Introduction
Measuring B- and T-cell responses against particular antigens is pivotal to understand how the adaptive immune response develops in the course of an infection, or after vaccination. However, examination of B and T-cell responses in experimental studies is difficult for several reasons. Under experimental conditions, several groups or batches of animals must be examined considering individual variations. In the case of large animals, implies housing and management in special facilities that have limited allocation capacity. Under farm conditions, although more animals can be examined, the preservation and processing of samples become the main challenge, as farms are usually far from research institutes. Also, it is not always possible to obtain samples, such as lymphocytes from lymph nodes, that can only be collected after killing the animal, due to ethical and economic reasons. Thus, separation of peripheral blood mononuclear cells (PBMC) after bleeding an animal is a common strategy that allows sampling numerous animals at the same time. This is convenient when performing a longitudinal follow-up study (e.g., to evaluate the variation of the adaptive response over time) and elucidating B- and T-cell responses by means of cryopreservation of PBMC. Besides, frozen replicas of PBMC from a given animal can be used to retest or to perform additional tests if necessary.
Cryopreserved PBMC are extensively used in human immunology. However, PBMC, as other cell types, are sensitive to the freezing process. It is well-known that cells can be damaged during freezing, mainly by the intracellular formation of ice crystals -which can mechanically damage the cell-, or by the osmotic imbalance between the intra- and extra-cellular space, resulting in dehydration and shrinkage. In addition, channels formed by the residual unfrozen medium outside the cells could also damage them (1). The impact of cryopreservation on T and B-cell subsets of PBMC continues to be a controversial issue. For human PBMC, some reports have not observed substantial changes between fresh and frozen cells in terms of phenotype proportions and functionality, such as the antigen-specific responses or the response to mitogens (2–6). Nevertheless, others claimed that cryopreservation alters the proportions of human PBMC subsets (7, 8), the antigen-specific IFN-γ responses using whole PBMC or specific T-cell subsets (9, 10), and impairs the proliferation after mitogen or antigen stimulation as well (3).
In the case of pigs, frozen PBMC are frequently used as well; however, few articles have analyzed the effects of cryopreservation (11, 12). Koch et al. (11) indicated that cryopreservation could impair some immunological functions, such as proliferation after PHA-stimulation, while Li et al. (12) found that impact on PBMC stimulated by PMA was disparate, from a significantly lower production of IL-6 in frozen cells to a significantly higher production of IFN-γ in frozen PBMC compared to fresh PBMC.
The present study aimed at evaluating the impact of cryopreservation on porcine PBMC in terms of viability, T- and B-cell subset proportions and functionality after mitogen- or antigen-specific stimulation. In a preliminary study, three cryopreservation media were compared to assess the best method to allow the highest survival rate.
Materials and Equipment
Kits
Porcine IgG ELISPOTBASIC kit (HRP) including capture antibody MY91/145, biotinylated antibody MT78/145, streptavidin-peroxidase and TMB substrate, 3151-2H, Mabtech, Nacka Strand, Sweden CellTrace™ Violet Cell Proliferation Kit, C34557, Thermo Fisher Scientific, Waltham, Massachusetts, US
Cell Media
RPMI Medium 1640 with HEPES wo L-Gln (RPMI-1640), H3BE04-558F, Lonza, Basilea, Switzerland
L-Glutamine 200 mM, 25030024, Thermo Fisher Scientific, Waltham, Massachusetts, US
Non-essential amino acids solution (100x), 11140035, Thermo Fisher Scientific, Waltham, Massachusetts, US
Sodium Pyruvate (100 mM), S-8636, Millipore Sigma, Saint Louis, Missouri
2-Mercaptoethanol, M6250-10ML, Merck, Darmstadt, Germany
Penicillin-Streptomycin (10,000 U/mL), 15140122, Thermo Fisher Scientific, Waltham, Massachusetts, US
Gentamicin (50 mg/mL), 15750045, Thermo Fisher Scientific, Waltham, Massachusetts, US
Fetal Bovine Serum (FBS), 35-079-CV, Corning, New York, US
Supplemented medium-1 (SM1): RPMI 1640 plus: 1mM L-Glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, 5 mM 2-mercaptoethanol, 50,000 IU/l penicillin 1, 50 mg/l streptomycin, 50 mg/l gentamicin and 10% FBS
Supplemented medium-2 (SM2): RPMI 1640 containing 10% FBS
Buffers and Others
Cytiva HyClone™ Phosphate Buffered Saline (PBS), 10462372, Thermo Fisher Scientific, Waltham, Massachusetts, US
Dulbecco’s phosphate-buffered saline (DPBS), 14040133, Thermo Fisher Scientific, Waltham, Massachusetts, US
Horse Serum, New Zealand origin, 16050122, Thermo Fisher Scientific, Waltham, Massachusetts, US
Sodium bicarbonate (NaHCO3), 1.06329, Merck, Darmstadt, Germany
Sodium carbonate monohydrate (Na2Co3), 230952-100gr, Merck, Darmstadt, Germany
Bovine Serum Albumin (BSA), A7906-100G, Merck, Darmstadt, Germany
Tween® 20, P1379, Merck, Darmstadt, Germany
Supplemented DPBS (blocking solution for flow cytometry): DPBS containing 5% FBS and 5% horse serum
Carbonate–bicarbonate buffer (sterile): 4.3 g NaHCO3 and 5.3 g Na2Co3 in 1 L distilled water (pH 9.4)
Standard diluent buffer: 5 g BSA and 1 mL Tween-20 in 1L PBS (pH 7.4)
Wash buffer for ELISPOT: 1 ml Tween 20 in 1 L distilled water (pH 7.4)
Wash buffer for flow cytometry: DPBS containing 2% FBS
Histopaque 1.077, 10771-500ML, Merck, Darmstadt, Germany
Trypan blue, T6146, Merck, Darmstadt, Germany
Cryopreservation Products
Homemade freezing medium: 90% FBS and 10% Dimethyl sulfoxide Hybri-Max™ (DMSO), D2650-100ML, Merck, Darmstadt, Germany
PSC Cryopreservation kit, A2644601, Thermo Fisher Scientific, Waltham, Massachusetts, US
CryoStor® CS10, 07955, Stemcell Technologies, Vancouver, Canada
Mitogens and Others
Concanavalin A (ConA) from Canavalia ensiformis, C2010, Merck, Darmstadt, Germany
Lectin from Phaseolus vulgaris (red kidney bean) (PHA), L1668-5MG, Merck, Darmstadt, Germany
R848 included in the Porcine IgG ELISPOTBASIC kit, 3151-2H, Mabtech, Nacka Strand, Sweden
Recombinant Porcine IL-2 Protein, 652-P2-020/CF, R&D Systems, Minneapolis, Minnesota, USA
Antibodies, Streptavidin and Substrate
Antibodies for IFN-γ ELISPOT
Purified mouse Anti-Pig IFN-γ porcine, clone P2G10, 559961, final dilution 1:100, BD Biosciences Pharmingen, San Jose, California, US
Biotin Mouse Anti-Pig IFN-γ, clone P2C11, 559958, final dilution 1:1000, BD Biosciences Pharmingen, San Jose, California, US
Antibodies for Flow Cytometry
Mouse anti pig CD3, clone PPT3, MCA5951GA, final dilution 1:200, Bio-Rad, Hercules, California, US
PE Rat Anti-Mouse IgG1, clone A85-1, 562027, final dilution 1:800, BD Biosciences Pharmingen, San Jose, US
FITC mouse anti-pig CD4α, clone 74-12-4, 559585, final dilution 1:100, BD Biosciences Pharmingen, San Jose, US
Alexa Fluor® 647 Mouse Anti-Pig CD8α, clone 76-2-11, 561475, final dilution 1:100, BD Biosciences Pharmingen, San Jose, US
Mouse Anti-Porcine CD21-PE, clone BB6-11C9.6, 4530-09, final dilution 1:100, SouthernBiotech, Birmingham, Alabama, USA
Streptavidin and Substrate
Streptavidin HRP (ELISA GD), SNN2004, Thermo Fisher Scientific, Waltham, Massachusetts, US
TMB substrate for ELISpot, 3651-10, Mabtech, Nacka Strand, Sweden
Field Virus and Vaccine
Porcine reproductive and respiratory syndrome virus (PRRSV) strain 3267 (batch 40) belonged to our PRRSV bank (13), UAB, Barcelona, Spain. It was produced and titred in porcine alveolar macrophages obtained from three-weeks old PRRSV-negative piglets.
PORCILIS® PRRS, modified live-virus PRRSV vaccine, MSD Animal Health, Madison, New Jersey, Us
Others
SepMate™ 50 (IVD), 85450, Stemcell Technologies, Saint Égrève, France
Frosty™ Freezing Container, 5100-0001, Thermo Fisher Scientific, Waltham, Massachusetts, US
96-Well, Cell Culture-Treated, U-Shaped-Bottom Microplate, 3799, Corning, New York, US
MultiScreen-HA filter plate MAHAS4510, MAHAS4510, Merck, Darmstadt, Germany
Software and Equipment
FCS Express 7, de novo Software, Glendale, California, US
Statsdirect Statistical software 3.3.5, Statsdirect LTD, Birkenhead, United Kigndom
GraphPad Prism 9.1.2, GraphPad Software Inc., San Diego, California, US
MACSQuant Analyzer 10, Miltenyi Biotec, Bergisch Gladbach, Germany
Methods
Preliminary Screening of Freezing Media
Three products/methodologies for freezing porcine PBMC were compared: Homemade freezing medium; PSC Cryopreservation kit, and CryoStor CS10. Fresh and frozen cells were assessed for viability and functionality by means of trypan blue staining and the IFN-γ ELISPOT (using PHA as a mitogenic stimulus) and IgG ELISPOT (using a R848+IL-2 cocktail).
Blood samples from eight commercial pigs of six weeks of age were collected in heparin tubes by jugular venipuncture. PBMC were isolated within 4h of collection by density-gradient centrifugation with Histopaque 1.077 in SepMate™ tubes. After two washes with PBS (400 x g, 10 min), PBMC of each animal were resuspended in SM1.
Cells collected in conical sterile tubes were stored overnight at 4°C. Afterwards, PBMC were counted using trypan blue staining and then were adjusted to the desired working concentrations. Half of the PBMC were used as fresh samples for ELISPOT assays, and the other half were frozen to repeat the same assays one month later.
For cryopreservation procedure with homemade freezing medium, PBMC were gently resuspended in 1mL of the freezing medium kept on ice and then transferred to cryovials. Cryovials were immediately distributed in controlled-grade freezing devices (−1°C/minute) and stored overnight at -80°C. The next day, samples were transferred to a liquid nitrogen tank at -196°C.
For PSC Cryopreservation kit and CryoStor CS10, PBMC were frozen following the manufacturer’s instructions. Thus, 1 mL of PSC Cryomedium (chilled at 4°C) was added dropwise to the PBMC, while gently rocking the tube back and forth, followed by gentle resuspension of the PBMC pellet. Then, samples were transferred to cryovials that were frozen as above. For CryoStor CS10, PBMC were softly resuspended in 1 mL (chilled at 4°C) and transferred to cryovials. Cryovials were distributed in controlled-grade freezing devices previously refrigerated at 4°C. Fifteen minutes after storing the cryovials at -80°C they were softly agitated. The next day, they were transferred to a liquid nitrogen tank at -196°C. In all cases, the concentration of PBMC was set at 20-25 x 106 PBMC/cryovial.
One month later, all samples were thawed by immersion in a 37°C water bath. Then, PBMC were transferred to a conical sterile tube and diluted 1/10 in SM1. Samples were centrifuged (400 x g, 10 minutes), the supernatant was removed and PBMC were washed one more time under the same conditions. Finally, PBMC were resuspended again in SM1. For PSC Cryopreservation kit, samples were resuspended according to the manufacturer’s instructions; in this case, RevitaCell™ was added to the SM1 (100 μL in 10mL of medium). In all cases, cells were allowed to rest overnight at 37°C (5% CO2) with the cap loosened (14, 15). The next morning, PBMC were counted as above and adjusted to the desired working concentration.
The viability of fresh and frozen cells was assessed in a Neubauer chamber after trypan blue staining. Recovery rates were calculated for frozen cells as follows: (n° viable cells after thawing/n° viable cells before freezing) x 100. The functionality of fresh and frozen T and B-cells was assessed by means of the IFN-γ and IgG ELISPOT assays, respectively.
The IFN-γ ELISPOT was performed as previously described (16). Briefly, pre-wetted (200 μL/well PBS, 1 min) filter plates were coated with the P2G10 IFN-γ monoclonal antibody (diluted in carbonate–bicarbonate buffer at 1 μg/mL; 50 μL/well) and incubated overnight at 4°C. Plates were then washed with sterile PBS (200 μL/well) and blocked for 1h at 37°C with 100 μL/well of SM2. After removal of the blocking solution, PBMC were dispensed; 50,000 PBMC/well in 100 μL volume for PHA-stimulated wells (plus PHA 10 μg/ml in 100 μL SM1) or 500,000 PBMC/well in 100 μL volume plus 100 μL SM1 for negative control wells. After 20h of incubation (37°C; 5% CO2), PBMC were removed by washing plates five times using wash buffer (200 μL/well) allowing wells to soak well for 1 min in each wash step. Then, biotinylated detection antibody P2C11 was added at 0.5 μg/mL (50 μL/well diluted in the standard diluent) and incubated for 1h at 37°C. After that incubation, plates were washed as above and the reaction was revealed by incubation of plates with streptavidin-peroxidase diluted in standard diluent (final concentration 0.5 μg/mL; 50 μL/well; incubation 1h at 37°C), washing plates as above and addition of insoluble TMB (50 μL/well; 20 minutes, in the dark at room temperature). All tests were run in triplicates. A stereomicroscope binocular was used to read the spots. Frequencies of IFN-γ secreting cells (SC) were calculated by subtracting the counts of spots in unstimulated cells, from the counts in PHA-stimulated ones. Results were expressed as responding cells/106 PBMC.
The IgG ELISPOT was carried out using a commercial kit. PBMC were split into two 1 mL aliquots in conical sterile polypropylene tubes. One of the aliquots was stimulated for 72 h (37°C, 5% CO2) with the polyclonal activator R848 and recombinant porcine IL-2 at 1 µg/mL and 10 ng/mL, respectively (16, 17). The other aliquot was kept as an unstimulated control. Aliquots were cultivated for three days prior to plating into ELISPOT. Then, cells were washed, resuspended in SM1, re-counted and adjusted to 100,000 cells/well. Plates previously pre-wetted (200 μL/well PBS, 1 min), coated with the MT421 monoclonal antibody and incubated overnight at 4°C, were washed and blocked. After removal of the blocking solution, PBMC were dispensed. Antibodies and streptavidin concentrations, as well as times of incubation, washing procedures, etc. were carried out following the manufacturer’s instructions. Captured IgG was visualized by the addition of the biotinylated antibody followed by the addition of streptavidin-peroxidase and insoluble TMB. All tests were run in triplicates. Frequencies of IgG-SC were calculated by subtracting the counts of spots in unstimulated cells, from the counts in stimulated ones. Results were expressed as responding cells/106 PBMC. In all ELISPOT experiments, IgG and IFN-γ, all reagents were filtered (0.2 μm) before use.
Evaluation of the Cryopreservation Impact on PBMC: Phenotyping and Responses to Mitogens and Specific Antigens
Five 4-week-old piglets (ear tags number 51, 53, 57, 59, 66) were immunized against PRRSV by vaccinating them with a modified live vaccine (PORCILIS PRRS®, 2 ml, intramuscular). Blood samples were collected in heparin tubes one month later. PBMC were obtained by gradient centrifugation as above; half of the PBMC were used as fresh samples for a set of analyses and the other half were frozen to repeat the same analyses after one month of frozen storage. Since results obtained during the preliminary screening pointed out that Cryostor CS10 provided the best results, it was selected as the freezing medium. The viability of fresh and frozen cells was assessed by trypan blue staining in a Neubauer chamber.
Phenotype of T and B-Cells
For phenotype characterization, fresh or frozen PBMC were initially incubated with supplemented PBS for 20 min on ice. Then, for T-cells labelling was carried out with antibodies against CD3, CD4α, and CD8α. Briefly, PBMC were incubated with the primary antibody anti-CD3 followed by an antibody anti-mouse IgG1-PE. In the next step, antibodies anti-CD4α:FITC and anti-CD8α:Alexa Fluor 647 were added. B-cells were labelled separately with an antibody anti-CD21conjugated to PE. Between each step, cells were washed twice with washing buffer and centrifuged at 500 x g for 5 min. All tests were run in triplicates. Samples were acquired on a MACSQuant Analyzer 10. Fluorescence minus one (FMO) controls, matched isotype controls, and background caused by secondary antibodies were used for gating and analysis. The acquired data were analysed using FCS Express 7.
Proliferation of T and B-Cells
For proliferation, fresh or frozen PBMC were labelled with CellTrace Violet at 5 μM according to the manufacturer’s instructions. Then, cells were suspended in SM1 at 2 × 106 cells/ml with 100 μl/well plated in a 96-well U-bottom plate. Another 100 μl of SM1 or SM1 containing PRRSV isolate 3267 (MOI 0.5), PHA or ConA (10 μg/ml) were added as stimuli. After 5 days, cells were harvested and stained with anti-CD3, anti-CD4α, anti-CD8α and anti-CD21 antibody as described above. All tests were run in duplicates.
Frequencies of IFN-γ-Secreting Cells After Recall Antigen or Mitogen Stimulation Using ELISPOT
IFN-γ ELISPOT was done as described above, using both fresh and frozen PBMC. In this section, besides the PHA response, the antigen-specific (PRRSV) IFN-γ-SC frequencies were also measured. For this purpose, 500,000 cells/well were stimulated with the PRRSV strain 3267 diluted in SM1 at a multiplicity of infection (MOI) of 0.1. PHA-stimulated cultures and PBMC incubated in SM1 were included as positive and negative controls, respectively as described above. Frequencies of PRRSV-specific and PHA-stimulated IFN-γ-SC were calculated by subtracting the counts of spots in unstimulated cells from the counts in stimulated ones. Results were expressed as responding cells/106 PBMC.
IgG ELISPOT
IgG ELISPOT to measure polyclonal responses were performed as described above.
Statistical Analysis
Statistics were performed using StatsDirect v2.7.7. Mann-Whitney U- and Wilcoxon’s signed ranks, or Kruskal-Wallis (Dwass–Steel–Chritchlow–Fligner method for multiple comparisons) non-parametric tests were used for comparisons of means between two or more sets of data, respectively.
Discussion
The use of frozen PBMC in pig immunology is a common procedure. Most often this is a need arising from the complex logistics required for processing samples collected in a farm located far from the analysis laboratory. However, there is very little information regarding the performance of frozen porcine PBMC compared to freshly isolated ones.
In the present study, the first step was to compare several cryopreservation media. The results of our comparison indicated that the viability after recovery was similar for all three specific freezing media (≥89% on average) (
Table 1
), although cells frozen with PSC Cryopreservation kit showed a trend to perform worse in the IFN-γ ELISPOT (
Supplementary Table 1
). It has been reported that the viability of PBMC below 70-80% seriously impaired the results of functional tests (19–21). Regarding recovery rates, no obvious impact was observed for any of the cryopreservation procedures (
Table 1
), obtaining similar results to those described by Liang and collaborators (2019) (22), who found a mean of 73.7% when comparing five methods. With our results, homemade freezing medium, PSC cryopreservation kit, and CryoStor CS10 fulfilled requirements of viability and recovery. In the following experiments, CryoStor CS10 was used for the sake of convenience.
The additional analyses using a recall antigen or different mitogens showed that the impact of freezing was more evident when the recall responses were examined, as indicated by the reduction of proliferation and the number of spots produced in IFN-γ ELISPOT (
Figures 1C
and
2B
, respectively). Since the viability of CD172+ cells, which comprised the majority of antigen presenting cells, did not differ from that of CD3+ cells or the whole PBMC, the poorer recall response was supposed not caused by extra extent damage of CD172+ cells. Such similar studies using human or mice PBMC are still controversial (3–10, 23). Ford et al. (10) reported a decrease in the frequency of antigen-specific IFN-γ producing CD4+ T-cells in malaria vaccine studies. According to the authors, the impairment affected mostly short-term IFNγ-producing effector memory CD4+ T-cells, while the potentially central memory cells seemed to be retained in cryopreserved PBMC. This could be consistent with our observations using porcine cells. Firstly, animals used in the present study were vaccinated against PRRSV one month before sampling, so it would be expectable to have recently developed memory cells (not long-lived yet). Secondly, the effector memory T-cells are thought to be the main source of IFN-γ among human CD4+ memory T-cells (revised by 24). Some preliminary evidence was also observed via in vitro re-stimulation of PBMC from PRRSV-infected pigs (25).
A pre-stimulation step before performing the assay has been suggested to improve the sensitivity of the recall responses in the IFN-γ ELISPOT, particularly using frozen cells (26). Interestingly, in a subsequent experiment in which we compared the performance of frozen PBMC from PRRSV-vaccinated pigs using pre-stimulation and no-pre-stimulation, we observed a significant increase in the frequency of PRRSV-specific IFN-γ-SC from two animals, while no improvement was observed from the rest (data not shown). Therefore, as in Smith et al. (26), the improvement observed performing the pre-stimulation step depended on the individual.
It is worth noting that, in our case, the proliferative response of double positive CD4/CD8α T-cells to the recall antigen was largely impaired when PBMC were frozen (
Figure 1C
). Porcine CD4/CD8 double positive cells account for a large proportion of memory cells (27), which have been demonstrated as the primary source of IFN-γ when responded to the PRRSV recall stimulation (28). The expression of CD27 further divides them into central (TCM, CD4+/CD8α+/CD27+) and effector memory T-cells (TEM, CD4+/CD8α+/CD27–) (27). As reported by Kick et al. (25), the response of memory cells might be associated with the clearance of PRRSV viremia when pigs were challenged. Upon in vitro re-stimulation, TCM proliferated at a higher level, while TEM are more potent in producing IFN-γ and TNF-α. A specific impairment of the function of these memory cells after freezing may undermine their role in response to different antigens, at least to PRRSV. But other studies on human PBMC indicated that freezing did not have a substantial effect on the recall responses (4–6). Further confirmation would require a specific analysis of the naïve, TCM and TEM compartments using animals at different times of immunisation (short- and long-term after antigen exposure).
When using mitogens for T-cells or polyclonal activation of B-cells, the picture was different (
Figures 2A
,
4
, respectively;
Supplementary Table 2
). The impact of freezing seemed to be of minor importance, if any, for polyclonally activated PBMC used in the IFN-γ of IgG ELISPOT. Li et al. (12) suggested that freezing of porcine PBMC could even result in a slight increase in the cytokine production upon PMA stimulation.
In our case, when PHA was used, the proliferation of T-cells was slightly affected. But the individual variation was noticeable, and in some cases, cells that were frozen showed even a higher proliferation (
Figure 1C
). In contrast, Koch et al. (11) reported a decreased proliferation, as measured by 3H-thymidine incorporation, but, in Koch’s study, PBMC were not rested after thawing. In others papers it has been shown that resting PBMC after thawing improved the proportion of T-cells, particularly CD4 (15).
In striking contrast, proliferation after stimulation with Con A was significantly impaired in cells that were frozen (
Figure 1C
). The precise mechanisms by which PHA and Con A induce T-cell proliferation are poorly understood. Besides being lectins and acting by crosslinking, activation of T-cells by Con A and PHA might happen via distinct molecular machinery. For example, CD28 was assumed to be a co-stimulatory signal during Con A stimulation (29), which has not been proved for PHA. The precise reason by which freezing affected cultures activated with one or the other lectin cannot be ascertained in the present study.
The last effect related to the freezing was a decrease in the proportion of CD21+ cells. Reimann et al. (3) determined that freezing resulted in a decrease of the proportion of CD19+ cells in PBMC from HIV patients. The causes of such loss were not resolved at that moment and are also unable to be determined through our results. But, in any case, the loss of CD21+ cells did not significantly affect the IgG ELISPOT.
To conclude, our results suggest that frozen porcine PBMC are mostly suitable for immunophenotyping and functional testing as long as mitogens are used. For recall antigen stimulation, freezing had a significant impact on the magnitude of the response, although responding animals could be identified. Nevertheless, same PBMC condition (fresh or frozen) should be used within a given study/trial to ensure comparability of the results. Given that freezing is a common treatment when working with porcine PBMC, it would be advisable to further characterize the response of naïve and memory cells at different times of the immune response.