key: cord-0964274-r8sw0wf2
authors: Giménez, Estela; Albert, Eliseo; Torres, Ignacio; Remigia, María José; Alcaraz, María Jesús; Galindo, María José; Blasco, María Luisa; Solano, Carlos; Forner, María José; Redón, Josep; Signes‐Costa, Jaime; Navarro, David
title: SARS‐CoV‐2‐reactive interferon‐γ‐producing CD8+ T cells in patients hospitalized with coronavirus disease 2019
date: 2020-07-02
journal: J Med Virol
DOI: 10.1002/jmv.26213
sha: df446519c7a1187d036c6f7a4cf95fdfb7511530
doc_id: 964274
cord_uid: r8sw0wf2

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) T‐cell immune responses in patients with coronavirus disease 2019 (COVID‐19). Both CD4+ and CD8+ T cells may be instrumental in resolution of and protection from SARS‐CoV‐2 infection. Here, we tested 25 hospitalized patients either with microbiologically documented COVID‐19 (n = 19) or highly suspected of having the disease (n = 6) for presence of SARS‐CoV‐2‐reactive CD69+ expressing interferon‐γ (IFN‐γ) producing CD8+ T cells using flow‐cytometry for intracellular cytokine staining assay. Two sets of overlapping peptides encompassing the SARS‐CoV‐2 Spike glycoprotein N‐terminal 1 to 643 amino acid sequence and the entire sequence of SARS‐CoV‐2 M protein were used simultaneously as antigenic stimulus. Ten patients (40%) had detectable responses, displaying frequencies ranging from 0.15 to 2.7% (median of 0.57 cells/µL; range, 0.43‐9.98 cells/µL). The detection rate of SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells in patients admitted to intensive care was comparable (P = .28) to the rate in patients hospitalized in other medical wards. No correlation was found between SARS‐CoV‐2‐reactive IFN‐γ CD8+ T‐cell counts and SARS‐CoV‐2 S‐specific antibody levels. Likewise, no correlation was observed between either SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells or S‐specific immunoglobulin G‐antibody titers and blood cell count or levels of inflammatory biomarkers. In summary, in this descriptive, preliminary study we showed that SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells can be detected in a non‐negligible percentage of patients with moderate to severe forms of COVID‐19. Further studies are warranted to determine whether quantitation of these T‐cell subsets may provide prognostic information on the clinical course of COVID‐19.

On 11 March 2020 the World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic. 1 As of June 19 more than 8 500 000 cases of COVID-19 have been reported worldwide, causing over 454 000 deaths. 2 commonly results in pneumonia, which can evolve to into acute respiratory distress syndrome, leading to respiratory or multiorgan failure. 3, 4 Elucidation of immune responses conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to develop an effective vaccine prototype, which is urgently needed to blunt the progression of the pandemic. A number of studies have focused on characterizing SARS-CoV-2-specific antibody kinetic profiles. [5] [6] [7] [8] [9] [10] Nevertheless, there is scant information on T-cell responses against SARS-CoV-2 in patients with COVID-19. CD4+ and CD8+ T cells targeting structural viral proteins appear to confer broad and longlasting protection against SARS-CoV. 11 Several clusters of cytotoxic T lymphocyte (CTL) epitopes restricted by HLA-class I specificities commonly found in Caucasians (ie, HLA-A02) have been mapped within the spike (S) and the membrane (M) of SARS-CoV proteins. 11 Since SARS-CoV shares high sequence identity with SARS-CoV-2, 12 it is reasonable to expect an analogous scenario in the SARS-CoV-2

infection. There is limited information on the features of T-cell responses against SARS-CoV-2; several studies demonstrated the presence of SARS-CoV-2 -reactive T cells in a large number of patients with COVID-19 and also in unexposed inidviduals, although the potential functionality of these cells was not comprehensively investigated. [13] [14] [15] Here, we developed a flow cytometry for intracellular cytokine staining (ICS) assay to enumerate peripheral blood SARS-CoV-2-reactive interferon γ (IFN-γ)-producing CD8+ T cells, which was used to assess virus-elicited T-cell immunity in patients with moderate to severe COVID-19.

Twenty-five nonconsecutive patients ( Responses ≥0.1% were considered specific.

Clinical laboratory investigation included complete blood count and serum levels of ferritin, Dimer-D, and C reactive protein (CRP). Data on serum interleukin-6 (IL-6) levels were not available.

Frequency comparisons for categorical variables were carried out using the Fisher exact test. Differences between medians were compared using the Mann-Whitney U test. The Spearman's rank test was used for analysis of correlation between continuous variables. Two-sided exact P values were reported. A P-value <.05 was F I G U R E 1 Enumeration of SARS-CoV-2-S1/M-reactive CD69+-expressing IFN-γ-producing CD8+ T cells by flow cytometry for intracellular staining in patients with COVID-19. Panel A depicts the gating strategy. Panel B includes plots from patients with detectable responses, while panel C shows plots from 10 patients testing negative. Dot-plot figures were built with Flow-Io software (BD Biosciences). COVID-19, coronavirus disease 2019; IFN-γ, interferon-γ; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 considered statistically significant. The analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL).

Patients in this cohort were admitted to our center at a median of 7 days (range, 0-28 days) after onset of symptoms. Twenty-two patients presented with pneumonia and imaging findings on chest-X-ray or computed tomography-scans compatible with COVID-19. The remaining three patients, clinically suspected of COVID-19 with no evidence of pneumonia, were admitted due to either aggravation of baseline chronic conditions (n = 2) or venous thrombosis. Median hospitalization time of patients was 18 days (range, 4-52 days). Seven patients needed intensive care unit (ICU), of whom two died.

As shown in Table 1 , diagnosis of COVID-19 was reached in 18 patients by RT-PCR in upper or lower respiratory tract specimens, either at initial screening (n = 11) or after repeat testing (n = 7). Of these, 14 also had serological evidence of SARS-CoV-2 infection. One patient repeatedly tested negative by RT-PCR, but exhibited IgG seroconversion. Finally, in six patients no microbiological evidence of SARS-CoV-2 infection was obtained. These latter patients tested negative by a multiplexed RT-PCR assay targeting prevalent respiratory viruses and bacteria in upper or lower respiratory tract specimens.

Enumeration of peripheral blood SARS-CoV-2-S1/M-reactive CD69+-IFN-γ CD8+ T cells was carried out at a median of 27 days from onset of symptoms (range, 2-47 days). Eight patients were rescreened within the next 5 days. Ten patients (40%) had detectable responses (Table 1 and 

We next investigated the potential relationship between SARS-CoV-2-reactive IFN-γ CD8+ T-cell counts, S-specific IgG-antibody titers and blood levels of a number of prognostic laboratory parameters of COVID-19 progression, including total leukocyte, lymphocyte and neutrophil counts, and markers of inflammation (or coagulation), such as ferritin, CRP, and Dimer-D. To this end, only patients with microbiological confirmation of COVID-19 were included in these analyses. A proinflammatory state was noticed in most patients in this series, while cytopenias were less frequently observed ( Table 2) .

Patients with or without detectable SARS-CoV-2-reactive IFN-γ CD8+ T cells were comparable regarding these parameters ( Table 2 ).

In addition, no correlation was found between either SARS-CoV-2reactive IFN-γ CD8+ T cells or S-specific IgG-antibody titers and blood cell count or levels of any of these biomarkers (Rho < 0.2 and P > .5 in all correlation analyses).

There is a knowledge gap regarding the immune mechanisms that confer protection against SARS-CoV-2. On the basis of experimental evidence gathered on SARS-CoV and MERS-CoV infections, 18, 19 it is assumed that both CD4+ and CD8+ T cells play a major role in virus clearance and long-term protection against SARS-CoV-2 infection.

Although plausible, data supporting this assumption is lacking. In this context, to the best of our knowledge, only a few studies have assessed the frequency of SARS-CoV-2-reactive CD4+ T cells (by flow cytometry) or T-cells (by IFNγ-ELISPOT) in peripheral blood from patients with COVID-19 or COVID-19 convalescent individuals, respectively. [13] [14] [15] Here, we optimized a flow cytometry ICS method for quantitation of SARS-CoV-2-reactive-activated (CD69+ expressing)-CD8+ T cells producing IFN-γ upon antigenic stimulation. This assay uses whole blood as a matrix, thus circumventing the need for peripheral blood mononuclear cells separation, and a combination of two peptide mixes composed of overlapping peptides spanning the S1 region of the S glycoprotein and the entire amino acid sequence of the M protein. We chose SARS-CoV-2 S1 and M proteins as antigenic stimuli because they are expected to contain highly immunogenic CTL epitopes restricted by HLA-class I specificities commonly found in Caucasian individuals (all patients in this series), based on sequence alignment with SARS-CoV homologous proteins. 11, 20 Moreover, amino acid sequences of these CTL epitopes are reasonably distinct from aligned sequences present in seasonal coronaviruses, thereby minimizing the likelihood of detecting cross-reactive CD8+ T cells. 11 In turn, by combining S1 and M peptide libraries, we Several findings arose from our study. First, SARS-CoV-2 IFN-γ CD8+ T cells targeting S1 and M proteins were detected in 40% of patients at a relatively late stage after onset of symptoms (median, 27 days). It is likely that these T-cell subsets could have been circulating long before; our study, however, was not designed to characterize their kinetics. It is relevant to note that the median age of our patients was high (62 years); although speculative, the rate of detection could arguably have been higher in younger people (no immunosenescence).

Braun and colleagues detected S1 and S2-reactive CD4+ T cells expressing cell-surface activation markers in 12 and 15 out of 18 patients (median age, 52 years old), respectively, presenting with mild to severe forms of COVID-19. 13 However, the potential antiviral functionality of these T-cell subsets was not explored. Ni et al identified SARS-CoV-2-specific IFN-γ T cells (measured by ELISPOT) targeting the nucleocapsid protein, the M protein or the S1 receptor binding domain (RBD) in up to 7 out of 14 individuals who had recovered from COVID-19. 14 Second, the frequency of SARS-CoV-2-reactive IFN-γ CD8+

T cells varied widely across patients in this series, and was unexpectedly high in some cases (eg, patient 7 in Table 1 Fifth, among patients with microbiologically confirmed COVID-19, we found no correlation between SARS-CoV-2-reactive IFN-γ CD8+ T cells and S-specific antibody levels, which in turn appear to strongly correlate with SARS-CoV-2 neutralizing activity of sera, 22 suggesting that SARS-CoV-2 targeted B and T-cell responses may follow divergent dynamics, as noticed in SARS CoV infection. 20 Our data is nevertheless in contrast with the study by Ni et al 14 who found a strong correlation between SARS-CoV-reactive IFN-γ T cells and S1-RBD-specific neutralizing antibodies in convalescent individuals (in our current study patients with active disease were included).

Sixth, lung inflammation is the main cause of life-threatening respiratory disorders at the severe stage of COVID-19. It is plausible that SARS-CoV-2-reactive T cells and certain specificities of SARS CoV-2-specific antibodies (ie, those mediating antibody-dependent enhancement-ADE-) may be mechanistically involved in promoting such a proinflammatory state. 23, 24 In this respect, we found no correlation between SARS-CoV-2-reactive IFN-γ CD8+ T cells, antibodies targeting the S protein (which contain both neutralizing and ADE epitopes) and serum levels of ferritin, CRP, and Dimer-D.

However, these findings should not be overinterpreted, and by no means rule out involvement of virus-driven immunopathogenic mechanisms in progression to acute respiratory distress syndrome.

Further prospective studies assessing how the dynamics of these parameters relate in sequential specimens are needed to shed light on this issue.

Besides the low number of patients in our series, the main limitation of the current study is that the specificity of SARS-CoV-2reactive IFN-γ-CD8+ T cells was not proven. Based on sequence analyses, SARS-CoV-2 and seasonal alpha and beta coronaviruses may share HLA-class I-restricted immunogenic epitopes mapping within S1 and M potentially eliciting cross-reactive T cells. 20 In support of this assumption, S-reactive CD4+ T cells could be detected in 34% of healthy control individuals who had seemingly not been infected by SARS-CoV-2, albeit at lower frequencies than in patients with COVID-19, and displaying a differential pattern of cellsurface activation markers. 13 Moreover, Grifoni et al 15 detected SARS-CoV-2-reactive CD4+ T cells in approximately 40% to 60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.

We also tested four healthy asymptomatic individuals with no evidence of active or past COVID-19 and found one of them to be reactive, although displaying a low frequency of SARS-CoV-2 reactive IFN-γ-CD8+ T cells (0.12%) (data not shown). Against the epidemiological framework of heavy SARS CoV-2 community transmission, as is currently confronting Spain, it may be unwise to recruit asymptomatic individuals as negative controls, even ones testing negative by RT-PCR or having no evidence of seroconversion, given that some of these subjects could have been exposed to and developed measurable T-cell responses. Unfortunately, cryopreserved blood specimens from healthy individuals with or without documented infection caused by seasonal coronaviruses had not been collected before SARS-CoV-2 appeared in our health department.

Studies aimed at assessing the specificity of IFN-γ CD8+ T cells detected by our assay have been designed and are to be initiated as soon as control is gained over our local epidemic outbreak.

In summary, in this descriptive, preliminary study we showed that SARS-CoV-2-reactive IFN-γ CD8+ T cells can be detected in a non-negligible percentage of patients with moderate to severe forms of COVID-19. As previously suggested, 13, 15 quantitation of these T-cell subsets, whether cross-reactive or specific, may provide prognostic information on the clinical course of COVID-19. Studies designed to address this issue are currently underway.

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How to cite this article

SARS-CoV-2-reactive interferon-γ-producing CD8+ T cells in patients hospitalized with coronavirus disease 2019

No public or private funds were used for the current study. The authors are grateful to all personnel who work at Clinic University Hospital, in particular to staff at the Microbiology laboratory for their commitment to the fight against COVID-19.

The authors declare that there are no conflict of interests. 

http://orcid.org/0000-0003-3010-4110