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摘要： Lung tissue resident memory (TRM) cells are thought to play crucial roles in lung host defense. We have recently shown that immunization with the adjuvant LTA1 (derived from the A1 domain of E. coli heat labile toxin) admixed with OmpX from K. pneumoniae can elicit antigen specific lung Th17 TRM cells that provide serotype independent immunity to members of the Enterobacteriaceae family. However, the upstream requirements to generate these cells are unclear. Single-cell RNA-seq showed that vaccine-elicited Th17 TRM cells expressed high levels of IL-1R1, suggesting that IL-1 family members may be critical to generate these cells. Using a combination of genetic and antibody neutralization approaches, we show that Th17 TRM cells can be generated independent of caspase-1 but are compromised when IL-1α is neutralized. Moreover IL-1α could serve as a molecular adjuvant to generate lung Th17 TRM cells independent of LTA1. Taken together, these data suggest that IL-1α plays a major role in vaccine-mediated lung Th17 TRM generation.

关键词： TaABI5-D3   原核表达   重组蛋白   多克隆抗体   小麦  

摘要： 碱性亮氨酸拉链转录因子ABI5 (abscisic acid-insensitive 5)是脱落酸介导的种子萌发的关键调控因子，而TaABI5基因与小麦穗发芽密切相关。前期研究表明，TaABI5的多拷贝基因成员TaABI5-D3具有编码完整TaABI5蛋白的能力，其开放阅读框为1 191 bp，编码396个氨基酸，分子量为42.796 kDa。本研究通过构建原核表达载体pET-28a-TaABI5-D3，并在大肠杆菌中进行表达，最终得到纯化的His-TaABI5-D3重组蛋白。该重组蛋白以包涵体的形式存在，最佳表达条件为：16 ℃、0.6 mmol/L IPTG、150 r/min过夜诱导12 h。进一步将His-TaABI5-D3重组蛋白进行纯化后免疫Balb/C小鼠制备多克隆抗体，Western blotting结果显示：抗体的特异性完好。该研究结果为下一步明确TaABI5蛋白在小麦籽粒中的功能奠定了基础。

关键词： 嵌合抗原受体T(CAR-T)细胞   结肠腺癌(COAD)   细胞间充质上皮转化因子(c-Met)   生物信息学  

摘要： 目的 探索细胞间充质上皮转化因子（c-Met）可以作为嵌合抗原受体T（CAR-T）细胞治疗结肠癌有效靶点的可能性。方法 通过生物信息学方法分析c-Met在结肠腺癌（COAD）中的特异性表达及其临床意义；使用免疫组织化学验证结肠癌临床患者肿瘤组织中c-Met的表达，采用流式细胞术检测结肠癌细胞株HCT116中c-Met的表达；使用慢病毒感染人外周血单个核细胞（PBMC）中原代T细胞，制备靶向c-Met的二代CAR-T细胞，并观察c-MetCAR-T细胞对HCT116的抑制效果。结果 免疫组织化学与生物信息学显示c-Met在COAD中高表达，其中相对低表达的患者生存预后较好，在正常结肠组织中低表达或不表达。流式细胞术显示c-Met在HCT116细胞中也高表达。c-MetCAR-T细胞能够靶向表达抗原的肿瘤细胞，并且在抗原刺激下，CAR-T细胞特异性增殖，起到杀伤癌细胞与释放白细胞介素2（IL-2）和γ干扰素（IFN-γ）的生物学作用。结论 c-Met有成为COAD潜在治疗靶点的潜能；c-Met CAR-T细胞在体外对结肠癌细胞具有抑制作用。

关键词： Gastric cancer   IFN-gamma   Immunotherapy   PBMC   STING   cGAS  

摘要： Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-β levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-β was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

摘要： PURPOSE:Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN:We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS:Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS:These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

关键词： Droplet‐based microfluidics   Heterogeneity   Plasmacytoid dendritic cells   Quorum sensing   Type I interferon  

摘要： Type I interferons (IFN-Is) are key in fighting viral infections, but also serve major roles beyond antiviral immunity. Crucial is the tight regulation of IFN-I responses, while excessive levels are harmful to the cells. In essence, immune responses are generated by single cells making their own decisions, which are based on the signals they perceive. Additionally, immune cells must anticipate the future state of their environment, thereby weighing the costs and benefits of each possible outcome, in the presence of other potentially competitive decision makers (i.e., IFN-I producing cells). A rather new cellular communication mechanism called quorum sensing describes the effect of cell density on cellular secretory behaviors, which fits well with matching the right amount of IFN-Is produced to fight an infection. More competitive decision makers must contribute relatively less and vice versa. Intrigued by this concept, we assessed the effects of immune quorum sensing in pDCs, specialized immune cells known for their ability to mass produce IFN-Is. Using conventional microwell assays and droplet-based microfluidics assays, we were able the characterize the effect of quorum sensing in human primary immune cells in vitro. These insights open new avenues to manipulate IFN-I response dynamics in pathological conditions affected by aberrant IFN-I signaling.

关键词： 急性淋巴细胞白血病   MTHFR(1298A>C)   MTHFR(677C>T)   甲氨蝶呤  

摘要： 目的 研究MTHFR(1298A>C)和MTHFR(677C>T)位点多态性与甲氨蝶呤(methotrexate,MTX)血药浓度和不良反应的相关性。方法 选取2014年10月—2021年12月上海市儿童医院收治的185例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患儿，收集MTHFR(1298A>C)和MTHFR(677C>T)基因分型、不良反应和血药浓度等实验室检查指标结果。结果 185例患儿使用MTX后的总体不良反应发生率为95.1%。MTHFR(1298A>C)2种基因型不良反应发生率除粒细胞缺乏差异有统计学意义(P=0.006)外，其他均无统计学意义；MTHFR(677C>T)3种基因型的ALL患儿不良反应发生率除粒细胞缺乏(P=0.041/0.012)、消化道反应(P=0.037/0.011)和黏膜毒性(P=0.039/0.016)差异有统计学意义外，其他均无统计学意义；MTHFR(677C>T)3种基因型的ALL患儿在24 h的MTX血药浓度差异有统计学意义(P=0.021);MTHFR(677C>T)3种基因型的ALL患儿进行亚叶酸钙翻倍解救发生率差异有统计学意义(P=0.007/0.002)。结论 MTHFR(1298A>C)和MTHFR(677C>T)多态性可能不是预测儿童ALL进行MTX化疗的良好指标，结局提示了翻倍解救的重要性，即翻倍解救可以明显降低黏膜毒性和骨髓毒性高发患儿的此类不良反应发生率。

关键词： Adverse drug reactions   Anaphylaxis   Biomarker   Drug hypersensitivity   HLA  

摘要： BACKGROUND:Oxaliplatin is commonly used to treat gastrointestinal malignancies. However, its applications are limited due to potential adverse drug reactions (ADRs), particularly severe anaphylactic shock. There is no method to predict or prevent ADRs caused by oxaliplatin. Therefore, we aimed to investigate the genetic HLA predisposition and immune mechanism of oxaliplatin-induced ADRs. METHODS:A retrospective review was performed for 154 patients with ADRs induced by oxaliplatin during 2016-2021 recorded in our ADR notification system. HLA genotyping was conducted for 47 patients with oxaliplatin-induced ADRs, 1100 general population controls, and 34 oxaliplatin-tolerant controls in 2019-2023. The in vitro basophil activation test (BAT) was performed and oxaliplatin-specific IgE levels were determined. RESULTS:The incidence of oxaliplatin-induced ADRs and anaphylactic shock in our cohort was 7.1% and 0.15%, respectively. Of the 154 patients, 67.5% suffered rash/eruption; 26.0% of the patients who could not undergo oxaliplatin rechallenge were considered to show oxaliplatin-induced immune-mediated hypersensitivity reactions (HRs). The genetic study found that the HLA-DRB∗12:01 allele was associated with oxaliplatin-induced HRs compared to the general population controls (sensitivity = 42.9%; odds ratio [OR] = 3.4; 95% CI = 1.4-8.2; P = 0.008) and tolerant controls (OR = 12; 95% CI = 2.3-63.7; P = 0.001). The in vitro BAT showed higher activation of CD63 basophils in patients with oxaliplatin-induced HRs compared to the tolerant controls (P < 0.05). Only four patients (8.5%) with oxaliplatin-induced ADRs were positive for oxaliplatin-specific IgE. CONCLUSIONS:This study found that 26.0% of patients with oxaliplatin-induced ADRs could not undergo oxaliplatin rechallenge. HLA-DRB∗12:01 is regarded as a genetic marker for oxaliplatin-induced hypersensitivity.

关键词： 慢性粒细胞白血病   稳定性   Hopf分支   周期解   细胞分化时滞  

摘要： 为了研究慢性粒细胞白血病干细胞分化时滞对白血病干细胞，白血病成熟细胞和效应T细胞之间的动力学行为影响，建立了带有干扰素-α和伊马替尼治疗项的慢性粒细胞白血病免疫时滞模型。通过对改进的时滞模型稳定性分析，依据规范型和中心流形理论，得到Hopf分支的临界条件与性质。并给出干扰素-α和伊马替尼联合效应T细胞控制慢性粒细胞白血病的条件，如在药物干预下，调节分化时滞会使慢性粒细胞白血病干细胞与白血病成熟细胞的数量呈现稳定不增状态，或由大振幅周期波动转向为小振幅波动状态。最后，数值模拟验证了所得理论。