研究單一過敏原誘發口服耐受性抑制鍵結過敏原致敏小鼠呼吸道發炎反應的免疫調節機轉

Abstract

口服耐受性被認為由數種機制達成，包含特異性T細胞凋亡、特異性T細胞無應性、特異性調節性T細胞生成，然而詳細完整的機制尚未了解透徹。本研究目的為探討在多種過敏原致敏的個體，透過餵食單一過敏原而誘發單一特異性口服耐受性，是否具調節不同特異性過敏反應的功能。本實驗利用化學鍵結兩種實驗常用抗原—卵清蛋白 (ovalbumin) 和 β-乳球蛋白 (β-lactoglobulin)，以此鍵結抗原 (BLG-conjugated OVA，B-O) 致敏小鼠，探究餵食其中一種抗原，是否能保護小鼠免於另一抗原誘發的呼吸道發炎反應。實驗發現餵食卵清蛋白有效避免β-乳球蛋白誘發的抗原特異性反應，包含血清中特異性免疫球蛋白E (BLG-specific IgE)、呼吸道過度反應 (airway hyperresponsiveness)、肺部細胞浸潤、呼吸道發炎反應。餵食β-乳球蛋白也可減緩卵清蛋白誘發的過敏反應。利用帶有單一T細胞受體小鼠 (DO11.10 mouse)，發現餵食卵清蛋白可增進CD4+CD25+ T細胞的非抗原特異性抑制功能。轉送帶有卵清蛋白短鏈 (OVA323-339 peptide) 的B細胞到DO11.10小鼠，可促使脾臟細胞和脾臟CD4+ T細胞對卵清蛋白短鏈產生無應性，暗示脾臟B細胞可能呈獻卵清蛋白短鏈誘發調節性T細胞生成，而參與口服耐受性。口服抗原活化的CD4+CD25+ T細胞與B細胞誘發的調節性T細胞 (Treg-of-B cells) 具數個相似處，包含兩者皆表達誘導T細胞共刺激分子 (inducible T-cell co-stimulator，ICOS) 和程序性細胞死亡蛋白質1 (programmed cell death 1，PD1)、分泌介白素10 (interleukin-10，IL-10) 和轉化生長因子β (transforming growth factor-β，TGF-β)、具有非抗原特異性抑制功能。此結果暗示脾臟B細胞誘發之調節性T細胞，有可能參與在口服耐受性複雜的非特異性抑制機制中。單一過敏原產生的口服耐受性具潛力調節相關性過敏原的不當反應，此效果可見於調節性T細胞與不當反應的特異性T細胞共同存在的環境中，在鍵結抗原誘發的過敏性氣喘模式中，此非特異性調節潛力可更清楚呈現。 另外，我們探究重複給予呈獻抗原的B細胞，對B細胞誘發的調節性T細胞 (Treg-of-B cells) 生成的影響。我們發現在第三輪B細胞誘發的調節性T細胞 (Treg-of-B3rd cells) 中，表現介白素10 (IL-10) 的細胞比例增加，介白素的表達量也有增加。同時，介白素10也在第三輪B細胞誘發的調節性T細胞的抑制功能中扮演角色。第三輪B細胞誘發的調節性T細胞和B細胞誘發的調節性T細胞相比，仍然維持表達誘導T細胞共刺激分子 (ICOS)、程序性細胞死亡蛋白質1 (PD1)、毒殺T淋巴球相關抗原4 (cytotoxic T-lymphocyte associated antigen 4, CTLA4) 和抑制功能。我們也發現主要表達介白素10的細胞群，大多侷限在表達肌肉腱膜纖維肉瘤癌基因同源物 (avian musculo-aponeurotic fibrosarcoma oncogene homolog, c-Maf) 的細胞群中。在培養過程中，介白素10、、介白素4、轉化生長因子β、介白素27都有逐漸增加的趨勢。此結果顯示，多次給予呈獻抗原的B細胞具有能力誘發T細胞成為介白素10表達的細胞，可能透過可能透過介白素27與和轉化生長因子β的調控。 總結地說，我們的研究成果顯示B細胞誘發的調節性T細胞，具有非抗原特異性抑制功能，而且可能參與在複雜的口服耐受性機制中。另一部分，多次給予呈獻抗原的B細胞具有能力，透過活化肌肉腱膜纖維肉瘤癌基因同源物使T細胞成為表達介白素10的細胞。在未來研究，會著重在肌肉腱膜纖維肉瘤癌基因同源物的表達與否和B細胞誘發的調節性T細胞的抑制能力是否有關，以及肌肉腱膜纖維肉瘤癌基因同源物是否有協同其他蛋白共同調控介白素10的基因表現 。

Although oral tolerance has been considered the result of a number of protective mechanisms, the real mechanisms of oral tolerance are yet to be defined. The present study was aimed to determine whether a single allergen-induced oral tolerance would affect the immune responses to other related allergens that were primed together with the orally treated allergen. We investigated whether oral administration of a single antigen would protect the conjugated antigen-sensitized mice from the other antigen induced airway inflammation. We found that oral administration of ovalbumin (OVA) was able to prevent the immune responses to β-lactoglobulin (BLG) in BLG-conjugated OVA (B-O) sensitized mice and vice versa. Oral administration of antigen enhanced the suppressive potential of CD4+CD25+ T cells to inhibit non-antigen-specific T cell proliferation. The B cell-induced regulatory T (referred to as Treg-of-B) cells showed several similarities with orally treated CD4+CD25+ T cells, including the expression of inducible T-cell co-stimulator (ICOS) and programmed cell death 1 (PD1), the production of interleukin (IL)-10 and transforming growth factor (TGF)-β, and the non-antigen-specific suppressive ability, and therefore might play a role in the sophisticated mechanism of oral tolerance. The results suggested that single allergen induced oral tolerance had the ability to modulate the immune responses to other related allergens and was able to be more clearly detected in our conjugated antigen-induced asthmatic model. In addition, we investigated the effect of re-encounter the antigen-presenting B cells on the generation of Treg-of-B cells. We found that the IL-10-producing population and production of IL-10 increased in Treg-of-B3rd cells. IL-10 also played a role in the suppressive function of Treg-of-B3rd cells. Treg-of-B3rd cells maintained expressions of ICOS, PD1, and cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and the suppressive abilities as that of Treg-of-B1st cells. The major population of IL-10-producing Treg-of-B3rd cells was confined to the avian musculo-aponeurotic fibrosarcoma oncogene homolog (c-Maf)-expressing population. The levels of IL-10, IL-4, IL-27, and TGF-β increased gradually during the generation of Treg-of-B3rd cells. These results suggested that re-encounter the antigen-presenting B cells converted CD4+CD25- T cells into IL-10-producing Treg-of-B3rd cells and the pathway of IL-27/TGF-β-c-Maf might be involved. In summary, our results suggested that Treg-of-B cells had a non-antigen-specific suppressive function and might play a role in the sophisticated mechanism of oral tolerance. Moreover, re-encounter the antigen-presenting B cells induced IL-10-producing CD4+ Treg-of-B3rd cells via activation of c-Maf. In the future, the upstream signaling activated generation of IL-10-producing Treg-of-B cells, the relationship between c-Maf and the suppressive function of Treg-of-B cells required further research and the binding proteins of c-Maf in the regulation of IL-10 or other genes required more investigations as well.