T-cell dysregulation in chronic lymphocytic leukemia associates with low response rates to autologous T cell-based therapies. How CLL affects T-cell responses remains unknown. Although valuable data can be obtained by studying primary patient cells in vitro to some extent, we felt that dynamic (in vivo) studies were obligatory to gain mechanistic insights into the impact of CLL cell presence on antigen-specific responses. In this paper we investigated epigenetic, transcriptional and functional consequences of CLL on the CD8+ T-cell response in vitro using primary patient cells and in vivo, using an adoptive transfer murine model.
We show that in CLL patients, CD8+ T cells in resting state are skewed towards KLRG1+CD127- short lived effector cells (SLEC) at the expense of KLRG1-CD127+ (memory precursor effector cells; MPEC). In vitro stimulation of these cells leads to rapid induction of an effector gene profile which is rescued upon CLL cell depletion, indicating a reversible, CLL-induced phenotype hinting at epigenetic regulation. This hypothesis was investigated in-depth in a novel, adoptive transfer mouse model combining the well-known Eµ-TCL1 mouse with the OT-I model. We first let mice develop leukemia by transferring TCL1 splenocytes. Upon full development of CLL, the mice are injected with OT-I cells (CD8+ T cells that specifically recognize the ovalbumin (OVA) peptide SINFEKL) and simultaneously receive an mCMV-OVA infection. This model allows the study of antigen-specific CD8+ T cell responses whilst CLL is present in the mouse, thereby acting as bystander cells and not direct targets to the T cells.
OT-I cells from CLL mice were compared to their WT counterparts and at day 7 of infection the percentages of total OT-I cells (CD45.1+CD8+) nor the percentage effector cells (CD62L-CD44+) were different, indicating that the magnitude of this initial response was not affected by CLL. However, if we zoom in on the effector cells, CLL induced skewing towards SLECs and a lower proportion of OT-I cells were MPEC. This was associated with increased expression of the effector-related transcription factor T-bet and reduced expression of the memory-related transcription factor Bcl-6. Upon restimulation in vitro, OT-I cells from CLL mice displayed a reduced expression of IFNγ, TNFα and the degranulation marker CD107a. This shows that CLL induces some form of unresponsiveness to repeated stimuli.
Chromatin and transcriptome profiling of the OT-I cells at day 7 of infection revealed strong epigenetic modifications leading to activation of an effector and silencing of a memory profile through the presence of CLL. Also OT-I cells from CLL mice resembled PD1hi and PD1int CD8+ T cells as defined by other studies in TCL1 mice. When looking at chromatin accessibility, key genes were altered by CLL. To assess whether early memory formation is impaired by CLL and if CD8+ T cells from CLL mice are less responsive to repeated stimuli, a memory recall experiment was designed. OT-I cells were harvested after 7 days of primary infection and transferred into new WT mice. After a period of rest, the mice were re-infected with mCMV-OVA(N4). The OT-I cells originating from the CLL mice were less able to expand as the percentage and absolute numbers of OT-I cells was reduced, confirming a dysfunctional memory response. However, the remaining cells did express more effector cytokines upon in vitro restimulation. Interestingly, this is similar to what is observed in CLL patients where we and others have shown similar or even elevated levels of cytokine expression upon in vitro activation.
Altogether we have shown that presence of CLL induces a short-lived effector phenotype and impaired memory responses by epigenetic reprogramming during the primary challenge.
To finish this behind-the-scenes look at our paper, we will share a few interesting facts on how this paper came to be. The first experiment for this study was done already in 2017 by first author Anne and Rianne. We started in Amsterdam and later collaborated with the group in Rijeka, Croatia where many of the follow-up experiments were performed by Inga, Mia and Felix. In 2019, Fleur joined the team and that is when we decided to dive into the epigenetic and transcriptional regulation of this model, which also prompted the collaboration with the data science department within the institute. So all in all, a long-term project with international and national collaborative partners that has now resulted in this comprehensive work. We are excited to see what the future will bring and we hope to further contribute to the knowledge on T-cell regulation in CLL with this work and our future projects.