Guldenpfennig Caitlyn, Guan Yue, Cseri Bela, Lopez Elida, Teixeiro Emma, Daniels Mark
Department of Molecular Microbiology and Immunology NextGen Precision Health, University of Missouri, Columbia, SC, United States.
Front Immunol. 2025 Jul 2;16:1630599. doi: 10.3389/fimmu.2025.1630599. eCollection 2025.
Upon antigen recognition, naive CD8 T cells must induce c-JUN N-terminal kinase (JNK), NF-κB, and Akt signaling to drive differentiation and generate a heterogeneous effector response. While the roles of these three pathways individually in mediating essential cellular responses for CD8 T cell differentiation are well established, the mechanisms of signal integration and crosstalk between these pathways to produce a diverse and heterogeneous response to infection remain poorly understood. Here, we establish the critical role of the Plenty of SH3 Domains (POSH) scaffold protein in coordinating signals from all three pathways to support CD8 T cell differentiation and fate.
Using novel conditional T cell POSH knockout reporter mouse models (as POSHfl/fl CD4-Cre eGFP, POSHfl/fl GzmB-Cre eGFP), we determined the phenotype of T cells in the thymus and periphery through flow cytometry. Polyclonal and OT1 TCR transgenic POSH cKO CD8 T cells were stimulated in vitro and analyzed by flow cytometry to assess cell fate. JNK, NF-κB, and Akt pathways were examined via flow cytometry and immunoblotting. Purified OT1 CD8 T cells from these mice were adoptively transferred and subsequently challenged with VSV-OVA infection; their phenotype, effector function, and signaling were then assessed ex vivo by flow cytometry.
We demonstrate that POSH is essential for proper induction of the JNK, NF-κB, and Akt pathways. Furthermore, the absence of these signals due to POSH deficiency results in reduced differentiation into short-lived effector cells (SLECs), delayed proliferation, and decreased survival of memory precursor cells (MPECs) during the contraction phase.
Collectively, these data identify POSH as a key regulator of CD8 T cell fate and enhance our understanding of the complex mechanisms governing signal integration during CD8 T cell responses to infection.
幼稚CD8 T细胞在识别抗原后，必须诱导c-JUN氨基末端激酶（JNK）、核因子κB（NF-κB）和Akt信号传导，以驱动分化并产生异质性效应反应。虽然这三条信号通路各自在介导CD8 T细胞分化所必需的细胞反应中的作用已得到充分证实，但这些信号通路之间信号整合和相互作用以产生对感染的多样化和异质性反应的机制仍知之甚少。在此，我们确定了富含SH3结构域（POSH）的支架蛋白在协调来自这三条信号通路的信号以支持CD8 T细胞分化和命运方面的关键作用。
使用新型条件性T细胞POSH基因敲除报告小鼠模型（如POSHfl/fl CD4-Cre eGFP、POSHfl/fl GzmB-Cre eGFP），我们通过流式细胞术确定胸腺和外周T细胞的表型。体外刺激多克隆和OT1 TCR转基因POSH基因敲除CD8 T细胞，并通过流式细胞术进行分析，以评估细胞命运。通过流式细胞术和免疫印迹检测JNK、NF-κB和Akt信号通路。从这些小鼠中纯化的OT1 CD8 T细胞进行过继转移，随后用VSV-OVA感染进行攻击；然后通过流式细胞术在体外评估它们表型、效应功能和信号传导。
我们证明POSH对于正确诱导JNK、NF-κB和Akt信号通路至关重要。此外，由于POSH缺陷导致这些信号缺失，会使向短命效应细胞（SLEC）的分化减少、增殖延迟，并在收缩期导致记忆前体细胞（MPEC）存活减少。
总体而言，这些数据确定POSH是CD8 T细胞命运的关键调节因子，并增进了我们对CD8 T细胞感染反应期间信号整合复杂机制的理解。
