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Comparative Study
. 2021 Nov 8;13(11):2239.
doi: 10.3390/v13112239.

Lasting Changes to Circulating Leukocytes in People with Mild SARS-CoV-2 Infections

Allison E Kennedy  1   2   3 , Laura Cook  4   5 , Jessica A Breznik  1   2   3 , Braeden Cowbrough  1   2   3 , Jessica G Wallace  1   2   3 , Angela Huynh  3 , James W Smith  3 , Kiho Son  3   6 , Hannah Stacey  1   2   7 , Jann Ang  1   2   7 , Allison McGeer  8   9 , Brenda L Coleman  8   9 , Maggie Larché  3 , Mark Larché  3 , Nathan Hambly  3   6 , Parameswaran Nair  3   6 , Kjetil Ask  3   6 , Matthew S Miller  1   2   7 , Jonathan Bramson  1   3 , Megan K Levings  4   10   11 , Ishac Nazy  3   12 , Sarah Svenningsen  3   6 , Manali Mukherjee  3   6 , Dawn M E Bowdish  1   2   3   6
Affiliations
Comparative Study

Lasting Changes to Circulating Leukocytes in People with Mild SARS-CoV-2 Infections

Allison E Kennedy et al. Viruses. .

Abstract

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratory infections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.

Keywords: COVID-19; SARS-CoV-2; immune activation; immunophenotype; inflammation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
CD4+ and CD8+ T-cell responses to the M, N, and S peptide pools after mild SARS-CoV-2 infection. (a) The number of SARS-CoV-2-specific T-cells is measured as a percent of CD4+ T-cells expressing both CD25 and OX40, or CD8+ T-cells expressing both CD69 and CD137, after activation with the S, M, or N peptide pools 1–3 months and 6–9 months after infection. The polyclonal activator Cytostim is used as a positive control. (b) All COVID-19 seropositive participants had an increase in CD25+OX40+CD4+ T-cells in response to at least one of the M, N, or S antigens 1–3 months after mild COVID-19 infection, compared to seronegative individuals recovered from other mild respiratory infections. Each participant is indicated by a single data point: other respiratory infection n = 11; 1–3 months post COVID-19 infection n = 11; 6–9 months post COVID-19 infection n = 8. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars represent the mean ± standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 2
Figure 2
Transient increases in soluble mediators of inflammation occur after mild SARS-CoV-2 infection. (a) CRP levels in serum were higher 1–3 months after mild COVID-19 infection, and there was a trend towards remaining elevated 6–9 months after mild COVID-19 infection, compared to levels observed in seronegative individuals after other respiratory infections. Serum TNF levels (b) and IL-6 levels (c) were higher 1–3 months after COVID-19 infection, but returned to levels seen in individuals who had other respiratory infections by 6–9 months. Each participant is indicated by a single data point: other respiratory infection n = 11; 1–3 months post-COVID n = 9–12; 6–9 months post-COVID n = 8. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presented as mean ± standard deviation. * p < 0.05; ** p < 0.01.
Figure 3
Figure 3
Evidence of prolonged T-cell activation after mild SARS-CoV-2 infection. The polyclonal activator Cytostim was used to measure T-cell responses. COVID-19 seropositive individuals had a higher proportion of CD4+ (a) and CD8+ (b) activated T-cells 1 to 3 months after infection, compared to seronegative individuals after other respiratory infections. (c) The number of CD4+ T-cells that responded to polyclonal stimulation correlated with CRP in COVID-19 seropositive individuals. (d–g) Activation markers OX40, CCR6, CCR4, and CD69 on CD4+ T-cells were higher 1–3 months after COVID-19 infection, compared to after other respiratory infections, after Cytostim exposure. Each participant is indicated by a single data point: other respiratory infection n = 7–11; 1–3 months post COVID-19 infection n = 11–12; 6–9 months post COVID-19 infection n = 5–8. Multiple group comparisons in (a,b) and (d–g) were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presented as mean ± standard deviation. Data in C was assessed by Spearman’s rank correlation. * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
Transient changes in circulating lymphocytes occur 1–3 months after COVID-19 infection. Absolute numbers of circulating CD45+ T-cells (a), CD4+ T-cells (b), and CD8+ T-cells (c) were not different after 1–3 months or 6–9 months post-symptom presentation in seropositive individuals recovered from mild COVID-19 infection, compared to seronegative individuals recovered from other respiratory infections; however, NK cell numbers (d) were lower in the 1–3 months post-COVID-19 infection group. At 1–3 months post recovery from COVID-19 there was an increase in CD45RA−CCR7+ central memory CD4+ T-cells (e), and an increase in CD45RA+CCR7−CD57+CD28− terminally differentiated CD8+ T-cells (f), compared to individuals recovered from other respiratory infections, but these differences were not apparent in individuals who had recovered from COVID-19 6–9 months prior. (g) Levels of circulating regulatory T-cells (measured as a % of CD4+ T-cells) were higher in individuals 1–3 months post COVID-19 infection. Each participant is indicated by a single data point: other respiratory infection n = 11; 1–3 months post COVID-19 infection n = 11–13; 6–9 months post COVID-19 infection n = 8. ns—not statistically significant. Multiple group comparisons were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; bars are presented as mean ± standard deviation. * p < 0.05.
Figure 5
Figure 5
Evidence of sustained cellular inflammation after mild SARS-CoV-2 infection. (a) There was a trend towards a decreasing ratio of myeloid to lymphoid cells after SARS-CoV-2 infection, compared to individuals recovered from other infections, which was driven by a decrease in circulating neutrophils (b). Although total monocyte numbers did not change after infection (c), surface expression of the migratory markers CX3CR1 and CCR2 decreased transiently (d). Concurrent increases in surface expression of the migration and activation marker CD11b implies that the monocytes were activated. (e) Correlation analysis (Spearman’s correlation) of SARS-CoV-2 specific CD4+ and CD8+ T-cell responses with measures of monocyte activation and migratory potential. Multiple group comparisons in (a–d) were tested using Welch’s One-Way ANOVA and the Games–Howell post-hoc test; in (a–c) bars are presented as mean ± standard deviation, and each dot indicates a participant. The spread of expression of monocyte surface markers in d was visualized by concatenating uncompensated CD45+CD19−CD3−CD56−CD11b+HLADR+CD14+ events in FlowJo for each infection group prior to overlaying geometric mean fluorescence intensity expression data from all participants onto the same histogram plot. Other respiratory infection (grey) n = 11, 1–3 months after COVID-19 infection (red) n = 14, 6–9 months from COVID-19 infection (pink) n = 8. ns—not statistically significant. Data in (e) were assessed with the rcorr function in the Hmisc package in R, and only statistically significant associations are shown. * p < 0.05.
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