本論文的研究目的為探討poly(lactic-co-glycolic acid)(PLGA)奈米粒子透過小鼠骨髓中的Lin2-CD11b+Gr-1highLy-6Clow細胞(Gr-1high)與Lin2-CD11b+Gr-1low -Ly-6Chigh細胞(Gr-1low),交叉呈現PLGA奈米粒子之抗原所造成OT-1 CD8+ T細胞的影響,以及使用500 nm polystyrene Yellow-Green Fluoresbrite microspheres (YG-MP)來研究的生體分佈動力學。 我們發現Gr-1high與Gr-1low細胞可在in vitro環境將PLGA奈米粒子內包覆之ovalbumin (OVA)抗原,經由交叉呈現使OT-1 CD8+ T細胞產生增生反應、表現CD25與CD69、分泌IL-2、TNF-α、IFN-γ等活化反應,並且可在in vitro環境使OT-1 CD8+ T細胞活化為殺手型T細胞,產生perforin與granzyme B,並進行專一性細胞毒殺反應。此外我們也發現Gr-1low細胞的交叉呈現能力比Gr-1high細胞更好。將PLGA/OVA NPs注射到小鼠體內後,也會產生抗原專一性免疫反應,包括對OVA專一性IgG1、IgG2a、IgG2b抗體的產生、以及OT-1 CD8+ T細胞的增生反應。YG-MP在小鼠體內生體分佈的結果顯示在血液與骨髓中最主要吞噬YG-MP的細胞為B220-CD11b+Gr-1highLy-6Clow顆粒球,在脾臟則為B220+CD11b- B細胞。YG-MP主要分佈在marginal zone以及紅髓區(red pulp)。 綜合以上結果,我們確認了Gr-1+細胞,包括Gr-1high與Gr-1low細胞,具有交叉呈現PLGA/OVA NPs的抗原的作用。
We attempt in this study to investigate the effect of ovalbumin (OVA) encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on cross-presentation of soluble antigens, mediated by Gr-1+ cells from mouse bone marrow, including Lin2‾CD11b+Gr-1highLy-6Clow (Gr-1high) and the Lin2‾CD11b+Gr-1lowLy-6Chigh (Gr-1low) subpopulations, to OT-1 CD8+ T cells. We also examined the biodistribution kinetics of 500 mm polystyrene nanoparticles, Yellow-Green Fluoresbrite microspheres (YG-MPs), in the mouse immune system by flow cytometry. Our results showed that PLGA/OVA NPs-primed Gr-1+ cells stimulated the proliferation of OT-1 CD8+ T cells in vitro, whereas Gr-1low cells exhibited higher capability of antigen presentation than that of Gr-1high cells. Treatment of Gr-1+ cells with PLGA/OVA NPs upregulated the expression of activation markers CD25 and CD69, as well as the pro-inflammatory mediators, such as IL-2, TNF-α, and IFN-γ. PLGA/OVA NPs-primed Gr-1+ cells also induced the intracellular expression of perforin and granzyme B, and promoted antigen-specific cytotoxic lymphocyte (CTL) effect in vitro. Injection of mice with PLGA/OVA NPs induced the production of antigen-specific antibodies and stimulated the proliferation of OT-1 CD8+ T cells in vivo. Flow cytometric analysis showed that the major phagocytes of YG-MPs are B220‾CD11b+Gr-1highLy-6Clow granulocytes in the blood and bone marrow, and B220+CD11b‾ B cells in the spleen. Immunostaining and microscopic examination illustrated that YG-MPs are mainly distributed in the marginal zone and red pulp of the spleen. In summary, this study demonstrated that treatment of Gr-1+ cells with PLGA/OVA NPs induced cross-presentation of soluble antigens both in vitro and in vivo.