CBP絲胺酸磷酸化在輔助型T細胞分化與iNKT細胞產生細胞激素中所扮演的角色

Abstract

CREB (cAMP response element-binding protein) 結合蛋白 (CBP) 是轉錄共活化因子，其參與了許多蛋白質的交互作用。CBP的基因突變或功能缺陷被證實與魯賓斯坦-泰必氏綜合症（Rubinstein-Taybi Syndrome；RTS）及急性骨髓性白血病等疾病有相關聯性。利用小鼠模式證實在胸腺細胞中條件式基因剔除CBP會降低週邊血中的CD4+ 和CD8+ T 細胞數。此外，胸腺CBP條件式基因剔除會導致CD8+ T細胞展現出作用型、記憶型或先天性免疫T細胞的特性，並且在接受刺激後快速地產生干擾素伽瑪(IFN-γ)。先前研究證實IκB磷酸激酶複合體(kinase complex)中的一個蛋白單體IKKα會磷酸化CBP上1382和1386位置的絲胺酸，進而改變CBP的結合傾向由NF-κB至p53。然而，CBP絲胺酸磷酸化在輔助型T細胞分化與iNKT細胞產生細胞激素中所扮演的角色仍未被深入探討。 在本研究中，我們利用CBP絲胺酸磷酸化突變小鼠(CBPS1383/1387Am/m; AA)證實野生型(WT)及AA小鼠腸繫膜淋巴結輔助型T細胞在經過刺激後所產生的細胞激素並沒有顯著差異。WT和AA小鼠的處女T細胞分化成第一型和第二型輔助型T細胞的能力也相似。這些結果顯示CBP的突變並不會影響輔助型T細胞的分化。此外，WT及AA小鼠脾臟、週邊淋巴結及腸繫膜淋巴結中的iNKT細胞族群及次族群沒顯著差異，暗示CBP突變不會影響iNKT細胞的發育及分化。在α-GalCer的刺激下，WT和AA小鼠iNKT細胞所產生的干擾素伽瑪 (IFN-γ)，介白素-4 （IL-4）及介白素-17 (IL-17) 也不具顯著差異。總結來説，我們的研究顯示CBP絲胺酸磷酸化的突變並沒有參與輔助型T細胞的分化及iNKT細胞產生細胞激素的調控。

半乳糖凝集素-3 (Galectin-3) 是含有碳水化合物識別區 (CRD) 的半乳糖苷結合蛋白質，參與了許多免疫細胞的調控，其中包括了巨噬細胞、單核細胞、嗜中性白血球、T細胞及B細胞。Galectin-3的功能十分多樣化，如參與細胞活化、黏附、遷移、去顆粒、細胞凋亡及細胞激素的分泌。然而，Galectin-3在不變的自然殺手T細胞 (iNKT cells) 中調控免疫反應及細胞激素產生所扮演的角色尚未釐清。在被刺激的情況下，iNKT細胞會很迅速的產生細胞激素如干擾素伽瑪 (IFN-γ)、腫瘤壞死因子 (TNF)、介白素 (Interleukin) IL-2、IL-3、IL-4、IL-5、IL-9、IL-10、IL-13、IL-17、IL-21及顆粒球巨噬細胞株刺激因子（GM-CSF）。此外，不同的iNKT次族群如第一型iNKT細胞 (iNKT1)、第二型iNKT細胞 (iNKT2)及第十七型iNKT細胞 (iNKT17) 負責產生不同的細胞激素。 在本研究中，我們欲探討Galectin-3在iNKT細胞中細胞激素產生所扮演的角色。結果顯示在α-GalCer的刺激下，野生型 (WT) 及半乳糖凝集素-3缺失型 (Gal-3 KO) 小鼠的胰臟、週邊淋巴結及腸繫膜淋巴結的iNKT細胞所產生的干擾素伽瑪 (IFN-γ)、介白素-4（IL-4）及介白素-17 (IL-17) 皆相似不具顯著差異，除了Gal-3 KO小鼠腸繫膜淋巴結iNKT細胞比WT iNKT細胞產生較少的IFN-γ。此外，我們的結果也顯示WT及Gal-3 KO小鼠脾臟、週邊淋巴結及腸繫膜淋巴結iNKT細胞的次族群在統計上沒有顯著差異。 總結來說，我們的研究結果顯示Galectin-3不影響iNKT細胞產生細胞激素。然而，我們不排除Galectin-3調控iNKT細胞其他功能的可能性。

CREB (cAMP response element-binding protein) binding protein (CBP) is a transcriptional coactivator that plays an important role in the protein-protein interaction. Genetic alteration or functional inactivation of CBP had been showed to associate with Rubinstein-Taybi syndrome (RTS) and acute myeloid leukemia. In mouse model, condition knock-out of CBP in thymocytes showed reduced number of peripheral blood CD4+ and CD8+ T cells. Besides, condition knock-out of CBP in thymus results in CD8+ thymocytes with effector-, memory-, or innate-like T-cell phenotype and producing the effector cytokine IFN-γ rapidly upon stimulation. Previous study showed that subunit of IκB kinase complex, IKKα can phosphorylate CBP at Ser-1382 and Ser-1386 and switch the CBP binding preference from NF-κB to p53. However, the roles of CBP serine phosphorylation in TH cell differentiation and iNKT cell cytokine production remain unclear. In this study, CBP serine phosphorylation mutant mice (CBPS1383/1387Am/m; AA) are employed and we found out that cytokines production by T helper cells in mesenteric lymph nodes (mLNs) of WT mice and AA mice were comparable after stimulation. Furthermore, the abilities of naïve T cells to differentiate into TH1 and TH2 cells were similar in WT and AA mice. These results together indicated that CBP mutant does not affect the TH cell differentiation. Furthermore, population and subpopulation of iNKT cells in the spleen (SP), peripheral lymph nodes (pLNs) and mesenteric lymph nodes (mLNs) of WT and AA mice were comparable, indicated that CBP mutant did not affect the development and differentiation of iNKT cells. Upon α-GalCer stimulation, IFN-γ, IL-4 and IL-17 produced by iNKT cells in WT and AA mice were comparable. In conclusion, our study showed that CBP serine phosphorylation mutant does not play a role in the T helper cell differentiation and the cytokine production by iNKT cells.

Galectin-3 (Gal-3), a β-galactoside-binding animal lectin carrying carbohydrate-recognition domains (CRD) is involved in the regulation of many immune cells including macrophage, monocyte, neutrophils, T and B lymphocytes. Functions of Gal-3 are diverse such as cell activation, adhesion, migration, degranulation, apoptosis and cytokine secretion. However, the role of Gal-3 in invariant natural killer T (iNKT) cells-mediated immune responses and cytokine production is not yet elucidated. Upon stimulation, iNKT cells rapidly produce variety of cytokines including IFN-γ, tumor necrosis factor (TNF), IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21 and GM-CSF. Moreover, different subsets of iNKT cells, i.e. iNKT1, iNKT2 and iNKT17, produce distinct cytokines. In this study, I aim to clarify the role of Gal-3 in cytokine production by iNKT cells. Our results demonstrate that upon α-GalCer stimulation, IFN-γ, IL-4 and IL-17 produced by iNKT cells in spleen (SP), peripheral lymph nodes (pLNs) and mesenteric lymph nodes (mLNs) of wild type (WT) mice and galectin-3 knock out (Gal-3 KO) mice are comparable except in mLN of Gal-3 KO mice, which produced less IFN-γ compared to WT mice. Furthermore, our data indicate that similar pattern of iNKT subpopulation in the SP, pLN and mLN of WT and Gal-3 KO mice after stimulation. This work indicates that Galectin-3 do not play a role in iNKT cell cytokine production. However, we do not exclude the possibility that there is a regulatory role in iNKT cells mediated by Galectin-3.