Epigenetic immune cell counting in human blood samples for immunodiagnostics

Abstract

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.

Tregopathies: Monogenic diseases resulting in regulatory T-cell deficiency

“…52 In patients with IPEX syndrome, most lymphocyte subsets have a normal distribution, with the exception of the amount of CD4 1 T cells, eosinophils, and IgE, values of which are frequently found to be increased. Although the frequency of FOXP3 1 Treg cells, when characterized by surface phenotype, varies from zero to comparable or even greater than in healthy control subjects, 52,53,[64][65][66] the amount of DNA demethylation at FOXP3 TSDR is consistently higher in patients with IPEX syndrome, even at birth, 20 which is unique to this syndrome. Despite sometimes normal FOXP3 expression level, Treg cells of patients with IPEX syndrome are unable to suppress Teff cells.…”

“…Therefore the most precise method to count tTreg cells is through quantification of TSDR demethylation. 20,21 Abbreviations used allo-HSCT: Allogeneic hematopoietic stem cell transplantation APC: Antigen-presenting cell BACH2: BTB domain and CNC homolog 2 CTLA-4: Cytotoxic T lymphocyte-associated antigen Treg cells also express inhibitory molecules on the surface (CTLA-4, PD-1, and TIGIT) and secrete immunosuppressive cytokines (IL-10 and TGF-b) through which they exert their regulatory function. A number of other surface markers (CD39, CD73, GARP, GITR, ICOS, and CD95) and transcription factors (BACH2, HELI-OS, and EOS) also play a functional role in FOXP3 1 Treg cells.…”

“…14 Epigenetic quantitative PCR to detect lineage-specific demethylated regions for CD4 1 and CD8 1 T cells, Treg cells, B cells, NK cells, monocytes, and neutrophils has been recently developed and proposed as a potential immune diagnostic test for newborn diagnosis of primary immunodeficiencies, including IPEX syndrome. 20 The mutations, main clinical presentations, Treg cell phenotype and function, and functional experiments to confirm the mutation effect in Tregopathies, along with accompanying references, are summarized in Table I. Table I also includes IL-10/IL-10R deficiency; functional assays used to differentiate between IL-10 and IL-10R defects are reviewed here.…”

Circulating Immune Cell Composition and Cancer Risk: A Prospective Study Using Epigenetic Cell Count Measures

“…In general, We developed and validated a series of epigenetic assays for the quantification of various leukocyte sub-populations in blood. These assays can be employed and quantitated in a variety of substrates, including DNA extracted from non-intact leukocytes (6). Applying these assays to DNA extracted from stored buffy coat samples of the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort, we reported a relationship between a higher ratio of Foxp3+ to total CD3+ T-lymphocytes ("ImmunoCRIT" immune tolerance ratio) and increased risk of developing lung, colorectal, and ER-negative breast cancers (7).…”

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots

“…In addition, TREC/KREC analysis has the potential to miss several SCID subtypes (e.g., ADA, ZAP70, and MHC deficiency) and NBS for other PIDDs does not currently exist due to the lack of screening methods (17,(19)(20)(21)(22). Nevertheless, recent advances have demonstrated the ability to screen metabolites related to Adenosine Deaminase (ADA) deficient SCID and purine nucleoside phosphorylase deficiency, and epigenetic markers for immune cell profiles (23)(24)(25).…”