研究CD43在CD8+ T 淋巴球活化過程中之作用

Study of the function of CD43 in CD8+ T lymphocytes activation

劉雅惠(Yae-Huei Liou)

指導教授 ： 廖南詩 博士

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2003年)

摘要

CD43是一種穿膜醣蛋白，大量表現在T淋巴球的表面上。先前的研究認為CD43參與T細胞的活化，但其為促進或抑制活化的角色，仍具爭議。在本實驗室先前的研究中，利用固定抗體在細胞培養盤上的刺激方式刺激T細胞，發現在給予抗T細胞受體及IL-2的情況下，抗CD43抗體的刺激會進一步增加CD8+ T淋巴球的增殖，卻不影響CD4+ T淋巴球的增殖，建議CD43在CD8+ T 淋巴球的活化過程中有輔助功能。此外，在脾臟CD8+ T淋巴球上CD43的表現量高於CD4+ T淋巴球，CD43的特殊表現模式及其對CD8+ T淋巴球增殖的可能影響，促使我進一步研究CD43在CD8+ T淋巴球活化中的角色。我的實驗設計是以非抗CD43抗體刺激的方式來研究CD43在CD8+ T淋巴球活化過程中的作用。首先，我利用非抗原專一性的體外刺激方式，以除去T細胞的正常脾臟細胞外加不同濃度抗CD3抗體來刺激CD8+ T淋巴球，分別比較野生型小鼠及CD43基因剔除(CD43-/-)小鼠CD8+ T淋巴球的增生程度，發現在低濃度抗CD3抗體刺激下，野生型CD8+ T淋巴球的增生程度高於CD43-/-CD8+ T淋巴球。然而在試混合淋巴球反應(mixed lymphocytes reaction, MLR)中，CD43的缺損並不會降低CD8+ T 淋巴球的活化以及增殖。此外，利用專一性抗原(antigen-specific)??鏈及骨髓衍生性樹突細胞(BM-Dc)刺激CD8+ T淋巴球，我們發現在不成熟BM-Dc刺激下，可以偵測到CD43輔助的作用，但是在成熟BM-Dc刺激下則否。我進一步利用外加CTLA-4-IgFc的方式來證明mature BM-Dc與immature BM-Dc作用的差異，並非源自mature BM-Dc上大量B7分子的作用遮蔽住CD43促進CD8+ T淋巴球增殖的功能，而且在本文測試系統下，CD43的作用無法獨立於CD28與B7所造成的costimulation之外。 先前的研究指出，在CD4+ T淋巴球與抗原呈現細胞相互作用時，CD43會被排除在兩個細胞所共同形成的免疫突觸(Immunological Synapse, IS)結構之外，但在CD8+ T淋巴球上則是未知。我在OT-1 TCR transgenic系統下以專一性抗原刺激CD8+ T淋巴球活化，觀察CD43的分佈，卻發現有超過半數的活化CD8+ T淋巴球，其CD43與TCRb共同座落在兩細胞所形成的交界處，這也意味著CD43可能參與CD8+ T淋巴球的活化。

關鍵字

CD43 ； 免疫突觸 ； 骨髓衍生性樹突細胞

並列摘要

CD43 is a transmembrane sialogycoprotein expressed on the surface of a variety of hematopoietic cell, including T lymphocyte. Previous studies suggested that CD43 might be involved in T lymphocyte activation. Both positive and negative effects of CD43 are reported, but its definite function remains controversial. Our earlier study showed that costimulation of naïve CD8+ T lymphocytes with plate-bound a-CD43 monoclonal antibody significantly enhances the proliferation response to TCR stimulation in the presence of exogenous IL-2. This result suggests that CD43 might help the activation of CD8+ T lymphocytes. Others and we also found that the expression of CD43 in splenic CD8+ T lymphocytes is uniformly higher than that on CD4+ T lymphocytes. Three results prompted me to further investigate the role of CD43 in the activation of CD8+ T lymphocytes without using a-CD43 antibody stimulation. Three stimulation systems were used in my study. Firstly, CD8+ T lymphocytes were stimulated with T lymphocyte-depleted splenocytes plus various amounts of anti-CD3 antibody. We found that the proliferation of wild type CD8+ T lymphocytes was higher than that of CD43-/- CD8+ T lymphocytes at low concentrations of anti-CD3 antibody. Secondly, CD8+ T lymphocytes were stimulated with allogeneic spleen cells. We found that the deficiency of CD43 did not attenuate the proliferation of CD8+ T lymphocytes. Thirdly, by using OT-1 TCR transgenic system, the CD8+ T lymphocytes were stimulation by antigen-specific peptide-pulsed dendritic cells. We found that the proliferation of wild type CD8+ T lymphocytes was higher than that of CD43-/- CD8+ T lymphocytes. Since CD43 is a large and highly negatively charged protein that extends in a linear conformation outward from the cell membrane, it was suggested that CD43 might function as a barrier for T lymphocyte-APC interaction. Several studies demonstrated that CD43 was excluded from the immunological synapse between CD4+ T lymphocyte and APC. We observed that the exclusion of CD43 from the T/APC contact site during CD8+ T lymphocyte activation is not obligatory in OT-1 TCR transgenic system. These results suggest that CD43 may play a positive role in regulation of CD8+ T lymphocyte activation.

並列關鍵字

CD43 ； Immunological synapse ； BM-Dc

參考文獻

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