Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients

Abstract

Background: Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known.

Methods: Fifty-seven patients were included in this case-control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane- SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay.

Results: Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4⁺ T cells reactive against at least one SARS-CoV-2 protein. CD8⁺ T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals.

Conclusions: This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection.

Keywords: Antigen-spcific T cells; Immunosuppression; Renal transplantation; Sars-CoV-2.

Fig. 1

T cell response in unexposed and COVID-19 patients. Percentage of unexposed RTx patients, healthy or exposed COVID-19 patients with a detectable CD4 (a) or CD8 (b) T cell response after stimulation with S, M or N OPPs. A detectable CD4 and CD8T cell response was defined as a minimum of 0.01% CD4⁺CD154⁺CD137⁺ or CD8⁺CD137⁺ T cells after background of unstimulated cells was subtracted

Fig. 2

Pre-existing SARS-CoV-2 reactive T cells can be detected in renal transplant patients. Isolated PBMCs from RTx patients (20), healthy individuals (20) and COVID-19 patients (17) were stimulated for 16 h with 1 µg/ml SARS-CoV-2 OPPs from M (n = 11–19), N (n = 17–20) or S (n = 17–20) proteins and afterwards stained with antibodies against the depicted markers for flow cytometry analysis. a Antigen-specific T helper cells were identified as Live/Dead-Marker⁻CD3⁺CD4⁺CD137⁺CD154⁺ and antigen-specific cytotoxic T cells were identified as Live/Dead-Marker⁻CD3⁺CD8⁺CD137⁺. Frequency of overall SARS-CoV-2 reactive CD4⁺CD137⁺CD154⁺ (b, left panel) and CD8⁺CD137⁺ (c, left panel) T cells specific to the M, N or S protein combined and subdivision in cells reactive towards the M, N or S protein among all CD4⁺ or CD8⁺ cells, respectively. Groups were compared using the Kruskal–Wallis test (indicated by “p- = ”); pairwise comparison was done using the two-sided, unpaired post-hoc Mann–Whitney U test. p-values < 0.05 are underlined in red

Fig. 3

RTx patients and healthy individuals have equal frequencies of pre-existing SARS-CoV-2 reactive T cells expressing Th1 cytokines or granzyme B. Isolated PBMCs from RTx patients (20), healthy individuals (20) and COVID-19 patients (17) were stimulated for 16 h with 1 µg/ml SARS-CoV-2 OPPs from M (n = 11–19), N (n = 17–20) or S (n = 17–20) proteins. Expression of Th1 cytokines IFNγ, IL-2 or TNF and granzyme B in antigen-specific CD4⁺CD137⁺CD154⁺ a and CD8⁺CD137⁺ b among all CD4 + or CD8 + cells, respectively. c Fraction of patients with SARS-CoV-2 reactive T cells co-expressing 2–4 cytokines or granzyme b among all antigen-specific T cells. Groups were compared using the Kruskal–Wallis test (indicated by “p- = ”); pairwise comparison was done using the two-sided, unpaired post-hoc Mann–Whitney U test. p-values < 0.05 are underlined in red. (H healthy individuals, CoV COVID-19 patients)

Fig. 4

Differences in the frequency of SARS-CoV-2 M, N or S protein reactive T cells. Isolated PBMCs from RTx (20), healthy individuals (20) and COVID-19 patients (17) were stimulated for 16 h with 1 µg/ml SARS-CoV-2 OPPs from M (n = 11–19), N (n = 17–20) or S (n = 17–20) proteins. Comparison of the frequencies of SARS-CoV-2 reactive a CD4⁺CD137⁺CD154⁺ and b CD8⁺CD137⁺ T cells after stimulation with the M, N or S proteins in RTx patients and healthy individuals. Groups were compared using the Kruskal–Wallis test (indicated by “p- = ”); pairwise comparison was done using the two-sided, unpaired post-hoc Mann–Whitney U test. p-values < 0.05 are underlined in red

Fig. 5

Comparison of SARS-CoV-2 reactive memory cell phenotypes. Isolated PBMCs from RTx patients (20), healthy individuals (20) and COVID-19 patients (17) were stimulated for 16 h with 1 µg/ml SARS-CoV-2 OPPs from M (n = 11–19), N (n = 17–20) or S (n = 17–20) proteins and analyzed by flow cytometry. a Identification of antigen-specific memory T cells: After gating on SARS-CoV-2 reactive CD4⁺CD137⁺CD154⁺ and CD8⁺CD137⁺ T cells, memory cells were identified by expression of CD45RA and CCR7 as naïve (CD45RA⁺CCR7⁺), central memory (CM, CD45RA⁻CCR7⁺), effector memory (EM, CD45RA⁻CCR7⁻) and TEMRA (CD45RA⁺CCR7⁻) cells. Comparison of SARS-CoV-2 reactive memory b CD4⁺CD137⁺CD154⁺ and c CD8⁺CD137⁺ T cells in RTx patients, healthy individuals and COVID-19 patients. Groups were compared using the Kruskal–Wallis test (indicated by “p- = ”); pairwise comparison was done using the two-sided, unpaired post-hoc Mann–Whitney U test. p-values < 0.05 are underlined in red.(H healthy individuals, CoV COVID-19 patients)