ObjectiveThe DRB1 locus is strongly associated with both susceptibility and resistance to rheumatoid arthritis (RA). DRB1 alleles encoding the VKA or VRA epitope in positions 11, 71 and 74 confer the highest risk of developing RA, while the allele encoding VEA is protective. We therefore investigated the feasibility of creating antigen‐specific tolerance without inducing alloreactivity by replacing lysine with glutamic acid at position 71 in DRβ1*04:01.Materials and MethodsIndividual DRB1 alleles and the DRB1*04:01K71E allele were cloned into T2 cell lines to measure binding of biotinylated peptides. Transgenic animals expressing DRB1*04:01, DRB1*01:01 or DRB1*04:01K71E were injected with collagen to measure T cell proliferation. Skin and bone marrow transplants between DRB1*04:01K71E and DRB1*04:01 mice were performed to determine if the single amino acid change at position 71 would be recognized as foreign. DRB1*04:01 mice transplanted with DRB1*04:01K71E bone marrow were injected with collagen to test if resistance to collagen sensitization could be transferred.ResultsReplacing lysine (K) at position 71 in DRβ1*04:01 with glutamic acid (E) blocked collagen peptide binding and rendered the DRB1*04:01K71E mice resistant to collagen sensitization. Skin and bone marrow transplants from DRB1*04:01K71E mice were not rejected by DRB1*04:01 mice, suggesting the single E71 difference was not recognized as allogeneic. Bone marrow from DRB1*04:01K71E mice adoptively transferred antigen‐specific tolerance to collagen to DRB1*04:01 mice.ConclusionThese studies demonstrate that editing a single amino acid in DRβ1*04:01 blocks collagen peptide binding without inducing alloreactivity and could therefore represent a gene therapy approach to induce antigen‐specific passive tolerance.

中文翻译：

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在 DRβ1*04：01 的第 71 位取代谷氨酸可诱导胶原蛋白特异性耐受，而不会诱导同种异体反应


目的DRB1 位点与类风湿性关节炎 （RA） 的易感性和耐药性密切相关。编码 VKA 或 VRA 表位的 DRB1 等位基因位于第 11 、 71 和 74 位，发生 RA 的风险最高，而编码 VEA 的等位基因具有保护性。因此，我们研究了通过在 DRβ1*04：01 中用谷氨酸取代赖氨酸来产生抗原特异性耐受性而不诱导同种异体反应的可行性。将单个 DRB1 等位基因和 DRB1*04：01K71E 等位基因克隆到 T2 细胞系中，以测量生物素化肽的结合。向表达 DRB1*04：01、DRB1*01：01 或 DRB1*04：01K71E 的转基因动物注射胶原蛋白以测量 T 细胞增殖。在 DRB1*04：01K71E 和 DRB1*04：01 小鼠之间进行皮肤和骨髓移植，以确定第 71 位的单个氨基酸变化是否会被识别为外来氨基酸。向移植 DRB1*04：01K71E 骨髓的 DRB1*04：01 小鼠注射胶原蛋白，以测试是否可以转移对胶原蛋白致敏的抵抗力。结果用谷氨酸 （E） 取代 DRβ1*04：01 第 71 位的赖氨酸 （K） 阻断了胶原蛋白肽的结合，使 DRB1*04：01K71E 小鼠对胶原蛋白致敏产生抗性。DRB1*04：01K71E 小鼠的皮肤和骨髓移植未被 DRB1*04：01 小鼠排斥，表明单个 E71 差异未被识别为同种异体。来自 DRB1*04：01K71E 小鼠的骨髓将对胶原蛋白的抗原特异性耐受性过继转移到 DRB1*04：01 小鼠。结论这些研究表明，在 DRβ1*04：01 中编辑单个氨基酸可阻断胶原蛋白肽结合而不诱导同种异体反应，因此可能代表一种诱导抗原特异性被动耐受的基因治疗方法。