Epigenetic quantification of tumor-infiltrating T-lymphocytes

Abstract

The immune system plays a pivotal role in tumor establishment. However, the role of T-lymphocytes within the tumor microenvironment as major cellular component of the adaptive effector immune response and their counterpart, regulatory T-cells (Treg), responsible for suppressive immune modulation, is not completely understood. This is partly due to the lack of reliable technical solutions for specific cell quantification in solid tissues. Previous reports indicated that epigenetic marks of immune cells, such as the Treg specifically demethylated region (TSDR) within the FOXP3 gene, may be exploited as robust analytical tool for Treg-quantification. Here, we expand the concept of epigenetic immunophenotyping to overall T-lymphocytes (oTL). This tool allows cell quantification with at least equivalent precision to FACS and is adoptable for analysis of blood and solid tissues. Based on this method, we analyse the frequency of Treg, oTL and their ratio in independent cohorts of healthy and tumorous ovarian, colorectal and bronchial tissues with 616 partly donor-matched samples. We find a shift of the median ratio of Treg-to-oTL from 3-8% in healthy tissue to 18-25% in all tumor entities. Epigenetically determined oTL frequencies correlate with the outcome of colorectal and ovarian cancers. Together, our data show that the composition of immune cells in tumor microenvironments can be quantitatively assessed by epigenetic measurements. This composition is disturbed in solid tumors, indicating a fundamental mechanism of tumor immune evasion. Epigenetic quantification of T-lymphocytes serves as independent clinical parameter for outcome prognosis.

Figure 1

Epigenetic profiling of the CD3 and GAP DH loci. (A) Genomic localization and organization of the genes. Transcripts are shown depending on their orientation above or below the chromosomal bar. Amplicons used for epigenetic analysis are indicated as red boxes. (B) Epigenetic profiling obtained from bisulfite-sequencing of amplicons shown in (A). Each line represents the bisulfite-conversion status of CpGs in the amplicon tested on purified cell types indicated left. Individual squares represent single CpG-positions. Blue squares correspond to the CpG-variant and yellow squares to the TpG-variant. The color code is shown on the right-hand side. (C) Amplification profiles of bisulfite-conversion specific qPCRs. Upper parts: qPCR specific for TpG-variant tested on dilutions of 12,500, 2,500, 500, 100 and 20 plasmid copies representing TpG (red) and CpG template variants (blue). Lower parts: The same experiment using qPCR specific for the CpG-variant. Linearity of qPCRs is shown inside each graph by plotting CP-values over log-concentration of template.

Figure 2

CD3⁺ and FOXP3⁺ cell counting using epigenetic qPCR and FACS . Peripheral blood was collected from 17 donors. Cell frequency was determined by FACS (X-axis) and qPCR analysis (Y-axis). (A) Cell frequencies determined by FACS describe the percentage of CD3⁺ cells per all nucleated cells. qPCRs were performed applying the CD3-specific qPCR system. (B) Cell frequencies determined by FACS describe the percentage of CD4⁺CD25⁺CD127⁻ cells. qPCRs were performed using the FOXP3-specific qPCR system. (C) Cell frequencies determined by FACS describe the percentage of CD4⁺CD25⁺CD127⁻ cells within CD3⁺ cells. R indicates Spearman rank correlation coefficient for FACS compared to qPCR measurement. Statistical significance is indicated as follows: **p ≤ 0.01; ***p ≤ 0.001.

Figure 3

Correlation of CD3⁺ T cells with Treg in various tissues. CD3⁺ T cell levels (X-axis) are plotted against Treg levels (Y-axis). Cell frequencies were derived from measurements of the TpG variants in the CD3 intergenic region and the FOXP3 TSDR. Analysis was performed for whole blood, ovarian (OT), lung (BT) and colorectal tissue (CT-II) as well as for ovarian (OvCa), bronchial (BCa) and colorectal (CRC-II) cancer. Correlation between the two parameters is indicated by the Spearman rank correlation coefficient Rho, and the p value is given as parameter for significance. Each dot indicates a single sample measured with both the CD3 and FOXP3 qPCR assay.

Figure 4

Frequency of tissue-infiltrating lymphocytes. Boxplots showing percentages of (A) Treg, (B) CD3⁺ T cells, (C) Treg within CD3⁺ T cells in healthy and cancerous tissues. N indicates the number of patients included in each plot. The box depicts the middle 50% of the distribution. The line in the box represents the median. Whiskers extend to include 95% of all data. Outliers are indicated by circles. The statistical significance is indicated as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5

Cumulative survival of colorectal and ovarian cancer patients. Samples from each tumor entity were divided into two groups at their median value of tumor-infiltrating overall T-lymphocytes (oTL) as measured by CD3-specific qPCR. Levels above median (high oTL frequencies), green; levels below median (low oTL frequencies), blue. (A) Progression free survival (PFS). In the left (CRC-I; median = 26.1%; N_high = 7; N_low = 12) and middle (CRC-II; median = 23.9%; N_high = 17; N_low = 22) part colorectal cancer samples are shown. The right part shows ovarian cancer samples (OvCa; median = 7.76%; N_high = 23; N_low = 23). (B) Overall survival (OS). Left part: CRC-I cohort (N_high = 3; N_low = 7); middle part: CRC-II cohort (N_high = 11; N_low = 19); Right part: OvCa cohort (N_high = 14; N_low = 18).

Figure 6

Quantification of tumor-infiltrating T-lymphocytes using immunohistochemistry. (A) illustrates a representative immuno-staining of colorectal cancer (CRC) and normal colonic mucosa (colon tissue; CT) sections prepared from tissue micro arrays. Overall T-lymphocytes (oTL; red, membranous) and regulatory T cells (Treg; red, nuclear) were stained using antibodies directed against CD3 and FOXP3, respectively. H&E represents hematoxylin/eosin staining. Original magnification: x20; inset: x80. (B) Kaplan-Meier plots illustrating cumulative survival of patients with colorectal cancer. Both, oTL and Treg were counted in cores of 1 mm in diameter and were used to perform PFS and OS analysis. Tumor samples were divided into two groups at their median level of oTL (median = 195). Levels above the median (“high”) are represented by a green line and levels below the median (“low”) are represented by a blue line. Sample numbers are as follows: oTL: N_high = 23, N_low = 15 for PFS; N_high = 17, N_low = 14 for OS. Treg: N_high = 19, N_low = 18 for PFS; N_high = 16,N_low = 15 for OS.