High Frequencies of PD-1 + TIM3 + TIGIT + CTLA4 + Functionally Exhausted SARS-CoV-2-Specific CD4 + and CD8 + T Cells Associated with Severe Disease in Critically ill COVID-19 Patients

  1. Pierre-Gregoire Coulon
  2. Swayam Prakash
  3. Nisha R. Dhanushkodi
  4. Ruchi Srivastava
  5. Latifa Zayou
  6. Delia F. Tifrea
  7. Robert A. Edwards
  8. J. Figueroa Cesar
  9. Sebastian D. Schubl
  10. Lanny Hsieh
  11. Anthony B. Nesburn
  12. Baruch D. Kuppermann
  13. Elmostafa Bahraoui
  14. Hawa Vahed
  15. Daniel Gil
  16. Trevor M. Jones
  17. Jeffrey B. Ulmer
  18. Lbachir BenMohamed

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Abstract

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4 + and CD8 + T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4 + and CD8 + T cells between severely ill and asymptomatic COVID-19 patients and correlated this with α-CCCs and β-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: ( i ) Presented a broad leukocytosis and a broad CD4 + and CD8 + T cell lymphopenia; ( ii ) Developed low frequencies of functional IFN- γ -producing CD134 + CD138 + CD4 + and CD134 + CD138 + CD8 + T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; ( iii ) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1 + TIM3 + TIGIT + CTLA4 + CD4 + and PD-1 + TIM3 + TIGIT + CTLA4 + CD8 + T cells; and ( iv ) Displayed similar frequencies of co-infections with β-CCCs strains but significantly fewer co-infections with α-CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4 + and CD8 + T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with α-CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4 + and CD8 + T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with α-CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4 + and CD8 + T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.

KEY POINTS

  • A broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4 + and CD8 + T-cells were associated with severe disease onset in COVID-19 patients.

  • High frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4 + and CD8 + T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients.

  • Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4 + and CD8 + T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens.

  • The cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4 + and CD8 + T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients.

  • Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the α-CCCs strains.

  • Compared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4 + and CD8 + T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the β-CCCs strains.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.30.478343: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: All subjects were enrolled under an approved Institutional Review Board–approved protocol (IRB#-2020-5779).
    Consent: A written informed consent was obtained from participants prior to inclusion.
    Sex as a biological variableForty-one percent were females, and 59% were males with an age range of 19-91 (median 56 and average 55).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Wells of 96-well Multiscreen HTS Plates (Millipore, Billerica, MA) were pre-wet with 30% ethanol for 60 seconds and then coated with 100 µl primary anti-IFN-γ antibody solution (10 µg/ml of 1-D1K coating antibody from Mabtech, Cincinnati, OH) OVN at 4°C.
    anti-IFN-γ
    suggested: None
    Subsequently, we used the following anti-human antibodies for surface-marker staining: anti-CD45 (BV785, clone HI30 – BioLegend)
    anti-human
    suggested: None
    anti-CD45
    suggested: (BioLegend Cat# 368528, RRID:AB_2715888)
    Software and Algorithms
    SentencesResources
    A total of ∼200,000 lymphocyte gated PBMCs (140,000 alive CD45+) were acquired by Fortessa X20 (Becton Dickinson, Mountain View, CA) and the subsequent analysis performed using FlowJo software (TreeStar, Ashland, OR).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    (1) Corresponding CCCs peptides were determined after proteins sequences alignments of all four homologous CCCs proteins plus the SARS-CoV-2 related one using various Multiple Sequences Alignments (MSA) algorithms ran in JALVIEW, MEGA11 and M-coffee software’s (i.e. ClustalO, Kalign3 and M-coffee – the latter computing alignments by combining a collection of Multiple Alignments from a Library constituted with the following algorithms: T-Coffee, PCMA, MAFFT, ClustalW, Dialigntx, POA, MUSCLE, and Probcons).
    T-Coffee
    suggested: (T-Coffee, RRID:SCR_011818)
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    Probcons
    suggested: (ProbCons, RRID:SCR_011813)
    In addition, we confirmed our results with global and local Pairwise alignments (Needle and Water algorithms ran in Biopython).
    Biopython
    suggested: (Biopython, RRID:SCR_007173)
    Statistical analyses: To assess the potential linear negative relationship between COVID-19 severity and the magnitude of each SARS-CoV-2 epitope-specific T cell response, correlation analysis using GraphPad Prism version 8 (La Jolla, CA) were performed to calculate the Pearson correlation coefficients (R), the coefficient of determination (R2) and the associated P-value (correlation statistically significant for P ≤ 0.05).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The slope (S) of the best-fitted line (dotted line) was calculated in Prism by linear-regression analysis.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Therefore, there are limitations with the current vaccines. First, by applying a strong selection pressure on Spike only, this will likely shape virus evolution towards the appearance of variants with mutations in Spike that can escape vaccine-induced antibody protection (100-102). Second, although the Spike protein seems to generate a T-cell response(34), excluding other viral antigens from the vaccine that could contain immunodominant T cell epitopes (35, 36, 44, 103) may lead to (i) a limited repertoire of CD8+ T cell responses and (ii) generate a CD4+ T helper / Tfh response that might not sustain the B-cell memory efficiently (multiple studies underscore the correlation between T and B responses: (25, 35, 36, 75, 89), leading to a reduction in antibody production over time (104, 105). These concerns seem especially relevant in the long term(106) and in the elderly and immunocompromised patients, populations known to be already at risk of developing severe COVID-19 (41, 107, 108). The positive correlation between functional SARS-CoV-2 specific CD4+ and CD8+ T cells and better disease outcome in asymptomatic COVID-19 patients supports the importance of developing CoVs vaccines that target, not only antibody responses, but also early functional SARS-CoV-2 specific CD4+ and CD8+ T cell responses. Moreover, these vaccines may benefit from a combination with immune checkpoint blockade to reverse the exhaustion of SARS-CoV-2 specific CD4+ and CD8+ T cells in individuals who are...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.01.30.478343v1 on bioRxiv