Regulatory T (Treg) cells play a critical role in suppressing the immune response, thereby maintaining immune homeostasis and self-tolerance. Dysregulation in Treg cell type, frequency, or function is implicated in a variety of conditions, including autoimmune disease and cancer. There is still much to learn about Treg effector functions and molecular mechanisms in both health and disease. Thus, the ability to identify, quantify and characterize these cells is essential.
Role of Treg cells in the immune system
Treg cells exert their regulatory effects via competition for growth factors, soluble factors, and inhibitory receptors. This variety of mechanisms translates into myriad approaches by which drugs can target and influence Treg cells to shift the balance of the immune system. It is also why Treg dysregulation is observed in so many disease indications (see Figure 1).
Discovery of Treg cells
Mouse models, genetic mapping, and cell isolation all played a role in the discovery of Treg cells. The first observations came from a mouse strain called “scurfy”, where X-linked mutations in the transcription factor Forkhead box P3 gene (FoxP3) were linked to pronounced autoimmune pathology.1 In 2001, it was suggested that FoxP3 gene mutations cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, the human equivalent of mouse scurfy syndrome.2
Meanwhile, researchers found that transferring CD4+CD25- T cells into athymic nude mice resulted in evidence of autoimmune disease. Reconstitution of CD4+CD25+ cells prevented these developments in a time- and dose-dependent manner, suggesting these cells are important for immunologic self-tolerance.3
Ultimately, CD4+CD25+ cells were found to be FoxP3+ Treg cells, with the following key observations:
- FoxP3 is the main transcription factor programming the development and function of Treg cells.4
- While FoxP3 might be necessary for identifying Treg cells, it is not sufficient as activated T cells may express FoxP3 transiently. Thus, identification of Treg cells using flow cytometry would require several protein markers used in parallel—CD4, CD25, FoxP3, and low levels of CD127.5
- Treg-specific demethylated region (TSDR), a marker based on the methylation status of the FoxP3 gene, specifically characterizes Treg cells without cross-reacting with activated T cells.6,7
These last two observations led to two technological approaches that are now widely used to monitor Treg cells.
Methods for monitoring Treg cells
Currently, there are two primary approaches to monitoring Treg cells
- Flow cytometry (protein-based). This approach uses fluorescent antibodies, usually targeted at surface markers, to label cells. For Tregs, the two main markers are CD4 and CD25. CD127 is commonly used as a negative marker and intracellular staining of FoxP3 is usually included as well.
- Epigenetic cell counting (DNA-based). Precision for Medicine’s proprietary immune monitoring platform, Epiontis ID, uses the absence of genomic DNA methylation signals at the TSDR region of the FoxP3 gene to identify Treg cells. In this approach, the DNA within a biological sample is treated with bisulfite, which converts all unmethylated cytosines into uracils causing chemically-induced based mutations that affect base pairing. Subsequently, quantitative real-time polymerase chain reaction (PCR) is performed to detect and measure only DNA copies from Treg cells.
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Figure 2. Assay principle for epigenetic cell counting
Epiontis ID is fully automated, highly standardized, and very robust and reproducible over time, so it can support on-study measurements and comparison of data across different studies or with pre-existing healthy subject data. As of Q3 2022, assays for 36 markers have been fully validated on this platform (see Figure 3).
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Any combination of these markers can be put into a custom panel for immune monitoring applications. To date, the most commonly used assay is the FoxP3 assay for Treg cells, which has been used in more than 100 clinical studies. Studies have demonstrated that quantification of Treg cell number by epigenetic cell counting correlates well with flow cytometry (see Figure 4).8
Adapted from Baron U, et al. Sci Transl Med. 2018;10(452):eaan3508.
Using Epiontis ID to monitor Treg cells: Case studies
Case study 1: Autoimmune disease
In autoimmune diseases, the immune system shifts to pro-inflammatory immune cell populations. In a recent phase 2 trial, patients with systemic lupus erythematosus (SLE) were treated with iberdomide, a cereblon ligand. Treatment response was measured using the SLE Responder Index and additional exploratory biomarker analysis was performed at baseline and weeks 4, 12, and 24 using both epigenetic immunophenotyping and flow cytometry.
Epigenetic immunophenotyping showed that Treg frequency increased following treatment in a dose-dependent manner, while T follicular helper cells increased slightly and T helper 17 (Th17) cells remained stable throughout treatment (see Figure 5).9 This case study not only demonstrated how cell populations that can be challenging to measure by flow cytometry can be monitored with Epiontis ID, but also suggested a potential mechanism of action for how iberdomide induces immune tolerance.
Case study 2: Cancer
In cancer, the immune system often shifts to low inflammatory activity. A study published in Epigenetics in 2013 coined the term immunoCRIT, the ratio of suppressive Treg cells to total T cells, as a measure of immune tolerance. In this study, Epiontis ID was used to characterize Treg infiltration in tissue or tumor samples. It was found that that immunoCRIT is elevated in tumor samples and this elevation becomes more pronounced as the cancer progresses.10 Thus, epigenetic immunophenotyping could potentially be useful for setting thresholds for stratifying patient cohorts or predicting response to treatment.
Case study 3: IPEX syndrome
IPEX is caused by a mutation in the FoxP3 gene, while IPEX-like disease is a similar, sometimes less severe, condition that is not caused by FoxP3 mutations. In a study involving patients with IPEX and IPEX-like disease, Epiontis ID was used to measure the levels of Treg cells. Patients with IPEX were found to have high levels of TSDR demethylated cells that did not display a suppressive function due to the FoxP3 gene mutation. IPEX-like patients had low levels of TSDR demethylated cells, much lower than healthy subjects and somewhat lower than patients with other autoimmune diseases.
Flow cytometric analysis is difficult in IPEX as FoxP3 protein levels vary by gene mutation. However, TSDR demethylation analysis using Epiontis ID, coupled with FoxP3 gene mutation analysis, may improve diagnostic accuracy and help to optimize treatment of patients with IPEX.
Selecting the best approach for Treg monitoring
When deciding which method of Treg monitoring is most appropriate for an immune monitoring project, researchers should consider the phase and size of the study, the cell types of interest, the sample type, and the timeline (see Figure 6).
In research, preclinical, and small early clinical studies, flow cytometry is usually recommended. As soon as human samples are introduced, Epiontis ID can be added for early bridging purposes. In large, multi-center, late-stage trials, Epiontis ID offers ease and cost-effectiveness as it can use frozen samples and does not require fresh blood or peripheral blood mononuclear cell (PBMC) processing. If specialty applications such as receptor occupancy or protein phosphorylation are required, flow cytometry is necessary.
Flow cytometry would also be the only option for cell types that are not yet available with Epiontis ID, but certain cell types—such as Tregs and Th17 cells—are easier to measure epigenetically. It is also more feasible to measure blood and tissue in parallel using Epiontis ID. Finally, since Epiontis ID assays are fully validated and can be combined as necessary, there is no need to allocate time or resources to developing and validating a custom panel.
Conclusion
Both flow cytometry and epigenetic immunophenotyping can be used to monitor Treg cells in clinical studies. Understanding the limitations and advantages of each technology platform is essential for selecting the most appropriate approach for Treg cell monitoring based on study phase and size, key cell types of interest, and sample type. Given the vast diversity of Treg cells, future research will focus on the identification and validation of molecular markers for defining and differentiating among subtypes to better understand their complex roles in immunity and their potential as therapeutic targets.
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References
1. Russell WL, Russell LB, Gower JS. Exceptional inheritance of a sex-linked gene in the mouse explained on the basis that the X/O sexchromosome constitution is female. Proc Natl Acad Sci. 1959;45:554-560.
2. Wildin RS, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001;27(1):18-20.
3. Sakaguchi S, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155(3):1151-1164.
4. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4(4):330-336.
5. Liu W, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006;203(7):1701-1711.
6. Floess S, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007;5(2):e38.
7. Baron U, et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur J Immunol. 2007;37(9):2378-2389.
8. Baron U, et al. Epigenetic immune cell counting in human blood samples for immunodiagnostics. Sci Transl Med. 2018;10(452):eaan3508.
9. Merrill JT, et al. Phase 2 trial of iberdomide in systemic lupus erythematosus. N Engl J Med. 2022;386(11):1034-1045.
10. Türbachova I, et al. The cellular ratio of immune tolerance (immunoCRIT) is a definite marker for aggressiveness of solid tumors and may explain tumor dissemination patterns. Epigenetics. 2013;8(11):1226-1235.
