Favorable prognostic influence of T-box transcription factor Eomesodermin in metastatic renal cell cancer patients

Abstract

T-box transcription factors, T-box expressed in T cells (T-bet) encoded by Tbx21 and Eomesodermin (Eomes), drive the differentiation of effector/memory T cell lineages and NK cells. The aim of the study was to determine the prognostic influence of the expression of these transcription factors in peripheral blood (pB) in a cohort of 41 metastatic (m) RCC patients before receiving sorafenib treatment and to analyze their association with the immunophenotype in pB. In contrast to Tbx21, in the multivariate analysis including clinical features, Eomes mRNA expression was identified as an independent good prognostic factor for progression-free survival (PFS, p = 0.042) and overall survival (OS, p = 0.001) in addition to a favorable ECOG performance status (p = 0.01 and p = 0.008, respectively). Eomes expression correlated positively not only with expression of Tbx21 and TGFβ1 mRNA, but also with mRNA expression of the activation marker ICOS, and with in vivo activated HLA-DR(+) T cells. Eomes expression was negatively associated with TNFα-producing T cells. On protein level, Eomes was mainly expressed by CD56(+)CD3(-) NK cells in pB. In conclusion, we identified a higher Eomes mRNA expression as an independent good prognostic factor for OS and PFS in mRCC patients treated with sorafenib.

Keywords: Effector T cells; Eomesodermin; Memory T cells; Natural killer cells; Renal cell carcinoma; T-box expressed in T cells.

Fig. 1

Correlation of mRNA expression of Eomes and Tbx21 with mRNA expression of TGFβ1 and ICOS and with HLA-DR⁺ T cells and cytokine-producing T cells. mRNA expression levels of Tbx21, Eomes, TGFβ1, and ICOS in PBMCs were determined by quantitative RT-PCR. The relative amount was expressed as ratio marker (pg/µl)/HMBS (pg/µl). The sample concentration was calculated using a plasmid standard curve. mRNA levels of all markers were within the respective detection range in all patient samples. HLA-DR surface expression on CD8⁺ and CD4⁺ T cells was determined by flow cytometry. IFNγ- and TNFα- producing T cells were measured by intracellular flow cytometry after in vitro stimulation of PBMCs by PMA/ionomycin

Fig. 2

Kaplan–Meier curves for Tbx21 mRNA, Eomes mRNA, ECOG, and Eomes⁺/CD56⁺CD3⁻ NK cells. mRNA was derived from PBMCs. mRNA expression levels were divided into quartiles with low (1. quartile), intermediate low (2. quartile), intermediate high (3. quartile), and high (4. quartile) mRNA expression. Eomes⁺/CD56⁺CD3⁻ NK cells were determined by intracellular flow cytometry and divided into two groups based on the median frequency of Eomes⁺ cells (51.2 %) in the CD56⁺CD3⁻ population. Survival curves were compared with the log-rank test. a PFS progression-free survival, b OS overall survival

Fig. 3

Expression of Eomes in peripheral lymphocyte subpopulations of mRCC patients. Eomes expression was determined by intracellular flow cytometry staining in lymphocyte subpopulations as indicated. The box/whisker graphs display 25–75 % (box) and 10–90 % (whisker). The line in the box represents the median value. a Exemplary flow cytometry plots from one patient showing Eomes expression in CD56⁺CD3⁻ NK cells and CD8⁺CD3⁺ T cells. b Frequency of Eomes-expressing cells in CD3⁺ T cells, CD8⁺CD3⁺ T cells, CD4⁺CD3⁺ T cells, and CD56⁺CD3⁻ NK cells. c Frequency of Eomes-expressing CD8⁺ T cells within CD45RA⁺CCR7⁺ T_naive cells, CD45RA⁺CCR7⁻ T_E, CD45RA⁻CCR7⁻ T_EM, and CD45RA⁻CCR7⁺ T_CM. d Eomes median fluorescence intensity (MFI) values in CD8⁺ memory/effector subpopulations and in CD56⁺CD3⁻ NK cells