2020-12-12 | 文獻閱讀

Applying high-dimensional single-cell technologies to the analysis of cancer immunotherapyPembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trialPharmacologic Screening Identifies Metabolic Vulnerabilities of CD8+ T CellsRestoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist AntibodiesLysine acetylation of NKG2D ligand Rae-1 stabilizes the protein and sensitizes tumor cells to NKG2D immune surveillanceLow-dose carboplatin reprograms tumor immune microenvironment through STING signaling pathway and synergizes with PD-1 inhibitors in lung cancerErlotinib plus bevacizumab vs erlotinib monotherapy as first-line treatment for advanced EGFR mutation-positive non-squamous non-small-cell lung cancer: Survival follow-up results of the randomized JO25567 studyNeoadjuvant immune checkpoint inhibitors in cancer, current state of the artOutcomes in Patients With Non-small-cell Lung Cancer With Brain Metastases Treated With Pembrolizumab-based TherapyTargeting tumor-associated macrophages as an antitumor strategyCircular RNA in cancer development and immune regulationMethylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs
1 將高維單細胞技術應用於癌症免疫治療分析
標題：Applying high-dimensional single-cell technologies to the analysis of cancer immunotherapy
期刊：Nature Reviews Clinical Oncology
時間：2020-12-04
DOI：http://dx.doi.org/10.1038/s41571-020-00449-x
Abstract
Advances in molecular biology, microfluidics and bioinformatics have empowered the study of thousands or even millions of individual cells from malignant tumours at the single-cell level of resolution. This high-dimensional, multi-faceted characterization of the genomic, transcriptomic, epigenomic and proteomic features of the tumour and/or the associated immune and stromal cells enables the dissection of tumour heterogeneity, the complex interactions between tumour cells and their microenvironment, and the details of the evolutionary trajectory of each tumour. Single-cell transcriptomics, the ability to track individual T cell clones through paired sequencing of the T cell receptor genes and high-dimensional single-cell spatial analysis are all areas of particular relevance to immuno-oncology. Multidimensional biomarker signatures will increasingly be crucial to guiding clinical decision-making in each patient with cancer. High-dimensional single-cell technologies are likely to provide the resolution and richness of data required to generate such clinically relevant signatures in immuno-oncology. In this Perspective, we describe advances made using transformative single-cell analysis technologies, especially in relation to clinical response and resistance to immunotherapy, and discuss the growing utility of single-cell approaches for answering important research questions.
Fig.1 免疫腫瘤學中單細胞分析的工作流程
Fig.2 將單細胞分析納入涉及免疫療法的臨床試驗的一般生物樣本框架
2 Pembrolizumab聯合化療與安慰劑聯合化療治療以前未經治療的局部復發性不可手術或轉移性三陰性乳腺癌（KEYNOTE-355）：一項隨機，安慰劑對照，雙盲，3期臨床試驗
標題：Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial
期刊：Lancet
時間：2020-12-03
DOI：http://dx.doi.org/10.1016/S0140-6736(20)32531-9
Abstract
Background
Pembrolizumab monotherapy showed durable antitumour activity and manageable safety in patients with metastatic triple-negative breast cancer. We aimed to examine whether the addition of pembrolizumab would enhance the antitumour activity of chemotherapy in patients with metastatic triple-negative breast cancer.
Methods
In this randomised, placebo-controlled, double-blind, phase 3 trial, done in 209 sites in 29 countries, we randomly assigned patients 2:1 with untreated locally recurrent inoperable or metastatic triple-negative breast cancer using a block method (block size of six) and an interactive voice-response system with integrated web-response to pembrolizumab (200 mg) every 3 weeks plus chemotherapy (nab-paclitaxel; paclitaxel; or gemcitabine plus carboplatin) or placebo plus chemotherapy. Randomisation was stratified by type of on-study chemotherapy (taxane or gemcitabine–carboplatin), PD-L1 expression at baseline (combined positive score [CPS] ≥1 or <1), and previous treatment with the same class of chemotherapy in the neoadjuvant or adjuvant setting (yes or no). Eligibility criteria included age at least 18 years, centrally confirmed triple-negative breast cancer; at least one measurable lesion; provision of a newly obtained tumour sample for determination of triple-negative breast cancer status and PD-L1 status by immunohistochemistry at a central laboratory; an Eastern Cooperative Oncology Group performance status score 0 or 1; and adequate organ function. The sponsor, investigators, other study site staff (except for the unmasked pharmacist), and patients were masked to pembrolizumab versus saline placebo administration. In addition, the sponsor, the investigators, other study site staff, and patients were masked to patient-level tumour PD-L1 biomarker results. Dual primary efficacy endpoints were progression-free survival and overall survival assessed in the PD-L1 CPS of 10 or more, CPS of 1 or more, and intention-to-treat populations. The definitive assessment of progression-free survival was done at this interim analysis; follow-up to assess overall survival is continuing. For progression-free survival, a hierarchical testing strategy was used, such that testing was done first in patients with CPS of 10 or more (prespecified statistical criterion was α=0·00411 at this interim analysis), then in patients with CPS of 1 or more (α=0·00111 at this interim analysis, with partial alpha from progression-free survival in patients with CPS of 10 or more passed over), and finally in the intention-to-treat population (α=0·00111 at this interim analysis). This study is registered with ClinicalTrials.gov, NCT02819518, and is ongoing.
Findings
Between Jan 9, 2017, and June 12, 2018, of 1372 patients screened, 847 were randomly assigned to treatment, with 566 patients in the pembrolizumab–chemotherapy group and 281 patients in the placebo–chemotherapy group. At the second interim analysis (data cutoff, Dec 11, 2019), median follow-up was 25·9 months (IQR 22·8–29·9) in the pembrolizumab–chemotherapy group and 26·3 months (22·7–29·7) in the placebo–chemotherapy group. Among patients with CPS of 10 or more, median progression-free survival was 9·7 months with pembrolizumab–chemotherapy and 5·6 months with placebo–chemotherapy (hazard ratio [HR] for progression or death, 0·65, 95% CI 0·49–0·86; one-sided p=0·0012 [primary objective met]). Median progression-free survival was 7·6 and 5·6 months (HR, 0·74, 0·61–0·90; one-sided p=0·0014 [not significant]) among patients with CPS of 1 or more and 7·5 and 5·6 months (HR, 0·82, 0·69–0·97 [not tested]) among the intention-to-treat population. The pembrolizumab treatment effect increased with PD-L1 enrichment. Grade 3–5 treatment-related adverse event rates were 68% in the pembrolizumab–chemotherapy group and 67% in the placebo–chemotherapy group, including death in <1% in the pembrolizumab–chemotherapy group and 0% in the placebo–chemotherapy group.
Interpretation
Pembrolizumab–chemotherapy showed a significant and clinically meaningful improvement in progression-free survival versus placebo–chemotherapy among patients with metastatic triple-negative breast cancer with CPS of 10 or more. These findings suggest a role for the addition of pembrolizumab to standard chemotherapy for the first-line treatment of metastatic triple-negative breast cancer.
3 藥理學篩選可鑑定CD8+ T細胞的代謝漏洞
標題：Pharmacologic Screening Identifies Metabolic Vulnerabilities of CD8+ T Cells
期刊：Cancer Immunology Research
時間：2020-12-04
DOI：http://dx.doi.org/10.1158/2326-6066.CIR-20-0384
Abstract
Metabolic constraints in the tumor microenvironment constitute a barrier to effective anti-tumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type-specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity-promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis, but impaired anti-tumor CD8+ T cell responses. Our screen also revealed high T cell-specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and ER stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of anti-tumor CD8+ T-cell function and potential therapeutic targets.
4 T細胞效應子功能的恢復，Treg的耗竭和腫瘤細胞的直接殺傷：a-TIGIT拮抗劑抗體作用的多種機制
標題：Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies
期刊：Molecular Cancer Therapeutics
時間：2020-12-04
DOI：http://dx.doi.org/10.1158/1535-7163.MCT-20-0464
Abstract
TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti–PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti–PD(L)-1-like restoration of αβ T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP–enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.
5 NKG2D配體Rae-1的賴氨酸乙醯化可穩定蛋白質並使腫瘤細胞對NKG2D免疫監視敏感
標題：Lysine acetylation of NKG2D ligand Rae-1 stabilizes the protein and sensitizes tumor cells to NKG2D immune surveillance
期刊：Cancer Letters
時間：2020-12-03
DOI：http://dx.doi.org/10.1016/j.canlet.2020.12.002
Highlights
NKG2D ligands are shed from tumor cell surface by matrix metalloproteinases (MMPs) to escape immune surveillance.
Shedding of NKG2D ligand Rae-1 from tumor cells can be prevented by acetyltransferases GCN5 and PCAF.
GCN5 and PCAF stabilize Rae-1 by posttranslational acetylation at Lysines 80 and 87 to reinvigorate NKG2D antitumor cytotoxicity.
GCN5 correlates with human NKG2D ligand ULBP1 at protein levels in human tumor tissue array.
High expression of GCN5 and PCAF is associated with increased overall survival rate in many cancers.
Abstract
Shedding, loss of expression, or internalization of natural killer group 2, member D (NKG2D) ligands from the tumor cell surface leads to immune evasion, which is associated with poor prognosis in patients with cancer. In many cancers, matrix metalloproteinases cause the proteolytic shedding of NKG2D ligands. However, it remained unclear how to protect NKG2D ligands from shedding. Here, we showed that the shedding of the mouse NKG2D ligand Rae-1 can be prevented by two critical acetyltransferases, GCN5 and PCAF, which acetylate the lysine residues of Rae-1 to avoid shedding both in vitro and in vivo. In contrast, mutations at lysines 80 and 87 of Rae-1 abrogated this acetylation and thereby desensitized tumor cells to NKG2D-dependent immune surveillance. Notably, the protein levels of GCN5 correlated with the expression levels of the human NKG2D ligand ULPB1 in a human tumor tissue microarray and, more importantly, with prolonged overall survival in many cancers. Our results suggest that the acetylation of Rae-1 protein at lysines 80 and 87 by GCN5 and PCAF protects Rae-1 from shedding so as to activate NKG2D-dependent immune surveillance. This discovery may shed light on new targets for NKG2D immunotherapy in cancer treatment.
6 小劑量卡鉑通過STING信號通路重編程腫瘤免疫微環境並與PD-1抑制劑協同作用
標題：Low-dose carboplatin reprograms tumor immune microenvironment through STING signaling pathway and synergizes with PD-1 inhibitors in lung cancer
期刊：Cancer Letters
時間：2020-12-02
DOI：http://dx.doi.org/10.1016/j.canlet.2020.11.049
Highlights
Low-dose carboplatin increases CD8+ T cell infiltration and PD-L1 expression.
Low-dose carboplatin is safe and turns the 「cold」 tumor into 「hot」 tumor.
Low-dose carboplatin potentiates the anti-tumor effect of PD-1 inhibitors.
Carboplatin induces DNA Damage and activates the STING signaling pathway.
STING knockdown in tumor reverses the anti-tumor effect of low-dose carboplatin.
Abstract
Although the combination of chemotherapy and immunotherapy is a hot topic in lung cancer, little is understood regarding the possible mechanisms behind their synergy. Moreover, safety is a major concern for clinicians while performing chemotherapy. Therefore, it is important to determine the appropriate dose and period of chemotherapy for combining it with immunotherapy, and investigate the underlying synergistic mechanism. Here, we showed that carboplatin can induce DNA damage and activate the canonical STING/TBK1/IRF3 pathway and non-canonical STING-NF-κB signaling complex. Further, low-dose carboplatin changed the 「cold」 tumor into a 「hot」 tumor via the signaling hub STING, augmenting CD8+ T-cell infiltration, increasing PD-L1 expression, and hence potentiating the anti-tumor effect of PD-1 inhibitors; importantly, there were no adverse effects. Furthermore, knocking down STING in tumor cells effectively reversed PD-L1 upregulation and STING pathway activation, and reduced the anti-tumor effect of low-dose carboplatin and carboplatin-PD-1 inhibitor combination. Our findings collectively reported a previously unexplored role of low-dose carboplatin targeting in the STING pathway and provided an economical, useful and safe option for improving the efficacy of PD-1 inhibitors in lung cancer.
7 厄洛替尼聯合貝伐單抗vs厄洛替尼單藥治療晚期EGFR突變陽性非鱗狀非小細胞肺癌的一線治療：隨機JO25567研究的生存隨訪結果
標題： Erlotinib plus bevacizumab vs erlotinib monotherapy as first-line treatment for advanced EGFR mutation-positive non-squamous non-small-cell lung cancer: Survival follow-up results of the randomized JO25567 study
期刊：Lung Cancer
時間：2020-12-04
DOI：http://dx.doi.org/10.1016/j.lungcan.2020.11.020
Highlights
First long-term OS data of erlotinib + bevacizumab vs erlotinib in this population.
Both treatment arms showed a similar median OS of approximately 4 years.
Results of EGFR and VEGF inhibitor combination therapies are eagerly awaited.
Abstract
Objectives
The JO25567 randomized Phase II study demonstrated a statistically significant progression-free survival (PFS) benefit with erlotinib plus bevacizumab compared with erlotinib monotherapy in chemotherapy-naïve Japanese patients with epidermal growth factor receptor mutation-positive (EGFR+) non-small-cell lung cancer (NSCLC). Here we present updated PFS and final overall survival (OS) data after a median follow-up of 34.7 months.
Materials and methods
Patients with stage IIIB/IV or postoperative recurrent NSCLC were randomized to receive oral erlotinib 150 mg once daily (n = 77) or erlotinib in combination with intravenous bevacizumab 15 mg/kg every 21 days (n = 75) until disease progression or unacceptable toxicity. OS was analyzed using an unstratified Cox proportional hazards model.
Results
Consistent with the primary analysis, addition of bevacizumab to erlotinib was associated with a significant improvement in PFS (hazard ratio [HR] 0.52; 95 % confidence interval [CI]: 0.35–0.76; log-rank two-sided P = 0.0005; median 16.4 months vs 9.8 months, respectively). In contrast, a significant improvement in OS was not seen (HR 0.81; 95 % CI, 0.53–1.23; P =  0.3267; median 47.0 months vs 47.4 months, respectively). Post-study therapy was similar between the treatment arms and EGFR mutation type did not affect OS outcomes. The 5-year OS rate was numerically higher with erlotinib plus bevacizumab vs erlotinib monotherapy (41 % vs 35 %). Updated safety analyses confirmed the previously reported manageable tolerability profile, with no new safety issues.
Conclusion
Addition of bevacizumab to first-line erlotinib did not show significant improvement in OS in Japanese patients with stage IIIB/IV or postoperative recurrent EGFR+ NSCLC. Both treatment arms showed a similar median OS benefit (as long as 4 years), irrespective of individual patient characteristics. Results from ongoing studies evaluating the combination of EGFR and VEGF signaling inhibitors are eagerly awaited.
8 癌症中的新輔助免疫檢查點抑制劑，當前技術水平
標題：Neoadjuvant immune checkpoint inhibitors in cancer, current state of the art
期刊：Critical Reviews in Oncology/Hematology 
時間：2020-12-04
DOI：http://dx.doi.org/10.1016/j.critrevonc.2020.103172
Highlights
Preclinical data support the use of immunotherapy (IO) in the neoadjuvant setting.
Two clinical trials reported higher efficacy of neoadjuvant IO versus adjuvant IO.
Neoadjuvant trials are a unique opportunity to identify predictive biomarkers.
Neoadjuvant IO present limits: unconventional responses, toxicity and resistance.
Abstract
Immunotherapy has been a revolution in cancer management in the metastatic setting. This has led to a prompt evaluation of such therapies in earlier stages. This article discusses the still limited amount of data finding the rationale to assess such therapy in this setting and reviews preclinical and clinical data available. Overall, neoadjuvant immunotherapy is a promising approach for the treatment of cancers and the rationale supporting its use is strong. Neoadjuvant immunotherapy resulted, in the majority of clinical trials, in improved pathologic complete response rates with a favorable toxicity profile and no delay in surgery. Various regimens were effective: inhibitory immune check-point blockers (IICPB) alone, combination of PD-1 and CTLA-4 inhibitors, combination of chemotherapy (CT) and IICPB, phased CT and IICPB (either IICPB before CT or IICPB after CT). Yet the question whether neoadjuvant immunotherapy will benefit to patients in terms of disease-free and, ultimately, overall survival remains unknown.
9 派姆單抗治療的非小細胞肺癌伴腦轉移患者的預後
標題：Outcomes in Patients With Non-small-cell Lung Cancer With Brain Metastases Treated With Pembrolizumab-based Therapy
期刊：Clinical Lung Cancer
時間：2020-11-11
DOI：http://dx.doi.org/10.1016/j.cllc.2020.10.017
Abstract
Background
Patients with metastatic non–small-cell lung cancer (mNSCLC) and untreated brain metastases (BM) have been excluded from most trials of immune checkpoint inhibitors (ICIs). Real-world evidence on efficacy and survival outcomes of ICIs in patients with BM is limited.
Patients and Methods
We conducted a single-center retrospective study of patients with mNSCLC treated with pembrolizumab with or without chemotherapy and compared progression-free survival (PFS) and overall survival (OS) between patients with and without BM using Kaplan-Meier and Cox methodology. We also characterized systemic and intracranial objective response rate (ORR) and treatment details, including timing of cranial irradiation.
Results
Between Augutst 2013 and December 2018, 570 patients with mNSCLC treated with pembrolizumab-based therapy were analyzed. Of 126 (22.1%) patients with BM, 96 (76.2%) had treated BM (local therapy prior to pembrolizumab), and 30 (23.8%) had untreated BM. Of patients with untreated BM, 17 (56.7%) underwent radiation within 30 days after pembrolizumab initiation. In the remaining 13 (43.3%) treated with pembrolizumab-based therapy alone, intracranial ORR was 36.4%. Patients with and without BM did not have significantly different systemic ORR (27.8% vs. 29.7%; P = .671), PFS (mPFS 9.2 vs. 7.7 months; P = .609), or OS (mOS 18.0 vs. 18.7 months; P = .966). Factors associated with improved survival on Cox analysis included female gender, performance status, adenocarcinoma histology, and first-line therapy.
Conclusions
Patients with BM did not have inferior survival to patients without BM after treatment with pembrolizumab-based therapy. In the current era, BM may not automatically confer inferior survival, and should not exclude patients from receiving pembrolizumab-based therapy.
10 靶向腫瘤相關巨噬細胞作為抗腫瘤策略
標題：Targeting tumor-associated macrophages as an antitumor strategy
期刊：Biochemical Pharmacology
時間：2020-12-03
DOI：http://dx.doi.org/10.1016/j.bcp.2020.114354
Abstract
Tumor-associated macrophages (TAMs) are the most widely infiltrating immune cells in the tumor microenvironment (TME). Clinically, the number of TAMs is closely correlated with poor outcomes in multiple cancers. The biological actions of TAMs are complex and diverse, including mediating angiogenesis, promoting tumor invasion and metastasis, and building an immunosuppressive microenvironment. Given these pivotal roles of TAMs in tumor development, TAM-based strategies are attractive and used in certain tumor therapies, including inhibition of angiogenic signalling, blockade of the immune checkpoint, and macrophage enhancement phagocytosis. Several attempts to develop TAM-targeted agents have been made to deplete TAMs or reprogram the behaviour of TAMs. Some have shown a favourable curative effect in monotherapy, combination with chemotherapy or immunotherapy in clinical trials. Additionally, a new macrophage-based cell therapeutic technology was recently developed by equipping macrophages with CAR molecules. It is expected to break through barriers to solid tumor treatment. Although TAM-related studies have yielded positive antitumor outcomes, further investigations are needed to better characterize TAMs, which will benefit further establishment of novel strategies for tumor therapy. Here, we concisely summarize the functions of TAMs in the TME and comprehensively introduce the latest TAM-based regimens in cancer treatment.
Fig.1 腫瘤相關巨噬細胞（TAM）通過多種機制驅動腫瘤進展，包括刺激血管生成，促進腫瘤細胞轉移和抑制抗腫瘤免疫力
Fig.2 基於TAM的治療策略
11 環狀RNA在癌症發展和免疫調節中的作用
標題：Circular RNA in cancer development and immune regulation
期刊：Journal of Cellular and Molecular Medicine
時間：2020-12-05
DOI：http://dx.doi.org/10.1111/jcmm.16102
Abstract
Circular RNAs (circRNAs) are a class of single‐stranded RNAs with closed loop structures formed by covalent bonds of head and tail. Exploration of circRNAs is continually increasing; however, their functional relevance largely remains to be elucidated. In general, they are stable, abundant, conserved and expressed in tissue‐specific manner. These distinct properties and their diverse cellular actions indicate that circRNAs modulate transcription and translation, and may even function as translation templates. Growing evidence reveals that circRNAs contribute to various physiological and pathological processes, including the initiation and progression of cancer. In this review, we present the current knowledge about circRNAs in cancer development, as well as their potential for use as biomarkers and even therapeutic targets. CircRNA’s role in immune regulation and antitumour immunotherapy is also discussed. In addition, possible challenges in antitumour therapy are raised, and current progress and future perspectives are provided.
Fig.1 環狀RNA功能
Fig.2 癌症中的環狀RNA
12 甲基化敏感限制性內切酶定量聚合酶鏈反應可快速，準確，精確地檢測人類原發性Treg中調節性T細胞（Treg）特異甲基化區域的甲基化狀態
標題：Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs
期刊：Journal of Immunology
時間：2020-12-04
DOI：http://dx.doi.org/10.4049/jimmunol.1901275
KeyPointAbstract
Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.