COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity

Abstract

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3⁺ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4⁺ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB⁺ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8⁺ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8^hi and CD8^lo T cells within the CD8⁺ population in HD are similar, a significant increase was observed in CD8^lo T cell frequency in COVID-19 patients. CD8^lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8^hi T cells. Interestingly, the frequency of CD8^lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8^hi/CD8^lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8^hi/CD8^lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

Keywords: CD8 + T cells; COVID-19; SARS-CoV-2; T cells dysfunction; cytotoxic CD4 + T cells.

Figure 1

Frequency of different lymphocyte subsets from HD and COVID-19 patients of an Argentine cohort. Percentage of CD19⁺ cells, CD3⁺ cells, CD8⁺ T cells, total CD4⁺ T cells, Tconv (CD4⁺ Foxp3^-) and Treg (CD4⁺ Foxp3⁺) T cells from PBMC samples from the entire cohort of hospitalized patients (A) or patients of the first and second wave analyzed separately (B). Dots show individual measurements of groups: HD (black), MD COVID-19 patients (blue), and SD COVID-19 patients (red), and black lines show the mean frequency of each population +/- SEM. One-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test or the Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used for statistical analysis when the measured variable had or had not met the normality assumptions, respectively. HD, healthy donors; MD, patients with moderate disease; SD, patients with severe disease.

Figure 2

Frequency of inhibitory receptors and cytokine-producing CD4⁺ T cells from HD and COVID-19 patients. Radar plots depict the normalized average frequencies of Tconv (CD4⁺Foxp3^-) and Treg (CD4⁺Foxp3⁺) expressing exhaustion, cytotoxic markers, and cytokines, from PBMCs of HD and SD and MD patients of the first and second waves (A). Frequency of GZMB⁺, CD107a⁺, CD39⁺, PD-1⁺ and TNF⁺, IFN-γ and IL-2-producing Tconv cells, and GZMB⁺, CD107a⁺, CD39⁺, PD-1⁺ and TIGIT⁺ Treg during the first and second wave of COVID-19. Dots show individual measurements of groups: HD (black), MD patients (blue), and SD patients (red), and black lines show the mean frequency of each population +/- SD (B, C). In (B, C) one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparisons test or the Kruskal-Wallis test followed by Dunn’s multiple comparisons test were used when the measured variable had or had not met the normality assumptions, respectively.

Figure 3

Frequency of CD8 high and low subpopulations and exhaustion markers from PBMC of HD and COVID-19 patients. Representative dot plots and graphs show frequencies of CD8^hi (filled circle) or CD8^lo T cells (open circle) from PBMCs of the entire cohort of HD and MD and SD COVID-19 patients. Dots show individual measurements. Data presented as mean +/- SD (A). Frequencies of TIGIT⁺, CD39⁺, and PD-1⁺ within CD8^hi (filled circle) and CD8^lo T cells (open circle) (B). Dots show individual measurements and lines indicate paired data. Two-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparisons test were calculated in (A, B) for statistical analysis.

Figure 4

Assessment of cytokine-producing CD8^lo and CD8^hi T cells and their state of differentiation. Representative dot plots (left) and accumulated data (right) line graphs show frequencies of TNF, IL-2, and IFN-y-producing CD8^hi (filled circle) and CD8^low T cells (open circle) from PBMCs of the entire cohort of HD and MD and SD COVID-19 patients (A). Frequency of GZMB and CD107a-producing CD8^hi or CD8^lo T cell populations of the entire cohort of HD and MD and SD COVID-19 patients (B). Lines indicate paired data in (A, B) Differentiation profile of CD8^hi and CD8^lo subpopulations (C). Two-way analysis of variance (ANOVA) followed by Bonferroni (A, B) or Tukey (C) multiple comparisons test was calculated for statistical analysis.

Figure 5

Determination of immune variables that enabled segregation of COVID-19 patients based on disease severity. Frequencies of CD8^hi and CD8^lo T cells of discharged and deceased COVID-19 patients (A) and their respective INDEX (B). Frequencies of GZMB-producing Treg and CD19⁺ lymphocytes in discharged or deceased COVID patients (C). Linear discriminant analysis (LDA) of parameters measured from the entire cohort. Patients were grouped in HD (gray), MD COVID-19 (blue), and SD COVID-19 (red), and graphs show their distribution in the canonical axes 1 and 2 (D). P values were determined by the t-Test in A and by Mann-Whitney test (B,C). Data presented as mean +/- SEM.