Evaluation of Oropharyngeal responses to SARS-CoV-2

采集了2020年末至2021年在国立儿童医院接受扁桃体切除术的110名儿童的外周血、扁桃体组织和腺样体组织，目的是：1)评估既往新冠肺炎的患病率和感染的流行病学危险因素；2)评估口咽组织中SARS-CoV-2特异性免疫反应的性质；3)比较新冠肺炎恢复期儿童和无新冠肺炎病史儿童的黏膜组织和血液中的免疫状况。所有患者在手术前均为SARS-CoV-2聚合酶链式反应阴性。我们通过血清学测试和流式细胞术鉴定识别SARS-CoV-2刺突蛋白RBD区域的B细胞，确定了24名有新冠肺炎病史的参与者。在这些参与者中，只有11人之前进行了SARS-CoV-2的聚合酶链式反应阳性检测(手术前25-303天)，并且知道他们之前的感染情况。在大流行早期被发现为新冠肺炎康复者的参与者更有可能是西班牙裔，并生活在贫困率为10%的邮政编码地区(Mudd等人)。喉镜，2022年)。 用高维流式细胞术和CITE-SEQ，我们在来源于健壮的双生中心(GC)反应的组织中发现了SARS-CoV-2特异性记忆B细胞，有体细胞超突变和类别转换的证据；我们还在扁桃体和腺样体中发现了重叠的克隆，表明细胞在这两个口咽组织之间迁移。我们还发现，在新冠肺炎恢复期受试者中，参与GC和抗病毒反应的B和T细胞群持续扩张，包括GC B细胞、GC-Tfh中的CD57+PD-1和CD57+PD1+CD8TRM细胞，以及CXCR3+(干扰素-γ相关)Tfh群。这些变化在腺样体中最为明显，外周血中观察到的变化较少。通过CITE-SEQ，我们在恢复期样本中发现了更多扩增的CD8+和CD4+T细胞克隆，其中一些具有已被报道为SARS-CoV-2特异性的CDR3序列。最后，在急性感染后10个月，我们发现了持续的病毒RNA。综上所述，我们的结果表明SARS-CoV-2感染后几周到几个月的上呼吸道持续的SARS-CoV-2特异性免疫(Xu等人。免疫系统。2023年)。然而，肌肉注射疫苗是否在口咽淋巴组织中引起类似的组织免疫仍然是一个重要的问题。 在过去的一年里，我们对接种了SARS-CoV-2mRNA疫苗的儿童扁桃体、腺样体和外周血中的SARS-CoV-2特异性B细胞反应进行了表征，并将这些样本与感染SARS-CoV-2或同时感染和免疫的儿童的样本进行了比较。值得注意的是，我们在感染和接种疫苗后的儿童扁桃体和腺样体中都发现了SARS-CoV-2特异性B细胞。这些细胞主要是记忆B细胞，但也有一小部分GC B细胞，这表明肌肉接种后和感染后的上呼吸道都可以找到并保持免疫记忆。尽管如此，疫苗接种和感染在粘膜组织和血液中产生了不同数量和质量的反应，记忆B细胞表达与不同功能反应相关的不同标记。我们的工作为疫苗接种后的组织特异性免疫提供了证据，但也强调了感染后产生的SARS-CoV-2特异性B细胞与接种后产生的B细胞之间的差异，这将为评估粘膜疫苗提供重要的见解(Xu等人，正在修订中)。

Peripheral blood, tonsil tissues, and adenoid tissues were obtained from 110 children who underwent tonsillectomy at Children's National Hospital from late 2020 to 2021 with the goal of: 1) evaluating the prevalence of prior COVID-19 and epidemiologic risk factors for infection; 2) evaluating the nature of SARS-CoV-2-specific immune responses in the oropharyngeal tissue; and 3)comparing the immune profile in the mucosal tissue and blood of COVID-19-convalescent children and children without prior COVID-19. All patients had to be PCR negative for SARS-CoV-2 prior to surgery. We identified 24 participants with evidence of prior COVID-19 through serologic testing and identification of B cells that recognize the RBD region of the SARS-CoV-2 spike protein by flow cytometry. Only 11 of these participants had a prior positive PCR test for SARS-CoV-2 (25-303 days prior to surgery) and were aware of their prior infection. Participants found to be COVID-19-convalescent during this early period of the pandemic were more likely to be Hispanic and to live in a zip code with a poverty level >10% (Mudd et al. Laryngoscope, 2022). With high dimensional flow cytometry and CITE-seq, we identified SARS-CoV-2-specific memory B cells in the tissue derived from robust geminal center (GC) responses, with evidence of somatic hypermutation and class switching; we also noted overlapping clones in the tonsil and adenoid suggesting migration of cells between these two oropharyngeal tissues. We also found persistent expansion of B and T cell populations involved in the GC and anti-viral response including GC B cells, CD57+PD-1hi GC-Tfh, and CD57+PD1+ CD8 TRM cells, as well as CXCR3+ (IFN-gamma-associated) Tfh populations in COVID-19-convalescent subjects. These changes were most notable in the adenoids, with fewer changes observed in peripheral blood. With CITE-seq, we found more expanded CD8+ and CD4+ T cell clones in convalescent samples, some of which had CDR3 sequences that have been reported to be SARS-CoV-2 specific. Lastly, we found persistent viral RNA by ddPCR as long as 10 months after acute infection. In summary, our results indicate persistent SARS-CoV-2-specific immunity in the upper respiratory tract weeks to months after infection (Xu et al Nat. Immunol. 2023). However, whether intramuscular vaccination elicits similar tissue immunity in the oropharyngeal lymphoid tissue remained an important question. In the last year, we characterized SARS-CoV-2-specific B cell responses in tonsils, adenoids, and peripheral blood of children who had been immunized with a SARS-CoV-2 mRNA vaccine and compared these samples with those from children infected with SARS-CoV-2 or both infected and immunized. Notably, we found SARS-CoV-2-specific B cells in the tonsils and adenoids of children both post-infection and post-vaccination. These were primarily memory B cells but also a small portion of GC B cells, indicating that immune memory can be found and maintained in the upper respiratory tract after intramuscular vaccination as well as after infection. Nonetheless, vaccination and infection generated different quantities and qualities of responses in both the mucosal tissues and blood, with memory B cells expressing different markers associated with distinct functional responses. Our work provides evidence for tissue-specific immunity post-vaccination, but also highlights differences between SARS-CoV-2 specific B cells generated post-infection and post-vaccination, which will provide important insight for evaluating mucosal vaccines (Xu et al, under revision).