key: cord-0749830-6k0goyxf
authors: Nguyen, Thi H.O.; Rowntree, Louise C.; Petersen, Jan; Chua, Brendon Y.; Hensen, Luca; Kedzierski, Lukasz; van de Sandt, Carolien E.; Chaurasia, Priyanka; Tan, Hyon-Xhi; Habel, Jennifer R.; Zhang, Wuji; Allen, Lily; Earnest, Linda; Mak, Kai Yan; Juno, Jennifer A.; Wragg, Kathleen; Mordant, Francesca L.; Amanat, Fatima; Krammer, Florian; Mifsud, Nicole A.; Doolan, Denise L.; Flanagan, Katie L.; Sonda, Sabrina; Kaur, Jasveen; Wakim, Linda M.; Westall, Glen P.; James, Fiona; Mouhtouris, Effie; Gordon, Claire L.; Holmes, Natasha E.; Smibert, Olivia C.; Trubiano, Jason A.; Cheng, Allen C.; Harcourt, Peter; Clifton, Patrick; Crawford, Jeremy Chase; Thomas, Paul G.; Wheatley, Adam K.; Kent, Stephen J.; Rossjohn, Jamie; Torresi, Joseph; Kedzierska, Katherine
title: CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naïve precursor frequency and T cell receptor promiscuity
date: 2021-04-15
journal: Immunity
DOI: 10.1016/j.immuni.2021.04.009
sha: ed7f8b48ddba48fa15a5259379d5f0b2ca0a3d3e
doc_id: 749830
cord_uid: 6k0goyxf

To better understand primary and recall T cell responses during COVID-19, it is important to examine unmanipulated SARS-CoV-2-specific T cells. Using peptide-HLA tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed towards subdominant epitopes – B7/N257, A2/S269 and A24/S1208 – CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequency in pre-pandemic samples, and at increased frequency during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults and elderly individuals predominantly displayed a naïve phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N105 +CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells, and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

To better understand primary and recall T cell responses during COVID-19, it is important to 78 examine unmanipulated SARS-CoV-2-specific T cells. Using peptide-HLA tetramers for 79 direct ex vivo analysis, we characterized CD8 + T cells specific for SARS-CoV-2 epitopes in 80 COVID-19 patients and unexposed individuals. Unlike CD8 + T cells directed towards 81 subdominant epitopes -B7/N 257 , A2/S 269 and A24/S 1208 -CD8 + T cells specific for the 82 immunodominant B7/N 105 epitope were detected at high frequency in pre-pandemic samples, Identification of SARS-CoV-2 CD8 + T cell epitopes allows accurate determination of the 126 magnitude and phenotype of SARS-CoV-2-specific CD8 + T cells directly ex vivo in COVID-127 19 patients and pre-pandemic PBMCs. It also allows us to precisely define the persistence of 128 long-term memory CD8 + T cells and their recall following SARS-CoV-2 re-infection and/or 129 vaccination. Ex vivo frequencies of SARS-CoV-2-specific CD8 + T cells appear to be present 130 mainly in the range of ∼1x10 -5 to 5x10 -5 in the CD8 + T cell population, which is ∼2-10-fold 131 lower than the frequency of long-term memory CD8 + T cells specific for influenza or EBV. 132 HLA-A*02:01-restricted SARS-CoV-2 epitopes appear to exhibit the lowest frequency of 133 ∼1x10 -5 in the CD8 + T cell population (Habel et 

We recruited a total of 21 COVID-19 subjects, including 8 acute hospitalized patients and 13 164 convalescent COVID-19 patients (Fig.1A, Table S1 ). Of the 8 patients hospitalised, 1 ICU 165 and 3 ward patients required oxygen support. Ten of the convalescents were community 166 SARS-CoV-2 infections. All COVID-19 patients seroconverted for SARS-CoV-2 antibodies. 167

The median age of COVID-19 patients was 54 years and 33% were females. As controls, we 168 analysed pre-pandemic PBMC and tonsil samples from 31 subjects across three age groups: 4 169 children (median age 6 years, range 3-15), 20 adults (median age 46 years, range 24-63) and 170 7 elderly (median age 72 years, range 65-76), 37.6% were females. Additionally, we tested 171 pre-existing B7/CD8 + T cell populations in lung and spleen tissues from 9 HLA-B7 172 individuals (median age 46 years, range 22-63). 173

Within this study, a traveller cohort included three male SARS-CoV-2 cases (a 19-174

year-old CA3 index case with moderate COVID-19, one 21-year-old CA2 individual with 175 mild symptoms and one 24-year-old CA1 individual who remained asymptomatic; Table S1 ). 176

Four asymptomatic suspected cases (SU1, SU2, SU3, SU4; all 20 years old) were also 177 investigated. All were returned travellers. RBD and Spike IgG antibody levels were 178 significantly (p<0.05) higher in cases (log 10 median titre 3.195 RBD IgG, 3.237 Spike IgG) 179 compared to suspected cases (log 10 median titre 1.762 RBD IgG, 1.994 Spike IgG) and 180 healthy unexposed individuals (log 10 median titre 2.024 RBD IgG, 2.239 Spike IgG) 181 ( Fig.S1A ). IgM titres in COVID-19 cases were significantly elevated compared to those 182 found in healthy unexposed individuals (Fig.S1A) . RBD-and Spike-reactive B cell responses 183 were concordant with the antibody titres and showed significantly increased frequencies of 184 RBD-and Spike-specific B cells in COVID-19 cases when compared to the suspected cases 185 reactive T cells by ICS for intracellular IFN-γ, TNF, MIP-1β and CD107a (Fig.1B, Fig.S3A ) . 197 In agreement with the antibody and B cell data, SARS-CoV-2-reactive CD4 + and CD8 + T 198 cells were detected in COVID-19 cases, but not the suspected cases, with CD4 + T cells 199 generally dominating over CD8 + T cell populations, as previously reported (Habel et al., 200 2020) . No differences were observed between the responses against S, N and M proteins for 201 both CD4 + and CD8 + T cells (Fig.1B, right panel) . The exception was CA2 with highly 202 prominent CD8 + T cell responses directed towards the N peptide pool (IFN-γ production up to 203 ∼25% of CD8 + T cells), markedly above IFN-γ-producing CD4 + T cells (range 0.24-1.15%) 204 ( Fig.1C) . As CA2 was HLA-B*07:02 + and HLA-A*02:01 + (Table S1) PE and APC fluorophores, as well as dual-TAME with B7/N 105 tetramer-PE and an irrelevant 222 B7/EBV-tetramer (EBNA-3 379-387 , RPPIFIRRL) conjugated to APC (Fig.S4AB ). Following 223 TAME (Fig.2) , CD8 + T cells specific for the immunodominant B7/N 105 epitope could be 224 readily detected ex vivo in all COVID-19 patients ( Fig.2A, Fig.S4C ) at a mean frequency of 225 6.88x10 -4 (n=11; Fig.2D ), with the B7/N 105 + CD8 + T cells being easily detected without 226 enrichment ( Fig.2A) . B7/N 105 -specific CD8 + T cell pools in COVID-19 patients were 227 numerically immunodominant when compared to three subdominant SARS-CoV-2-specific 228 CD8 + T cell populations directed at B7/N 257 , A2/S 269 and A24/S 1208 epitopes (by 38.03-, 229 21.54-and 8.92-fold respectively, p<0.05; Figure 2D ). 230

The frequency of B7/N 105 -specific CD8 + T cells in COVID-19 patients was 231 significantly higher than that in adult (mean 3.00x10 -5 ; p=0.0017) and elderly (mean 6.76x10 -232 5 , p=0.0023) pre-pandemic PBMC samples (Fig.2BD) , suggesting clonal expansion after 233 SARS-CoV-2 infection. The values for pre-pandemic children tonsil samples (mean 2.76x10 -234 tetramer + CD8 + T cells with age (Nguyen et al., 2017b) . Moreover, the magnitude of the 237 immunodominant B7/N 105 in combined pre-pandemic adult and elderly PBMCs (mean 238 4.64x10 -5 ) was higher than that for the subdominant B7/N 257 (mean 1.52x10 -6 , p<0.0001), the 239 previously described A2/S 269 frequencies (Habel et al., 2020) (mean 1.65x10 -6 , p=0.0022) and 240 A24/S 1208 , although not statistically significant (mean 9.5x10 -6 , p=0.2484 Mann-U) (Fig.2D) , 241

suggesting that the immunodominance of B7/N 105 -specific CD8 + T cell responses in COVID-242 19 reflects higher precursor frequencies in unexposed individuals. Although the frequency of 243 SARS-CoV-2-specific B7/N 105 tetramer-positive CD8 + T cells in children's pre-pandemic 244 tonsils (mean 2.76x10 -4 ) appeared higher than that found in matched PBMCs (mean 2.5x10 -5 ) 245 (Table S2; tetramers represented a small but distinct population in COVID-19 patients (n=5, Fig.3A) . 354

The TCRαβ repertoire displayed a considerable level of diversity by both circos and bubble 355 plots ( Fig.6A-C) , although common TRBV (43% of total 175 TCRs: TRBV2 at 11%, 356 TRBV7-9 at 13%, TRBV20-1 at 20%), TRBJ (68% of total TCRs: TRBJ2-2 at 59%, TRBJ2-357 7 at 9%), TRAV (32% of total TCRs: TRAV12-1 at 23%, TRAV12-2 at 3%, TRAV14/DV4 358 at 6%) and TRAJ (24% of total TCRs: TRAJ43 at 18%, TRAJ30 at 6%) gene segments were 359

found across different COVID-19 patients ( Fig.6AB; Table S3 ). More importantly, two key 360

TCRα motifs within the CDR3α loop were found across the COVID-19 patients (Fig.6D) . of TCRαβ pairs per group, to calculate diversity scores for single alpha and beta chains and 380 paired TCRαβ clonotypes (Fig.7A, Table S4 ). As observed in our earlier analyses, A2/S 269 381 TCR repertoire (paired TCRdiv=147.9) was less diverse when compared to both pre-382 pandemic and COVID-19 B7/N 105 repertoires, in which diversity was mainly driven by the 383 beta chain. Pre-pandemic B7/N 105 -specific TCRs were extremely diverse (paired 384

TCRdiv=730.4), and then narrowed following primary COVID-19 infection with different 385 individuals (paired TCRdiv=299.9), although the latter were still considered highly diverse. 386

To contextualize the COVID-19 TCR diversity with other well-established acute (influenza 387 A) and chronic viral infections (EBV and CMV), the TCR diversity against the B7/N 105 388 epitope was reminiscent of the relatively diverse TCR repertoires against the chronic and 389 immunodominant A2/CMV-pp65 495-503 epitope. Independent analysis of the common CDR3 390 motifs was also conducted using TCRdist (Dash et al., 2017; Valkenburg et al., 2016) to 391 generate highly significant alpha and beta amino acid motifs for A2/S 269 and B7/N 105 TCRs 392 (Fig.7B, Fig.S5 ). Similar to our initial analysis (Fig.6D) , COVID-19 A2/S 269 TCR repertoire 393 generated two dominant alpha motifs (TRAV12-2/12-1-TRAJ43/30), where the gene pairing 394 of TRAV12-1 with TRAJ43 was highly significant (p<2.3E-07, Fig.S6 ). In comparison, 395 COVID-19 B7/N 105 TCR repertoire encompassed several alpha and beta chain motifs which, 396 surprisingly did not overlap with the pre-pandemic B7/N 105 motifs. 397

The probability of generating (P gen ) TCR alpha and beta chains were then calculated 398 using TCRdist, which correlates with the number of insertions and deletions within the CDR3 399 region (Fig.7C) . Within the TCR alpha chain, P gen values were similar for COVID-19 and 400 well-established IAV, EBV and CMV epitopes. The probability of generating beta chains in 401 the B7/N 105 groups were comparable between pre-pandemic and COVID-19, but both 402 COVID-19 and pre-pandemic B7/N 105 's P gen were significantly lower than A2/S 269 403 (p adj =0.0012 and 0.0435, respectively), and was similar to the A2/CMV repertoire. The lower 404 beta P gen values for B7/N 105 were supported by both B7/N 105 groups having significantly 405 more beta N-insertions than A2/S 269 , however lower numbers of N-deletions were 406 significantly only observed for the COVID-19 B7/N 105 TCR repertoire, but not for pre-407 pandemic B7/N 105 or A2/S 269 . Taken together, the lower probability of generating B7/N 105 408 TCRs, by way of more insertions, reflects the extreme nature in diversity for both pre- conclusion was that the COVID-19 CD8 + T cell response was not significantly shaped by 433 pre-existing immunity to endemic coronaviruses. However, as 100% of the paediatric and 434 81.3% of adult pre-pandemic donors in our study had a prototypical naïve (and not even stem 435 cell memory; T SCM ) B7/N 105 -specific CD8 + T cell phenotype directly ex vivo, this suggests that 436 either the HCoV-OC43/HKU1 peptide is not presented on the infected cell surface (for 437 example due to different processing of the peptide within the cell), the SARS-CoV-2-derived 438 N 105-113 peptide is not cross-reactive with the corresponding N-derived peptides originating 439 from other human coronaviruses, or our pre-pandemic adult and paediatric donors were not 440 exposed to those circulating coronaviruses. While we cannot exclude that these CD8 + T cells 441 are naïve antigen-experienced T cells that express a naïve CD45RA + CD27 -CD95phenotype, 442 our previous study indicated that the SARS-CoV2 A2/S 269 -specific CD8 + T cells with naïve 443 CD45RA + CD27 -CD95phenotype could not respond to the peptide stimulation (Habel et all single alpha and beta chains, were generated with the TCRdist pipeline, and 679 contextualized using publicly available data from A2/EBV-BMLF1 280-288 , A2/M1 58-66 680 (influenza A), and A2/CMV-pp65 495-503 TCR repertoires, which were not included in the 681 statistical analysis. Statistically analysis between COVID-19 A2/S 269 , B7/N 105 and B7/N 105 682 pre-pandemic (PP) repertoires for variations in P gen , insertions, and deletions are further 683 described in Methods using linear mixed models and P-values were adjusted (p adj ) for 684 multiple testing using the Benjamini & Hochberg FDR method. See also Table S4, Figure S5  685 and Figure S6 . (Table S1 ). The traveller cohort included 3 COVID-19 cases (CA1-3) at 703 convalescence and 4 suspected cases (SU1-4). Acute and convalescent COVID-19 patients 704 were recruited via the Alfred Hospital, Austin Hospital, University of Melbourne or James 705 Cook University. Eight of the donors were admitted to hospital during their active infection 706 (Table S1 ). Healthy pre-pandemic blood donors were recruited via the University of 707

Melbourne or buffy packs obtained from the Australian Red Cross LifeBlood (West 708 Melbourne, Australia) (Table S1 ). Healthy COVID-19-unexposed and pre-pandemic tonsils, 709 spleens and lungs were also obtained. 

Peripheral blood was collected in heparinized tubes and peripheral blood monocular cells 714 (PBMCs) were isolated via Ficoll-Paque separation

Experiments conformed to the Declaration of Helsinki Principles and the Australian 717

National Health and Medical Research Council Code of Practice. Written informed consents 718 were obtained from all blood donors prior to the study. Lung and spleen tissues were 719 obtained from deceased organ donors after written informed consents from the next of kin

Written informed consents were obtained from participants' parental or guardians for 721 underage tonsil tissue donors. The study was approved by the Alfred Hospital (#280/14)

Austin Health (HREC/63201/Austin-2020); the University of Melbourne (#2057366

#2056901.1, #2056689, #2056761, #1442952, #1955465, and #1443389), the Australian Red

the Tasmanian Health and Medical (ID H0017479) and the

Peptides and peptide-HLA class I tetramers

Overlapping synthetic peptides spanning the SARS-CoV-2 Nucleocapsid (N) and Membrane 730 (M) proteins, and the immunogenic regions of Spike (S) were purchased from Miltenyi 731

Biotec and reconstituted in 80% DMSO. SARS-CoV-2 peptides shown to bind HLA

HLA-A*02:01 and HLA-A*24:02 (B7/N 66-74 FPRGQGVPI

QYIKWPWYI) were purchased from GenScript and 735 reconstituted in DMSO. Tetramers were generated from soluble, biotinylated HLA-B*07:02 736 or HLA-A*24:02 monomers. Briefly, HLA α-heavy chain with C-terminal BirA biotinylation 737 motif and β2-microglobulin were expressed and purified as inclusion

6M Guanidine HCl and refolded with either N 105

or S 1208 (for HLA-A*24:02) peptide

Following dialysis in 10mM Tris, HLA monomers 742 were purified via DEAE and HiTrapQ ion exchange chromatography

BirA ligase in 50mM Bicine pH 8.3, 10mM ATP, 10mM magnesium acetate and

Following S200 gel permeation chromatography fully biotinylated HLA monomers 745 were stored at -80 °C and conjugated to fluorescently-labeled streptavidin (SA)

BD Biosciences) at an 8:1 monomer to SA molar ratio to form pMHC-I tetramers

Ex vivo tetramer enrichment

-50x10 6 ) were stained with B7/N 105 -PE, B7/N 257 -APC

A2/S 269 -PE or A24/S 1208 -APC tetramers at room temperature for 1 hr in MACS buffer (PBS 753 with 0.5% BSA and 2 mM EDTA). Cells were then incubated with anti-PE and

microbeads (Miltenyi) and tetramer + cells were enriched using magnetic separation (Nguyen 755

#563964), anti-CCR7-AF700 (#561143), anti-CD14-APC-H7 (#560180

H7 (#560177), anti-CD45RA-FITC (#555488), anti-CD8-PerCP-Cy5.5 (#565310)

CD95-PE-CF594 (#562395), anti-PD1-PE-Cy7 (#561272) (BD Biosciences)

BV510 (#317332), anti-HLA-DR-BV605 (#307640) (BioLegend

Live/Dead near-infrared (#L10119, Invitrogen) stain for 30 mins, washed, resuspended in 762 MACS buffer and analysed by flow cytometry. Lung cells were stained with tetramer for 1 hr 763

PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described 789

Thermo Fisher Scientific) to trimeric S protein biotinylated using 791

recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labelled to APC using an APC 792

PBMCs were surface stained with Aqua viability 793 dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U

IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG 795 BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 796 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048)

CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829)

Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII 799

Intracellular cytokine staining (ICS)

On d10, cells were stimulated with 804 peptides for 5-6 hrs in the presence of GolgiPlug and GolgiStop

10U/mL IL-2, and SARS-CoV-2-reactive T cells were quantified using anti-IFN-γ-V450

(#560371), anti-TNF-AF700 (#557996), anti-MIP-1b-APC (#560686) (BD Biosciences), and 807 anti-CD107a-AF488

Spike-specific ELISA for detection of IgM, IgG and IgA antibodies was 812 performed as described

Enumeration of 876 human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies

A serological assay to 880 detect SARS-CoV-2 seroconversion in humans

Isolation and rapid sharing of the 2019 novel 885 coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia

Virus-888 specific memory CD8 T cells provide substantial protection from lethal severe acute 889 respiratory syndrome coronavirus infection

TCR clonotypes modulate the 892 protective effect of HLA class I molecules in HIV-1 infection

Towards identification of immune and genetic correlates of 896 severe influenza disease in Indigenous Australians

Immunological memory to SARS-CoV-2 assessed for greater 899 than six months after infection

Quantifiable predictive features 902 define epitope specific T cell receptor repertoires

Unbiased Screens Show CD8(+) T 905 Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside 906 outside the Spike Protein

Allele frequency net database (AFND) 2020 update: gold-standard data classification, open 910 access genotype data and new query tools

Targets of T Cell 913 Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed 914 Individuals

circlize Implements and 916 enhances circular visualization in R

Suboptimal SARS-CoV-2-919 specific CD8(+) T cell response associated with the prominent HLA-A*02

Humoral and circulating follicular helper T cell 923 responses in recovered patients with COVID-19

The ABC of Major Histocompatibility Complexes 926 and T Cell Receptors in Health and Disease

Human CD8+ T cell cross-reactivity across 929 influenza A, B and C viruses

Integrated Immune Dynamics Define 932 Correlates of COVID-19 Severity and Antibody Responses

Circulating TFH cells, serological 935 memory, and tissue compartmentalization shape human influenza-specific B cell immunity

A virus specific CD8+ T cell immunodominance hierarchy 939 determined by antigen dose and percursor frequencies

Resonant inelastic x-ray scattering study of [Formula: see text]-RuCl3: a progress report

Age-related dysregulation in CD8 946 T cell homeostasis: kinetics of a diversity loss

Direct link between mhc polymorphism, T cell avidity, and diversity in immune defense

Molecular, cellular, and antigen requirements for development of age-associated T cell clonal 952 expansions in vivo

Magnitude and Kinetics of CD8+ 955 T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point

Maintenance of the EBV-specific CD8+ TCRalphabeta 959 repertoire in immunosuppressed lung transplant recipients

Understanding CD8(+) T-cell responses toward the native 962 and alternate HLA-A*02:01-restricted WT1 epitope

Perturbed CD8(+) T cell 965 immunity across universal influenza epitopes in the elderly

Long-lived memory T lymphocyte responses against SARS coronavirus 968 nucleocapsid protein in SARS-recovered patients

Broad and strong memory CD4(+) and CD8(+) T cells 971 induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

Public clonotype usage 975 identifies protective Gag-specific CD8+ T cell responses in SIV infection

A Shared TCR Bias 979 toward an Immunogenic EBV Epitope Dominates in HLA-B*07:02-Expressing Individuals

Single-Cell Approach to 983 Influenza-Specific CD8(+) T Cell Receptor Repertoires Across Different Age Groups

Tissues, and Following Influenza Virus Infection. Front Immunol 9

Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: 987 rapid induction, prolonged 1contraction, and formation of functional memory

SARS-CoV-2 991 epitopes are recognized by a public and diverse repertoire of human T cell receptors

Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, 996 and Test Setup

Lack of peripheral memory B cell responses 999 in recovered patients with severe acute respiratory syndrome: a six-year follow-up study

Breadth of concomitant immune 1003 responses prior to patient recovery: a case report of non-severe COVID-19

Molecular basis for universal 1007 HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses

Early priming minimizes the age-related 1011 immune compromise of CD8 T cell diversity and function

Recovery from severe H7N9 disease is associated with diverse 1014 response mechanisms dominated by CD8(+) T cells

Recovery from severe H7N9 disease is associated with diverse 1017 response mechanisms driven by CD8+ T cells

Clonally diverse CD38(+)HLA-1021 DR(+)CD8(+) T cells persist during fatal H7N9 disease

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute 1025 respiratory distress syndrome

Aging and 1028 cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in 1029 humans

ggplot2: Elegant Graphics for Data Analysis

Recovery 1034 from the Middle East respiratory syndrome is associated with antibody and T-cell responses

Sci Immunol 2. recall responses during COVID-19. Nguyen et al. analyze ex vivo CD8 + T cells specific for SARS-CoV-2 epitopes and find that immunodominant B7/N 105 -specific CD8 + T cells are present at high frequencies in blood samples from unexposed, acute COVID-19, and convalescence individuals

HIGHLIGHTS • Analyses of SARS-CoV-2-specific CD8 + T cells ex vivo using peptide-HLA tetramers • Tetramer-specific CD8 + T cells in unexposed individuals display a naïve phenotype • B7/N 105

T cells are seen in high numbers during COVID-19 and persist longterm • High naïve frequency and TCR plasticity underpin dominant

per epitope included as a covariate and subject included as a random effect in order to control 853 for unintentional differences in sequencing effort and non-independence of the data across 854 subjects, respectively. Log10 of P gen was modelled using a Gaussian distribution, whereas 855 insertions and deletions were analyzed using a generalized model for the negative binomial 856 distribution. P-values were adjusted (p adj ) for multiple testing using the Benjamini & 857Hochberg FDR method. We also used the TCRdist framework on a subset of the data to 858 characterize repertoire diversity using TCRdiv, which was contextualized using publicly 859 available data from A2/EBV-BMLF1 280-288 , A2/M1 58-66 (influenza A), and A2/CMV-pp65 495-860 503 repertoires. For this analysis, we only considered cells that had functional, paired alpha 861 and beta sequences, and we randomly down-sampled repertoires such that each were derived 862 from an equivalent number of donors and had an equivalent number of TCRs for comparison 863 (i.e., A2/S 269 , B7/N 105 COVID-19, A2/EBV, A2/M1 and A2/CMV). The subsampled and full 864 repertoires are detailed in Table S4 . 865 866

Statistical significance of nonparametric datasets (two-tailed) were determined using 868GraphPad Prism v9 software. Mann-Whitney (unpaired) and Wilcoxin (paired) tests were 869 used for comparisons between two groups. Kruskal-Wallis (unmatched) test with Dunn's 870 multiple comparisons was used to compare more than two groups. Tukey's multiple 871 comparison test compared row means between more than two groups. 872 873 874