Bellone M, Ostlie N, Karachunski P, Manfredi A A, Conti-Tronconi B M
Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul 55108.
J Immunol. 1993 Jul 15;151(2):1025-38.
Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.
通过注射电鳐烟碱型乙酰胆碱受体（TAChR）在C57BL/6小鼠中诱导实验性自身免疫性重症肌无力。我们在此研究TAChR分子上隐蔽性CD4 +表位的存在，以及它们与存活于克隆清除后的潜在自身反应性CD4 +细胞的关系。用天然或变性TAChR免疫的C57BL/6小鼠的CD4 +细胞，在体外与20个氨基酸残基长的重叠合成肽进行反应，筛选TAChRα、γ和δ亚基的序列。仅α亚基上的三个表位被一致识别。用大剂量（纳摩尔）TAChR免疫的小鼠仅清楚地识别免疫显性序列Tα150 - 169。抗TAChR CD4 +细胞与小鼠α亚基序列或与人γ和δ亚基的任何合成序列均无交叉反应，而人γ和δ亚基与相应的小鼠亚基非常相似。为便于识别隐蔽性表位，我们给小鼠注射与TAChRα、γ和δ亚基序列相对应的合成肽池。除了三个免疫显性α亚基表位外，在序列Tα304 - 322、Tγ105 - 124、Tγ120 - 139、Tγ401 - 420、Tγ357 - 376、Tδ16 - 35、Tδ61 - 80、Tδ121 - 140和Tδ301 - 320内的其他表位也被CD4 +细胞识别。如此致敏的CD4 +细胞与哺乳动物序列α304 - 322、γ105 - 124、γ120 - 139和δ301 - 320发生交叉反应。用大剂量（约40纳摩尔）的单个TAChR合成隐蔽性表位免疫小鼠。对五个隐蔽性表位（上述加上δ121 - 140）致敏的CD4 +细胞与自身序列发生交叉反应。我们通过用递增剂量的肽（约1.2至约20纳摩尔）免疫小鼠，并检测CD4 +细胞的体外抗肽反应，确定了体内CD4 +细胞对强免疫显性表位肽Tα150 - 169以及隐蔽性表位肽Tγ120 - 139和Tδ301 - 320致敏的剂量依赖性。所测试的表位之间未发现差异。3至10微克的剂量诱导强烈的CD4 +致敏，并且致敏细胞对相关肽的体外反应的剂量依赖性相当。通过用天然TAChR检测肽致敏的CD4 +细胞，研究了体外TAChR加工过程中隐蔽性表位的产生：仅产生了两个隐蔽性表位。
