Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins

Abstract

(1) Background: statins have been considered an attractive class of drugs in the pharmacological setting of COVID-19 due to their pleiotropic properties and their use correlates with decreased mortality in hospitalized COVID-19 patients. Furthermore, it is well known that statins, which block the mevalonate pathway, affect γδ T lymphocyte activation. As γδ T cells participate in the inflammatory process of COVID-19, we have investigated the therapeutical potential of statins as a tool to inhibit γδ T cell pro-inflammatory activities; (2) Methods: we harvested peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild clinical manifestations, COVID-19 recovered patients, and healthy controls. We performed ex vivo flow cytometry analysis to study γδ T cell frequency, phenotype, and exhaustion status. PBMCs were treated with Atorvastatin followed by non-specific and specific stimulation, to evaluate the expression of pro-inflammatory cytokines; (3) Results: COVID-19 patients had a lower frequency of circulating Vδ2+ T lymphocytes but showed a pronounced pro-inflammatory profile, which was inhibited by in vitro treatment with statins; (4) Conclusions: the in vitro capacity of statins to inhibit Vδ2+ T lymphocytes in COVID-19 patients highlights a new potential biological function of these drugs and supports their therapeutical use in these patients.

Keywords: COVID-19; SARS-CoV-2 infection; mevalonate pathway; statin; γδ T cells.

Figure 1

Frequency of Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and in healthy subjects. (A) Distribution of percentages of total lymphocytes, T (CD3+), Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy subjects. Shown is mean ± SD. (B) Representative dot-plots showing the frequency of Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and healthy subjects. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 2

Phenotypic analysis of circulating Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and in healthy subjects. Distribution of ex vivo memory subsets of Vδ1+ (A) and Vδ2+ (C) T lymphocytes based on the expression of CD27 and CD45RA in hospitalized and recovered COVID-19 patients, compared to healthy donors. Median is shown. Healthy donors were represented as triangle, COVID-19 patients as circle and COVID-19 recovered as rhombus. Representative dot-plots showing Vδ1+ (B) and Vδ2+ (D) T lymphocyte memory subset distribution in hospitalized, recovered, and healthy subjects. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

Analysis of exhaustion-marker expression by circulating Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and healthy subjects. Expression of TIM-3 and PD-1 on Vδ1+ (A) and Vδ2+ (B) T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy donors. Shown are percentage (left panels) and MFI (right panels) ± SD. (C) Representative dot-plots of exhaustion-marker expression by Vδ1+ and Vδ2+ T lymphocytes from hospitalized and recovered COVID-19 patients, compared to healthy donors. (D) Frequency of exhausted Vδ1+ and Vδ2+ T cells per 1 × 10⁶ T lymphocytes. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Expression of pro-inflammatory cytokines by Vδ1+ T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy subjects. Cumulative histograms representing the expression of pro-inflammatory cytokines by Vδ1+ T lymphocyte. Shown are percentage (left panels), MFI (central panel, log₁₀ scale), and iMFI (right panels) ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Expression of pro-inflammatory cytokines by Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy subjects. Cumulative histograms representing the expression of pro-inflammatory cytokines. Shown are percentage (left panels), MFI (central panel, log₁₀ scale), and iMFI (right panels) ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 6

Intracellular cytokine expression by Vδ2+ T lymphocytes in COVID-19 patients and healthy donors upon zoledronate stimulation. Intracellular levels of IFN-γ, TNF-α, and IL-17, in terms of percentage and iMFI, expressed by Vδ2+ T cells, are represented as histogram plots. Shown is mean ± SD. * p < 0.05, ** p < 0.01, *** p< 0.001, and **** p < 0.0001.

Figure 7

Inhibition of Vδ2+ T cell cytokine expression by atorvastatin upon zoledronate stimulation (A) Intracellular levels of TNF-α, IFN-γ, and IL-17 expressed by Vδ2+ T cells from COVID-19 patients upon stimulation with zoledronate in presence of atorvastatin. Data are represented as histogram plots. Each histogram shows mean ± SD. Purple histogram represented Zoledronate condition, lilac histogram represented Zoledronate + 10 μM statin condition, and pink histogram represented Zoledronate + 50 μM statin condition. (B) Representative counterplots showing pro-inflammatory cytokine expression by Vδ2+ T lymphocytes. * p < 0.05, ** p < 0.01, *** p< 0.001, and **** p < 0.0001.

Figure 8

Inhibition of Vδ2+ T cell cytokine expression by atorvastatin upon Iono/PMA stimulation. (A) Intracellular levels of TNF-α, IFN-γ, and IL-17 expressed by Vδ2+ T cells from COVID-19 patients upon stimulation with Iono/PMA in presence of atorvastatin. Data are represented as histogram plots, showing mean ± SD. Blue histogram represented Iono/PMA condition, light blue histogram represented Iono/PMA + 10 μM statin condition, and light blue powder histogram represented Iono/PMA + 50 mM statin condition. (B) Representative counterplots showing pro-inflammatory cytokine expression by Vδ2+ T lymphocytes. * p < 0.05, *** p< 0.001, and **** p < 0.0001.