भ्रष्टाचार बनाम मेजबान रोग के प्रेरण और<em> Vivo में</em> टी सेल निगरानी एक MHC-मिलान murine मॉडल का उपयोग
Summary
Murine अस्थि मज्जा प्रत्यारोपण एक व्यापक रूप से इस्तेमाल तकनीक प्रतिरक्षाविज्ञानी मनुष्यों में भ्रष्टाचार बनाम मेजबान रोग संचालन तंत्र का अध्ययन है. टी सेल की तस्करी पैटर्न की निगरानी करने की क्षमता<em> Vivo में</em> भ्रष्टाचार बनाम मेजबान रोग के दौरान विकास और टी सेल प्रतिक्रिया के स्थायीकरण के विस्तृत विश्लेषण के लिए अनुमति देता है.
Abstract
Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo.
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity).
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant.
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.
Protocol
Discussion
उत्प्रेरण के लिए प्रोटोकॉल यहाँ प्रस्तुत चूहों में GVHD murine GVHD के एक नैदानिक प्रासंगिक मॉडल का प्रतिनिधित्व करता है. मूलतः बर्गर एट अल द्वारा 1994 में स्थापित, C57BL / 6 BALB.B तनाव संयोजन में MHC मिलान GVHD निर्भर CD4, CD8…
Declarações
The authors have nothing to disclose.
Acknowledgements
हम ऐलिस Gaughan और जिओ Jing वैंग जिसका बकाया तकनीकी समर्थन, बौद्धिक इनपुट, और नैतिक समर्थन इन अध्ययनों को आगे बढ़ने में महत्वपूर्ण भूमिका निभाई थी ऋणी हैं. इन अध्ययनों NIH अनुदान AI036532 गाह समर्थित थे.
Materials
| Name of Reagent | Company | Catalogue Number | Comments |
| RPMI 1640 | Invitrogen | 12633-012 | |
| Fetal Calf Serum | Invitrogen | 10439016 | |
| 40 μM Cell Strainer | BD Biosciences | 352340 | |
| CD3e-Biotin | Miltenyi Biotech | 130-093-021 | |
| Anti-Biotin Microbeads | Miltenyi Biotech | 130-091-147 | |
| CD8a Microbeads | Miltenyi Biotech | 130-049-401 | Used to deplete CD8 T cells from spleen. |
| CD8a Purification Antibody Cocktail | Miltenyi Biotech | 130-095-236 | Used to purify CD8 T cells from spleen. |
| D-Luciferin | Caliper Life Sciences | 122796 |
Referências
- Cooke, K. R. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin. Blood. 88, 3230-3239 (1996).
- Berger, M. T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens. Transplantation. 57, 1095-1102 (1994).
- Nimer, S. D. Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial. Transplantation. 57, 82-87 (1994).
- Korngold, R., Sprent, J. Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences. Journal of Immunology. 135, 3004-3010 (1985).
- Cao, Y. A. Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation. 80, 134-139 (2005).
- Asady, R. E. l. TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease. J. Exp. Med. 201, 1647-1657 (2005).
- Larson, R. S., Springer, T. A. Structure and function of leukocyte integrins. Immunol Rev. 114, 181-217 (1990).
- Karecla, P. I. Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7). Eur. J. Immunol. 25, 852-856 (1995).
- Cepek, K. L. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin. Nature. 372, 190-193 (1994).
- Malarkannan, S. The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161, 3501-3509 (1998).
