determination-immune-cell-identity-purity-using-epigenetic-based

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have ...

Precision for Medicine

Experimental and clinical evidence indicate that inflammatory processes in atherogenesis and the development of cardiovascular complications are promoted by a loss of regulatory T cell (Treg)-mediated immunological tolerance to plaque antigens. Yet, the association between alterations of systemic Treg frequency and cardiovascular disease incidence remains uncertain.

The Ratio of Regulatory (FOXP3+) to Total (CD3+) T Cells Determined by Epigenetic Cell Counting and Cardiovascular Disease Risk: A Prospective Case-cohort Study in Non-diabetics.

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3 T, CD8 T, CD4 T, and FOXP3 regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

Epigenetic immune cell counting in human blood samples for immunodiagnostics.

Adoptive transfer of regulatory T cells (FOXP3 Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after  expansion. : We isolated  Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vβ diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their  and  potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. : LAP repurification results in >90% LAPFOXP3 Tregs, leaving behind FOXP3 and FOXP3 nonTregs within the LAP population. After 4-week expansion, the LAP Tregs were >1 billion cells, highly suppressive and anergic , >90% demethylated in the TSDR and able to maintain TCR Vβ diversity. In the xGVHD model, exogenous CD25PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP Tregs increased median survival to 47 days, while the LAP parent (CD25) and LAP nonTregs had median survival of 39 and 31 days, respectively. : These preclinical data together provide evidence that LAP Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP Tregs to prevent or treat GVHD and other autoimmune diseases.

Sven Olek

Epiontis GmbH

Precision for Medicine

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR