Biwott Kipchumba, Lkhamkhuu Algirmaa, Ghaffar Nimrah, Papp Albert Bálint, Tarban Nastaran, Goda Katalin, Bacso Zsolt
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Doctoral School of Molecular Cell and Immune Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Int J Mol Sci. 2025 Jun 26;26(13):6123. doi: 10.3390/ijms26136123.
Ruxolitinib, a clinically approved JAK1/2 inhibitor used in the treatment of hematologic malignancies and inflammatory conditions, has been shown to interfere with the function of cytotoxic T lymphocytes (CTLs). Previous studies supported the involvement of the multidrug resistance transporter P-glycoprotein (Pgp/ABCB1) in CTL biology; however, the nature of its regulation remains unclear. To address this, we investigated the impact of ruxolitinib on Pgp expression and function in human CD8 T cells. We demonstrate that CD8 T lymphocytes express Pgp dynamically at both the mRNA and protein levels across naïve, short-term, and long-term activation states. Ruxolitinib increased the calcein accumulation in human Pgp-overexpressing NIH-3T3 cells and in CTLs and directly modulated Pgp function by increasing its basal ATPase activity in a concentration-dependent manner (10-100 μM), similar to the effect of the known Pgp substrate/modulator verapamil. Although measurable ATPase stimulation and transport inhibition were observed at supratherapeutic concentrations of ruxolitinib, its Pgp-mediated efflux may also occur at therapeutically relevant concentrations. In contrast, at therapeutically relevant plasma concentrations (1-3 μM), ruxolitinib significantly stabilized the mRNA expression of Pgp during early T-cell receptor (TCR) activation and inhibited the TCR-induced upregulation of Pgp, CD8, and PD-1 surface markers, suggesting its interference with activation-associated differentiation. At these same concentrations, ruxolitinib also impaired CCL19-directed transmigration of CTLs across human umbilical vein endothelial cell (HUVEC) monolayers, indicating disruption of lymphoid homing cues. Collectively, these findings demonstrate that ruxolitinib modulates Pgp at both the transcriptional and functional levels, with distinct concentration dependence. The ability of ruxolitinib to alter CTL activation and migration at clinically relevant plasma concentrations highlights the need for careful evaluation of JAK inhibitor-mediated immunomodulation and its implications for vaccination, transplantation, and T cell-based immunotherapies.
芦可替尼是一种临床批准用于治疗血液系统恶性肿瘤和炎症性疾病的JAK1/2抑制剂，已被证明会干扰细胞毒性T淋巴细胞（CTL）的功能。先前的研究支持多药耐药转运蛋白P-糖蛋白（Pgp/ABCB1）参与CTL生物学过程；然而，其调控的本质仍不清楚。为了解决这个问题，我们研究了芦可替尼对人CD8 T细胞中Pgp表达和功能的影响。我们证明，CD8 T淋巴细胞在幼稚、短期和长期激活状态下，在mRNA和蛋白质水平上均动态表达Pgp。芦可替尼增加了人Pgp过表达的NIH-3T3细胞和CTL中的钙黄绿素积累，并通过以浓度依赖的方式（10-100μM）增加其基础ATP酶活性直接调节Pgp功能，类似于已知的Pgp底物/调节剂维拉帕米的作用。尽管在芦可替尼的超治疗浓度下观察到了可测量的ATP酶刺激和转运抑制，但在治疗相关浓度下也可能发生其Pgp介导的外排。相比之下，在治疗相关的血浆浓度（1-3μM）下，芦可替尼在早期T细胞受体（TCR）激活期间显著稳定了Pgp的mRNA表达，并抑制了TCR诱导的Pgp、CD8和PD-1表面标志物的上调，表明其干扰了与激活相关的分化。在这些相同浓度下，芦可替尼还损害了CTL跨人脐静脉内皮细胞（HUVEC）单层的CCL19定向迁移，表明淋巴归巢信号被破坏。总体而言，这些发现表明芦可替尼在转录和功能水平上调节Pgp，具有明显的浓度依赖性。芦可替尼在临床相关血浆浓度下改变CTL激活和迁移的能力突出了仔细评估JAK抑制剂介导的免疫调节及其对疫苗接种、移植和基于T细胞的免疫疗法的影响的必要性。
