TL;DR – How eccDNA talks to the immune system

Extrachromosomal circular DNA (eccDNA) is a circular DNA molecule that can form during infection, tissue damage, and immune activation. In immune and infection models, eccDNA can act as an innate immune trigger, shape T-cell responses, and serve as a readout of DNA damage and chromatin stress. This article explains eccDNA innate immunostimulatory activity, summarizes evidence from infection and T-cell systems, and shows how to design eccDNA immune studies using Circle-seq, eccDNA methylation sequencing, and eccDNA qPCR quantification.

Why eccDNA and the immune system are now linked in every infection study

eccDNA is a circular DNA molecule outside chromosomes that increasingly appears in immune and infection research.

Immunologists historically focused on cytokines, surface markers, and gene expression to understand infection and T-cell responses. These readouts remain essential, but they rarely capture the underlying DNA damage and chromatin stress that drive immune signaling. eccDNA sits exactly at this interface.

When cells face viral or bacterial infection, they experience replication stress, oxidative damage, and nuclease activity. Small DNA fragments can be excised and ligated into circular molecules, forming eccDNA in infected cells, immune cells, and surrounding tissues. Because these circles can relocate to the cytosol or extracellular space, they are well positioned to interact with innate immune sensors.

For infection biologists and immuno-oncology teams, this means:

• eccDNA complements RNA-seq, ATAC-seq, and cytokine panels.
• eccDNA profiles help separate generic inflammation from specific DNA damage patterns.
• eccDNA innate immunostimulatory activity can reveal why some infection models show strong interferon signatures while others do not.

Traditional readouts tell you what the immune system is doing. eccDNA immune profiling adds context on why the immune response looks that way in a given infection or T-cell system.

In our work with immunology, immuno-oncology, and infectious disease clients, eccDNA immune profiling is now used alongside standard immune assays to uncover mechanisms that would otherwise remain hidden.

How eccDNA is generated during infection and tissue damage

During infection and tissue damage, eccDNA forms from DNA breaks, replication stress, and mis-repaired chromatin fragments.

Mechanisms of eccDNA formation under genomic stress. Schematic overview of multiple routes that generate eccDNA, including chromothripsis, breakage–fusion–bridge cycles, replication-based events, and episome-like excision, illustrating how DNA damage and repair errors can give rise to circular DNA molecules. (Deng E. et al. (2024) Biomolecules)

Biogenesis of eccDNA under infectious stress

eccDNA biogenesis in infection arises from stressed DNA replication and repair processes.

Viral and bacterial infections raise the burden on the host genome. Viral replication factories, bacterial toxins, and reactive oxygen species can all generate double-strand breaks and stalled replication forks. When repair is incomplete or mis-directed, small DNA segments can be excised and circularised instead of neatly re-integrated.

Key routes of eccDNA formation in infection models include:

• End-joining of small double-strand break fragments.
• Excision of repetitive or transposable elements under replication stress.
• Rearrangement-linked circles from antigen receptor loci in developing or activated T cells.

The spectrum of eccDNA in infected cells therefore reflects both the pathogen's biology and the host's DNA repair landscape.

Release and localisation: intracellular vs extracellular eccDNA

eccDNA can remain nuclear, move into the cytosol, or be released as extracellular DNA.

Not all eccDNA is immunostimulatory. Its immune impact depends strongly on location:

• Nuclear eccDNA often behaves as a chromatin by-product, with modest direct immune effects.
• Cytosolic eccDNA can be detected by DNA sensors such as cGAS, acting as a danger signal.
• Extracellular eccDNA can appear in plasma, bronchoalveolar lavage, or vesicles, reflecting tissue damage or immune activation at a distance.

In infection models, bursts of cytosolic or extracellular eccDNA often track with peaks in inflammatory cytokines and cell death. For eccDNA immune projects, choosing which compartment to sample (cells vs plasma vs vesicles) is a strategic design decision, not a technical detail.

eccDNA as an innate immune trigger: DAMP and cGAS–STING signaling

eccDNA can function as a damage-associated molecular pattern (DAMP) that activates innate immune pathways.

eccDNA as a damage-associated molecular pattern (DAMP)

As a DAMP, eccDNA signals that cells have experienced severe genomic stress or injury.

Innate immune systems evolved to sense misplaced or altered nucleic acids. eccDNA fits this category well: it is self-DNA, but in an abnormal circular form and often in the wrong compartment.

Compared with linear genomic or mitochondrial DNA, eccDNA:

• Can be enriched for repetitive or regulatory sequences.
• May carry distinct methylation and chromatin marks.
• Can accumulate preferentially in stressed or transformed cells.

When eccDNA accumulates outside the nucleus, innate immune cells may perceive it as a "help" or "danger" signal, triggering controlled inflammation that can become pathogenic if sustained.

Cytosolic eccDNA and the cGAS–STING pathway

Cytosolic eccDNA can engage the cGAS–STING pathway and drive interferon responses.

The cGAS–STING axis is a central DNA-sensing pathway in innate immunity. When cGAS encounters DNA in the cytosol, it produces cGAMP, activating STING and downstream transcription factors such as IRF3 and NF-κB. This cascade leads to type I interferon and inflammatory cytokine production.

cGAS–STING activation by diverse nucleic acid ligands, including extrachromosomal DNA. (Guo J. et al. (2025) Biomedicines)

In infection and tumor models:

• DNA damage and defective repair can push eccDNA into the cytosol.
• Cytosolic eccDNA can act similarly to viral DNA in triggering cGAS–STING.
• The resulting interferon signature shapes how T cells and myeloid cells behave in the tissue.

For immuno-oncology teams, this is especially relevant. Treatments that boost DNA damage in tumor cells may increase eccDNA load and cGAS–STING activation, enhancing anti-tumor immunity but also raising the risk of systemic inflammation.

Beyond cGAS–STING: TLRs, inflammasomes, and other DNA sensors

eccDNA may also interact with other DNA sensors, including TLRs and inflammasome components.

While cGAS–STING receives most attention, infection models suggest additional sensing layers:

• Endosomal TLR9 may detect eccDNA with unmethylated CpG motifs.
• Inflammasome pathways can be indirectly engaged when eccDNA induces cell stress and secondary danger signals.
• Other cytosolic sensors may respond to eccDNA complexes or eccDNA–protein aggregates.

The net effect depends on sequence content, methylation, and how eccDNA is packaged. This complexity is one reason eccDNA innate immunity is now a serious topic for detailed epigenomic sequencing and immune profiling, not just an interesting side note.

To help teams navigate these options, a short comparison between DNA ligands is useful:

eccDNA in viral and bacterial infection models

In viral and bacterial infection models, eccDNA reflects host–pathogen conflict and can correlate with innate immune activation.

eccDNA in viral infection models

Viral infection often increases eccDNA production and reshapes eccDNA content.

DNA and RNA viruses drive intense replication and immune pressure. In several viral infection systems, researchers have reported:

• Increased eccDNA levels in infected cells compared with uninfected controls.
• Enrichment of eccDNA derived from antiviral genes, repetitive elements, or viral integration sites.
• Associations between eccDNA burden, interferon signatures, and cytotoxic T-cell infiltration.

In chronic infection models, persistent eccDNA may mark ongoing low-grade DNA damage and immune activation. For viral immunology groups, eccDNA infection readouts can help distinguish transient antiviral bursts from sustained, tissue-damaging inflammation.

eccDNA in bacterial and mycobacterial infection

Bacterial and mycobacterial infections can also reshape eccDNA landscapes in host tissues.

Intracellular pathogens such as Mycobacterium or Listeria induce strong macrophage responses and DNA damage. In these systems, eccDNA has been detected:

• In infected macrophages undergoing oxidative stress.
• In granulomatous tissue, where chronic inflammation and cell turnover are high.
• In circulation, potentially reflecting tissue destruction and immune activity.

By profiling eccDNA infection patterns in these models, researchers can monitor how host DNA damage evolves across early infection, chronic disease, and treatment response.

Extracellular eccDNA in infection microenvironments

Extracellular eccDNA in fluids like plasma can serve as a minimally invasive marker of immune activation.

During infection, eccDNA can be released into:

• Plasma or serum.
• Bronchoalveolar lavage fluid in lung infection models.
• Supernatants of infected cell cultures or organoids.

Measuring eccDNA in these compartments enables:

• Time-course monitoring in animal infection models.
• Comparison between different treatment arms or vaccine regimens.
• Non-terminal sampling in longitudinal studies.

Key takeaways from eccDNA infection models include:

• eccDNA levels often rise with DNA damage and inflammation.
• eccDNA sequence content may highlight pathways under selective pressure.
• Combining eccDNA immune readouts with RNA-seq and cytokines offers a more complete view of host–pathogen interactions.

eccDNA in T-cell development, activation, and immunotherapy

In T-cell biology, eccDNA marks antigen receptor rearrangements, proliferation, and stress during activation or therapy.

eccDNA from antigen receptor rearrangements and T-cell development

T-cell receptor rearrangements naturally generate eccDNA fragments during development.

As thymocytes rearrange TCR genes, excised DNA segments can circularise, forming TCR excision circles. These are a classical form of eccDNA, historically used as markers of recent thymic emigrants.

T-cell receptor excision circles as a readout of thymic output. (Söderström A. et al. (2022) Frontiers in Immunology)

For immunologists, this has two implications:

• eccDNA T-cell markers already have precedent in immune monitoring.
• New eccDNA sequencing tools can extend this logic beyond TCR loci to broader genomic regions.

By combining eccDNA analysis with TCR/BCR repertoire sequencing, teams can study both clonal structure and the underlying DNA rearrangements or stress.

eccDNA during T-cell activation, effector function, and exhaustion

Activated T cells can accumulate eccDNA as they proliferate and face DNA damage.

Upon antigen encounter, T cells enter rapid cell cycles, reshape their chromatin, and produce effector molecules. These demands increase the risk of replication stress and DNA breaks. Under such conditions:

• Activated effector T cells may show increased eccDNA load.
• Exhausted T cells in chronic infection or tumors may carry characteristic eccDNA patterns.
• Differences in eccDNA profiles can correlate with cytokine output and survival.

These eccDNA T-cell signatures provide a complementary view to flow cytometry markers like PD-1 or TIM-3.

Implications for immuno-oncology and adoptive T-cell therapies

eccDNA analysis can support quality control and mechanistic studies in immuno-oncology and adoptive cell therapy.

In CAR-T or TCR-engineered products, intense ex vivo stimulation and genome editing can modify DNA damage and eccDNA formation. Carefully designed eccDNA immune studies could:

• Monitor DNA stress in manufactured T-cell products.
• Explore how pre-existing eccDNA patterns relate to in vivo expansion or toxicity.
• Help interpret cGAS–STING activation and inflammatory events in tumor microenvironments.

A concise Q&A helps clarify positioning:

Q: How is eccDNA profiling different from TCR sequencing in T-cell studies?

TCR sequencing tracks clonal receptor usage, while eccDNA profiling captures broader DNA damage, rearrangements, and innate immune signaling potential. Used together, they provide a more complete picture of T-cell biology in infection and immunotherapy.

Experimental strategies to profile eccDNA in immune and infection models

eccDNA immune projects typically combine discovery-scale sequencing, epigenetic characterisation, and targeted validation.

Example workflow for eccDNA analysis from tissue or blood. Conceptual pipeline illustrating how tumor tissue or blood samples are processed to isolate eccDNA, followed by enrichment, rolling-circle amplification, sequencing, and qPCR-based detection of eccDNA-associated oncogenes, providing a template for immune and infection models that use plasma or serum. (Deng E. et al. (2024) Biomolecules)

Discovery: Circle-seq and eccDNA sequencing in immune models

Circle-seq is a sequencing workflow that enriches and profiles eccDNA across the genome.

For immunology and infection labs, eccDNA Sequencing (Circle-seq) is often the starting point. When designed for eccDNA immune projects, a Circle-seq workflow can:

• Enrich circular DNA from infected cell lines, primary immune cells, or plasma.
• Map eccDNA species associated with viral or bacterial infection, T-cell activation, or innate immune signaling.
• Provide candidate immunostimulatory eccDNA sequences for downstream validation.

Our eccDNA Sequencing (Circle-seq) service supports immune and infection models with:

• Validated eccDNA enrichment protocols compatible with PBMCs, sorted T-cell subsets, infected cell lines, and biofluids.
• Bioinformatics modules focused on cGAS–STING pathway genes, antiviral loci, and inflammatory signatures.
• Reporting formats optimized for integration with RNA-seq and cytokine data.

Mechanistic readouts: eccDNA methylation and chromatin state

eccDNA methylation sequencing measures epigenetic marks on circular DNA that influence immunostimulatory activity.

DNA methylation can alter how DNA sensors perceive a molecule. Hypomethylated DNA often appears more "foreign" to the innate immune system. By using eccDNA methylation sequencing, researchers can:

• Determine whether eccDNA species in infection models are hypo- or hyper-methylated.
• Link methylation patterns to observed interferon or cytokine profiles.
• Explore how treatments, such as DNA methylation inhibitors, reshape eccDNA immunogenicity.

Our eccDNA methylation sequencing workflows are designed to:

• Preserve circular molecules through bisulfite or enzymatic methylation protocols.
• Generate base-level methylation calls on eccDNA linked to immune pathways.
• Integrate results with Circle-seq discovery data in a unified report.

Quantitative validation: eccDNA qPCR for immune-related circles

eccDNA qPCR quantification provides targeted, high-throughput measurement of selected eccDNA species.

Once candidate immunostimulatory eccDNA species are identified, eccDNA qPCR is a practical way to validate and track them across conditions:

• Custom primer design ensures specificity for eccDNA junctions.
• qPCR enables fast screening across many infection time points, donors, or treatment arms.
• MIQE-aligned reporting supports reproducible quantitative analysis.

Our eccDNA qPCR Quantification Service helps immune and infection teams by:

• Designing custom eccDNA panels focused on infection, T-cell activation, or innate immunity loci.
• Providing guidance on reference controls, standard curves, and limits of detection.
• Delivering data in formats ready for statistical analysis and figure generation.

To choose between methods, many teams benefit from a simple comparison:

Designing eccDNA immune studies: sample types, controls, and project workflow

Well-designed eccDNA immune studies align sample types, controls, and sequencing methods with clear biological questions.

Choosing sample types for eccDNA–immune studies

Sample selection determines which part of the immune landscape eccDNA will report on.

Common options include:

• Sorted immune cell subsets

  Ideal for mechanistic questions in T-cell or myeloid biology.

• Infected cell lines or organoids

  Useful for controlled infection models and screening experiments.

• Primary tissues

  Capture complex microenvironments in animal models or ex vivo systems.

• Plasma, serum, or BAL fluid

  Enable minimally invasive longitudinal sampling of eccDNA infection markers.

In practice, many successful eccDNA immune projects combine at least two compartments, such as sorted T cells plus plasma, to link local DNA damage with systemic signals.

Essential experimental controls and confounders

Appropriate controls are critical to avoid over-interpreting eccDNA as a generic damage marker.

Practical control strategies include:

• Uninfected or mock-infected cells, matched for culture conditions.
• Time-matched vehicle controls in drug or vaccine studies.
• DNase-treated samples to test dependence on DNA for immune effects.
• Technical replicates to monitor library preparation and sequencing variability.
• Spike-in controls for normalisation where appropriate.

It is also important to track potential confounders:

• Cell viability and death modality (apoptosis vs necrosis).
• Batch effects in sample processing or sequencing.
• Differences in sample storage, freeze–thaw cycles, or shipping conditions.

From study design to data interpretation: a stepwise workflow

A structured workflow helps immunology teams move from concept to interpretable eccDNA data.

A typical eccDNA immune project can follow these steps:

1. Define the biological question

   For example: "Does our viral infection model activate cGAS–STING via eccDNA?"

2. Select sample types and time points

   Decide on cell types, tissues, and biofluids, plus infection or treatment timeline.

3. Choose methods and sequencing depth

   Combine Circle-seq discovery, eccDNA methylation sequencing, and eccDNA qPCR as needed.

4. Plan controls and QC readouts

   Define uninfected controls, DNase conditions, and viability metrics.

5. Execute library preparation and sequencing

   Use validated workflows optimized for eccDNA in immune and infection samples.

6. Perform bioinformatics and integration

   Map eccDNA species, annotate immune-relevant loci, and integrate with RNA-seq or cytokine data.

7. Validate key findings

   Apply eccDNA qPCR quantification and functional assays where appropriate.

Study design checklist for eccDNA immune projects

• Clear question linking eccDNA to infection, T-cell biology, or innate immunity.
• At least one discovery-scale method (e.g., Circle-seq).
• Defined plan for targeted validation (eccDNA qPCR).
• Proper negative and technical controls.
• Integration with other immune readouts (RNA-seq, flow cytometry, cytokines).

Partnering with a specialised eccDNA team for immune projects

Partnering with an experienced eccDNA sequencing provider helps immunology labs design robust eccDNA infection and T-cell studies.

Our team supports immunology researchers, infectious disease labs, CROs, and immuno-oncology groups with:

• Study design support for eccDNA immune profiling in viral, bacterial, and T-cell models.
• Optimized protocols for low-input immune samples and challenging biofluids.
• Integrated packages combining eccDNA Sequencing (Circle-seq), eccDNA methylation sequencing, and eccDNA qPCR Quantification Service.
• Bioinformatics focused on innate immune pathways, cGAS–STING signaling, and T-cell biology.

Ready to plan an eccDNA immune project?

You can discuss sample types, controls, and a tailored sequencing strategy with our eccDNA sequencing specialists and move from concept to data-driven insight in your infection or T-cell models.

FAQs: eccDNA in immune and infection research

eccDNA immune projects raise practical questions on methods, sample types, and interpretation.

Q1. How much material do I need for eccDNA immune profiling in T cells?

For most eccDNA sequencing projects, we recommend starting from enough cells to yield microgram-scale genomic DNA, especially when working with sorted T-cell subsets. For low-input situations, enrichment and carefully optimized protocols can still provide informative eccDNA immune readouts, but pilot tests are valuable.

Q2. Can I use archived plasma or serum to study eccDNA in infection?

Yes, many eccDNA infection studies use frozen plasma or serum. However, consistent pre-analytics are important: harmonized collection tubes, processing times, and freeze–thaw history will improve comparability. When possible, prospectively collected samples with defined time points relative to infection are ideal.

Q3. How do I know whether eccDNA is driving immune activation or just reflecting cell death?

eccDNA often tracks with cell death, but not all eccDNA species are equal. Combining eccDNA sequencing with functional assays (e.g., adding purified eccDNA to immune cells), DNase controls, and methylation analysis can help distinguish passive by-products from eccDNA species with strong innate immunostimulatory activity.

Q4. Is eccDNA profiling compatible with other omics readouts in the same study?

Yes. Many teams design integrated projects that include eccDNA Sequencing (Circle-seq), RNA-seq, and cytokine profiling. This helps link eccDNA innate immunity signals to transcriptional changes, pathway activation, and functional outcomes in infection and T-cell models.

Q5. Are eccDNA immune assays suitable for clinical decision-making?

Current eccDNA immune profiling workflows, including Circle-seq, eccDNA methylation sequencing, and eccDNA qPCR quantification, are designed for research use only. They are well suited for preclinical infection models, mechanistic immunology, and biomarker discovery, but not for direct clinical diagnosis or treatment decisions.