Karsinoembriyonik antijene özgül immün yanıtların HLA-A 0201 transjenik fare modelinde ölçülmesi ve değerlendirilmesi

Amaç: Karsinoembriyonik antijen (CEA) geni taşıyan bir rekombinant vektör aracılığı ile immünize edilen transjenik farelerde, CEA’e karşı oluşan özgül T lenfosit yanıtını ölçmek ve deneysel parametreleri irdeliyerek değerlendirmek. Gereç ve Yöntem: Rekombinant vektör, ALVAC-CEA ile immünize edilen HLA-A*0201 (A2) transjenik farelerden dalak hücreleri izole edildi. Kültürlerde özgül CD8+ sitotoksik T lenfositleri aktive etmek ve çoğaltmak için, hücreler iki farklı CEA peptidi ile veya kontrol peptid ile uyarıldı. CEA’e özgül T lenfosit yanıtlarının ölçülmesinde standart bir değerlendirme işlemi için optimum parametreleri bulmak amacıyla, faklı peptid konsantrasyonları ve farklı hücre sayıları test edildi. T lenfosit cevabı, IFN-γ salgısı olarak ELISPOT yöntemi ile ölçüldü. Bulgular: ALVAC-CEA ile aşılanan transjenik fareler, CEA’e özgül olan etkin T lenfosit yanıtları oluşturdu. Oluşan yanıtların optimal seviyelerde ölçülebilmesi için, hücre sayısı ve uyarıcı peptid miktarı gibi parametre değerlerinin kullanılan yöntem açısından önemli olduğu saptandı. Sonuç: CEA’e özgül yanıtların ölçülmesinde deneysel parametreler tam olarak optimize edilmelidir. Özgül T lenfosit yanıtlarının oluşumunda etkin bir vektör olan ALVAC-CEA, CEA eksprese eden tümörlerin tedavisinde kullanılabilir.

Measurement and evaluation of CEA-specific immune responses in a HLA-A 0201 transgenic mice model

Aim: To measure specifi c T cell responses against to carcinoembryonic antigen (CEA) in transgenic mice that were immunized by a recombinant vector expressing CEA gene, and to evaluate experimental parameters. Materials and Methods: Splenocytes were isolated from spleens of HLA-A2 transgenic mice that were immunized with a recombinant vector, ALVAC-CEA. Cells were pulsed with an irrelevant peptide for controls or with two diff erent CEA-specifi c peptides to activate and expand CEA-specifi c CD8+ cytotoxic T lymphocytes in cultures. Various concentrations of peptides and various cell numbers were tested to fi nd optimum parameters for a standard evaluation procedure of measuring CEA-specifi c T cell responses. Responding T cells were assessed by IFN-γ secretion in ELISPOT assay. Results: Transgenic mice immunized by ALVAC-CEA developed effi cient specifi c T-cell responses to CEA. Parameters such as peptide concentration and cell number used in in vitro experimental procedure were found to be signifi cant in terms of obtaining optimal measurements. Conclusion: Experimental parameters should be optimized to measure CEA-specifi c T cell responses. ALVAC-CEA is an effi cient vector to induce specifi c T cell responses. Therefore, it can be a very useful vaccine in immunotherapy of cancers that express CEA.

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1. Horig H, Medina FA, Conkright WA, et al. Strategies for cancer therapy using carcinoembryonic antigen vaccines. Expert Rev Mol Med 2000; 2:1-24.
2. Kass E, Schlom J, Thompson J, et al. Induction of protective host immunity to carcinoembryonik (CEA), a self antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Cancer Research 1999; 59:676-683.
3. Haga S, Watanabe O, Shimizu T, et al. The clinical value of tissue carcinoembryonic antigen in breast cancer. Surgery Today 1991; 21:278-283.
4. Neil L. Berinstein. Carcinoembryonic antigen as a target for therapeutic anticancer vaccines: a review. Journal of Clinical Oncol. 2002; 20:2197-2207.
5. Borenstein SH, Graham J, Zhang X-L et al. CD8+ T Cells are necessary for recognition of allelic, but not locus-mismatched or xeno-, HLA Class I transplantation antigens. Journal of Immunol. 2000; 165:2341-2353.
6. Schmitz J, Reali E, Hodge JW, et al. Identification of an interferon-γ-inducible carcinoembryonic antigen (CEA) CD8+ T-cell epitope, which mediates tumor killing in CEA transgenic mice. Cancer Research 2002; 62:5058–5064.
7. Wada S, Tsunoda T, Baba T, et al. Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2. Cancer Research 2005; 65: 4939-4946.
8. Kan-Mitchell J, Bisikirska B, Wong-Staal F, et al. The HIV-1 HLA-A2-SLYNTVATL is a help-independent CTL epitope. Journal of Immunology 2004; 172: 5249–5261.
9. Arlen P, Tsang KY, Marshall JL, et al. The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol Immunother. 2000; 49: 517-529.
10. Linnemann T. Wiesmüller KH, Gellrich S. et al. A T-cell epitope determined with random peptide libraries and combinatorial peptide chemistry stimulates T cells specific for cutaneous T-cell lymphoma. Annals of Oncology 2000; 11, Supplement 1,95-99.
11. Von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clinical Cancer Research 2000; 6: 2219-2228.
12. Vella AT, Dow S, Potter TA, et.al. Cytokineinduced survival of activated T cells in vitro and in vivo (interleukin 4/interleukin 2/interleukin 7/interleukin 15). Proc. Natl. Acad. Sci. USA. 1998; 95: 3810-3815.
13. Cornish GH, Sinclair LV, and Cantrell DA. Differential regulation of T-cell growth by IL-2 and IL-15. Blood. 2006; 108: 600-608.