Cytokine-independent detection of antigen-specific germinal center T follicular helper (Tfh) cells in immunized non-human primates using a live cell Activation Induced Marker (AIM) technique

A range of current candidate AIDS vaccine regimens are focused on generating protective HIV neutralizing antibody responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective antibody responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GC) are the engines of antibody affinity maturation. GC T follicular helper (GC Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as antigen-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining (ICS). Cytokine production by GC Tfh cells may be intrinsically limited in comparison to other T helper effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify antigen-specific GC Tfh cells. RNAseq was performed using TCR stimulated GC Tfh cells to identify candidate markers based on differentially expressed genes and other criteria. Validation experiments determined CD25 (IL2Ra) and OX40 to be highly upregulated activation induced markers on the surface of GC Tfh cells after stimulation. In comparison to ICS, the activation induced marker (AIM) assay identified > 10-fold more antigen-specific GC Tfh cells in HIV Env protein immunized macaques (BG505 SOSIP). Additionally, the AIM assay detected antigen-specific CXCR5+ and CXCR5- CD4 T cells in peripheral blood. Thus, AIM demonstrates that antigen-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function. Overall design: Total RNA obtained from GC Tfh cells from 3 rhesus macaque lymph node cell samples stimulated with Staphylococcal Enterotoxin B compared with 2 unstimulated cell samples.

当前一系列候选艾滋病疫苗方案均聚焦于诱导产生具有保护性的HIV中和抗体应答。其中多数研究依赖于恒河猴动物模型。目前，关于保护性抗体应答的形成机制以及如何提升其效力，仍存在诸多关键认知空白。生发中心（Germinal Centers, GC）是抗体亲和力成熟的核心引擎。GC滤泡辅助性T（Germinal Center T Follicular Helper, GC Tfh）CD4阳性T细胞是GC形成与维持的必要条件。然而，在蛋白免疫后开展疫苗特异性GC Tfh细胞的相关研究一直颇具挑战，这是因为传统的细胞内细胞因子染色（Intracellular Cytokine Staining, ICS）技术难以有效识别抗原特异性GC Tfh细胞。与其他T辅助效应细胞相比，GC Tfh细胞的细胞因子分泌能力可能存在内在限制：这是因为GC Tfh细胞的生物学功能是为生发中心内的单个B细胞提供辅助支持，而非分泌大量细胞因子浸润周边组织。为验证这一假说，我们开发了一种不依赖细胞因子的抗原特异性GC Tfh细胞识别方法。研究人员通过经T细胞受体（T Cell Receptor, TCR）刺激的GC Tfh细胞开展RNA测序（RNA Sequencing, RNAseq），基于差异表达基因及其他筛选标准确定候选标记物。验证实验证实，CD25（IL-2受体α链，IL2Ra）与OX40是刺激后GC Tfh细胞表面显著上调的激活诱导标记物（Activation Induced Marker, AIM）。与ICS技术相比，AIM检测法在经HIV Env蛋白免疫的恒河猴（BG505 SOSIP）样本中，可检出超过10倍数量的抗原特异性GC Tfh细胞。此外，AIM检测法还可在外周血中检测到抗原特异性CXCR5阳性与CXCR5阴性的CD4阳性T细胞。综上，AIM检测法证实，抗原特异性GC Tfh细胞本质上仅能分泌极少量细胞因子，这或许是其生物学功能的核心特征之一。整体实验设计：从3份经葡萄球菌肠毒素B（Staphylococcal Enterotoxin B, SEB）刺激的恒河猴淋巴结细胞样本中分离得到GC Tfh细胞的总RNA，并与2份未受刺激的细胞样本进行对比分析。