Expanded CLL CD8+ T-Cells Are Driven Into a Senescent KLRG1+ Effector Memory Phenotype
Authors: Göthert Joachim Rudolf , Eisele Lewin , Klein-Hitpass Ludger , Weber Stefanie , Führer Anja , Sellmann Ludger , Roeth Alexander , Duehrsen Ulrich , Pircher Hanspeter , Dürig Jan 
Published: October 2019

Abstract
Abstract 913
Altered numbers and functions of T-cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we determined numbers of blood lymphocyte subsets of CLL patients in a longitudinal manner. We found that dynamic expansions of the peripheral blood CD4+ and CD8+ T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Additionally, we performed gene expression profiling (GEP) of blood CD3+ T-cells of CLL patients and normal donors. We identified a list of 135 genes that had significantly increased expression and 11 genes that had significantly decreased expression in CLL T-cells. The up-regulated genes included killer cell lectin-like receptor familiy members KLRA1, KLRC2, KLRD1 (CD94), KLRK1 and KLRF1 as well as CD244 (NK-cell receptor 2B4), CD160 (NK cell receptor BY55), PRF1 (perforin 1) and CRTAM (class-I MHC-restricted T-cell associated molecule). These up-regulated genes are known to be preferentially expressed by CD8+ T-cells with an effector memory phenotype. We used Gene Set Enrichment Analysis (GSEA) to investigate whether CLL T-cell genes correlate with a previously published gene expression signature of effector memory CD8+ T-cells (Willinger et al., Journal of Immunolgy 175[9], 2005). This analysis revealed a highly significant enrichment of CLL T-cell genes within the effector memory CD8+ T-cell signature (p<0.0001, FDR q<0.001). Next, we studied the CLL CD8+ T-cell compartment using flow cytometry. As already implied by GEP and GSEA, the flow cytometric analysis revealed a relative shift of subsets within the CD8+ T-cell compartment. Compared to normal donors we observed a decreased proportion of naïve CD8+ T-cells (CD45RA+CCR7+) and an increased proportion of CD8+ effector memory cells (CD45RA-CCR7-) in the CLL cohort as compared to normal donor controls. When absolute cell numbers were calculated on the basis of these results it became evident that the elevation in the absolute number of overall CD8+ T-cells in CLL was primarily attributable to the expansion of the effector memory subset of CD8+ T-cells. Subsequently, we compared the killer cell lectin-like receptor G1 (KLRG1) surface expression of CLL and normal donor CD8+ effector memory T-cells. KLRG1 marks cells, which have undergone extensive proliferation and lack replicative potential. We observed that the absolute increments of effector memory CD8+ T-cells in human CLL patients were mainly due to the expansion of the senescent KLRG1 expressing subset of cells. In order to test whether the CD8+ effector memory expansion is a general biologic CLL phenomenon we studied the CD8+ T-cell compartment of a murine transgenic CLL model (7-month-old TCL1 transgenic mice). It was previously described that TCL1 CD5+CD19+ B-cell hyperplasia first emerges in the peritoneal cavity of TCL1 transgenic mice. Therefore, we specifically studied how CD8+ T-cell subsets respond to arising CLL in the peritoneal cavity of TCL1 transgenic mice. Strikingly, we found that our observation of effector memory shifted CD8+ T-cells in human CLL was phenocopied in the peritoneal cavity of TCL1 transgenic mice. The proportion of peritoneal naïve CD8+ T-cells (CD62L+CD44-) was significantly decreased while the proportion of CD8+ effector memory T-cells (CD62L-CD44+) was significantly increased. Moreover, we observed a more than two-fold increase of KLRG1+ effector memory CD8+ T-cells in the peripheral blood and spleens of TCL1 transgenic mice compared to wild-type controls. In summary, we were able to show that human as well as mouse CLL CD8+ T-cells are driven into a senescent effector memory phenotype. This might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment.

Disclosures:
No relevant conflicts of interest to declare.