प्रेरित pluripotent स्टेम सेल (आईपीएस) से टी lymphocytes की पीढ़ी टी immunotherapy सेल आधारित के लिए भ्रूण स्टेम कोशिकाओं का उपयोग करने का एक वैकल्पिक दृष्टिकोण देता है. विधि से पता चलता है कि या तो द्वारा का उपयोग<em> इन विट्रो में</em> या<em> Vivo में</em> प्रेरण सिस्टम, आईपीएस कोशिकाओं दोनों पारंपरिक और प्रतिजन विशेष टी लिम्फोसाइटों में अंतर करने में सक्षम हैं.
Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.
अधिनियम आधारित चिकित्सा के लिए, पुनः जलसेक vivo में अत्यधिक प्रतिक्रियाशील एजी विशेष टी कोशिकाओं की बड़ी संख्या की इन विट्रो पीढ़ी में एक इष्टतम दृष्टिकोण है. हालांकि हमारे विधि में इन विट…
The authors have nothing to disclose.
हम प्रदान करने के लिए डॉ. Shinya Yamanaka (क्योटो विश्वविद्यालय) धन्यवाद आईपीएस MEF एनजी 20D-17 सेल लाइन का समर्थन करने के लिए OT1-2A डॉ. Dario Vignali (सेंट जूदास बच्चों अनुसंधान अस्पताल) • pMig द्वितीय का निर्माण, डा. जुआन कार्लोस Zuniga सेल OP9 DL1 लाइन का समर्थन करने के लिए (इम्यूनोलॉजी विभाग, टोरंटो विश्वविद्यालय के) – Pflucker, और इस अध्ययन के डिजाइन की मदद करने के लिए डॉ. केंट ई Vrana (औषधि विभाग, पेन स्टेट यूनिवर्सिटी कॉलेज ऑफ मेडिसिन के). इस परियोजना, राष्ट्रीय कैंसर संस्थान, Barsumian ट्रस्ट और मेलेनोमा रिसर्च फाउंडेशन (जे सांग) से अनुदान K18CA151798 संख्या के साथ अनुदान के तहत वित्त पोषित है.
Name of the reagent | Company | Catalogue number |
C57BL/6J mice | Jackson Laboratory | 000664 |
B6.129S7-Rag1tm1Mom/J | Jackson Laboratory | 002216 |
Anti-CD3 (2C11) antibody | BD PharMingen | 553058 |
Anti-CD28 (37.51) antibody | BD PharMingen | 553295 |
Anti-CD3 (17A2) antibody | BioLegend | 100202 |
Anti-CD4 (GK1.5) antibody | BioLegend | 100417 |
Anti-CD8 (53-6.7) antibody | BioLegend | 100714 |
Anti-CD25 (3C7) antibody | BioLegend | 101912 |
Anti-CD44 (1M7) antibody | BioLegend | 103012 |
Anti-CD117 (2B8) antibody | BioLegend | 105812 |
Anti-TCR-β (H57597) antibody | BioLegend | 109220 |
Anti-IL-2 (JES6-5H4) antibody | BioLegend | 503810 |
Anti-IFN-γ (XMG1.2) antibody | BioLegend | 505822 |
DMEM | Invitrogen | ABCD1234 |
α-MEM | Invitrogen | A10490-01 |
FBS | HyClone | SH3007.01 |
Brefeldin A | Sigma | B7651 |
Polybrene | Sigma | 107689 |
GeneJammer | Integrated Sciences | 204130 |
RNA kit | Qiagen | 74104 |
DNA kit | Qiagen | 69504 |
CD8 Isolation Kit | Miltenyi Biotec | 130-095-236 |
ACK lysis buffer | Lonza | 10-548E |
mFlt-3L | PeproTech | 250-31L |
mIL-7 | PeproTech | 217-17 |
Gelatin | Sigma | G9391 |
FITC-anti-OVA antibody | Rockland Immunochemicals | 200-4233 |
Permeabilization buffer | Biolegend | 421002 |
BSA | Sigma | A7906 |
Formaldehyde | Sigma | F8775 |
0.4 μm filter | MIllipore | |
Moflo Cell Sorter | Dake Cytomation | |
Calibur Flow Cytometer | BD | |
LSR II Flow Cytometer | BD | |
Mouse restrainer | Braintree Scientific |