Role of estrogen signaling in fibroblastic reticular cells for innate and adaptive immune responses in antigen-induced arthritis.

Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three-dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19-expressing cells (Ccl19-CreERαfl/fl) were generated and tested. Ccl19-CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28-day-long antigen-induced arthritis model to enable analyses of differentiated T- and B-cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19-CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question.

CCL17 and CCL19 are markers of disease progression in alveolar echinococcosis.

OBJECTIVES: Alveolar echinococcosis (AE) represents one of the deadliest helminthic infections, characterized by an insidious onset and high lethality. METHODS: This study utilized the Gene Expression Omnibus (GEO) database, applied Weighted Correlation Network Analysis (WGCNA) and Differential Expression Analysis (DEA), and employed the Matthews Correlation Coefficient (MCC) to identify CCL17 and CCL19 as key genes in AE. Immunohistochemistry and immunofluorescence co-localization techniques were used to examine the expression of CCL17 and CCL19 in liver tissue lesions of AE patients. Additionally, a mouse model of multilocular echinococcus larvae infection was developed to study the temporal expression patterns of these genes, along with liver fibrosis and inflammatory responses. RESULTS: The in vitro model simulating echinococcal larva infection mirrored the hepatic microenvironment post-infection with multilocular echinococcal tapeworms. Quantitative RT-PCR analysis showed that liver fibrosis occurred in AE patients, with proximal activation and increased expression of CCL17 and CCL19 over time post-infection. Notably, expression peaked during the late stages of infection. Similarly, F4/80, a macrophage marker, exhibited corresponding trends in expression. Upon stimulation of normal hepatocytes by vesicular larvae in cellular experiments, there was a significant increase in CCL17 and CCL19 expression at 12 h post-infection, mirroring the upregulation observed with F4/80. CONCLUSION: CCL17 and CCL19 facilitate macrophage aggregation via the chemokine pathway and their increased expression correlates with the progression of infection, suggesting their potential as biomarkers for AE progression.

Sleep promotes T-cell migration towards CCL19 via growth hormone and prolactin signaling in humans.

Sleep strongly supports the formation of adaptive immunity, e.g., after vaccination. However, the underlying mechanisms remain largely obscure. Here we show in healthy humans that sleep compared to nocturnal wakefulness specifically promotes the migration of various T-cell subsets towards the chemokine CCL19, which is essential for lymph-node homing and, thus, for the initiation and maintenance of adaptive immune responses. Migration towards the inflammatory chemokine CCL5 remained unaffected. Incubating the cells with plasma from sleeping participants likewise increased CCL19-directed migration, an effect that was dependent on growth hormone and prolactin signaling. These findings show that sleep selectively promotes the lymph node homing potential of T cells by increasing hormonal release, and thus reveal a causal mechanism underlying the supporting effect of sleep on adaptive immunity in humans.

Genetically light-enhanced immunotherapy mediated by a fluorinated reduction-sensitive delivery system.

The lack of safe and efficient therapeutic agent delivery platforms restricts combined therapy's effect, and combined cancer therapy's multi-component delivery effect needs improvement. The novel gene delivery system SS-HPT-F/pMIP-3ß-KR was proposed to construct fluorine-containing degradable cationic polymers SS-HPT-F by a mild and simple amino-epoxy ring-opening reaction. By modifying the fluorinated alkyl chain, the delivery efficiency of the plasmid was greatly improved, and the cytoplasmic transport of biomolecules was completed. At the same time, a combination plasmid (MIP-3ß-KillerRed) was innovatively designed for the independent expression of immune and photodynamic proteins. Which was efficiently transported to the tumor site by SS-HPT-F. The MIP-3ß is expressed as an immune chemokine realize the immune mobilization behavior. The photosensitive protein KillerRed expressed in the tumor killed cancer cells under irradiation and released the exocrine immune factor MIP-3ß. The immunogenic cell death (ICD) produced by photodynamic therapy (PDT) also induced the immune response of the organism. The synergistic effect of PDT and MIP-3ß mobilized the immune properties of the organism, providing light-enhanced immune combination therapy against malignant tumors. Therefore, in subcutaneous tumor-bearing and metastatic animal models, the carrier tumor growth and mobilize organism produce an immune response without systemic toxicity. This work reports the first efficient gene delivery system that achieves light-enhanced immunotherapy.

Downregulation of dermatopontin in cholangiocarcinoma cells suppresses CCL19 secretion of macrophages and immune infiltration.

OBJECTIVE: The tumor microenvironment (TME) in cholangiocarcinoma (CHOL) is typically characterized by a low level of immune infiltration, which accounts for the dismal prognosis of this patient population. This study sought to investigate the mechanisms underlying the reduced infiltration of immune cells into the CHOL TME. METHODS: We constructed a Least Absolute Shrinkage and Selection Operator (LASSO) regression model to identify prognosis-related differentially expressed genes (DEGs). The 'Corrplot' package was employed to analyze the correlation between dermatopontin (DPT) and immune infiltration in CHOL. The Tumor and Immune System Interaction Database (TISIDB) was used to evaluate the association between DPT and immunology. Single-cell analysis was conducted to localize CCL19 secretions. Western blot and qPCR were utilized to detect DPT expression, while immunofluorescence was performed to investigate the cellular localization of DPT. Additionally, ELISA analysis was employed to assess the alteration in CCL19 secretion in cancer-associated fibroblasts (CAFs) and macrophages. RESULTS: Our findings revealed that CHOL patients with low DPT expression had a poorer prognosis. Enrichment analysis demonstrated a positive correlation between DPT levels and the infiltration of immunomodulators and immune cells. Moreover, high DPT levels were associated with enhanced anti-PD-1/PD-L1 immunotherapeutic responses. Furthermore, DPT expression impacted the landscape of gene mutations, showing a negative association with tumor grade, stage, and lymph node metastasis. Based on the results of protein peptides analysis and cell experiments, it was inferred that the downregulation of DPT in CHOL cells effectively suppressed the secretion of CCL19 in macrophages. CONCLUSIONS: DPT is a novel prognosis-related biomarker for CHOL patients, and this study provides preliminary insights into the mechanism by which DPT promotes the infiltration of immune cells into the CHOL TME.

CCL19-Positive Lymph Node Stromal Cells Govern the Onset of Inflammatory Arthritis via Tropomyosin Receptor Kinase.

OBJECTIVE: The study objective was to assess the role of CCL19+ lymph node stromal cells of the joint-draining popliteal lymph node (pLN) for the development of arthritis. METHODS: CCL19+ lymph node stromal cells were spatiotemporally depleted for five days in the pLN before the onset of collagen-induced arthritis (CIA) using Ccl19-Cre × iDTR mice. In addition, therapeutic treatment with recombinant CCL19-immunoglobulin G (IgG), locally injected in the footpad, was used to confirm the results. RNA sequencing of lymph node stromal cells combined with T cell coculture assays using tropomyosin receptor kinase (Trk) family inhibitors together with in vivo local pLN small interfering RNA (siRNA) treatments were used to elucidate the pathway by which CCL19+ lymph node stromal cells initiate the onset of arthritis. RESULTS: Spatiotemporal depletion of CCL19+ lymph node stromal cells prevented disease onset in CIA mice. These inhibitory effects could be mimicked by local CCL19-IgG treatment. The messenger RNA sequencing analyses showed that CCL19+ lymph node stromal cells down-regulated the expression of the tropomyosin receptor kinase A (TrkA) just before disease onset. Blocking TrkA in lymph node stromal cells led to increased T cell proliferation in in vitro coculture assays. Similar effects were observed with the pan-Trk inhibitor larotrectinib in cocultures of lymph node stromal cells of patients with rheumatoid arthritis and T cells. Finally, local pLN treatment with TrkA inhibitor and TrkA siRNA led to exacerbated arthritis scores. CONCLUSION: CCL19+ lymph node stromal cells are crucially involved in the development of inflammatory arthritis. Therefore, targeting of CCL19+ lymph node stromal cells via TRK could provide a tool to prevent arthritis.

[Antitumor Study of Neoantigen-reactive T Cells Co-expressing IL-7 and CCL19 
in Mouse Lung Cancer].

BACKGROUND: Neoantigen reactive T cell (NRT) has the ability to inhibit the growth of tumors expressing specific neoantigens. However, due to the difficult immune infiltration and the inhibition of tumor microenvironment, the therapeutic effect of NRT in solid tumors is limited. In this study, we designed NRT cells (7×19 NRT) that can express both interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19) in mouse lung cancer cells, and evaluated the difference in anti-tumor effect between 7×19 NRT cells and conventional NRT cells. METHODS: We performed next-generation sequencing and neoantigen prediction for mouse Lewis lung carcinoma (LLC), prepared RNA vaccine, cultured NRT cells, constructed retroviral vectors encoding IL-7 and CCL19, transduced NRT cells and IL-7 and CCL19 were successfully expressed, and 7×19 NRT was successfully obtained. The anti-tumor effect was evaluated in vivo and in vitro in mice. RESULTS: The 7×19 NRT cells significantly enhanced the proliferation and invasion ability of T cells by secreting IL-7 and CCL19, achieved significant tumor inhibition in the mouse lung cancer and extended the survival period of mice. The T cell infiltration into tumor tissue and the necrosis of tumor tissue increased significantly after 7×19 NRT treatment. In addition, both 7×19 NRT treatment and conventional NRT treatment were safe. CONCLUSIONS: The anti-solid tumor ability of NRT cells is significantly enhanced by the arming of IL-7 and CCL19, which is a safe and effective genetic modification of NRT.

CCL19-producing fibroblasts promote tertiary lymphoid structure formation enhancing anti-tumor IgG response in colorectal cancer liver metastasis.

Tertiary lymphoid structures (TLSs) are associated with enhanced immunity in tumors. However, their formation and functions in colorectal cancer liver metastasis (CRLM) remain unclear. Here, we reveal that intra- and peri-tumor mature TLSs (TLS+) are associated with improved clinical outcomes than TLS- tumors. Using single-cell-RNA-sequencing and spatial-enhanced-resolution-omics-sequencing (Stereo-seq), we reveal that TLS+ tumors are enriched with IgG+ plasma cells (PCs), while TLS- tumors are characterized with IgA+ PCs. By generating TLS-associated PC-derived monoclonal antibodies in vitro, we show that TLS-PCs secrete tumor-targeting antibodies. As the proof-of-concept, we demonstrate the anti-tumor activities of TLS-PC-mAb6 antibody in humanized mouse model of colorectal cancer. We identify a fibroblast lineage secreting CCL19 that facilitates lymphocyte trafficking to TLSs. CCL19 treatment promotes TLS neogenesis and prevents tumor growth in mice. Our data uncover the central role of CCL19+ fibroblasts in TLS formation, which in turn generates therapeutic antibodies to restrict CRLM.

Single-cell profiling reveals the metastasis-associated immune signature of hepatocellular carcinoma.

AIM: Metastasis is the leading cause of mortality in hepatocellular carcinoma (HCC). The metastasis-associated immune signature in HCC is worth exploring. METHODS: Bioinformatic analysis was conducted based on the single-cell transcriptome data derived from HCC patients in different stages. Cellular composition, pseudotime state transition, and cell-cell interaction were further analyzed and verified. RESULTS: Generally, HCC with metastasis exhibited suppressive immune microenvironment, while HCC without metastasis exhibited active immune microenvironment. Concretely, effector regulatory T cells (eTregs) were found to be enriched in HCC with metastasis. PHLDA1 was identified as one of exhaustion-specific genes and verified to be associated with worse prognosis in HCC patients. Moreover, A novel cluster of CCR7+ dendritic cells (DCs) was identified with high expression of maturation and migration marker genes. Pseudotime analysis showed that inhibition of differentiation occurred in CCR7+ DCs rather than cDC1 in HCC with metastasis. Furthermore, interaction analysis showed that the reduction of CCR7+ DCs lead to impaired CCR7/CCL19 interaction in HCC with metastasis. CONCLUSIONS: HCC with metastasis exhibited upregulation of exhaustion-specific genes of eTregs and inhibition of CCL signal of a novel DC cluster, which added new dimensions to the immune landscape and provided new immune therapeutic targets in advanced HCC.