Wang M, Bregenholt S, Petersen J S
Islet Discovery Research, Novo Nordisk A/S, Bagsvaerd, Denmark.
Scand J Immunol. 2003 Sep;58(3):342-9. doi: 10.1046/j.1365-3083.2003.01308.x.
The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose-dependent, with maximum at 30-300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells. Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-gamma(IFN-gamma) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner. This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.
霍乱毒素（CTB）的无毒B亚基已被用作黏膜疫苗接种实验系统中的佐剂。然而，其佐剂作用背后的机制仍不清楚。在此，我们使用体外系统来阐明抗原特异性T细胞中的这些机制。利用来自钥孔戚血蓝蛋白（KLH）免疫小鼠的脾细胞，初步实验表明，重组CTB（rCTB）对2周前免疫小鼠分离出的脾细胞的KLH特异性增殖没有影响。然而，rCTB强烈增强了4周前用KLH免疫小鼠脾细胞的KLH特异性增殖。这种佐剂作用是剂量依赖性的，在30 - 300 ng/ml rCTB时达到最大值。在更高剂量的CTB下，由于细胞凋亡的诱导，这种作用会下降。使用抗体清除和共培养系统，我们表明rCTB直接共刺激KLH特异性CD4⁺和CD8⁺ T细胞增殖，但不刺激B细胞。酶联免疫斑点（ELISPOT）分析显示，rCTB还将KLH特异性CD4⁺ T细胞介导的白细胞介素-2（IL-2）、IL-4和干扰素-γ（IFN-γ）的产生提高了四到五倍。表征rCTB在体内的佐剂作用证实了上述结果，即当rCTB在二次但不是初次KLH免疫方案中用作经典佐剂时，它介导了体外T细胞反应增加两倍。这些数据共同表明，rCTB可以通过以剂量依赖性方式直接共刺激抗原致敏的CD4⁺和CD8⁺ T细胞而作为一种强效佐剂。这一新见解可能对未来基于细菌毒素的疫苗的合理设计具有重要价值。
