T cell receptor beta chain (TCRβ) diversity (Dβ) gene segments are highly conserved across evolution, with trout Dβ1 sequence identical to human and mouse Dβ1. A key conserved feature is enrichment for glycine in all three Dβ reading frames (RFs). Previously, we found that replacement of mouse Dβ1 with a typical immunoglobulin D_H sequence, which unlike Dβ is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRβ complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dβ sequence in place of glycine would also influence T cell biology, we targeted the TCRβ locus with a novel glycine-deficient DβDKRQ allele that replaces Dβ1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DβDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of β selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer’s patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dβ sequence is used to shape the pre-immune TCRβ repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.

T 细胞受体 β 链 (TCRβ) 多样性 (Dβ) 基因片段在进化过程中高度保守，鳟鱼 Dβ1 序列与人类和小鼠 Dβ1 相同。一个关键的保守特征是所有三个 Dβ 阅读框 (RF) 中甘氨酸的富集。_{之前，我们发现用典型的免疫球蛋白 D H}序列替换小鼠 Dβ1 （与 Dβ 不同，富含酪氨酸）会导致胸腺选择后 TCRβ 互补决定区 3 (CDR-B3) 中酪氨酸的使用增加，从而改变T 细胞数量、CDR-B3 多样性和 T 细胞功能。为了测试将带电氨基酸掺入到 Dβ 序列中代替甘氨酸是否也会影响 T 细胞生物学，我们用一种新型甘氨酸缺陷型 D β DKRQ 等位基因靶向 TCRβ 基因座，该等位基因用带电氨基酸取代了所有Dβ1编码序列。三个阅读框。使用D β DKRQ开发 T 细胞，表达的 TCR CDR-B3 耗尽了酪氨酸和甘氨酸，并富含种系编码的赖氨酸、精氨酸和谷氨酰胺。在从 DN3bc 到 DN4 阶段过渡期间发生的 β 选择过程中，总胸腺细胞数量下降。在所有随后的胸腺阶段和脾脏中，常规胸腺细胞和 T 细胞数量仍然减少。相比之下，派尔氏淋巴结和大肠中的调节性 T 细胞数量有所增加。就功能后果而言，T 细胞对卵清蛋白免疫显性表位的反应性降低。这些发现支持了这样的观点，即 Dβ 序列的自然选择用于塑造免疫前 TCRβ 库，影响常规 T 细胞和调节性 T 细胞的发育，并影响表位识别。