Biological Sciences Graduate Program, University of Maryland, College Park, MD, United States.
Institute for Physical Science and Technology, University of Maryland, College Park, MD, United States.
Front Immunol. 2022 Mar 16;13:779888. doi: 10.3389/fimmu.2022.779888. eCollection 2022.
Cytotoxic T lymphocytes (CTLs) play an integral role in the adaptive immune response by killing infected cells. Antigen presenting cells (APCs), such as dendritic cells, present pathogenic peptides to the T cell receptor on the CTL surface and co-stimulatory signals required for complete activation. Activated CTLs secrete lytic granules containing enzymes that trigger target cell death at the CTL-target contact, also known as the immune synapse (IS). The actin and microtubule cytoskeletons are instrumental in the killing of CTL targets. Lytic granules are transported along microtubules to the IS, where granule secretion is facilitated by actin depletion and recovery. Furthermore, actomyosin contractility promotes target cell death by mediating mechanical force exertion at the IS. Recent studies have shown that inflammatory cytokines produced by APCs, such as interleukin-12 (IL-12), act as a third signal for CTL activation and enhance CTL proliferation and effector function. However, the biophysical mechanisms mediating such enhanced effector function remain unclear. We hypothesized that the third signal for CTL activation, IL-12, modulates cytoskeletal dynamics and force exertion at the IS, thus potentiating CTL effector function. Here, we used live cell total internal reflection fluorescence (TIRF) microscopy to study actomyosin and microtubule dynamics at the IS of murine primary CTLs activated in the presence of peptide-MHC and co-stimulation alone (two signals), or additionally with IL-12 (three signals). We found that three signal-activated CTLs have altered actin flows, myosin dynamics and microtubule growth rates as compared to two signal-activated CTLs. We further showed that lytic granules in three-signal activated CTLs are less clustered and have lower velocities than in two-signal activated CTLs. Finally, we used traction force microscopy to show that three signal-activated CTLs exert greater traction forces than two signal-activated CTLs. Our results demonstrate that activation of CTLs in the presence of IL-12 leads to differential modulation of the cytoskeleton, thereby augmenting the mechanical response of CTLs to their targets. This indicates a potential physical mechanism which the third signal can enhance the CTL response.
细胞毒性 T 淋巴细胞 (CTL) 通过杀死受感染的细胞在适应性免疫反应中发挥着重要作用。抗原呈递细胞 (APC)，如树突状细胞，将病原体肽呈递给 CTL 表面的 T 细胞受体，并提供完全激活所需的共刺激信号。激活的 CTL 分泌含有酶的裂解颗粒，这些酶在 CTL-靶标接触处引发靶细胞死亡，也称为免疫突触 (IS)。肌动蛋白和微管细胞骨架在 CTL 靶标的杀伤中起着重要作用。裂解颗粒沿着微管运输到 IS，在那里通过肌动蛋白耗竭和恢复促进颗粒分泌。此外，肌球蛋白收缩力通过在 IS 处介导机械力施加来促进靶细胞死亡。最近的研究表明，APC 产生的炎性细胞因子，如白细胞介素-12 (IL-12)，作为 CTL 激活的第三信号，增强 CTL 的增殖和效应功能。然而，介导这种增强的效应功能的生物物理机制仍不清楚。我们假设 CTL 激活的第三信号 IL-12 调节 IS 处的细胞骨架动力学和力施加，从而增强 CTL 的效应功能。在这里，我们使用活细胞全内反射荧光 (TIRF) 显微镜研究了在肽-MHC 和共刺激单独存在（两个信号）或另外存在 IL-12（三个信号）激活的小鼠原代 CTL 的 IS 处的肌动蛋白和微管动力学。我们发现，与两个信号激活的 CTL 相比，三个信号激活的 CTL 具有改变的肌动蛋白流、肌球蛋白动力学和微管生长速率。我们进一步表明，三个信号激活的 CTL 中的裂解颗粒比两个信号激活的 CTL 聚类程度更低，速度更慢。最后，我们使用牵引力显微镜显示，三个信号激活的 CTL 施加的牵引力大于两个信号激活的 CTL。我们的结果表明，在 IL-12 存在的情况下激活 CTL 会导致细胞骨架的差异调节，从而增强 CTL 对其靶标的机械反应。这表明了一种潜在的物理机制，即第三信号可以增强 CTL 反应。
