IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor.

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.

Chemical Constituents and Antioxidant, Anti-Inflammatory and Anti-Tumor Activities of Melilotus officinalis (Linn.) Pall.

Two new p-hydroxybenzoic acid glycosides, namely p-hydroxybenzoic acid-4-O-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 1) and 4-O-α-l-rhamnopyran-osyl-(1 → 6)-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 2), and seven known compounds, compound 3, 6, 7 (acid components), compound 8, 9 (flavonoids), compound 4 (a coumarin) and compound 5 (an alkaloid), were isolated from the 70% ethanol aqueous extract of the aerial parts of Melilotus officinalis (Linn.) Pall. The structures of all compounds were elucidated by use of extensive spectroscopic methods Infrared Spectroscopy (IR), High resolution electrospray ionization mass spectrometry (HR-ESI-MS), and ¹H and 13C-NMR). Sugar residues obtained after acid hydrolysis were identified by high-performance liquid chromatography (HPLC). The antioxidant activity of all the compounds was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The anti-inflammatory effects of the compounds were also evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. All compounds were shown to inhibit LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, in LPS-stimulated RAW 264.7 cells. The inhibitory effect of all the compounds on MCF-7 cells was determined by Cell Counting Kit-8 (CCK-8) method. The results showed that compounds 1, 2, 7, 8, 9 exhibited better antioxidant activity compared to the other compounds. compounds 1-9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, of which compound 7 was the most effective against inflammatory factors. compounds 1 and 2 have better antitumor activity compared to other compounds. Further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.

Functional assessment of the cell-autonomous role of NADase CD38 in regulating CD8+ T cell exhaustion.

Exhausted CD8+ T cells with limited effector functions and high expression of multiple co-inhibitory receptors are one of the main barriers hindering antitumor immunity. The NADase CD38 has received considerable attention as a biomarker of CD8+ T cell exhaustion, but it remains unclear whether the increased CD38 directly promotes T cell dysfunctionality. Here, we surprisingly found that although Cd38 deficiency partially reverses NAD+ degradation and T cell dysfunction in vitro, the terminal exhausted differentiation of adoptively transferred CD8+ T cells in tumor is not impacted by either deficiency or overexpression of CD38. Monitoring the dynamic NAD+ levels shows that NAD+ levels are comparable between tumor infiltrated WT and Cd38 -/- OT-1 cells. Therefore, our results suggest that decreased NAD+ are correlated with T cell dysfunction, but deficiency of CD38 is not enough for rescuing NAD+ in tumor infiltrated CD8+ T cells and fails to increase the efficacy of antitumor T cell therapy.

The intrinsic role and mechanism of tumor expressed-CD38 on lung adenocarcinoma progression.

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8+ T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca2+ and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.

Growth retardation of fetal rats exposed to nicotine in utero: possible involvement of CYP1A1, CYP2E1, and P-glycoprotein.

To elucidate the possible metabolic mechanism of intrauterine growth retardation induced by nicotine, this study determines the effects of prenatal nicotine exposure on fetal development and cytochrome P4501A1 (CYP1A1), CYP2E1, and P-glycoprotein (Pgp) expression in maternal liver and placenta. Pregnant rats were given 1.0 mg/kg nicotine subcutaneously twice a day from gestational day (GD) 8 to GD 15, 18, or 21. In nicotine-treated groups, fetal developmental parameters including body weight were significantly lower. The activities of CYP1A1 and CYP2E1 in maternal liver microsomes in nicotine-treated groups increased significantly with progressing gestation when compared with the corresponding control, but returned to the level similar to the control in late pregnancy. Nicotine-treated groups induced pathological changes and increased malondialdehyde (MDA) content in the placenta when compared with the control. The gene expressions of CYP1A1 and CYP2E1 in the placenta increased significantly in nicotine-treated groups on GD 15 and GD 18, but returned to the level similar to the corresponding control on GD 21. In nicotine group, there was a decrease of mdr1a expression on GD 15, GD 18, and GD 21, with the most significant decrease on GD 15. In contrast, no significant difference was found in mdr1b mRNA expression between the nicotine-treated animals and the corresponding control. In comparison with the corresponding control, the placental Pgp protein significantly decreased on GD 15 and GD 18. Our results showed that prenatal nicotine exposure resulted in inhibition of fetal growth significantly. The induction of CYP2E1 and CYP1A1 gene expression by nicotine in the maternal liver and placenta may be involved with the observed increase in oxidative stress and lipid peroxidation. The inhibition of the placental Pgp expression by nicotine may also contribute to an increased susceptibility of the fetus to environmental toxins.

Tumor-Repopulating Cells Induce PD-1 Expression in CD8+ T Cells by Transferring Kynurenine and AhR Activation.

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8+ T cells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8+ T cells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8+ T cells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive T cell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications.