For analyzing C. albicans- or superantigen (SEB)-reactive CD4⁺ T cells, blood was sampled from healthy German Landrace pigs (3–12 months) either by taking blood from the external jugular vein or by heart puncture after sedation with ketamine hydrochloride and azaparone (20 mg/kg BW; Ursotamin; Serumwerk Bernburg AG and 2 mg/kg BW; Stresnil; Janssen-Cilag GmbH).

For analyzing A. suum-reactive CD4⁺ T cells during acute, primary parasite infection, German landrace piglets were aged 8–10 weeks and orally inoculated with a dose of 12–15,000 A. suum eggs/pig. Parts of spleen and lung were sampled from infected piglets after sedation with ketamine hydrochloride and azaparone (20 mg/kg BW; Ursotamin; Serumwerk Bernburg AG and 2 mg/kg BW; Stresnil; Janssen-Cilag GmbH) and euthanizing the animals by intracardial injection with 10 mg/kg BW of tetracaine hydrochloride, mebezonium iodide, and embutramide (T61, Intervet, Germany).

For analyzing A. suum-reactive CD4⁺ T cells during trickle infection, commercial Rattlerow Seghers hybrid piglets were aged for 4 weeks. Animals were orally inoculated with 80 Ascaris eggs per day for seven consecutive weeks. At the end of this study, all piglets were first sedated with Stresnil (Janssen-Cilag GmbH) and subsequently euthanized by electric stunning followed by exsanguination, and parts of the spleen and lung were sampled.

For analyzing S. suis-reactive CD4⁺ T cells after Ide_Ssuis immunization, German Landrace piglets at the age of 5–12 weeks were intramuscularly injected as described (42) with 0.4 mg recombinant His-tagged Ide_Ssuis (rIde_Ssuis, prime) and boosted with 0.25 mg rIde_Ssuis 2 weeks later, supplemented with 20% (vol/vol) Emulsigen (MVP Technologies, Omaha, NE, USA) as adjuvant. Placebo control animals were exclusively injected with PBS, supplemented with 20% (vol/vol) Emulsigen. Fourteen days post-booster immunization, heparinized blood samples were taken from the Vena cava cranialis.

Infective A. suum eggs were produced as previously described (43). In brief, A. suum eggs were obtained by culturing female adult worms from the slaughter house overnight in worm culture medium [BSS supplemented with 1% Glucose (AppliChem), 200 U/ml Penicillin and 200 µg/ml Streptomycin (PAN-Biotech), Gentamycin (50 µg/ml, PAN-Biotech), and Amphotericin B (0.25 µg/ml, PAN-Biotech)]. Released eggs were collected, washed several times in water, and placed in 0.1% formalin-containing distilled water for embryonation (4 weeks). Embryonation rates were checked weekly and by reaching 95% fertilized eggs used for infection. For generation of worm antigens (Asc Lys) and worm excretory–secretory products (Asc ES), A. suum L3 larvae were recovered from the lungs of infected animals using a modified Baermann funnel. Therefore, lungs were cut into 1–2 cm chunks and placed in 37°C pre-warmed saline (0.9%) onto a gaze-lined funnel. Vital worms were isolated from the flow through, washed extensively in antibiotic containing worm culture media, and were either snap-frozen for production of L3 worm lysate (Asc Lys) or cultured in worm culture media for 1–2 weeks to collect excretory–secretory products containing worm culture supernatants. Worm culture supernatants were further concentrated using centrifugal protein concentrators with a 5-kDa MWCO (Vivaspin, Sartorius) to obtain the final, concentrated Asc ES. LPS contaminations of Asc Lys and Asc ES were analyzed by LAL test using Endosafe^®-PTS™ and cartridges (Charles River Laboratories International, Inc.). Calculated as final concentration when applied in vitro LPS values ranged between 0.812 and 1.624 ng/ml for Asc Lys and 0.0261 and 0.0522 ng/ml for Asc ES.

Mononuclear cells from porcine peripheral blood were isolated by density centrifugation of diluted (1:2 in 0.9% NaCl) blood using Pancoll solution (density 1.077 g/ml, PAN-Biotech). Splenic cells were isolated by passing spleen tissue through a 70-µm mesh, followed by erythrocyte lysis with ACK buffer containing 150 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM Na₂EDTA. Gradient isolation of lung lymphocytes was performed after enzymatic tissue digestion. In brief, lung tissue was rinsed with warm HBSS (PAN-Biotech) using a 20-gauge needle penetrating several areas to bleed out the organ sample. Pre-digestion was performed by introducing collagenase VIII (Sigma-Aldrich) and collagenase D (Sigma-Aldrich) in warm HBSS (PAN-Biotech) supplemented with 2% FCS (PAN-Biotech) and 0.6% BSA (AppliChem) using again a 20-gauge needle. The organ was cut into little pieces, transferred to Liberase TM/DH (1:2, Sigma-Aldrich) and DNase I (0.15 mg/ml, Sigma-Aldrich) containing HBSS, and digested at 37°C for 30 min. Ice-cold HBSS was used to stop digestion, and the suspension was filtered using a 70-µm cell strainer to remove cell clumps and undissociated tissue. After washing, the cell pellet was resuspended in 40% Percoll solution (GE healthcare™) and layered onto 70% Percoll. After gradient isolation at 400 × g for 20 min lymphocytes were obtained at the interface. Porcine CD14⁺ monocytes were purified from PBMC or splenocytes using magnetic bead separation (CD14 MicroBeads, Miltenyi Biotec).

For detection of SEB or C. albicans-reactive Th cells from healthy pigs, isolated PBMCs were rested overnight in complete IMDM (PAN-Biotech) supplemented with 10% FCS (PAN-Biotech), 100 U/ml Penicillin, and 100 µg/ml Streptomycin (PAN-Biotech) and stimulated on the following day with superantigen (SEB, 1 µg/ml, Sigma-Aldrich) or PMA (20 ng/ml, Sigma-Aldrich) plus ionomycin, (1 µg/ml, Sigma-Aldrich) or C. albicans lysate (40 µg/ml, GREER^®) for 6 h and in presence of BrefeldinA (3 µg/ml, eBioscience) during the last 4 h of restimulation. C. albicans lysate was produced by dissolving statically grown cellular antigen, defatted, powdered, and dried by GREER^® in PBS. For detection of A. suum-reactive Th cells, splenocytes and lung parenchymal cells were rested overnight in complete IMDM (PAN-Biotech) supplemented with 10% FCS, 100 U/ml Penicillin, and 100 µg/ml Streptomycin and stimulated on the following day with 20 µg/ml of Ascaris L3 worm lysate (Asc Lys) or L3 excretory–secretory products (Asc ES) for 6 h and in presence of BrefeldinA (3 µg/ml, eBioscience) during the last 4 h of restimulation.

Cells were stained for flow cytometry analyses (BD FACS Canto II, BD FACS Diva software, FlowJo v9 software by Tree Star) with the following antibodies specific for pig species: anti-TCR1δ-unlab (clone PGBL22A, IgG1, Kingfisher Biotech), anti-CD4a-Pe-Cy7 or -AlexaFluor^® 647 or -PE (clone 4-12-4, IgG2b, BD Biosciences), anti-CD3ε-PerCP-Cy5.5 or -PE-Cy7 (clone BB23-8E6-8C8, IgG2a, BD Biosciences), anti-CD8α-FITC or -AlexaFluor^® 647 or -PE (clone 76-2-11, IgG2a, BD Biosciences), and IFN-γ-PE or -PerCP-Cy5.5 (clone P2G10, IgG1, BD Biosciences).

The following cross-reactive primary or secondary antibodies were used: anti-human CD14-Viogreen (clone Tük4, IgG2a, Miltenyi Biotec), anti-human CD154-Vioblue or, -PE (clone 5C8, IgG2a, Miltenyi Biotec), anti-human TNF-α-Pacific Blue or -Brillant Violet 605 (clone Mab11, IgG1, BioLegend) anti-human IL-17A-AlexaFluor^® (clone CZ8-23G1, IgG1, Miltenyi Biotec), and anti-mouse IgG1-FITC (clone M1-14D12, IgG, eBioscience). Fixable viability dyes were used in eFluor^® 780 or 506 and CFSE as proliferation dye (all from eBioscience). Intracellular antigens were stained after fixation and permeabilization of cells (Transcription Factor Staining Buffer Set, eBioscience or Cytofix/Cytoperm™, BD).

CFSE-labeled porcine PBMC from healthy animals were expanded in complete IMDM supplemented with 10% FCS (PAN-Biotech), 100 U/ml Penicillin, and 100 µg/ml Streptomycin (PAN-Biotech) in the presence of either C. albicans lysate (20 µg/ml, GREER) or SEB (1 µg/ml, Sigma-Aldrich) for 7 days followed by two resting days. In parallel, autologous CD14⁺ monocytes were seeded in complete IMDM at an initial density of 2.5 × 10⁵ cells/well in 96-well plates and differentiated into monocyte-derived dendritic cells (MoDC) in the presence of recombinant pig GM-CSF (20 ng/ml, R&D) and interleukin (IL)-4 (10 ng/ml, R&D) for 7 days. MoDC were primed with C. albicans antigen (if not indicated elsewhere: 40 µg/ml) or left unprimed for 1 day and matured with LPS (100 ng/ml), recombinant pig TNF-α (25 ng/ml) and pig recombinant IL-1β (10 ng/ml) for another day. For C. albicans-specific restimulation, expanded pig PBMC were harvested, washed, and 400,000 cells/200 μl/well were added to primed and matured MoDC for 6 h. Brefeldin A (3 µg/ml, eBioscience) was added for the last 4 h of autologous restimulation. Notably, for analyzing superantigen-reactive T cells, SEB-expanded cells were restimulated with unprimed, matured MoDC in the presence of SEB antigen (1 µg/ml).

For ex vivo detection of Ide_Ssuis-reactive Th cells, PBMCs were separated from heparinized blood by density gradient centrifugation on Biocoll (Merck-Biochrome). Samples were cryo-conserved in FCS, 10% DMSO at −80°C, or liquid nitrogen. Thawed PBMCs were cultivated in RPMI (Merck-Biochrome), supplemented with 10% FCS, 100 U/ml Penicillin, and 100 µg/ml Streptomycin. Following a resting time (2–4 h), PBMCs were stimulated for 18 h with 5 µg/ml rIde_Ssuis or with control-antigen (ctr-ag) Sfb I (fibronectin-binding protein of Streptococcus pyogenes), and in presence of Brefeldin A (2 µg/ml, Enzo Life Sciences) for the last 4 h. Recombinant Ide_Ssuis was expressed in E. coli and purified as described previously (42). As control antigen, the His-tagged C-terminal part of the S. pyogenes SfbI protein expressed in E. coli M15 pSTH 12 was used (44). LPS contaminations of rIde_Ssuis and rSfbI were analyzed by LAL test using Endosafe^®-PTS™ and cartridges (Charles River Laboratories International, Inc.). Final LPS concentration applied in vitro ranged between 0.118 and 0.236 ng/ml for rSfbI and 0.95 and 1.9 ng/ml for rIde_Ssuis. 3–5 × 10e5 CD3⁺CD4⁺ cells/sample were measured with BD LSRFortessa, and data analysis was performed using FlowJo V10.1 (Tree Star Inc., OR, USA) and GraphPad PRISM^®V5.01 software (La Jolla, CA, USA).

Serum IgG antibodies specific for rIde_Ssuis were determined in an ELISA as described previously (42).

The detection of CD154 as an early activation marker for visualizing antigen-reactive T cells by flow cytometry has been established in human and mouse studies and refined during the last years to properly identify Th cell responses to pathogens, environmental-, tumor-, neo-, or auto-antigens ranging from complex antigenic mixtures to proteins, peptide pools, or even single peptides (24, 45). To establish CD154 marker detection for antigen-reactive T cells in swine, we analyzed the Th cell response toward a complex antigenic mixture of C. albicans (Cand) and SEB, a bacterial superantigen known to cross-link MHC class II α-chain and TCRβ-chain. In addition, phorbol myristate acetate and ionomycin (P/I) was used as polyclonal stimulus downstream TCR triggering. Figure 1A shows CD154 expression ex vivo in CD4⁺ T cells from freshly isolated porcine PBMC of healthy piglets detected after 6 h of stimulation using a cross-reactive human mAb (clone 5C8). The addition of BrefeldinA during the last 4 h of stimulation prevents its transport to the cell surface and allows for intracellular staining of CD154 after fixation of cells. As shown in Figure 1A and summarized in Figure 1B, porcine CD4⁺ T cells display a defined population of CD154-expressing T cells following antigen-dependent (Cand, SEB) and TCR-independent (P/I) stimulation when compared to the unstimulated control (w/o). The frequency of C. albicans-specific T cells, identified as CD154⁺ cells upon antigenic stimulation, varies widely ranging from 0.07 to 0.32% (Figure 1B) of total CD4⁺ T cells from individual pigs. Similarly, the frequency of SEB-responding cells varies between individual pigs (range 0.21–1.07% of total CD4⁺, Figure 1B).

To assess whether CD154 expression assigns C. albicans-reactive T cells to the naïve or activated/memory T cell compartment, we analyzed CD8α coexpression on CD154⁺ and CD154⁻ cells after stimulation with C. albicans (Figure 1C). Our data indicates that Candida-specific CD154⁺ cells are enriched in the double-positive CD4⁺CD8α⁺ compartment, a subpopulation that in the pig is known to include activated and memory T helper cells (8).

A reliable marker for antigen-reactive T cells should be associated with a functional cytokine response. We therefore costained for TNF-α and IFN-γ production, known to be produced by human antigen-specific T cells in response to C. albicans (46, 47). Figure 1D clearly illustrates CD154 and cytokine coexpression upon SEB stimulation with most of the cytokine-producing cells expressing CD154, independent of the type of cytokine.

Furthermore, gamma delta T cells, identified as CD3⁺TCR1δ⁺ (48), do not similarly respond with CD154 upregulation to TCR-dependent (SEB or C. albicans lysate) or TCR-independent (P/I) stimulation when compared to CD3⁺CD4⁺ T helper cells (Figure 1E). We therefore conclude that in swine, TCR triggering induces expression of CD154 in CD4⁺ T helper cells detectable after 6 h of stimulation. Moreover, most cytokine-expressing cells coexpressed CD154 following SEB stimulation indicating that functional T cells are reliably identified by CD154 expression. Thus, we could establish CD154 as a good and promising marker for cytokine-producing antigen-reactive T cells in the pig.

The low frequency of antigen-specific T cells makes it difficult to study CD154 expression kinetics and its dependence on antigen dose in a detailed fashion. Therefore, it is necessary to expand the population of antigen-reactive T cells. Figure 2A illustrates a strategy that combines in vitro expansion of antigen-reactive PBMC with an autologous restimulation of expanded T cells using antigen-primed MoDC. PBMCs were CFSE labeled and expanded in the presence of 40 µg/ml of C. albicans lysate. In parallel, purified CD14⁺ blood monocytes were differentiated into dendritic cells (MoDC) using GM-CSF and IL-4 for 7 days. Rested T cells were finally restimulated with autologous, antigen primed, and matured MoDC (Figure 2A, lower panel). C. albicans-reactive, expanded T cells showed robust expression of CD154 when restimulated with antigen-primed MoDC (Figure 2B, lower left panel). Subgroup analysis further revealed that expression of CD154 was restricted to proliferated CD4⁺ cells (including double-positive CD4⁺CD8α⁺), with non-proliferative CD4⁺ T cells or proliferated CD4⁻CD8α⁺ T cells being negative for CD154 expression (Figure 2B). Figure 2C proofs CD154 expression of C. albicans-reactive, expanded T cells to be dependent on MoDC restimulation. In addition, coculturing C. albicans expanded T cells with unprimed (w/o) MoDC exhibited a rather low background expression suggesting that the observed CD154 expression of CD4⁺ T cells clearly depends on the presence of the specific antigen (Figure 2D). Combining expansion with antigen-primed MoDC restimulation and CD154 detection thereby precisely identified and enhanced the analysis of the antigen-reactive T cell population specific for a commensal pathogen, such as C. albicans.

For optimizing timing and dosing in the described strategy of expansion and autologous restimulation (Figures 2A,B), we first investigated whether the antigen dose had an impact on CD154 expression. Superantigen (SEB) expanded T cells where therefore restimulated for 5 h with autologous MoDC in the absence or presence of SEB (0.001–1 µg/ml, Figure 3A) and analyzed intracellular CD154 expression of proliferative CFSE^lowCD4⁺ T cells. We found an increase of CD154⁺ Th cells with increasing SEB concentrations, strongly indicating superantigen dose dependency for CD154⁺ expression. By contrast, expanded PBMC exposed to SEB (1 µg/ml) but in the absence of APCs (no MoDC, Figure 3B) did not respond with CD154 expression, consistent with the idea that CD154 expression requires TCR–MHC interaction. Similarly, a dose-dependent effect of CD154 expression was observed when restimulating C. albicans expanded PBMC with C. albicans-primed MoDC (2.5–40 µg/ml, Figure 3C). We next assessed the time course of CD154 expression in porcine T cells. Restimulating superantigen-expanded PBMC with MoDC in the presence of 1 µg/ml SEB, identified CD154⁺ antigen-responsive cells starting as early as 4 h and with increasing frequencies for all analyzed time points (0–8 h, Figures 3D,E). By contrast, before (0 h) or 2 h after restimulation, CD154 expression was negligible in proliferative CD4⁺ cells (Figure 3D and summarized in Figure 3E). A similar kinetic, but lower in ratios (Cand, 4–8 h: 7.72–21.8% mean CD154⁺ vs. SEB 4–8 h: 25.27–47.33% mean CD154⁺) was observed when analyzing C. albicans expanded PBMC restimulated with C. albicans-primed MoDC (Figures 3F,G). Notably, CD154 expression did not reach a plateau within 8 h of restimulation in both, superantigen or C. albicans-reactive CD4⁺ T cells. Based on these data, we defined an optimal priming dose of 40 µg/ml lysate and 6 h of restimulation time being acceptable for detecting C. albicans-specific T cells following MoDC restimulation. In summary, CD154 expression in swine CD4⁺ T cells was induced after short-term contact to antigen-loaded DC (4–8 h) and was clearly dependent on the antigen dose.

T cell receptor stimulation-activated T cells start to produce a specific set of cytokines upon recognition of their cognate antigen, if they are not anergic. Quantity and quality of cytokine production greatly determines the immune response toward a specific pathogen. However, in the absence of ongoing infection, pathogen-specific cytokine producing cells from either memory or naive compartment are rare and their detection is hampered by a very low signal-to-noise ratio (22). To investigate whether the use of expansion, restimulation, and CD154 expression enhances resolution for detecting antigen-reactive cytokine producing T helper cells in healthy pigs above background signals, we first evaluated the ex vivo cytokine response of C. albicans-stimulated and non-stimulated (w/o) T cells referring to the population of either total CD4⁺ cells (Figure 4A) or depicting CD154/cytokine coexpressing CD4⁺ T cells (Figure 4B). As expected, sample heterogeneity, staining background, and limitations in processing large cell numbers restricted the analysis of rare, antigen-reactive functional subgroups and did not allow to identify precise signals above non-stimulated background (Figures 4A,B). However, porcine CD4⁺ T cells expanded in the presence of C. albicans lysate and cocultured with either unprimed (w/o) or antigen-primed (Cand) MoDC markedly improved resolution of antigen-responsive CD154⁺/cytokine coproducing CD4⁺ T cells (Figures 4C,D). Thus, functional subgroup analysis of antigen-reactive cells in the absence of an active infection was facilitated by increasing their numbers in vitro by expansion followed by autologous restimulation and analysis of cytokine/CD154 coexpression.

Understanding how pathogen-specific T cells orchestrate the host immune response in the early phase of an acute infection is essential for diagnostics and therapy. We therefore focused on the intestinal parasitic roundworm infection with A. suum, known to have a major economic impact on pig industry (49, 50). Before the parasite colonizes the intestine and completes its life cycle, it migrates along the hepato-tracheal route causing tissue damage in liver and lung. In light of its significant importance on growth performance, data on the parasite-specific adaptive immune-response in swine are sparse and urgently needed.

We therefore asked whether CD154 expression analysis can be applied to readily identify Ascaris-specific T cells in affected and systemic organs of acutely infected vs. naive pigs. We analyzed ex vivo CD154⁺ Th cells of pigs experimentally infected with A. suum and necropsied at lung stage of infection (7 dpi). Lung lymphocytes were isolated and stimulated with a lysate of A. suum lung-stage L3 larvae (Asc Lys) for 6 h. We found an increase, yet not significant, in the frequency of antigen-induced CD154⁺ lung T cells in infected pigs that was absent in naive control animals (Figure 5A). While more A. suum-specific CD4⁺ T cells were detected in lungs of infected compared to naive animals (Figures 5A,B, upper panel, median 0.0665 vs. 0.185, p = 0.1, Mann–Whitney test), we observed only a marginal difference in lymphocytes isolated from the spleens of respective animals (Figure 5B, lower panel, median 0.0513 vs. 0.124, p = 0.2, Mann–Whitney test). Of note, IFN-γ-coproducing CD154⁺CD4⁺ T cells were only found in Asc Lys stimulated lung parenchymal cells of infected pigs (Figure 5A, indicated by italic numbers).

In addition to antigen-reactive T cells responding to whole worm lysate as antigen source, we aimed to determine frequencies of T cells reactive to excretory–secretory (ES) worm products, a complex mixture of released compounds with various functions, some of them known for their immunogenicity (51, 52). We therefore evaluated the frequency of A. suum-specific T cells in a slightly different infection scenario that more closely resembles the situation in contaminated finishing barns. Rattlerow Seghers hybrid piglets were trickle infected over 7 weeks (80 eggs/day), with incoming larvae continuously migrating to the lung for the entire infection period. To investigate against which antigenic compartment the Ascaris-specific response is directed, lung lymphocytes were analyzed for CD154 ex vivo after short-term stimulation with either A. suum lysate (Asc Lys) or purified and concentrated Ascaris-derived ES (Asc ES) products (Figure 5C). Although whole worm lysate induced a significant population of CD154⁺-expressing lung CD4⁺ T cells (w/o vs. Asc Lys median 0.0564 vs. 0.253, p = 0.0079, Mann–Whitney test), the frequency of A. suum-reactive cells stimulated with Asc ES was significantly higher (Figures 5C,D, Asc Lys vs. Asc ES median 0.253 vs. 0.694, p = 0.0159, Mann–Whitney test). Again, we found no substantial increase in CD154⁺-expressing splenic T cells stimulated with either Asc Lys or Asc ES (Figure 5E).

Collectively, these data demonstrate that even without prior expansion CD154-expression identified a population of A. suum-specific T cells in acute parasite infection (7 dpi) or following trickle infection and that worm-specific T cells preferentially accumulated in affected lung tissues.

To validate whether CD154 expression on swine Th cells can be used to monitor CD4⁺ T cells reactive for a single recombinant protein in a vaccination approach, we analyzed CD154 responses in immunized pigs. Here, we focused on an experimental immunization to S. suis regarded as one of the most important pig pathogens (35). All investigated strains of S. suis degrade porcine IgM (53). The responsible protease, Ide_Ssuis, is a highly protective antigen against S. suis serotype 2 as shown in a vaccine trial with the recombinant protein (42).

Figure 6A illustrates the experimental design for Ide_Ssuis vaccination in German Landrace pigs. The animals were immunized with either recombinant Ide_Ssuis or placebo (plac, adjuvant only) intramuscularly with a booster immunization 2 weeks later (Figure 6A). Two weeks after the boost, PBMCs and serum samples were obtained from both groups. Following stimulation of PBMCs (for 18 h) with medium alone, rIde_Ssuis or with an unrelated recombinant protein obtained from the same expression system (ctr-ag rSfbI) pre-gated activated T helper cells (CD4⁺CD8α⁺) were further assessed for intracellular CD154 and cytokine coexpression. Ide_Ssuis immunized animals showed a significant increase of IFN-γ and TNF-α secreting CD154⁺ T cells compared to placebo animals (Figures 6B,C) when stimulated with rIde_Ssuis, indicating an antigen-reactive T helper cell response toward the vaccination target. Stimulation of PBMCs with ctr-ag also induced T helper cell reactivity in some of the animals but was not restricted to the immunized group. To control for polyclonal activation by residual LPS contamination during in vitro activation, the T cell response to 1 µg/ml LPS was analyzed in parallel and showed only background levels compared to Ide_Ssuis protein (data not shown). A successful vaccination requires activation of T helper cells capable of costimulating B cells for generating antibodies that protect against the disease of interest. We analyzed Ide_Ssuis-specific IgG antibody titers in immunized animals 2-week post-vaccination to correlate frequency of CD154⁺/IFN-γ producing T helper cells with the Ide_Ssuis-specific IgGs, since especially IFN-γ is important to support the IgG class-switch of B cells (54). Indeed, we found a significant correlation of Ide_Ssuis-specific IgG and frequencies of Ide_Ssuis-specific, IFN-γ producing CD154⁺ Th cells in immunized animals (Figure 6D). By contrast, no correlation was observed when plotting Ide_Ssuis IgG levels against ctr-ag-specific CD154⁺ Th cells (Sfbl, Figure 6E). These data suggest that the analysis of CD154⁺ expression can be used to identify immunization-responsive cells and can therefore be applied to validate the induction of a T helper cell response toward a given vaccination target in swine.