Background:Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations.Methods:Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2⁺ (C-C chemokine receptor 2) and CCR2⁻ macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection.Results:Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2⁺ and CCR2⁻ macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2⁺ and CCR2⁻ macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2⁻ macrophages reduced allograft survival, depletion of donor CCR2⁺ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2⁺ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival.Conclusions:Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2⁺ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart–based interventions.

中文翻译：

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供体巨噬细胞调节心脏移植后的排斥反应

背景：心脏移植后的细胞排斥会导致显着的发病率和死亡率。目前的免疫抑制策略并不完善，主要针对受体 T 细胞，并且会产生副作用。先天免疫反应在 T 细胞的招募和激活中发挥着重要作用。针对供体先天免疫反应将代表免疫反应级联中最早的干预机会。对心脏移植中供体免疫细胞类型和功能的了解有限，目前还没有针对这些细胞群的治疗方法。方法：利用遗传谱系追踪、细胞消融和条件基因删除，我们检查了供体单核吞噬细胞的多样性和巨噬细胞在小鼠移植心脏急性细胞排斥过程中的功能。我们在移植后的多个时间点对供体和受体的巨噬细胞和单核细胞进行了单细胞 RNA 测序。根据我们的成像和单细胞 RNA 测序数据，我们在移植前使用选择性细胞消融策略评估了供体 CCR2 ⁺（CC 趋化因子受体 2）和 CCR2 ^{-巨噬细胞的功能相关性。}最后，我们进行了功能验证，供体巨噬细胞通过 MYD88（骨髓分化初级反应蛋白 88）发出信号以促进细胞排斥。结果：供体巨噬细胞持续存在于排斥的移植心脏中，并与受体单核细胞来源的巨噬细胞共存。单细胞 RNA 测序鉴定了供体 CCR2 ⁺和 CCR2 ^-巨噬细胞群，并揭示了受体单核细胞、巨噬细胞和树突状细胞之间的显着多样性。时间分析表明，供体 CCR2 ⁺和 CCR2 ^-巨噬细胞在转录上是不同的，经历了显着的形态变化，并在移植后表现出独特的激活特征。尽管选择性消耗供体 CCR2-^巨噬细胞会降低同种异体移植物的存活率，但供体 CCR2 ⁺巨噬细胞的消耗会延长同种异体移植物的存活率。通路分析显示，供体 CCR2 ⁺巨噬细胞通过激活 B 细胞信号传导的 MYD88/核因子 kappa 轻链增强子被激活。供体巨噬细胞中 MYD88 的缺失导致抗原呈递细胞募集减少，抗原呈递细胞向 T 细胞呈递抗原的能力降低，同种异体移植反应性 T 细胞的出现减少，并延长同种异体移植物的存活时间。结论：供体和移植物的不同群体受体巨噬细胞共存于移植心脏内。供体 CCR2 ⁺巨噬细胞是同种异体移植排斥的关键介质，供体巨噬细胞中 MYD88 信号传导的缺失足以抑制排斥并延长同种异体移植存活。这凸显了基于供体心脏的干预措施的治疗潜力。