Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

Activation and subsequent differentiation of T cells following antigenic stimulation are triggered by highly coordinated signaling events that lead to instilling cells with a discrete metabolic and transcriptional feature. Compelling studies indicate that intracellular nicotinamide adenine dinucleotide (NAD⁺) levels have profound influence on diverse signaling and metabolic pathways of T cells, and hence dictate their functional fate. CD38, a major mammalian NAD⁺ glycohydrolase (NADase), expresses on T cells following activation and appears to be an essential modulator of intracellular NAD⁺ levels. The enzymatic activity of CD38 in the process of generating the second messenger cADPR utilizes intracellular NAD^+, and thus limits its availability to different NAD⁺ consuming enzymes (PARP, ART, and sirtuins) inside the cells. The present review discusses how the CD38-NAD⁺ axis affects T cell activation and differentiation through interfering with their signaling and metabolic processes. We also describe the pivotal role of the CD38-NAD⁺ axis in influencing the chromatin remodeling and rewiring T cell response. Overall, this review emphasizes the crucial contribution of the CD38⁻NAD⁺ axis in altering T cell response in various pathophysiological conditions.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells’ fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER–mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory–like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. Significance: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Effector CD8+ T cells rely primarily on glucose metabolism to meet their biosynthetic and functional needs. However, nutritional limitations in the tumor microenvironment can cause T-cell hyporesponsiveness. Therefore, T cells must acquire metabolic traits enabling sustained effector function at the tumor site to elicit a robust antitumor immune response. Here, we report that IL12-stimulated CD8+ T cells have elevated intracellular acetyl CoA levels and can maintain IFNγ levels in nutrient-deprived, tumor-conditioned media (TCM). Pharmacological and metabolic analyses demonstrated an active glucose–citrate–acetyl CoA circuit in IL12-stimulated CD8+ T cells supporting an intracellular pool of acetyl CoA in an ATP-citrate lyase (ACLY)–dependent manner. Intracellular acetyl CoA levels enhanced histone acetylation, lipid synthesis, and IFNγ production, improving the metabolic and functional fitness of CD8+ T cells in tumors. Pharmacological inhibition or genetic knockdown of ACLY severely impaired IFNγ production and viability of CD8+ T cells in nutrient-restricted conditions. Furthermore, CD8+ T cells cultured in high pyruvate–containing media in vitro acquired critical metabolic features of IL12-stimulated CD8+ T cells and displayed improved antitumor potential upon adoptive transfer in murine lymphoma and melanoma models. Overall, this study delineates the metabolic configuration of CD8+ T cells required for stable effector function in tumors and presents an affordable approach to promote the efficacy of CD8+ T cells for adoptive T-cell therapy. Significance: IL12-mediated metabolic reprogramming increases intracellular acetyl CoA to promote the effector function of CD8+ T cells in nutrient-depleted tumor microenvironments, revealing strategies to potentiate the antitumor efficacy of T cells.

PD1 blockade therapy, harnessing the cytotoxic potential of CD8 ⁺ T cells, has yielded clinical success in treating malignancies. However, its efficacy is often limited due to the progressive differentiation of intratumoral CD8 ⁺ T cells into a hypofunctional state known as terminal exhaustion. Despite identifying CD8 ⁺ T cell subsets associated with immunotherapy resistance, the molecular pathway triggering the resistance remains elusive. Given the clear association of CD38 with CD8 ⁺ T cell subsets resistant to anti-PD1 therapy, we investigated its role in inducing resistance. Phenotypic and functional characterization, along with single-cell RNA sequencing analysis of both in vitro chronically stimulated and intratumoral CD8 ⁺ T cells, revealed that CD38-expressing CD8 ⁺ T cells are terminally exhausted. Exploring the molecular mechanism, we found that CD38 expression was crucial in promoting terminal differentiation of CD8 ⁺ T cells by suppressing TCF1 expression, thereby rendering them unresponsive to anti-PD1 therapy. Genetic ablation of CD38 in tumor-reactive CD8 ⁺ T cells restored TCF1 levels and improved the responsiveness to anti-PD1 therapy in mice. Mechanistically, CD38 expression on exhausted CD8 ⁺ T cells elevated intracellular Ca ²⁺ levels through RyR2 calcium channel activation. This, in turn, promoted chronic AKT activation, leading to TCF1 loss. Knockdown of RyR2 or inhibition of AKT in CD8 ⁺ T cells maintained TCF1 levels, induced a sustained anti-tumor response, and enhanced responsiveness to anti-PD1 therapy. Thus, targeting CD38 represents a potential strategy to improve the efficacy of anti-PD1 treatment in cancer.

In the tumor microenvironment (TME), regulatory T cells (T _regs ) adapt their metabolism to thrive in low-glucose, high-lactate conditions, but the mechanisms remain unclear. Our study identifies CD38 as a key regulator of this adaptation by depleting nicotinamide adenine dinucleotide (oxidized form) (NAD ⁺ ), redirecting lactate-derived pyruvate toward phosphoenolpyruvate and bypassing the tricarboxylic acid (TCA) cycle. This prevents accumulation of α-ketoglutarate, which destabilizes T _regs by inducing hypermethylation at the Foxp3 locus. Restoring NAD ⁺ with nicotinamide mononucleotide reverses this adaptation, pushing T _regs back to the TCA cycle and reducing their suppressive function. In YUMM1.7 melanoma-bearing mice, small-molecule CD38 inhibition selectively destabilizes intratumoral T _regs , sparking robust antitumor immunity. These findings reveal that targeting the CD38-NAD ⁺ axis disrupts T _regs metabolic adaptation and offers a strategy to enhance antitumor responses.