key: cord-0864397-4kfok5mg
authors: Arcanjo, Angélica; Pinto, Kamila Guimarães; Logullo, Jorgete; Leite, Paulo Emílio Corrêa; Menezes, Camilla Cristie Barreto; Freire-de-Lima, Leonardo; Diniz-Lima, Israel; Decoté-Ricardo, Debora; Rodrigues-da-Silva, Rodrigo Nunes; Freire-de-Lima, Celio Geraldo; Filardy, Alessandra Almeida; Lima-Junior, Josué da Costa; Bertho, Alvaro Luiz; De Luca, Paula Mello; Granjeiro, José Mauro; Barroso, Shana Priscila Coutinho; Conceição-Silva, Fátima; Savino, Wilson; Morrot, Alexandre
title: Critically ill COVID-19 patients exhibit hyperactive cytokine responses associated with effector exhausted senescent T cells in acute infection
date: 2021-08-24
journal: J Infect Dis
DOI: 10.1093/infdis/jiab425
sha: 9230f784b8f1ee80d524623a823186a81e917d15
doc_id: 864397
cord_uid: 4kfok5mg

COVID-19 can progress to severe pneumonia with respiratory failure and is aggravated by the deregulation of the immune system causing an excessive inflammation including the cytokine storm. We herein report that severe acutely infected patients have high levels of both type-1 and type-2 cytokines. Our results show abnormal cytokine levels upon T cell stimulation, in a non-polarized profile. Furthermore, our findings indicate that this hyperactive cytokine response is associated with a significantly increased frequency of late-differentiated T cells with particular phenotype of effector exhausted/senescent CD28 (-)CD57 (+) cells. Interestingly, we demonstrated for the first time an increased frequency of CD3 (+)CD4 (+)CD28 (-)CD57 (+) T cells with expression of programmed death 1 (PD-1), one of the hallmarks of T cell exhaustion. These findings reveal that COVID-19 is associated with acute immunodeficiency, especially within the CD4 (+) T cell compartment and points to possible mechanisms of loss of clonal repertoire and susceptibility to viral relapse and reinfection events.

COVID-19 is a devastating disease caused by the SARS-CoV-2 coronavirus infection, originally classified as a severe acute respiratory syndrome coronavirus (SARS-CoV). Most SARS-CoV-2 infected individuals are asymptomatic or exhibit an influenza-like inflammatory reaction. However, 5-20% of infected subjects develop a mild to severe condition whose major symptoms range from shortness of breath, vascular thrombosis and pulmonary obstruction (1) . The severity of the infection is related to the presence of reduced immunological repertoire in elderly patients and the presence of comorbidities such as diabetes, obesity and cardiovascular dysfunction associated with increased expression of the angiotensin-converting enzyme 2 (ACE2) receptor, used by the virus to infect epithelial cells in the upper and lower airways (2) . The binding of SARS-CoV-2 to ACE2 occurs through its spike (S) protein and viral entry is enhanced by the type II transmembrane serine protease TMPRSS2, which cleaves a portion of the S protein, exposing its fusion domain (3) .

The high mortality rate seen in COVID-19 is related to the unregulated activation of the immune system. Patients who evolve to the severe form of the infection have a high neutrophil/ lymphocyte rate, acute pulmonary neutrophilic infiltration showing elevated serum cytokines, ferritin, haemophagocytosis, D-dimer, and soluble CD25 (the IL-2 receptor alpha chain) (4, 5) . The presence of activated neutrophils and macrophages in the target tissues has been associated with induction of neutrophil extracellular traps (6) and of thrombocytogenesis, promoting vascular collapse, respiratory distress and multiorgan failure, which are related to the so-called cytokine release syndrome (CRS), including excessive productions of granulocyte and macrophage colony stimulating factor (GM-CSF), interleukin (IL) -2, IL-6, IL-7, IL-10, tumor necrosis factor α (TNF-α) and granulocyte colony stimulating factor (G-CSF) (7) .

A c c e p t e d M a n u s c r i p t 5 The cytokine storm syndrome is most commonly triggered by viral infections and occurs in 3.7-4.3% of severe cases of sepsis; being associated with a hyperinflammatory response. The clinical characteristics of the syndrome consist of sustained elevated fever, abnormally high levels of serum ferritin and triglycerides, pancytopenia, disseminated intravascular coagulation, liver dysfunction and splenomegaly (8) . Other changes are also present, such as decreased or absent NK cell activity, elevated serum levels of interleukin receptor chains, as well as hemophagocytosis, defined as phagocytosis of blood cells such as erythrocytes, leukocytes or platelets (9) . In general, the predisposing factors for the development of the cytokine storm consist of a different combination, varying from viral escape mechanisms, preventing the antiviral immune response, associated with genetic defects or acquired in host defense and other immunological abnormalities, such as low levels of interferon. All of this culminates in impaired viral clearance, leading to unregulated activation of the immune system and Severe Acute Respiratory Syndrome (SARS) (8) .

The underlying molecular mechanisms implicated in inducing the cytokine storm in critically ill patients with COVID-19 remain poorly understood. Importantly, the presence of high serum levels of IL-2 and CD25s (soluble IL-2 receptor a chain) in severe COVID-19 patients possible implies the participation of T cells in this immunopathology. Both IL-2 and CD25s are produced by activated T cells, suggesting a possible event of hyper reactivation of T cell responses in severe patients (10, 11 patients were suspended at a density of 1x10 6 /mL in complete RPMI medium. Cells were incubated in the presence of anti-CD28 (2 µg/ml) plus specific COVID-19 peptide mix (2 µg/ml of each MHCI peptide FL9, FL19, FF9 and FWF9) and anti-CD28 for 6h at 37°C in a 5% CO 2 incubator, followed by an additional 3 h in the presence of Brefeldin A (10 µg/mL).

Lymphocytes were stained for cell membrane markers (anti-CD28, anti-CD57, anti-CD8, anti-CD4, anti-CD3), and permeabilized for intracellular perforin staining using purified mouse anti-human perforin (BD Pharmingen) and anti-mouse IgG AlexaFluor488 (Sigma).

Samples were acquired using BD LSR Fortessa and the data were analyzed using Flow Jo and DIVA softwares. The selection of T-cell epitopes spanning the Spike protein sequence was made using the IEDB analysis resource Consensus tool, which combines predictions from ANN aka NetMHC, SMM and Comblib. Considering lengths of 9 mers, the prediction Figure 5B ). Interestingly, we also found that VEGF was present at significantly increased levels in sera from patients with severe respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as compared to healthy controls ( Figure 5C ).

Recent studies have reported an association between VEGF and programmed death-ligand 1 (PDL-1) in T cell exhaustion pathways in several malignancies (14) . This issue is particularly relevant considering that the low count of CD4 + and CD8 + lymphocytes is a hallmark finding in COVID-19 disease, and both T cell subtypes are shown to express significantly higher PD-1 levels in COVID-19 patients (15) . These findings suggest a higher susceptibility of these Figure 6D ). 

A Systematic Review of COVID-19 Epidemiology Based on Current Evidence

ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

D-dimer levels on admission and all-cause mortality risk in COVID-19 patients: a meta-analysis

Blood molecular markers associated with COVID-19 immunopathology and multi-organ damage

The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19)

Deciphering the COVID-19 cytokine storm: Systematic review and meta-analysis

COVID-19 and multiorgan failure: A narrative review on potential mechanisms

A review of the main histopathological findings in coronavirus disease 2019

Analysis of the changes of inflammatory cytokine levels in patients with critical coronavirus disease 2019 undergoing invasive mechanical ventilation

Impaired natural killer cell counts and cytolytic activity in patients with severe COVID-19

Suitability of 3D human brain spheroid models to distinguish toxic effects of gold and poly-lactic acid nanoparticles to assess biocompatibility for brain drug delivery

Human T cell activation with phytohemagglutinin. The function of IL-6 as an accessory signal

VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers

Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients

Cellular aging and senescence characteristics of human Tlymphocytes

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway

Using IL-2R/lymphocytes for predicting the clinical progression of patients with COVID-19

Bystander activation and autoimmunity

Protein kinases and the hypoxiainducible factor-1, two switches in angiogenesis

Early thrombolytic failure in a patient with massive pulmonary embolism combined with multiple organ dysfunction syndrome: what next?

CD8+ hyperactivation and senescence correlate with early carotid intima-media thickness in HIV+ patients with no cardiovascular disease

The Many Faces of Innate Immunity in SARS-CoV-2 Infection

An Overview of Current Knowledge of Deadly CoVs and Their Interface with Innate Immunity

Hypercoagulopathy in Severe COVID-19: Implications for Acute Care

The authors confirm contribution to the paper as follows: 

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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