मानवीय इन विट्रो में इम्यून असंतुलन का एक प्रारंभिक राज्य की मान्यता के लिए स्क्रीनिंग उपकरण के रूप में दमन
Summary
Tregs प्रतिरक्षा प्रणाली की प्रबल दमन कर रहे हैं. अद्वितीय सतह मार्करों की एक कमी के लिए उन्हें परिभाषित है, इसलिए, Tregs की परिभाषा मुख्य रूप से कार्य कर रहे हैं. यहाँ हम एक अनुकूलित वर्णन<em> इन विट्रो में</em> परख जोखिम में विषयों में प्रतिरक्षा असंतुलन की पहचान के लिए T1D विकसित करने में सक्षम.
Abstract
Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic β-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.
Protocol
Discussion
केवल Tregs के लिए अनूठी विशेषता के रूप में विषयों के बीच, दमनकारी समारोह मज़बूती से और समान परीक्षण किया जाना चाहिए एक ही के भीतर और विभिन्न अध्ययनों के बीच रोग के विकास के विभिन्न चरणों पर. हम दमन हमारे इस ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस अध्ययन अधिकतम McGee किशोर Diabetesat विस्कॉन्सिन और Wisconsin के बच्चों के अनुसंधान संस्थान के मेडिकल कॉलेज के लिए राष्ट्रीय अनुसंधान केंद्र द्वारा समर्थित किया गया. funders अध्ययन डिजाइन में कोई भूमिका नहीं थी, डेटा संग्रह और विश्लेषण, या पांडुलिपि की तैयारी.
Materials
| Name of the reagent or instrument | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Ficoll-Paque PLUS | Amersham Pharmacia Biotech | 17-1440-03 | |
| DPBS-1X | Gibco | 14190-144 | |
| Trypan Blue | Invitrogen | 15250-061 | |
| anti-CD4 microbeads | Miltenyi | 130-045-101 | |
| Pre-separation filters | Miltenyi | 130-041-407 | |
| LS column | Miltenyi | 130-042-401 | |
| EDTA | Invitrogen | 15575-020 | |
| BSA | Sigma-Aldrich | B4287 | |
| Anti-human CD4-APCCy7 (clone RPA-T4) | BD Pharmingen | 557852 | |
| Anti-human CD25-PE (clone M-A251; IL-2Rα) | BD Pharmingen | 555432 | |
| Anti-human CD8-FITC (clone RPA-T8) | BD Pharmingen | 555366 | |
| Anti-human CD14-FITC (clone M5E2; LPS receptor) | BD Pharmingen | 555397 | |
| Anti-human CD32-FITC (clone FLI8.26; FcγR-type II) | BD Pharmingen | 555448 | |
| Anti-human CD116-FITC (clone M5D12; GM-CSFRα chain) | BD Pharmingen | 554532 | |
| Dynalbeads M-450 tosylactivated | Invitrogen | 140-13 | |
| Anti-human CD3 | Ancell | 144-024 | |
| Buffer1 | Homemade | 0.1M Na2B4O7 pH7.6 | |
| Buffer2 | Homemade | PBS/2mM EDTA/ 0.1% BSA pH7.4 | |
| Buffer3 | Homemade | 0.2M Tris/0.1% BSA pH8.5 | |
| Complete RPMI media | Homemade | RPMI 1640 media 2 mM L-glutamine 5 mM HEPES 100 U/μg/ml peni/strept 0.5 mM sodium pyruvate | |
| [3H] thymidine | Perkin Elmer | NET027Z005MC | |
| human pooled AB serum | Atlanta Biologicals | S40110 | |
| Multiscreen harvest plate | Millipore | MAHFC1H60 | |
| Microscint 20 | Perkin Elmer | 6013621 |
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