Chronic lymphocytic leukemia (CLL) cells circulating in peripheral blood (PB) differ from the leukemic fraction in lymph nodes (LNs) with respect to cell division and drug sensitivity. CD40 stimulation of PB CLL cells in vitro results in chemoresistance and provides a partial model for the LN microenvironment. The TLR9 ligand CpG induces proliferation in immunoglobulin variable heavychain-unmutated CLL, but apoptosis in immunoglobulin variable heavy-chain-mutated CLL. To juxtapose proliferative with antiapoptotic signals, we investigated the effects of CpG in the context of CD40 ligation in mutated versus unmutated CLL cells in this study. Prolonged CD40 ligation induced classical, followed by alternative nuclear factor-jB (NF-jB), activity in both subgroups, correlating with enhanced Bfl-1 and Bcl-X L levels, respectively. A dichotomy in NF-jB signaling occurred on combined CD40/TLR9 triggering. This induced declining p52 and Bcl-X L levels, and reversed chemoresistance only in mutated cells, whereas unmutated cells proliferated, maintained p52 and Bcl-X L and remained chemoresistant. The pivotal contribution of Bcl-X L to chemoresistance was shown by the BH3 mimetic ABT-737 and RNA interference. Finally, in ex vivo LN samples, p52, p65 and Bcl-X L levels were highly expressed, corroborating the in vitro findings. Thus, a distinction in NF-jB activation and drug susceptibility in mutated versus unmutated (LN-like) CLL cells was uncovered, which was causally linked to Bcl-X L levels.
In chronic lymphocytic leukemia (CLL), acquired T-cell dysfunction impedes development of effective immunotherapeutic strategies, through as-yet unresolved mechanisms. We have previously shown that CD8+ T cells in CLL exhibit impaired activation and reduced glucose uptake after stimulation. CD8+ T cells in CLL patients are chronically exposed to leukemic B cells, which potentially impacts metabolic homeostasis resulting in aberrant metabolic reprogramming upon stimulation. Here, we report that resting CD8+ T cells in CLL have reduced intracellular glucose transporter 1 (GLUT1) reserves, and have an altered mitochondrial metabolic profile as displayed by increased mitochondrial respiration, membrane potential, and levels of reactive oxygen species. This coincided with decreased levels of peroxisome proliferator-activated receptor γ coactivator 1-α, and in line with that, CLL-derived CD8+ T cells showed impaired mitochondrial biogenesis upon stimulation. In search of a therapeutic correlate of these findings, we analyzed mitochondrial biogenesis in CD19-directed chimeric antigen receptor (CAR) CD8+ T cells prior to infusion in CLL patients (who were enrolled in NCT01747486 and NCT01029366 [https://clinicaltrials.gov]). Interestingly, in cases with a subsequent complete response, the infused CD8+ CAR T cells had increased mitochondrial mass compared with nonresponders, which positively correlated with the expansion and persistence of CAR T cells. Our findings demonstrate that GLUT1 reserves and mitochondrial fitness of CD8+ T cells are impaired in CLL. Therefore, boosting mitochondrial biogenesis in CAR T cells might improve the efficacy of CAR T-cell therapy and other emerging cellular immunotherapies.
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