Effect of hypoxia-induced mIL15 expression on expansion and memory progenitor stem-like TILs in vitro.

The adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) has proven clinically beneficial in patients with non-small cell lung cancer refractory to checkpoint blockade immunotherapy, which has prompted interest in TIL-adoptive cell transfer. The transgenic expression of IL15 can promote the expansion, survival, and function of T cells ex vivo and in vivo and enhance their anti-tumor activity. The effect of expressing mIL15 regulated by hypoxia in the tumor microenvironment on the expansion, survival, and stem-like properties of TILs has not been explored. Using TILs expanded from the tumor tissues of lung cancer patients, TILs with or without mIL15 expression (TIL-mIL15 or UN-TIL) were generated by lentiviral transduction. To reflect the advantages of mTIL15, the cells were divided into groups with IL2 (TIL-mIL15+IL2) or without IL2 (TIL-mIL15-IL2). Compared to UN-TIL cells, mIL15 expression had a similar capacity for promoting TIL proliferation and maintaining cell viability. Our experimental findings indicate that, compared to UN-TIL and TIL-mIL15+IL2 cells, the expression of mIL15 in TIL-mIL15-IL2 cells promoted the formation of stem-like TILs (CD8+CD39-CD69-) and led to significant decreases in the proportion and absolute number of terminally differentiated TILs (CD8+CD39+CD69+). RNA-Seq data revealed that in TIL-mIL15-IL2 cells, the expression of genes related to T cell differentiation and effector function, including PRDM1, ID2, EOMES, IFNG, GZMB, and TNF, were significantly decreased, whereas the expression of the memory stem-like T cell marker TCF7 was significantly increased. Furthermore, compared to UN-TIL and TIL-mIL15+IL2 cells, TIL-mIL15-IL2 cells showed significantly lower expression levels of inhibitory receptors LAG3, TIGIT, and TIM3, which was consistent with the RNA-Seq results. This study demonstrates the superior persistence of TIL-mIL15-IL2 cells, which may serve as a novel treatment strategy for lung cancer patients.

Sun Z ，Xu A ，Wu Z ，Lan X ，Gao G ，Guo B ，Yu Z ，Shao L ，Wu H ，Lv M ，Wang Y ，Zhao Y ，Wang B ... -  《Frontiers in Immunology》

Characterization of tumor-infiltrating lymphocytes and their spatial distribution in triple-negative breast cancer.

The tumor immune microenvironment, particularly tumor-infiltrating lymphocytes (TILs), plays a critical role in disease progression and treatment response in triple-negative breast cancers (TNBCs). This study was aimed to characterize the composition of TILs and investigate their clinicopathological and prognostic significance with a special focus on the spatial distribution of TILs in TNBCs. We analyzed TNBC samples through PanCancer Immune Profiling using NanoString nCounter assays to identify immune-related genes that are expressed differentially in relation to TIL levels and evaluated protein expression of selected markers through immunohistochemical staining on tissue microarrays. For a comprehensive assessment of the expression of cytotoxic T lymphocyte (CTL) and natural killer (NK) cell markers, a CTL-NK score was devised based on CD8+, CD56+, CD57+, GNLY+, and GZMB+ TIL levels. Gene expression analysis revealed significant upregulation of CTL and NK cell-associated genes including GNLY, KLRC2, and GZMB in TIL-high TNBCs. Immunohistochemical validation confirmed that TNBCs with higher TILs had a greater amount of CD56+, CD57+, GNLY+, and GZMB+ TILs not only in absolute number but also in proportion relative to CD4+ or CD8+ TILs. High TIL and its subset (CD4+, CD8+, CD56+, CD57+, GNLY+, and GZMB+ TIL) infiltration correlated with favorable clinicopathological features of tumor. In survival analysis, high CTL-NK score was found to be an independent prognostic factor for better disease-free survival (DFS) of the patients. Furthermore, uniformly high TIL infiltration was linked to better DFS, whereas cases with heterogeneous TIL infiltration showed no difference in survival compared to those with uniformly low TIL infiltration. Our study showed that CTL and NK cell-associated gene expression and protein levels differ significantly according to TIL levels and that CTL-NK score and distribution of TILs within tumors have a prognostic value. These findings emphasize the importance of CTLs and NK cells as well as the spatial uniformity of TIL infiltration in clinical outcome of TNBC patients, providing valuable insights for refining prognostic assessments and guiding immunotherapeutic strategies.

Han E ，Choi HY ，Kwon HJ ，Chung YR ，Shin HC ，Kim EK ，Suh KJ ，Kim SH ，Kim JH ，Park SY ... -  《-》

Differential predictive value of resident memory CD8(+)T cell subpopulations in patients with non-small-cell lung cancer treated by immunotherapy.

A high density of resident memory T cells (TRM) in tumors correlates with improved clinical outcomes in immunotherapy-treated patients. In most clinical studies, TRM are defined by the CD103 marker. However, it is clearly established that not all TRM express CD103, but can be defined by other markers (CD49a, CD69, etc). The frequency of these subpopulations of TRM expressing or not CD103 varies according to the location of the cancer. Little is known about their functionality and their predictive impact on response to immunotherapy. In preclinical models, only some subpopulations of TRM are associated with cancer vaccine efficacy. Multiparametric cytometry analyses were used to demonstrate the presence of TRM subpopulations in the lung in mice after vaccination and in fresh ex vivo human non-small cell lung cancer (NSCLC). An analysis of the T-cell repertoire of these TRM was conducted to search for their relationships. Multiplex immunofluorescence techniques were used to quantify intratumor infiltration of TRM subpopulations in two cohorts of patients with NSCLC. The impact on the clinical outcome of the TRM tumor infiltration was also investigated. We identified two main TRM subpopulations in tumor-infiltrating lymphocytes derived from patients with NSCLC: one co-expressing CD103 and CD49a (double positive (DP)), and the other expressing only CD49a (simple positive (SP)); both exhibiting additional TRM surface markers like CD69. Despite higher expression of inhibitory receptors, DP TRM exhibited greater functionality compared with SP TRM. Analysis of T-cell receptor (TCR) repertoire and expression of the stemness marker TCF1 revealed shared TCRs between populations, with the SP subset appearing more progenitor-like phenotype. In the training cohort, PD-L1 (Programmed Death-Ligand 1) and TCF1+CD8+T cells predict response to anti-PD-1. In patient with NSCLC validation cohorts, only DP TRM predicted PD-1 blockade response. Multivariate analysis, including various biomarkers associated with responses to anti-PD-(L)1, such as total CD8, TCF1+CD8+T cells, and PD-L1, showed that only intratumoral infiltration by DP TRM remained significant. This study highlights the non-equivalence of TRM subpopulations. The population of TRM co-expressing CD103 and CD49a appears to be the most functional and has the most significant capacity for predicting response to immunotherapy in multivariate analysis in patients with NSCLC.

Paolini L ，Tran T ，Corgnac S ，Villemin JP ，Wislez M ，Arrondeau J ，Johannes L ，Ulmer J ，Vieillard LV ，Pineau J ，Gey A ，Quiniou V ，Barennes P ，Pham HP ，Gruel N ，Hasan M ，Libri V ，Mella S ，De Percin S ，Boudou-Rouquette P ，Caidi A ，Cremer I ，Blons H ，Leroy K ，Laurent-Puig P ，De Saint Basile H ，Gibault L ，Ravel P ，Mami-Chouaib F ，Goldwasser F ，Fabre E ，Damotte D ，Tartour E ... -  《Journal for ImmunoTherapy of Cancer》

CD4(-)/CD8(-) double-negative tumor-infiltrating lymphocytes expanded from solid tumor tissue suppress the proliferation of tumor cells in an MHC-independent way.

Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4-/CD8- double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4-CD8- TILs (double-negative TIL, DN-TILs) derived from solid tumor. DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3+ TILs, which expanded more than 800-folds in 2 weeks, consisting of a mixture of alpha-beta (αβ) and gamma-delta (γδ) T-cell receptor (TCR)-expressing cells (with the majority being αβ-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL.

Lu J ，Huang C ，He R ，Xie R ，Li Y ，Guo X ，Zhang Q ，Xu Q ... -  《-》

Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.

A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear. The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy. To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed. It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell. Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.

Haoyue W ，Kexiang S ，Shan TW ，Jiamin G ，Luyun Y ，Junkai W ，Wanli D ... -  《-》