HIV感染者滤泡辅助T细胞病毒储存库对免疫重建不良的影响

Following the early application of antiretroviral therapy (ART), viral load (VL) is suppressed and CD4+ T cell count can recover in most of HIV infected individuals, but still up to 30% of HIV infected individuals who are receiving ART do not exhibit a marked increase in the CD4+ count, despite achieving suppression of the VL. It is referred to as poor immune reconstitution (PIR). Until now mechanisms responsible for such PIR are still unknown. Immune reconstitution outcome may be influenced by the size and persistence of HIV latent reservoir. As one subset of CD4+T cells, follicular T helper cells (Tfh) promote B cell differentiation and provide help to humoral immunity. It was demonstrated that Tfh is an important HIV reservoir and PD-1 is a unique marker for PIR. Our previous study found that it is difficult to induce a high level of neutralizing antibody production by the HIV envelopes, and this may be associated with increase of PD-1+ T cells and non-specific Tfh cells, and activation of non-specific immune responses. Taken together, we speculate that the size of the Tfh latent reservoir will expand before and after PIR. And increased cell frequency and diminished functions especially the help effect on humoral immunity of Tfh will be observed after PIR. In this proposal, we will analyze the effects of the frequency and functions of Tfh cells and characterization of Tfh latent reservoir on immune reconstitution in HIV infected individuals, and investigate the mechanisms in order to prevent PIR and optimize treatment strategies.

随着抗逆转录病毒治疗(ART)早期应用，多数HIV感染者的病毒载量（VL）被控制，CD4+T细胞数恢复正常。但仍有约30%的感染者，ART后VL被控制，CD4细胞数持续不升，发生免疫重建不良。目前免疫重建不良的原因及机制不明确。HIV储存库的规模及持续存在可能与之相关。作为CD4+T细胞，滤泡辅助T细胞(Tfh)促进B细胞分化辅助体液免疫。已证明，Tfh是HIV的重要储存库；PD-1是免疫重建不良的特有标志。申请团队前期发现多数HIV包膜难以诱导高水平中和抗体，与增加PD-1+T细胞数量、增加非特异性Tfh数量、激活非特异性免疫应答相关。综上我们推测：免疫重建不良发生前后Tfh病毒储存库增大；Tfh数量增高，但功能下降，对体液免疫的辅助降低。本研究将分析HIV感染者PBMC中Tfh数量、功能及Tfh病毒储存库特征对免疫重建不良的影响，探讨相关机制，为预防免疫重建不良发生及优化治疗策略提供依据。

临床治疗HIV感染者时常遇到免疫重建不良的问题，本研究拟阐明免疫重建不良发生的机制。. 本研究追踪MSM人群治疗超过24个月的HIV感染者291人，将达到病毒学抑制且ART24个月后CD4+T细胞数<350cells/µL的感染者判定为免疫重建不良者（INR），而≥350cells/µL为免疫重建良好者（IR）。分析两组人口学特征及临床指标差异、血浆病毒序列及储存库前病毒特征、抗体水平、T和B细胞亚群及记忆B细胞BCR特征，同时鉴定ART前HIV感染者的病毒学亚型及原发耐药流行情况。. 本研究发现MSM人群HIV感染者免疫重建不良的发生率为15.12%。比较IR和INR血浆病毒env序列特点发现：两组个体的HIV包膜序列变异率相近；INR组X4嗜性毒株占比高、包膜有更长的V2区氨基酸长度、更多的V3区正电荷数、更少的V3区和V4区N糖基化位点数，其辅助受体结合区变异性更大，且中和抗体PGT128和PGT145表位变异率更高。分析感染者血浆抗体水平发现，IR及INR ART前CD4+T细胞数与VL均无显著相关性，但当患者治疗前免疫力较差时，呈显著负相关；感染者血浆针对我国优势亚型（CRF01_AE和B）病毒中和活性高于针对其他亚型的假病毒。对B亚型假病毒的中和活性高与免疫应答良好相关；ART前血浆结合抗体水平与ART后免疫应答无相关性。记忆B细胞BCR的HCDR3特征的研究结果显示，与INR组相比，IR组HCDR3长度及频率更趋于正态分布；V基因中IGHV1-69基因和J基因中IGHJ2基因使用频率明显增加；并且具有较低的SHM中HCDR3突变率、V和J区的突变率、插入和置换频率。发现两组之间13个V-J基因组合使用频率具有显著性差异。未经ART的MSM人群HIV感染者流行的HIV亚型分布及原发耐药流行结果显示，CRF01_AE为主要优势流行亚型；这些感染者病毒存在较高频率的耐药突变，其中以新近感染者中最高（16.67%）；CRF01_AE与CRF07_BC/B亚型组相比，ART前具有更低的CD4/CD8T细胞比值。. 本研究还比较了两组病毒储存库特征、PBMC的转录组特征，分析感染T、B细胞的异质性，尤其是滤泡辅助T细胞，揭示免疫重建不良发生的机制，为提早预防其发生、有针对性的优化治疗方案奠定基础。