T cell deficiencies are commonly treated by intravenous hematopoietic stem/progenitor cell (HSPC) transplantation. However, this approach often leads to delayed and incomplete T cell reconstitution, partly due to impaired thymic function. In contrast, intrathymic delivery of HSPCs enables rapid, thymus-autonomous T cell development. Using a ZAP-70-deficient mouse model of severe immunodeficiency, we show that intrathymic transplantation of wild-type HSPCs results in robust thymic engraftment, with mature T cells and FOXP3+ regulatory T cells (Treg) detectable within four weeks. This reconstitution parallels the regeneration of a functional thymic medulla and the emergence of mature medullary thymic epithelial cells (mTECs). Importantly, we identify an early wave of donor-derived RORγT+ ILC3s that correlates with medullary regeneration. While dispensable for steady-state thymopoiesis, ILC3s accelerate medulla formation and support optimal T cell differentiation in this immunodeficient setting. These findings uncover a critical role for ILC3–TEC crosstalk in thymic repair and highlight intrathymic HSPC transplantation as a strategy to enhance immune reconstitution in immunodeficient hosts. ### Competing Interest Statement The authors have declared no competing interest.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized treatment for hematologic malignancies, yet many patients fail to achieve durable remission. Insufficient in vivo CAR T-cell expansion consistently correlates with treatment failure across clinical trials. Robust anti-tumor T-cell responses require extensive bioenergetic support, with metabolic fitness emerging as a key determinant of CAR T-cell potency. However, the metabolic pathways that support CAR T cell persistence and function in vivo after infusion into patients remain poorly defined. To address this gap, we developed a comprehensive immuno-metabolic pipeline combining single-cell metabolic assays and plasma metabolomics to analyze CAR T-cells from patients enrolled in CD22 and CD19/CD22 CAR clinical trials for relapsed/refractory B-ALL (NCT02315612, NCT03448393, NCT05098613). Initial high-throughput profiling of healthy donor–derived CD19, CD22, and CD33 CAR T-cells, incorporating either CD28 or 4-1BB costimulatory domains, revealed construct-specific metabolic and functional phenotypes. Across constructs and donors, protein translation—measured by puromycin incorporation—emerged as a robust marker of metabolic activity and cytokine polyfunctionality. Based on these data, we developed protein translation-based single-cell assays to map metabolic dependencies across major metabolic pathways—including glycolysis, oxidative phosphorylation (OXPHOS), glutamine metabolism, and fatty acid oxidation—in patient-derived CAR T-cells pre- and post-infusion. Pre-infusion CD22 and CD19/CD22 CAR T-cells exhibited a highly glycolytic phenotype with minimal reliance on oxidative phosphorylation (n=20 samples). In contrast, post-infusion peripheral blood CAR T cells underwent marked metabolic reprogramming, characterized by a reduced glycolytic dependence and increased reliance on oxidative phosphorylation (OXPHOS) and glutamine uptake (n=29 samples, day 7–14 post infusion). To further delineate metabolic heterogeneity among CAR T-cell subsets, we combined translation-based assays with spectral cytometry in the CD22 CAR cohort (n=12 patients). Notably, in pre-infusion samples, enrichment of CAR T-cell clusters with high OXPHOS dependence correlated with higher expansion, a memory-like phenotype, and complete remission. In post-infusion samples, globally elevated protein translation was associated with higher expansion, and OXPHOS-dependency characterized a CAR T-cell cluster exhibiting a stem-memory phenotype (CCR7High CD62LHigh CD127High TCF1High). Collectively, these data identify OXPHOS-driven translation and amino acid metabolism as key metabolic programs sustaining in vivo CAR T-cell function. Building on these findings, we explored the amino acid environment of post-infusion CAR T-cells. Plasma metabolomics from CD22CAR trial patients (n=20) revealed significant depletion of glutamine and arginine in individuals experiencing cytokine release syndrome (CRS). While such an amino acid-scarce environment may limit CAR T-cell function, this effect could potentially be overcome by augmented expression of metabolite solute carrier (SLC) transporters. Supporting this hypothesis, reanalysis of published scRNA-seq data from post-infusion CD19 CAR T-cells (Haradhvala et al., Nat Med, 2022) showed that CD8+CAR+ T-cells from complete responders expressed higher levels of SLCs, particularly those mediating amino acid uptake. Functional perturbation studies further confirmed the role of amino-acid SLCs: knockdown of either the glutamine (SLC1A5) or arginine (SLC7A1) transporter impaired OXPHOS, reduced stem-memory frequency, and diminished cytotoxicity upon repeated antigen challenge. Guided by these results, we engineered “MetaboArm” CAR T cells co-expressing SLC transporters to enhance amino acid uptake and improve metabolic fitness. Constructs incorporating glutamine or arginine transporters—SLC1A5, SLC7A1, or SLC38A9—significantly increased OXPHOS activity and enhanced anti-leukemic efficacy both in vitro and in vivo. Together, this study establishes the first clinical-trial–based metabolic atlas of post-infusion CAR T cells, identifying amino acid–driven OXPHOS via SLC transporters as a central determinant of therapeutic efficacy and guiding rational metabolic engineering of next-generation CARs.

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation in CpG context (5mCpG) and cytosine hydroxymethylation (5hmCpG) are DNA modifications that identify stable cell phenotypes, but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a method to profile Th cell identity using Cas9-targeted single-molecule nanopore sequencing. Targeting as few as 10 selected genomic loci, we were able to distinguish major in vitro polarized murine T cell subtypes, as well as intermediate phenotypes, by their native DNA 5mCpG patterns. Moreover, by using off-target sequences, we were able to infer transcription factor activities relevant to each cell subtype. Detection of 5mCpG and 5hmCpG was validated on intestinal Th17 cells escaping transforming growth factor β control, using single-molecule adaptive sampling. A total of 21 differentially methylated regions mapping to the 10-gene panel were identified in pathogenic Th17 cells relative to their nonpathogenic counterpart. Hence, our data highlight the potential to exploit native DNA methylation profiling to study physiological and pathological transition states of Th cells.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is one of the most common forms of blood cancer worldwide. Outcomes for patients with relapsed or refractory (r/r) DLBCL have remained dismal. Chimeric antigen receptor (CAR) T-cell therapy targeting the B-cell surface marker CD19 has recently emerged as a novel, effective approach capable of producing durable responses in r/r DLBCL patients. Indeed, nearly all r/r DLBCL patients in the United States are considered candidates for FDA-approved CAR-T therapy. However, CAR-T therapy has a number of major shortcomings. First, it has limited efficacy. The median progression-free survival (PFS) of r/r DLBCL patients treated with CAR-T therapy is roughly six months. Second, it has major toxicities. CAR-T therapy is associated with risk of life-threatening side effects such as cytokine release syndrome (CRS) and CAR-T cell-related encephalopathy syndrome (CRES). Third, it is highly expensive. Strategies for identifying, a priori, those patients likely to respond to this therapy could provide an important tool for clinical decision-making and improving outcomes in patients with r/r DLBCL. Methods & Results: We enrolled a real-world cohort of 161 r/r DLBCL patients treated with CD19-directed CAR-T therapy and applied whole exome and transcriptome sequencing on their pre-treatment biopsies to identify predictors of outcome. A number of clinical variables were associated with worse PFS or overall survival (OS) including a higher International Prognostic Index (IPI) score at diagnosis, involvement of multiple extranodal sites, poor performance status at the time of apheresis, high-grade CRES, and the need for ICU admission. Conversely, lower IPI score, activated B cell type (ABC) subtype of DLBCL, need for tocilizumab administration, and achievement of complete remission trended with more favorable survival outcomes. As expected, the most frequently mutated genes were involved in histone modification (e.g. KMT2D, CREBBP, EP300), B-cell signaling (e.g. MYD88, PIM1, CD79B) and DNA damage-response/repair (e.g. TP53, DDX3X, BCL6, ATM). Gene mutations associated with the least favorable prognosis were SOCS1, BCL7A, and MYC, while HIST1H1E, CD79B, and MPEG1 mutations were associated with a favorable prognosis. We further developed a supervised approach for analysis and validation of gene expression profiles and their association with outcome. We first divided the patients into independent training and test sets. In the training set, we applied the Cox proportional hazards model to identify individual genes that were associated with PFS in CAR-T therapy treated patients. We identified two gene expression signatures that were associated with superior outcomes and poor outcomes, respectively. These two signatures were combined to generate a single prognostic score that comprised the genomic model that was validated in the independent test set. The first “low-risk” signature appeared to be derived predominantly from tumor cells with a number of B-cell related genes including CD19, PAX5, CD79A and CXCR5 as well as genes associated with the NF-kB pathway. The second signature included macrophage-related genes such as CD163, FCGR1A (CD64) and MSR1, as well as genes associated with MYC targets, ferroptosis and iron transport. We found that the model distinguished patients with significantly different outcomes. Patients in the lowest quartile of prognostic scores had a median PFS of 2.8 months, while those in the highest quartile of scores had a significantly longer median PFS of 18 months (Wilcoxon rank-sum test p< 0.001). 73% of patients identified as having favorable risk achieved complete remission. Conclusion: The management of CAR-T treated patients remains challenging owing to limitations in efficacy, cost and side effects. Our study indicates that genomic features of the tumor prior to treatment are significantly associated with response to CAR-T therapy in DLBCL. We anticipate that the translation of these findings into clinical practice will enable the development of precision medicine approaches using CAR-T therapy in in this challenging disease.

Tumor‐infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T cells have demonstrated remarkable success in the treatment of relapsed/refractory melanoma and hematological malignancies, respectively. These treatments have marked a pivotal shift in cancer management. However, as “living drugs,” their effectiveness is dependent on their ability to proliferate and persist in patients. Recent studies indicate that the mechanisms regulating these crucial functions, as well as the T cell's differentiation state, are conditioned by metabolic shifts and the distinct utilization of metabolic pathways. These metabolic shifts, conditioned by nutrient availability as well as cell surface expression of metabolite transporters, are coupled to signaling pathways and the epigenetic landscape of the cell, modulating transcriptional, translational, and post‐translational profiles. In this review, we discuss the processes underlying the metabolic remodeling of activated T cells, the impact of a tumor metabolic environment on T cell function, and potential metabolic‐based strategies to enhance T cell immunotherapy.

Approximately 25% of cancers are preceded by chronic inflammation that occurs at the site of tumor development. However, whether this multifactorial oncogenic process, which commonly occurs in the intestines, can be initiated by a specific immune cell population is unclear. Here, we show that an intestinal T cell subset, derived from interleukin-17 (IL-17)-producing helper T (TH17) cells, induces the spontaneous transformation of the intestinal epithelium. This subset produces inflammatory cytokines, and its tumorigenic potential is not dependent on IL-17 production but on the transcription factors KLF6 and T-BET and interferon-γ. The development of this cell type is inhibited by transforming growth factor-β1 (TGFβ1) produced by intestinal epithelial cells. TGFβ signaling acts on the pretumorigenic TH17 cell subset, preventing its progression to the tumorigenic stage by inhibiting KLF6-dependent T-BET expression. This study therefore identifies an intestinal T cell subset initiating cancer.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.

Quantification of mitochondrial DNA (mtDNA) following transfer of MSC mitochondria to GSCs (A) Detection of MSC mtDNA in GSCs. MSC mtDNA concentrations are expressed relative to GSC mtDNA. Mean with SEM, t test with Welch correction, *p < 0.05. (B) Total mtDNA concentrations in GSCs at different time points after MSC mitochondria transfer, expressed relative to GSC genomic DNA. Mean + SEM and multiple t tests, *p < 0.05.

TMZ dose-response assay Caspase-GloⓇ 3/7 assay Cytotox assay Extracellular Flux Assays Cell Proliferation Western Blot Flow Cytometry Metabolite usage Mitoplates Mass Spectrometry Quantification 13C stable isotope tracing experiments Preparation and quantification of MSC mtDNA in GSCs Imaging

MSC mitochondria increase GSC survival in response to TMZ. A and F, Time lines for the response of GSCs to TMZ, at day 5 (B–E) and at 48 hours (G–H). B, Survival of GSCs in response to TMZ (dose–response 6–400 μmol/L). Framed TMZ concentration of 50 μmol/L used in all subsequent experiments. C, Effect of TMZ on the transfer of mitochondria from MSCs to GSCs, as analyzed by flow cytometry. D and E, Effects of MSC mitochondria on GSC survival in response to TMZ. D, GSC caspase 3/7 activity (n = 16, three independent experiments). E, Cytotox assay (Incucyte). Representative images and quantification of Cytotox labeled GSCs (n = 15, 3 independent experiments). G and H, Effects of MSC mitochondria on GSC survival in response to TMZ (48 hours). H, GSC cell death. FACS analysis of Zombie violet-stained GSCs. Representative data and quantification from seven independent experiments with mean and SEM values. H, GSC cell number (n = 84 from seven independent experiments). Tukey boxplots with Kruskal–Wallis test; **, P < 0.01; ***, P < 0.001. C–E and G, Statistical analysis by one-way ANOVA; *, P < 0.05; ***, P < 0.001.

Metabolites produced by GSCs following MSC mitochondria acquisition and TMZ treatment

Metabolomics analysis of resected GBM from patients, pre- and post-TMZ treatment shows increased nucleotides concentrations. A, Metabolomics analysis were performed on GBM resected tumors from 8 patients, before treatment and after radiotherapy and several cycles (3 to 13) of TMZ treatment. B, MRI profiles and H&E-stained tissue sections of the analyzed GBM tumors, before and after TMZ treatment. Tumor areas at first and second resections are framed. Scale bar, 100 μm. C, Mass spectrometry metabolomics analysis of the resected GBM. The C12/C13 metabolite ratios normalized to tissue protein concentrations are indicated. Out of the 8 patients, 6 followed a similar trend (solid lines), while 2 followed an opposite trend (dotted lines). Statistical analysis by ratio paired Student t test (AMP, GMP, UMP) and Wilcoxon matched-pairs signed-rank test (CMP) on metabolomics data from the 6 patients.

MSC mitochondria modify metabolite fluxes in GSCs. [U-13C]-glucose and [U-13C]-glutamine isotope profiling of GSCs with and/or without MSC mitochondria and treated or not with TMZ (48 hours; n = 3). A, Glycolysis intermediates. B, Isotopologues of TCA cycle metabolites from 13C-glucose (24 and 48 hours). C, Isotopologues of TCA cycle metabolites from 13C-glutamine (48 hours). M3/M4 and M5/M3 ratios of isotopologues in GSCs with/out MSC mitochondria. Mean + SEM. Two-tailed unpaired t tests; ***, P < 0.001. Colors refer to the number of carbon originating from [U-13C]-glucose (A, B) and [U-13C]-glutamine (C).

Chimeric antigen receptor (CAR) T-cells have shown remarkable success in the treatment of hematological malignancies, but many patients still relapse. One common adverse on-target effect of CAR T-cells is cytokine release syndrome. Importantly though, the impact of fever, a hallmark of this syndrome, on CAR T-cell function is not known. We find that exposure of CAR T-cells to hyperthermia (40°C) significantly decreased their subsequent cytotoxicity against NALM6 leukemic cells, both ex-vivo and in-vivo. This was associated with reduced secretion of IL-2, IFNg, and IL-8 by CAR T-cells and high dimensional analyses revealed the induction of a terminally differentiated T cell phenotype. Gene profiling assays highlighted a negative enrichment of mTORC1, glycolysis, and oxidative phosphorylation gene sets in CAR T-cells subjected to hyperthermia and metabolome analyses unveiled significant reductions in urea cycle metabolites. Notably, pharmacological supplementation of arginine markedly enhanced the ex-vivo and in-vivo cytotoxicity of hyperthermia-exposed CAR T-cells. Moreover, in the absence of hyperthermia, short-term arginine supplementation enhanced the metabolic fitness of CAR T-cells, augmenting their long-term in-vivo persistence and cytotoxicity. We identify arginine metabolism as a critical pathway in CAR T-cells rendered dysfunctional by exposure to hyperthermia and pharmacological arginine support will inform future iterations of CAR T-cell interventions.

Cocultures of red MitoTracker-labeled MSCs and green CellTracker-labeled GSCs

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/CAT1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a post-translational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors--by inhibition of deoxyhypusine synthase--abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This impacted pathway is critical for eIF5A-regulated erythropoiesis as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins were especially sensitive to the loss of hypusine and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in del(5q) myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPC, synchronizing mitochondrial metabolism with erythroid differentiation.

MSC mitochondria modify the metabolic response of GSCs to TMZ. MSC mitochondria were transferred by Mitoception to GSCs which were subsequently treated with TMZ (50 μmol/L) for 48 hours. A, Time line. Effects of TMZ in the presence/absence of MSC mitochondria on GSC OCRs (B and C) and ECARs (D and E). All values were normalized to GSC cell numbers. B, Representative plot of GSC OCR in basal conditions, treated with TMZ, MSC mitochondria or both, and after sequential addition of oligomycin, FCCP and rotenone/antimycin. Mean values and SEM (n = 4). C, Tukey boxplots showing basal respiration and maximal respiration (n = 25 from four independent experiments). One-way ANOVA; **, P < 0.01; ***, P < 0.001. D, Representative plot of GSC ECAR in basal conditions, treated with TMZ, MSC mitochondria or both, and after sequential addition of glucose, oligomycin, oxamate, and 2-deoxyglucose. Mean values and SEM (n = 5). E, Tukey boxplots showing GSC basal glycolysis (n = 25 from four independent experiments). One-way ANOVA; **, P < 0.01; ***, P < 0.001. F, OCR versus ECAR values of GSCs treated with TMZ, with MSC mitochondria or with both. Mean and SEM values. G–J, GSC mitochondrial mass and ROS production. GSCs labeled with MitoTracker and MitoSox were analyzed by FACS, following the acquisition of MSC mitochondria and 48 hours TMZ treatment. G, GSC total mitochondrial mass. Representative experiment and relative mitochondria mean fluorescence intensity values represented as mean ± SEM (n = 7). H, Expression of COX IV protein. Representative Western blots for COX IV and β−actin expression (MW markers in kDa). Quantifications (n = 3) represented as mean ± SEM. I, GSC ROS production as measured with Mitosox. Representative data and quantification from independent experiments (n = 9) with mean and SEM values. J, Ratios of GSC ROS production over mitochondrial mass (n = 7). G–J, One-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

EDITORIAL article Front. Immunol., 26 June 2023Sec. Multiple Sclerosis and Neuroimmunology Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1236217