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T-cell dysfunction in CLL
early CLL studies biological inference was occasionally predicated on the selection of an inaccurate control.102 In general, T cells from CLL patients are compared to age- matched healthy controls but a more detailed characteri- zation of normal T-cell states for accurate comparisons is required. Perhaps machine learning approaches could assist in making these decisions.103
Studying single-cell epigenomic and transcriptomic pro- files have gained attention in research on CLL cells and non-malignant T cells, however no such studies are avail- able on CLL T cells yet. In a similar approach like deter- mining the cell-of-origin of CLL,4 lineage tracing might address whether a hierarchical pathway of differentiation towards dysfunction exists in CLL T cells. If such a hierar- chical pathway exists, it could assist in disease monitoring of patients. Currently, treatment decisions are made based on CLL characteristics but possibly the development of T- cell dysfunction over time provides clues on disease pro- gression and the need for treatment in the future.
Single-cell approaches also allow increased resolution for individual populations of cells with a distinct function- al or dysfunctional phenotype. These single-cell maps are becoming available for many different T-cell states in a healthy and malignant setting104 and comparison to CLL T cells might allow better understanding of the states of these cells. An unanswered question in the field is to what extent CLL T cells are truly exhausted, because cytokine expression seems intact. This residual function could indi- cate a progenitor exhaustive state that is likely more reversible than a terminally exhaustive state. A combined analysis of key exhaustion-related features such as expres- sion of inhibitory receptors, transcription factors such as TOX and TCF-1, and chromatin remodeling, could help answering this question.
High-dimensional flow or mass cytometry is also an excellent means to study single-cell phenotypes. Hartmann et al.105 described a method to characterize the metabolic regulome of CD8+ T cells with mass cytometry and their method also allowed integration with imaging to study spatial organization of different metabolic programs in the tissue. Although it is not easy to obtain human CLL LN, this technology would permit detailed characteriza- tion of the metabolic program within the CLL TME and potentially answer questions on the role of T cells in that environment. It would also allow simultaneous study of the CLL B cells and the T cells within the same patient. We believe that is essential for determining how these cellular interactions regulate T- and B-cell function in CLL.
As mentioned in the introduction, understanding the molecular mechanisms of CLL-induced T-cell dysfunction is essential to improve CAR T-cell therapy, and the in vitro generation of CAR T cells provides a window-of-opportu- nity to optimize these cells. Additional genetic editing of targets such as TCF-1, PGC1α and PAC1 might improve the stemness and memory phenotype, mitochondrial fit-
ness and the response to hypoxia. In addition, the differ- ential roles of CD4+ and CD8+ T cells in CLL might call for a different approach for CAR generation of the two sub- sets. Although both subsets display features of exhaus- tion, for CD4+ cells the prevention of Treg expansion is important whilst for the CD8+ the cytotoxic capacity needs to be improved. Current approaches do not distin- guish between CD4+ and CD8+ cells during the expansion phase and the CAR construct is the same, it might be worthwhile to explore a subset-specific approach in CLL.
Concluding remarks
In summary, we have described the features of T-cell dysfunction and can conclude that CD4+ and CD8+ T cells have both overlapping as well as distinct roles in CLL. Both show features of exhaustion and whilst CD4+ T cells possibly play a tumor-supportive role in the TME, CD8+ T cells have reduced cytotoxic capacity. Transcriptional control of T cells is executed by several key transcription factors that are known for their role in effector T-cell function but, depending on the context, could also steer cells into dysfunction and exhaustion. Epigenetic marks and their regulators play a major role in determining the context in which transcription factors operate and it is likely that T-cell dysfunction caused by metabolic perturbations in the TME is epigenetically mediated. The dynamics and plasticity of these tran- scriptional profiles of dysfunctional T cells are still an active topic of debate in healthy as well as CLL T cells. For CLL, plasticity of T-cell function and phenotype is important for immunotherapeutic approaches such as CAR T-cell therapy. Although in vitro generation of CAR T cells can be optimized, the signals they receive once transferred back into the patients and entering the CLL TME are important to take into account. Therefore, inte- grating molecular studies of CLL cells and T cells is nec- essary to unravel factors leading to T-cell dysfunction, an understanding that can improve re-invigoration of T cells by autologous T-cell-based immunotherapy.
Disclorures
JCS has previously received funding from Roche and currently receives funding from Bloodwise, the Kay Kendall Leukaemia Fund and Cancer Research UK; APK is funded by a Dutch Research Council (NWO) VIDI grant and an European Research Council (ERC) Consolidator grant; FSP and EE declare no conflicts of interest.
Contributions
FSP designed the set-up of this review and did an extensive lit- erature review; FSP, JCS, EE and APK contributed to writing and editing the manuscript and all authors read and approved the final version of this manuscript.
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