key: cord-0428426-53fqlujz authors: Wagner, K. I.; Mateyka, L. M.; Jarosch, S.; Grass, V.; Weber, S.; Schober, K.; Hammel, M.; Burrell, T.; Kalali, B.; Poppert, H.; Beyer, H.; Schambeck, S.; Holdenrieder, S.; Stroetges-Achatz, A.; Haselmann, V.; Neumaier, M.; Erber, J.; Priller, A.; Yazici, S.; Roggendorf, H.; Odendahl, M.; Tonn, T.; Witter, K.; Dick, A.; Mijocevic, H.; Protzer, U.; Knolle, P. A.; Pichlmair, A.; Crowell, C. S.; Gerhard, M.; D Ippolito, E.; Busch, D. H. title: Recruitment of highly functional SARS-CoV-2-specific CD8+ T cell receptors mediating cytotoxicity of virus-infected target cells in non-severe COVID-19 date: 2021-07-23 journal: nan DOI: 10.1101/2021.07.20.21260845 sha: fb382586f44f08f43ff8f040916b538fb9e80e26 doc_id: 428426 cord_uid: 53fqlujz T cell immunity is crucial for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and has been widely characterized on a quantitative level. In contrast, the quality of such T cell responses has been poorly investigated, in particular in the case of CD8+ T cells. Here, we explored the quality of SARS-CoV-2-specific CD8+ T cell responses in individuals who recovered from mild symptomatic infections, through which protective immunity should develop, by functional characterization of their T cell receptor (TCR) repertoire. CD8+ T cell responses specific for SARS-CoV-2-derived epitopes were low in frequency but could be detected robustly early as well as late - up to twelve months - after infection. A pool of immunodominant epitopes, which accurately identified previous SARSCoV- 2 infections, was used to isolate TCRs specific for epitopes restricted by common HLA class I molecules. TCR-engineered T cells showed heterogeneous functional avidity and cytotoxicity towards virus-infected target cells. High TCR functionality correlated with gene signatures of T cell function and activation that, remarkably, could be retrieved for each epitope:HLA combination and patient analyzed. Overall, our data demonstrate that highly functional HLA class I TCRs are recruited and maintained upon mild SARS-CoV-2 infection. Such validated epitopes and TCRs could become valuable tools for the development of diagnostic tests determining the quality of SARS-CoV-2-specific CD8+ T cell immunity, and thereby investigating correlates of protection, as well as to restore functional immunity through therapeutic transfer of TCR-engineered T cells. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new beta coronavirus responsible for the Coronavirus Disease 2019 (COVID- 19) , a pathological condition that can progress to severe pneumonia and a fatal outcome 1, 2 . The adaptive immune system plays a critical role in SARS-CoV-2 infections. In most cases, T and B cells react quickly and in a coordinated manner to the infection [3] [4] [5] and develop a robust memory pool detectable months after exposure regardless of disease severity [6] [7] [8] . In line with that, individuals who recovered from COVID-19 experienced low reinfection rates, in particular for symptomatic infections [9] [10] [11] . In contrast, severe clinical manifestations occurred in the less frequent scenario where immune responses were suboptimal -i.e. in elderly individuals where antigen presentation is less efficient and the pool of naïve T cells is scarce 4, 12 . Despite the increasing understanding of SARS-CoV-2 immunity, a clear correlate of protection is still missing. This is of paramount importance as it would allow for the identification of individuals with minimal risk of reinfection as well as the minority of patients with high risk of developing severe symptoms due to lack of adequate immunity. In addition, it would provide relevant platforms or tools for the validation of candidate vaccines, including immunity towards virus variants. Antibody titers have been extensively used to describe SARS-CoV-2 adaptive immunity as their detection is suitable for high-throughput testing and intrinsically mirrors an adequate recruitment of CD4 + T cells. However, the role of antibodies in protective immunity is still controversial. Seropositive convalescent individuals showed lower risk of reinfections 11,13,14 but neither total nor neutralizing antibody titers protected from severe symptoms during primary infections 4, 15 . In addition, the resolution of SARS-CoV-2 infections in individuals unable to produce antibodies 16 further indicated that other immune compartments are rather essential for protective immunity. Early induction of SARS-CoV-2-specific T cells was shown to associate with milder diseases and less prolonged virus shedding 4, 17 and, remarkably, depletion of CD8 + T cells abrogated protectiveness against re-challenge in pre-clinical models 18 . Altogether, this evidence strongly supports a key role of T cells in the control and resolution of SARS-CoV-2 infections. This should hold true in particular for CD8 + T cells due to their unique contribution in protecting from intracellular pathogens 19 by the direct killing of target cells. Of similar importance, T cells persist longer than waning antibodies 8, 20 , thus being more informative about the long-term maintenance of functional SARS-CoV-2 immunity. SARS-CoV-2-specific T cell immunity has been widely characterized quantitatively 6, 8, 12, 21, 22 . However, little is known about their quality, and thereby determinants of protection. This lack of knowledge in the COVID-19 field mainly derives from the broad use of single dose peptide mixes (15-mers) for T cell analyses, which are often preferred as they can be designed easily to cover entire open reading frames (ORFs). Their usage was highly valuable to gain information on the magnitude and breadth of SARS-CoV-2 T cell responses in a remarkably short time 8, 22 but at the expense of precision, i.e. specificity and quality of single-epitope responses. Indeed, the epitopes responsible for the observed T cell reactivity are often unknown and peptide mixes are usually used at a single high concentration, which hinders discrimination of high (protective) and low (suboptimal) functional T cells. This information has become extremely relevant as i) high frequency of cross-reactivity with common cold coronaviruses was reported, even though mainly for CD4 + T cells [23] [24] [25] [26] [27] , and ii) suboptimal -but high-frequency -T cell responses were observed in severe COVID-19 patients due to recruitment of low functional cross-reactive memory T cells rather than highly specific naïve T cells 12 . Finally, CD8 + T cell responses are still under-represented, and thereby less investigated, as 15-mer peptides primarily stimulate CD4 + T cells 25 . Altogether, this evidence underlines how the detection and magnitude of T cell responses is not always equivalent to functionality. Thus, we decided to investigate in depth the quality of SARS-CoV-2-specific CD8 + T cells through the analyses of their T cell receptor (TCR) repertoire. A TCR is the fingerprint of a T cell and determines its specificity, functionality and fate. Furthermore, preclinical studies have shown that highly functional TCRs drive the establishment of protective immunity in infectious diseases, as they dominate primary infections 28 and react faster to recall infections 29 . By using SARS-CoV-2 immunodominant epitopes restricted to the most common Human Leucocyte Antigen (HLA) class I molecules, we accessed the SARS-CoV-2 epitope-specific TCR repertoire in convalescent individuals who experienced mild symptoms and for whom, thereby, a protective immunity should have established. We identified highly functional TCRs that, when engineered in primary T cells, conferred high sensitivity to epitope stimulation and cytolytic capacity toward target cells which were infected with viable SARS-CoV-2 virus. Interestingly, TCR functionality correlated with a gene signature of recent activation that we could retrieve from each epitope:HLA combination analyzed. Overall, we show that highly functional, and thereby protective, TCRs are recruited in CD8 + T cell responses against SARS-CoV-2 during non-severe disease. To study SARS-CoV-2-specific CD8 + T cell responses, we designed a pool of SARS-CoV-2 peptides (9-mer) predicted to be immunogenic for the most common HLA class I molecules (HLA-A*01:01, A*02:01, A*03:01, A*11:01, A*24:02, B*07:02, B*08:01, B*35:01). This HLA combination covers 73% of the European Caucasian population (cumulative HLA allele frequency, allelefrequencies.net, similar to 30 ). Briefly, we performed in silico predictions for peptides of 8-11 amino acid length derived from all ORFs of the SARS-CoV-2 genome and analyzed for their MHC Class I binding affinity to the selected set of HLAs. Peptides with high prediction for stable HLA binding were further screened for additional parameters (i.e. proteasomal cleavage, immunogenicity and transport to the cell surface), which are important prerequisites for a proper antigen surface presentation. We finally selected 40 candidates, among which nine showed 100% homology with SARS-CoV and 31 were unique for SARS-CoV-2 (Supplementary Table 1 ). No homology was found with published epitopes (IEBD databank) from "common cold" coronaviruses (229E, NL63, HKU1, OC43, degree of homology higher than 70%). SARS-CoV-2 T cell responses contract and become barely detectable ex vivo within a few weeks after infection 31 . Our cohort of convalescent individuals (PCR+ with mild course of the disease and no need of hospitalization, referred to as mild COVID-19 from now on) comprised of blood sampled at least 30-50 days after infection. Therefore, we tested the sensitivity of ex vivo responses to a commercially available peptide mix of the Spike (S) protein (Peptivator S, 15-mer mix) and, as expected, we detected low frequency but reliable T cell responses in most mild COVID-19, despite mainly in CD8 -T cells ( Supplementary Fig. 1 ). Stimulation with our 9mer pool, however, indicated detectable T cell responses in few individuals where the size of frequencies and robustness of detection were in addition suboptimal ( Supplementary Fig. 1 ), primarily due to the small size of our pool (4-fold lower number of peptides than Peptivator S pool). Thus, in order to detect such extremely low frequency SARS-CoV-2 reactive CD8 + T cell populations, we adapted an expansion protocol where T cells are stimulated with autologous pulsed PBMCs and in vitro expanded prior re-challenge and T cell functional analyses 32 (Fig. 1A ). We successfully observed SARS-CoV-2 T cell responses after expansion within a set of mild COVID-19 subjects; furthermore, as expected by the design of the 9-mer peptide pool, primarily CD8 + but not CD8 -T cells responded to the stimulation ( Supplementary Fig. 2 ). After validating the robustness of our 9-mer pool in stimulating SARS-CoV-2-specific CD8 + T cell responses, we next studied such T cell responses across four distinct cohorts: 53 mild COVID-19; 28 asymptomatic seropositive individuals; 37 asymptomatic individuals who continuously tested seronegative for SARS-CoV-2 IgG antibodies throughout the observation period; 28 unexposed individuals for whom blood was collected before the outbreak (Table 2) . Mild COVID-19 and asymptomatic seropositive individuals showed strong, although variable, CD8 + T cell responses with 81% and 78% response rate, respectively (detection limit set to 0.1% IFN-γ + CD8 + /CD8 -T cells after background subtraction). In contrast, CD8 + T cell responses were observed in a small proportion of asymptomatic seronegative and unexposed individuals, with very weak responses especially for pre-pandemic donors ( Fig. 1 B-C) . T cell responses could develop in absence of antibody production 6 , supporting our findings in the asympomatic seronegative individuals. Reactivity in pre-pandemic individuals, instead, has been explained as cross-reactive T cells 26, 27, 33 or associated to an exceptionally high naïve precursor frequency 34 . As before, CD8 + T cells dominated the overall T cell responses after in vitro peptide expansion ( Fig. 1 C and Supplementary Fig. 3 ). In order to exclude a bias in T cell responses due to a different representation of HLA class I molecules among the different cohorts, we characterized the HLA class I haplotype of each participant via either genomic sequencing or HLA-specific antibody staining, according to sample availability. For the HLA class I molecules included in the SARS-CoV-2 epitope prediction, the coverage among the four cohorts was at comparable levels for the majority of HLA class I molecules ( Supplementary Fig. 4 ). Except for pre-pandemic donors, blood was collected over several months, thus allowing us to investigate the longevity of SARS-CoV-2 CD8 + T cell immunity. We first looked at CD8 + T cell responses and antibody titers at early time points after diagnosis (PCR test) for mild COVID-19 and after enrollment in the study for asymptomatic seropositive cohorts. We observed concomitantly detectable levels of SARS-CoV-2-specific IgG and CD8 + T cells in the majority of the individuals (Fig. 1D ), in line with previous reports describing coordinated responses of the humoral and T cell arms in individuals who resolved the infection without the development of severe symptoms 4 . Despite the fact that antibody titers rapidly waning over time (Fig. 1E ), SARS-CoV-2-specific CD8 + T cells remained relatively stable (Fig. 1F ), similar to other reports 8, 20 , and, remarkably, were detected up to twelve months post-infection (Fig. 1G) . Overall, we could show that the designed 9-mer pool can be used to detect SARS-CoV-2 CD8 + T cell responses regardless symptom severity, and that long-lasting CD8 + T cell immunity establishes upon primary infection. Before investigating the functionality of SARS-CoV-2-specifc TCR repertoires, we searched in our 9-mer pool for immunodominant epitopes, in order to study CD8 + T cell responses specific for single epitopes that might have high relevance in SARS-CoV-2 infection. To do so, we applied a two-step deconvolution process. First, the 9-mer pool was split into four distinct sub-pools, each composed of 8-12 peptides, which we used to re-stimulate PBMCs previously expanded on the entire 9-mer pool ( Supplementary Fig. 5 ). In a second step, peptides from reactive sub-pools were tested individually on the same donors. We performed this deconvolution on mild COVID-19 responders (Fig. 1C) for whom HLA class I genotype was available (n = 34). We found 19 immunogenic peptides eliciting IFN-γ secretion in CD8 + T cells, with a certain variability in the magnitude of responses and the in number of responders ( Fig. 2A and Supplementary Fig. 6 ). Except for ORF1_LTN and ORF1_HSI, immunogenicity All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint of the remaining epitopes was reported also in other studies 8, 24, 27, 33, 35, 36 (Supplementary Table 1 ). Interestingly, we often observed CD8 + T cell responses against multiple epitopes deriving from the same SARS-CoV-2 ORF (e.g. donor #11 and #12) as well as from different ORFs (e.g. donor #14 and #24) ( Fig. 2A) in individual donors, indicating that broad and polyclonal CD8 + T cell response are elicited upon infection. We also assessed the specificity of the identified immunogenic epitopes to SARS-CoV-2 through evaluation of single-peptide responses in the few pre-pandemic responders. We confirmed CD8 + T cell reactivity and identified immunogenic epitopes only for four of them. In addition, we observed responses to only one epitope per donor and at low magnitude ( Fig. 2A, donors [35] [36] [37] [38] [39] [40] [41] [42] , overall highlighting how less consistent and robust these responses are comparable to mild COVID-19. All four epitopes stimulating CD8 + T cells in pre-pandemic individuals were found immunogenic also in mild COVID-19 ( Fig. 2A) . Despite a similar frequency in the unexposed individuals (except for B35/ORF1_VPF), ORF1_DTD and ORF3_FTS epitopes showed remarkably high immunodominance in mild COVID-19 ( Fig. 2B ), which could be explained by an unusually high precursor naïve frequency similar to the findings of Nguyen et al. 34 In contrast, similar percentages of responders were found in mild COVID-19 and unexposed for A2/N_LLL and B35/OFR1_VPF epitopes (Fig. 2B) , which would more support the hypothesis of cross-reactive epitopes with limited relevance in SARS-CoV-2 infections. Additional analyses would be necessary to decipher comprehensively the source of pre-pandemic responses. Next, to understand the relevance of the identified immunogenic epitopes in SARS-CoV-2 T cell immunity, we quantified their immunodominance by contextualizing CD8 + T cell responses with regard to the HLA background of the donors (in other words, we analyzed the number of responders to HLA-matched predicted epitopes). Among the 19 immunogenic SARS-CoV-2 epitopes, eleven showed immunodominance of at least 50% (Fig. 2C , marked in red). By combining the responses of individual epitopes restricted to the same HLA class I molecule, we achieved a response rate of 100% for HLA-A*01:01, HLA-A*11:01 and B*35:02 and 63% for HLA-A*03:01 but only 40% for HLA-A*02:01, 33% for HLA-B*08:01 and less than 15% or no responses for the remaining HLAs ( Supplementary Fig. 7A ). To increase HLA coverage, we further tested a second pool of SARS-CoV-2-derived 9-mers composed of a mixture of newly predicted and published epitopes (Supplementary Table 1 ). We confirmed additional 27 immunogenic epitopes, of which 17 displayed an immunodominance higher than 50% ( Fig. 2D and Supplementary Fig. 7B ); furthermore, we sharply increased the response rate for HLA- Overall, we identified a pool of immunodominant SARS-CoV-2 epitopes that could specifically identify individuals who had been exposed to the virus. Indeed, despite sporadic responses in All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint non-infected individuals, the unique pattern of multiple-epitope responses makes convalescent COVID-19 individuals uniquely distinguishable. Previous analyses have revealed several promising CD8 + T cell epitopes with high immunodominance and specificity to SARS-CoV-2, but without providing any information on the quality of the detected T cell responses. To fill this gap in knowledge, we identified and functionally characterized SARS-CoV-2 epitope-specific TCR repertoires. We first focused on two highly immunodominant SARS-CoV-2 epitopes restricted to frequent HLA class I molecules (A1/ORF1_VTN and A3/ORF3a_FTS). PBMCs from HLA-matched, mild COVID-19 individuals were expanded on the 9-mer pool and re-challenged with either A1/ORF1_VTN or A3/ORF3a_FTS epitope prior to flow-cytometric cell sorting followed by single-cell RNA sequencing (scRNA-seq). In addition to CD8 + IFN-γ + T cells, which should be enriched in freshly re-activated T cells specific for the investigated epitopes, we also sorted CD8 + IFN-γ -T cells where TCRs specific for either other immunogenic epitopes of the 9-mer pool or completely unrelated pathogens could be found (Fig. 3A) . In this way, on the one hand, most of the information about SARS-CoV-2-specific TCR repertoire of a donor would be retrieved without losing the focus on the epitopes of highest interest. On the other hand, the potential of gene signatures to infer TCR specificity and functionality may be explored. In total, four donors (two donors for each selected peptide) were investigated ( Supplementary Fig. 8 ). Analysis of clonotypic expansion and Leiden clustering revealed that the most expanded TCR clonotypes were present in clusters 0, 1 and 5 ( Fig. 3B-C) , indicating that these three clusters may contain activated and expanded SARS-CoV-2-specific T cells. Particularly cluster 1 contained cells with high, and for some markers unique, expression of effector molecules (IFNG, GZMB and IL-2) and activation markers (XCL1, CD69 and CRTAM) (Fig. 3D ). The XCL1 chemokine is produced by activated T cells during infections and inflammatory responses and interacts with XCR1 receptor on dendritic cell, thereby promoting dendritic cellmediated cytotoxic immune response 37 . CRTAM is expressed on activated T cells 38, 39 and coordinates cell polarity during activation, which was shown crucial for production of effector cytokines 40 . Together with the up-regulation of IFNG, GZMB, IL-2 and CD69 genes, this signature of recent activation may suggest an enrichment of T cells specific for A1/ORF1_VTN and A3/ORF3a_FTS epitopes in cluster 1. Pooled samples were deconvoluted according to single nucleotide polymorphisms 41 and assigned to an individual donor by gender and HLA class I genotype (Supplementary Fig. 9 A-C). All donors showed a polyclonal TCR repertoire ( Fig. 3E ) with the dominating clonotypes distributed among clusters 0, 1 and 5 (Fig. 3F) . The TCR repertoire for cluster 1 (IFNG + cluster) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint was highly diverse, in particular in case of ORF3a_FTS and showed a higher fraction of clonally expanded TCRs (Fig. 3E ). In summary, we found that SARS-CoV-2-specific TCR repertoires are highly polyclonal with some clonotypes showing a prominent signature of recent activation, presumably reflecting fresh re-stimulation. To investigate the quality of the identified SARS-CoV-2-specific TCR repertoires, we next reexpressed candidate TCRs for functional characterization. We selected TCRs from both We first tested the specificity of our transgenic TCRs against ORF1_VTN and ORF3a_FTS epitopes via epitope-HLA multimer staining. All TCRs selected from cluster 1 (IFNG + cluster) showed strong staining toward the corresponding HLA-matched multimer (relevant multimer), except for A3/TCR 32 that stained weaker. No staining was instead observed for irrelevant multimers i.e. HLA-A*03:01 or HLA-A*01:01 multimer loaded with a different epitope (SARS-CoV-2 ORF1_KLF and pp50245-253, respectively), thus confirming specific epitope recognition ( Fig. 4A-B) . Instead, none of the TCRs selected from clusters 0 and 5 (IFNGcluster) reacted to ORF1_VTN and ORF3a_FTS multimers, but A3/TCR 40 showed specificity for ORF1_KLF ( Fig. 4A-B ). To evaluate whether the remaining IFNGclusters-derived TCRs could recognize SARS-CoV-2 epitopes at all, we pulsed autologous PBMCs (from the same donor the TCR was isolated from) with SARS-CoV-2 9-mer pool and used them to stimulate TCR-engineered T cells; cytokine release was observed only for A1/TCR 3399 ( Supplementary Fig. 10 ). As CMV and EBV infections have high prevalence in the human population and usually induce big-size clonal responses 43, 44 , we also stimulated our engineered T cells with CMV and EBV peptide pools but no cytokine release was observed ( Supplementary Fig. 10 ). Altogether, HLA multimer staining confirmed that cluster 1 was enriched in freshly re-stimulated TCRs and that signature of recent activations are indicative of TCR specificity to recent epitope re-challenge. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. In summary, we showed that the TCR repertoire specific for SARS-CoV-2 ORF1_VTN and ORF3a_FTS epitopes contains highly functional and cytotoxic TCRs and that expression of genes related to recent activation is indicative of epitope specificity. The validated TCRs compose a minor part of the total repertoire we isolated out of ORF1_VTN and ORF3a_FTS specific T cells. Expression of IFNG, among other genes, was indicative of epitope specificity, as it reflected reactivity after fresh re-stimulation prior to sorting and sequencing. Therefore, we searched our transcriptomic dataset for gene signatures that could associate with T cell functionality in order to accurately predict functionality also for nonvalidated TCRs. Taking into consideration that TCRs were isolated from in vitro expanded CD8 + T cells after fresh re-stimulation, we analyzed the predictive potential of an available gene signature related to CD8 + T cell activation. CD8 + activation score was enriched in cells expressing freshly restimulated TCRs, but it showed only a trend of correlation with T cell functionality among the reactive TCRs (IFN-γ EC50) (Fig. 5A ). To improve the sensitivity of prediction, we defined two additional gene signatures more specific for our dataset and based on gene expression of cells expressing the TCRs that we selected for re-expression and characterization; a "reactivity signature" composed of genes differentially expressed between epitope-reactive TCRs and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. Fig. 11A ), and a "functionality signature" comprising genes that best correlated with IFN-γ EC50 (Supplementary Fig. 11B -C). Epitope-reactive TCRs showed high scores for both signatures and, remarkably, functionality score accurately predicted T cell functionality. The percentage of IFNG + cells, but not the clonotype size, also clearly identified epitope-reactive TCRs but was not sufficient to resolve low (TCR 32) and highly functional TCRs (TCR 13 and TCR 28 among others) (Fig. 5A) . When applied to the entire identified TCR repertoires, high activation and functionality scores clearly separated and identified SARS-CoV-2-specific T cells responding to the recent re-stimulation, which were almost all belonging to the IFNG + cluster (Fig. 5B ). More importantly, within the reactive TCRs, We finally expanded this TCR repertoire analyses to additional nine immunodominant SARS-CoV-2 epitopes (Fig. 2D ) restricted to five different HLA class I molecules in eight mild COVID-19 individuals. Each donor was stimulated with a specific epitope of interest and expanded in vitro prior to re-stimulation and sorting of CD8 + IFN-γ + cells. Each individual was additionally labeled with a DNA-tagged antibody in order to make it distinguishable when pooled with other donors for scRNA-seq (Fig. 6A ). Despite sorting, transcriptomic data revealed heterogeneous expression of genes related to T cell function and activation, which resulted in different enrichments in the reactivity, functionality and activation scores (Fig. 6B ). High reactivity scores were broadly observed, suggesting that the majority of the isolated repertoire should be specific to SARS-CoV-2. In addition, a fraction of those TCRs was particularly enriched also in the functionality score, as well as in the activation score, indicating the presence of TCRs of presumably high functionality (Fig. 6B) . For a better evaluation, we correlated the reactivity and functionality scores for each clonotype of the newly identified repertoires, and we used the in vitro characterized TCRs (Fig. 4-5 ) as controls. We selected the functionality score over the activation score due to its higher accuracy in predicting TCR functionality (Fig. 5A ). We observed a bimodal distribution, with the functional transgenic TCRs (TCR 13, TCR 28, TCR 43, TCR 3456 and TCR 3398) occupying the score high cluster and the low-avidity (TCR 32)/nospecific TCRs (TCR 18, TCR 40, TCR 82, TCR 3409 and TCR 3399) showing low scores. All other clonotypes distributed within the reactivity/functionality landscape and a relevant number of clonotypes overlaid with highly functional transgenic TCRs (Fig. 6C) , thus further corroborating our initial observations. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint As a next step, we wanted to understand the functional landscape of epitope-specific TCR repertoire. To do so, we deconvoluted the two sample pools and assigned each sample to one donor according to DNA barcodes ( Supplementary Fig. 12 ). Most of the donors showed again a highly polyclonal TCR repertoire (Fig. 6D) . Remarkably, highly functional (defined by high gene signature scores) TCRs were predicted for all donors and analyzed SARS-CoV-2 epitopes (Fig. 6E) . Overall, we could show that polyclonal CD8 + T cell responses are elicited against immunodominant SARS-CoV-2 epitopes and that highly functional TCRs are recruited in mild COVID-19, despite some variability according to HLA-epitope combination. The importance of CD8 + T cells in respiratory virus infections is well recognized. CD8 + T cells are recruited to the lungs within 8-10 days from infection [45] [46] [47] , contribute to viral clearance [48] [49] [50] , and generate a pool of memory cells that protect from re-infection 46, 48 . Also in SARS-CoV infections, CD8 + T cells provided substantial protection in preclinical studies 51 and long-lasting memory SARS-CoV-specific CD8 + T cells have been detected up to 17 years after infection in humans 24 . T cell immunity against SARS-CoV-2 shares many of the above-mentioned aspects. Early recruitment of T cells prevented severe disease 15 and was usually followed by the establishment of a robust pool of functional memory T cells 6,27 detectable up to 6-10 months after infection 20, [52] [53] [54] . In addition to this existing body of evidence, we further showed that SARS-CoV-2-specific CD8 + T cells are detectable up to 12 months from infections, a hint pointing towards long-lasting immunity similar to SARS-CoV. Of course, continuous follow-up is necessary to strengthen the interpretation of even longer maintenance of CD8 + T cell immunity. However, whether and how CD8 + T cells may mediate protective immunity in SARS-CoV-2 infections needs to be investigated in more detail. Despite encouraging preclinical data showing loss of protection following CD8 + T cell depletion 55 , evidence of the protective role of CD8 + T cells in humans is still scarce. SARS-CoV-2-reactive CD8 + T cells have often been All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. 61, 62 , especially now that precise genetic All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint engineering offers the flexibility of generating increasingly sophisticated but near-tophysiological autologous T cell products 42 . Furthermore, accumulated evidence supports a model where late recruitment of T cells 15, 63 , presumably due to a delayed activation of type I interferon response 64 Remarkably, we predicted highly functional TCRs for each of the eleven immunodominant SARS-CoV-2 epitopes analyzed, despite a certain degree of functional heterogeneity. Together with the observed polyclonality, our data indicate that functional and diverse CD8 + T cell immunity should normally establish in non-severe SARS-CoV-2 infections. Polyclonality and high functionality are hallmarks of a protective TCR repertoire 67 , thus strongly supporting a similar role also in the context of SARS-CoV-2 infections. Nevertheless, the extension of TCR repertoire analyses to settings of severe infections remains a fundamental next step to assign CD8 + T cell responses a role as correlates of protection. Overall, our data provide first evidence that SARS-CoV-2-specific CD8 + T cells persists up to 12 months after infection and are composed of a polyclonal and highly functional TCR repertoire capable of mediating direct killing of virus-infected cells. In addition, we provide tools -epitopes and TCRs -indicative of functional responses, useful for appropriately investigating and potentially diagnosing correlates of protection in severe patients. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. All data generated or analyzed during this study are included in this article, its supplementary information files and/or are available from the corresponding authors upon reasonable request. The notebooks containing all steps of data processing and analysis can be found online (https://github.com/SebastianJarosch/2021_Wagner_Mateyka_Jarosch_COVID_scRNAseq) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint HKU1 (NC_006577), HCoV-NL63 (NC_005831) and HCoV-229E (NC_002645). Peptides unique to SARS-CoV-2 with highest scores in various prediction tools were selected for further validation in an in-house peptide pool. The peptide pool was supplemented with IEDB published SARS-CoV-2 epitopes and epitopes homologous to SARS-CoV-1 (NC_004718) and MERS (NC_019843) (Supplementary Table 1 ). For all time points of blood donation, a serum sample was taken and analyzed for anti-SARS-COV-2 IgG using the iFlash Immunoassay analyzer, following the manufacturer´s protocol. Briefly, the serum samples were incubated with samples treatment solution and SARS-CoV-2 antigen-coated paramagnetic microparticles to form a complex. Unbound material was washed from the solid phase and a second incubation step with Acridinium-labeled anti-human IgG conjugate followed. After washing, the pre-Trigger and Trigger solutions were added to the reaction mix and the resulting chemiluminescent reaction was measured as relative light units by the iFlash optical system. A cutoff was calculated from SARS-CoV-2 IgG calibrators. PBMCs were isolated from whole blood by gradient density centrifugation according to For intracellular cytokine release assay of ex vivo or post-expansion natural T cells, PBMCs were stimulated with 1 µg/ml peptide pool, 1 µg/ml peptide or 1 µg/ml PepTivator SARS-CoV-2 Protein S pool. For TCR-engineered T cells, K562 antigen presenting cells (retrovirally transduced with HLA-A1 or HLA-A3) were irradiated (80 Gy), loaded with different peptide All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Analyzer. After import of sequence raw data in the uType software, the sequences were analyzed for HLA type creation by aligning to recent IMGT HLA allele database. All detailed information for the strategy of scRNA seq data pre-processing can be found in the uploaded notebooks. All analyses have been performed using SCANPY 68 . Briefly, cells with less than 200 genes were excluded as well as genes present in less than three cells. Cells with more than 20 % mitochondrial genes were excluded and cut-offs for the maximum number of counts (experiment 1: 50.000, experiment 2: 40.000) and number of genes (experiment 1: 7.000, experiment 2: 6.000) were selected individually for the two experiments. Counts were normalized per cell, logarithmized and the variance was scaled to unit variance and zero mean. The number of counts, percentage of mitochondrial genes and cell cycle score was regressed out before highly variable genes were identified and filtered. The data was batch corrected using batch-balanced k nearest neighbors (bbknn) for the individual donors. Donor reallocation All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. ; https://doi.org/10.1101/2021.07.20.21260845 doi: medRxiv preprint was performed using scSplit 41 and hla-genotyper/gene score for Y chromosome genes (https://www.uniprot.org/docs/humchry.txt) for the first experiment and souporcell 69 in combination with barcoded antibodies for the second experiment. Since HLA prediction from RNA sequencing data is not very accurate, an HLA score was introduced to allocate donors to clusters derived from scSplit demultiplexing. In principle, HLA matching was scored considering all predictions and all original genotypes in order to find the best match between prediction and genotype. Detailed information can be found in the uploaded notebooks. Clonotype analysis was performed using scirpy 70 . Cells belonging to one clonotypes were defined to have identical αand βchain CDR3 nucleotide sequences and both pairs of TRA/TRB sequences were considered in case additional chains have been present. DNA constructs for CRISPR/Cas-9-mediated HDR at TRAC locus were designed in silico with the following structure: 5′ homology arm (300-400 base pairs (bp), P2A, TCR-β (including mTRBC with additional cysteine bridge 71 ), T2A, TCR-α (including mTRAC with additional cysteine bridge 71 ), bGHpA tail, 3′ homology arm (300-400 bp). All HDR DNA template sequences were synthesized by Twist. CRISPR/Cas9-mediated TCR knock-out and knock-in (KI) with subsequent editing verification via TCR KI staining and FACS was performed as described 42 on PBMCs isolated from whole blood of healthy donors and cultured at 180 IU/ml IL-2 . TCR sequences for re-expression are listed in Supplementary Fehler! Verweisquelle konnte nicht gefunden werden.. All pMHC monomers were produced in-house as previously described 30, 72, 73 . Briefly, recombinantly expressed and purified human HLA-A*01 and HLA-A*03 heavy chains and β2m were denatured in urea and subsequently refolded into heterodimeric pMHC complexes in presence of an excess of respective synthetic peptide. Re-folded pMHC monomers were purified using size exclusion chromatography, concentrated and stored at -80 °C. pMHC biotinylation was performed as described 30 . Briefly, monomeric pMHCs were activated via a tyrosine tubulin ligase-mediated addition of an azo-tyrosine group and functionalized via click chemistry in presence of DBCO-PEG4-Biotin. For multimer staining, 0.4 µg biotinylated pMHC was multimerized on 0,1 µg fluorophoreconjugated StrepTavidin backbone in a final volume of 50 µl. Up to 5x10 6 cells were incubated All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. COVID-19 donors were in vitro expanded via co-culture with autologous PBMCs pulsed with 10 µg/µl 9-mer peptide pool for 10-12 days, and freshly re-stimulated with 1 µg/µl crude A1/ORF1_VTN and A3/ORF3a_FTS epitopes, respectively, for 4 h prior to sorting. For each donor, 2.500 CD8 + IFN-γ + and 10.000 CD8 + IFN-γ -T cells were sorted. Donors with same HLA background were pooled prior processing for single-cell RNA sequencing. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. A ORF1_DTD S_LTD ORF3_FTS ORF1_GTD ORF1_AMD ORF1_LTN S_KIA N_LQL S_VLN S_VVF S_ALN N_ILL N_LLL S_KVG S_RLF ORF1_KLF ORF1_VTN ORF1_ASM ORF1_STF ORF1_TTI M_LSY S_GYQ N_LSP S_PYR ORF1_NYM S_VYS ORF1_WSM M_SYF ORF1_YFM ORF1_KPN ORF1_KPV ORF1_VPM ORF1_FVK ORF1_SLS ORF1_YLK ORF1_VPF ORF1_FAV ORF1_LVA ORF1_NVL ORF1_HSI 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 23, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV We thank Max Koch for the processing of blood for PBMCs isolation. We gratefully acknowledge Catharina Gerhards, Margot Thiaucourt and Laura Mirbach for their contribution in the logistic organization of blood sample delivery.This study was supported by the EIT Health CoViproteHCt #20877, the German National Network of University Medicine of the Federal Ministry of Education and Research (BMBF; NaFoUniMedCovid19, 01KX2021; COVIM) and the DFG SFB1321 (Modeling and Targeting