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. 2023 Feb 4;12(4):516.
doi: 10.3390/cells12040516.

Prospective Evaluation of CD45RA+/CCR7- Effector Memory T (TEMRA) Cell Subsets in Patients with Primary and Secondary Brain Tumors during Radiotherapy of the Brain within the Scope of the Prospective Glio-CMV-01 Clinical Trial

Ilka Scheer  1   2 , Ina Becker  1   2 , Charlotte Schmitter  2   3 , Sabine Semrau  2   3 , Rainer Fietkau  2   3 , Udo S Gaipl  1   2   3 , Benjamin Frey  1   2   3 , Anna-Jasmina Donaubauer  1   2   3
Affiliations

Prospective Evaluation of CD45RA+/CCR7- Effector Memory T (TEMRA) Cell Subsets in Patients with Primary and Secondary Brain Tumors during Radiotherapy of the Brain within the Scope of the Prospective Glio-CMV-01 Clinical Trial

Ilka Scheer et al. Cells. .

Abstract

Radiotherapy (RT) of the brain is a common treatment for patients with high-grade gliomas and brain metastases. It has previously been shown that reactivation of cytomegalovirus (CMV) frequently occurs during RT of the brain. This causes neurological decline, demands antiviral treatment, and is associated with a worse prognosis. CMV-specific T cells are characterized by a differentiated effector memory phenotype and CD45RA+ CCR7- effector memory T (TEMRA) cells were shown to be enriched in CMV seropositive individuals. In this study, we investigated the distribution of TEMRA cells and their subsets in the peripheral blood of healthy donors and, for the first time, prospectively within the scope of the prospective Glio-CMV-01 clinical trial of patients with high-grade glioma and brain metastases during radiation therapy as a potential predictive marker. First, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of TEMRA cells in a longitudinal manner. The CMV serostatus and age were considered as influencing factors. We revealed that patients who had a reactivation of CMV have significantly higher amounts of CD8+ TEMRA cells. Further, the distribution of the subsets of TEMRA cells based on the expression of CD27, CD28, and CD57 is highly dependent on the CMV serostatus. We conclude that the percentage of CD8+ TEMRA cells out of all CD8+ T cells has the potential to serve as a biomarker for predicting the risk of CMV reactivation during RT of the brain. Furthermore, this study highlights the importance of taking the CMV serostatus into account when analyzing TEMRA cells and their subsets.

Keywords: TEMRA cells; brain metastases; cytomegalovirus (CMV); glioblastoma; infection; radiotherapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Gating strategy for the assessment of CD8+ and CD4+ CD45RA+ CCR7- effector memory T cells (TEMRA) and their subsets. T cells were gated as CD3+ leukocytes and divided into CD8+ and CD4+ T cells. Both CD8+ and CD4+ T cells were divided into naive T cells (TN), central memory T cells (TCM), effector memory T cells (TEM), and effector memory T cells re-expressing CD45RA (TEMRA) based on the expression of CD45RA and CCR7. TEMRA cells were defined as CD45RA+ CCR7-. Both CD8+ and CD4+ TEMRA cells were divided into further subsets based on the expression of CD27 and CD28 and on the expression of CD28 and CD57. Gates for CCR7, CD45RA, CD27, and CD57 were set using fluorescence minus one (FMO) controls.
Figure 2
Figure 2
Distribution of T cell subsets in healthy donors. TN, TCM, TEM, and TEMRA cells are depicted as percentages of CD8+ T cells in (A), and of CD4+ T cells in (B). Individuals with cytomegalovirus (CMV) seropositivity are depicted in red (n = 10) and individuals that are seronegative for CMV are depicted in blue (n = 11). For statistical analysis, a non-parametric two-tailed Mann–Whitney U-test was used (*: p < 0.05; ***: p < 0.001).
Figure 3
Figure 3
Amounts of TEMRA cells before, during, and after radiotherapy (RT). TEMRA cells are depicted as percentages of CD8+ T cells in (A), and of CD4+ T cells in (B). TEMRA cells were measured in CMV seropositive patients, depicted in red, before RT (n = 11), halfway through RT (n = 12), and after RT (n = 7), as well as in CMV seronegative patients, depicted in blue, before RT (n = 13), halfway through RT (n = 11), and after RT (n = 12). For statistical analysis, the Kruskal–Wallis test was used to compare the amount of TEMRA cells halfway through and after RT to the amount before RT. For the analysis between the CMV seronegative and seropositive group within each time point, a non-parametric two-tailed Mann–Whitney U-test was applied (**: p < 0.01).
Figure 4
Figure 4
Comparison of TEMRA cells in healthy donors and patients with or without CMV reactivation. TEMRA cells are depicted as percentages of CD8+ T cells in (A), and of CD4+ T cells in (B). TEMRA cells were measured in CMV seropositive healthy donors (n = 10), CMV seropositive patients before radiation therapy without reactivation (n = 9), and patients in the CMV seropositive group who had a reactivation of CMV (n = 3). For the statistical analysis, a non-parametric two-tailed Mann–Whitney U-test was applied (*: p < 0.05; **: p < 0.01).
Figure 5
Figure 5
TEMRA cell subsets based on the expression of CD27 and CD28. (A): CD8+ TEMRA cells in healthy donors were divided into four subsets based on the expression of CD27 and CD28. TEMRA cell subsets are depicted as percentages of CD8+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 10) and individuals that are seronegative for CMV are depicted in blue (n = 11). (B): CD8+ TEMRA cells in patients before RT were divided into four subsets based on the expression of CD27 and CD28. They are depicted as percentages of CD8+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 11) and individuals that are seronegative for CMV are depicted in blue (n = 13). (C): CD4+ TEMRA cells in healthy donors were divided into four subsets based on the expression of CD27 and CD28. They are depicted as percentages of CD4+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 10) and individuals that are seronegative for CMV are depicted in blue (n = 11). (D): CD4+ TEMRA cells in patients before RT were divided into four subsets based on the expression of CD27 and CD28. They are depicted as percentages of CD4+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 11) and individuals that are seronegative for CMV are depicted in blue (n = 13). For the statistical analysis, a non-parametric two-tailed Mann–Whitney U-test was used (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).
Figure 6
Figure 6
TEMRA cell subsets based on the expression of CD57 and CD28. (A): CD8+ TEMRA cells in healthy donors were divided into four subsets based on the expression of CD57 and CD28. They are depicted as percentages of CD8+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 10) and individuals that are seronegative for CMV are depicted in blue (n = 11). (B): CD8+ TEMRA cells in patients before RT were divided into four subsets based on the expression of CD57 and CD28. They are depicted as percentages of CD8+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 11) and individuals that are seronegative for CMV are depicted in blue (n = 13). (C): CD4+ TEMRA cells in healthy donors were divided into four subsets based on the expression of CD57 and CD28. They are depicted as percentages of CD4+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 10) and individuals that are seronegative for CMV are depicted in blue (n = 11). (D): CD4+ TEMRA cells in patients before RT were divided into four subsets based on the expression of CD57 and CD28. They are depicted as percentages of CD4+ TEMRA cells. Individuals with CMV seropositivity are depicted in red (n = 11) and individuals that are seronegative for CMV are depicted in blue (n = 13). For the statistical analysis, a non-parametric two-tailed Mann–Whitney U-test was used (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).
Figure 7
Figure 7
Influence of age on the percentage of TEMRA cells. A Spearman correlation and a simple linear regression were performed to correlate the percentage of CD8+ TEMRA cells out of all CD8+ T cells to age, depicted as circles, (A–F) and the percentage of CD4+ TEMRA cells out of all CD4+ T cells to age, depicted as squares, (G–L). CMV seronegative individuals are depicted in blue, CMV seropositive individuals are depicted in red, and CMV seronegative and seropositive individuals combined are depicted in purple. (A) shows CMV seronegative healthy donors (n = 11). (B) shows CMV seropositive healthy donors (n = 10). (C) shows all healthy donors independent of CMV serostatus (n = 21). (D) shows CMV seronegative patients before the start of RT (n = 13). (E) shows CMV seropositive patients before the start of RT (n = 11). (F) shows all patients before the start of RT independent of CMV serostatus (n = 24). (G) shows CMV seronegative healthy donors (n = 11). (H) shows CMV seropositive healthy donors (n = 10). (I) shows all healthy donors independent of CMV serostatus (n = 21). (J) shows CMV seronegative patients before the start of RT (n = 13). (K) shows CMV seropositive patients before the start of RT (n = 11). (L) shows all patients before the start of RT independent of CMV serostatus (n = 24).
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This work was funded by the Bundesministerium für Bildung und Forschung (BMBF; GREWIS-alpha, 02NUK050E).