Summary T-cell metabolic dysfunction is increasingly recognized as a hallmark of poor antitumor responses. Still, how distinct metabolic states shape T-cell function and which signaling pathways sustain them remains unclear. Here, we show that T cells from patients with chronic lymphocytic leukemia (CLL) adopt a hyperactivated phenotype characterized by high cytokine production, aberrant proliferation and a bias toward glycolysis, supported by sustained glutamine-driven mTORC1 activity. In parallel, T cells from these patients display alterations in mitochondrial network and cristae architecture, which further limit oxidative phosphorylation. mTORC1 inhibition reduces glucose dependence, restores mitochondrial metabolic engagement and normalizes T-cell activation and proliferation. Together, these findings identify metabolic disbalance as a central contributor to the hyperactivated state of T cells in CLL. We propose a model in which reduced OXPHOS reflects both mitochondrial defects that pre-exist in T cells from patients before TCR engagement, and a failure to engage mitochondrial metabolism upon activation supported by the actionable target mTORC1. Highlights T cells from patients with chronic lymphocytic leukemia display high cytokine production and excessive division cycles upon CD3/28 engagement. mTORC1-sustained glycolysis and defects in mitochondrial structure compromise OXPHOS in hyperactivated T cells. Exogenous glutamine uptake sustains non-lysosomal mTORC1 activity in T cells.

Studying how microenvironmental cues influence metabolic reprogramming can uncover mechanisms driving tumor progression. Using an in vitro model with proliferative stimuli of the in vivo lymph node niche (LN)-including interleukin-21 (IL-21)-we examined metabolic rewiring in chronic lymphocytic leukemia (CLL) cells. We found that the metabolic intermediates of upper glycolysis and its branching pathways are key in fulfilling metabolic demands of proliferating CLL cells. Among branching pathways, the pentose phosphate pathway (PPP) was the most transcriptionally upregulated in proliferating CLL cells. Increased expression of PPP genes was detected ex vivo at the bulk and single-cell level in the LN-resident and -emigrating CLL cells, with more consistency across enzymes of the nonoxidative PPP branch. Expression of the latter correlated with shorter failure-free survival in CLL patients. At the cellular level, metabolomics and 13C-glucose tracing confirmed high activity of the non-oxidative PPP in proliferating CLL cells. IL-21 regulated the expression of PPP enzymes, with STAT3 serving as the primary downstream effector. CRISPR/Cas9-mediated silencing of PPP enzymes revealed that, in vitro, proliferating CLL cells from most patients were not dependent on these enzymes. In contrast, silencing transketolase (TKT)-the rate-limiting enzyme of the non-oxidative PPP-abolished tumor engraftment in vivo, demonstrating that CLL cells rely on this pathway within the tumor microenvironment. These findings uncover a CLL-specific metabolic reprogramming wherein IL-21-STAT3 drives PPP activity and identify the nonoxidative PPP as a critical in vivo vulnerability of leukemic cells in the murine CLL model.

Autologous cellular immunotherapies, which rely on cytotoxic T lymphocytes (CTLs), are increasingly applied in different B-cell malignancies. The general assumption is that CTLs exert their cytolytic function through granzymes that induce apoptosis in the target cell. However, the killing mechanism of immunotherapeutic CTLs is not clearly elucidated. Using T-cell redirecting bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T-cells we assessed which cell death pathways were activated in B cell line models as well as in primary material from CLL patients. We demonstrate that for cytotoxic T-cell killing of malignant B-cells by any of the treatment strategies, caspase activity was not essential. We could also exclude a role for TNF or TRAIL-mediated pathways. Using electron microscopy, CAR T-cell and BsAb-mediated cell death showed a mixed apoptotic/necroptotic phenotype. This was corroborated by knockout and chemical inhibition of the essential necroptosis proteins RIPK1, RIPK3, and MLKL. Necroptotic death of target cells correlated with the release of HMGB1 in the supernatant as well as various immunomodulatory molecules from the T-cells. Besides known immune activators IFN-y, IL-17 and IL-6, also anti-inflammatory IL-10 and IL1-RA were released. Moreover, the quantity of these immunomodulators was differentially affected after application of apoptosis versus necroptosis inhibitors. Together, these data demonstrate that (CAR) T-cell-mediated killing of lymphoma cells has a necroptotic arm which is correlated with modulation of the wider immune response. They imply that manipulation of the apoptotic versus necroptotic balance in immunotherapy could affect engagement of the autologous immune response.

Autologous T-cell therapies show limited efficacy in chronic lymphocytic leukemia (CLL), where acquired immune dysfunction prevails. In CLL, disturbed mitochondrial metabolism has been linked to defective T-cell activation and proliferation. Recent research suggests that lipid metabolism regulates mitochondrial function and differentiation in T cells, yet its role in CLL remains unexplored. This comprehensive study compares T-cell lipid metabolism in CLL patients and healthy donors, revealing critical dependence on exogenous cholesterol for human T-cell expansion following TCR-mediated activation. Using multi-omics and functional assays, we found that T cells present in viably frozen samples of patients with CLL (CLL T cells) showed impaired adaptation to cholesterol deprivation and inadequate upregulation of key lipid metabolism transcription factors. CLL T cells exhibited altered lipid storage, with increased triacylglycerols and decreased cholesterol, and inefficient fatty acid oxidation (FAO). Functional consequences of reduced FAO in T cells were studied using samples from patients with inherent FAO disorders. Reduced FAO was associated with lower T-cell activation but did not affect proliferation. This implicates low cholesterol levels as a primary factor limiting T-cell proliferation in CLL. CLL T cells displayed fewer and less clustered lipid rafts, potentially explaining the impaired immune synapse formation observed in these patients. Our findings highlight significant disruptions in lipid metabolism as drivers of functional deficiencies in CLL T cells, underscoring the pivotal role of cholesterol in T-cell proliferation. This study suggests that modulating cholesterol metabolism could enhance T-cell function in CLL, presenting novel immunotherapeutic approaches to improve outcome in this challenging disease.

Background:Anti-CD20 antibodies are first-line treatments for B cell malignancies. Natural killer (NK) cells are important mediators of anti-CD20 antibody efficacy in humans through antibody-dependent cellular cytotoxicity (ADCC). In B cell malignancies, the lymph nodes are a critical site of pathology and the T cell-derived signals CD40L and IL-4 within the lymph node microenvironment can mediate tumour proliferation, survival and resistance to pro-apoptotic therapy. CD40L and IL-4 have recently been shown to inhibit NK cell activation against chronic lymphocytic leukaemia (CLL) cells via the HLA-E:NKG2A immune checkpoint axis. However, the effect of these signals on NK cell-mediated ADCC of malignant B cells is unclear. Methods:Using a combination of clinical samples, murine models, flow cytometry, immunoblotting, immunohistochemistry, ELISA, bioinformatics and functional assays, we examined the impact of lymph node-mimicking conditions on NK cell-mediated ADCC against malignant B cells. Exogenous CD40L and IL-4 were used to mimic T-B cell interactions in 2D malignant B cell cultures, in addition to a 3D spheroid model of T cell-dependent CLL proliferation. Results:CD40L and IL-4 increased HLA-E expression on the surface of primary CLL cells and non-Hodgkin's lymphoma (NHL) cell lines, and this decreased NK cell-mediated ADCC via ligation of the inhibitory receptor NKG2A. High HLA-E surface expression was observed in lymph node FFPE sections of CLL and NHL patients and in a 3D ex vivo lymph node-mimicking model of CLL. NKG2A blockade potentiated NK cell-mediated ADCC against malignant B cells treated with CD40L and IL-4 and improved anti-CD20 antibody therapy in a murine model of B cell lymphoma. Conclusion:These results reveal a novel mechanism of resistance to anti-CD20 therapy in B cell malignancies and demonstrate that the combination of anti-NKG2A with anti-CD20 could improve the treatment of patients with CLL or NHL.

Chronic lymphocytic leukemia is a malignant lymphoproliferative disorder for which primary or acquired drug resistance represents a major challenge. To investigate the underlying molecular mechanisms, we generate a mouse model of ibrutinib resistance, in which, after initial treatment response, relapse under therapy occurrs with an aggressive outgrowth of malignant cells, resembling observations in patients. A comparative analysis of exome, transcriptome and proteome of sorted leukemic murine cells during treatment and after relapse suggests alterations in the proteasome activity as a driver of ibrutinib resistance. Preclinical treatment with the irreversible proteasome inhibitor carfilzomib administered upon ibrutinib resistance prolongs survival of mice. Longitudinal proteomic analysis of ibrutinib-resistant patients identifies deregulation in protein post-translational modifications. Additionally, cells from ibrutinib-resistant patients effectively respond to several proteasome inhibitors in co-culture assays. Altogether, our results from orthogonal omics approaches identify proteasome inhibition as potentially attractive treatment for chronic lymphocytic leukemia patients resistant or refractory to ibrutinib. The molecular mechanisms underlying resistance to therapy in Chronic lymphocytic leukemia (CLL) remain to be explored. Here, the authors perform multi-omics analysis in a mouse model of ibrutinib resistance and suggest proteasome inhibition for overcoming it.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remains elusive. Using a nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and prosurvival signaling via changes in the phosphoproteome, indicating the existence of constitutive CXCR4 oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B cells to venetoclax is of clinical relevance. Taken together, we established a noncanonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that their selective targeting has clinical potential.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

Chronic lymphocytic leukemia (CLL) cells circulate between peripheral (PB) blood and lymph node (LN) compartments, and strictly depend on microenvironmental factors for proliferation, survival and drug resistance. All cancer cells display metabolic reprogramming and CLL is no exception – though the inert status of the PB CLL cells has hampered detailed insight into these processes. We summarize previous work on reactive oxygen species (ROS), oxidative stress, and hypoxia, as well as the important roles of Myc, and PI3K/Akt/mTor pathways. In vitro co-culture systems and gene expression analyses have provided a partial picture of CLL LN metabolism. New broad omics techniques allow to obtain molecular and also single-cell level understanding of CLL plasticity and metabolic reprogramming. We summarize recent developments and describe the new concept of glutamine addiction for CLL, which may hold therapeutic promise.

The field of immunometabolism cannot be considered ‘emerging’ anymore; it is at the moment one of the most active and rapidly evolving areas of biomedical research. Its hottest zone is cancer immunometabolism. This is partly due to the clinical application of immunotherapy, with either antibodies (checkpoint blockade) or cellular therapies (e.g., CAR‐T cells). In addition, the proliferating tumor cells create a nutrient‐deprived microenvironment that impairs the metabolic fitness and functionality of infiltrating immune cells such as T cells, NK cells, and macrophages. The key concepts are bidirectional metabolic signaling, plus the conviction that a better understanding of these processes will improve current immunotherapies, and foster new tools and targets for treatment. This collection of reviews will address various exciting aspects from junior and established scientists in the field.

The tumor suppressor p53 has been described to control various aspects of metabolic reprogramming in solid tumors, but in B cell malignancies that role is as yet unknown. We generated pairs of p53 functional and knockout (KO) clones from distinct B cell malignancies (acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B cell lymphoma, and multiple myeloma). Metabolomics and isotope tracing showed that p53 loss did not drive a common metabolic signature. Instead, cell lines segregated according to cell of origin. Next, we focused on glutamine as a crucial energy source in the B cell tumor microenvironment. In both TP53 wild-type and KO cells, glutamine deprivation induced cell death through the integrated stress response, via CHOP/ATF4. Lastly, combining BH3 mimetic drugs with glutamine starvation emerged as a possibility to target resistant clones. In conclusion, our analyses do not support a common metabolic signature of p53 deficiency in B cell malignancies and suggest therapeutic options for exploration based on glutamine dependency.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remain elusive. Using a newly established nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and pro-survival signaling via changes in the phosphoproteome, indicating the existence of basal CXCR4-oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B-cells to venetoclax is of clinical relevance. Taken together, we established a new, non-canonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that selective targeting thereof has clinical potential. ### Competing Interest Statement SMA and RH are employed by QVQ, a CRO offering Nb services and reagents. A provisional patent application has been filed on the research described in this manuscript (P135924EP00).

Autologous T-cell-based therapies, such as CAR-T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) which correlates with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aims to investigate mechanisms by which CLL cells suppress (CAR) T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion and that specifically re-stimulation of (CAR-) T cells in presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, co-culture of T cells with CD40-activated CLL cells did not result in T-cell dysfunction and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40-signaling cascade revealed that the SRC-kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of SIGLEC-10-ligands CD24 and CD52, which was prevented by dasatinib; suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced (CAR) T-cell dysfunction upon co-culture with resting CLL cells. These results demonstrate that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T cell interactions. Targeting CD24- and CD52-mediated CLL-T cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.

Background: Chronic lymphocytic leukemia (CLL) remains incurable despite availability of targeted therapies. Successful autologous cell-based anti-cancer therapies require functionality and longevity of effector cells, features that highly depend on complex metabolic processes. A key feature of CLL is the suppression of T-cell function, but the underlying mechanism is still poorly understood. We previously found signs of impaired metabolic plasticity upon stimulation of T cells from CLL patients (van Bruggen, Blood 2019). However, in-depth analysis of the metabolic signature of CLL T cells, and how it differs from healthy T cells, is lacking. Aims: With this study, we aim to analyze mitochondrial and glycolytic metabolism of T cells from healthy individuals and CLL patients in real-time, and characterize their metabolic signatures and fuel dependencies upon T cell receptor (TCR) engagement. Methods: Peripheral blood mononuclear cells (PBMCs) of age-matched healthy donors (HD) and untreated CLL patients were analyzed by flow cytometry, extracellular flux analysis, LC-MS metabolomics and C13 fractional labelling upon stimulation with αCD3/αCD28 antibodies. Results: Upon stimulation, T cells from CLL patients showed decreased activation levels and reduced increase in mitochondrial mass, as compared to HD T cells. Accordingly, extracellular flux analysis revealed an impairment to upregulate mitochondrial activity upon stimulation in T cells from all patients analyzed, including reduced spare respiratory capacity, an important indicator of mitochondrial and (CAR)-T cell fitness. The response in glycolysis, a key pathway for early T-cell activation, varied between patients. These findings pointed towards mitochondrial dysfunction rather than overall metabolic impairment at the basis of T-cell dysfunction in CLL. Metabolomics analyses on stimulated T-cells from CLL and HD indicated a global decrease in metabolite abundance in CLL, with several intermediates of the tricarboxylic acid cycle (TCA) as significant hits. This deficient mitochondrial metabolism in CLL T cells could be caused by hindered activity of mitochondrial enzymes or decreased fuel utilization. Glucose and glutamine are major fuels of the TCA cycle and, together with fatty acids, are used for biomolecule synthesis and energy production. Fractional labelling showed that mitochondrial enzymes are functional in CLL T cells and identified glutamine as the key contributor to the TCA cycle in stimulated T cells derived from both HD and CLL. These observations, together with our recent finding that activated CLL cells predominantly depend on glutamine (Chen, Blood 2022), hint at a compromised availability of glutamine for the surrounding T cells in CLL. Counterintuitively, activation and proliferation studies revealed that HD T cells are more susceptible to glucose and glutamine deprivation than CLL-derived T cells, which might be due to the fact that HD T cells rely on highly active metabolism sustained by these fuels, whereas the metabolic state of CLL T cells is already lower in non-restricted nutrient conditions. Summary/Conclusion: CLL T cells have a distinct metabolic profile upon stimulation, which is characterized by defective mitochondrial content and activity. Despite finding glutamine as the main fuel for the TCA cycle in both HD and CLL T cells, the metabolic rate of CLL T cells is lower, which ultimately contributes to T-cell dysfunction.Keywords: Mitochondria, Chronic lymphocytic leukemia

Despite being a B-cell malignancy, dysfunction of the T-cell compartment is a key feature of chronic lymphocytic leukemia (CLL). This leads to complications in acquired immunity and low success rates in autologous T cell-based therapies in these patients, particularly chimeric antigen receptor T cells (CAR-T). Despite exhibiting features of exhaustion, T cells from CLL patients are not reinvigorated by immune checkpoint inhibitors. Although T-cell dysfunction in CLL is heterogeneous, a consistent feature among all CLL patients is T-cell subset skewing towards effector T cells at the expenses of the central memory compartment (Martens et al., Leukemia 2023) and intact ability to produce inflammatory cytokines. A common feature of senescent T cells includes this maintained ability to produce pro-inflammatory cytokines alongside dysfunctions such as cell-cycle arrest, a decline in proliferation, and reduced activation. Notably, senescence often coincides with impaired mitochondrial function which is known to play a crucial role in the development of memory phenotypes and the efficacy of CAR-T cells (Van der Windt et al., Immunity 2012; Van Bruggen et al., Blood 2019). Given these associations, our aim is to comprehensively investigate the interplay between T-cell dysfunction, senescence, and mitochondrial impairment in CLL patients through a multifaceted approach encompassing analysis of T-cell metabolism, (epi)genetics, and functional phenotyping. Metabolic profiling of T cells from CLL patients and age-matched healthy donors (HD) was performed by extracellular flux analysis, flow cytometry, metabolomics and 13C tracing. A decreased mitochondrial respiration upon T-cell receptor stimulation was observed in all CLL patients. On the other hand, glycolytic activity correlated with T-cell activation status, which varied among patients. We identified alterations in the utilization of the three main mitochondrial fuels in T cells from CLL patients: i) induction of pseudohypoxia by CLL cells, leading to a re-routing of glucose-derived pyruvate into lactate rather than into the mitochondria; ii) a reduced rate of conversion of glutamine into glutamate resulting in decreased utilization of this fuel within the mitochondria; and iii) impaired lipid uptake, along with diminished fatty acid oxidation. Accordingly, mitochondria from CLL T cells showed intrinsic features of dysfunction including depolarization and increased production of reactive oxygen species (ROS). So far, these findings strongly demonstrate a misbalance in metabolic fueling resulting in mitochondrial dysfunction, aligning more closely with senescence rather than classical T-cell exhaustion. To further reinforce this observation, we assessed the expression of cell-surface proteins that characterize senescent T cells (e.g., absence of CD27/CD28 and presence of PD1/CD57/KLRG1). Indeed, we found an increased proportion of senescent cells in the T-cell compartment of CLL patients, as compared to HD. In response to stimulation, CLL T cells showed a strikingly distinct cell-cycle pattern whereby a larger proportion of cells resided in S and G2/M phases, indicating cell-cycle arrest. Cell cycle arrest is often induced by DNA damage. Transcriptomic and epigenomic profiling of T cells of CLL patients indeed revealed a molecular profile consistent with inflammation and an increased p53/DNA damage response upon stimulation. These features are known to be induced by dysfunctional mitochondria and ROS in senescent cells, underscoring a direct link between mitochondrial metabolism and senescence. Altogether, our results strongly point towards senescence as the dominant dysfunctional T-cell state in CLL patients, with impaired mitochondrial metabolism at its basis. The distinctive mechanisms leading to T-cell senescence, as opposed to classical exhaustion, underscore the necessity for different strategies to reinvigorate T cells in the context of immunotherapy. Therefore, interventions aimed at ameliorating T-cell dysfunction in CLL should prioritize enhancement of mitochondrial metabolism to attenuate DNA damage and the induction of senescence. This would be especially beneficial to improve the fitness of autologous CAR-T cells for these patients.

PDF file - 793K, Nucleofection with Noxa siRNA or control siRNA was performed on CLL cells in the presence or absence of the proteasoom inhibitor GSI. Protein lysates were probed for Mcl-1, Noxa and -actin. As a control, lysates of untransfected CLL cells in the presence or absence of GSI are shown.

PDF file - 986K, A. ABT-737 resistant CLL cells were stimulated with CD40L for 72 hours (n=4). The black line shows apoptosis levels from CLL cells detached from the feeder layer and subsequently treated with different concentrations of ABT-737 (0.001-10 M). CLL cells treated with ABT-737 in the presence of CD40-expressing feeder cells are depicted in gray. B. ABT-737 sensitive CD40-stimulated CLL cells treated with ABT-737 after detachment from feeder cells (black line) and while continuously in contact with CD40-expressing feeder cells (gray line) (n=5).

PDF file - 866K, CLL cells were stimulated with CD40L for 48 hrs. After detachment, cells were incubated with 10 M or 100 M fludarabine Q-VD (12,5 M), NAC (5mM). Protein lysates were probed for Noxa, Mcl-1 and -actin.