Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Dec 1;7(12):1391-402.
doi: 10.4161/epi.22675. Epub 2012 Oct 29.

Epigenetic biomarkers of T-cells in human glioma

John K Wiencke  1 , William P Accomando, Shichun Zheng, Joe Patoka, Xiaoqin Dou, Joanna J Phillips, George Hsuang, Brock C Christensen, E Andres Houseman, Devin C Koestler, Paige Bracci, Joseph L Wiemels, Margaret Wrensch, Heather H Nelson, Karl T Kelsey
Affiliations

Epigenetic biomarkers of T-cells in human glioma

John K Wiencke et al. Epigenetics. .

Abstract

Immune factors are thought to influence glioma risk and outcomes, but immune profiling studies to further our understanding of the immune response are limited by current immunodiagnostic methods. We developed a new assay to capture glioma immune biology based on quantitative methylation specific PCR (qMSP) of two T-cell genes (CD3Z: T-cells, and FOXP3: Tregs). Flow cytometry of T-cells correlated well with the CD3Z demethylation assay (r = 0.93; p < 2.2 × 10 (-16) ), demonstrating the validity of the assay. Furthermore, there was a high correlation between qMSP and immunohistochemistry (IHC) in quantifying tumor infiltrating T-cells (r = 0.85; p = 3.4 × 10 (-11) ). Applying our qMSP methods to archival whole blood from 65 glioblastoma multiforme (GBM) cases and 94 non-diseased controls, GBM cases had highly statistically significantly lower T-cells (p = 1.7 × 10 (-9) ) as well as Tregs (p = 5.2 × 10 (-11) ) and a modestly lower ratio of Tregs/T-cells (p = 0.024). Applying the methods to 120 excised glioma tumors, we observed that tumor infiltrating CD3+ T-cells were positively correlated with glioma tumor grade (p = 5.7 × 10 (-7) ), and that Tregs were enriched in tumors compared with peripheral blood indicating active chemoattraction of suppressive Tregs into the tumor compartment. Poorer patient survival was correlated with higher levels of tumor infiltrating T-cells (p = 0.01) and Tregs (p = 0.04). DNA methylation based immunodiagnostics represent a new generation of powerful laboratory tools offering many advantages over conventional methods that will facilitate large clinical epidemiologic studies and capitalize on stored archival blood and tissue banks.

PubMed Disclaimer

Figures

None
Figure 1. DNA methylation in MACS purified human leukocyte samples at immune cell-specific DMRs. (A) Illumina Infinium HumanMethylation27 data for top 100 putative T cell-specific DMRs identified from LME model. Beta values are scaled (zero centered) by gene locus. (B) MethyLight data for FOXP3 and CD3Z gene regions illustrating immune lineage-specific DNA methylation patterns. MACS purified cell type abbreviations: B, B lymphocytes; Gran, granulocytes; Neut, neutrophils; Mono, monocytes; NK, CD56+ natural killer cells; CD16+NK, CD16+CD56dim natural killer cells; CD16-NK, CD16-CD56bright natural killer cells; NKT, CD3+CD56+ natural killer T-cells; t, CD3+ T lymphocytes; Tc, CD3+CD8+ T lymphocytes (cytotoxic T-cells); Th, CD3+CD4+ T lymphocytes (helper T-cells); Treg, CD3+CD4+CD25+FOXP3+ regulatory T-cells
<b>Figure 2</b>.
Figure 2.
CD3+ T-cell detection by qMSP. (A) Schematic showing methylation specific primers and probe targeting six CpGs (lollipops) in a region of the CD3Z gene identified as demethylated in CD3+ T-cells. (B) Real time PCR Ct values decrease linearly with 10-fold increase in bisulfite converted CD3+ T-cell DNA concentration. Bisulfite converted universal methylated DNA was used to keep total amount of DNA in all samples constant. At least five replicates of each sample are plotted. (C) Evaluation of CD3+ T-cell level by flow cytometry is highly correlated with T-cell quantification by CD3Z qMSP in whole blood specimens from glioma patients and healthy donors. (D) CD3+ T-cell count by imunohistochemical staining correlates with T-cell quantification by CD3Z qMSP in excised tumors across histological subtypes. Pearson correlations and F-test p values are shown in B‒D.
<b>Figure 3</b>.
Figure 3.
T-cells and Tregs in the peripheral blood of glioma patients and healthy donors. (A) Case control comparison of T-cells measured via CD3Z demethylation assay. (B) Case control comparison of Tregs measured via FOXP3 demethylation assay. (C) Case control comparison of Treg percent of T-cells determined by the ratio of FOXP3/CD3Z demethylation. In each panel, the displayed p value is from a Wilcoxon rank sum test between non-diseased control bloods and GBM patient bloods. Each data point represents the average of all replicate qMSP measurements for a single individual. Box plots superimposed on the data points cover the 2nd and 3rd quartile range with a line drawn at the median value, and whiskers that extend to the data point that is no more than 1.5 times the length of the box away from the box. The notches surrounding each median line extend to ± 1.58 IQR/sqrt(n), such that if the notches from two boxplots do not overlap there is strong evidence for a significant difference in the two medians.
<b>Figure 4</b>.
Figure 4.
Association between cigarette smoking and peripheral blood T-cells and Tregs in glioma patients and healthy donors. (A) Comparison of peripheral blood T-cell levels, determined by CD3Z demethylation, among never, former and current cigarette smokers stratified by glioma case status. (B) Comparison of peripheral blood Treg levels, determined by FOXP3 demethylation, among never, former and current cigarette smokers stratified by glioma case status. (C) Comparison of peripheral blood Treg percent of T-cells, determined by ratio of FOXP3 to CD3Z demethylation, among never, former and current cigarette smokers stratified by glioma case status. In each panel the displayed p-values are from Wilcoxon rank sum tests between the two groups indicated. Each data point represents the average of all replicate qMSP measurements for a single individual. Box plots superimposed on the data points cover the 2nd and 3rd quartile range with a line drawn at the median value, and whiskers that extend to 1.5 times the length of the box.
<b>Figure 5</b>.
Figure 5.
Levels of T-cell and Treg infiltrates in excised glioma tissue. (A) T-cell levels, determined by CD3Z demethylation, in solid glioma samples stratified by tumor grade. (B) Treg levels, determined by FOXP3 demethylation, in solid glioma samples stratified by tumor grade. (C) Treg percent of T-cells, determined by ratio of FOXP3 to CD3Z demethylation, in solid glioma samples stratified by tumor grade. In each panel the displayed p-value is from a Wilcoxon rank sum test between lower grade tumors (Grades I, II and III) and Grade IV tumors. Each data point represents the average of all replicate qMSP measurements for a single individual. Box plots superimposed on the data points cover the 2nd and 3rd quartile range with a line drawn at the median value, and whiskers that extend to 1.5 times the length of the box.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ohgaki H, Kleihues P. Epidemiology and etiology of gliomas. Acta Neuropathol. 2005;109:93–108. doi: 10.1007/s00401-005-0991-y. - DOI - PubMed
    1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007;114:97–109. doi: 10.1007/s00401-007-0243-4. - DOI - PMC - PubMed
    1. Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol. 2007;170:1445–53. doi: 10.2353/ajpath.2007.070011. - DOI - PMC - PubMed
    1. Kleihues P, Burger PC, Scheithauer BW. The new WHO classification of brain tumours. Brain Pathol. 1993;3:255–68. doi: 10.1111/j.1750-3639.1993.tb00752.x. - DOI - PubMed
    1. Wiemels JL, Wilson D, Patil C, Patoka J, McCoy L, Rice T, et al. IgE, allergy, and risk of glioma: update from the San Francisco Bay Area Adult Glioma Study in the temozolomide era. Int J Cancer. 2009;125:680–7. doi: 10.1002/ijc.24369. - DOI - PMC - PubMed

Publication types

MeSH terms