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Double-stranded DNA (dsDNA) in the cytoplasm of eukaryotic cells is abnormal and typically indicates the presence of pathogens or mislocalized self-DNA. Multiple sensors detect cytosolic dsDNA and trigger robust immune responses via activation of type I interferons. Several cancer immunotherapy treatments also activate cytosolic nucleic acid sensing pathways, including oncolytic viruses, nucleic acid-based cancer vaccines, and pharmacological agonists. We report here that cytosolic dsDNA introduced into malignant cells can robustly upregulate expression of CCL22, a chemokine responsible for the recruitment of regulatory T cells (Tregs). Tregs in the tumor microenvironment are thought to repress anti-tumor immune responses and contribute to tumor immune evasion. Surprisingly, we found that CCL22 upregulation by dsDNA was mediated primarily by interferon regulatory factor 3 (IRF3), a key transcription factor that activates type I interferons. This finding was unexpected given previous reports that type I interferon alpha (IFN-α) inhibits CCL22 and that IRF3 is associated with strong anti-tumor immune responses, not Treg recruitment. We also found that CCL22 upregulation by dsDNA occurred concurrently with type I interferon beta (IFN-β) upregulation. IRF3 is one of two transcription factors downstream of the STimulator of INterferon Genes (STING), a hub adaptor protein through which multiple dsDNA sensors transmit their signals. The other transcription factor downstream of STING, NF-κB, has been reported to regulate CCL22 expression in other contexts, and NF-κB has also been associated with multiple pro-tumor functions, including Treg recruitment. However, we found that NF-κB in the context of activation by cytosolic dsDNA contributed minimally to CCL22 upregulation compared with IRF3. Lastly, we observed that two strains of the same cell line differed profoundly in their capacity to upregulate CCL22 and IFN-β in response to dsDNA, despite apparent STING activation in both cell lines. This finding suggests that during tumor evolution, cells can acquire, or lose, the ability to upregulate CCL22. This study adds to our understanding of factors that may modulate immune activation in response to cytosolic DNA and has implications for immunotherapy strategies that activate DNA sensing pathways in cancer cells., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (T FH ) cells and, consequently, receive more contact-dependent T cell help 1 . As these GC B cells and T FH cells do not maintain long-lasting contacts in the chaotic GC environment 2-4 , it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on T FH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of T FH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from T FH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

Sphingosine-1-phosphate (S1P) and its receptors have been implicated in functions of Langerhans cells and atopic dermatitis. In this study, we investigated the roles of S1P receptor type 2 (S1P 2 ) in a mouse model of atopic dermatitis, which was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) on ventral skin on D0, followed by repeated DNCB challenge on both ears from D7 to D49. Wild-type mice with atopic dermatitis displayed severe inflammation and mast cell accumulation in ear tissues and elevated IgE levels in serum. Furthermore, the mice showed significantly increased sizes of draining lymph nodes, high levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes and high levels of chemokines CCL17 and CCL22 in ears. Administration of JTE-013, a selective antagonist of S1P 2 (3 mg/kg, i.p, from D19 to D49) before DNCB challenge significantly suppressed DNCB-induced atopic responses in ears and lymph nodes. JTE-013 administration also significantly decreased the lymph nodes sizes, the levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes, and the levels of chemokines CCL17 and CCL22 in ears. Furthermore, the inflammatory responses of atopic dermatitis were greatly ameliorated in S1pr2 gene-deficient mice. As CCL17 and CCL22 are CCR4 ligands, acting as Th2-attracting chemokines, we investigated CCL17 and CCL22 expression in bone marrow-derived dendritic cells (BMDCs) from wild-type and S1pr2 gene-deficient mice. Addition of IL-4 (10 ng/mL) markedly increased the levels of CCL17 and CCL22, but IL-4-induced CCL17 and CCL22 expression was significantly blunted in BMDCs from S1pr2 gene-deficient mice. Furthermore, pretreatment with JTE-013 (1-30 μM) dose-dependently suppressed this induction in BMDCs from wild-type mice. Our results demonstrate that blockage of S1P 2 ameliorates not only DNCB-induced atopic dermatitis symptoms but also Th2 cell-attracting capacity of dendritic cells, suggesting S1P 2 as a potential therapeutic target for atopic dermatitis.

In tumor microenvironment, macrophages as a polarized M2 population promote tumor progression via releasing multiple cytokines and chemokines. A brown seaweed fucose-rich polysaccharide, fucoidan has antitumor activity and immune modulation through affecting tumor cells and lymphocytes. Here, we focused on the effect of fucoidan on macrophages especially M2 subtype. Our results demonstrated that fucoidan down-regulated partial cytokines and chemokines, especially a M2-type chemokine CCL22. Furthermore, fucoidan inhibited tumor cells migration and CD4 + T lymphocytes, especially Treg cells, recruitment induced by M2 macrophages conditioned medium through suppression of CCL22. Mechanismly, fucoidan inhibited CCL22 via suppressing p65-NF-κB phosphorylation and nuclear translocation. In addition, p38-MAPK and PI3K-AKT also affected the expression of CCL22 through differential modulation of NF-κB transcriptional activity. Taken together, we reveal an interesting result that fucoidan can inhibit tumor cell migration and lymphocytes recruitment by suppressing CCL22 in M2 macrophages via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor immunotherapy.

Macrophages play an important role in the development of age-related macular degeneration (AMD). In this study, the spatial and temporal changes and the polarization of macrophages in murine laser-induced choroidal neovascularization (CNV) were investigated, and the polarized M1 and M2 biomarkers in the aqueous humors of neovascular AMD (nAMD) patients were studied. Macrophages, the main infiltrating inflammatory cells in CNV lesions, were evidenced by a significant increase in F4/80 mRNA expression and by the infiltration of F4/80+ cells in the lesions and the vicinity of laser-induced CNV. The mRNA expressions of M1-related markers were dramatically upregulated in the early stage, while the M2-related markers were slightly upregulated in the middle stage and sustained until the late stage. The results of immunostaining showed a similar early-but-transient M1 pattern and a delayed-but-sustained M2 pattern in laser-induced CNV. In addition, a higher M2/M1 ratio was found in both the murine models (Arg-1/iNOS and CCL22/CXCL10) and the aqueous humors of nAMD patients (CCL22/CXCL10) than in the controls. Our results suggested that the dynamic patterns of M1 and M2 were different in both the experimental and clinical CNV. The M2 macrophages were predominant and may play a more important role in the development of CNV.

Approximately 10% of gastric carcinomas are associated with Epstein-Barr virus (EBV) and are defined as EBV-associated gastric carcinomas (EBVaGCs). EBVaGCs are known to be accompanied by massive CD8(+) cytotoxic T cell (CTL) infiltration; however, adoptive cellular immunotherapy based on EBV-specific CD8(+) CTLs has been explored with limited success. Because regulatory T cells (Tregs) are regarded as a critical hurdle in anti-tumour immunity, we assessed the distribution of Tregs in 45 cases of EBVaGC and 45 cases of EBV-negative gastric carcinoma (EBVnGC) with matched clinicopathological parameters by immunohistochemistry. We showed that Tregs were significantly increased in EBVaGC compared to EBVnGC (15.92 ± 11.45/HPF vs. 8.45 ± 6.16/HPF, p = 0.001). In addition, we explored the accumulation mechanisms of Tregs in EBVaGC by using EBV (+) gastric carcinoma cell lines SNU719 and GT39 as ex vivo models. When peripheral blood mononuclear cells (PBMCs) were co-cultured with EBV (+) gastric carcinoma cell lines, the Treg frequency increased, and they underwent phenotypic and functional changes. The enhanced recruitment by CCL22 produced by EBVaGC cells, the decreased emigration due to CCR7 downregulation on the Treg surface, the higher proliferation rate, and the lower apoptosis rate of Tregs at tumour sites may promote the accumulation of Tregs in EBVaGC.

Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4(+)T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.