PMC

Expansion and restimulation increases resolution in the phenotypical profile of commensal-specific T cells. Cytokine-producing cells of porcine PBMC were directly analyzed ex vivo after Candida albicans restimulation, gated as described in Figure A and plotted as panel (A) cytokine-producing cells of total CD4⁺ T cells or panel (B) cytokine/CD154⁺ coproducing cells of CD4⁺ T cells (n = 10–11). PBMCs were further expanded for C. albicans antigen and analyzed for cytokine/CD154⁺ expression following restimulation with Cand-primed monocyte-derived dendritic cell (MoDC) (40 µg/ml). Panel (C) illustrates representative cytokine/CD154 analysis of expanded C. albicans-specific T cells restimulated with either umprimed (w/o) or C. albicans-primed MoDC. Cytokine analysis of C. albicans-expanded T cells are summarized in panel (D) (TNF-α: n = 10, unpaired t-test, p = 0.0166, IFN-γ: w/o n = 6, Cand n = 8, Mann–Whitney test, p = 0.0047, interleukin-17 w/o n = 4, Cand n = 8, Mann–Whitney test, p = 0.073).

Ascaris suum specific CD4⁺ T cells in acute and trickle infection ex vivo. (A) Representative contour plots showing ex vivo analysis of CD154 expression in lung lymphocytes of naive vs. infected piglets gated on CD4⁺ T cells (according to gating strategy of Figure A) when left untreated or stimulated with parasite lysate of L3 larvae (Asc Lys, 20 µg/ml). Italic numbers indicate frequency of IFN-γ-coproducing CD154⁺CD4⁺ T cells. (B) Frequencies of CD154⁺ cells among CD4⁺ T cells stimulated with Asc Lys in cells isolated from lung or spleen of naive vs. infected piglets (n = 3, lung: median 0.0665 vs. 0.185, p = 0.1; spleen: median 0.0513 vs. 0.124, p = 0.2, Mann–Whitney test). (C) Representative plots from ex vivo lung analysis of trickle infected piglets when stimulated with worm lysate (Asc Lys, 20 µg/ml) or excretory–secretory worm products (Asc ES, 20 µg/ml) or left untreated from n = 5 piglets, summarized in (D) (w/o vs. Asc Lys: median 0.0564 vs. 0.253, p = 0.0079 and Asc Lys vs. Asc ES: median 0.253 vs. 0.694, p = 0.0159, Mann–Whitney test). (E) Summarized CD154⁺ frequencies of lymphocytes isolated from the spleen of trickle infected piglets, Mann–Whitney test.

Pig antigen-responding T cells can be expanded and visualized after monocyte-derived dendritic cell (MoDC) restimulation. (A) Experimental setup. Purified blood CD14⁺ monocytes were differentiated into MoDC for 7 days in the presence of GM-CSF and interleukin-4. MoDCs were primed with 40 µg/ml Candida albicans lysate and maturation was triggered by TNF-α and LPS treatment for 1 day. In parallel, PBMCs were CFSE-labeled and expanded in the presence of 20 µg/ml Candida antigen for 7 days followed by a 2-day resting phase. Expanded lymphocytes were restimulated with autologous, primed MoDC (MoDC:T cell ratio, 1:5) and CD154 expression was assessed according to the gating strategy in (B). CD154 expression of CD4⁺CFSE^low (proliferated cells, lower left plot) was compared to unproliferated CD4⁺CFSE^high (lower middle, red plot) and proliferated CD4⁻CD8α⁺CFSE^low T cells (lower right, blue plot). (C) As control, CD154⁺ frequency of Cand expanded PBMC without DC restimulation (w/o DC ctrl.) is shown exemplarily [gated on CD4⁺CFSE^low as depicted in panel (B)]. (D) CD154⁺ T cell frequencies after restimulating expanded T cells with unprimed (w/o) or Candida antigen-primed (Cand) MoDC (n = 6, Mann–Whitney test, **p = 0.0043).

CD154 identifies porcine, antigen-reactive CD4⁺ T cells. T cell receptor stimulation (TCR)-independent and TCR-dependent activation induced CD154 expression in pig CD4⁺ T cells detected by flow cytometry and intracellular staining. (A) Gating strategy and representative zebra plots showing the CD154 signal from ex vivo PBMC gated on CD4⁺ T cells that were either unstimulated (w/o) or stimulated with Candida albicans lysate (Cand, 40 µg/ml), staphylococcal Enterotoxin B (SEB, 1 µg/ml), or PMA/ionomycin (P/I) for 6 h. Panel (B) summarizes w/o, Cand, and SEB stimulatory conditions for PBMC from n = 5 to 6 animals (w/o vs. Cand, p = 0.0271, Student’s t-test and w/o vs. SEB, p = 0.0043, Mann–Whitney test). (C) Analysis of CD8α coexpression of Candida-reactive CD154⁺CD4⁺ T cells or non-responding CD154⁻CD4⁺ T cells after Cand stimulation of PBMC. Concatenated contour plots (from n = 3 animals) are illustrated and numbers in gates identify CD8α negative (left rectangle gate) or positive (right rectangle gate) CD4⁺ T cells. (D) Flow cytometry of CD154/Cytokine coexpression analysis in either Cand (40 µg/ml) or SEB (1 µg/ml) stimulated PBMC for TNF-α, IFN-γ, and interleukin-17A. (E) Analysis of CD154 expression in gamma delta T cells (identified by TCR1δ⁺ expression and pregated on live CD3/duplet exclusion/FSC-SSC properties) upon antigen-specific (Cand, 40 µg/ml), superantigen-specific (SEB, 1 µg/ml), and TCR unspecific (PMA/ionomycin) stimulation.

Antigen dose and timing regulate CD154 expression of porcine CD4⁺ T cells. (A) Superantigen [staphylococcal Enterotoxin B (SEB)] expanded T cells were restimulated with autologous monocyte-derived dendritic cell (MoDC) for 5 h in the absence (left plot) or presence of varying concentrations of SEB (0.001–1 µg/ml) and assessed intracellularly for CD154 expression of proliferating CD4⁺ T cells according to gating strategy of Figure B. (B) As control, SEB expanded T cells were confronted with 1 µg/ml SEB in the absence of MoDC. (C) Candida albicans antigen expanded T cells were restimulated for 6 h with MoDC that were primed with varying concentrations of C. albicans antigen (2.5–40 µg/ml) or left unprimed (left plot). Representative plots for two independent titration experiments are shown. (D) Time course of CD154 expression after restimulating SEB-expanded cells with MoDC in the presence of 1 µg/ml SEB (0–8 h). Representative plots from n = 3 pigs are shown and summarized in panel (E). (F) Time course of CD154 expression following restimulation of C. albicans-expanded cells with C. albicans antigen-primed MoDC for 0–8 h. Representative plots from n = 3 pigs are shown and summarized in panel (G). Data in panels (E,G) are presented as mean ± SEM of CD154⁺ T cells over time (for data points lacking error bars, SEM values are smaller than circles representing means).

Ide_Ssuis-reactive Th cells are increased in PBMCs of immunized piglets identified by CD154 expression and cytokine production and the frequency of CD154⁺IFN-γ⁺ Th cells correlates with Ide_Ssuis-specific IgG. (A) Piglets were primed with rIde_Ssuis followed by booster immunization 2 weeks later. Blood samples for the investigation of antigen-specific Th cells were taken 2 week post-booster immunization. (B,C) To detect antigen-reactive Th cells, PBMCs derived from rIde_Ssuis-immunized (imm; n = 8) or placebo-treated (plac; n = 7) piglets were ex vivo restimulated for 18 h with 5 µg/ml rIde_Ssuis or ctr-antigen (rSfb I), respectively, in presence of Brefeldin A (2 µg/ml) for the last 4 h. The frequency of antigen-specific Th cells was calculated as the difference of specified cells from antigen-restimulated and medium-cultivated PBMC, respectively. Statistical analysis was performed with Kruskal–Wallis test (**p = 0.0063) and Dunn’s multiple comparison test (*p ≤ 0.05). Ide_Ssuis specific-IgGs were measured by ELISA with serum from the same piglets before and after immunization, using an independent Ide_Ssuis-IgG positive reference serum. The correlation between Ide_Ssuis-IgG and frequency of Ide_Ssuis induced CD154⁺IFN-γ⁺ Th cells (D) or frequency of ctr-ag induced CD154⁺IFN-γ⁺ (E) was estimated by Pearson correlation (*p ≤ 0.05).