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Abstract：
The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB+ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB+ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB+ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB+ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB+ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB+ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB+/- natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB+ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB+ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.
摘要：
我们研究的目的是确定表达颗粒酶B的调节性B细胞（GZMBB细胞）是否在具有肾移植耐受性的移植患者的血液中富集。为了实现这一目标,我们分析了两个单细胞RNA测序（scRNAseq）数据集：（1）外周血单核细胞（PBMC），包括肾移植患者的GZMB+B细胞，即，接受常规免疫抑制治疗的移植物功能稳定的患者(STA，n=3),无药耐受患者(TOL，n=3),和抗体介导的排斥反应患者(ABMR，n=3),和(2)来自这些组的离体诱导的GZMB+B细胞。在患者的PBMC中，我们首先发现天然GZMB+B细胞富含天然杀伤(NK)细胞(如NKG7和KLRD1)和调节性B细胞(如GZMB,IL10和CCL4)。我们对天然GZMB+B细胞进行了假颞叶轨迹分析，发现它们是高度分化的B细胞，其轨迹与常规记忆B细胞非常不同，并与转录因子KLF13相关。通过具体分析TOL中的GZMB+天然B细胞，我们发现这些细胞具有与HLA分子表达减少相关的非常特异的转录组特征,凋亡，以及血液中的炎症反应（通常），并且该特征在离体诱导后是保守的，随着与迁移过程相关的基因的诱导，例如CCR7、CCL3或CCL4。对这些GZMB+/-天然B细胞与PBMC中存在的所有免疫细胞之间的受体/配体相互作用的分析也表明GZMB+B细胞是携带最多配体并且与其他免疫细胞相互作用最多的B细胞。特别是在宽容的患者中。最后,我们表明这些GZMB+B细胞能够在炎症条件下渗入移植物，因此表明它们可以在发生免疫事件的地方起作用。
