has been made in development of immunoassays for detection of antibody responses to SARS-CoV-2, clinical assays for detection of cellular immune response have lagged.
Interferon-gamma (IFN-γ) release assay (IGRA) are in vitro blood diagnostics used in clinical laboratories to measure IFN-γ released by antigen-specific T-cells after overnight stimulation with pathogen-specific peptides. Here, we describe clinical performance and kinetics of a whole blood IGRA ( Figure 1A ) for simple and high throughput detection of cellular immune response to SARS-CoV-2.
All participants provided written informed consent. Freshly collected blood in lithium heparin tube was (1) left unstimulated as negative control, stimulated with (2) a single SARS-CoV-2 peptide pools for CD4+ T cells and (3) two SARS-CoV-2 peptide pools for CD8+ T cells, and (4) stimulated with mitogen as positive control. A single CD4+ T cell megapool (CD4+ pool) consisted of 221 predicted HLA class II CD4+ T cell epitope peptides covering the entire viral proteome except for the spike protein which was covered with 253 15-mer peptides overlapping by 10-residues and two CD8+ T cell megapools (CD8+ pools A and B) together consisted of 628 predicted HLA class I CD8+ T cell epitopes from the entire SARS-CoV-2 proteome [5, 6] . IFN-γ concentration in the plasma fraction was measured with an automated ELISA instrument in international units (IU) per mL. IFN-γ response was defined as peptide stimulated minus unstimulated. The Mann Whitney U test was used to compare median IFNγ responses between groups. The Wilcoxon signed-rank test of medians was used to compare differences between paired results. The receiver operating characteristic curve was A c c e p t e d M a n u s c r i p t 4 used to derive an IFN-γ response cutoff at the Youden maximum index value which assigns equal weight to sensitivity and specificity.
In total, 82 adult COVID-19 convalescent patients including 64 from an outpatient COVID-19 treatment trial of interferon-λ (IλT) and 18 from Stanford Blood Center convalescent plasma donors (CPD), and 48 uninfected healthy adult blood donors with negative ELISAs for SARS-CoV-2 IgG, were tested with the IGRA. IFN-λ has been shown to not have a negative impact on adaptive response to SARS-CoV-2 [7] . Among all convalescents, the median age However, the sensitivity was 70% (95% CI 56-81) in 50 IλT convalescents on day 31 ( Figure   S2 ). Stimulating T cells with a commercially available peptide pool consisting of spike, S1, nucleocapsid, and membrane protein (Miltenyi Biotek, Bergisch Gladbach, Germany) in 20 healthy blood donors and 24 IλT convalescents (16 on day 17 and 8 on day 31), the median IFN-γ response was 0.02 IU/mL (IQR 0.01-0.05) in healthy blood donors and 3.59 IU/mL (IQR 1.31-6.58) in convalescents ( Figure 1E ). The IFN-γ response in 23 convalescents was 19-fold higher with Miltenyi peptide pool than with CD4+ T cell peptide pool described above In summary, using a simple and deeply-studied IGRA method used for diagnosis of latent Mycobacterium tuberculosis infection [10, 11] , we show that whole blood stimulation with a SARS-CoV-2 peptide pool can sensitively detect a cellular immune response to SARS-CoV-2 and accurately distinguish between convalescents and uninfected healthy blood donors.
Given the relative ease of performing whole blood IGRA and the widespread use of IGRA, SARS-CoV-2 IGRA can be implemented in clinical laboratories to identify individuals with reactive T cells to SARS-CoV-2. The assay may be further enhanced by using more sensitive and commercially available peptide pools with defined specificity for SARS-CoV-2 vs. seasonal coronavirus [1, 3] . SARS-CoV-2 IGRA may prove useful in identifying individuals with cellular immunity and thus serv` e as a powerful diagnostic tool in managing the COVID-19 pandemic.
M a n u s c r i p t 