key: cord-0741708-coyz6wm5
authors: Rowntree, Louise C; Petersen, Jan; Juno, Jennifer A; Chaurasia, Priyanka; Wragg, Kathleen; Koutsakos, Marios; Hensen, Luca; Wheatley, Adam K; Kent, Stephen J; Rossjohn, Jamie; Kedzierska, Katherine; Nguyen, Thi HO
title: SARS‐CoV‐2‐specific CD8(+) T‐cell responses and TCR signatures in the context of a prominent HLA‐A*24:02 allomorph
date: 2021-06-04
journal: Immunol Cell Biol
DOI: 10.1111/imcb.12482
sha: 9fe0ccc943510b267b042c626d47f9dd9e598a2c
doc_id: 741708
cord_uid: coyz6wm5

In‐depth understanding of human T cell‐mediated immunity in COVID‐19 is needed if we are to optimize vaccine strategies and immunotherapies. Identification of SARS‐CoV‐2 T cell epitopes and generation of peptide‐HLA tetramers facilitates direct ex vivo analyses of SARS‐CoV‐2‐specific T cells and their T cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS‐CoV‐2 epitopes restricted by HLA‐A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike‐derived peptides generated CD8(+)IFN‐γ(+) responses above background, S(1208‐1216) (QYIKWPWYI), S(448‐456) (NYNYLYRLF) and S(193‐201) (VFKNIDGYF), with S(1208) generating immunodominant CD8(+)IFN‐γ(+) responses. Using peptide‐HLA‐I tetramers, we performed direct ex vivo tetramer enrichment for HLA‐A*24:02‐restricted CD8(+) T cells in COVID‐19 patients and pre‐pandemic controls. The precursor frequencies for HLA‐A*24:02‐restricted epitopes were within the range previously observed for other SARS‐CoV‐2 epitopes for both COVID‐19 patients and pre‐pandemic individuals. Naïve A24/SARS‐CoV‐2‐specific CD8(+) T cells increased ~7.5‐fold above the average precursor frequency during COVID‐19, gaining effector and memory phenotypes. Ex vivo single‐cell analyses of TCRαβ repertoires found that the A24/S(448) (+)CD8(+) T cell TCRαβ repertoire was driven by a common TCRβ chain motif, while the A24/S(1208) (+)CD8(+) TCRαβ repertoire was diverse across COVID‐19 patients. Our study provides an in depth characterisation and important insights into SARS‐CoV‐2‐specific CD8(+) T cell responses associated with a prominent HLA‐A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID‐19 and could be exploited in vaccine or immunotherapeutic approaches.

The coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronvirus 2 (SARS-CoV-2) has, as of June 2021, infected more than 170 million people, caused at least 3.5 million deaths 1 and paralysed economies globally. International research efforts have led to the development of successful COVID-19 vaccine candidates, which have proven to be safe and effective in phase 2/3 clinical trials, especially against severe hospitalisation and fatal disease outcomes 2, 3 . While rolling out the vaccination programs globally, it is still important to develop an in-depth understanding on immune responses to SARS-CoV-2

This article is protected by copyright. All rights reserved infection so that immunopathology can be managed, immunotherapies optimized and universal next generation vaccines designed rationally.

producing anti-viral cytokines like IFN-, TNF and IL-2, and establishing long-term and generally broadly cross-reactive immunological memory. Early studies demonstrated that CD8 + T cells become activated prior to recovery from COVID-19 4, 5 , and survive into convalescence 6, 7 , indicating an involvement of CD8 + T cells in SARS-CoV-2 clearance and recovery. Both in vitro peptide stimulation assays and peptide-MHC-I tetramer approaches provided further evidence on activation, IFN- production and clonal proliferation of SARS-CoV-2-specific CD8 + T cells [8] [9] [10] .

Activated CD69 + CD137 + CD8 + T cells directed towards overlapping megapeptide pools derived from spike (S), membrane (M), nucleocapsid (N) and ORF proteins could be detected in ~70% of acute and convalescent COVID-19 patients. Cross-reactive CD8 + T cell responses towards peptides derived from circulating human coronaviruses (hCoV) have also been proposed in some individuals 11, 12 .

To provide a better understanding of SARS-CoV-2-specific CD8 + T cells in acute COVID- 19 , their persistence into long-term memory and subsequent recall following vaccination and/or infection, specific CD8 + T cell epitopes need to be identified. Several SARS-CoV-2 CD8 + T cell epitopes have recently been defined for HLA class I alleles such as HLA-A*01:01, A*02:01, A*03:01, A*11:01, HLA-B*07:02, B*27:05, B*40:01 and B*44:03 8, 11, 13, 14 . Identification of these SARS-CoV-2 CD8 + T cell specificities allowed us to determine the precursor frequency of SARS-CoV-2 tetramer-specific CD8 + T cells in pre-pandemic samples, acute COVID-19 and convalescence 8, 9 . Ex vivo phenotypic analysis revealed that immunodominant B7/N 105 + CD8 + T cell responses originated from high frequency naïve pools found across HLA-B*07:02-expressing individuals 9 . In contrast, A2/S 269 + CD8 + T cells were of suboptimal frequency and phenotypes 8 .

The question remains whether SARS-CoV-2-specific CD8 + T cell responses generally reflect the immunodominant B7/N 105 + CD8 + or subdominant A2/S 269 + CD8 + T cell responses. To answer this question, a broad range of SARS-CoV-2 CD8 + T cell specificities restricted across several dominant HLAs need to be identified and analysed directly ex vivo.

In our study, we utilized a combination of peptide prediction and in vitro peptide stimulation to identify novel SARS-CoV-2 epitopes restricted by one of the most frequent HLA class I, especially in Indigenous and Asian populations, HLA-A*24:02. Of the peptides screened, three spike-derived peptides generated CD8 + IFN-γ + responses above background, S 1208-1216

This article is protected by copyright. All rights reserved (QYIKWPWYI), S 448-456 (NYNYLYRLF) and S 193-201 (VFKNIDGYF), with S 1208 generating the strongest CD8 + IFN-γ + responses. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for A24/S 1208 + CD8 +9 and A24/S 448 + CD8 + T cells in HLA-A*24:02-expressing COVID-19 patients and pre-pandemic controls. We found that CD8 + T cells directed at both HLA-A*24:02-restricted epitopes had similar frequencies and activation phenotypes in COVID-19 patients, while the frequencies in pre-pandemic samples were comparable to those of A2/S 269 + CD8 + T cells 8 . A24/S 448 + CD8 + T cell and A24/S 1208 + CD8 + T cell responses had contrasting TCR repertoires, where the A24/S 448 + CD8 + TCR repertoire was driven by a common TCR chain motif, while the A24/S 1208 + CD8 + TCRαβ repertoire was diverse across COVID-19 patients.

In this study, we recruited 8 HLA-A*24:02-expressing individuals; 4 HLA-A*24:02 + convalescent COVID-19 patients from community infections (median age 63 years old, range 52-75, 100% male, range 71-217 days post symptom onset) and 4 HLA-A*24:02 + pre-pandemic healthy controls (median age 36 years old, range 24-59, 100% male).

Using the COVID-19 donors, we probed for SARS-CoV-2-specific CD8 + T cells (Figure 1c ). S 1208 generated the strongest CD8 + IFNγ + response in three of four donors tested (mean 9.73%), with S 448 and S 193 responses observed in two (mean 3.85%) and one (3.37%) donors, respectively ( Figure 1d ). Only one donor showed responses to the M and N pool, with very small IFN-γ production (~1%) to 4 of the 6 M peptides tested that was not able to be narrowed to a single epitope.

This article is protected by copyright. All rights reserved Peptide sequence conservation analysis for the S-derived SARS-CoV-2 immunogenic peptides was extended to previously circulating coronaviruses. SARS-CoV-2 S 1208, S 448 and S 193 peptide sequences were compared to reference protein sequences for the hCoV strains NL63, 229E, HKU1 and OC43 (Figure 2a) . The SARS-CoV-2/S 1208 epitope shared 66% sequence identity with all of the hCoV strains. All hCoV strains displayed differences at position 1 and 9 (anchor residue), in addition to variation at either position 3 (HKU1 and OC43) or 8 (NL63 and 229E). Sequence conservation was lower for S 448 and S 193 peptides, where sequency identity ranged between 44 and 55%. More of these amino acid variations occurred at position 2 (anchor residue) and we therefore predict the analogous hCoV peptides are unlikely to be HLA-A*24:02 ligands, though this is yet to be formally determined.

To further analyse the SARS-CoV-2-specific A24/CD8 + populations, tetramer-associated magnetic enrichment 15, 16 was performed to determine the ex vivo frequencies of A24/S 1208 + CD8 +9 and A24/S 448 + CD8 + T cells in HLA-A*24:02 + COVID-19 patients and pre-pandemic controls ( Figure 2b ). The precursor frequency of A24/S 448 + CD8 + cells in COVID-19 convalescent donors (mean 6.3x10 -5 , n = 4) was significantly higher than that observed in pre-pandemic healthy individuals (mean 8.44x10 -6 , n = 4, P = 0.0286) (Figure 2c , left panel). The precursor frequencies of A24/S 448 + CD8 + T cells were comparable to the previously observed frequencies of A24/S 1208 + CD8 + T cells (mean 7.71x10 -5 for COVID-19 convalescents and 9.50x10 -6 for prepandemic donors 9 ), with most donors having similar frequencies of both epitopes ( Figure 2c , right panel). Furthermore, the precursor frequencies for both SARS-CoV-2 HLA-A24 epitopes were within the range previously observed for other SARS-CoV-2 epitopes for both COVID-19 patients and pre-pandemic individuals 8, 9 . Finally, the percentage of both the A24/S 448 + CD8 + and A24/S 1208 + CD8 + T cells increased, though not significantly, after 12 days of stimulation with the S peptide pool in 2 and 3 COVID-19 donors, respectively ( Figure 2d ). Evidently, the SARS-CoV-2specific CD8 + T cells were primed by SARS-CoV-2 in the COVID-19 individuals and are thus, at least under in vitro conditions, capable of clonal expansion. Overall, our data suggest that naïve A24/SARS-CoV-2-specific CD8 + T cells can be expanded 7.5-fold following COVID-19.

This article is protected by copyright. All rights reserved

The activation profiles of A24/S 448 + CD8 + T cells tested directly ex vivo from COVID-19 patients and pre-pandemic healthy individuals were assessed by CD27, CD45RA and CD95 staining to determine the prevalence of the naïve (T Naïve ) (CD27 + CD45RA + CD95 -), stem cell memory (T SCM ) (CD27 + CD45RA + CD95 + ), central memory (T CM )-like (CD27 + CD45RA -), effector memory (T EM )like (CD27 -CD45RA -), and effector memory CD45RA (T EMRA ) (CD27 -CD45RA + ) subsets ( Figure   2b ). The phenotype of the A24/S 448 + CD8 + T cells was highly varied across the convalescent COVID-19 donors, with the majority of the tetramer-positive CD8 + T cells being T SCM (n = 2), T EMRA (n = 1) or T Naïve (n = 1) (Figure 2e) . Conversely, the prevalence of the naïve phenotype (mean of 73%) was significantly enriched in pre-pandemic individuals compared to convalescent COVID-19 patients (P = 0.0008). This phenotypic pattern in both the COVID-19 patients and prepandemic healthy individuals, aligns with what has been previously observed for other SARS-CoV-2-specific CD8 + T cells, including A24/S 1208 + CD8 + T cells 9 . The pre-pandemic individuals displayed a mainly naïve profile (mean of 61%), while the A24/S 1208 -specific CD8 + T cells in the COVID-19 donors were more skewed toward T Naive (n = 2), T CM (n = 1), or T EMRA (n = 1) phenotypes 9 .

CD8 + T cell immunodominance, functionality and protection are all impacted by the molecular signature of the TCR repertoire [17] [18] [19] . Thus, using direct ex vivo tetramer staining and human singlecell TCR multiplex RT-PCR 15, 20 , we determined the TCR clonal composition and diversity.

We dissected the TCR repertoires for A24/S 448 and A24/S 1208 -specific CD8 + T cells in PBMCs from 4 HLA-A*24:02-expressing COVID-19 patients, examining the TCR sequence for a total of 48 SARS-CoV-2-specific CD8 + T cells (Figure 3a ; Table 1 ).

The overall diversity of the TCRαβ sequences was analysed, firstly focussing on the TRBV region ( Figure 3b ). For A24/S 448 + CD8 + T cells, all donors had TRBV2 gene usage where one key TCRβ motif within the CDR3β loop was found across the COVID-19 patients, despite the fact that a limited number of A24/S 448 -specific sequences were examined per donor (1-10 cells/donor) ( Table 1) . This motif was dominated by the TRBV2/TRBJ2-7 CAS(S/A)XXXGYEQYF (where X denotes any amino) sequence found in all COVID-19 patients (40% of total TCR repertoire). This same CDR3β loop motif was also identified when TRBJ2-7 or 2-1 linked with 5 different TRBV

This article is protected by copyright. All rights reserved segments, TRBV5-4, 6-1, 6-4, 7-2 and 10-1 found across 2 of the 4 COVID-19 patients (40% of total TCR repertoire). Similar CDR3β loops were also identified in a third donor, composed of TRBV4-1/TRBJ2-7 gene segments. Overall, the sequences with this CDR3β loop comprised 85% of the A24/S 448 -specific repertoire (Figure 3c ). The CDR3β loop motif paired with a range of different TRAV/TRAJ gene segments (Figure 3d ) and therefore A24/S 448 + CD8 + T cell specificity was likely driven by the TCRβ chain rather than the TCRα chain.

In contrast, the A24/S 1208 -specific CD8 + TCRαβ repertoire (4-11 cells/donor) was generally diverse, with almost all sequences identified only once and no TCR clonotypes overlapping between individuals. Each donor had distinct usage of TRAV, TRBV and TRAJ gene segments, with no common motifs within CDR3 and CDR3 sequences. Interstingly, the TRBJ2-7 and 2-1 were enriched in the A24/S 1208 -specific CD8 + TCRαβ repertoire, represented in 26% and 22% of the sequenced TCR chains, respectively ( Figure 3c) . Overall, the common motif suggests more rigid requirements for TCR clones recognizing the A24/S 448 epitope compared to the A24/S 1208 epitope. The lack of TCR plasticity may explain the low naïve precursor frequency observed for A24/S 448 , similar to what has been previously observed for A2/S 269 8 , however more data may be needed to determine the driving factors for A24/S 1208 recognition. 

This article is protected by copyright. All rights reserved Using peptide prediction tools in combination with an in vitro peptide stimulation, we identified SARS-CoV-2 epitopes restricted by HLA-A*24:02, namely three spike-derived peptides, S 1208-1216 (QYIKWPWYI), S 448-456 (NYNYLYRLF) and S 193-201 (VFKNIDGYF), with S 1208 generating the strongest CD8 + IFN-γ + responses. Both A24/S 1208-1216 and A24/S 448-456 have been recently independently identified by other groups 10, 11, 23 . Although all three SARS-CoV-2derived peptides shared 44-66% sequence identity with hCoVs, the variations occur in either the anchor residues or likely TCR contact residues, suggesting a potential to escape pre-existing CD8 + T cell immunity and supported by our observation of predominatly naïve A24/S 1208 and A24/S 448specific CD8 + T cells in pre-pandemic individuals. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for HLA-A*24:02-restricted CD8 + T cells in COVID-19 patients and pre-pandemic controls. The precursor frequencies for HLA-A*24:02-restricted epitopes were within the range previously observed for other SARS-CoV-2 epitopes like HLA-A*02:01-restricted S 269 for both COVID-19 patients and pre-pandemic individuals 8 . Thus, it appears that 5-10-fold expansion of SARS-CoV-2-specific CD8 + T cell responses is prototypical for the primary COVID-19, with the exception of B7/N 105 + CD8 + T cell responses 9 . Similarly, the phenotypic pattern of the A24/S 448 + and A24/S 1208 + CD8 + T cells aligns with previous SARS-CoV-2 epitopes, with pre-pandemic individuals displaying a prototypical naïve phenotype compared to the more varied T CM phenotype observed in COVID-19 patients 8, 9 . A24/S 448 + CD8 + T cell and A24/S 1208 + CD8 + T cell responses had contrasting TCR repertoires. While A24/S 448 + CD8 + T cell TCR repertoire was driven by a common TCR chain motif, the A24/S 1208 + CD8 + TCRαβ repertoire was diverse across COVID-19 patients. As in our recent study 9 , it appears that TCR diversity might be linked with the prominence of SARS-CoV-2 CD8 + T cell responses during the primary infection. The common TCR chain motif recognizing the A24/S 448 and the associated lack of TCR plasticity is reminiscent of the TCR requirements for the subdominant A2/S 269 epitope 9, 24 and is in contrast to the diverse TCR repertoire and promiscuity in TCR-TCR pairing of the immunodominant HLA-B*07:02-restricted N 105 -specific CD8 + T cells 9 .

Overall, our study provides important insights into SARS-CoV-2-specific CD8 + T cell responses associated with a highly frequent HLA-A*24:02 allele and thus contributes to our knowledge on the experimentally verified SARS-CoV-2 CD8 + T cell epitopes. Assembling a comprehensive dataset on the key CD8 + T cell epitopes restricted by the prominent HLA alleles is needed to depict 'universal' HLAs in COVID-19 capable of presening peptides which elicit

This article is protected by copyright. All rights reserved broadly cross-reactive immunity across a range of emerging variants. Our previous work defined universal HLA class I alleles (A*02:01, A*03:01, B*08:01, B*18:01, B*27:05 and B*57:01) in influenza, mounting robust CD8 + T cell responses against any human influenza A virus circulating over the last century, including the pandemic strains as well as the avian H5N1 and H7N9 viruses 25 . Identification of such universal CD8 + (and CD4 + ) T cell epiopes in COVID-19 is needed for the rational design of the next generation universal COVID-19 vaccines, especially for high risk groups like Indigenous people.

This study recruited 4 convalescent COVID-19 individuals from community infections (mild: Guanidine HCl and refolded with either S 1208 or S 448 peptide, as essentially as described 8 . Purified,

This article is protected by copyright. All rights reserved fully biotinylated HLA-A24 monomers were stored at -80 °C and conjugated to fluorescentlylabeled streptavidins (SA), PE-SA or APC-SA (BD Biosciences, Franklin Lakes, NJ, USA) at an 8:1 monomer to SA molar ratio to form peptide/MHC class I tetramers.

PBMCs samples were stimulated with A24/SARS-CoV-2 predicted peptides from 2 peptide pools (1 µM) for 12 days in RF-10 medium (+20 U mL -1 IL-2) 27 

For tetramer-associated magnetic enrichment (TAME), cells (5-32x10 6 Hamburg, Germany) using the FACSAriaIII (BD Bioscience) for TCR analyses. Plates were kept cold, centrifuged then stored at -80°C. Amplification of paired CDR3 and CDR3 regions were performed using multiplex-nested RT-PCR, as described previously 15, 16 . TCR sequences were analyzed using IMGT/V-QUEST and flow cytometry data were analyzed using FlowJo v10 software. Data visualization was performed in R using packages for circular layout 29 and graphics generation 30 .

This article is protected by copyright. All rights reserved THON and KK led and supervised the study. THON, KK, and LCR designed the experiments. 

This article is protected by copyright. All rights reserved 

An interactive web-based dashboard to track COVID-19 in real time

Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

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

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19

Suboptimal SARS-CoV-2−specific CD8 + T cell response associated with the prominent HLA-A*02:01 phenotype

CD8 + T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

SARS-CoV-2-specific CD8 + T cell responses in convalescent COVID-19 individuals

Unbiased screens show CD8 + T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein

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

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

Characterization of pre-existing and induced SARS-CoV-2-specific CD8 + T cells

Accepted Article This article is protected by copyright. All rights reserved 15

Molecular basis for universal HLA

A*0201-restricted CD8 + T-cell immunity against influenza viruses

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

Magnitude and kinetics of CD8 + T cell activation during hyperacute HIV infection impact viral set point

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

Public clonotype usage identifies protective Gagspecific CD8 + T cell responses in SIV infection

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

CD8 + T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition

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

Preexisting CD8 + T-cell immunity to the H7N9 influenza A virus varies across ethnicities

NetCTLpan: pan-specific MHC class I pathway epitope predictions

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

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

Circlize implements and enhances circular visualization in R

Elegant Graphics for Data Analysis

The authors declare no competing interests.

This article is protected by copyright. All rights reserved