探討B細胞誘發調節性T細胞外泌體之抑制作用

Abstract

調節性T細胞是一群具有免疫抑制能力的T細胞。透過表達Foxp3轉錄因子，他們抑制致病性T細胞增殖或過度活化以維持免疫系統的穩定狀態。因此，調節性T細胞也被用於治療過敏、自體免疫疾病等免疫療法。在我們實驗室先前的研究中發現了當B細胞和CD4+CD25- T細胞共培養後會誘導出一群不表現Foxp3的調節性T細胞，我們將它們命名為「B細胞誘導之調節性T細胞 (Treg-of-B)」。在體外增殖的實驗中我們證明B細胞誘導之調節性T細胞具有抑制CD4+ T細胞增生的能力，我們將重心放在探討其誘發免疫抑制的機制上。首先，我們利用流式細胞儀分析分離自小鼠脾臟的B細胞及CD4+CD25- T細胞，確認細胞純度達90%以上，接著也分析了培養後的B細胞誘導之調節性T細胞標誌，結果證明我們建立了穩定且良好的培養系統用於體外生成B細胞誘導之調節性T細胞。另一方面，有研究指出調節性T細胞的外泌體參與在其免疫抑制能力中，因此，我們也想了解B細胞誘導之調節性T細胞來源之外泌體是否對抑制T細胞增殖有所貢獻。培養B細胞誘導之調節性T細胞後我們以額外的抗CD3和抗CD28抗體或是骨髓源性巨噬細胞再刺激B細胞誘導之調節性T細胞，並透過超高速離心法純化外泌體。這些外泌體除了表現特異性標誌如CD9和CD63外，也表達調節性T細胞相關抑制分子，如LAG3、PD-1和CD39，並進一步用於研究對CD4+ T細胞增生的影響。結果顯示，B細胞誘導之調節性T細胞來源之外泌體僅能些微降低Th1細胞增殖，但無法有效抑制稚幼CD4+ T細胞和Th2細胞。然而，B細胞誘導之調節性T細胞來源之外泌體被證明能顯著損害Th1細胞產生IFN-γ。這些數據支持B細胞誘導之調節性T細胞來源之外泌體在第一型免疫反應疾病中的潛在治療能力。

Regulatory T cells are a group of T cells with immunosuppressive abilities. By expressing the Foxp3 transcription factor, they perform immunoregulatory functions and suppress the proliferation or hyperactivation of pathogenic T cells to maintain homeostasis. Therefore, regulatory T cells were used as immunotherapy to treat allergies, autoimmune diseases, etc. In our previous studies, it was found that when B cells and CD4+CD25- T cells were co-cultured, it could induce a subtype of regulatory T cells which did not express Foxp3, named "B-cell-induced regulatory T cells," also called Treg-of-B cells. In in vitro proliferative experiments, we demonstrated that Treg-of-B cells have the ability to inhibit the proliferation of CD4+ T cells. We focused on exploring the mechanism of their immunosuppressive capability. First, we analyzed B cells and CD4+CD25- T cells isolated from mice spleen by flow cytometry and confirmed that the cell purity was over 90%. Then we also examined the Treg-of-B markers after culture. The results proved that we have established a stable and sound culture system for in vitro generation of Treg-of-B cells. On the other hand, some studies have pointed out that the exosomes of regulatory T cells are involved in their immunosuppressive ability. Therefore, we want to understand whether the exosomes derived from Treg-of-B cells contributed to the suppression of T cell proliferation. After culturing Treg-of-B cells, we restimulated them with plate-bond anti-CD3 and anti-CD28 antibodies or bone marrow-derived macrophages and then purified exosomes by ultracentrifugation. In addition to expression of specific signs such as CD9 and CD63, Treg-of-B cell-derived exosomes expressed regulatory T cell-related inhibitory molecules, such as LAG3, PD-1, and CD39. They were further used to investigate the effect on CD4+ T cell proliferation. The results showed that Treg-of-B cell-derived exosomes could only slightly reduce the proliferation of Th1 cells but not effectively inhibit naïve CD4+ T cells and Th2 cells. Nonetheless, Treg-of-B cell-derived exosomes were revealed to significantly impair IFN-γ production by Th1 cells. These data supported the potential therapeutic capacity of Treg-of-B cell-derived exosomes in type 1 immune diseases.