A quest for universal anti-SARS-CoV-2 T cell assay: systematic review, meta-analysis, and experimental validation.

"we extracted datasets from these 94 studies which measured the spike-specific t cell response by performing elispot aim and ics for further analysis and evaluation as given in supplementary data 1 - 4 .; all the research articles that performed aim/ics/elispot assay (n = 131) are summarized in supplementary data 5 .; the most commonly used sets of markers for cd4 + t cells were ox40 and cd137 and cd69 and cd137 for cd8 + t cells in case of covid-19 studies (supplementary data 1 4 5 ) .; therefore we shortlisted 18 (12 studies - for covid-19 convalescent subjects and 8 studies - for vaccinated subjects) research articles containing 89 different datasets (supplementary data 1 ) in which ox40 and cd137 cd69 and cd137 were used to measure spike-specific cd4+ and cd8 + t cells response respectively.; the range of central tendencies for cd4 + aim was0 02%-1% (mean) 0 07%-0 8% (median) and 0 065%-0 4% (gm) while the central tendencies for cd8 + aim were 0 01%-0 25% (mean) 0 09%-0 15% (median) and 0 03%-0 05% (gm) (fig 2a b ; table 2 supplementary data 1 ).; for the full list of descriptive statistics for each study please refer to supplementary data 1 .; we calculated the range of central tendencies for aim assays in vaccinated individuals which were found to be 0 125%-0 48% (gm) and 0 2%-0 9% (mean) for cd4+ cells and 0 03%-0 3% (gm) for cd8+ cells (fig 2c d table 2 supplementary data 1 ).; the range of readouts from aim assays reported in studies using alternative markers to identify spike-specific t cells are summarized in supplementary fig 3 ( supplementary data 5 ) .; for covid-19 vaccinated individuals the median range of 136 datasets from 28 research articles - were found to be 5-1200 sfus/million (fig 3a ; supplementary data 2 ).; seventy out of 136 datasets used whole spike peptide pools followed by s1 peptide pools in 32/136 datasets (supplementary data 2 ).; in addition we found a broad range of responses in studies using elispot assays to examine t cell response in subjects with co-morbidities or active covid-19 (summarized in supplementary data 2 ).; for a full list of descriptive statistics for each study see supplementary data 2 .; expression (supplementary data 3 ).; + was found to be 0 01%-0 1% (median) and 0 03%-0 8% (mean) (fig 4a supplementary data 3 ) while the central tendencies for cd8 + ifn?; + were 0 016%-0 1% (median) and 0 02%-0 22% (mean) (fig 4b supplementary table 2 supplementary data 3 ).; for a full list of descriptive statistics for each dataset see supplementary data 3 .; most studies including those we reviewed in this paper used cd137 + cd69+ surface markers (21/59) (supplementary fig 1c supplementary data 5 ).; spots evaluated by the elispot assay ( p = 0 008; r = 0 366 n = 51 fig 6 supplementary data 6 ).; + intracellular expression ( p = 0 014 r = 0 32 n = 58 fig 6 supplementary data 6 ).; our correlation matrix indicated that cd25 + cd69+ but not cd137 + cd69+ cd8 + aim significantly correlated with both cd4 + aim (cd137 + ox40+ ( p = 0 007 r = 0 35 n = 58 supplementary data 6 ) and cd25 + ox40+ ( p = 0 0009 r = 0 424 n = 58 supplementary data 6 ).; + response (fig 6 supplementary data 6 ).; one such variability is the time of stimulation which has ranged from 4-6 h in some ics assays to 18-48 h in aim ics and elispot assays (supplementary data 2 5 ).; for similar reasons studies using the stimulation index to depict aim+ cells are excluded from fig 1 but included in supplementary data 5 .; supplementary information file supplementary data 1 supplementary data 2 supplementary data 3 supplementary data 4 supplementary data 5 supplementary data 6 reporting summary publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."

"Competing interests The authors declare no competing interest."

"This research has been conducted with the significant contribution and expertise of THSTI, NCR Biotech Science Cluster BIOREPOSITORY and DBT India Consortium”. We acknowledge Ms. Tarini Vohra for her help in PBMC isolation and conducting T cell assays. We thank Ms. Jyotsana Kaushal for her help in T cell expansion. We thank Dr. Deepak Kumar Rathore for their help in FACS Sorting. This study was supported by the Mission COVID Suraksha grant (BT/CS0010/CS/02/20) from BIRAC, India. We sincerely acknowledge the entire team of Immunology Core Lab of THSTI for supporting this study. P.K.G. is recipient of JC Bose Fellowship from Science and Engineering Research Board, Government of India. A.A. is recipient of Ramachandran National Bioscience Award from Department of Biotechnology, Government of India. Author contributions: A.B. & A.Z. have contributed equally to this study. Conceptualization: A.A., P.K.G.; Data curation: A.A.; Funding acquisition: A.A., P.K.G.; Investigation: A.B., A.Z., S.S., D.R.M.; Methodology: A.B., A.Z., S.V., P.S., R.C., A.A.; Project administration: A.A., P.K., S.B., P.K.G., A.K.P., R.T.; Visualization: A.B., A.Z., R.C.; Writing: original draft: A.B., A.Z., A.A.; Writing: review & editing: A.A., P.K.G."