T细胞是人体重要的一类免疫细胞，在抵抗病原微生物以及维持机体的正常免疫功能中发挥重要作用。CD4⁺ T细胞是人类免疫缺陷病毒（Human immunodeficiency virus，HIV）感染的主要靶细胞，随着疾病进展，机体的CD4⁺ T细胞会大量减少，以致引发一系列的机会性感染，最终进展为艾滋病（Acquired immune deficiency syndrome，AIDS）。HIV能整合到细胞的基因组中，以HIV前病毒DNA的形式藏匿在组织细胞内从而不易被体内的免疫系统所识别，最终形成病毒潜伏库，HIV潜伏库是当今艾滋病难以实现治愈的重要障碍。

程序性细胞死亡蛋白2（Programmed cell death protein 2，PDCD2）是一种高度保守的真核蛋白，广泛分布于人体的多种组织，在细胞凋亡中发挥重要的调节作用。PDCD2存在多种剪切形式，其不同剪切异构体的功能机制仍不明确，而且PDCD2及其剪切异构体对T细胞以及HIV潜伏感染T细胞的凋亡作用机制仍需深入探讨。

本研究中，我们构建了含有人PDCD2即PDCD2(H)、猴PDCD2即PDCD2(RM)、三种剪切形式包括PDCD2(HS3)、PDCD2(HS4)、PDCD2(HS7)基因的重组慢病毒表达质粒，通过慢病毒包装及感染的方式在T淋巴细胞系来源的Jurkat和HIV潜伏感染细胞ACH2中分别过表达这些基因，建立了多个稳定细胞株，研究PDCD2及其剪切异构体在凋亡诱导剂刺激下的细胞凋亡作用。实验结果表明在无任何刺激剂的情况下，Jurkat和ACH2细胞的凋亡率不因细胞内PDCD2表达量的增加而改变。通过摸索FasL、TRAIL和TNF-α三种凋亡诱导剂刺激Jurkat、ACH2与HeLa细胞株的凋亡作用，发现Jurkat和ACH2细胞对TRAIL（25 ng/ml）刺激的凋亡作用最为敏感。进而，在TRAIL刺激下，与对照组细胞相比，高表达PDCD2基因的Jurkat和ACH2细胞凋亡率显著增高，有统计学差异（* P<0.05）。此外，在TRAIL刺激下，高表达PDCD2 基因的ACH2细胞上清中HIV病毒载量显著升高（*P<0.05），说明PDCD2的高表达可以促进TRAIL介导下HIV潜伏感染细胞中HIV病毒的释放。

此外，本研究表明人PDCD2与猴PDCD2即使有12个氨基酸序列存在差异，同源性96.5%，但两者对TRAIL诱导的细胞凋亡作用无差异。接下来，我们也对PDCD2的C结构域在凋亡中发挥的作用进行了初步探究。在未刺激的条件下，人PDCD2全长及其剪切异构体PDCD2(HS3)、PDCD2(HS4)、PDCD2(HS7)的过表达不会影响Jurkat/ACH2细胞的凋亡。但在TRAIL刺激下，PDCD2(HS3)不能有效诱导Jurkat/ACH2细胞的凋亡，说明完全缺失PDCD2_C结构域的蛋白分子不能发挥其促细胞凋亡作用。PDCD2(HS7)包含了C结构域靠近信号肽第226至292号氨基酸序列，该剪切异构体的表达可以促进Jurkat/ACH2细胞的凋亡，其结果与全长PDCD2(H)所诱导的细胞凋亡一致。

综上，本研究观察到PDCD2能在TRAIL刺激下促进T细胞和HIV潜伏感染T细胞的凋亡，同时能促进HIV潜伏感染细胞病毒的释放，提示PDCD2在诱导HIV潜伏感染细胞凋亡及病毒活化的过程中发挥重要作用。此外，PDCD2中C结构域的完全缺失会影响PDCD2发挥的促细胞凋亡作用，但C结构域包含的第226至292号氨基酸序列可使PDCD2恢复其促凋亡功能，提示该序列可能是决定PDCD2细胞凋亡作用的主要功能区域。

T cell is one of the important types of immunocytes in human, which plays a vital role in defending against pathogens and maintaining normal immune function. CD4⁺ T cells are the main target cells of human immunodeficiency virus (HIV) infection. The decline of CD4⁺ T cells is the hallmark of disease progression with the series of opportunistic infections. HIV could be integrated into the host genome and be harbored in immune cells, which is difficult to be recognized by the immune system and contributing to the formation of HIV reserviors. HIV reservoirs is the main barrier of achieving HIV cure.

Programmed cell death protein 2 (PDCD2) is a highly conserved eukaryotic protein widely expressed in human tissues and plays a crucial role in apoptosis. There are multiple splicing isoforms of PDCD2, and the function or mechanism of different splicing isoforms remains unclear. The apoptosis mechanism of PDCD2 and its splicing isoforms in T cells and HIV latently infected T cells needs to be further explored.

In this study, we constructed recombinant lentiviral vectors containing human PDCD2(H), rhesus macaque PDCD2(RM) and three splicing isoforms of human PDCD2 including PDCD2(HS3), PDCD2(HS4) and PDCD2(HS7). These genes were overexpressed in T cell line Jurkat and HIV latently infected cells ACH2 through lentivirus packaging and infection. We focus on the apoptosis of PDCD2 and its splicing isoforms under the stimulation of apoptosis inducers by establishing several stable cell lines. Apoptosis was not significantly increased in both Jurkat cells and ACH2 cells stably expressing PDCD2 compared with untransfected control cells. We further explored the apoptosis of Jurkat, ACH2 and HeLa cell lines induced by FasL, TRAIL and TNF-α, and found that 25 ng/ml TRAIL was the most sensitive concentration to induce the apoptosis in Jurkat and ACH2 cells. We also found that apoptosis was significantly elevated in Jurkat or ACH2 cells overexpressing PDCD2 induced by TRAIL when compared to the treated mock-control group (*P<0.05). Interestingly, viral load in the supernatant was much higher in ACH2 cells overexpressing PDCD2 (*P<0.05), implying that high expression of PDCD2 could promote the HIV release mediated by TRAIL in infected cells with HIV latency.

Although there were differences in 12 amino acids sequences with a 96.5% homology between PDCD2(H) and PDCD2(RM), no difference in apoptosis induced by TRAIL was found between them. To investigate the function of different splicing isoforms of PDCD2, we next explored the role of C domain of PDCD2 (PDCD2_C) in apoptosis induced by TRAIL. We found that PDCD2(H) and its three splicing isoforms including PDCD2(HS3), PDCD2(HS4) and PDCD2(HS7) did not affect the apoptosis of Jurkat or ACH2 cells. However, PDCD2(HS3) could not increase the apoptosis of Jurkat or ACH2 cells induced by TRAIL, demonstrating that PDCD2 protein completely defecting PDCD2_C could not promote apoptosis. Meanwhile, PDCD2(HS7) that contained amino acid from 226 to 292 of PDCD2_C closed to signal peptide could promote the apoptosis of Jurkat or ACH2 cells stimulated by TRAIL, which was consistent with apoptosis of PDCD2(H).

Collectively, we found that PDCD2 overexpression enhanced the apoptosis of T cells with or without HIV latent infection induced by TRAIL, and increased the viral reactivation and release from HIV latently infected cells, highlighting the important role of PDCD2 in apoptosis of HIV latently infected cells induced by TRAIL and the potential reactivation of HIV-1 reservoirs. Importantly, we also found that PDCD2 protein without C domain could not trigger the apoptosis, but the apoptosis could be restored when PDCD2 possessed the part of PDCD2_C containing amino acid from 226 to 292, suggesting this sequence may be the most important motif of the PDCD2 pro-apoptosis function.