Division of Innate and Comparative Immunology, Center for Human Systems Immunology, Duke University, Durham, North Carolina, United States of America.
Department of Surgery, Duke University, Durham, North Carolina, United States of America.
PLoS Pathog. 2024 May 13;20(5):e1012223. doi: 10.1371/journal.ppat.1012223. eCollection 2024 May.
Natural killer-like B (NKB) cells are unique innate immune cells expressing both natural killer (NK) and B cell receptors. As first responders to infection, they secrete IL-18 to induce a critical cascade of innate and adaptive immune cell infiltration and activation. However, limited research exists on the role of NKB cells in homeostasis and infection, largely due to incomplete and erroneous evaluations. To fill this knowledge gap, we investigated the expression of signaling and trafficking proteins, and the in situ localization and transcriptome of naïve NKB cells compared to conventionally-defined NK and B cells, as well as modulations of these cells in SIV infection. Intracellular signaling proteins and trafficking markers were expressed differentially on naïve NKB cells, with high expression of CD62L and Syk, and low expression of CD69, α4β7, FcRg, Zap70, and CD3z, findings which were more similar to B cells than NK cells. CD20+NKG2a/c+ NKB cells were identified in spleen, mesenteric lymph nodes (MLN), colon, jejunum, and liver of naïve rhesus macaques (RM) via tissue imaging, with NKB cell counts concentrated in spleen and MLN. For the first time, single cell RNA sequencing (scRNAseq), including B cell receptor (BCR) sequencing, of sorted NKB cells confirmed that NKB cells are unique. Transcriptomic analysis of naïve splenic NKB cells by scRNAseq showed that NKB cells undergo somatic hypermutation and express Ig receptors, similar to B cells. While only 15% of sorted NKB cells showed transcript expression of both KLRC1 (NKG2A) and MS4A1 (CD20) genes, only 5% of cells expressed KLRC1, MS4A1, and IgH/IgL transcripts. We observed expanded NKB frequencies in RM gut and buccal mucosa as early as 14 and 35 days post-SIV infection, respectively. Further, mucosal and peripheral NKB cells were associated with colorectal cytokine milieu and oral microbiome changes, respectively. Our studies indicate that NKB cells gated on CD3-CD14-CD20+NKG2A/C+ cells were inclusive of transcriptomically conventional B and NK cells in addition to true NKB cells, confounding accurate phenotyping and frequency recordings that could only be resolved using genomic techniques. Although NKB cells were clearly elevated during SIV infection and associated with inflammatory changes during infection, further interrogation is necessary to acurately identify the true phenotype and significance of NKB cells in infection and inflammation.
自然杀伤样 B (NKB) 细胞是独特的先天免疫细胞，表达自然杀伤 (NK) 和 B 细胞受体。作为感染的第一反应者，它们分泌 IL-18 以诱导先天和适应性免疫细胞浸润和激活的关键级联反应。然而，由于评估不完整和错误，NKB 细胞在稳态和感染中的作用的研究有限。为了填补这一知识空白，我们研究了信号和运输蛋白的表达，以及与传统定义的 NK 和 B 细胞相比，幼稚 NKB 细胞的原位定位和转录组，以及 SIV 感染对这些细胞的调节。幼稚 NKB 细胞上表达的细胞内信号蛋白和运输标记物存在差异，高表达 CD62L 和 Syk，低表达 CD69、α4β7、FcRg、Zap70 和 CD3z，这些发现与 B 细胞更相似，而不是 NK 细胞。通过组织成像，在恒河猴（RM）的脾脏、肠系膜淋巴结 (MLN)、结肠、空肠和肝脏中鉴定出 CD20+NKG2a/c+ NKB 细胞，NKB 细胞计数集中在脾脏和 MLN 中。通过单细胞 RNA 测序 (scRNAseq)，包括 B 细胞受体 (BCR) 测序，对分选的 NKB 细胞进行的单细胞 RNA 测序首次证实 NKB 细胞是独特的。scRNAseq 分析脾脏幼稚 NKB 细胞的转录组表明，NKB 细胞经历体细胞超突变并表达 Ig 受体，与 B 细胞相似。虽然仅 15%的分选 NKB 细胞显示 KLRC1（NKG2A）和 MS4A1（CD20）基因的转录表达，但仅 5%的细胞表达 KLRC1、MS4A1 和 IgH/IgL 转录本。我们观察到，早在 SIV 感染后 14 天和 35 天，RM 肠道和口腔颊黏膜中 NKB 细胞的频率就增加了。此外，粘膜和外周 NKB 细胞与结直肠细胞因子环境和口腔微生物组变化有关。我们的研究表明，CD3-CD14-CD20+NKG2A/C+ 细胞上的 NKB 细胞门控包括转录组常规的 B 和 NK 细胞以及真正的 NKB 细胞，这使得准确表型和频率记录受到干扰，只能通过基因组技术来解决。尽管 NKB 细胞在 SIV 感染期间明显升高，并与感染期间的炎症变化有关，但需要进一步研究以准确识别 NKB 细胞在感染和炎症中的真正表型和意义。
