Lymphocyte proliferation in common variable immunodeficiency (CVID) patients by carboxyfluorescein succinimidylester (CFSE)

Year 2011, Volume: 1 Issue: 3, 171 - 177, 31.01.2014

Ayşegül İzgi Tunç Akkoç İsmail Öğülür Ayzer Tevetoğlu Elif Karakoç Aydıner Safa Barış Nerin Bahçeciler Işıl Barlan

Abstract

Objective: Common variable immunodeficiency (CVID) is a heterogeneous disease in the group of predominantly antibody deficiencies, which is defined by hypogammaglobulinemia and normal or low level of B cells, and characterized by increased susceptibility to recurrent bacterial infections, autoimmune disorders, and malignancies. In this study, we aimed to investigate the proliferation of the B and T lymphocytes in patients with CVID.
Method: The proliferation of CD19 + , CD21 + , CD27 + B cells, and CD3 + , CD4 + , CD8 + T cells were investigated by Carboxyfluorescein Succinimidyl Ester (CFSE) method. CVID patients (n=13) and control group (n=5) were enrolled in the study. B and T cells were isolated from periferal blood samples with the negative selection procedure called Rosette Sep andthe isolated lymphocytes were stained in dark using CFSE. The cells were stimulated with anti- CD2, CD3, CD28 (CDmix) and cultured for four days in order to determine T cell proliferation. The proliferation of CD3 + , CD4 + , CD8 + T cells was analyzed by flow cytometry. Isolated B cells were cultured for four days along with anti-IgM+IL-4. The proliferation of CD19 + , CD27 + and CD21 + B cells were analyzed with flow cytometry. 
Results: First proliferation of T lymphocytes and CD19 + B lymphocytes and second proliferation of CD4 + T lymphocytes after stimulation were increased in CVID patients compared to healthy controls. 
Conclusion: In CVID patients T cell and B cells proliferation seem to be normal.

Key words: CVID, CFSE, Lymphocytes proliferation, T lymphocyte isolation, B lymphocyte isolation

Keywords

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Sık değişken immün yetmezlik hastalarında Carboxyfluorescein succinimidylester (CFSE) yöntemi ile lenfosit proliferasyonu

Abstract

Amaç: Sık Değişken İmmün Yetmezlik (SDİY); hipogamaglobülinemi ve normal / düşük B hücre ile tanımlanan antikor eksikliği hastalıkları grubundandır ve tekrarlayan bakteriyel infeksiyonlar, otoimmün hastalıklar ve malignite ile karakterize edilmektedir. Bu çalışmada, SDİY hastalarındaki T ve B lenfositlerinin proliferasyonlarının incelenmesi amaçlanmıştır.
Yöntem: T ve B hücre alt gruplarında hastalığın immünopatogenezinde önemli rol oynadığı düşünülen CD19 + , CD21 + , CD27 + B hücreleri ve CD3 + , CD4 + , CD8 + T hücrelerinin Carboxyfluorescein SuccinimidylEster (CFSE) ile hücre proliferasyon yanıtlarına bakıldı. Çalışmaya SDİY tanısı almış hastalar (n=13) ve kontrol grubu için SDİY hastalarıyla yaş uyumu olan sağlıklı bireyler (n=5) alındı. Tüm deneklerden alınan kan örneklerine, negatif seçim yöntemi (Rosette Sep) ile T ve B hücre izolasyonu yapıldı. Daha sonra izole edilen lenfositler CFSE-FITC ile karanlık ortamda işaretlendi ve T hücre proliferasyonu için hücreler anti-CD2, anti-CD3, anti-CD28 (CDmix) ile uyarılarak 4 günlük kültürleri yapıldı. Kültür sonunda hücreler anti-CD3-PE, anti-CD4-PE, anti-CD8-APC ile boyandıktan sonra CD3 + , CD4 + , CD8 + T hücrelerinin proliferasyonu akım sitometrisi ile bakıldı. B hücre proliferasyonu için hücreler anti-IgM + IL-4 ile 4 gün kültür edildikten sonra anti-CD19-APC-Cy7, anti- CD27-PE, anti-CD21-APC ile boyandı ve CD19 + , CD27 + ve CD21 + B hücrelerinin proliferasyonu akım sitometrisi ile bakıldı. 
Bulgular: T lenfositlerinin ve CD19 + B hücrelerinin proliferasyonlarının 1. bölünmesinde ve CD4 + T hücrelerinin 2. bölünmesinde, CDmix uyaranlı SDİY’li hastalarda, CDmix uyaranlı kontrolden anlamlı olarak daha fazla prolifere olduğu gözlendi. 
Sonuç: T hücreleri ve B hücrelerinin proliferasyonlarının SDİY hastalarında normal olduğu gösterildi.

Anahtar Kelimeler : SDİY, CFSE, Lenfosit proliferasyonu, T lenfosit izolasyonu, B lenfosit izolasyonu

Keywords

SDİY, CFSE, Lenfosit proliferasyonu, T lenfosit izolasyonu, B lenfosit izolasyonu

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