For former mate vivo ELISpots, peptides were put into 2??105 PBMCs per test at 2?g?ml?1 for 16C18?h. and Fight (10.1101/2021.05.11.21256877). Abstract NP105C113-B*07:02-specific CD8+ Lisinopril (Zestril) T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105C113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated Rabbit Polyclonal to AGR3 with T cell receptor usage, transcriptome signature and disease severity (acute gene usage, with long CDR3 loops preferentially expressed in NP105C113-B*07:02-specific T cell receptor (TCR), has been observed in both unexposed and COVID-19-recovered individuals10. The present study suggested a role for cross-reactive responses in COVID-19 based on pre-existing immunity to seasonal coronaviruses or other pathogens. However, a subsequent study suggested that the immunodominant NP105C113-B*07:02 CD8+ T cell responses are unlikely to arise from pre-existing cross-reactive memory pools, but Lisinopril (Zestril) rather represent a high frequency of naive T cell precursors found across HLA-B*07:02-expressing individuals7. In this study, we present? an in-depth analysis to explore correlations across NP105C113-B*07:02-specific T cell responses, TCR repertoires and disease severity. We saw stronger overall T cell responses in individuals recovered from severe COVID, which may be explained by high exposure to viral protein; however, we found an immunodominant epitope response (HLA-B*07:02 NP105C113-specific CD8+) which significantly associated with mild cases. Importantly, this epitope is one of the most dominant CD8+ T cell epitopes reported so far by us and others. We examined potential mechanisms of protection using single-cell transcriptome analysis, and functional evaluation of expanded T cell clones bearing the same TCRs as those identified in single-cell analysis. We also assessed the ability of T cell lines and clones to mount effective effector function against cells infected with live SARS-CoV-2 virus and vaccinia virus-expressing SARS-CoV-2 proteins. We found that NP105C113-B*07:02 is the dominant NP response in HLA-B*07:02-positive patients with mild symptoms, with high frequency and higher magnitude when compared with severe cases. Single-cell analysis revealed that preserved beneficial functional phenotypes are associated with protection from severe illness and have better overall antiviral function. In addition, NP105C113-B*07:02-specific T cells can recognize the naturally processed epitope in live virus and recombinant vaccinia virus-infected cells, which correlates with antiviral efficacy. Results NP105C113-B*07:02-specific T cell responses are stronger in patients recovered from mild COVID-19 infection A previous study has identified five dominant CD8+ epitopes targeting NP, including the most dominant epitope NP105C113 (amino acid sequence SPRWYFYYL) restricted by HLA-B*07:02 (ref. 3). This present study includes 52 individuals who recovered from COVID-19, comprising 30 mild cases and 22 severe cases (including 4 with critical illness; clinical features summarized in Supplementary Table 1 and Extended Data Fig. 1aCc). All the patients were HLA typed and 19 (36.5%) were HLA-B*07:02 positive (10 mild and 9 severe cases; Extended Data Fig. ?Fig.1d).1d). We proceeded to carry out ex vivo interferon (IFN)- ELISpot assays using HLA-B*07:02-positive convalescent samples 1C3?months postinfection. Of HLA-B*07:02 individuals, 79% (15/19) showed responses to this epitope, which accounted for 29% of individuals from the overall cohort (15/52) (Fig. ?(Fig.1a),1a), including 90% (9/10) of individuals recovered from mild and 67% (6/9) from severe disease (Fig. ?(Fig.1b).1b). This further confirms the dominance of this NP105C113-B*07:02 T cell response in our cohort, in particular in individuals recovered from mild illness. In addition, individuals recovered from mild disease made significantly stronger responses to this epitope, compared with those who had recovered from severe disease (Fig. ?(Fig.1c;1c; value was calculated: *value was calculated. (d) Breakdown of patient numbers and severity; HLA-B*07:02 negative (mild N?=?20 light grey, N?=?13 dark grey); HLA-B*07:02 positive responders (mild N?=?9 red, severe N?=?6 blue); HLA-B*07:02 positive non-responders (mild N?=?1 Lisinopril (Zestril) light red, severe N?=?3 light blue). Strong cytotoxicity and inhibitory receptor expression are associated with disease severity To explore the mechanisms underlining this association, we sorted NP105C113-B*07:02-specific T cells at a single-cell level with peptide major histocompatibility complex class I (MHC-I) pentamers using Lisinopril (Zestril) flow cytometry. We performed single-cell analysis using SmartSeq2 for peripheral Lisinopril (Zestril) blood mononuclear cell (PBMC) samples from four convalescent patients, including two who recovered from mild COVID-19 infection (C-COV19-005, age 56?years and C-COV19-046, age 76?years) and two who recovered.