Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection

Abstract

African swine fever (ASF) caused by the African swine fever virus (ASFV) is a fatal and highly contagious disease of domestic pigs characterized by rapid disease progression and death within 2 weeks. How the immune cells respond to acute ASFV infection and contribute to the immunopathogenesis of ASFV has not been completely understood. In this study, we examined the activation, apoptosis, and functional changes of distinct immune cells in domestic pigs following acute infection with the ASFV CADC_HN09 strain using multicolor flow cytometry. We found that ASFV infection induced broad apoptosis of DCs, monocytes, neutrophils, and lymphocytes in the peripheral blood of pigs over time. The expression of MHC class II molecule (SLA-DR/DQ) on monocytes and conventional DCs as well as CD21 expression on B cells were downregulated after ASFV infection, implying a potential impairment of antigen presentation and humoral response. Further examination of CD69 and ex vivo expression of IFN-γ on immune cells showed that T cells were transiently activated and expressed IFN-γ as early as 5 days post-infection. However, the capability of T cells to produce cytokines was significantly impaired in the infected pigs when stimulated with mitogen. These results suggest that the adaptive cellular immunity to ASFV might be initiated but later overridden by ASFV-induced immunosuppression. Our study clarified the cell types that were affected by ASFV infection and contributed to lymphopenia, improving our understanding of the immunopathogenesis of ASFV.

Keywords: ASFV; T cell early activation; apoptotic; cytokine; immunopathogenesis.

Figure 1

Virulent ASFV infection led to predominant reduction of B cells, CD4 T cells, monocytes, and DCs in periphery blood. At the indicated time points, PBMCs were prepared and stained for distinct leukocyte subsets and were analyzed by FCM. The absolute amount of each leukocyte subset per milliliter of blood was calculated based on the percentage of the specific cell type in live PBMCs. The numbers of monocyte (A), cDC1 (B), cDC2 (C), neutrophil (D), lymphocyte (E), B cell (F), γδ T cell (G), CD3 T cell (H), CD4 T cell (I), CD4⁺CD8⁺ T cell (J), CD8 T cell (K), and NK cell (L) in 1 mL blood. Data shown are mean ± SD. *p < 0.05. **p < 0.01.

Figure 2

Apoptosis of DCs, monocytes, and neutrophils in vivo after ASFV infection. At the indicated time points, PBMCs of ASFV-infected and ASFV-uninfected piglets were prepared and stained for the apoptosis of cDC1, cDC2, monocytes, and neutrophils and were analyzed by FCM. Representative zebra plots (left panel) and kinetic changes (right panel) of the percentage of apoptotic (Annexin V⁺) cDC1 (A), cDC2 (B), monocytes (C), and neutrophils (D). FVD780⁺ cells, dead cells. Data shown are mean ± SD. *p < 0.05.

Figure 3

Apoptosis of T cell subsets in vivo after ASFV infection. At the indicated time points, PBMCs of ASFV-infected and ASFV-uninfected piglets were prepared and stained for apoptotic T cell subsets and were analyzed by FCM. Kinetic changes of the percentage of apoptotic (Annexin V⁺) B cell (A), NK cell (B), γδ T cell (C), CD4 T cell (D), CD4⁺CD8⁺ T cell (E), and CD8⁺ T cell (F). FVD780⁺ cells, dead cells. Data shown are mean ± SD. *p < 0.05; **p < 0.01.

Figure 4

Downregulation of MHC II on APCs after ASFV infection. At the indicated time points, PBMCs of piglets infected with or without ASFV were prepared and stained for monocyte and DCs and were analyzed by FCM. MFI of MHC II on monocyte (A), cDC1 (B), and cDC2 (C) was calculated for assessment of the expression level on each subset (upper panel). Each point represents data from a single piglet, while bars represent the mean of each group. A representative histogram of the MHC II⁺ APCs at 5 dpi (lower panel); the black line represents uninfected piglets and the orange line represents infected piglets, while the blue line means MHC II-FMO control. Data shown are mean ± SD. ns, no statistical significance. *p < 0.05; **p < 0.01.

Figure 5

Downregulation of CD21 on B cells after ASFV infection. At the indicated time points, PBMCs of ASFV-infected or ASFV-uninfected piglets were separated and stained for FCM analysis of B cells. A representative histogram of CD21⁺ B cell at 5 dpi (left panel), and the black line represents uninfected piglets and the orange line represents infected piglets, while the closed blue histogram means FITC-CD21-FMO control. Dynamic changes of CD21 MFI on B cells (right panel). Each point represents data from a single pig, while the bars represent the mean of each group. Data shown are mean ± SD. ns, no statistical significance. **p < 0.01; ***p < 0.001.

Figure 6

Early activation of different lymphocyte subsets after ASFV infection in blood and organs. Single-cell suspensions of peripheral blood, mLN, lung, and spleen were prepared at 3 dpi and stained for lymphocyte subsets and were analyzed by FCM. The percentages of CD69⁺ lymphocyte subsets in PBMC (A), mLN (B), lung (C), and spleen (D) were compared between ASFV-uninfected and ASFV-infected groups. Data shown are mean ± SD. *p < 0.05.

Figure 7

ASFV infection induced cytokine-secreting T cells and NK cells. PBMC of both ASFV-infected and ASFV-uninfected piglets were separated at 5 dpi and stained for lymphocyte subsets, followed by intracellular cytokine staining for TNF-α, IFN-γ, and IL-2, and then, the cells were analyzed by FCM. Representative pseudocolor plots (left panel: the upper one, uninfected; the lower one, infected) and increased frequencies of IFN-γ⁺, IFN-γ⁺TNF-α⁺ or IL-2⁺ CD4 T (A), CD8 T (B), CD4⁺CD8⁺ T (C), NK (D) and γδ T cells (E,F) (right panel). Data shown are mean ± SD. *p < 0.05.

Figure 8

ASFV infection impaired the capability of T cells to produce cytokines. At the indicated time points, PBMC of ASFV-infected and uninfected piglets were separated and stimulated with mitogen and were stained for lymphocyte subsets, followed by an intracellular cytokine staining step and cells were determined by FCM. Representative pseudocolor plots (A) and the differential expression of cytokines in CD4⁺CD8⁺ T cell (B). Representative pseudocolor plots (left panels) and the differential expression of cytokines in CD4⁺CD8⁺ T cell (C), CD4 T cell (D) and CD8 T cell (E) (right panels). Each point represents data from a single pig while bars represent the mean of each group. Data shown are mean ± SD. ns, no statistical significance. *p < 0.05; **p < 0.01.