概要: Pre-existing memory T-cells against SARS-CoV-2 are present in a fraction of unexposed individuals and their induction by common cold corona viruses (CCCoVs) infection is suggested. Here we demonstrate that SARS-CoV-2-reactive T-cells were present in the memory compartment of virtually all unexposed individuals but possessed only low functional avidity. They harbored multiple, highly variable cross-reactivities that were not restricted to CCCoVs. Cross-reactivity to CCCoV was almost absent in COVID-19 patients. This was irrespective of strong T-cell memory against CCCoV in all donors. In severe but not mild COVID-19, SARS-CoV-2-specific T-cells also displayed low functional avidity and reduced clonal expansion, despite strongly increased frequencies. Our data question a major protective role of CCCoV for COVID-19. Instead, we suggest that a low avidity pre-existing T-cell memory may contribute to the excessive but low avidity T-cell responses, which we identified here as a hallmark of severe COVID-19
项目整体设计: Single-cell sequencing of 6 unexposed and 14 COVID-19 patients
PBMCs were stimulated for 7h with peptide pools of SARS-CoV-2 and reactive CD154+CD4+ T cells were isolated
提取方案:
CD154+ cells were isolated by MACS and further purified by FACS sorting on a MACSQuant Tyto (Miltenyi Biotec) based on dual expression of CD154 and CD69. Sorted CD154+ T cells were removed from the sorting chamber into pre-coated low-bind collection tubes, 1ml RPMI-1640 medium supplemented with 5% AB Serum was added, and cells were centrifuged for 5 min at 400 x g, 4°C. The supernatant was carefully removed leaving 10-30μl to reach a maximum concentration of 1000 cells /μl.
建库方案:
Single-cell suspensions were loaded on a ChromiumChip G (10x Genomics) according to the manufacturer’s instructions for processing with the Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit v1.1. TCR single-cell libraries were subsequently prepared from the same cells with the Chromium Single Cell V(D)J Enrichment Kit, Human T Cell. Chromium Next GEM Single Cell 5′ Library
测序信息
分子类型:
Poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina NovaSeq 6000
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-
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文章
Low-Avidity CD4+ T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19.
