Analysis of Co-inhibitory Receptor Expression in COVID-19 Infection Compared to Acute Plasmodium falciparum Malaria: LAG-3 and TIM-3 Correlate With T Cell Activation and Course of Disease

Abstract

Coronavirus disease 2019 (COVID-19) which is caused by the novel SARS-CoV-2 virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. The virus induces a strong T and B cell response but little is known about the immune pathology of this viral infection. Acute Plasmodium falciparum malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of T cells. In malaria, T cells express a variety of co-inhibitory receptors which might be a consequence of their activation but also might limit their overwhelming function. Thus, T cells are implicated in protection as well as in pathology. The outcome of malaria is thought to be a consequence of the balance between co-activation and co-inhibition of T cells. Following the hypothesis that T cells in COVID-19 might have a similar, dual function, we comprehensively characterized the differentiation (CCR7, CD45RO) and activation status (HLA-DR, CD38, CD69, CD226), the co-expression of co-inhibitory molecules (PD1, TIM-3, LAG-3, BTLA, TIGIT), as well as the expression pattern of the transcription factors T-bet and eomes of CD8⁺ and CD4⁺ T cells of PBMC of n = 20 SARS-CoV-2 patients compared to n = 10 P. falciparum infected patients and n = 13 healthy controls. Overall, acute COVID-19 and malaria infection resulted in a comparably elevated activation and altered differentiation status of the CD8⁺ and CD4⁺ T cell populations. T effector cells of COVID-19 and malaria patients showed higher frequencies of the inhibitory receptors T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte-activation gene-3 (LAG-3) which was linked to increased activation levels and an upregulation of the transcription factors T-bet and eomes. COVID-19 patients with a more severe disease course showed higher levels of LAG-3 and TIM-3 than patients with a mild disease course. During recovery, a rapid normalization of these inhibitory receptors could be observed. In summary, comparing the expression of different co-inhibitory molecules in CD8⁺ and CD4⁺ T cells in COVID-19 vs. malaria, there is a transient increase of the expression of certain inhibitory receptors like LAG-3 and TIM-3 in COVID-19 in the overall context of acute immune activation.

Keywords: COVID-19; LAG-3; PD-1; Plasmodium falciparum; SARS-CoV-2; T cells; TIM-3; malaria.

Figure 1

(A–D) Frequency of CD3⁺ T cells and distribution of naïve and memory subsets as well as the activation status of T cells in COVID-19 and malaria patients compared to healthy donors. (A) Frequency of CD3⁺, CD8⁺, and CD4⁺ T cells in healthy donors (HD) and COVID-19 and malaria patients. (B) Frequency of CD69⁺, HLA-DR⁺CD38⁺ CD8⁺, and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. (C) T cell distribution illustrated in donut charts showing the mean frequency of naive (CCR7⁺/CD45RO⁻), central memory CM (CCR7⁺/CD45RO⁻), transitional memory TM (CCR7⁻/CD45RO⁺/CD27⁺), effector memory EM (CCR7⁻/CD45RO⁺/CD27⁻), and terminal effector memory EMRA (CCR7⁻/CD45RO⁻) CD8⁺, and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. Frequencies in COVID-19 and malaria patients were compared with HD. (D) Representative dot plots of PBMC gated on either CD8⁺ or CD4⁺ T cells for T cell distribution. P-values were calculated by Mann–Whitney test. P-values smaller than 0.05 were considered significant, where *, **, ***, and **** indicate p-values between 0.01 to 0.05, 0.001 to 0.01, 0.0001 to 0.001 and <0.0001 respectively.

Figure 2

(A–C) Activation status of naïve and memory subsets of CD8⁺ and CD4⁺ T cells in COVID-19 and malaria patients compared to healthy donors. (A) Frequency of CD69 and co-expression of HLA-DR and CD38 in different CD8⁺ and CD4⁺ T cell differentiation subsets in healthy donors and COVID-19 and malaria patients. (B) Frequency of CD226 on CD8⁺ and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. (C) Representative dot plots of PBMC gated on either CD8⁺ or CD4⁺ T cells for T cell activation. P-values were calculated by Mann–Whitney test. P-values smaller than 0.05 were considered significant, where *, **, and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01, and 0.0001 to 0.001, respectively.

Figure 3

(A–C) Frequency and co-expression of inhibitory receptors on CD8⁺ and CD4⁺ T cells in COVID-19 and malaria patients compared to healthy donors. (A) Frequency of co-inhibitory receptors PD1, TIGIT, LAG-3, TIM-3, and BTLA on CD8⁺ and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. Representative dot plots of the PD1, TIGIT, TIM-3, LAG-3, and BTLA frequency gated on either CD8⁺ or CD4⁺ T cells. (B) Frequency of LAG-3 and TIM-3 on the CD8⁺ and CD4⁺ T cell differentiation subsets in healthy donors and COVID-19 and malaria patients. (C) Co-expression of PD1/LAG-3 and PD1/TIM-3 illustrated in donut charts showing the mean frequency of CD8⁺ and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. Frequencies in COVID-19 and malaria were compared with HD. P-values were calculated by Mann–Whitney test. P-values smaller than 0.05 were considered significant, where *, **, ***, and **** indicate p-values between 0.01 to 0.05, 0.001 to 0.01, 0.0001 to 0.001 and <0.0001 respectively.

Figure 4

(A–D) Expression profile of inhibitory and stimulatory receptors as well as the transcription factors T-bet and eomes in COVID-19 and malaria patients. (A) Frequency of HLA-DR/CD38 on PD1⁻LAG-3⁻, PD1⁻TIM-3⁻, PD1⁺LAG-3⁺, and PD1⁺TIM-3⁺ CD8⁺ and CD4⁺ T cells in COVID-19 and malaria patients. (B) Frequency of CD69 on PD1⁻LAG-3⁻, PD1⁻TIM-3⁻, PD1⁺LAG-3⁺ and PD1⁺TIM-3⁺ CD8⁺ and CD4⁺ T cells in COVID-19 and malaria patients. (C) Frequency of T-bet and eomes in CD8⁺ and CD4⁺ T cells in healthy donors and COVID-19 and malaria patients. Representative dot plots of the T-bet and eomes frequency gated on either CD8⁺ or CD4⁺ T cells. (D) Frequency of T-bet and eomes in PD1⁻LAG-3⁻ and PD1⁺LAG-3⁺ CD8⁺ and CD4⁺ T cells in COVID-19 and malaria patients. P-values were calculated by Wilcoxon test for paired analysis and Mann–Whitney test for unpaired analysis. P-values smaller than 0.05 were considered significant, where *, **, ***, and **** indicate p-values between 0.01 to 0.05, 0.001 to 0.01, 0.0001 to 0.001 and <0.0001 respectively.

Figure 5

(A–C) Correlation of the expression of inhibitory and stimulatory receptors on T cells with disease course and CRP levels in COVID-19 patients. (A) Comparison of LAG-3 and TIM-3 frequency on CD8⁺ and CD4⁺ T cells between mild and severe COVID-19 patients. (B) Correlations between CRP levels and frequencies of LAG-3, TIM-3, and CD69 on CD8⁺ and CD4⁺ T cells in COVID-19 patients. (C) Frequency of PD1, LAG-3, and TIM-3 on CD8⁺ and CD4⁺ T cells measured at different time points in three COVID-19 patients. T0 indicates the first sample date. T1 indicates the second sample date (13, 15, or 20 days after T0). P-values were calculated by Mann–Whitney test. For bivariate correlation analysis the Spearman correlation was applied. P-values smaller than 0.05 were considered significant, where * indicate p-values between 0.01 to 0.05.