The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-γ and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-γ secretion assessed using ELISA showed a significantly higher titer (**p < 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.
Veterinary vaccination represents a crucial aspect of animal husbandry and health, as it contributes to improving food security and animal welfare by conferring protection against diseases that affect the livestock sector globally1. An effective in vitro method to assess the immunogenicity of possible vaccine candidates would help accelerating the process of vaccine development and production. It is, therefore, necessary to expand the field of immune assays with innovative methodologies based on in vitro studies, as this would help to unveil the complexity of the immune processes related to immunization and pathogen infection. Currently, in vivo animal immunization and challenge studies, which require periodic sampling (e.g., blood and spleen), are used to measure the immunogenicity of candidate vaccines and adjuvants. These assays are expensive, time-consuming, and have ethical implications, because in most cases, animal euthanasia is carried out by the end of the trials.
As an alternative to in vivo assays, peripheral blood mononuclear cells (PBMCs) have been used to evaluate vaccine-induced immune responses in vitro2. PBMCs are a heterogeneous population of cells composed of 70%-90% lymphocytes, 10%-20% monocytes, and a limited number of dendritic cells (DCs, 1%-2%)3. PBMCs harbor antigen-presenting cells (APCs) such as B cells, monocytes, and DCs, which constantly patrol the organism searching for signs of infection or tissue damage. Locally secreted chemokines facilitate the recruitment and activation of APCs to these sites by binding to cell surface receptors. In the case of monocytes, chemokines direct their fate to either differentiate into DCs or macrophages4. As soon as DCs encounter and capture a pathogen, they migrate to secondary lymphoid organs, where they can present the processed pathogen peptide antigens using major histocompatibility complex (MHC) class I or class II surface proteins to CD8+ T cells or CD4+ T cells, respectively, thus triggering an immune response5,6.
The key role played by DCs in orchestrating a protective immune response against various pathogens makes them an interesting research target for understanding intracellular immune mechanisms, especially when designing vaccines and adjuvants against infectious agents7. Since the fraction of DCs that can be obtained from PBMCs is rather small (1%-2%), monocytes have instead been used to generate DCs in vitro8. These monocyte-derived DCs (MoDCs) were initially developed as a possible treatment strategy in cancer immunotherapy9. More recently, MoDCs have been used for vaccine research10,11,12, and classical monocytes are the predominant subtype (89%) for MoDC production13. The production of MoDCs in vitro has previously been achieved through the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) given in combination with other cytokines such as interleukin-4 (IL-4), tumor necrosis factor α (TNF-α), or IL-1314,15,16.
The success of an in vitro MoDC assay relies on the capability of antigen-stimulated mature MoDCs to modulate the extent and type of the immune response specific to the type of antigen detected17. The type of pathogen recognized and presented by MoDCs determines the differentiation of CD4+ T helper (Th) cells into either Th1, Th2, or Th17 effector cells and is characterized by a pathogen-specific secretory cytokine profile. A Th1 response is elicited against intracellular pathogens and results in the secretion of interferon-gamma (IFN-γ) and tumor necrosis factor beta (TNF-β), which modulates phagocytic-dependent protection. A Th2 response is triggered against parasitic organisms and is characterized by IL-4, IL-5, IL-10, and IL-13 secretion, which initiates phagocytic-independent humoral protection. Th17 offers neutrophil-dependent protection against extracellular bacterial and fungal infections mediated by the secretion of IL-17, IL-17F, IL-6, IL-22, and TNF-α18,19,20,21. Based on previous studies, it has been noted that not all pathogens fall within the expected cytokine profile. For example, dermal MoDCs, in response to Leishmania parasitic infection, stimulate IFN-γ secretion from CD4+ T cells and CD8+ T cells, thus inducing a protective proinflammatory Th1 response22.
It has also been shown that, in chicken MoDCs primed with Salmonella lipopolysaccharide (LPS), can induce a variable response against Salmonella typhimurium by activating both Th1 and Th2 responses, whereas Salmonella gallinarum induces a Th2 response alone, which could explain the higher resistance of the latter toward MoDC clearance23. The activation of MoDCs against Brucella canis (B. canis) has also been reported in both canine and human MoDCs, meaning this could represent a zoonotic infection mechanism24. Human MoDCs primed with B. canis induce a strong Th1 response that confers resistance to severe infection, whereas canine MoDCs induce a dominant Th17 response with a reduced Th1 response, subsequently leading to the establishment of chronic infection25. Bovine MoDCs show an enhanced affinity for foot-and-mouth disease virus (FMDV) conjugated with immunoglobulin G (IgG) as compared to non-conjugated FMDV alone, as the MoDCs form a viral-antibody complex in response to the former10. Taken together, these studies show how MoDCs have been used to analyze the complexity of immune responses during pathogen infection. The adaptive immune responses can be evaluated by the quantification of specific markers associated with lymphocyte proliferation. Ki-67, an intracellular protein detected only in dividing cells, is regarded as a reliable marker for proliferation studies26, and similarly, CD25 expressed on the surface of T cells during the late phase of activation corresponds to lymphocyte proliferation27,28.
This study demonstrates a standardized method for the in vitro generation of cattle MoDCs followed by their application in an in vitro immune assay used for testing the immunogenicity of vaccines. A commercially available rabies vaccine (RV) was used to validate the efficacy of this assay. T lymphocyte activation and proliferation were measured by flow cytometry, real-time quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA) through the analysis of well-established cell activation markers such as Ki-67 and CD25 and the secretion of IFN-γ28,29,30,31. No animal or human experimental trials are performed during the MoDC assay.
Blood collection is performed by a certified veterinarian service in accordance with the ethical guidelines of the Austrian Agency for Health and Food Safety(AGES) and in compliance with the accepted animal welfare standards32. The study received ethical approval from the Austrian Ministry of Agriculture. The experimental design for MoDCs generation and its subsequent application is illustrated in Figure 1.
1. Production of naive MoDCs
NOTE: Whole blood samples were obtained from a single pathogen-free calf by jugular vein puncture with heparinized vacutainers (eight 9 mL blood tubes were used for this study). Transport the blood in an ice box. Store the samples at 2-4 °C for later use, or process them immediately. Keep the blood rotating to avoid blood clotting. Sterilize the vacutainers with 70% ethanol. All the following experiments were performed with one biological sample and six technical replicates.
2. MoDC endocytic activity assay
NOTE: The antigen uptake assay or endocytic activity assay measures the ability of naive MoDCs to internalize foreign material. Perform the assay using naive MoDCs cultured with 3% w/v cytokine cocktail and with 5 days of incubation, as previously described34.
3. Generation of antigen-pulsed MoDCs
NOTE: A commercially available and clinically approved vaccine against rabies virus (RV) can induce the differentiation of naive MoDCs into mature antigen-presenting MoDCs. Use 0.1% (~1 µL) of a single RV vaccination dose to generate antigen-pulsed MoDCs. Furthermore, it is preferred to produce RV-pulsed MoDCs in the same culture plate used (in step 1.3.8) to generate naive MoDCsbecause transferring the naive MoDCs to a new 24-well plate will negatively affect them.
4. MoDC-lymphocyte co-culture
NOTE: The in vitro MoDC-lymphocyte co-culture system determines the ability of MoDCs to prime antigen-specific lymphocytes. The different treatment groups of cells after 16 days of co-culture include specific, non-specific, and control. The specific group is defined as lymphocytes co-cultured with RV-pulsed MoDCs; the non-specific group is defined as lymphocytes co-cultured with non-antigen-pulsed MoDCs; and the control group is defined as lymphocytes cultured without MoDCs.
5. Flow cytometric analysis
NOTE: Stain the cells with appropriate markers/mAb prior to running the samples on a flow cytometer. Refer to the Table of Materials for details on the reagents (staining mAb and isotype controls), kit, instrument, and software used for the flow cytometry analysis.
6. Messenger RNA (mRNA) expression analysis
7. Enzyme-linked immunosorbent assay
8. Statistical analysis
This methodology describes the in vitro generation of cattle MoDCs for the evaluation of candidate vaccine antigens prior to performing in vivo studies. Figure 1 illustrates the experimental scheme of bovine MoDC generation and the application of the MoDCs for the in vitro assay. Using the magnetic-based cell sorting technique, it was possible to collect approximately 26 million CD14+ myocytes from the harvested PBMCs, which were previously isolated from 50 mL of cattle blood. The eluted cell fraction free of CD14+ monocytes was rich in lymphocytes and was used as a source of naive CD4+ and CD8+ T cells (CD14− lymphocyte cell fraction).
The naive monocyte fraction was composed of 98% CD14+ monocyte cells, as observed after CD14 cell staining and flow cytometry (Figure 2A,B). The purified naive monocyte cells, when subjected to culturing in the presence of the 3% cytokine cocktail (GM-CSF and IL-4) with a subsequent incubation of 5 days, differentiated into a DC-like phenotype. From a starting culture of 26 million CD14+ monocytes, a total of approximately 12 million MoDCs could be obtained after 5 days of incubation. The naive MoDCs were functionally capable of antigen uptake, as observed using the flow cytometry analysis of FITC-dextran (Figure 3). Furthermore, the naive MoDCs were phenotypically characterized by assessing the expression of MHC class II and co-stimulatory CD86 and CD40 cell surface markers, which validated the DC-like phenotype (Figure 4).
The antigen-pulsed stimulation of naive MoDCs was achieved by culturing them in the presence of inactivated RV for 2 days. The activation of naive lymphocytes (the CD14− cell fraction) was achieved by antigen-pulsed MoDC-lymphocyte co-culturing with the subsequent supplementation of IL-2. During the MoDC-lymphocyte co-culture on day 9, a morphological change was observed in the RV-pulsed MoDCs, as they showed dendrite extension, which is a characteristic of MoDC maturation (Figure 5). On day 14, lymphocyte activation was enhanced by restimulating the co-culture through the addition of newly produced RV-pulsed MoDCs from the same animal.
Compared to the non-pulsed MoDC-lymphocyte co-culture, a significant increase (p < 0.01) in lymphocyte proliferation was demonstrated by the upregulation of the Ki-67 and CD25 activation markers on both the CD4+ and CD8+ T cells on day 16 in the pulsed MoDC-lymphocyte co-culture (Figure 6). The CD8+ T cells from the mature RV-pulsed MoDC co-cultures exhibited an eight-fold upregulation (p < 0.01) of Ki-67 when compared to the non-specific group (Figure 7A). The CD4+ T cells in the same co-culture showed a seven-fold increase (p < 0.01) in Ki-67 compared to controls (Figure 7B). This demonstrates the ability of RV-primed MoDCs to successfully present the RV antigen to naive lymphocytes and subsequently activate them in an in vitro condition, similar to what happens in a living animal. In addition to analyzing the cells using flow cytometry, the co-cultures were also subjected to qPCR and ELISA to quantify the RV-specific lymphocyte activation using RNA transcription (Ki-67 and IFN-γ) and extracellular secretion (IFN-γ), respectively (Figure 8). These additional detection methods can also be used as confirmatory tests to further validate the results of flow cytometry. The RNA expression for Ki-67 and IFN-γ demonstrated by qPCR and the IFN-γ levels demonstrated by ELISA showed similar patterns of increase, indicating lymphocyte proliferation, in the antigen-specific co-culture as compared to the non-specific treatment group. Therefore, the qPCR and ELISA results correlated with the flow cytometry results. The qPCR showed a >30% increase in IFN-γ expression and a >5% increase in Ki-67 expression in all the co-cultures using GAPDH as a calibrator (Figure 8A,B). A significantly higher concentration of secreted IFN-γ (**p > 0.01) was measured with ELISA using culture supernatants from the RV-pulsed MoDC-lymphocyte co-culture compared to the non-specific treatment group (Figure 8C).
Figure 1: Experimental design of the bovine MoDC-based in vitro assay. (A) Harvesting and cell sorting of the CD14+ monocyte and CD14− lymphocyte cell fractions from bovine PBMCs. Cattle blood is processed by density gradient centrifugation to collect PBMCs, followed by magnetic-based cell sorting using immunomagnetic cell separation columns and the subsequent culturing of the harvested CD14+ naive monocyte cell fraction in supplemented RPMI 1640 medium. (B) The production of MoDCs using 3% cytokine cocktail (GM-CSF + IL-4) with 5 days of incubation and MoDC-lymphocyte co-culture. On day 0, the monocytes are cultured in the presence of the cytokine cocktail and incubated for 48 h to induce differentiation. On day 2, the culture is restimulated with the same cytokine cocktail, followed by incubation for 72 h, which leads to the production of naive MoDCs. On day 5, the vaccine antigen (rabies vaccine) is added to the naive MoDC cell culture, followed by 48 h incubation. On day 7, the co-culturing of antigen-pulsed MoDCs with naive lymphocytes (the CD14− cell fraction) is performed, followed by incubation. On day 9, IL-2 is added to the co-culture. On day 14, the enrichment/restimulation of the activated/primed lymphocytes is performed by the addition of antigen-pulsed MoDCs, followed by incubation for 48 h. Lastly, on day 16, the cells and culture supernatant are harvested for the lymphocyte proliferation assay. Abbreviations: MODCs = bovine monocyte-derived dendritic cells; PBMCs = peripheral blood mononuclear cells; GM-CSF = granulocyte-macrophage-colony-stimulating factor. Please click here to view a larger version of this figure.
Figure 2: Morphology and purity of bovine CD14+ monocytes harvested from PBMCs using anti-CD14-conjugated microbeads. (A) Morphology of bovine monocytes incubated for 4 h at 37 ˚C in complete culture medium to remove microbeads, fixed on poly-lysine-coated slides, and stained with modified Giemsa stain. Scale bar = 50 µm. (B) Flow cytometry histogram showing the eluted CD14+ cell fraction with a 98.6% purity level observed using anti-CD14 antibody (red) and FITC-conjugated mouse IgG1 isotype control antibody (green). Abbreviations: PBMCs = peripheral blood mononuclear cells; FITC cont = fluorescein isothiocyanate control. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 3: Endocytic activity of bovine MoDCs generated in vitro. Flow cytometry histogram showing the uptake of the tracer molecule (FITC-dextran) by day 5 naive MoDCs. MoDCs incubated at 37 °C for 60 min with the tracer molecule (blue) and MoDCs incubated on ice with the tracer molecule (grey) used as a background control. Abbreviations: MODCs = bovine monocyte-derived dendritic cells; FITC = fluorescein isothiocyanate. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 4: Phenotyping of in vitro-generated bovine MoDC-specific cell surface markers. Flow cytometry histograms for (A) gating strategies of MoDCs and for (B) day 5 naive MoDCs stained with three different DC-specificmAbs (blue), including the following: anti-sheep MHC II mAb, anti-bovine CD86 mAb, and anti-bovine CD40 mAb. All compared with their corresponding isotype controls (red). Abbreviations: DC = dendritic cells; MODCs = bovine monocyte-derived DCs; MHC II = major histocompatibility complex II. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 5: Signs of maturation of in vitro-produced MoDCs during MoDC-lymphocyte co-culture. Observation of the characteristic extended dendritic structure by antigen-pulsed MoDCs with inverted microscopy on day 9 of the MoDC-lymphocyte co-culture. (A) Mature MoDCs within a highly confluent area of co-cultured lymphocytes. (B) Mature MoDCs are easily distinguishable in an area with fewer lymphocytes in co-culture. Scale bars = 50 µm. Abbreviation: MODCs = bovine monocyte-derived dendritic cells. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 6: Sequential gating strategy adopted for dot plots against Ki-67 and CD25 expression by lymphocytes in MoDC-lymphocyte co-culture. (A) The full gating strategy for cells harvested from day 16 MoDC-lymphocyte co-culture. (B) The Ki-67 expression from CD4+-gated lymphocytes and (C) from CD8+ lymphocytes compared with the FMO control (without mAb) and mouse IgG1-k isotype control mAb. The CD25 expression on gated (D) CD8+ and (E) CD4+ lymphocytes compared with mouse IgG1 isotype control mAb. The treatment group specifically represents lymphocytes co-cultured with RV-pulsed MoDCs, whereas the control represents lymphocytes cultured in the absence of MoDCs. Abbreviations: MODCs = bovine monocyte-derived dendritic cells; FMO = fluorescent minus one; RV = rabies vaccine. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 7: Flow cytometry data analysis of Ki-67 and CD25 expression by CD4+ and CD8+ lymphocytes after priming with the antigen. Ki-67 and CD25 expression from day 16 co-culture. The treatment group (specific) is defined as lymphocytes cultured with RV-pulsed MoDCs; the non-specific group corresponds to lymphocytes cultured with non-antigen-pulsed MoDCs; the control group corresponds to lymphocytes cultured without MoDCs. The horizontal bars represent the mean of six technical replicates. (A) Intracellular expression of the Ki-67 marker by CD8 T cells and (B) by CD4 T cells. (C) Cell surface expression of the CD25 marker by CD8 T cells and (D) by CD4 T cells. Abbreviations: MODCs = bovine monocyte-derived dendritic cells; RV = rabies vaccine. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Figure 8: Th1 marker (IFN-γ and Ki-67) activation by MoDCs primed with the rabies virus antigen, as detected through qPCR and ELISA. Data taken from one animal with six technical replicates. The horizontal bars represent the mean. Three PCR replicates shown as fold changes compared to naive lymphocytes after normalization with a GAPDH reference gene. (A) IFN-γ expression, (B) Ki-67 expression. (C) Comparison of IFN-γ secretion in the culture supernatant between three treatment groups, as detected by ELISA. The specific group is defined as lymphocytes cultured with RV-pulsed MoDCs; the non-specific group corresponds to lymphocytes cultured with non-antigen-pulsed MoDCs; the control group corresponds to lymphocytes cultured without MoDCs. Abbreviations: MODCs = bovine monocyte-derived dendritic cells; RV = rabies vaccine. This figure has been modified from Kangethe et al., 201811. Please click here to view a larger version of this figure.
Reagents | Final Concentration | Volume Per reaction |
dNTPs Mix (10 mM) | 1,000 µM | 1 µL |
Random Hexamer Primers (50 ng/µL) | 25 µM | 1 µL |
RNA template | 0.1 – 1 µg/µL | χ µL (as required) |
RNAse Free water | – | χ µL (as required) |
Final Reaction Volume | – | 10 µL |
Table 1: Master mix composition (RNA primer mix).
Reagents | Final Concentration | Volume Per reaction |
RT buffer 10x | 1x | 2 µL |
MgCl2 25 mM | 5 mM | 4 µL |
DTT 0.1 M | 10 mM | 2 µL |
RNAse inhibitor 40 U/µL | 2 U | 1 µL |
Table 2: The 2x PCR reaction mix. Abbreviations: RT = reverse transcriptase; DTT = dithiothreitol.
Cytokine | Species | Accession Number | Sequence | Length | Tm | |||
IFN-γ | Bos taurus | FJ263670 | F- GTGGGCCTCTCTTCTCAGAA | 234 | 80.5 | |||
R- GATCATCCACCGGAATTTGA | ||||||||
Ki-67 | Bos taurus | XM_015460791.2 | F-AAGATTCCAGCGCCCATTCA | 148 | 86.5 | |||
R-TGAGGAACGAACACGACTGG | ||||||||
GAPDH | Bos taurus | Sassu et al., 2020 | F-CCTGGAGAAACCTGCCAAGT | 214 | 85.5 | |||
R-GCCAAATTCATTGTCGTACCA |
Table 3: Primer sets used for amplification50.
Reagents | Final Concentration | Volume Per reaction |
Supermix | 1x | 5 µL |
Forward Primer (5 µM) | 250/ 125 nM | 1 µL |
Reverse Primers (5 µM) | 250/ 125 nM | 1 µL |
cDNA 1:10 diluted | 1.25 ng | 2 µL |
Nuclease-free Water | – | 1 µL |
Final Reaction Volume | – | 10 µL |
Table 4: qPCR master mix
Tube No. | Concentration of Standard | Serial Dilution | |||||
1 | 50 ng/mL | 50 µL of standard + 350 µL of wash buffer | |||||
2 | 12.5 ng/mL | 150 µL from tube 1 + 450 µL of wash buffer | |||||
3 | 6.25 ng/mL | 250 µL from tube 2 + 250 µL of wash buffer | |||||
4 | 3.13 ng/mL | 250 µL from tube 3 + 250 µL of wash buffer | |||||
5 | 1.56 ng/mL | 250 µL from tube 4 + 250 µL of wash buffer | |||||
6 | 0.78 ng/mL | 250 µL from tube 5 + 250 µL of wash buffer | |||||
7 | 0.2 ng/mL | 150 µL from tube 6 + 450 µL of wash buffer | |||||
8 | 0.1 ng/mL | 250 µL from tube 7 + 250 µL of wash buffer | |||||
9 | 0.025 ng/mL | 100 µL from tube 8 + 300 µL of wash buffer |
Table 5: IFN-γ standard dilution series
Supplementary File 1: Protocols for the synthesis of complementary DNA, real-time quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Please click here to download this File.
Supplementary Figure S1: The antigen uptake ability MoDCs with different concentrations of the cytokine cocktail and with 3 days or 5 days of culture. Different concentrations (5% w/v and 3% w/v) of the cytokine cocktail containing GM-CSF and IL-4 were tested either with 3 days or 5 days of culture to assess the best combination to generate high-performance antigen uptake MoDCs (using the tracer molecule FITC-dextran). Please click here to download this File.
Supplementary Figure S2: CD4 and CD8 priming with diptheria toxoid (DT) and Bluetongue virus serotype 4 (BTV). The expression of Ki-67 by CD8 cells after pulsing with (A) DT and (C) BTV and the expression of Ki-67 by CD4 cells after pulsing with (B) DT and (D) BVT at day 16. Comparisons were made with cultures pulsed with DT and BVT (specific), (C,D) with CD40L, and with non-specific priming and control treatments. The horizontal bars indicate the mean. *p < 0.05, **p < 0.01 according to a Mann-Whitney test. Please click here to download this File.
This study demonstrates a standardized in vitro method for generating and phenotyping bovine MoDCs and their subsequent use in measuring the vaccine immunogenicity of a commercial vaccine (e.g., RV). Bovine MoDCs can be used as a tool for screening potential vaccine antigens against cattle diseases and predicting their potential clinical impact based on immune responses before proceeding toward in vivo animal trials. The MoDCs generated were identified based on their morphological, phenotypic, and functional characteristics. We showed that the MoDCs derived from cattle CD14+ monocytes exhibited features seen in DCs, such as extended dendrites, the expression of cell surface markers for antigen presentation such as MHC II, the expression of co-stimulatory molecules on MoDCs such as CD40 and CD86, endocytic activity, and the ability to activate naive lymphocytes.
The cell culture media used in this experiment (DMEM and RPMI) are widely used for cell differentiation and cultivation, with RPMI being more frequently adopted for monocyte differentiation36. RPMI supplementation with either FBS or HS (horse serum) does not affect monocyte differentiation37. Recombinant GM-CSF and IL-4 supplementation provides an inflammatory condition that triggers monocyte differentiation and is routinely used for the production of functionally viable MoDCs capable of antigen uptake and presentation to immune cells38,39. In this study, RPMI 1640 medium supplemented with 10% FBS (complete culture medium), 1% penicillin-streptomycin, and the addition of a commercially available cytokine cocktail (GM-CSF plus IL-4) induced bovine monocyte differentiation, resulting in the production of viable MoDCs. When the generated MoDCs were incubated with RV before co-culturing with naive lymphocytes, they induced a T cell-dependent immune response specific against RV, proving that mature MoDCs can adequately present RV antigens to lymphocytes that have never come across RV. This is a crucial characteristic of adaptive immunity that we can now replicate in the lab.
We observed that a higher concentration of cytokine cocktail (5%) coupled with a longer incubation time (5 days) produced MoDCs with lower endocytic ability for antigen uptake than those treated with a lower concentration of cytokine cocktail (3%) with the same incubation time (5 days), as shown in Supplementary Figure S1. Hence, we conclude that a lower concentration of cytokine cocktail (e.g., 3%) with 5 days of incubation is optimal to produce functionally potent MoDCs. Cell surface markers such as MHC II, CD40, and CD86 are present on APCs, and their upregulation indicates functional cell activation40,41,42. We demonstrated enhanced expression of MHC II, CD40, and CD86 after monocyte differentiation into naive MoDCs using the cytokine cocktail (Figure 4).
Optimizing the ratio of naive lymphocytes and MoDCs in a culture system is a key factor that influences the outcome of the MoDC assay, with different MoDC to lymphocyte ratios inducing varied lymphocyte responses43. However, after evaluating MoDC to lymphocyte ratios of 1:10-1:40, we observed that increasing the concentration of MoDCs did not increase the lymphocyte activation, and we eventually settled on an optimal ratio of 1:20, which is similar to previously reported studies44,45. It should be noted that the eluted naive lymphocyte fraction might contain non-specifically activated T cells; therefore, it is imperative to have a control group corresponding to only naive lymphocyte culture.
Activated CD4+ and CD8+ T cells express specific markers and secrete a variety of cytokines, and their quantification indicates immune activation. The intracellular expression of Ki-67, a well-characterized immune activation marker, was upregulated in this MoDC-lymphocyte co-culture29,46. Similarly in this study, the enhanced IFN-γ secretion by lymphocytes indicated a Th1 response elicited against the RV antigen30,31,47. IL-2 expression by CD4+ T cells is also a good marker for visualizing T cell activation; however, its incorporation on day 9 of the MoDC-lymphocyte co-culture made it an unsuitable target for measuring lymphocyte proliferation for this study48. The upregulation of CD25 in the presence of IL-2 has been previously shown to determine the priming of both CD4+ and CD8+ T cells and was used in this study to measure the priming of naive lymphocytes by RV-pulsed MoDCs28.
Cytotoxic CD8 T cells are major effector cells against intracellular vaccine antigens or viral pathogens49. By measuring lymphocyte activation markers (Ki-67 and CD25) and cytokine expression (IFN-γ), we found that the antigen-loaded MoDCs had significant (p < 0.01) activation of both CD8 and CD4 T cell responses, with the CD8 response and Th1 polarization being more prevalent against the RV antigen. An important factor that needs to be considered when designing an MoDC assay for adjuvant-conjugated vaccines is adjuvant-induced lymphocyte responses. We recommend using a control group (adjuvant control) composed of only adjuvant to eliminate any background signals. This setup can also be used to measure the effect of different adjuvants that can amplify lymphocyte responses toward a low-immunogenic antigen, which is crucial for protection during in vivo studies.
There are some limitations in this study that we want to highlight. The experimental data for each assay were derived from a single animal with six technical replicates. Having a larger number of biological replicates would be beneficial to minimize errors and provide enhanced statistical reliability and results with higher accuracy. Nevertheless, in a previous publication11, this assay was also tested and validated using a diptheria toxoid (Supplementary Figure S2A,B) and a commercial vaccine against Bluetongue virus serotype 4 (Supplementary Figure S2C,D) with three and two biological animals, respectively. Furthermore, comparing the results obtained from this in vitro study with an in vivo study would help to additionally validate the authenticity of this immune assay, which, in turn, would accelerate the process of implementation of this in vitro MoDC assay as a routine test in vaccine production and quality control. Lastly, the expression of CD14 in MoDCs was not investigated, as the literature is inconsistent regarding this aspect, especially when comparing MoDCs from different animal species.
In conclusion, we report an in vitro bovine MoDC assay that can be used to measure vaccine-induced immunogenicity. This MoDC assay can also be evaluated using various methods, including flow cytometry, ELISA, and qPCR. Having multiple methods for evaluating activation markers is crucial in areas with limited resources where a flow cytometer may not be readily available. This assay can be included as a quality control step by national veterinary laboratories that produce large batches of bovine vaccines. In addition, it can be used to identify potential vaccine antigens during the development of new bovine vaccines and even to select which adjuvant to use before an animal trial. All these factors will contribute toward a more ethical and affordable approach to developing and using bovine vaccines.
The authors have nothing to disclose.
We thank Dr. Eveline Wodak and Dr. Angelika Loistch (AGES) for their support in determining the health status of the animals and for providing BTV, Dr. Bernhard Reinelt for providing bovine blood, and Dr. Bharani Settypalli and Dr. William Dundon of the IAEA for useful advice on the real-time PCR experiments and language editing, respectively.
ACK Lysing Buffer | Gibco, Thermo Fisher | A1049201 | Ammonium-Chloride-Potassium buffer for lysis of residual RBCs in harvested PBMC Fraction |
BD Vacutainer Heparin Tubes | Becton, Dickinson (BD) and Company | 366480 | 10 mL, additive sodium heparin 158 USP units, glass tube, 16 x 100 mm size |
Bovine Dendritic Cell Growth Kit | Bio-Rad, UK | PBP015KZZ | Cytokine cocktail composed of recombinant bovine IL-4 and GM-CSF |
Bovine IFN-γ ELISA Kit | Bio-Rad | MCA5638KZZ | Kit use for measuring IFN-γ expression in culture supernatant |
CD14 Antibody | Bio-Rad | MCA2678F | Mouse anti-bovine CD14 monoclonal antibody, clone CC-G33, isotype IgG1 |
CD25 Antibody | Bio-Rad | MCA2430PE | Mouse anti bovine CD25 monoclonal antibody, clone IL-A11, isotype IgG1 |
CD4 Antibody | Bio-Rad | MCA1653A647 | Mouse anti bovine CD4 monoclonal antibody, clone CC8, isotype IgG2a |
CD40 Antibody | Bio-Rad | MCA2431F | Mouse anti-bovine CD40 monoclonal antibody, clone IL-A156, isotype IgG1 |
CD8 Antibody | Bio-Rad | MCA837F | Mouse anti bovine CD8 monoclonal antibody, clone CC63, isotype IgG2a |
CD86 Antibody | Bio-Rad | MCA2437PE | Mouse anti-bovine CD86 monoclonal antibody, clone IL-A190, isotype IgG1 |
CFX96 Touch Real-Time PCR Detection System | Bio-Rad | – | Thermal cycler PCR machine |
Corning Centrifuge Tube | Falcon Corning | 352096 & 352070 | 15 mL and 50 mL, high-clarity poypropylene conical bottom, graduated, sterial, seal screw cap, falcon tube |
Cytofix/Cytoperm Plus | BD Bio Sciences | 555028 | Fixation/permeabilization kit with BD golgiPlug, use for flow cytometer cell staining |
Ethanol | Sigma Aldrich | 1009832500 | Absolute for analysis EMSURE ACS,ISO, Reag. Ph Eur |
Fetal Bovine Serum (FBS) | Gibco, Thermo Fisher | 10500064 | Qualified, heat inactivated |
Ficoll Plaque PLUS | GE Health care Life Sciences, USA | 341691 | Lymphocyte-isolation medium |
FlowClean Cleaning Agent | Beckman Coulter, Life Sciences | A64669 | 500 mL |
FlowJo | FlowJo, Becton, Dickinson (BD) and Company, LLC, USA | – | Flow cytometer Histogram software |
FlowTubes/ FACS (Fluorescence-activated single-cell sorting) Tube | Falcon Corning | 352235 | 5 mL, sterial, round bottom polystyrene test tube with cell strainer snap cap, use in flow cytometry analysis |
Fluoresceinisothiocynat-Dextran | Sigma Aldrich, Germany | 60842-46-8 | FITC-dextran MW |
Gallios Flow Cytometer | Beckman Coulter | – | Flow cytometer machine |
Hard-Shell 96-Well PCR Plates | Bio-Rad | HSP9601 | 96 well, low profile, thin wall, skirted, white/clear |
Human CD14 MicroBeads | Miltenyi Bioteck, Germany | 130-050-201 | 2 mL microbeads conjugated to monoclonal anti-human CD14 antibody isotype IgG2a, used for selection of bovine monocytes from PBMCs |
Kaluza | Beckman Coulter, Germany | – | Flow cytometer multicolor data analysis software |
MACS Column | Miltenyi Bioteck, Germany | 130-042-401 | Magnetic activated cell sorting or immune magentic cell separation colum for separation of various CD14 cell population based on cell surface antigens |
MHC Class II DQ DR Polymorphic Antibody | Bio-Rad | MCA2228F | Mouse anti-sheep MHC Class II DQ DR Polymorphic:FITC, clone 49.1, isotype IgG2a, cross reactive with bovine |
Microcentrifuge Tube | Sigma Aldrich | HS4325 | 1.5 mL, conical bottom, graduated, sterial tube |
Microsoft Power Point | Microsoft | – | The graphical illustrations of experimental design |
Mouse IgG1 Negative Control:FITC for CD14, CD40 Antibody | Bio-Rad | MCA928F | Isotype control CD14 and CD40 monoclonal antibody |
Mouse IgG1 Negative Control:PE for CD86 Antibody | Bio-Rad | MCA928PE | Isotype control CD86 monoclonal antibody |
Mouse IgG1 Negative Control:RPE for CD25 Antibody | Bio-Rad | MCA928PE | Isotype control CD25 monoclonal antibody |
Mouse IgG2a Negative Control:FITC for MHC Class II Antibody | Bio-Rad | MCA929F | Isotype control for MHC class II monoclonal antibody |
Nobivac Rabies | MSD Animal Health, UK | – | 1 µL/mL of cell cultured inactivated vaccine containing > 2 I.U./mL Rabies virus strain |
Optical seals | Bi0-Rad | TCS0803 | 0.2 mL flat PCR tube 8-cap strips, optical, ultraclear, compatible for qPCR machine |
Penicillin-Streptomycin | Gibco, Thermo Fisher | 15140122 | 100 mL |
Phosphate Buffer Saline (PBS) | Gibco, Thermo Fisher | 10010023 | pH 7.4, 1x concentration |
Prism – GraphPad 5 Software | Dotmatics | – | Statistical software |
Purified Anti-human Ki-67 antibody | Biolegend, USA | 350501 | Monoclonal antibody, cross reactive with cow, clone ki-67 |
Purified Mouse IgG1 k Isotype Ctrl Antibody | Biolegend | 400101 | Isotype control for Ki-67 monoclonal antibody |
READIDROP Propidium Iodide | BD Bio Sciences | 1351101 | Live/dead cell marker used for flow cytometry, amine reactive dye |
Recombinant Human IL-2 Protein | R&D System, USA | 202-IL-010/CF | Interleukin-2, 20 ng/ml |
RNeasy Mini Kit | Qiagen | 74106 | Kit use for extraction of total RNA; RLT buffer = lysis buffer; RW1 buffer = stringent guanidine-containing washing buffer; RDD buffer = DNase buffer; RPE buffer = mild wash buffer; RNaseOUT = RNase inhibitor. |
RPMI 1640 Medium | Sigma Aldrich | R8758 | Cell culture media with L-glutamine and sodium bicarbonate |
SMART-servier medical art | Les Laboratories Servier | – | Licensed under a creative commons attribution 3.0 unported license |
SsoAdvanced Universal SYBR Green Supermix | Bio-Rad | 172-5270 | 2x qPCR mix conatins dNTPs, Ss07d fusion polymerase, MgCl2, SYBR Green I supermix = supermix, ROX normalization dyes. |
SuperScript III First-Strand Synthesis System | Invitrogen, Thermo Fisher | 18080051 | Kit for cDNA synthsis |
Tissue Culture Test plate 24 | TPP, Switzerland | 92024 | 24 well plate, sterilized by radiation , growth enhanced treated, volume 3.18 mL |
Trypan Blue Solution | Gibco, Thermo Fisher | 15250061 | 0.4%, 100 mL, dye to assess cell viability |
UltraPure DNase/RNase-Free Distilled Water | Invitrogen, Thermo Fisher | 10977023 | 0.1 µm membrane filtered distilled water |
VACUETTE Heparin Blood Collection Tubes | Thermo Fisher Scientific | 15206067 | VACUETTE Heparin Blood Collection Tubes have a green top and contain spray-dried lithium, sodium or ammonium heparin on the inner walls and are usedin clinical chemistry, immunology and serology. The anticoagulant heparin activates antithrombin, which blocks the clotting cascade and thus produces a whole blood/plasma sample. |
Water | Sigma Aldrich | W3500-1L | Sterile-filtered, bioReagent suitable for cell culture |