Abstract: 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.

Abstract: Background In chronic lymphocytic leukemia (CLL), acquired T cell dysfunction impedes development of effective immunotherapeutic strategies through yet unresolved mechanisms. As an intricate relationship exists between cellular metabolism and T cell function, we hypothesized that the interaction of T cells with CLL cells leads to metabolic deregulation, resulting in T cell dysfunction. We have indeed recently shown impaired induction of glycolysis upon stimulation in CLL patient-derived T cells (Siska et al., 2016). Whether metabolic alterations are already present prior to activation and to what extent CLL cells contribute to impaired metabolic fitness of T cells is unknown. Furthermore, the clinical relevance of impaired metabolic fitness of CD8 T cells in CLL is still elusive. Methods CLL Patients were selected for CLL treatment naivety, and a white blood cell count greater than 20×109 cells/L. Age-matched healthy donors (HD) were used as controls. CAR T cell infusion products were generated from relapsed/refractory (R/R) CLL patients enrolled in two clinical trials of single-agent CTL019 therapy at the University of Pennsylvania (registered at clinicaltrials.gov; NCT01029366, and NCT01747486). T cells were analyzed either directly ex vivo, or after culture in the presence or absence of CD3 and CD28 antibodies by flow cytometry or XF96 Seahorse. Results Following stimulation, CD8+ T cells from CLL patients displayed reduced expression of activation markers, and showed reduced degranulation, while PD1 expression was higher. This was accompanied by reduced surface expression of the glucose transporter GLUT1, and reduced glucose uptake despite proficient glucose availability. Impaired glucose uptake by T cells seemed mediated by CLL cells as this was fully restored by depleting CLL cells prior to activation. This phenomenon is likely to be mediated by a soluble factor produced by CLL cells since reduced glucose uptake was still evident after physical separation of T cells and CLL cells in transwell experiments. Because mitochondrial metabolism is instrumental to engage the first steps of glycolysis upon T cell stimulation (van der Windt et al. 2013), and mitochondrial reactive oxygen species (ROS) promote glucose uptake by promoting Glut1 expression (Liemburg-Apers et al. 2015), we next focused on mitochondrial function. We found that ex vivo CLL-derived CD8+ T cells displayed increased basal respiration (measured by O2 consumption rate, an indicator of oxidative phosphorylation), which was accompanied by an increased mitochondrial membrane potential and elevated mitochondrial ROS produced by CLL-derived CD8+ T cells (Fig. 1A). Spare respiratory capacity (SRC) is the extra mitochondrial capacity available in a cell to produce energy under conditions of increased work or stress such as T cell activation. SRC was reduced in ex vivo CLL-derived CD8+ T cells, indicating a lower ability to deal with enhanced bio-energetic demand. While total ex vivo CLL-derived CD8+ T cells showed similar mitochondrial mass compared to HD, antigen-experienced CLL-derived CD8+ T cells displayed reduced mitochondrial mass compared to HD. This latter coincided with decreased levels of PGC-1α, the master regulator of mitochondrial function and biogenesis, and its target anti-oxidant SOD2. Consistent with these data, reduced mitochondrial biogenesis occurred in T cells derived from CLL patients upon T cell stimulation (Fig. 1B). The clinical significance of these findings was studied by measuring mitochondrial mass in CD19-directed chimeric antigen receptor (CAR) CD8+ T cells within the infusion product of R/R CLL patients included in the CAR T cell trial. Subjects with a complete response had increased mitochondrial mass compared to non-responders (Fig. 1C). Also, a clear positive correlation was found between mitochondrial mass and T cell persistence as well as with memory T cells that were negative for exhaustion markers. Conclusion This is the first report describing a skewed mitochondrial metabolic profile in CLL CD8+ T cells prior to stimulation, which is maintained after stimulation and is then accompanied by impaired glucose uptake. Decreased mitochondrial fitness correlates with impaired CAR-T cell persistence and poor responses. Therefore, boosting mitochondrial biogenesis in T cells in CLL might improve the efficacy of CAR-T cell therapy and other emerging cellular immunotherapies. Disclosures Fraietta: Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Eldering:Celgene: Research Funding. Levin:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Rathmell:Calithera: Research Funding. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy. Kater:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract: The efficacy of autologous (αβ) T-cell–based treatment strategies in chronic lymphocytic leukemia (CLL) has been modest. The Vγ9Vδ2-T cell subset consists of cytotoxic T lymphocytes with potent antilymphoma activity via a major histocompatibility complex–independent mechanism. We studied whether Vγ9Vδ2-T cells can be exploited as autologous effector lymphocytes in CLL. Healthy control Vγ9Vδ2-T cells were activated by and had potent cytolytic activity against CLL cells. However, CLL-derived Vγ9Vδ2-T cells proved dysfunctional with respect to effector cytokine production and degranulation, despite an increased frequency of the effector-type subset. Consequently, cytotoxicity against malignant B cells was hampered. A comparable dysfunctional phenotype was observed in healthy Vγ9Vδ2-T cells after coculture with CLL cells, indicating a leukemia-induced mechanism. Gene-expression profiling implicated alterations in synapse formation as a conceivable contributor to compromised Vγ9Vδ2-T–cell function in CLL patients. Dysfunction of Vγ9Vδ2-T cells was fully reversible upon activation with autologous monocyte-derived dendritic cells (moDCs). moDC activation resulted in efficient expansion and predominantly yielded Vγ9Vδ2-T cells with a memory phenotype. Furthermore, ibrutinib treatment promoted an antitumor T helper 1 (TH1) phenotype in Vγ9Vδ2-T cells, and we demonstrated binding of ibrutinib to IL-2-inducible kinase (ITK) in Vγ9Vδ2-T cells. Taken together, CLL-mediated dysfunction of autologous Vγ9Vδ2-T cells is fully reversible, resulting in potent cytotoxicity toward CLL cells. Our data support the potential use of Vγ9Vδ2-T cells as effector T cells in CLL immunotherapy and favor further exploration of combining Vγ9Vδ2-T-cell–based therapy with ibrutinib.

Abstract: Quiescent T cells primarily use oxidative phosphorylation (OXPHOS) to generate ATP, while in response to activation T cells switch to high rates of aerobic glycolysis, also known as the Warburg effect. While less efficient in overall ATP production, this switch to aerobic glycolysis is essential to produce cellular biomass needed for proliferation, and is required for T cell effector functions. In chronic lymphocytic leukemia (CLL) acquired T cell dysregulation occurs independent of treatment with functional impairment and exhaustion of T cells. As tumor-imposed metabolic restrictions in mouse models can mediate T cell hyporesponsiveness during cancer, we hypothesized that in the context of CLL T cell metabolism might be altered. Comparison of gene expression profiles of T cells from patients with CLL and age-matched healthy donors (HD) revealed a highly significant increase in the expression of genes in the OXPHOS pathway (P=3.4*10-15) in the CD8 T cell compartment. In corroboration with these array results, we found that in naïve CD8 T cells in CLL, mitochondrial mass and respiration were significantly increased. In addition, increased mitochondrial ROS production was observed in the naïve CD8 T cell subset. Using Seahorse EFA technology on sorted CD8 T cells from CLL and age-matched HD, we found increased oxygen consumption rates in CLL derived CD8 T cells, indicating increased OXPHOS, while the spare respiratory capacity was lower in these cells, indicating impeded ability to cope with cellular stress. Extracellular acidification rates (ECAR; indicating glycolysis), and uptake of fluorescently labeled glucose were similar in CD8 T cells from CLL patients and HD. Next, we studied the metabolic plasticity of CLL derived T cells by stimulation of PBMCs from CLL patients and age-matched HD using anti-CD3/CD28 antibodies. Two days after stimulation, CLL derived T cells had diminished expression of activation markers CD25 and CD38, which correlated with reduced uptake of fluorescently labeled glucose. Moreover, preliminary Seahorse analysis of the immediate response to stimulation with anti-CD3/CD28 showed a reduced increase in ECAR in CLL derived CD8 T cells, indicating an impairment of the glycolytic switch in these cells. Taken together, these results demonstrate that the metabolic fitness of CD8 T cells is impaired in CLL at resting state and after activation. Boosting T cell metabolism in CLL might therefore improve existing immunotherapies in CLL such as CAR-T cell therapy. This work is funded by VENI and VIDI grants from the Dutch Organisation for Scientific Research, and a Marie Curie Career Integration Grant from the European Union. Disclosures Kater: Celgene: Research Funding.