1044 - T cells from chronic lymphocytic leukemia patients are not exhausted but show signs of senescence, providing alternative clues for improvement

Introduction
Chronic lymphocytic leukemia (CLL) is currently an incurable disease and there is an unmet need for novel therapeutic strategies. Utilization of T cells to target CLL is a promising strategy, but so far, has not been successful in the majority of CLL patients. Exhaustion of T cells has been proposed as contributing factor to the acquired T-cell dysfunction patients develop. However, we have demonstrated that reduced levels of activation and metabolic impairments of CLL T cells are rescued upon in vitro elimination of CLL cells (van Bruggen et al., 2022), which indicates that the cells are not (terminally) exhausted. We hypothesize that instead of exhaustion, senescence is the dominant dysfunctional T-cell state in CLL patients. Senescent T cells share common features with senescent somatic cells such as cell-cycle arrest, decline in proliferation and activation, but are able to produce high amounts of pro-inflammatory cytokines, features that have been previously described for CLL T cells. Here we study phenotypical and molecular characteristics of senescence in CLL T cells.
Methods
We assessed expression of cell-surface proteins that characterize senescent T cells (e.g. absence of CD27/CD28 and presence of PD1/CD57/KLRG1) with flow cytometry. To measure cell-cycle progression, T cells were stimulated through CD3/CD28 for 5 days and, using a DNA dye, the different cell-cycle phases were measured with flow cytometry. To study the direct effect of CLL cells on autologous T cells, T cells were stimulated in the presence or absence of their CLL cells. On day 2 the CD4+ T cells were FACS-sorted and mRNA sequencing was performed to investigate the activity of molecular pathways.
Results
We found that CLL patients have increased proportions of CD27-CD28- T cells compared to age-matched healthy donors (HD) (Fig. 1A, B). In accordance with previous findings (Martens et al., 2023), expression of KLRG1 was increased on CLL T cells and furthermore, a CD27-CD28--PD1+CD57+KLRG1¬+ subset was more abundant within the CLL T cells compared to HD. Senescent cells are typically cell-cycle arrested and when stimulating T cells in vitro through CD3/CD28, we observed a strikingly distinct cell-cycle pattern whereby a larger proportion of CLL T cells resided in S and G2/M phases (Fig. 1C). RNA sequencing on CD4+ T cells stimulated with CD3/CD28 in the presence or absence of their autologous CLL cells revealed that CLL cells induced a clear inflammatory gene signature, indicated by upregulation of TNF and NFκB pathways. Pathways related to cell cycle such as E2F transcription factors and G2/M progression were repressed in presence of CLL. A potential cause for cell-cycle arrest and senescence is DNA damage. In our transcriptome data we observed activity of the p53 pathway when T cells were stimulated with CLL cells present as well as upregulated expression of ATM and CDKN1A (encoding p21) (Fig. 1D, E); a possible indication of an activated DNA damage response. In addition, our group has also demonstrated that CLL T cells were specifically impaired in mitochondrial metabolism whilst glycolysis was largely intact, that CLL T cells have decreased TCA intermediates and increased reactive oxygen species (ROS) upon stimulation (Goedhart & Simon-Molas, EHA 2023), which are all indicators of senescence. Potential intervention in MAPK or ATM signaling to alleviate senescence in the CLL T cells is to be investigated.
Conclusion
We conclude that the dominant T-cell dysfunctional state in CLL is more fitting with senescence rather than exhaustion. These two T-cell states arise by distinct mechanisms and also require different means of reinvigoration in the context of immunotherapy. MAPK/p38 signaling is specifically activated in senescent cells, which could also be activated by DNA damage. Further study is warranted on how these signals can be interrupted to restore function of T cells in for example chimeric antigen receptor (CAR) T-cell therapy.