ELISpot-functional-T-cell-assay

ELISpot / FluoroSpot / Functional T-cell assay

一行要約

ELISpot / FluoroSpot は cytokine 分泌を per-cell 解像度で検出する古典的免疫機能 assay で、antigen-specific T-cell の頻度・活性を定量する gold standard。Intracellular cytokine staining (ICS), AIM (activation-induced marker), MHC tetramer staining と並び、neoantigen vaccine / TCR-T / TIL therapy / IO trial の pharmacodynamic biomarker および CAR-T 製品 release assay で必須の機能評価ツール。

原理

(1) ELISpot: PVDF 膜にcapture 抗体 (anti-IFN-γ / -IL-2 / -TNF-α) を coat → PBMC + antigen (peptide pool / tumor lysate) で 18-24 h stimulation → 分泌 cytokine が膜近傍に固定 → detection 抗体 + enzyme で spot 形成 → automated reader で per-cell quantification (spot-forming unit, SFU)。(2) FluoroSpot: 蛍光検出で 2-3 cytokine 同時 (polyfunctional T-cell)。(3) ICS: Brefeldin A で cytokine 分泌阻害 → 細胞内 staining → flow cytometry で phenotype × function。(4) AIM: stimulation 後の CD137 / OX40 / CD69 で specific T-cell 同定 (cytokine 分泌不要)。(5) MHC tetramer: HLA-peptide tetramer で antigen-specific T-cell の TCR 直接同定 (neoantigen-specific TCR-seq の前提)。

主要エビデンス / 適用領域

• Neoantigen vaccine: pre-/post-vaccination ELISpot で neoantigen-specific T-cell 誘導確認 (mRNA vaccine, peptide vaccine 試験で routine biomarker)
• CAR-T release assay: 製造後 CD19/BCMA-CAR の potency assay で IFN-γ ELISpot 標準
• TIL therapy 製品評価: 拡張 TIL の tumor reactivity (autologous tumor coculture + IFN-γ ELISpot)
• TCR-T: candidate TCR の specificity / avidity assessment
• IO pharmacodynamic biomarker: anti-PD-1 治療下の peripheral T-cell function 変化
• Vaccine 試験 (mRNA-4157 / V940): KEYNOTE-942 で adjuvant 黒色腫 mRNA vaccine 効果機序解析

適用分野と限界

• 強み: per-cell 解像度, low-frequency (1/10⁵-10⁶) detection, 凍結 PBMC 適用可能, regulatory pathway 確立 (CAR-T release で FDA 承認標準), low cost
• 限界: phenotype 不明 (CD4 vs CD8 区別は ICS / AIM 必要), antigen-specificity は peptide pool dependent, T-cell only (B-cell / NK には ELISpot 別 protocol), 96-well 標準で throughput 制限, automated counting standardization 課題

Open Questions

• Multi-cytokine FluoroSpot の polyfunctional signature と臨床応答の相関
• High-throughput ELISpot (384-well, microfluidic chip) 開発
• TCR-pMHC binding 予測 との統合 (in silico → ELISpot validation)
• Standardization across centers: clinical trial network での inter-lab reproducibility

関連エンティティ

• 補完手法: Flow-cytometry-CyTOF / CITE-seq / TCR-seq / scRNA-seq
• 関連薬剤: mRNA-vaccine / Cancer-vaccine / CAR-T-therapy / TCR-T-therapy / TIL-therapy / PD-1-inhibitor
• 関連細胞: CD8-T-cell / CD4-T-helper-cell / CAR-T-cell / Exhausted-CD8-T-cell / Tpex
• 関連サイトカイン: IFN-gamma / IL-2 / TNF-alpha / Granzyme-B

関連論文

直近 30 件のみ表示中 (関連 77 件)。 全件は MOC ページ または Obsidian (source Markdown) から辿れます。

Basic-Extracellular vesicles

• Shin et al. NatMater 2023 — Curvature-sensing peptide inhibits tumour-derived exosomes for enhanced cancer immunotherapy
• Merlotti et al. SciImmunol 2023 — Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent

Basic-Immunotherapy

• Kureshi et al. CancerCell 2026 — Dendritic cell redundancy enables priming of anti-tumor cd4+ t cells in pancreatic cancer
• Pinto et al. CancerGeneTher 2026 — Preclinical development of a mutant KRAS targeting therapeutic cancer vaccine
• George et al. CancerRes 2026 — Immune checkpoint blockade augments lymphodepleting chemotherapy-induced antitumor immunity by expanding effector CD8…
• Liu et al. Cell 2020 — Detecting tumor antigen-specific T cells via interaction-dependent fucosyl-biotinylation
• Sellars et al. Cell 2022 — Cancer vaccines Building a bridge over troubled waters
• Malekzadeh et al. ClinCancerRes 2020 — Antigen experienced T cells from peripheral blood recognize p53 neoantigens
• Gupta et al. NatBiotechnol 2026 — Immune-remodeling mRNAs expressing IRF8 or NIK generate durable antitumor immunity in multiple cancer models
• Pires et al. NatMater 2026 — IL-12-releasing nanoparticles for effective immunotherapy of metastatic ovarian cancer
• Gonzalez-Kozlova et al. NatMed 2026 — Humoral IgG1 responses to tumor antigens underpin clinical outcomes in immune checkpoint blockade
• LopezDeRodas et al. NatRevClinOncol 2021 — Tumour antigen-induced T cell exhaustion-the archenemy of immune-hot malignancies
• Rojas et al. Nature 2023 — Personalized RNA neoantigen vaccines stimulate t cells in pancreatic cancer
• Foord et al. SciTranslMed 2021 — Characterization of ascites-and tumor-infiltrating gammadelta T cells reveals distinct repertoires and a beneficial
• Lowery et al. Science 2022 — Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers

Basic-Liquid-biopsy

• Chen et al. Nature 2026 — Cell-free chromatin state tracing reveals disease origin and therapy responses

Basic-Neutrophil and myeloid cell

• Zuo et al. CancerCell 2026 — IL-36gamma armored CAR T cells reprogram neutrophils to induce endogenous antitumor immunity
• Zhao et al. NatBiotechnol 2026 — A logic-gated trispecific engager enhances macrophage killing of cancer cells in solid tumors
• Aloe et al. TranslLungCancerRes 2021 — Emerging and multifaceted role of neutrophils in lung cancer

Basic-Others

• Tang et al. CancerCell 2026 — Mapping cell type-resolved transcriptomic profiles to patient survival in pancreatic cancer
• Hao et al. Cell 2021 — Integrated analysis of multimodal single-cell data
• Rohatgi et al. NatRevClinOncol 2020 — Cancer vaccine induces potent T cell responses-but is it enough

Basic-Tumor microenvironment

• Podaza et al. CellRepMethods 2026 — Tumor immune microenvironment reconstitution in patient-derived organoids enables therapy modeling for NSCLC

Clinical-Novel modalities

• Ott et al. Cell 2020 — A phase ib trial of personalized neoantigen therapy plus anti-PD-1 in patients with advanced melanoma, non-small cell
• Mandal et al. NatCancer 2023 — Structural surfaceomics reveals an AML-specific conformation of integrin beta2 as a CAR T cellular therapy target
• Creelan et al. NatMed 2021 — Tumor-infiltrating lymphocyte treatment for anti-PD-1-resistant metastatic lung cancer a phase 1 trial
• Hu et al. NatMed 2021 — Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma
• Blass et al. NatRevClinOncol 2021 — Advances in the development of personalized neoantigen-based therapeutic cancer vaccines
• Wagner et al. NatRevClinOncol 2021 — Immunogenicity of CAR T cells in cancer therapy
• Nicholas et al. npjPrecisOncol 2026 — Immunopeptidomics-guided identification of functional neoantigens in non-small cell lung cancer