Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry

Abstract

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25⁺CD69⁺CD40L⁺TNF-α^- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25⁺CD69⁺TNF-α^- T cells were identified. Within CD8α⁺ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

Keywords: African swine fever virus; CD25; CD40L; CD69; ICOS; SEB; activation-induced markers; pig.

Figure 1

Expression of AIMs in blood-derived CD3⁺ T cells after stimulation with PMA/ionomycin. (A) Representative FCM plots depicting expression of CD25, CD69, CD40L and TNF-α in CD3⁺ T cells when PBMC samples were unstimulated (Medium, top row) or stimulated with PMA/ionomycin (PMA, bottom row) for 18 h. Surface staining was performed for antibodies against CD3, CD4, CD8α, CD25 and CD69. Intracellular staining was performed for CD40L and TNF-α. Gates shown are representative of gating for total CD25⁺, total CD69⁺, total CD40L⁺ and total TNF-α⁺ T cells applied to PMA-stimulated samples and used in Boolean gating to create doughnut charts. (B) Doughnut charts of AIM phenotypes in PMA-stimulated samples generated by Boolean gating. Each doughnut represents the PBMC sample of one pig. Different phenotypes are indicated by different colors with all AIM phenotypes co-expressing TNF-α summarized in gray. CD25⁻CD40L⁻CD69⁻TNF-α⁻ T cells are not shown. (C) Live CD3⁺ T cells from unstimulated (Medium), SEB-stimulated and PMA-stimulated cultures were clustered using the t-SNE algorithm with generated clusters shown in a colored overlay (left side). Relative expression levels of CD69, CD25 and CD40L within clusters (right side) are colored from high (red) to low (blue).

Figure 2

Expression of AIMs in blood-derived CD4/CD8α-defined T cell subsets after stimulation with SEB. (A) Representative FCM plots depicting expression of CD25, CD69, CD40L and TNF-α in CD4⁻CD8α⁺, CD4⁺CD8α⁺ and CD4⁺CD8α⁻ T cells. PBMC samples were unstimulated (Medium, left columns) or stimulated with SEB (SEB, right columns) for 18 h. Cells were pre-gated in the following order: live, single and CD3⁺. Gates shown indicate gating applied to calculate frequencies of AIM⁺ and TNF-α⁺ cells. (B) Frequencies of CD25⁺, CD69⁺, CD40L⁺ and TNF-α⁺ cells within CD4/CD8α-defined T cell subsets in unstimulated (Medium) and SEB-stimulated samples. Each dot represents data from one animal (n = 5). Asterisks indicate significant differences between groups (^*p ≤ 0.05, ^**p ≤ 0.01).

Figure 3

Expression of AIMs in blood-derived CD4/CD8α-defined T cell subsets after stimulation with ASFV Estonia 2014. (A) Representative FCM plots depicting expression of CD25, CD69, CD40L, ICOS and TNF-α in CD4⁻CD8α⁺, CD4⁺CD8α⁺ and CD4⁺CD8α⁻ T cells. PBMC samples were incubated with mock inoculum (Mock, left columns) or ASFV Estonia 2014 (ASFV, right columns) for 18 h. Cells were pre-gated in the following order: live, single and CD3⁺. Gates shown indicate gating applied to calculate frequencies of AIM⁺ and TNF-α⁺ cells. Dot size was enlarged to improve visibility of AIM⁺ and TNF-α⁺ cells. (B) Frequencies of CD25⁺, CD69⁺, CD40L⁺, ICOS⁺ and TNF-α⁺ cells within CD4/CD8α-defined T cell subsets in mock-inoculated (Mock) and ASFV Estonia 2014 (ASFV)-stimulated samples. Each dot represents data from one animal (n = 5). Asterisks indicate significant differences between groups (^*p ≤ 0.05, ^**p ≤ 0.01).

Figure 4

Relative distribution of AIM phenotypes within CD4/CD8α-defined T cell subsets in SEB-stimulated vs. ASFV-stimulated PBMC cultures. (A) Doughnut charts of AIM phenotypes in SEB-stimulated samples generated by Boolean gating in CD4⁻CD8α⁺ (top row), CD4⁺CD8α⁺ (middle row) and CD4⁺CD8α⁻ (bottom row) T cells. Each doughnut represents the PBMC sample of one pig. Different phenotypes are indicated by different colors with all AIM phenotypes co-expressing TNF-α summarized in gray. CD25⁻CD40L⁻CD69⁻TNF-α⁻ and CD25 single⁺ T cell phenotypes are not shown. (B) Doughnut charts of AIM phenotypes in ASFV Estonia 2014-stimulated samples generated by Boolean gating in CD4⁻CD8α⁺ (top row), CD4⁺CD8α⁺ (middle row) and CD4⁺CD8α⁻ (bottom row) T cells. Each doughnut represents the PBMC sample of one pig. Different phenotypes are indicated by different colors with all AIM phenotypes co-expressing TNF-α summarized in gray. CD25⁻CD40L⁻CD69⁻TNF-α⁻ and CD25 single⁺ T cell phenotypes are not shown.

Figure 5

Induction of AIM⁺ CD4/CD8α-defined T cell phenotypes after ASFV Estonia 2014 stimulation. (A,B) Frequencies of selected AIM⁺ phenotypes within CD4⁻CD8α⁺ and CD4⁺CD8α⁺ T cells in mock-inoculated (Mock) vs. ASFV Estonia 2014 (ASFV)-stimulated samples. Boolean gating of AIMs was performed excluding (A) or including analysis of ICOS expression (B). Each dot represents data from one animal (n = 5). Asterisks indicate significant differences between groups (^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001).