should account for expected effect sizes based on published literature

• Pre-register experimental endpoints and analysis methods to avoid post-hoc biases

Model Selection:

• CT26 colon cancer models have demonstrated responsiveness to VISTA blockade, particularly in combination with PD-L1 inhibition

• Consider models with varying degrees of immunogenicity to assess the impact of VISTA across the spectrum of "hot" to "cold" tumors

• For mechanistic studies, include experiments in relevant knockout strains to confirm specificity

Outcome Measurements:

• Beyond tumor growth, assess survival, immune cell infiltration, and activation status

• Consider multi-parameter flow cytometry or spatial profiling of the tumor microenvironment

• Include assessments of potential immune-related adverse events, particularly when combining multiple checkpoint inhibitors

Carefully designed experiments with appropriate controls and comprehensive outcome assessments will yield the most interpretable and translatable results in VISTA-targeted studies.

What techniques are recommended for investigating VISTA signaling mechanisms given its unique cytoplasmic tail structure?

Investigating VISTA signaling presents unique challenges due to its atypical cytoplasmic tail structure. Unlike conventional immune receptors with ITIM/ITAM motifs, VISTA contains a conserved SH2-binding domain (YxxQ) . The following techniques are particularly well-suited for elucidating VISTA signaling mechanisms:

Phosphorylation Studies:

• Phospho-specific antibodies targeting the YxxQ motif to detect activation state

• Phospho-proteomics to identify changes in signaling networks following VISTA engagement

• Kinase inhibitor screens to identify enzymes responsible for VISTA phosphorylation

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation followed by mass spectrometry to identify binding partners

• Proximity ligation assays to visualize protein interactions in situ

• BiFC (Bimolecular Fluorescence Complementation) or FRET-based approaches to confirm direct interactions

Functional Domain Analysis:

• Structure-function studies using truncated or mutated VISTA constructs

• CRISPR-mediated precise editing of the YxxQ motif and other conserved regions

• Chimeric receptor approaches swapping the VISTA cytoplasmic tail with other receptors

Downstream Signaling Assessment:

• Calcium flux measurements following receptor engagement

• Phospho-flow cytometry to detect rapid signaling events

• Transcriptomic analysis to identify gene expression changes dependent on VISTA signaling

When conducting these studies, researchers should consider the exceptionally high conservation of the VISTA cytoplasmic tail (90.6% identity between mouse and human) , which suggests stringent structural requirements for its function. This conservation provides confidence that findings in mouse models are likely to translate to human biology.

How should researchers interpret contradictory findings between in vitro and in vivo studies of VISTA function in mouse models?

When encountering contradictions between in vitro and in vivo VISTA studies, researchers should consider several factors that might explain these discrepancies:

Microenvironmental Context:

• VISTA functions within a complex immune microenvironment that cannot be fully recapitulated in vitro

• The absence of spatial organization, tissue-specific factors, and three-dimensional architecture in standard in vitro systems may alter VISTA signaling dynamics

• Consider using more complex in vitro systems such as organoids or tissue slices that better preserve physiological context

Temporal Dynamics:

• In vivo immune responses evolve over days to weeks, while in vitro studies typically capture only snapshots

• Time-course experiments in both settings may reveal that apparent contradictions reflect different temporal phases of the same biological process

• When possible, implement live imaging approaches to capture dynamic changes

Cell-Cell Interactions:

• VISTA's function depends on interactions between multiple cell types, particularly between dendritic cells and T cells

• Single-cell type in vitro studies may miss critical intercellular communication

• Co-culture systems incorporating relevant interacting cell populations may better replicate in vivo findings

Experimental Controls:

• Differing activity of control antibodies or reagents between systems

• Variation in expression levels of VISTA or binding partners

• Differences in activation state of cells being studied

When publishing research on VISTA, transparently report any discrepancies between in vitro and in vivo findings rather than selectively presenting data that fits a particular hypothesis. These apparent contradictions often provide valuable insights into context-dependent functions and regulatory mechanisms.

What are the common technical challenges when working with mouse VISTA antibodies, and how can researchers overcome them?

Researchers working with mouse VISTA antibodies frequently encounter several technical challenges that can impact experimental outcomes. Understanding these issues and implementing appropriate solutions can significantly improve data quality and reproducibility:

Challenge: Cross-reactivity with Related Proteins

Solutions:

• Validate antibody specificity using VISTA knockout controls

• Perform side-by-side testing of multiple antibody clones (e.g., UMAB271, ORF.4)

• Confirm specificity through techniques like Western blotting prior to application in more complex assays

Challenge: Variability in Detection Sensitivity Across Applications

Solutions:

• Optimize antibody concentration for each specific application (starting with recommended dilutions such as 1:50 for IHC)

• Consider the use of signal amplification systems for low expression contexts

• Perform careful titration experiments to determine optimal concentration

Challenge: Epitope Masking in Tissue Sections

Solutions:

• Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

• Consider alternative fixation methods that better preserve VISTA epitopes

• Use freshly prepared tissue sections when possible, as epitope accessibility may diminish with storage

Challenge: Interference from Endogenous Fc Receptors

Solutions:

• Include appropriate Fc receptor blocking steps in protocols

• Consider using F(ab)2 fragments rather than whole antibodies when Fc effects are problematic

• Include proper isotype controls matched to the VISTA antibody (typically IgG1 for clones like UMAB271)

Challenge: Batch-to-Batch Variability

Solutions:

• Maintain records of antibody lot numbers used in experiments

• When possible, purchase sufficient quantities of a single lot for complete experimental series

• Include standard samples across experiments to normalize for batch effects

By systematically addressing these common challenges, researchers can ensure more consistent and reliable results when working with mouse VISTA antibodies across different experimental applications.

How can researchers effectively design experiments to distinguish the unique contributions of VISTA from other immunoregulatory molecules in complex immune responses?

Distinguishing VISTA's specific contributions from other immunoregulatory molecules requires thoughtfully designed experiments that isolate its functions while accounting for the complex network of immune checkpoints. The following experimental approaches are particularly effective:

Genetic Approaches:

• VISTA knockout mice compared with wild-type controls

• Conditional and cell-type specific deletion to determine context-dependent roles

• Knockin models with mutated signaling domains to distinguish receptor vs. ligand functions

Temporal Manipulation:

• Inducible systems to control VISTA expression/deletion at defined time points

• Sequential blockade experiments (e.g., anti-VISTA followed by anti-PD-L1 vs. the reverse order)

• Time-course analyses to track dynamic changes in immune response following VISTA perturbation

Combinatorial Blockade with Mechanistic Readouts:

When testing combinations such as anti-VISTA with anti-PD-L1 in mouse tumor models , include:

• Comprehensive immune phenotyping to identify cell populations specifically affected by each intervention

• Analysis of distinct signaling pathways downstream of different checkpoint molecules

• Gene expression profiling to identify unique transcriptional signatures

Ex Vivo Validation:

• Isolate cells from treated animals for functional assays (e.g., antigen-specific responses)

• Use ex vivo blockade or stimulation to confirm direct effects

• Cross-over experiments where cells from treated animals are transferred to untreated recipients

Controls to Consider:

• Multiple checkpoint blockade combinations to establish specificity

• Isotype controls that account for potential Fc-mediated effects

• Validation across different mouse strains to account for background genetic effects

The synergistic effects observed between anti-VISTA and anti-PD-L1 in mouse models highlight that these molecules likely operate through distinct but complementary mechanisms. Well-designed experiments that systematically isolate VISTA's contributions will be essential for developing optimal therapeutic strategies and understanding fundamental immune regulatory mechanisms.

What are the implications of VISTA's high evolutionary conservation for translational research using mouse models?

The exceptional evolutionary conservation of VISTA—with 76% sequence identity between mouse and human and 90.6% identity in the cytoplasmic tail —has profound implications for translational research. This conservation provides a strong foundation for the validity of mouse models in studying VISTA-related biology relevant to human health:

Translational Confidence:

• The high conservation suggests functional mechanisms identified in mice are likely preserved in humans

• Particularly for the cytoplasmic domain, signaling pathways and protein interactions identified in mouse models have high translational potential

• This contrasts with many other immune receptors that show greater divergence between species

Therapeutic Development Considerations:

• Mouse models are likely to provide more predictive preclinical data for VISTA-targeted therapies than for less conserved targets

• Antibodies targeting conserved epitopes may have cross-species reactivity, potentially allowing direct testing of clinical candidates in mouse models

• The success of combined anti-VISTA and anti-PD-L1 approaches in mouse tumor models may translate well to human clinical applications

Experimental Design Implications:

• "Humanized" VISTA knock-in mice may be less necessary than for other targets given the high conservation

• Focus on understanding fundamental biology rather than species-specific differences

• Consider evolutionary studies examining VISTA function across species to identify absolutely critical functional elements

The unusual genomic location of VISTA within an intron of the CDH23 gene across diverse species further supports the notion that VISTA represents an ancient immune regulatory mechanism with fundamental importance. This provides strong justification for continued investment in mouse models for VISTA research with high confidence in translational relevance.

How should researchers approach the study of VISTA in the context of its interactions with the broader immune checkpoint network?

Given VISTA's place within a complex network of immune checkpoints, researchers should adopt systematic approaches that consider both specific VISTA functions and broader network interactions:

Network Analysis Framework:

• Systems biology approaches to map interactions between VISTA and other checkpoint molecules

• Single-cell multi-omics to identify co-expression patterns and potential functional relationships

• Correlation of VISTA expression with other checkpoints across different immune contexts and disease states

Sequential and Combination Targeting:

• Examine temporal aspects of checkpoint expression (which comes first in immune responses?)

• Test ordered sequential blockade (anti-VISTA followed by anti-PD-L1 vs. the reverse)

• Investigate potential compensatory upregulation of other checkpoints following VISTA blockade

Cell Type-Specific Checkpoint Profiles:

• Map the checkpoint landscape on specific cell populations (e.g., tumor-infiltrating T cells vs. dendritic cells)

• Create comprehensive profiles of checkpoint molecule expression on matched cells from blood vs. tissue

• Determine how VISTA expression correlates with functional exhaustion markers compared to other checkpoints

Recommended Experimental Approaches:

• Multi-parameter flow cytometry panels including at least 3-5 major checkpoint molecules

• Multiplexed imaging to visualize co-expression patterns while preserving spatial information

• Computational modeling to predict network effects of VISTA perturbation

The demonstrated synergy between VISTA and PD-L1 blockade in mouse models provides a clear rationale for studying checkpoint molecules as an interconnected network rather than in isolation. Understanding the position of VISTA within this network will be essential for optimizing combination immunotherapy approaches and predicting resistance mechanisms.

What novel research approaches might help elucidate the structural basis for VISTA's immunoregulatory functions?

Despite significant advances in understanding VISTA's immunological functions, the structural basis for its activity remains incompletely characterized. The following cutting-edge approaches could provide crucial insights:

Cryo-Electron Microscopy (Cryo-EM):

• Determine the structure of VISTA in complex with binding partners

• Analyze conformational changes upon receptor-ligand engagement

• Map the structural implications of the conserved SH2-binding domain (YxxQ)

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Identify dynamic regions of VISTA that undergo conformational changes upon binding

• Map protein-protein interaction interfaces with high resolution

• Compare structural dynamics between mouse and human VISTA to identify conserved functional elements

AlphaFold and Computational Approaches:

• Leverage advanced AI-based structural prediction to model VISTA complexes

• Perform molecular dynamics simulations to understand the functional implications of the highly conserved cytoplasmic tail

• Use computational mutagenesis to predict the impact of specific amino acid changes

Structure-Guided Functional Validation:

• Design point mutations based on structural insights to test specific hypotheses

• Create chimeric proteins swapping domains between VISTA and related molecules

• Develop structure-based small molecule modulators as biological probes

CRISPR-Based Approach:

• Implement base editing or prime editing to introduce precise mutations in endogenous VISTA

• Create scanning mutagenesis libraries to map functionally critical residues

• Develop CRISPR activation/inhibition systems targeting VISTA regulatory elements