Spontaneous, persistent, T cell–dependent IFN-γ release in patients who progress to Long Covid

After acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a proportion of patients experience persistent symptoms beyond 12 weeks, termed Long Covid. Understanding the mechanisms that cause this debilitating disease and identifying biomarkers for diagnostic, therapeutic, and monitoring purposes are urgently required. We detected persistently high levels of interferon-γ (IFN-γ) from peripheral blood mononuclear cells of patients with Long Covid using highly sensitive FluoroSpot assays. This IFN-γ release was seen in the absence of ex vivo peptide stimulation and remains persistently elevated in patients with Long Covid, unlike the resolution seen in patients recovering from acute SARS-CoV-2 infection. The IFN-γ release was CD8+ T cell–mediated and dependent on antigen presentation by CD14+ cells. Longitudinal follow-up of our study cohort showed that symptom improvement and resolution correlated with a decrease in IFN-γ production to baseline levels. Our study highlights a potential mechanism underlying Long Covid, enabling the search for biomarkers and therapeutics in patients with Long Covid.


Figure S1
, having analysed PBMCs from unexposed (red), acutely infected and recovered by D180 (cyan) and Long COVID patients (green).IFN-γ and IL-2 were analysed on the same plates and although a change was seen for IFN-γ, no such change was observed for IL-2.IL-2 release was quantified as spot forming units per million PBMCs.Each donor was run in duplicate and zero results were set as 0.1 to allow their inclusion on a log scale.L.O.D. = limit of detection.Significance calculated by Kruskal-Wallis ANOVA.

Figure S3
Figure S3: IL-2 and IFN-γ release from peptide-and positive control-stimulated PBMCs show no general T cell disfunction in Long COVID patients.Data shown is from the same cohorts as Figure 1, having analysed PBMCs from unexposed (red), acutely infected and recovered patients at D28 (cyan), D90 (green and D180 (blue) and Long COVID patients (burgundy).These PBMCs were stimulated with either cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus peptides (CEF) (A&B) or with the positive control cocktail of anti-CD3 antibody, Staphylococcus Enterotoxin B (SEB), and Phytohemagglutinin (PHA) (C&D).IFN-γ and IL-2 responses were measured by FluoroSpot assay as spot forming units per million PBMCs.Each condition was run in duplicate and an unstimulated control was subtracted to remove background cytokine production.Zero results are set as 0.1 to allow their inclusion on a log scale.L.O.D. = limit of detection.Significance calculated by Kruskal-Wallis ANOVA, with Dunn's multiple comparison test between unexposed and each infected group.

Figure S7
Supplementary Figure 7. Representative dot plots from 1 donor are shown illustrating the gating strategy for generating the absolute count data, as has been used previously [13] [51].First the trucount bead population was identified (FITC vs APC) and then the trucount bead negative population (i.e.cells) were analysed by gating for single cells (FSC-A vs FSC-H), then CD45 hi lymphocytes (Non-Granulocytes) and Granulocytes were gated (CD45 vs SSC-A).Next, a Monocytes gate and Lymphocytes gate were discriminated by size and granularity (FSC-A vs SSC-A) and monocytes were gated by eliminating CD16hi cells (monos (non-gran gate on CD16 vs CD45 plot), the CD3 and CD19 negatives (CD3 vs CD19 plot) and finally the CD14 + monocyte population identified (CD14 vs SSC-A).From the lymphocytes gate CD3 + T cells, NK cells and B cells were identified (CD3 vs CD19 plot), the NK cells were then gated for CD56 + , CD56 + CD16 + and CD56 -CD16 + populations as All NK (CD16 vs CD56) and the proportion of NK cells expressing NKG2C was enumerated (CD56 vs NKG2C).The CD3 + T cells were gated from the NKT cells (CD56+ by CD56 vs CD3 plot), then the CD4 + and CD8 + expressing cells and double positive (DP) T cells and double negative (DN) T cells were identified.T regulatory (Treg) CD4 + T cells were identified as CD127lo and CD25hi (CD25 vs CD127 plot).Lastly both the CD4 + and CD8 + T cell populations were further subdivided into 4 memory populations defined by expression of CD27 and CD45RA, 4 differentiation populations defined by expression of CD57 and CD28 and HLA-DR expressing activated cells were identified (HLA-DR vs CD45RA plot).All gate and quadrant positions were identified using the FMO controls, the formula used to calculate the absolute cell counts from the event numbers in each gate or quadrant is illustrated.Figure S8: The gating strategy used to identify CD4 + and CD8 + T cells is shown (A) with representative plots from 1 donor sample.First a Time vs Side scatter gate was drawn, to identify the main flow of cells, then these cells were gated for forward scatter single cells (Forward scatter area (FSC-A) vs Forward scatter height (FSC-H)), side scatter single cells (Side scatter area (SSC-A) vs Side scatter height (SSC-H)), live B cell negative cells (SSC-W vs Live Dead Aqua dye & CD19 BV510), then lymphocytes were gated (FSC-A vs SSC-A (log scale)).CD3 positive cells were identified (CD3 BV650 vs CD14 FITC) and then CD8 + and CD4 + T cells gated (CD4 BV605 vs CD8 BV570).Representative plots showing IFNγ staining from CD3 + T cells, CD8 + and CD4 + T cell subsets from unstimulated and stimulated (polyclonal positive control) of a Healthy control (B) and long covid patient (C) are shown.This panel has been used before [52,53].This illustrates the increased production of IFNgamma in ex vivo long covid patient T cells compared to the healthy control volunteer.

Figure S9
Figure S9: IL-2 is produced in response to spike peptide stimulation after vaccination in patients with Long COVID.PBMCs were isolated from patients diagnosed with Long COVID before and after first vaccination and either stimulated with anti-spike peptide exvivo or mock treated with a media control.After 48 hours incubation IL-2 release from spike peptide-stimulated T cells was measured by FluoroSpot analysis, with negative control IL-2 subtracted from spike stimulated samples.For both datasets, IL-2 and IFN-γ were quantified as spot forming units per million PBMCs.Each donor was run in duplicate and zero results were set as 0.1 to allow their inclusion on a log scale.L.O.D. = limit of detection.Significance calculated by Wilcoxon Ranked Sign Test, ****p<0.0001.Pacific Blue FN50 IgG1 κ 310920 BioLegend Abbrv: BV = Brilliant Violet; BUV = Brilliant UV; Dz = Dazzle; AxF = Alexa Fluor Antibodies used for flow cytometry were validated for flow cytometry by Biolegend and Miltenyi biotech.Additionally, these antibodies have been used previously by Dr Sarah Jackson for other studies [13,53,54].

Figure S1 :
Figure S1: PBMCs from patients with Long COVID produce more interferon gamma.Representative images of wells of unstimulated PBMCs from 9 unexposed donors and 9 patients with Long COVID, taken from the same plate with the same exposures.

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