Early Notch signals induce a pathogenic molecular signature during priming of alloantigen-specific conventional CD4+ T cells in graft-versus-host disease

Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 hours after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both, and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv vs. Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection, as GVHD severity was similar in recipients of wild-type Tconv combined with Notch-deprived vs. wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and T helper polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with pathogenic effects of Notch in T cells at the earliest stages of GVHD. 4 samples per cohort (Notch blockade using Dll1/4 neutralizing antibodies vs isotype control antibodies - GD) were analyzed. Additional 4 samples contained 4C T cells retrieved from syngeneic recipients.移植物抗宿主病（Graft-versus-host disease, GVHD）是异基因造血细胞移植（allogeneic hematopoietic cell transplantation）后最严重的并发症。移植后最初48小时内传导的Notch信号可促进常规T细胞（conventional T cells, Tconv）产生促炎细胞因子，并抑制调节性T细胞（regulatory T cells, Tregs）的扩增。短期Notch抑制可诱导长期的GVHD防护效应。然而，目前尚不清楚Notch阻断是通过作用于Tconv、Tregs还是二者共同介导GVHD防护，以及同种抗原特异性T细胞中存在哪些早期Notch调控的分子事件。为解决上述科学问题，我们对T细胞移植物进行工程化改造，以实现Tconv与Tregs之间的选择性Notch阻断，并评估其在小鼠体内诱发GVHD的能力。仅在Tconv中实施Notch阻断即可介导GVHD防护，因为当受体小鼠接受野生型Tconv联合Notch缺陷型Tregs的移植物时，其GVHD严重程度与接受野生型Tconv联合野生型Tregs的受体小鼠无明显差异。为探究Notch信号对体内T细胞活化早期阶段的影响，我们构建了一种由克隆性同种抗原特异性4C CD4+ Tconv介导的新型急性GVHD模型。Notch缺陷型4C T细胞的早期活化、IL-2产生、增殖及T辅助细胞极化过程均得以保留。与之相反，Notch抑制会削弱IFN-γ与IL-17的产生，减弱mTORC1与ERK1/2的活化，并损害Myc调控基因子集的转录。该独特的Notch调控特征与T细胞白血病及发育中T细胞内已知的Notch靶标仅有极少重叠，凸显了Notch信号在成熟T细胞中的特异性调控作用。本研究结果揭示了GVHD早期阶段中，与T细胞致病效应相关的独特分子程序。本队列每组均设置4份样本（使用Dll1/4中和抗体进行Notch阻断组 vs 同型对照抗体组——GD），并完成了检测分析。另有4份样本为从同基因移植受体小鼠体内回收的4C T细胞。