SARS-CoV-2 T-cell response in COVID-19 convalescent patients with and without lung sequelae.

A specific T-cell response persists in the majority of COVID-19 patients 6 months after hospital discharge. This response is more prominent in those who required critical care during the acute COVID-19 episode but is reduced in patients with lung sequelae. https://bit.ly/3fBuVA4.

To the Editor:

Patients infected by SARS-CoV-2 may develop pneumonia (COVID-19), and require hospital admission and, eventually, critical care [1]. This has been related to a weaker innate immune response with impaired production of type I interferons (IFNs) [2]. In this setting, an antigen-specific T-cell response is needed for the elimination of SARS-CoV-2, as well as to develop long-lasting memory to respond to potential future SARS-CoV-2 infections [3, 4]. However, this response needs to be contained once the virus is eradicated to avoid further damaging the host.

Several studies have characterised the SARS-CoV-2 T-cell response in patients recovering from COVID-19 and showed that the intensity of the T-cell response relates to the severity of the acute pneumonia episode [5, 6]. Moreover, the severity of the disease is a risk factor for potential lung sequelae in COVID-19 survivors [7]. We recently reported that up to 57% of COVID-19 survivors present lung function abnormalities, particularly reduced carbon monoxide lung diffusion capacity (DLCO), 3 months after hospital discharge [8]. The relationship between the persistence of the specific T-cell response elicited during the acute COVID-19 episode and lung function abnormalities during follow-up is unknown.

To investigate these questions, we contrasted the in vitro T-cell response against the SARS-COV-2 spike (S) and the nucleocapsid (N) proteins, two well recognised viral antigens, in COVID-19 convalescent patients with normal and abnormal DLCO 6 months after hospital discharge.

This prospective, observational study included 25 adults who were hospitalised in our institution because of PCR-confirmed COVID-19 pneumonia and were studied at 6 months after hospital discharge. Participants were categorised according to their intensive care unit admission (or not) during the acute COVID-19 episode or by their DLCO 6 months after discharge (normal (≥80% pred) or abnormal (<80% pred)). The study was approved by the Ethical Review Board of our hospital (HCB/2020/0422), and all patients provided signed informed consent.

Demographic, clinical and biological characteristics were recorded on hospital admission and 6 months after discharge. At the latter time point, spirometry was performed and DLCO was measured (Medisoft, Sorinnes, Belgium) following international recommendations [9]. Likewise, blood was collected in EDTA tubes, and peripheral blood mononuclear cells (PBMCs) were isolated (Lymphoprep Abbott, Norway) and cryopreserved in fetal bovine serum (Gibco, US) and 10% dimethylsulfoxide. Pools of peptides covering the S and N proteins of SARS-CoV-2 were purchased from Miltenyi Biotec, USA (130-126-701 and 130-126-699 respectively). PBMCs from each donor were thawed, washed and: 1) an aliquot was stained with the antibodies listed below, to obtain the basal cell proportions; and 2) another aliquot was stimulated at 2×106 cells·mL−1 in X-Vivo plus 2% AB serum (Lonza, Belgium) with the S and N peptide pools (at 0.5 μg·mL−1) for 10 days. At day 10, cells were re-stimulated with 2.5 μg·mL−1 individual virus-specific peptide pools and (1/100) FastImmune (BD, USA) for 2 h following the addition of 10 μg·mL−1 brefeldin A (Sigma, Germany) for 4 h. Cells were stained with CD8-BV650, CD4-BV711, CD3-APC-R700, CD45-APC-H7, CD45RA–FITC, CD197-PECF594, CD196-PECy7, CXCR3–APC and IFNg-PE (BD) using Cytofix/Cytoperm (BD). All samples were acquired using a LSRFortessa SORP (BD) and analysed by FlowJo (FlowJo LLC, USA). Lymphocyte subpopulations were analysed as the proportion of CD4 or CD8. The expansion of specific populations is presented as fold change variations (i.e. the frequency of the population in stimulated PBMCS divided by the frequency of the population in unstimulated cells). Groups were compared using Mann–Whitney tests and analyses were performed using R version 3.6.1 or Prism 7 (GraphPad, La Jolla, CA, USA). A limitation of the study is the lack of lung function data prior the COVID-19 episode but only one patient in our study had a diagnosis of a lung condition (asthma) prior the COVID episode and this was not related to low DLCO (table 1).

TABLE 1

Clinical characteristics and T-cell response of COVID-19 patients 6 months after hospital discharge

	All (n=23)	No ICU (n=13)	ICU (n=10)	p-value	DLCO >80% pred (n=9)	DLCO <80% (n=14)	p-value
Age, years	60.0±10.5	60.2±9.4	59.8±12.3	0.9	55.4±9.6	62.9±10.4	0.13
Males	14 (61%)	8 (62%)	6 (60%)		6 (67%)	8 (57%)
BMI, kg·m−2	30.2±6.3	30.5±5.3	29.9±7.6	0.69	30.0±8.3	30.4±4.9	0.61
Previous lung disease #	1 (4%)	1 (8%)	0		1 (8%)	0
WHO disease severity score	4.5±1.4	3.6±0.8	5.7±1.2	<0.001	3.6±0.7	5.19±1.4	0.01
DLCO at 6 months, % pred	82.2±17.2	86.8±17.2	76.3±16.2	0.12	99.1±14.9	71.4±6.5	<0.001
Sequelae	14 (61%)	6 (46%)	8 (80%)		0 (0%)	14 (100%)
FEV1 at 6 months, % pred	95.4±13.1	99.4±13.7	90.3±10.4	0.14	104.9±12.4	89.3±9.6	<0.001
FVC at 6 months, % pred	91.5±14.2	95.2±14.1	86.8±13.6	0.12	104.1±12.7	83.5±8.0	<0.001
FEV1/FVC at 6 months, %	78.6±5.1	77.7±5.2	79.9±5.1	0.42	77.0±4.8	79.7±5.2	0.21
Response to SARS-CoV-2 S peptides
CD4 IFN-γ, %	5.1±4.4	3.2±3.4	7.5±4.6	0.05	4.0±3.3	5.8±5.0	0.61
CD8 IFN-γ, %	2.1±1.7	2.2±2.0	2.0±1.3	0.50	2.0±2.1	2.2±1.5	0.56
FC CD4 TEMRA, %	6.3±8.8	8.9±10.8	2.8±3.1	0.03	10.7±12.4	3.4±3.6	0.02
FC CD8 TEMRA, %	0.8±0.5	0.9±0.6	0.7±0.2	0.03	1.1±0.6	0.7±0.2	0.05
FC CD4 TCM, %	0.6±1.0	0.7±1.3	0.5±0.2	0.46	0.4±0.2	0.8±1.2	0.21
FC CD8 TCM, %	2.5±4.8	1.9±3.1	1.1±0.7	0.39	0.9±0.4	2.0±3.0	0.38
FC CD4 TEM, %	2.7±1.8	2.9±2.3	2.4±1.1	0.80	3.2±2.4	2.4±1.4	0.41
FC CD8 TEM, %	1.5±2.4	1.0±0.4	1.4±0.4	0.01	1.0±0.4	1.3±0.4	0.06
FC CD4 Th1, %	4.7±4.2	3.2±1.7	6.6±5.7	0.08	4.1±3.9	5.0±4.5	0.31
FC CD4 Th17, %	2.9±4.9	3.7±6.4	1.8±0.9	0.66	3.7±7.5	2.4±2.3	0.21
FC CD4 Th1/17, %	1.2±0.9	1.3± 1.0	1.1±0.6	0.85	1.3±1.1	1.1±0.7	0.66
Response to SARS-CoV-2 N peptides
CD4 IFN-γ, %	4.3±4.0	3.0±3.5	5.9±4.2	0.08	3.8±3.4	4.6±4.4	0.78
CD8 IFN-γ, %	8.0±9.0	7.5±9.5	8.8±8.7	0.85	13.2±10.9	4.7±5.8	0.01
FC CD4 TEMRA, %	4.2±4.1	5.4±4.7	2.6±2.5	0.07	6.0±4.1	3.0±3.7	0.02
FC CD8 TEMRA, %	0.7±0.6	0.8±0.7	0.5±0.2	0.04	0.9±0.8	0.5±0.2	0.28
FC CD4 TCM, %	0.7±1.1	0.8±1.4	0.5±0.2	0.71	0.4±0.2	0.9±1.4	0.41
FC CD8 TCM, %	2.3±5.1	1.9±3.2	0.9±0.7	0.62	0.8±0.3	1.9±3.1	0.26
FC CD4 TEM, %	2.6±1.8	2.8±2.2	2.4±1.1	0.99	3.2±2.4	2.2±1.2	0.15
FC CD8 TEM, %	1.5±2.4	1.2±0.5	2.3±1.3	0.04	1.6±1.2	1.8±1.0	0.31
FC CD4 Th1, %	5.6±5.1	3.6±2.0	8.1±6.7	0.04	4.2±3.7	6.4±5.7	0.27
FC CD4 Th17, %	2.1±2.8	2.7±3.8	1.4±0.6	0.66	2.8±4.2	1.7±1.6	0.28
FC CD4 Th1/17, %	0.8±0.4	0.8±0.4	0.8±0.5	0.80	0.9±0.6	0.8±0.3	0.99

Data are presented as mean±sd, unless otherwise stated. ICU: intensive care unit; DLCO: diffusing capacity of the lung for carbon monoxide; BMI: body mass index; WHO: World Health Organization; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; S: spike; IFN: interferon; FC: fold change; TEMRA: T-effector memory re-expressing CD45RA; TCM: T-central memory; TEM: T-effector memory; Th: T-helper; N: nucleocapsid. #: refers to a patient with a diagnosis of asthma prior to the COVID-19 episode. Statistically significant p-values are shown in bold.

We studied 23 patients (61% males) with a mean±sd age of 60.0±10.5 years; 10 of them (43%) needed critical care during the acute COVID-19 episode and 14 of them (61%) had DLCO <80% pred 6 months after discharge. Table 1 presents their main clinical and functional characteristics, and the study results.

A CD4 T-cell response to the S protein of SARS-CoV-2 (i.e. >2% of IFN-γ producing cells after stimulation) was found in 70% of the patients and a CD8 response in 43%. Conversely, 57% of the patients responded with CD4 and 70% with CD8 T-cells to the N protein of SARS-CoV-2. Overall, a T-cell specific response to either the S or N proteins was observed in 20 of the 23 patients studied (87%). Both the S and N peptides induced expansion of CD4 T-effector memory re-expressing CD45RA (TEMRA) and T-effector memory (TEM) cells with a T-helper (Th)1 (CXCR3) and Th17 (CD196) polarisation, whereas the S and N peptides expanded TEM and T-central memory CD8 cells (table 1).

The CD4 TEMRA and IFN-γ producing cells against the S peptide, and the CD8 TEM and TEMRA against the S and N peptides, were increased in patients requiring critical care during the acute COVID-19 episode (table 1). The CD8 IFN-γ response was reduced in patients with abnormal DLCO at convalescence, who also presented a reduced proportion of CD4 TEMRA cells (table 1).

This study shows that a T-cell specific response persists in the majority (87%) of COVID-19 patients 6 months after hospital discharge. This response is more prominent in those who required critical care during the acute COVID-19 episode, suggesting that the severity of the acute episode determined a more robust virus-specific T-cell expansion.

We also observed that the presence of reduced DLCO 6 months after discharge is related to a decrease in SARS-CoV-2 specific, IFN-γ producing CD8 T-cells. In addition, upon antigen stimulation, these patients presented a reduced expansion of cells with the TEMRA phenotype, suggesting a tighter control of the differentiation from memory cells towards the effector or the requirement of a costimulatory signal [10]. Further studies are required to elucidate these mechanisms and implications of these observations.