T-cell Metabolism Research Articles

Development of a Competitive Nutrient-Based T-Cell Immunotherapy Designed to Block the Adaptive Warburg Effect in Acute Myeloid Leukemia.

Background: T-cell-based adoptive cell therapies have emerged at the forefront of cancer immunotherapies; however, failed long-term survival and inevitable exhaustion of transplanted T lymphocytes in vivo limits clinical efficacy. Leukemia blasts possess enhanced glycolysis (Warburg effect), exploiting their microenvironment to deprive nutrients (e.g., glucose) from T cells, leading to T-cell dysfunction and leukemia progression. Methods: Thus, we explored whether genetic reprogramming of T-cell metabolism could improve their survival and empower T cells with a competitive glucose-uptake advantage against blasts and inhibit their uncontrolled proliferation. Results: Here, we discovered that high-glucose concentration reduced the T-cell expression of glucose transporter GLUT1 (SLC2A1) and TFAM (mitochondrion transcription factor A), an essential transcriptional regulator of mitochondrial biogenesis, leading to their impaired expansion ex vivo. To overcome the glucose-induced genetic deficiency in metabolism, we engineered T cells with lentiviral overexpression of SLC2A1 and/or TFAM transgene. Multi-omics analyses revealed that metabolic reprogramming promoted T-cell proliferation by increasing IL-2 release and reducing exhaustion. Moreover, the engineered T cells competitively deprived glucose from allogenic blasts and lessened leukemia burden in vitro. Conclusions: Our findings propose a novel T-cell immunotherapy that utilizes a dual strategy of starving blasts and cytotoxicity for preventing uncontrolled leukemia proliferation.

Enhanced tumor response to adoptive T cell therapy with PHD2/3-deficient CD8 T cells

While adoptive cell therapy has shown success in hematological malignancies, its potential against solid tumors is hindered by an immunosuppressive tumor microenvironment (TME). In recent years, members of the hypoxia-inducible factor (HIF) family have gained recognition as important regulators of T-cell metabolism and function. The role of HIF signalling in activated CD8 T cell function in the context of adoptive cell transfer, however, has not been explored in full depth. Here we utilize CRISPR-Cas9 technology to delete prolyl hydroxylase domain-containing enzymes (PHD) 2 and 3, thereby stabilizing HIF-1 signalling, in CD8 T cells that have already undergone differentiation and activation, modelling the T cell phenotype utilized in clinical settings. We observe a significant boost in T-cell activation and effector functions following PHD2/3 deletion, which is dependent on HIF-1α, and is accompanied by an increased glycolytic flux. This improvement in CD8 T cell performance translates into an enhancement in tumor response to adoptive T cell therapy in mice, across various tumor models, even including those reported to be extremely resistant to immunotherapeutic interventions. These findings hold promise for advancing CD8 T-cell based therapies and overcoming the immune suppression barriers within challenging tumor microenvironments.

Malic enzyme 2 maintains metabolic state and anti-tumor immunity of CD8+ T cells

The functional integrity of CD8+ Tcells is closely linked to metabolic reprogramming; therefore, understanding the metabolic basis of CD8+ Tcell activation and antitumor immunity could provide insights into tumor immunotherapy. Here, we report that ME2 is critical for mouse CD8+ Tcell activation and immune response against malignancy. ME2 deficiency suppresses CD8+ Tcell activation and anti-tumor immune response invitro and invivo. Mechanistically, ME2 depletion blocks the TCA cycle flux, leading to the accumulation of fumarate. Fumarate directly binds to DAPK1 and inhibits its activity by competing with ATP for binding. Notably, pharmacological inhibition of DAPK1 abolishes the anti-tumor function conferred by ME2 to CD8+ Tcells. Collectively, these findings demonstrate a role for ME2 in the regulation of CD8+ Tcell metabolism and effector functions as well as an unexpected function for fumarate as a metabolic signal in the inhibition of DAPK1.

CRISPR-based genetic screens advance cancer immunology.

CRISPR technologies have revolutionized research areas ranging from fundamental science to translational medicine. CRISPR-based genetic screens offer a powerful platform for unbiased screening in various fields, such as cancer immunology. Immune checkpoint blockade (ICB) therapy has been shown to strongly affect cancer treatment. However, the currently available ICBs are limited and do not work in all cancer patients. Pooled CRISPR screens enable the identification of previously unknown immune regulators that can regulate T-cell activation, cytotoxicity, persistence, infiltration into tumors, cytokine secretion, memory formation, T-cell metabolism, and CD4+ T-cell differentiation. These novel targets can be developed as new immunotherapies or used with the current ICBs as new combination therapies that may yield synergistic efficacy. Here, we review the progress made in the development of CRISPR technologies, particularly technological advances in CRISPR screens and their application in novel target identification for immunotherapy.

Unlocking Cell Therapy's Full Potential by Rewiring T-Cell Metabolism

Unlocking Cell Therapy's Full Potential by Rewiring T-Cell Metabolism

Fasting and Glucose Metabolism Differentially Impact Peripheral Inflammation in Human Type 2 Diabetes.

Cytokines produced by peripheral T-helper 1/17 cells disproportionately contribute to the inflammation (i.e., metaflammation) that fuels type 2 diabetes (T2D) pathogenesis. Shifts in the nutrient milieu could influence inflammation through changes in T-cell metabolism. We aimed to determine whether changes in glucose utilization alter cytokine profiles in T2D. Peripheral blood mononuclear cells (PBMCs), CD4+ T-cells, and CD4+CD25- T-effector (Teff) cells were isolated from age-matched humans classified by glycemic control and BMI. Cytokines secreted by CD3/CD28-stimulated PBMCs and Teff were measured in supernatants with multiplex cytokine assays and a FLEXMAP-3D. Metabolic activity of stimulated CD4+ T-cells was measured by a Seahorse XFe96 analyzer. In this study, we demonstrated that T-cell stimulated PBMCs from non-fasted people with T2D produced higher amounts of cytokines compared to fasting. Although dysglycemia characterizes T2D, cytokine production by PBMCs or CD4+ T-cells in T2D was unaltered by hyperglycemic media. Moreover, pharmacological suppression of mitochondrial glucose oxidation did not change T-cell metabolism in T2D, yet enhanced cytokine competency. In conclusion, fasting and glucose metabolism differentially impact peripheral inflammation in human T2D, suggesting that glucose, along with fatty acid metabolites per our previous work, partner to regulate metaflammation. These data expose a major disconnect in the use of glycemic control drugs to target T2D-associated metaflammation.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics

BackgroundChimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity.MethodsThis retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products.ResultsA pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response.ConclusionspH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Abstract 18: The polyamine-hypusine axis is a promising avenue to improve T-cell metabolism and persistence for adoptive cell therapy

Abstract There are significant barriers to the application of Adoptive Cell Therapy (ACT) to many cancer types. One of these barriers is a result of poor metabolic fitness of T cells, leading to poor proliferation, persistence, and dampened anti-tumor function. Leveraging T-cell metabolism to improve their function is becoming increasingly important to make significant strides in immunotherapy. Stat5a has been shown to be a master regulator of energy and metabolism in T cells, highlighting it as a promising avenue to improve T-cell metabolism for ACT. We have established that the expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells improves their persistence, polyfunctionality, and anti-tumor effects in a mouse model. This study further examines Stat5a and its downstream metabolic targets in the context of ACT. We carried out scRNAseq analysis and found that Stat5a directly interacts with genes involved in polyamine metabolism. More specifically, Stat5a activation increases the expression of ornithine decarboxylase (Odc1), spermidine synthase (Srm), deoxyhypusine synthase (Dhps), and deoxyhypusine hydroxylase (Dohh) in a subset of CD4+ T cells expressing CASTAT5. Cleavage Under Targets and Tagmentation (Cut&Tag) analysis of histone H3K27ac also demonstrated increased H3K27ac at promoter and enhancer regions of polyamine pathway genes in CASTAT5 compared to control CD4+ T cells. To further examine the function of Stat5a, the murine pro-B cell line, Ba/F3, is being used as a model to study Stat5a-mediated signaling. We have found that upon withdrawal of IL-3 from Ba/F3 cells, Stat5a activation is significantly downregulated. Our scRNA-seq-based metabolic flux estimation showed a significant reduction in metabolic fluxes and related genes, including polyamines, following IL-3 withdrawal. Correspondingly, Stat5a ChIP-Seq and Cut&Tag analyses of H3K27ac revealed Stat5a binding to active enhancer regions in the presence of IL-3 that are lost following its withdrawal. Untargeted metabolomics confirmed the marked Stat5a-mediated reduction in metabolite abundance after IL-3 deprivation, in a time-dependent manner. The polyamine-hypusine axis revealed in our studies is a promising avenue to improve T-cell function. This axis has been shown to regulate T-cell function, and potentially modulate mitochondrial respiration and T-cell activation. Hence, our studies have identified polyamine supplementation and gene modulation as promising strategies to improve the metabolism of Adoptive T cells in a Stat5a-mediated manner. Further studies will evaluate the effectiveness of this avenue in improving T-cell function, improving their efficacy for ACT, and improving patient outcomes. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Gang Zhou, Huidong Shi. The polyamine-hypusine axis is a promising avenue to improve T-cell metabolism and persistence for adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 18.

Acetyl-CoA carboxylase obstructs CD8+ T cell lipid utilization in the tumor microenvironment

The solid tumor microenvironment (TME) imprints a compromised metabolic state in tumor-infiltrating Tcells (TILs), hallmarked by the inability to maintain effective energy synthesis for antitumor function and survival. Tcells in the TME must catabolize lipids via mitochondrial fatty acid oxidation (FAO) to supply energy in nutrient stress, and it is established that Tcells enriched in FAO are adept at cancer control. However, endogenous TILs and unmodified cellular therapy products fail to sustain bioenergetics in tumors. We reveal that the solid TME imposes perpetual acetyl-coenzyme A (CoA) carboxylase (ACC) activity, invoking lipid biogenesis and storage in TILs that opposes FAO. Using metabolic, lipidomic, and confocal imaging strategies, we find that restricting ACC rewires Tcell metabolism, enabling energy maintenance in TME stress. Limiting ACC activity potentiates a gene and phenotypic program indicative of Tcell longevity, engendering Tcells with increased survival and polyfunctionality, which sustains cancer control.

Oleic acid triggers metabolic rewiring of T cells poising them for T helper 9 differentiation

T cells are the most common immune cells in atherosclerotic plaques, and the function of Tcells can be altered by fatty acids. Here, we show that pre-exposure of CD4+ Tcells to oleic acid, an abundant fatty acid linked to cardiovascular events, upregulates core metabolic pathways and promotes differentiation into interleukin-9 (IL-9)-producing cells upon activation. RNA sequencing of non-activated Tcells reveals that oleic acid upregulates genes encoding key enzymes responsible for cholesterol and fatty acid biosynthesis. Transcription footprint analysis links these expression changes to the differentiation toward TH9 cells, a pro-atherogenic subset. Spectral flow cytometry shows that pre-exposure to oleic acid results in a skew toward IL-9+-producing Tcells upon activation. Importantly, pharmacological inhibition of either cholesterol or fatty acid biosynthesis abolishes this effect, suggesting a beneficial role for statins beyond cholesterol lowering. Taken together, oleic acid may affect inflammatory diseases like atherosclerosis by rewiring Tcell metabolism.

Unlocking the potential of T-cell metabolism reprogramming: Advancing single-cell approaches for precision immunotherapy in tumour immunity.

As single-cell RNA sequencing enables the detailed clustering of T-cell subpopulations and facilitates the analysis of T-cell metabolic states and metabolite dynamics, it has gained prominence as the preferred tool for understanding heterogeneous cellular metabolism. Furthermore, the synergistic or inhibitory effects of various metabolic pathways within T cells in the tumour microenvironment are coordinated, and increased activity of specific metabolic pathways generally corresponds to increased functional activity, leading to diverse T-cell behaviours related to the effects of tumour immune cells, which shows the potential of tumour-specific T cells to induce persistent immune responses. A holistic understanding of how metabolic heterogeneity governs the immune function of specific T-cell subsets is key to obtaining field-level insights into immunometabolism. Therefore, exploring the mechanisms underlying the interplay between T-cell metabolism and immune functions will pave the way for precise immunotherapy approaches in the future, which will empower us to explore new methods for combating tumours with enhanced efficacy.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy

The manipulation of Tcell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ Tcells exvivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ Tcells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated Tcells synergized invivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ Tcells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance Tcell metabolism and anti-tumor activity.

Abstract 1889 Multi-omic Studies Reveal the Polyamine-Hypusine Axis as a Promising Avenue to Improve T-cell Persistence and Metabolism for Adoptive Cell Therapy

Abstract 1889 Multi-omic Studies Reveal the Polyamine-Hypusine Axis as a Promising Avenue to Improve T-cell Persistence and Metabolism for Adoptive Cell Therapy

CD8+ T-cell metabolism is related to cerebrovascular reactivity in middle-aged adults.

Cerebrovascular reactivity (CVR) decreases with advancing age, contributing to increased risk of cognitive impairment; however, the mechanisms underlying the age-related decrease in CVR are incompletely understood. Age-related changes to T cells, such as impaired mitochondrial respiration, increased inflammation, likely contribute to peripheral and cerebrovascular dysfunction in animals. However, whether T-cell mitochondrial respiration is related to cerebrovascular function in humans is not known. Therefore, we hypothesized that peripheral T-cell mitochondrial respiration would be positively associated with CVR and that T-cell glycolytic metabolism would be negatively associated with CVR. Twenty middle-aged adults (58 ± 5 yr) were recruited for this study. T cells were separated from peripheral blood mononuclear cells. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a marker of glycolytic activity) were measured using extracellular flux analysis. CVR was quantified using the breath-hold index (BHI), which reflects the change in blood velocity in the middle-cerebral artery (MCAv) during a 30-s breath-hold. In contrast to our hypothesis, we found that basal OCR in CD8+ T cells (β = -0.59, R2 = 0.27, P = 0.019) was negatively associated with BHI. However, in accordance with our hypothesis, we found that basal ECAR (β = -2.20, R2 = 0.29, P = 0.015) and maximum ECAR (β = -50, R2 = 0.24, P = 0.029) were negatively associated with BHI in CD8+ T cells. There were no associations observed in CD4+ T cells. These associations appeared to be primarily mediated by an association with the pressor response to the breath-hold test. Overall, our findings suggest that CD8+ T-cell respiration and glycolytic activity may influence CVR in humans.NEW & NOTEWORTHY Peripheral T-cell metabolism is related to in vivo cerebrovascular reactivity in humans. Higher glycolytic metabolism in CD8+ T cells was associated with lower cerebrovascular reactivity to a breath-hold in middle-aged adults, which is possibly reflective of a more proinflammatory state in midlife.

Somatostatin-SSTR3-GSK3 modulates human T-cell responses by inhibiting OXPHOS.

Somatostatin (SST) is a peptide hormone primarily synthesized in the digestive and nervous systems. While its impact on the endocrine system is well-established, accumulating evidence suggests a crucial role for SST and its analogues in modulating immune responses. Despite this, the precise mechanism through which SST regulates T cells has remained largely unknown. To elucidate the impact of SST on human T cells, we conducted a series of experiments involving cell culture assays, molecular analyses, and metabolic profiling. Human T cells were treated with SST, and various parameters including proliferation, cytokine production, and metabolic activities were assessed. Additionally, we employed pharmacological inhibitors and genetic manipulations to dissect the signaling pathways mediating SST's effects on T cells. We showed that SST diminishes T-cell proliferation by influencing IL-2 production and T-cell mitochondrial respiration, while having no discernible impact on TCR-induced glycolysis. Our findings also identified that the regulatory influence of SST on T-cell responses and metabolism is contingent on its receptor, SSTR3. Moreover, we demonstrated that SST governs T-cell responses and metabolism by acting through the T-cell metabolic checkpoint GSK3. Our study provides novel insights into the immunoregulatory function of SST in human T cells, highlighting the complex interplay between hormonal signaling and immune regulation. Understanding the molecular mechanisms underlying SST's effects on T cells may offer therapeutic opportunities for manipulating immune responses in various pathological conditions.

Reprogramming T-cell metabolism to enhance adoptive cell therapies.

Adoptive cell therapy (ACT) is an immunotherapeutic approach that involves isolating T cells from a patient, culturing them ex vivo, then reinfusing the cells back into the patient. Although this strategy has shown remarkable efficacy in hematological malignancies, the solid-tumour microenvironment (TME) has presented serious challenges for therapy efficacy. Particularly, the TME has immunosuppressive signalling and presents a metabolically challenging environment that leads to T-cell suppression. T-cell metabolism is an expanding field of research with a focus on understanding its inherent link to T-cell function. Here, we review the current model of T-cell metabolism from naïve cells through effector and memory life stages, as well as updates to the model from recent literature. These models of metabolism have provided us with the tools and understanding to explore T-cell metabolic and mitochondrial insufficiency in the TME. We discuss manipulations that can be made to these mitochondrial and metabolic pathways to enhance the persistence of infused T cells, overcome the metabolically challenging TME and improve the efficacy of therapy in ACT models. Further understanding and investigation of the impact of metabolic pathways on T-cell performance could contribute to improving therapy efficacy for patients.

De novo NAD+ synthesis contributes to CD8+ T cell metabolic fitness and antitumor function

The dysfunction and clonal constriction of tumor-infiltrating CD8+ Tcells are accompanied by alterations in cellular metabolism; however, how the cell-intrinsic metabolic pathway specifies intratumoral CD8+ Tcell features remains elusive. Here, we show that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) contributes to the maintenance of intratumoral CD8+ Tcell metabolic and functional fitness. De novo NAD+ synthesis is involved in CD8+ Tcell metabolism and antitumor function. KP-derived NAD+ promotes PTEN deacetylation, thereby facilitating PTEN degradation and preventing PTEN-dependent metabolic defects. Importantly, impaired cell-autonomous NAD+ synthesis limits CD8+ Tcell responses in human colorectal cancer samples. Our results reveal that KP-derived NAD+ regulates the CD8+ Tcell metabolic and functional state by restricting PTEN activity and suggest that modulation of de novo NAD+ synthesis could restore CD8+ Tcell metabolic fitness and antitumor function.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.

The Impact of Treatment-Free Intervals on T-Cell Exhaustion and Clonotypic Diversity in Myeloma Patients Treated with BCMA Bispecific Antibodies

Background: BCMAxCD3 bispecific antibodies (BsAb) have changed the treatment landscape in relapsed refractory multiple myeloma (RRMM). However, despite high response rates, the majority of patients continue to relapse. Infectious complications, mainly viral, represent another challenge with BsAb, seen in up to 90% of patients with half of them requiring hospitalization. Both issues, loss of response as well as infectious complications, can be linked to impaired T-cell function potentially emerging during BsAb therapy. Most BsAbs have been developed as weekly treatments and one hypothesis is that dose dense treatment could overstimulate T cells finally leading to exhaustion and dysfunctionality. Thus, we compared T cell profiles and their functionality from patients with weekly dosing to patients who switched to a monthly schedule due to toxicity or patient request. Methods: We longitudinally biobanked peripheral blood samples of 19 patients of whom 7 switched from a weekly (q1w) to a monthly (q4w) BsAb treatment schedule. Peripheral blood mononuclear cells (PBMCs) were analysed using 16-color flow cytometry, including T-cell subset phenotyping and the exhaustion marker PD1, TIM3, SLAMF6 and KLRG1. Furthermore, CITEseq, scTCRseq, and Seahorse metabolic studies were performed on selected patients. Results: The enrolled patients were heavily pretreated with a median number of 6 prior therapy lines. All patients responded to BCMA BsAb with VGPR or better and were on treatment for >3 month. Only one patient from the q4w cohort relapsed during the study period. High-risk cytogenetics, lines of previous therapy, and penta-refractory disease status were equally distributed between patients on weekly and monthly schedule. Infectious complications occurred in 10/19 (q1w) (52.6%) and 1/7 patients (q4w) (14.2%). First, we addressed the question if a monthly treatment schedule confers less T-cell exhaustion by comparing PD-1 checkpoint expression and T-cell subsets between q1w and q4w administration. Indeed, flow cytometry showed a significant decrease in the CD4+PD1+ (36.9 vs. 21.1% p<0.05, unpaired t-test) and in the CD8+PD1+ (29.44 vs. 16.54% p<0.05) T cells. We also observed a decrease in the frequency of the effector memory subsets (CD45RA-, CD62L-, TEM) of CD4+ (66.19 vs. 49.63% p<0.05) and CD8+ (53.7 vs. 40.32% p=0.09), further supporting a concept in which a treatment free interval leads to recovery of the T-cell system. We also had access to paired samples of five patients who switched from q1w (1 st sample) to q4w dosing (2 nd sample). Here, flow cytometry showed a decrease of exhausted CD4+PD1+ T cells (33.68 vs. 21.76% p=0.046, paired t-test) and CD8+PD1+ T cells (29.2% vs. 15.1% p=0.117), confirming our cross-sectional analysis. Likewise, expression of SLAMF6, a regulator of T cell exhaustion, also decreased from weekly to monthly schedule (SLAMF6/CD4+:20.7 vs. 15.2% p=0.06; and CD8+:17.8 vs. 11.3% p=0.1), but differences were not statistically significant probably due to sample size. Our analysis on T-cell metabolism did not reveal significant differences between weekly and monthly dosing schedules. After strict quality control, three of the paired samples were further analysed using CITE-seq and scTCRseq. In line with our flow results, patients showed a significant decrease in CD8+ pre-exhausted T-cell subset (CD8+TCF-1+CD27+) (3.55 vs. 1.54% p=0.02) and CD8+ terminal differentiated effector memory T-cells (CD8+ TEMRA) (4.66 vs. 3.57% p=0.04) from weekly to monthly schedules, again suggesting recovery of the T-cells after a longer treatment-free interval. We also observed a trend towards decrease in the CD4+ effector memory subset (CD4+TEM, 6.96 vs. 4.89% p=0.07). Interestingly, the number of hyper-expanded T cell clonotypes increased from weekly to monthly dosing in paired samples. This was somewhat unexpected but expansion could be interpreted as reactivation of a few previously exhausted T cells. Conclusions: The optimal dosing schedule of bispecific antibodies has yet to be determined. Our results support a more extended dose schedule to allow T-cell-recovery between BsAb administrations. Whether such a strategy will result in higher antitumor efficacy or reduced susceptibility to infections, has to be evaluated in future studies.

Multifactorial Basis of T Cell Dysfunction in CLL: Disrupted Mitochondrial Metabolism Induces T Cell Senescence

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.