Probiotic Bacillus Strains Enhance T Cell Responses in Chicken

Banning antibiotic growth promotors and other antimicrobials in poultry production due to the increasing antimicrobial resistance leads to increased feeding of potential alternatives such as probiotics. However, the modes of action of those feed additives are not entirely understood. They could act even with a direct effect on the immune system. A previously established animal-related in vitro system using primary cultured peripheral blood mononuclear cells (PBMCs) was applied to investigate the effects of immune-modulating feed additives. Here, the immunomodulation of different preparations of two probiotic Bacillus strains, B. subtilis DSM 32315 (BS), and B. amyloliquefaciens CECT 5940 (BA) was evaluated. The count of T-helper cells and activated T-helper cells increased after treatment in a ratio of 1:3 (PBMCs: Bacillus) with vital BS (CD4+: p < 0.05; CD4+CD25+: p < 0.01). Furthermore, vital BS enhanced the proliferation and activation of cytotoxic T cells (CD8+: p < 0.05; CD8+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BS increased the count of activated T-helper cells (CD4+CD25+: p < 0.1). UV-inactivated BS increased the proportion of cytotoxic T cells significantly (CD8+: p < 0.01). Our results point towards a possible involvement of secreted factors of BS in T-helper cell activation and proliferation, whereas it stimulates cytotoxic T cells presumably through surface contact. We could not observe any effect on B cells after treatment with different preparations of BS. After treatment with vital BA in a ratio of 1:3 (PBMCs:Bacillus), the count of T-helper cells and activated T-helper cells increased (CD4+: p < 0.01; CD4+CD25+: p < 0.05). Cell-free probiotic culture supernatants of BA as well as UV-inactivated BA had no effect on T cell proliferation and activation. Furthermore, we found no effect of BA preparations on B cells. Overall, we demonstrate that the two different Bacillus strains enhanced T cell activation and proliferation, which points towards an immune-modulating effect of both strains on chicken immune cells in vitro. Therefore, we suggest that administering these probiotics can improve the cellular adaptive immune defense in chickens, thereby enabling the prevention and reduction of antimicrobials in chicken farming.


Introduction
Poultry is the cheapest source of animal protein worldwide and the most common source of meat. In 2030, poultry meat is expected to represent about 41% of all the protein from meat sources [1]. Furthermore, increased poultry production in developing countries [2] is accompanied by an elevated overall usage of antimicrobials. However, there is growing concern about the resistance of pathogenic bacteria against antibiotics and other antimicrobials, the residual effects of antibiotics in meat products [3], and the public health risk from zoonotic pathogens such as Salmonella and Campylobacter. Consequently, the subtherapeutic usage of antibiotic growth promotors and other antimicrobials has been forbidden in farming in the EU since 2006 (Regulation (EC) No 1831/2003). In the last 55 four-to-six-week-old broiler chickens of the commercial layer Cobb500 (Cobb Germany Avimex GmbH, Wiedemar, Germany) were used. The birds were kept and fed as described previously [34]. In brief, all chickens were fed a starter diet from day 1 to day 14 post hatch and a grower diet afterwards (H. Wilhelm Schaumann GmbH, Pinneberg, Germany). The ration was fed on an ad libitum basis and water was always available. The chickens were kept in groups of approximately 20 broiler chickens in 4 m 2 floor pens. For experiments, 3 to 6 birds per week were stunned and blood was sampled in sodium citrate (Na-citrate) pre-filled polystyrene tubes (VACUETTE ® , Greiner Bio-One, Kremsmünster, Austria) by neck cutting.

Bacterial Strains and Culture
RPMI 1640 medium was inoculated with either B. amyloliquefaciens CECT 5940 (BA) or B. subtilis DSM 32315 (BS) and bacteria were cultured overnight at 37 • C and 120 rpm. To define the actual colony forming units per milliliter (cfu/mL), the bacterial growth was measured on a Tecan Infinite ® M200 Pro plate reader with Magellan™ v. 7.1 software (Tecan Group AG, Männedorf, Switzerland) and bacterial cultures were plated on tryptic-soy agar (TSA, Carl Roth) on Petri dishes (Greiner Bio-One, Frickenhausen, Germany) and counted the next day.

Probiotic Treatment/Co-culture
Co-culture experiments with probiotic bacterial strains were performed with 1×10 6 PBMCs with different preparations of either BS or BA in a total volume of 1 mL in 24-well plates (Eppendorf, Hamburg, Germany) for 24 h. All experiments were performed in RPMI 1640 medium containing 10% chicken serum without Pen/Strep at 41 • C with 5% CO 2 . For all co-culture experiments, concanavalin A (conA, Vector Laboratories, Newark, California, USA) was used as a positive control. PBMCs were treated at a concentration of 10 µg/mL conA ( Figure S1).

Treatment with Vital Probiotic Bacteria
To investigate the effect of vital probiotic bacteria, 1×10 6 PBMCs were co-cultured with either BS or BA in a ratio of 1:3 (PBMCs:Bacillus).

Treatment with Supernatants of Probiotic Culture
For treatment with cell-free supernatants of the probiotic bacterial cultures, 40 mL RPMI 1640 medium was inoculated with either BS or BA, and bacteria were cultured overnight at 37 • C and 100 rpm. The next day, the bacterial cultures were sterile-filtered using a vacuum filter system with a polyethersulfone membrane possessing a pore size of 0.22 µm (Corning, Corning, NY, USA). The supernatants of the cultures were then Microorganisms 2023, 11, 269 4 of 14 split into two new 50 mL tubes. For thermal inactivation of the secreted factors of the bacterial strains, one 50 mL tube was heat-treated (10 min at 80 • C), and the other 50 mL tube was left untreated. Heat-treated and untreated pure RPMI 1640 medium served as a negative control. 1×10 6 PBMCs were exposed to either 500 µL heat-treated supernatants, untreated supernatants, heat-treated RPMI 1640 medium, or untreated RPMI 1640 medium. Treatment occurred in a total volume of 1 mL for 24 h. All experiments with cell-free bacterial culture supernatants were performed in RPMI 1640 medium containing 10% chicken serum and without Pen/Strep at 41 • C with 5% CO 2 . To validate that cell-free supernatants were used, bacterial culture supernatants were plated on TSA agar plates before and after sterile filtering.

Treatment with UV-Inactivated Probiotic Bacteria
To investigate whether secreted factors or cell surface proteins could influence immune cell phenotypes, the bacteria were inactivated by UV light. Therefore, 20 mL RPMI 1640 medium was inoculated with either BS or BA and cultured overnight at 37 • C. On the next day, bacterial cultures were transferred into Petri dishes and irradiated with UV light (200 nm) for 5 h. Thereafter, the bacteria were harvested and stored at 4 • C overnight for coculture experiments. The next day, 1 × 10 6 PBMCs were treated with either UV-inactivated BS or BA in a ratio of 2:1 (PBMCs:Bacillus) in a total volume of 1 mL for 24 h. The number of bacteria used for treatment was determined before the UV inactivation. To validate the UV inactivation, bacterial cultures were plated on TSA agar plates before and after UV light exposure.

Statistical Analysis
The data obtained by flow cytometry was analyzed using a gating strategy ( Figure S3). The cell count of antibody-positive cells was calculated relative to the vital PBMCs measured on a BD FACSCanto™ II. Due to high variation in the immune response capacity, the results are displayed as the ∆ relative cell count, representing the difference of the relative cell count between the treatment and the respective untreated negative control for every biological replicate. Statistical analysis was performed by a one sample Student's t-test. All tests were performed using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences between groups were considered statistically significant at p < 0.05 (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). A statistical tendency is shown as +, p < 0.1.

Effects of Vital BS and BA on T and B Cells in a PBMC Composite
The effect of vital BS and BA on chicken adaptive immune cells was evaluated by measures of the proportions of the adaptive immune cell populations. In particular, the . https://doi.org/10.3390/xxxxx www.mdpi.com/journal/microorganism of CD4+ T-helper cells by 4.10% (p < 0.05, Figure 1a) and CD8+ cytotoxic T cells by 1.79% (p < 0.05, Figure 1b). Furthermore, the ∆ relative cell count of all CD3+ T cells increased by 7.83% ( Figure 1c). However, this result was not significant. Additionally, treatment with vital BS had no effect on Bu-1+ B cells (Figure 1d). After treatment with vital BA, the ∆ relative cell count of CD4+ T-helper cells in creased by 2.38% (p < 0.1, Figure 1a), while the count of CD8+ cytotoxic T cells (Figure 1b remained unaffected. The ∆ relative cell count of all CD3+ T cells increased by 2.64% (Fig  ure 1c), however, the result was not significant. The ∆ relative cell count of Bu-1+ B cells did not change after treatment with vital BA (Figure 1d).  The treatment of PBMCs with vital BS revealed an increase of the ∆ relative cell count of CD4+ T-helper cells by 4.10% (p < 0.05, Figure 1a) and CD8+ cytotoxic T cells by 1.79% (p < 0.05, Figure 1b). Furthermore, the ∆ relative cell count of all CD3+ T cells increased by  7.83% ( Figure 1c). However, this result was not significant. Additionally, treatment with vital BS had no effect on Bu-1+ B cells (Figure 1d).
The addition of vital BA resulted in an elevated number of the CD4+CD25+ activated T-helper cell proportion by 0.37% (p < 0.05, Figure 2a). The ∆ relative cell count of CD8+CD25+ activated cytotoxic T cells (Figure 2b) remained unaffected after treatment with vital BA, while all CD28+ αβ T cells increased by 3.81% (p < 0.05, Figure 2c).  After treatment with vital BA, the ∆ relative cell count of CD4+ T-helper cells increased by 2.38% (p < 0.1, Figure 1a), while the count of CD8+ cytotoxic T cells (Figure 1b) remained unaffected. The ∆ relative cell count of all CD3+ T cells increased by 2.64% (Figure 1c), however, the result was not significant. The ∆ relative cell count of Bu-1+ B cells did not change after treatment with vital BA (Figure 1d).
Corresponding to the increase of the ∆ relative cell count of CD4+ T-helper cells and CD8+ cytotoxic T cells, vital BS enhanced the count of CD4+CD25+ activated T-helper cells by 0.67% (p < 0.01, Figure 2a

Effects of Cell-Free Culture Supernatants of BS and BA on T and B cells in a PBMC Composite
After treatment with vital probiotic bacteria, we evaluated which component of the probiotics BS and BA is involved in the stimulation of the immune system, especially T cells. Therefore, in a first step, we analyzed the effects of cell-free bacterial culture supernatants of BS and BA on PBMCs by measuring the proliferation (Figure 3a

Effects of Cell-free Culture Supernatants of BS and BA on T and B cells in a PBMC Composite
After treatment with vital probiotic bacteria, we evaluated which component of the probiotics BS and BA is involved in the stimulation of the immune system, especially T cells. Therefore, in a first step, we analyzed the effects of cell-free bacterial culture supernatants of BS and BA on PBMCs by measuring the proliferation (Figure 3a-d) and the activation status (Figure 4a-b) of the adaptive immune cell populations, T and B cells.
PBMC treatment with cell-free culture supernatants of the probiotic BS did not affect the CD4+ T-helper cells (Figure 3a) and CD8+ cytotoxic T cells (Figure 3b). Furthermore, the ∆ relative cell count of all CD3+ T cells ( Figure 3c) and Bu-1+ B cells (Figure 3d) remained unaffected after treatment. Interestingly, we found a decreased number of CD4+ T-helper cells after treatment with the negative control, heat-treated cell-free culture supernatants of BS ( Figure S2a).
After treatment of PBMCs with cell-free culture supernatants of BA, we found no effect on the Δ relative cell count of CD4+ T-helper cells (Figure 3a), CD8+ cytotoxic T cells (Figure 3b), all CD3+ T cells (Figure 3c), and Bu-1+ B cells (Figure 3d). In addition, we found a decreased count of Bu-1+ B cells after exposure to heat-treated cell-free culture supernatants of BA ( Figure S2f). For CD4+CD25+ activated T-helper cells, the ∆ relative cell count was tendentially increased by 0.39% (p < 0.1, Figure 4a) after treatment with cell-free culture supernatants of BS, whereas CD8+CD25+ activated cytotoxic T cells (Figure 4b) were not affected. PBMC treatment with cell-free culture supernatants of the probiotic BS did not affect the CD4+ T-helper cells (Figure 3a) and CD8+ cytotoxic T cells (Figure 3b). Furthermore, the ∆ relative cell count of all CD3+ T cells (Figure 3c) and Bu-1+ B cells (Figure 3d) remained unaffected after treatment. Interestingly, we found a decreased number of CD4+ T-helper cells after treatment with the negative control, heat-treated cell-free culture supernatants of BS ( Figure S2a).
After treatment of PBMCs with cell-free culture supernatants of BA, we found no effect on the ∆ relative cell count of CD4+ T-helper cells (Figure 3a), CD8+ cytotoxic T cells (Figure 3b), all CD3+ T cells (Figure 3c), and Bu-1+ B cells (Figure 3d). In addition, we found a decreased count of Bu-1+ B cells after exposure to heat-treated cell-free culture supernatants of BA ( Figure S2f). After treatment of PBMCs with cell-free culture supernatants of BA, we found no effect on the Δ relative cell count of CD4+CD25+ activated T-helper cells (Figure 3a) and CD8+CD25+ activated cytotoxic T cells (Figure 3c-d).
(a) (b) Summarized, secreted factors, expected in the cell-free culture supernatants of BS, stimulated T-helper cell activation (CD4+CD25+). Furthermore, heat-treated cell-free culture supernatants, which are supposed to contain denaturated proteins, decreased the CD4+ T-helper cell count. Cell-free culture supernatants of BA had no effect on T and B cells. However, heat-treated cell-free culture supernatants decreased the count of Bu-1+ B cells.

Effects of UV-inactivated BS and BA on T and B Cells
As we could partially confirm the involvement of secreted factors in the stimulation of T-helper cells by BS and could further exclude the involvement of secreted factors in the stimulation of cytotoxic T cells by BS and of T-helper cells by BA, we investigated the effect of the bacterial cell surface. We hypothesized that direct contact of the bacterial cell surface and the adaptive immune cells constitutes the major component of adaptive immune cell activation. To this end, the bacteria were inactivated by UV light. After UV light exposure, we assumed that the bacteria possessed an intact cell surface, as reported earlier [36], which could be involved in a possible immunomodulatory effect. The proliferation (Figure 5a-d) and the activation status (Figure 6a-b) of the adaptive immune cell populations, T and B cells, were investigated.
After the treatment of PBMCs with UV-inactivated BS, we did not observe a difference in the Δ relative cell count of CD4+ T-helper cells (Figure 5a). In contrast, treatment with UV-inactivated BS significantly increased the CD8+ cytotoxic T cell count by 2.96% (p < 0.01, Figure 5b). Furthermore, the CD3+ T cell population was enhanced after treatment with UV-inactivated BS by 2.61% (Figure 5c), although, not significantly. The Bu-1+ B cell count remained unaffected after treatment with UV-inactivated bacteria (Figure 5d).
Treatment with UV-inactivated BA did not result in a change of the Δ relative cell count of CD4+ T-helper cells (Figure 5a), CD8+ cytotoxic T cells (Figure 5b), the CD3+ T cell population (Figure 5c), and Bu-1+ B cells (Figure 5d). For CD4+CD25+ activated T-helper cells, the ∆ relative cell count was tendentially increased by 0.39% (p < 0.1, Figure 4a) after treatment with cell-free culture supernatants of BS, whereas CD8+CD25+ activated cytotoxic T cells (Figure 4b) were not affected.
After treatment of PBMCs with cell-free culture supernatants of BA, we found no effect on the ∆ relative cell count of CD4+CD25+ activated T-helper cells (Figure 3a) and CD8+CD25+ activated cytotoxic T cells (Figure 3c,d).
Summarized, secreted factors, expected in the cell-free culture supernatants of BS, stimulated T-helper cell activation (CD4+CD25+). Furthermore, heat-treated cell-free culture supernatants, which are supposed to contain denaturated proteins, decreased the CD4+ Thelper cell count. Cell-free culture supernatants of BA had no effect on T and B cells. However, heat-treated cell-free culture supernatants decreased the count of Bu-1+ B cells.

Effects of UV-Inactivated BS and BA on T and B Cells
As we could partially confirm the involvement of secreted factors in the stimulation of T-helper cells by BS and could further exclude the involvement of secreted factors in the stimulation of cytotoxic T cells by BS and of T-helper cells by BA, we investigated the effect of the bacterial cell surface. We hypothesized that direct contact of the bacterial cell surface and the adaptive immune cells constitutes the major component of adaptive immune cell activation. To this end, the bacteria were inactivated by UV light. After UV light exposure, we assumed that the bacteria possessed an intact cell surface, as reported earlier [36], which could be involved in a possible immunomodulatory effect. The proliferation (Figure 5a-d) and the activation status (Figure 6a,b) of the adaptive immune cell populations, T and B cells, were investigated.
After the treatment of PBMCs with UV-inactivated BS, we did not observe a difference in the ∆ relative cell count of CD4+ T-helper cells (Figure 5a). In contrast, treatment with UV-inactivated BS significantly increased the CD8+ cytotoxic T cell count by 2.96% (p < 0.01, Figure 5b). Furthermore, the CD3+ T cell population was enhanced after treatment with UV-inactivated BS by 2.61% (Figure 5c), although, not significantly. The Bu-1+ B cell count remained unaffected after treatment with UV-inactivated bacteria (Figure 5d). We did not observe an effect on CD4+CD25+ activated T-helper cells (Figure 6a) after treatment with UV-inactivated BS. In contrast, the ∆ relative cell count of the CD8+CD25+ activated cytotoxic T cell population seemed to be increased by 0.19% (Figure 6b) after treatment with UV-inactivated BS, however, the result was not significant.
Treatment with UV-inactivated BA did not result in a change of the Δ relative cell count of CD4+CD25+ activated T-helper cells (Figure 6a) and CD8+CD25+ activated cytotoxic T cells (Figure 6b).  We did not observe an effect on CD4+CD25+ activated T-helper cells (Figure 6a) after treatment with UV-inactivated BS. In contrast, the ∆ relative cell count of the CD8+CD25+ activated cytotoxic T cell population seemed to be increased by 0.19% (Figure 6b) after treatment with UV-inactivated BS, however, the result was not significant.
Treatment with UV-inactivated BA did not result in a change of the Δ relative cell count of CD4+CD25+ activated T-helper cells (Figure 6a) and CD8+CD25+ activated cytotoxic T cells (Figure 6b).  Treatment with UV-inactivated BA did not result in a change of the ∆ relative cell count of CD4+ T-helper cells (Figure 5a), CD8+ cytotoxic T cells (Figure 5b), the CD3+ T cell population (Figure 5c), and Bu-1+ B cells (Figure 5d).
We did not observe an effect on CD4+CD25+ activated T-helper cells (Figure 6a) after treatment with UV-inactivated BS. In contrast, the ∆ relative cell count of the CD8+CD25+ activated cytotoxic T cell population seemed to be increased by 0.19% (Figure 6b) after treatment with UV-inactivated BS, however, the result was not significant.
Treatment with UV-inactivated BA did not result in a change of the ∆ relative cell count of CD4+CD25+ activated T-helper cells (Figure 6a) and CD8+CD25+ activated cytotoxic T cells (Figure 6b).
In summary, we observed that UV-inactivated BS increased the count of CD8+ cytotoxic T cells and seemed to stimulate cytotoxic T cell activation (CD8+CD25+). In contrast, UV-inactivated BA had no effect on CD4+ T-helper cell, CD8+ cytotoxic T cell, and Bu-1+ B cell responses in PBMCs of broiler chicken.

Discussion
The use of antimicrobials, especially antibiotics, in broiler production triggers the development of antimicrobial-resistant pathogenic bacteria with implications for human and animal health. Innovative feeding strategies, including the use of probiotics, have the potential to support animal health and, thus, may help to reduce the use of antimicrobials in poultry production. Proposed modes of probiotic action include immunomodulatory effects of specific strains. Primary cell culture systems represent a good alternative to animal experiments, more closely mimicking the in vivo model compared to conventional cell lines, to investigate the mechanisms of potentially immune-modulating feed additives. In this study, we investigated the immunomodulatory potential of two probiotic Bacillus strains, BS and BA, which are both commercially available probiotics for chicken farming, using an in vitro cell culture system with chicken immune cells [34].
We detected stimulating effects of both probiotic Bacillus strains on the proliferation (percentage of CD4+ T-helper cells) (Figure 1a), as well as on the activation (percentage of CD4+CD25+ activated T-helper cells) (Figure 2a) of T-helper cells. Furthermore, the count of activated CD28+ αβ T cells (Figure 2c) increased after treatment with both bacterial strains, which is in line with the increase of the CD4+ and the CD4+CD25+ activated T-helper cell proportion (Figures 1a and 2a). CD28, a costimulatory molecule important for T cell activation [37], is reported to be a late activation marker (48 h) and only for CD8+ cytotoxic T cells [38]. In our study, the CD28 signal increased significantly 24 h after treatment with BS and BA. These results hint towards an in vitro stimulation of CD4+ and CD4+CD25+ activated T-helper cells through vital BS and BA bacteria. CD4+CD25+ T cells are further reported to possess properties similar to that of mammalian regulatory T cells (Tregs) [39,40]. Therefore, chicken Tregs are required to maintain immune homeostasis and self-antigen tolerance. Interestingly, the CD4+CD25+ T cell population showed no downregulation of the cytotoxic T cell activity in our study and therefore did not seem to possess suppressing properties, as it was reported earlier [41]. The proliferation (percentage of CD8+ T cells) and activation (percentage of CD8+CD25+ T cells) of cytotoxic T cells increased after treatment with vital BS (Figures 1b and 2b). In another study, the applicability of chicken homologs to well-known mammalian T cell activation markers was investigated [38]. Next to CD28, CD5, MHC-II, CD44, and CD45, the marker CD25 was used to measure T cell activation. The frequencies of CD25+ T cells were increased after conA stimulation for 24 h, which demonstrated that CD25 could also be used for chicken PBMCs as an activation marker [38]. Therefore, we used CD25 as a T cell activation marker. In addition to the effect of vital bacteria, the activation of T-helper cells tended to increase after treatment with cell-free culture supernatants of BS (CD4+CD25+, Figure 4b). This result points towards an involvement of secreted factors of the bacteria, which remained in the supernatants, in the proliferation (CD4+) and activation (CD4+CD25+) of T-helper cells. Interestingly, we found a decrease in the CD4+ T-helper cell count after treatment with heat-treated cell-free culture supernatants of BS ( Figure S2a). This result possibly indicates important compounds for CD4+ T-helper cell survival, which were inactivated by heat in the cell-free bacterial culture supernatants. In the experiments with vital BS, we found stimulating effects on the proliferation (CD4+) and activation (CD4+CD25+) of T-helper cells (Figures 1a and 2a) as well as on the proliferation (CD8+) and activation (CD8+CD25+) of cytotoxic T cells (Figures 1b and 2b). This finding is in line with observations in a previous in vivo study in chicken with probiotic Lactobacillus acidophilus LA5 [42]. Moreover, B. subtilis spores, as adjuvants, were shown to enhance CD4+ and CD8+ T cell responses in vivo against avian influenza H9N2 [43]. In a necrotic enteritis challenge, B. subtilis DSM 32315, the same bacterial strain used in our study, was demonstrated to reduce pathology and improve the performance of broilers [23]. We show that UV-inactivated BS bacteria led to an increase of CD8+ cytotoxic T cells (Figure 5b), whereas the median after treatment with cell-free supernatants of both bacterial strains rather tended to decrease (Figure 3b). This result indicates an involvement of direct contact of the cell surface of BS with the immune cells for cytotoxic T cell activation. UV irradiation can denature the DNA of microorganisms, causing death or inactivation [44], however, the cell surface of UV-inactivated bacteria was reported to remain intact [36]. Additionally, the result obtained for all CD3+ T cells after treatment with UV-inactivated BS (Figure 5c) points to the same direction regarding an effect on the T cell population. However, probably partially due to a smaller sample size in this experiment, those results were not significant. In mice, B. subtilis was shown to induce anti-inflammatory M2 macrophages inhibiting T cell-mediated immune responses [45]. From our results, we suggest that BS may improve the performance of the animals against infection. Thus, BS helps to prevent and treat disease through the modulation of T cell responses in chicken.
B cells, another important cell type of the adaptive immune response, play a major role in the antibody-mediated humoral immune response. Probiotic effects on B cells have mainly focused on antibody production, like immunoglobulin A (IgA) production in the GIT [33,46,47]. Moreover, probiotics were reported to affect B cells by altering or increasing the number of antigen-specific B cells, as it was shown after in vitro stimulation of human PBMCs with different lactic acid bacterial (LAB) strains [48] and in chicken after vaccination with inactivated avian influenza H9N2 together with a B. subtilis spore adjuvant [43]. In pigs, B cell stimulation in PBMCs was also reported after in vivo administration of probiotic Enterococcus faecium NCIMB 10415 in piglets [49]. For Bu-1+ B cells, we found no effect of both vital probiotic Bacillus strains, BS and BA (Figure 1d). Furthermore, we found no differences after treatment with cell-free supernatants ( Figure 3d) and UVinactivated bacteria (Figure 5d). However, after treatment with heat-treated cell-free culture supernatants, we found a significant decrease in the Bu-1+ B cell population ( Figure S2f), possibly indicating an apoptosis-inducing effect of the denatured proteins or the loss of factors necessary for B cell survival.
This study provides evidence of a direct immunomodulatory effect of BS and BA on chicken T cells. Treatment with BS increased the proliferation (CD8+) and activation (CD8+CD25+) of cytotoxic T cells, which may enhance the cellular immune response, in particular by destroying pathogens through perforins and granzymes or inducing apoptosis of infected host cells [50]. Furthermore, treatment with both probiotic bacteria, BS and BA, increased the proliferation (CD4+) and activation (CD4+CD25+) of T-helper cells. However, the CD4+CD25+ T cell population could further represent Tregs, regulating and suppressing excessive T-helper cell 1 (Th1) and T-helper cell 2 (Th2) immune responses and maintaining immunological unresponsiveness and tolerance to self-antigens [40,50,51].

Conclusions
Our results point towards a possible involvement of secreted factors of BS in T-helper cell activation and proliferation, whereas BS stimulated cytotoxic T cells presumably through surface contact. For BA, more experiments are necessary to unravel the mechanism behind the action on T cells. All in all, we observed a T cell stimulation by both tested probiotic Bacillus strains in vitro. Therefore, we suggest that these probiotic Bacillus strains could improve the health of animals and their defense against infection and, thus, help to prevent and reduce the usage of antimicrobials in chicken farming.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11020269/s1, Figure S1: Influence of conA on activation and proliferation of T cells and B cells; Figure S2: Influence of heat-treated cell-free culture supernatants of B. subtilis DSM 32315 (grey) and B. amyloliquefaciens CECT 5940 (white) activation and proliferation of T and B cells; Figure S3: Example of the gating strategy for chicken PBMCs.