Human non-small cell lung cancer (NSCLC) is an inflammation-related disease. Although IL-17-induced NSCLC cell proliferation that can be regulated by transcription factors has been demonstrated, the role and mechanism of other regulatory molecules, such as long noncoding RNAs (lncRNAs), in cell proliferation remains unclear. In this study, we screened and verified the expression of aberrant lncRNAs in NSCLC cell lines (H1299 and PC9) stimulated with IL-17, and found that LINC01518 was not only overexpressed, but also enhanced cell proliferation through IL-17/IL-17RA. Further mechanism investigation discovered that IL-17-upregulated LINC01518 was mainly localized in the cytoplasm, and it could combine with miR-20a-5p through a "sponge" function, decreasing miR-20a-5p induction, while LINC01518 and miR-20a-5p co-overexpression could partially reverse the cell proliferation mediated by LINC01518 alone. LINC01518 increase or miR-20a-5p decrease could elevate E2F1 level boosting H1299 cell proliferation exposed to IL-17, while miR-20a-5p and E2F1 co-overexpression partially restored the cell proliferation inhibition from miR-20a-5p upregulation. Besides, the xenograft tumor experiments of mice confirmed that LINC01518 overexpression indeed promoted tumor growth, cell proliferation, miR-20a-5p downregulation, and E2F1 upregulation. While LINC01518 knockdown reduced tumor growth, cell proliferation, miR-20a-5p downregulation, and E2F1 upregulation induced by IL-17 stimulation. Taken together, these findings reveal that the LINC01518/miR-20a-5p/E2F1 axis contributes to IL-17-induced cell proliferation in NSCLC.

Rat Thy-1 nephritis (Thy-1 N) is a model for human mesangioproliferative glomerulonephritis (MsPGN). Although sublytic C5b-9 has been reported to affect pro-inflammatory cytokine expression in glomerular mesangial cells (GMCs), the induction of other pro-inflammatory mediators remains unclear. In this study, we demonstrated that SET and MYND domain-containing protein 4 (SMYD4), SRY-related HMG-box gene 7 (SOX7), S100 calcium-binding protein A8 (S100A8), and S100A9 were overexpressed in the renal tissues of Thy-1 N rats and in the GMCs stimulated by sublytic C5b-9. Mechanistically, SOX7 triggered S100A8/A9 gene transcription through binding to S100A8 promoter (+21 to + 31 nt) and S100A9 promoter (-1698 to -1688 nt), and SMYD4 mediated SOX7 methylation at K159, a new methylation site, enhancing SOX7 function and S100A8/A9 transcription. The animal experiments confirmed that silencing SMYD4, SOX7, or S100A8/A9 gene could inhibit GMC proliferation, extracellular matrix accumulation, and proteinuria in Thy-1 N rats. Moreover, silencing SMYD4 gene could inhibit renal SOX7-K159me2/3, and silencing SMYD4 or SOX7 gene could inhibit renal S100A8/A9 production. Meanwhile, the SOX7-K159 methylation inhibitory peptide exhibited the similar intervention effects as SMYD4 and SOX7 knockdown in Thy-1 N rats. In addition, the expression levels of SMYD4, SOX7, SOX7-K159me2/3, and S100A8/A9 all increased in the renal tissues of MsPGN patients. Collectively, our findings suggest that SMYD4-mediated SOX7-K159me2/3 partly contributes to sublytic C5b-9-induced S100A8/A9 expression in Thy-1 N rats, which provides a novel insight into human MsPGN pathogenesis.

Non-small cell lung cancer (NSCLC) is a disease related to inflammation. Proinflammatory cytokines such as interleukin 17 (IL-17) can induce cancer cell proliferation, metastasis and immune escape. Although NSCLC immune escape is partly due to the interaction between PD-1 and PD-L1 and PD-L1 expression can be upregulated in cancer cells upon stimulation with IL-17, the underlying mechanism of IL-17-triggered PD-L1 gene transcription in NSCLC cells remains elusive. RT‒PCR, real-time PCR, and IB were used to assess the levels of PD-L1, MEF2C, and TRIM31 in NSCLC tissues as well as in IL-17–stimulated H1299 or PC9 cells. Bioinformatics analysis, luciferase assays, and ChIP were utilized to investigate the transcriptional mechanism of the PD-L1 gene. Co-IP/IB was used to examine the interaction between MEF2C and PD-L1, including MEF2C ubiquitination. IHC staining was carried out to analyse the expression of IL-17RA, MEF2C, TRIM31, and PD-L1 in NSCLC tissue arrays. The corresponding plasmids were constructed and identified. An isograft model was used to verify the findings in vitro. PD-L1, MEF2C and TRIM31 expression levels were increased in NSCLC tissues and NSCLC cells exposed to IL-17. Mechanistically, MEF2C could bind to the − 778 to -475 nt and − 336 to -97 nt regions of the PD-L1 promoter. TRIM31 could mediate MEF2C K63-linked polyubiquitination at Lys 25, increasing MEF2C recruitment to the PD-L1 promoter and PD-L1 gene transcription. MEF2C, TRIM31 or PD-L1 gene silencing effectively suppressed MEF2C K63-linked polyubiquitination, PD-L1 induction and NSCLC growth in mice inoculated with Lewis lung cancer (LLC) cells transfected with the corresponding shRNA and treated with IL-17. IL-17 induces PD-L1 gene transcription in NSCLC cells through TRIM31-dependent MEF2C K63-linked polyubiquitination.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC).Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs)induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found thatIL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues andNSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduceIL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanisminvestigation revealed that STAT3 and p300 bound to the same region (-544 to-389 nt) of MMP19 promoter, andp300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and thecell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylationcould interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion.Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in thelung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLCmetastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and itsY705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy

BackgroundImmune checkpoint blockade has shown low response rates for advanced breast cancer, and combination strategies are needed. Microwave ablation (MWA) may be a trigger of antitumor immunity. This window-of-opportunity trial (ClinicalTrials.gov: NCT04805736) was conducted to determine the safety and feasibility of preoperative camrelizumab (an anti-PD-1 antibody) combined with MWA in the treatment of early-stage breast cancer.MethodsSixty participants were randomized to preoperatively receive single-dose camrelizumab alone (n = 20), MWA alone (n = 20), or camrelizumab+MWA (n = 20). A random number table was used to allocate interventions. The primary outcome was the safety and feasibility of MWA combined with camrelizumab.FindingsCamrelizumab and MWA were well tolerated alone and in combination without delays in prescheduled surgery. No treatment-related grade III/IV adverse events were observed. Different from in the single-dose camrelizumab or MWA group, participants showed stable counts of blood cells after combination therapy. After combination therapy, peripheral CD8+ T cells showed enhanced cytotoxic and effect-memory functions. Clonal expansional CD8+ T cells showed higher cytotoxic activity and effector memory- and tumor-specific signatures than emergent clones after combination therapy. Enhanced interactions between clonal expansional CD8+ T cells and monocytes were observed, suggesting that monocytes contributed to the enhanced functions of clonal expansional CD8+ T cells. Major histocompatibility complex (MHC) class I-related pathways and interferon signaling pathways were activated in monocytes by combination therapy.ConclusionsCamrelizumab combined with MWA was feasible for early-stage breast cancer. Peripheral CD8+ T cells were activated after combination therapy, dependent on monocytes with activated MHC class I pathways.FundingThis study was supported by the Natural Science Foundation of Jiangsu Province (BK20230017).

Minimally invasive thermal therapy is a successful alternative treatment to surgery in solid tumors with high complete ablation rates, however, tumor recurrence remains a concern. Central memory CD8+ T cells (TCM) play important roles in protection from chronic infection and cancer. Here we find, by single-cell RNA analysis of human breast cancer samples, that although the memory phenotype of peripheral CD8+ T cells increases slightly after microwave ablation (MWA), the metabolism of peripheral CD8+ T cells remains unfavorable for memory phenotype. In mouse models, glycolysis inhibition by 2-deoxy-D-glucose (2DG) in combination with MWA results in long-term anti-tumor effect via enhancing differentiation of tumor-specific CD44hiCD62L+CD8+ TCM cells. Enhancement of CD8+ TCM cell differentiation determined by Stat-1, is dependent on the tumor-draining lymph nodes (TDLN) but takes place in peripheral blood, with metabolic remodeling of CD8+ T cells lasting the entire course of the the combination therapy. Importantly, in-vitro glycolysis inhibition in peripheral CD8+ T cells of patients with breast or liver tumors having been treated with MWA thrice leads to their differentiation into CD8+ TCM cells. Our work thus offers a potential strategy to avoid tumor recurrence following MWA therapy and lays down the proof-of-principle for future clinical trials.

Metastasis is the major cause of cancer-related death, and lymph node is the most common site of metastasis in breast cancer. However, the alterations that happen in tumor-draining lymph nodes (TDLNs) to form a premetastatic microenvironment are largely unknown. Here, we first report the dynamic changes in size and immune status of TDLNs before metastasis in breast cancer. With the progression of tumor, the TDLN is first enlarged and immune-activated at early stage that contains specific antitumor immunity against metastasis. The TDLN is then contracted and immunosuppressed at late stage before finally getting metastasized. Mechanistically, B and follicular helper T (Tfh) cells parallelly expand and contract to determine the size of TDLN. The activation status and specific antitumor immunity of CD8 + T cells in the TDLN are determined by interleukin-21 (IL-21) produced by Tfh cells, thus showing parallel changes. The turn from activated enlargement to suppressed contraction is due to the spontaneous contraction of germinal centers mediated by follicular regulatory T cells. On the basis of the B-Tfh-IL-21-CD8 + T cell axis, we prove that targeting the axis could activate TDLNs to resist metastasis. Together, our findings identify the dynamic alterations and regulatory mechanisms of premetastatic TDLNs of breast cancer and provide new strategies to inhibit lymph node metastasis.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (−544 to −389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

目的:研究人胚肾293T(HEK 293T,简称293T)细胞中外源性肿瘤坏死因子受体相关因子(tumor necrosis factor receptor-associated factor 6,TRAF6)与 Krüppel样因子5(Krüppel-like factor 5,KLF5)的结合及TRAF6多聚泛素化修饰KLF5 的方式和修饰的位点.方法:将构建的Flag-TRAF6、HA-KLF5、泛素过表达质粒、shTRAF6小干扰质粒和TRAF6 C70A位点突变质粒行不同组合转染293T细胞48 h.用免疫沉淀(immunoprecipitation,IP)和免疫印迹(immunoblotting,IB)实验检查TRAF6与KLF5的结合以及KLF5 K63或K48多聚泛素化水平.此外,构建KLF5全部赖氨酸突变的质粒,分别与TRAF6质粒共转染293T细胞.用前述IP/IB检测KLF5 K63连接的多聚泛素化修饰,并确定KLF5 K63泛素化修饰的位点.结果:293T细胞中TRAF6能与KLF5结合;TRAF6过表达和基因沉默或TRA6酶活性缺失能相应上调或下调KLF5 K63的多聚泛素化;KLF5被TRAF6 K63多聚泛素化修饰的位点是其第99位和第100位的赖氨酸.结论:TRAF6能与KLF5相互作用,并对KLF5-K99和K100进行K63多聚泛素化修饰.

Mesangial proliferative glomerulonephritis (MsPGN) and its related rat model Thy-1 nephritis (Thy-1N) are associated with C5b-9 deposition and are characterized by proliferation of glomerular mesangial cell (GMC) and expansion of extracellular matrix (ECM) expansion, alongside overexpression of multiple growth factors. Although fibroblast growth factor 1 (FGF1), platelet-derived growth factor alpha (PDGF alpha), and transforming growth factor beta 1 (TGF-81) are well known for their proproliferative and profibrotic roles, the molecular mechanisms responsible for regulating the expression of these growth factors have not been thoroughly elucidated. In this study, we found that sublytic C5b-9 induction of sex-determining region Y-box 9 (SOX9) transactivated FGF1, PDGF alpha, and TGF-81 genes in GMCs, resulting in a significant increase in their mRNA and protein levels. Besides, sublytic C5b-9 induction of activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylated SOX9 at serine 181 and serine 64, which enhanced SOX9 ' s ability to transactivate FGF1, PDGF alpha, and TGF-81 genes in GMCs. Furthermore, we demonstrated that inhibiting ERK1/2 activation or silencing either ERK1/2 or SOX9 gene led to reduced SOX9 phosphorylation, decreased generation of FGF1, PDGF alpha, and TGF-81, and ameliorated glomerular injury in rat Thy-1N. Overall, these findings suggest that expression of FGF1, PDGF alpha, and TGF-81 is promoted by ERK1/2-mediated phosphorylation of SOX9, which may provide a valuable insight into the pathogenesis of MsPGN and offer a potential target for the development of novel treatment strategies for MsPGN.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

Interleukin-17 (IL-17), a potent proinflammatory cytokine, can trigger the metastasis of non-small cell lung cancer (NSCLC). However, the underlying mechanism involved in IL-17-induced NSCLC cell metastasis remains unclear. In this study, we found that not only the expression of IL-17, IL-17RA, and/or general control nonrepressed protein 5 (GCN5), SRY-related HMG-BOX gene 4 (SOX4), and matrix metalloproteinase 9 (MMP9) was increased in the NSCLC tissues and in the IL-17-stimulated NSCLC cells, but also IL-17 treatment could enhance NSCLC cell migration and invasion. Further mechanism exploration revealed that IL-17-upregulated GCN5 and SOX4 could bind to the same region (-915 to -712 nt) of downstream MMP9 gene promoter driving its gene transcription. In the process, GCN5 could mediate SOX4 acetylation at lysine 118 (K118, a newly identified site) boosting MMP9 gene expression as well as cell migration and invasion. Moreover, the SOX4 acetylation or MMP9 induction and metastatic nodule number in the lung tissues of the BALB/c nude mice inoculated with the NSCLC cells stably infected by corresponding LV-shGCN5 or LV-shSOX4, LV-shMMP9 plus IL-17 incubation were markedly reduced. Overall, our findings implicate that NSCLC metastasis is closely associated with IL-17-GCN5-SOX4-MMP9 axis.

目的:检查人胶质母细胞瘤(glioblastoma,GBM)组织和细胞系中ZBTB3的表达,并探讨ZBTB3对GBM细胞增殖和克隆形成的影响及其调控机制.方法:通过GEPIA2数据库分析GBM患者肿瘤组织中ZBTB3的表达情况.RT-PCR、qPCR和Western blot检测GBM细胞系(U251、U373、U87)中ZBTB3的mRNA和蛋白表达水平,筛选出ZBTB3表达最高的U87细胞.CCK-8和克隆形成实验检测沉默ZBTB3基因对U87细胞增殖和克隆形成的影响.用p38MAPK、AMPK、Akt1抑制剂处理U87细胞后,Western blot 检测 p38MAPK、AMPK、Akt1 的磷酸化水平,RT-PCR、qPCR 和 Western blot 测定 ZBTB3 的 mRNA 和蛋白表达水平,CCK-8和克隆形成实验检测细胞增殖和克隆形成.结果:GBM患者肿瘤组织中ZBTB3的表达显著高于正常组织.U251、U373和U87细胞中均可见ZBTB3的表达,其中U87细胞表达最高.沉默ZBTB3基因能明显抑制U87细胞的增殖和克隆形成.抑制AMPK既能显著降低U87细胞ZBTB3的表达水平,又可明显减弱U87细胞增殖和克隆形成.结论:GBM组织和细胞系中ZBTB3的表达显著上调,GBM细胞中AMPK活化并上调ZBTB3基因的表达,促进GBM细胞的增殖和克隆形成.

Background The suppression of chimeric antigen receptor (CAR) T cells by the tumor microenvironment (TME) is a crucial obstacle in the T-cell-based treatment of solid tumors. Extra domain B (EDB)-fibronectin is an oncofetal antigen expressed on the endothelium layer of the neovasculature and cancer cells. Though recognized as a T cell therapy target, engineered CAR T cells thus far have failed to demonstrate satisfactory in vivo efficacy. In this study, we report that targeting EDB-fibronectin by redirected TCR-CAR T cells (rTCR-CAR) bypasses the suppressive TME for solid tumor treatment and sufficiently suppressed tumor growth. We generated EDB-targeting CAR by fusing single-chain variable fragment to CD3ε, resulting in rTCR-CAR. Human primary T cells and Jurkat cells were used to study the EDB-targeting T cells. Differences to the traditional second-generation CAR T cell in signaling, immune synapse formation, and T cell exhaustion were characterized. Cytotoxicity of the rTCR-CAR T cells was tested in vitro, and therapeutic efficacies were demonstrated using xenograft models. Methods Results In the xenograft models, the rTCR-CAR T cells demonstrated in vivo efficacies superior to that based on traditional CAR design. A significant reduction in tumor vessel density was observed alongside tumor growth inhibition, extending even to tumor models established with EDB-negative cancer cells. The rTCR-CAR bound to immobilized EDB, and the binding led to immune synapse structures superior to that formed by second-generation CARs. By a mechanism similar to that for the conventional TCR complex, EDB-fibronectin activated the rTCR-CAR, resulting in rTCR-CAR T cells with low basal activation levels and increased in vivo expansion. Conclusion Our study has demonstrated the potential of rTCR-CAR T cells targeting the EDB-fibronectin as an anticancer therapeutic. Engineered to possess antiangiogenic and cytotoxic activities, the rTCR-CAR T cells showed therapeutic efficacies not impacted by the suppressive TMEs. These combined characteristics of a single therapeutic agent point to its potential to achieve sustained control of solid tumors.

目的:探讨长链非编码RNA01518(long intergenic non-protein coding RNA 01518,LINC01518)基因过表达或沉默对白介素(interleukin,IL)-17诱导非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响.方法:Western blot检测NSCLC 细胞系(PC9、H1299 和 H1975)IL-17 受体 A(IL-17 receptor A,IL-17RA)的表达后,用 IL-17 刺激 H1299 和 PC9 细胞不同时间,利用CCK-8实验测定细胞的增殖水平.LncRNA芯片筛查IL-17刺激H1299和PC9细胞3h后LncRNA上调的结果,从中选取某些上调的LncRNA进行RT-PCR和Real-time PCR验证.此外,将构建的pcDNA3.1/LINC01518或shLINC01518质粒转染H1299细胞,用CCK-8、EdU和克隆形成实验检查细胞的增殖情况.结果:3种NSCLC细胞系均有IL-17RA的表达.实验发现,IL-17刺激H1299和PC9细胞后能提高其增殖水平,同时受刺激的细胞内LINC01518的上调最为显著,且过表达LINC01518可提高H1299细胞的增殖水平,而沉默LINC01518基因则能抑制IL-17诱导的细胞增殖.结论:LINC01518能促进IL-17诱导的H1299细胞增殖.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

Minimally invasive thermal therapies have been attempted in the treatment of breast cancer, and the immune response induced by these therapies has not been fully reported. A clinical trial is performed to determine the effect of microwave ablation (MWA) in the treatment of early-stage breast cancer. The authors perform single-cell RNA sequencing on peripheral blood mononuclear cells (PBMCs) from six patients before and after ablation. NK and CD8+ T cells are activated by MWA of breast cancer, with the increased inhibitory signature of CD8+ T cells but not dysfunctional. Enhanced co-stimulatory signature of CD4+ T cells is observed and increased frequency of ICOS+ CD4+ T cells after MWA is confirmed by flow cytometric analysis. After ablation, T-cell clones expand with increased T-cell receptor diversities. Activated antigen receptor-mediated signaling pathways are found in B cells. Enhanced interactions between B cells and CD4+ T cells are found, indicating that B cells are important antigen-presenting cells that initiate CD4+ T cells in MWA-induced immune response. Blockade of CTLA-4 or PD-1 of post-MWA PBMCs show higher T-cell activity than that of pre-MWA PBMCs. This study provide global characteristics of MWA-induced systemic immune response and pave a way for the identification of potential targets to improve the immune response.

We explored use of chimeric antigen receptor T cell (CAR-T) for treatment of solid tumors, aiming at efficient killing of tumor cells while minimizing on-target off-tumor toxicities. We designed CAR targeting the extra domain B (EDB)-containing fibronectin, which is an oncofetal antigen widely expressed in tumor stroma and on the surface of cancer cells. Unlike membrane-spanning antigens, EDB-containing fibronectin is embedded in the extracellular matrix, and is enriched in new vasculatures and stromal tissues of tumor tissues. First, we made a second-generation EDB CAR (2G-EDB CAR) that killed EDB+ cells (U87MG, A549, and HUVEC) and showed tumor suppression in a U87MG xenograft tumor model. However, while in vitro the 2G-EDB CAR T cells were sufficiently cytotoxic, the in vivo inhibition of the tumor growth was moderate. Next we designed an EDB CAR that contained CD3ε to create 3ε-EDB CAR. We show that the 3ε-EDB CAR was incorporated into the T cell receptor (TCR) structure. The 3ε-EDB CAR completely displaced the native CD3ε in the TCR based on immune precipitation and Western analysis. The 3ε-EDB CAR was capable of inducing T cell activation and proliferation in the presence of EDB-fibronectin antigen. Further, the 3ε-EDB CAR T cells killed tumor cells containing EDB-fibronectin. In vivo, the 3ε-EDB CAR T cells demonstrated improved inhibition when compared to the 2G-EDB CAR T cells. To further enhance the EDB-targeting CAR, we added intracellular domain of CD28 to the 3ε-EDB CAR to create 3ε28-EDB CAR, and showed the 3ε28-EDB CAR was still incorporated into the TCR complex. The 3ε28-EDB CAR co-opted TCR complex and provided co-stimulatory signal upon binding of EDB antigens. Upon transduction of the 3ε28-EDB CAR into primary T cells, we observed upregulation of T cell activation markers CD25, CD69 and granzyme B in presence of the EDB antigen. We show that 3ε28-EDB CAR triggered the phosphorylation of CD3ζ, suggesting that EDB antigen stimulated co-opted TCR and signaled through the TCR-CD3 complex. T cells transduced with 3ε28-EDB CAR demonstrated stronger proliferation when activated by U87MG cells compared to 2G or 3ε-EDB CARs. Furthermore, 3ε28-EDB CAR T cells inhibited in vivo tumor growth in a dose-dependent manner, and most of the treated mice had no evidence of residual tumors. In conclusion, our results suggest that 3ε28-EDB CAR has great potential for treatment of solid tumors. Citation Format: Zhijie Zhang, Wen Qiu, Chang Liu, Muhan Wang, Yinghua Miao, Qingchao Ji, Rongchen Sun, Hongping Yin, Meijia Yang. T cell receptor modified with EDB-targeting ScFv and CD28 intracellular domain led to complete tumor regression in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5572.

Mycobacterium tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a dominant target antigen for cell-mediated immunity in the early phase of tuberculosis. The fms-like tyrosine kinase 3 ligand (FL) that induces potent immune response has been used as an adjuvant in vaccine development. In this study, a new recombinant plasmid (pIRES-epitope-peptides-FL) encoding three T cell epitopes of ESAT-6 and FL was constructed, and the immunogenicity of the DNA vaccine was assessed in C57BL/6 mice immunized with the plasmid DNA vaccine. Additionally, a strategy of intramuscular injection with the DNA vaccine (prime) and intranasal administration of the epitope peptides (boost) was employed to induce higher immune reaction of the mice. The results showed that mice vaccinated with the recombinant plasmid DNA vaccine and boosted with the peptides not only increased the levels of Th1 cytokines (IFN-γ and IL-12), the number of IFN-γ(+) T cells and activities of cytotoxic T lymphocytes as well as IgG, but also enhanced protection against Mycobacterium tuberculosis challenge. In conclusion, these data indicate that the novel recombinant pIRES-epitope-peptides-FL plasmid is a useful DNA vaccine for preventing Mycobacterium tuberculosis infection.