研究誘發Treg-of-B細胞和其抑制活性的機制

Abstract

過去研究指出，B 細胞在免疫系統上能扮演相向調控的角色。而近年來也不斷有文獻指出傳統B-2 細胞能有效提呈抗原給T細胞，並與CD4+CD25- T細胞共同培養之下，B-2細胞能活化以及誘導T細胞成為調節性T細胞，名為Treg-of-B2細胞，以達致免疫調控之效。該調節性T細胞的生成已經證實需要透過CD4+CD25- T細胞和B-2細胞之間的相互接觸，但誘發Treg-of-B2細胞和其抑制活性的機制仍有待釐清。已知活化T細胞需要透過T細胞上的T-細胞受器和抗原提呈细胞上的主要組織相容抗原決定位結合，另外T細胞表面上的共激分子與抗原提呈细胞表面上相應體的結合，則促使T 細胞進一步的活化。再者，細胞因子對於T細胞分化、成熟和功能上扮演著不可決少的角色，它能指導CD4+CD25- T 細胞成為不同的亞群:輔助性細胞或調節性細胞，而細胞因子信號是經由Janus激酶和信號轉導及轉錄激活(STAT)蛋白傳遞的。故本研究欲透過在B-2與CD4+CD25- T細胞共同培養之下，B-2細胞呈現抗體予CD4+CD25- T 細胞時，探討阻斷T細胞表面上的共激分子與B-2細胞表面上相應體的結合會否影響Treg-of-B2的生成。此外，在B-2細胞活化以及誘導CD4+CD25- T細胞成為調節性T細胞的生成過程中和其抑制免疫反應時所受到細胞因子信號以致磷酸化STAT蛋白也一併探究。 於卵蛋白胜肽片段(OVA323-339) 加上抗原呈現細胞的抗原特異性刺激系統下，我們首先從BALB/c 老鼠的脾臟將B-2 細胞分離出來，以OVA323-339加以刺激一天，再與從DO11.10 老鼠的脾臟分離出來的CD4+CD25- T 細胞共同培養三天。三天後重新分離B-2 細胞所誘導出來的CD4+CD25- T 細胞並分析其是否具有抑制能力和調節免疫特性。我們利用不同的中和抗體去阻斷CD4+CD25- T細胞表面上的共激分子與B-2細胞表面上相應體的結合，以利探討單一的共刺激信號阻断在Treg-of-B2細胞生成上的作用。另外，在探討誘發Treg-of-B2生成的過程和其抑制活性中所參與的磷酸化STAT蛋白方面，我們首先從BALB/c 老鼠的脾臟分離B-2 細胞和CD4+ CD25- T 細胞出來並在anti-CD3/CD28單株抗體刺激系統下共同培養三天。在誘發Treg-of-B2細胞生成的三天過程中，我們在特定的時間點收集Treg-of-B2細胞的細胞裂解物，以西方墨點法分析參與的磷酸化STAT蛋白。而在了解其抑制活性中所參與的磷酸化STAT蛋白方面，Treg-of-B2細胞經過三天培養後並分離出來作再度刺激，於不同的特定時間收集經過在anti-CD3/CD28單株抗體再度刺激下Treg-of-B2細胞的細胞裂解物，同樣地以西方墨點法分析參與的磷酸化STAT蛋白。 我們發現於卵蛋白胜肽片段 (OVA323-339) 抗原特異性刺激系統下，B-2細胞能成功的誘導CD4+ CD25- T 細胞為成為調節性T細胞，而該調節性T細胞 (Treg-of-B2細胞)會表現GITR、ICOS、PDL-1和LAG3並且會分泌大量的介白素-10，低量的介白素-4和γ干擾素。 另外我們證明了B-2細胞在作為抗原呈現細胞時，會表現與其他調節性T細胞生成有關的共激分子: GITRL、ICAM-1、ICOSL、CD70 和 OX40L。在單株中和抗體(anti-ICOSL, anti-ICAM-1, anti-CD70, anti-GITRL 或anti-OX40L)下，阻斷CD4+CD25- T細胞表面上的共激分子與B-2細胞表面上相應體的結合，沒能顯著地影響其生成、細胞因子的分泌以及其調節免疫效能。而我們發現在單株中和抗體 (anti-GITRL) 下生成的Treg-of-B2細胞上的GITR表達水平有明顯的下降。另一方面，在anti-CD3/CD28單株抗體刺激系統下，我們證實了在Treg-of-B2細胞生成過程中以及在其發揮抑制活性中參與的磷酸化STAT蛋白有:pStat1、pStat3、pStat5和pStat6，當中以pStat6最為顯著。 本篇的實驗結果支持了B-2細胞可以藉由誘導CD4+ CD25- T 細胞為成為調節性T細胞，以達負向調控免疫反應作用。我們首先證明了Treg-of-B2細胞並非藉由單一的共激通路去誘導CD4+CD25- T細胞成為具有抑制活性的細胞。更進一步分析，在單株中和抗體 (anti-GITRL) 下，以阻斷CD4+CD25- T細胞表面上的GITR與B-2細胞表面上GITRL的結合產生的共激信號，而生成的Treg-of-B2細胞上的GITR表達水平有明顯的下降，但因在單株中和抗體下生成的Treg-of-B2細胞仍俱有調節性功能，故推斷Treg-of-B2的GITR表達水平並沒有參與於負向調控免疫反應作用上。另一方面，Treg-of-B2細胞生成過程中以及在其發揮抑制活性中參與的磷酸化STAT蛋白有:pStat1、pStat3、pStat5和pStat6，當中以pStat6最為顯著。這研究結果使我們得知B-2細胞所誘導出的是一群獨特的調節性細胞亞群，因為它參與的磷酸化STAT蛋白有別於自然產生的調節性T細胞和其他經不同方法誘導而成的調節性T細胞。

B cells are capable to induce immunologic tolerance despite their primary role in humoral immunity. Accumulating evidence with a particular focus on defining conventional B-2 cells can serve as antigen presenting cells to convert naïve CD4+CD25- T cells into regulatory T cells (so-called Treg-of-B cells) in vitro. The B cells primed regulatory T were found to exert a suppressive function on T-cell proliferation. Previous study has already revealed that the mechanism involved in the generation of Treg-of-B cells was in a contact-dependent manner. There was a reversal of suppressive function of Tregs when B cells cultured with T cells were separated by a trans-well membrane. According to the two-signal model of T-cell activation, activation of naïve antigen-specific CD4+ T cells is thought to require at least both stimulation of the T cell receptor (TCR) (Signal 1), and stimulation of costimulatory molecules (Signal 2). The cytokine signal (Signal 3) is additional signal which plays critical role for generating a robust and specialized T-cell response. Many cytokine signaling is transmitted via Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway. In this study, we aim to (1) investigate whether costimulatory blockade by single blocking antibody could boost or dampen Treg-of-B-cell activation and (2) identify the phosphorylated STAT proteins which are involved in induction and suppressive function of Treg-of-B cells. To induce regulatory T cells stimulated by B-2 cells, naïve B cells were isolated from splenoctyes of BALB/c mice and then pulsed with OVA-peptide for a day. After that, OVA peptide-loaded B cells were co-cultured with naïve CD4+ T cells isolated from splenocytes of DO11.10 mice for 3 days without addition of exogenous cytokines. After a 3 day co-culture, CD4+ T cells were purified after depletion of B220+ cells. The suppressive function of isolated CD4+ T cells was further examined. The effect of costimulatory blockade on B cells generated regulatory T cell was examined by using single neutralizing blocking antibody. On the other hand, to study the involvement of phosphorylated STAT proteins during induction and suppressive function of Treg-of-B cells, naïve B cells were co-cultured with naïve CD4+ T cells which both isolated from splenocytes of BALB/c mice under the stimulation of anti-CD3/CD28 for 3 days. The cell lysates of CD4+ T cells and re-stimulated CD4+ T cells were harvested after depletion of B220+ cells during induction and suppressive function at various time points respectively. We found that OVA-peptide loaded B-2 cells can serve as antigen presenting cells to induce CD4+ T cells into regulatory T cells (Treg-of-B cells). We further demonstrated that B-2 cells expressed GITRL, ICAM-1, ICOSL, CD70 and OX40L as antigen presenting cells to CD4+ T cells. However, there was no reversal or enhancement of suppressive function of Treg-of-B cells on T-cell proliferation under the treatment of particular single blocking antibody during induction. Only under anti-GITR, the expression of GITR on Treg-of-B cells was significantly down-regulated. On the other hand, we identified that the phosphorylated Stat1, Stat3, Stat5 and Stat6 were involved in the induction and suppressive function of Treg-of-B cells. Our results confirmed that conventional B-2 cells can modulate immune responses through priming T cells into regulatory T cells. Also, we demonstrated that B cells generated regulatory T cells was not through single costimulatory pathways. We further demonstrated that the expression of GITR on Treg-of-B cells was not a critical molecule to exert suppressive function on T-cell proliferation. On the other hand, we have shown that phosphorylated Stat1, Stat3, Stat5 and Stat6 were involved in the induction and suppressive activity of Treg-of-B cells which is different from nTregs and other inducible Tregs. These findings might help us on identifying Treg-of-B cells into distinct subsets of Treg cells.