key: cord-0920350-35s77wup
authors: Nesterenko, Pavlo A.; McLaughlin, Jami; Tsai, Brandon L.; Burton Sojo, Giselle; Cheng, Donghui; Zhao, Daniel; Mao, Zhiyuan; Bangayan, Nathanael J.; Obusan, Matthew B.; Su, Yapeng; Ng, Rachel H.; Chour, William; Xie, Jingyi; Li, Yan-Ruide; Lee, Derek; Noguchi, Miyako; Carmona, Camille; Phillips, John W.; Kim, Jocelyn T.; Yang, Lili; Heath, James R.; Boutros, Paul C.; Witte, Owen N.
title: HLA-A∗02:01 restricted T cell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses
date: 2021-12-10
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.110167
sha: af4abdd93f0fcc881a867688a61d2982b0444bc3
doc_id: 920350
cord_uid: 35s77wup

Cross-reactivity and direct killing of target cells remain underexplored for SARS-CoV-2 specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA-polymerase (RdRp/NSP12) as highly conserved likely due to its critical role in the virus life cycle. We perform single-cell TCRαβ sequencing in HLA-A∗02:01 restricted, RdRp specific T cells from SARS-CoV-2 unexposed individuals. Human T cells expressing these TCRαβ constructs kill target cell lines engineered to express full length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long term immunity against SARS-CoV-2 and other coronaviruses.

Over 4 million people have died from COVID-19 as of August 2021 (World Health Organization). Many individuals are now immune as a result of successful vaccination 50 campaigns and protection afforded by the natural infection with SARS- CoV-2 (Anand et al., 2021; Baden et al., 2021; Lumley et al., 2020; Polack et al., 2020; Sadoff et al., 2021) . The virus continues to evolve and may escape immune responses generated against the original sequence (Harvey et al., 2021; Planas et al., 2021) . The BNT162b2 mRNA vaccine is 88% effective against the new Delta variant compared with 93.7% for the Alpha variant that was circulating 55

previously (Bernal et al., 2021) . Increased spread in vaccinated populations necessitates further understanding of the SARS-CoV-2 immune response.

This pandemic can only be controlled by herd immunity against contemporary strains of the virus. Vaccination against the wild type spike protein can prevent COVID-19 (Baden et al., 60 2021; Polack et al., 2020; Sadoff et al., 2021) . SARS-CoV-2 vaccines target the spike protein by generating neutralizing antibodies that prevent host cell infection (Khoury et al., 2021; Lumley et al., 2020) . SARS-CoV-2 variants often contain multiple mutations in the spike protein and can resist antibody neutralization creating the possibility that, upon further diversification, viral variants may escape current vaccine defenses (Hoffmann et al., 2021; Kuzmina et al., 2021; 65 Muik et al., 2021; Planas et al., 2021; Wang et al., 2021) . Cytotoxic T cells kill infected cells thereby directly limiting viral dissemination once the infection occurred (Hall et al., 1986; Harty et al., 2000; Jozwik et al., 2015; McMichael et al., 1983) . T cell recognition is not limited to surface proteins like the spike protein; more conserved proteins can be targeted. Internal SARS-CoV-2 proteins are more conserved than the spike and may present a therapeutic opportunity at 70 J o u r n a l P r e -p r o o f 4 generating T cell responses that can recognize many coronavirus strains (Grifoni et al., 2020a) . T cell vaccine strategies, targeting the nucleocapsid protein, are being explored to generate long term immunity against SARS- CoV-2 (Dutta et al., 2020; Gauttier et al., 2020; Sieling et al., 2021) . It remains unknown which epitopes elicit the most effective antiviral responses (Chen and John Wherry, 2020) . 75

Initial evidence for T cell control of respiratory infections was provided by children with genetic defects in T cell development (Hall et al., 1986) . Resident memory T cells, which are permanently localized in non-lymphoid tissues, including the lung, are thought to mediate antiviral responses (Jozwik et al., 2015) . In a human RSV infection disease severity was inversely 80 correlated with the preexisting T cells in the lung (Jozwik et al., 2015) . Adoptive transfer of highly functional T cell clones can reduce severity of viral diseases as well (Einsele et al., 2002; Feuchtinger et al., 2010a) . The mechanism of respiratory viral infection T cell control is thought to happen through FAS and perforin mediated lysis of infected cells (Topham et al., 1997) . The efficiency of lysis correlates with the ability to clear an infection (McMichael et al., 1983) . 85

Both convalescent donors and unexposed individuals have SARS-CoV-2 specific T cell responses (Le Bert et al., 2020; Braun et al., 2020; Grifoni et al., 2020b; Mateus et al., 2020; Peng et al., 2020; Tarke et al., 2021; Weiskopf et al., 2020) . CD8+ T cell responses have been identified as correlates of protection in SARS-CoV-2 infection (Chen and John Wherry, 2020; 90 Liao et al., 2020; McMahan et al., 2021) . Unexposed individuals may have T cell responses that were generated by common cold coronaviruses (HCoVs) and may be partially protective against SARS-CoV-2 encounter (Lipsitch et al., 2020; Mallajosyula et al., 2021; Mateus et al., 2020) . T J o u r n a l P r e -p r o o f 5 cells interact with target antigens through the T cell receptor (TCR), which is a heterodimer of alpha and beta chains. TCRs are inherently cross-reactive to maximize the breadth of ligand 95 recognition, however a single TCR is not guaranteed to recognize related antigens (Sewell, 2012) . Several cross-reactive CD8+ T cell responses are known, but specific TCRαβ clones that can drive such reactivity are not defined (Lineburg et al., 2021; Lipsitch et al., 2020; Mallajosyula et al., 2021; Mateus et al., 2020) . T cell memory is most often defined as ability to recognize synthetic peptide epitopes in functional assays or peptide-MHC multimer staining. 100

Recognition of processed epitopes derived from full length intracellular antigens is underexplored in SARS-CoV-2. Isolation of specific TCR clones permits unambiguous determination of reactivity and detailed characterization of immune responses such as cytotoxic potential and measurement of cross-reactivity against related viruses.

We employ recent technological advances in single-cell sequencing, DNA synthesis and gene transfer to recover antigen specific TCRαβ and subsequently characterize them in allogeneic T cells. The viral polymerase (NSP12/RdRp) was identified as highly conserved within SARS-CoV-2 and other human coronaviruses. RdRp reactive CD8+ T cells were then selected for TCRαβ droplet-based sequencing based on the intracellular level of TNFα and IFNγ via the 110 CLInt-seq, which allows for antigen specific TCR sequencing via commercially available Drop-Seq in cells that are stained for intracellular cytokines (Nesterenko et al., 2021) . TCRs were initially screened for single epitope recognition in a cell line system via the NFAT-GFP reporter system. Reactive TCRs were overexpressed in human PBMCs and killed antigen presenting cells that expressed the full length RdRp. Three TCR constructs were broadly reactive and cross-115 reacted with epitope homologues from HCoVs. J o u r n a l P r e -p r o o f 6 Results:

Antigens derived from highly conserved SARS-CoV-2 proteins should generate immune 120 responses effective against multiple variants. Human coronaviruses are separated by hundreds of years of evolution and serve as a model of the evolutionary constraints that may restrict variant emergence in SARS- CoV-2 (Forni et al., 2017; Killerby et al., 2018) . A group of coronavirus proteins involved in RNA synthesis, immune modulation, and structural machinery was selected for further analysis. Sequence identity was compared across all known human coronaviruses. All 125 proteins showed conservation within sub-classes: alpha coronaviruses (229E and NL63), beta coronaviruses (HKU1 and OC43) and both SARS viruses ( Figure 1A ). The RdRp was most conserved across all coronaviruses ( Figure 1A ). Across 893,589 SARS-CoV-2 samples sequenced, RdRp was well conserved and had few mutations compared to the spike protein ( Figure 1B , Table S4 ). 130

To generate TCR clones, we screened pooled peptide epitopes predicted to bind HLA-A*02:01 against HLA matched peripheral blood mononuclear cells (PBMCs) collected prior to December of 2019 ( Figure 2A ). We refer to these samples as unexposed to SARS-CoV-2. CD8+ T cells 135 that responded by production of TNFα and IFNγ were sorted from four different PBMC donors via fluorescence activated cell sorting (FACS) (Figure 2A ). Responses were low in all donors, around the level of background set based on DMSO control stimulation, as would be expected for donors who were not exposed to a specific pathogen.

Reactive cells were sorted for single-cell TCRαβ sequencing via a highly sensitive technique called CLInt-Seq. Clonally expanded TCR clones were synthesized and tested in an allogeneic cell-based system for evaluation of immune receptor activation ( Figure 2B) . A High throughput system for TCR reactivity profiling was established ( Figure S1A ). We utilized a Jurkat cell line that expressed the NFAT-zsGreen T cell activation reporter construct and the CD8 molecule to 145 stabilize MHC Class I interactions. This cell-based reporter system was then optimized with a well characterized TCR, clone 1G4, which is specific for the cancer antigen NY-ESO-1(D'Angelo et al., 2018) ( Figure S1B ). Comparison of TCR delivery by electroporation or viral integration resulted in similar extent of T cell activation ( Figure S1C ). SARS-CoV-2 specific TCRs were then electroporated or transduced into the Jurkat cell line and activation was 150 measured by FACS measurement of zsGreen. SARS-CoV-2 reactive epitopes were identified via epitope deconvolution using an array of sub-pools ( Figures 2C and S2) . Of 44 TCR constructs tested in this system, ten recognized the cognate peptide pool ( Figure 2D ). TCR clones that did not score as reactive in this assay, either did not reach the threshold of the reporter system or were originally expressed in T cells that did not recognize the queried peptide pool. Because the 155 responses sorted were around the level of background, non-reactive TCRs likely represent the background signal. Nine TCRs clearly recognized four unique epitopes of the RdRp ( Figure S2 and Table S1 ).

Processed antigen recognition is critical for vaccine induced priming of naïve T cell responses as well as for lysis of infected cells. To establish potential antiviral efficacy, seven RdRp specific J o u r n a l P r e -p r o o f 8 TCRs were overexpressed in HLA-A*02:01 positive human PBMCs via retroviral delivery ( Figure 3A and Table S2 ). Engineered PBMCs were co-cultured with a target cell line engineered to overexpress the full-length SARS-CoV-2 RdRp protein and HLA-A*02:01 ( Figure  165 3B). The engineered T cells were able to produce TNFα and IFNγ in response to recognition of processed antigens. (Figure 3C , 3D). Full length antigen recognition was significantly lower than peptide pulsing, as measured by T cell cytokine production, most likely due to the concentration of peptide during pulsing assays being supraphysiological ( Figure 3D ). CD4+ T cells the overexpressed the CoVTCRs also responded to peptide pulsing but did not recognize processed 170

antigen. Production of TNFα and IFNγ in CD4+ T cells ranged from 0.058-10.3%, depending on the TCR.

T cells control viral spread by killing virus infected cells. Cytotoxicity assays showed five out of seven TCRs can direct T cells to kill target cell lines ( Figure 3E ). Recognition of processed 175 epitopes was confirmed by supernatant IFNγ ELISA assay ( Figure 3F ). At 48 hours, processed antigen recognition was equivalent or better than the peptide pulsing control ( Figure 3F ).

We sought to determine how common RdRp specific T cells are. Recently a set of more than 160,000 TCRβ genes specific for SARS-CoV-2 was made publicly available (Nolan et al., 2020) . 180

This data was generated by peptide pool stimulation of PBMCs from 118 donors and subsequent TCRβ gene sequencing in reactive T cells (Klinger et al., 2015) . Unique epitopes were ranked by the count of cognate TCRβ sequences ( Figure S3A ). Three of the four epitopes we identified were frequently targeted by the SARS-CoV-2 specific TCRβ ( Figure S3A ). GLIPH2, an algorithm for grouping TCRs that recognize the same antigen (Huang et al., 2020) , showed three 185 TCRαβ constructs we defined grouped with other TCRs against ORF1ab, which contains the RdRp ( Figure S3B and Table S3 ). The epitope FV9 was frequently targeted, but its cognate TCR, CoVTCR 18, did not share sequence similarity with any ORF1ab specific TCRβ ( Figure   S3A and B). This TCR also lacked killing ability in the prior assay ( Figure 3E ). Peptide titration showed that this TCR only recognized antigen at high concentration of 10 µg/ml, confirming that 190 this TCR has low affinity for this specific target ( Figure S4 ). Two of the four TCRs against the RV9 epitope grouped with ORF1ab specific TCRβ ( Figure S3B ). CoVTCR 34, specific against RV9, was strongly cytotoxic but did not group with any TCRβ by GLIPH2 analysis.

We then queried RdRp TCR cross-reactivity against the HCoV epitopes. Epitope homologs were identified by alignment of RdRp sequences from all human coronaviruses. Each of the homologous epitopes was synthesized and TCR reactivity against each of the epitopes was 

This study provides a strong basis for considering the development of vaccines against either specific epitopes or the full length RdRp. Current vaccines provide strong protection against J o u r n a l P r e -p r o o f 10 COVID-19 caused by circulating variants of SARS-CoV-2. Continuous evolution of SARS-CoV-2 may necessitate updates to the vaccine's spike sequence, selection of a more conserved 210 antigen, or combination of both. One of the challenges of developing booster shots is the need to predict which variant will be the most common when the vaccine is administered. Failure to predict this accurately may decrease the efficacy of the booster. SARS-CoV-2 infection can be recognized by RdRp specific T cells as indicated by strong RdRp CD8+ T cell responses in convalescent donors (Tarke et al., 2021) . The RdRp sequence is particularly well conserved 215 within SARS-CoV-2 and among other human coronaviruses. Sequence conservation suggests that the critical functional role of this protein places restriction on its capacity to evolve. We show that RdRp specific T cells are cytotoxic against cells that express full length antigen, which suggests T cell responses against RdRp should help control SARS-CoV-2 infection and prevent COVID-19 disease. 220

Inducing broadly reactive T cell responses may be particularly important for generating lifelong immunity against SARS-CoV-2. T cells can recognize the target antigen even after accumulation of point mutations (Sewell, 2012) . While we identified RdRp as the most conserved protein, it too is likely to change, as evident from the accumulation of point mutations. Here, we defined 225 two RdRp epitopes that can elicit broadly coronavirus reactive T cell responses. T cells that recognize different human coronaviruses are likely to recognize novel mutation variants as they emerge, due to strong affinity for the antigen. Epitope TL9 reactive T cells have been previously identified as cross-reactive and associated with reduced disease severity, however specific TCR clone driving this response was not identified (Mallajosyula et al., 2021) . For the RV9 epitope 230 some TCRs were cross-reactive but others only recognized SARS-CoV-2, showing that TCRs J o u r n a l P r e -p r o o f 11 against the same antigen can have distinct reactivity. The cross-reactive TCRs against RV9 used the same V alpha chain TRAV38-2DV8, implicating that the usage of common alpha chain may allow cross reactivity. Specific TCR sequences that are known to allow for broad reactivity can be used as benchmarks for induction of such immunity. TCR based disease severity correlation 235 will require more TCR characterization to expand the scope of TCRs and HLA allele restrictions.

Induction of broadly reactive T cell responses, that are not affected by point mutations in the epitope sequence, as well as benchmarks for measurement of such responses can help guide development of T cell vaccines.

Several reports proposed the use of adoptive transfer of antigen specific T cells from convalescent donors, to treat severe COVID-19 disease (Basar et al., 2021; Ferreras et al., 2020; Keller et al., 2020) . Viral infections such as CMV and EBV have been previously treated by transfer of highly functional cytotoxic T cells (Einsele et al., 2002; Feuchtinger et al., 2010b; Papadopoulou et al., 2014) . It remains to be shown whether adoptive transfer of T cells can 245 control SARS-CoV-2 infection in pre-clinical models, which are complicated by the requirement to be done in the BSL-3 setting. Therapeutic T cell engineering is now routinely done for cancer treatment both in the context of clinical trials as well as FDA approved therapeutics (D'Angelo et al., 2018; Depil et al., 2020; Johnson et al., 2006) . There are several advantages to adoptive cell therapy with engineered T cells:1. Large number of antigen specific T cells can be readily 250 produced 2. Well validated TCR specificity 3. T cells have a younger phenotype. TCR engineered T cell also enlist additional CD4+ T cells, which are critical for establishing long term CD8+ T cell memory and antibody production (Sant and McMichael, 2012; Sun and Bevan, 2003) . Current approaches for adoptive T cell therapy are expensive and cumbersome (Depil et al., 2020) . Technological advances in gene delivery may make T cell engineering a practical 255 approach for viral disease treatment in specific groups of patients (Frank et al., 2020) .

We show that RdRp specific TCRs recognize processed epitopes in a reconstructed system of viral infection, however we do not show direct control of live SARS-CoV-2 virus. Such an 260 experiment is complicated by the requirement to be done in BSL3 setting. In addition, viruses developed complicated mechanisms to escape T cell effector function, which can make it difficult to detect activity and recognition. T cell function in SARS-CoV-2 is still being investigated and direct T cell suppression of viral replication has yet to be established. We did not show correlation between T cell responses we identified and disease severity. Such analysis 265 requires a large patient cohort and could indicate the importance of specific T cell responses. Isoplexis. ONW currently has consulting, equity, and/or board relationships with Trethera Corporation, Kronos Biosciences, Sofie Biosciences, Breakthrough Properties, Vida Ventures, Nammi Therapeutics, Two River, Iconovir, Appia BioSciences, Neogene Therapeutics, and Allogene Therapeutics. None of these companies contributed to or directed any of the research reported in this article. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Owen N. Witte (owenwitte@mednet.ucla.edu). 350

We generated unique TCR sequences. The full length TCR clone sequences are provided in this paper. Any cell line that we created and used is available to other investigators.

Data and code availability  Reactive TCR alpha/beta nucleotide sequences are provided in this paper.

 We have not created any original code.

 Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. 360

Cryo preserved peripheral blood mononuclear cells (PBMCs) were commercially purchased (Allcells and Hemacare) or obtained from the CFAR Virology Core Laboratory at the UCLA 365 AIDS Institute. PBMCs were thawed in a water bath set to 37C, transferred to 50 mL conical tube, 1 mL of warm R10 media was added drop wise and then 14 mL R10 were added. Cells were then centrifuged at 1300 RPM for 7 minutes. To isolate reactive T cells, peripheral blood mononuclear cells (PBMCs) (Allcells and Hemacare) were cultured in TCRPMI media: RPMI 1640 (Thermo Fisher), 10% FBS (Omega Scientific), 1X Glutamax (Thermo Fisher), 1X Sodium 370

Pyruvate (Thermo Fisher), 10 mM HEPES (Thermo Fisher), 1X non-essential amino acids (Thermo Fisher), 50μM β-mercaptoethanol (Sigma) and Penicillin-Streptomycin (Omega Scientific). PBMCs were cultured for 8 days in the presence of peptide pools at 1 µg/ml and 25U/ml of IL-2 (Peprotech) as previously described (Nesterenko et al., 2021) . PBMCs were then third-generation lentivirus packaging vector MNDU-3-ires-Strawberry. Lentivirus was produced as described previously (Seet et al., 2017) . Lentivirus was used to infect K562-HLA-A*02:01 cells that were described by us previously (Bethune et al., 2018) . To ensure stable expression of RdRp and HLA-A*02:01 in K562 cells, single cells were cloned by FACS deposition and selected for high levels of RFP and GFP. Stable RdRp protein level was confirmed by Western 395 blot using anti-Strep-tag II antibody (Abcam, 1:500) and goat anti-rabbit-IgG HRP(Bio-Rad, 1:5000). These single cell clones were used for the cytotoxicity assay.

RdRp HLA-A*02:01 restricted epitopes were identified by prior publications and or prediction by the netMHCpan4.0 (B et al., 2020; Grifoni et al., 2020a; Poran et al., 2020) . PBMCs were stimulated in 96 well U bottom plates with 10 µg/ml of peptide and 1 µg/ml of CD28/CD49d antibodies (BD Biosciences). For peptide titration assays, serial dilution was set up where the 405 original 10 µg/ml concertation was diluted 10 fold for every subsequent dilution. For recognition of processed antigen, PBMCs were stimulated with target cell lines without peptide at a 4:1 effector to target ratio. After one-hour, 1X Brefeldin A (Biolegend) was added. Cells were then incubated for 8 more hours after which they were either processed or moved to 4C. For analytical assays, cells were stained for surface markers CD3, CD4, CD8 and intracellular 410 markers TNFα and IFNγ using the Cytofix/Cytoperm kit (BD Biosciences).

For TCRαβ sequencing via the CLInt-Seq staining we followed our previously published protocol (Nesterenko et al., 2021) . Cells were stimulated as described above for intracellular 415 staining. PBS was made from 10X PBS and nuclease free water (Thermo Fisher). All buffers, except for DSP, contained RNAsin (Promega) at 1:400 dilution. Cells were washed twice with staining buffer: nuclease free PBS (Fisher scientific), 1% nuclease free BSA (Gemini) and 1:400

RNAsin plus (Promega). Cells were then stained for 15 min in buffer for surface antigens and subsequently washed once with staining buffer and twice with PBS. DSP was then added to each 

TNFα and IFNγ-producing CD8+ T cells were sorted by FACS into a 2 ml Eppendorf tube as 430 described previously (Nesterenko et al., 2021) . Sorted cells were then resuspended in 30-60 µL in .04% BSA solution with RNAsin, to reach a concentration of more than 100 cells/µL. To achieve such concentration, similarly processed Jurkat cells were added as a carrier cell population. 

TCRαβ were constructed from synthetic DNA fragments (IDT and Swift Biosciences). Some

TCRs were made as retroviral vectors as described previously (Nesterenko et al., 2021) . Some 440 constructs were built into the small pMAX vector (Lonza) designed for transfection-based expression. TCRs were infected into the Jurkat cell line using centrifugation at 1350G for 90 minutes at 30C with 5 µg/ml of polybrene (Sigma-Aldrich). DNA minipreps were prepared using the QIAprep miniprep kit (Qiagen) and concentration was routinely higher than 200 ng/µL. Cells were transfected with 2 µL of DNA, dissolved in water, using the Lonza 4D Nucleofactor 445 (Lonza) in 20 µL transfection media from the SE cell Line kit S (Lonza). Lonza pre-installed electroporation protocol for Jurkat clone E6.1 was then used. Cells were rested for 10 minutes and 80 µL of warm R10 media was added. Then total of 100 µL was transferred to a 24 well plate with 400 µL of warm R10 in each well. Cells were then incubated for 12 hours and afterwards they were used for functional assays over the course of three days. 

Retrovirus was produced as described previously (Nesterenko et al., 2021) . PBMCs were activated with CD3/CD28 beads (Thermo Fisher) in 24 well plates at 1:1 ratio. After 3 days, 460 TCR construct or empty construct NGFR control retrovirus was added to the cells with 5 µg/ml of polybrene (Sigma-Aldrich). Cells were centrifuged at 1350G for 90 minutes at 30C. After transduction, 1 ml from each well was removed and fresh media with 2X cytokines was added.

On the next day, infection was repeated, and the following day cells were washed. Cells were then cultured for two more days, at which point the CD3/CD28 beads were magnetically 465 removed. At this stage, cells were expanded further and used for downstream assays.

Transduction was always confirmed by FACS quantification of secondary transduction marker NGFR, and TCR surface expression was ensured by staining for the murine TCRβ constant region. Our TCR constructs use mouse constant regions to decrease mis-pairing with endogenous TCRs in PBMCs and allow surface TCR staining. 470

K562 target cell lines were co-cultured with TCR engineered PBMCs at a 2:1 effector to target ratio, in supplemented AIM V media without cytokines, with 1 µg/ml of CD28/CD49d antibodies (BD). For these experiments, we used K562 target cell line that stably expressed 475

RdRp as well as HLA-A*02:01. The RdRp negative cell line was used as a control for the assay.

The IncuCyte system (Sartorius) was used to quantify GFP surface. Because only the K562 target cells expressed GFP, loss of GFP was interpreted as decrease in the number of live cells.

48 hours after addition of effectors to targets, 50 µL of supernatant was collected for IFNγ J o u r n a l P r e -p r o o f 22 ELISA analysis as described previously (Nesterenko et al., 2021) using the OptEIA kit for IFNγ 480 detection (BD Biosciences). 

Coronavirus protein sequences were collected from NCBI Virus (Hatcher et al., 2017) . RefSeq 490 assembly accession numbers as follows: SARS-COV-2 (GCF_009858895.2), SARS-COV-1 (GCF_000864885.1), OC43 (GCF_003972325.1), NL63 (GCF_000853865.1), MERS (GCF_000901155.1), HKU1 (GCF_000858765.1), 229E (GCF_000853505.1). For missing protein accessions, we used BLAST(Altschul et al., 1990 ) to find the sequence most similar to the respective SARS-COV-2 protein sequence. We performed multiple sequence alignment and 495 calculated the percent identity matrix using the MUSCLE algorithm (Madeira et al., 2019) .

SARS-CoV-2 amino acid sequence variations representing 893,589 GISAID (Table S4) sequences were downloaded from CoV-Glue, an online web application for analysis of SARS-500

CoV-2 virus genome sequences, on 05-01-2021 (Shu and McCauley, 2017; Singer et al., 2020) .

The sequence of Wuhan-Hu-1 (NCBI, NC_045512.2) was used as a reference sequence for numbering, nucleotide location, and amino acid variations. CoV-GLUE excludes certain GISAID sequences; information about the total number of sequences retrieved from GISAID and the subset of sequences that passed CoV-GLUE exclusion criteria can be found here (cov-505 glue.cvr.gla.ac.uk/#/excludedSeqs). Up-to-date information on SARS-CoV-2 proteins and protein domains was queried from UniProt (https://www.uniprot.org/). Data was visualized using the Boutros Plotting Package (v6.0.3) for R(P'ng et al., 2019).

The SARS-CoV-2 specific TCRβ dataset (also known as ImmuneCODE MIRA) was downloaded from: https://clients.adaptivebiotech.com/pub/covid-2020. GLIPH2 analysis identifies antigen specificity groups based on enrichment of local motifs or global patterns differing by one amino acid in the TCRβ CDR3 amino acid sequences (Huang et al., 2020) .

CoV-2 antigen specific TCRs from ImmuneCODE MIRA dataset (Nolan et al., 2020) . TCRs from CD8 T cell experiments (minigene and class I peptide) were included and filtered for productive TCRβs. We filtered for GLIPH groups with V gene bias (p < 0.01) that contain both CoVTCRs and MIRA TCRs. We mapped the identity of the open reading frame (ORF) targeted by each MIRA TCR and counted the number of MIRA TCRs per ORF for each GLIPH group. 520

FlowJo wash used to analyze flow cytometry data and GraphPad Prism was used to generate plots and statistical analyses. Error bars represent standard deviation. Student's T test was used to compare the groups **p>0.01, ***p>0.001, p>0.0001. Protein conservation was visualized 525 using the Boutros Plotting Package (v6.0.0) for R (P'ng et al., 2019) . Epitope homologs were visualized with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Table S1. Summary of reactive TCR clones and cognate epitopes. Related to Figure 2 . 530 Table S2 . Nucleotide sequence of TCR clones that have been validated in PBMC. Related to -2 Vaccine. N. Engl. J. Med. 384, 403-416. Basar, R., Uprety, N., Ensley, E., Daher, M., Klein, K., Martinez, F., Aung, F., Shanley, M., Hu, 550 B., Gokdemir, E., et al. (2021) . Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy. Cell Rep. 36, 109432. Bernal, J.L., Andrews, N., Gower, C., Gallagher, E., Simmons, R., Thelwall, S., Stowe, J., Tessier, E., Groves, N., Dabrera, G., et al. (2021) . Braun, J., Loyal, L., Frentsch, M., Wendisch, D., Georg, P., Kurth, F., Hippenstiel, S., Dingeldey, M., Kruse, B., Fauchere, F., et al. (2020) . SARS-CoV-2-reactive T cells in healthy donors and patients with [270] [271] [272] [273] [274] Chen, Z., and John Wherry, E. (2020). T cell responses in patients with COVID-19. Nat. Rev. Immunol. 20, 529-536. D'Angelo, S.P., Melchiori, L., Merchant, M.S., Bernstein, D., Glod, J., Kaplan, R., Grupp, S., Tap, W.D., Chagin, K., Binder, G.K., et al. (2018) . Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 c259T Cells in Synovial Sarcoma. 570 Cancer Discov. 8, [944] [945] [946] [947] [948] [949] [950] [951] [952] [953] [954] [955] [956] [957] J o u r n a l P r e -p r o o f 26 Depil, S., Duchateau, P., Grupp, S.A., Mufti, G., and Poirot, L. (2020) . 'Off-the-shelf' allogeneic CAR T cells: development and challenges. Nat. Rev. Drug Discov. 19, 185-199. Dutta, N.K., Mazumdar, K., and Gordy, J.T. (2020) . The Nucleocapsid Protein of SARS-CoV-2:

a Target for Vaccine Development. J. Virol. 94. 575 Einsele, H., Roosnek, E., Rufer, N., Sinzger, C., Riegler, S., Löffler, J., Grigoleit, U., Moris, A., Rammensee, H.-G., Kanz, L., et al. (2002) . 

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Cross-reactive memory T cells and herd immunity to SARS-CoV-2

Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers

The EMBL-EBI search and sequence analysis tools APIs in 2019

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans

Correlates of protection against SARS-CoV-2 in rhesus macaques

Cytotoxic T-Cell Immunity to Influenza

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera

Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning

A large-scale database of T-cell receptor beta (TCRβ) sequences and binding associations from natural and synthetic exposure to SARS-CoV-2

BPG: Seamless, automated and interactive visualization of scientific data

Activity of Broad-Spectrum T Cells as Treatment 695 for AdV, EBV, CMV, BKV, and HHV6 Infections after HSCT

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

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Sequence-based prediction of SARS-CoV-2 vaccine targets using a mass spectrometry-based bioinformatics predictor 710 identifies immunogenic T cell epitopes

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19

Revealing the role of CD4+ T cells in viral immunity

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids

Why must T cells be cross-reactive?

GISAID: Global initiative on sharing all influenza data -from vision to reality

Antigen Vaccination of 1 Healthy Volunteers Induces a Ten-Fold Increase in Mean

Cell Responses 2 Equivalent to T-Cell Responses from Patients Previously Infected with SARS-CoV-2 3 4

CoV-GLUE: A Web Application for Tracking SARS-CoV-2 Genomic Variation

Defective CD8 T Cell Memory Following Acute Infection 730

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases

CD8+ T cells clear influenza virus by perforin or Fas-dependent processes

Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7

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

Human MHC Class I-restricted high avidity CD4+ T cells generated by co-transfer of TCR and CD8 mediate efficient tumor rejection in vivo