Abstract:
Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2 and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. In order to identify dolphin Treg cells, we tested a battery of antibodies to CD4, CD25 and FOXP3. In order to determine the functionality of dolphin Treg cells, we validated the use of human and porcine reagents to measure TGFß, the major Treg effector cytokine. In order to assess if the proportion of peripheral blood FOXP3+ dolphin lymphocytes were representative of Treg function we compared the number of FOXP3+ lymphocytes with the levels of serum TGFß and IL-10, the two major Treg effector cytokines, in each dolphin. In order to assess the capacity of dolphin Th cell subsets to respond to a Th1, Th2 or Treg stimulus, we tested the relative expression of effector cytokines upon in vitro stimulation with human recombinant cytokines. To understand the balance of cytokines in wild dolphins, we measured serum concentrations of 12 cytokines including the Th1 cytokines IL-2, IL-12 and IFNγ, the Th2 cytokines IL-4, IL-5 and IL-13, the Treg cytokines IL-10 and TGFß, and the inflammatory cytokines IL-1ß, IL-8, TNFα and GM-CSF, in 20 wild Sarasota dolphins.
This dataset contains information related to the percentage of total cells gated during flow cytometry based on antibody labeling, and the relative fluorescence from cells which have been labeled with different quantities of antibody A and B. It also includes the data about the various treatments of cells, various forms of Lymphocyte proliferation brought about by differing culturing conditions and Cytokine levels in pg/ml, and the information on Dolphin lymphocytes treated with cytokines associated with either a Th1, Th2, or Treg stimulus. Th1 = IL-12 and IFNy. Th2 = IL-4. Treg = IL-2. Furthermore, it contains data on gene expression via the comparative method.
* A revision to this dataset includes corrections to some ND and No amp cell values. No changes were made to data points with measured, numerical values.
Data Parameters and Units:
The worksheet “Immunophenotyping.gate” contains: File.Name (the unique laboratory identifier); Subject (sample origin; Entries starting with "FB" refer to the ID for a wild bottlenose dolphin in Sarasota, Florida); Antibody (a label used for flow cytometry); Percent.gated (Percent of total cells gated during flow cytometry based on antibody labeling).
The worksheet “Immunophenotyping.quadrant” contains: File.Name (the unique laboratory identifier); Subject (sample origin); Antibody (label used for flow cytometry); Quadrant.1_upper.left (the relative fluorescence from cells with relatively low antibody A labeling and relatively high antibody B labeling from two-color flow cytometry); Quadrant.2_upper.right (the relative fluorescence from cells with relatively high antibody A labeling and relatively high antibody B labeling from two-color flow cytometry); Quandrant.3_lower.left (the relative fluorescence from cells with relatively low antibody A labeling and relatively low antibody B labeling from two-color flow cytometry); Quadrant.4_lower.right (the relative fluorescence from cells with relatively high antibody A labeling and relatively low antibody B labeling from two-color flow cytometry).
The worksheets “TGFb.pig" & "TGFb.human” contains: Type (refers to Internal code for laboratory plate reader set up. B = blank. S = Standard curve. X = experimental sample); Wells (the well locations on 96-well plate); Subject (refers to sample origin; Entries starting with "FB" refer to the ID for a wild bottlenose dolphin in Sarasota, Florida); Treatment is Mitogen treatment of cells (NM = No mitogen. sub LPS = suboptimal LPS (0.05 micro gram /ml) pre-incubation. opt LPS = optimal LPS (5.0 µg/ml) pre-incubation. LPS (in pigs) = suboptimal LPS (0.1 micro gram /ml) pre-incubation. 1:4 = sample diluted four-fold.); TGF.beta.one_Obs.Conc_pg/ml (refers to Observed concentration of TGF beta 1 (pg/ml). OOR< = Out of Range Below).
The worksheet “Sarasota.2018.immune” contains: Subject (refers to Sample origin from a wild bottlenose dolphin in Sarasota, FL); Lymp.prolif_NM refers to Lymphocyte proliferation without mitogen stimulation (optical density reading from cell proliferation ELISA BrdU assay [OD]); Lymp.prolif_0.1ConA refers to Lymphocyte proliferation with 0.1 microgram /ml ConA; Lymp.prolif_1ConA refers to Lymphocyte proliferation with 1 microgram/ml ConA; Lymp.prolif_0.1PHA refers to Lymphocyte proliferation with 0.1 microgram /ml PHA; Lymp.prolif_1PHA refers to Lymphocyte proliferation with 1 microgram/ml PHA; Lymp.prolif_0.05LPS refers to Lymphocyte proliferation with 0.05 microgram /ml LPS; Lymp.prolif_5LPS refers to Lymphocyte proliferation with 5 microgram/ml LPS; PCL_net.FOXP3+lymp refers to Net FOXP3-positive lymphocytes as a proportion of cells labeled; PCL_net.CD4+/FOXP3+lymp refers to Net percent CD4-positive cells that are FOXP3-positive; PCL_CD4+lymp refers to Percent lymphocytes that are CD4-positive.; IL5_pg/ml, IL10_pg/ml, IL12_pg/ml, IL13_pg/ml, GMCSF_pg/ml, IFNg_pg/ml, TNFa_pg/ml refer to Cytokine levels in pg/ml (Measured using a human kit).; TGFb_pg/ml, IL1b_pg/ml, IL4_pg/ml, IL8_pg/ml refer to Cytokine levels in pg/ml. Measured using porcine reagents.
The worksheet “Sarasota.2018.PCR” contains: PCR.ID (refers to Laboratory identification number for PCR assay); Subject (refers to Sample origin from a wild bottlenose dolphin in Sarasota, FL).; Treatment refers to Dolphin lymphocytes were treated with cytokines associated with either a Th1, Th2, or Treg stimulus. Th1 = IL-12 and IFNy. Th2 = IL-4. Treg = IL-2.; Treatment.conc_ng/ml is Treatment concentration in ng/ml; IFNy_Ct, IL13_Ct, TGFb_Ct, IL4_Ct, IL10_Ct, HPRT1.54_Ct, HPRT1.63_Ct, S9.54_Ct, S9.63_Ct refer to Gene expression via the Comparative Ct method. No Amp = No amplification during PCR.; HPRT1.54_flag, HPRT1.63_flag, S9.54_flag and S9.63_flag refer to A warning that the housekeeping gene expression level is above the expected range, in which case, the data for the gene of interest is rejected.
Notes: NA = Not applicable; ND no data collected; ConA = concanavalin A; PHA= phytohemagglutinnin A; LPS= lipopolysaccharide; sub = suboptimal; opt = optimal.
Methods:
CD4 and CD25: The fluorescence of approximately 10,000 lymphocytes was read using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) and the automated CellQuest software (Becton Dickinson Immunocytometry System, San Jose, CA 95131, USA).
FOXP3: The fluorescence of approximately 10,000 lymphocytes was read using a BD Biosciences LSRFortessa X-20 Cell Analyzer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) and the FACSDiva software (Becton Dickinson Immunocytometry System, San Jose, CA 95131, USA).
TGFb: Tissue culture supernatant concentrations of TGFß following a 48 hour stimulation of porcine (n=3) or dolphin (n=5 for human reagents and n=6 for porcine reagents) PBMCs with LPS at sub-optimal (0.05 µg/ml for dolphin and 0.1µg/ml for pigs) and optimal (5.0 µg/ml for dolphin) concentrations. TGFß concentrations were measured using the Luminex platform and either porcine reagents (TGFb.pig) or human reagents (TGFb.human).
Cytokines: After the incubation and conjugation process, the plates were measured on the Bio-Plex 200 system (Bio-Rad, Hercules, CA 94547, USA), and analyzed using Bio-Rad Manager 5.0. The observed concentration (pg/ml or ng/ml) of each analyte for each sample was calculated using a curve fit generated for each analyte from seven or eight standards (depending on kit). If a sample concentration was extrapolated outside the standard curve and designated as “Value extrapolated beyond standard range” by the software, that sample concentration was accepted as the calculated value. If a sample concentration was reported as “out of range” by the software, that sample concentration was given a 0 pg/ml or 0 ng/ml value or the highest value on the standard curve, depending on whether it was below or above the measurable range. Prior to each use of the Bio-Plex 200 system, instrument calibration and validation procedure using the Bio-Rad Validation and Calibration kit (Bio-Rad, Hercules, CA 94547, USA) was performed to assure the instrument was performing properly, as per manufacturer’s instruction. The instrument passed both calibration and validation tests prior to each use.
Gene expression: Real-time PCR (qRT-PCR) reactions were performed using SYBR green (Thermo Fisher Scientific, Grand Island, NY 14072, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA 94547, USA). All samples were analyzed for housekeeping genes HPRT1 and S-9. Reactions containing water, but no cDNA, were used as negative controls. Product specificity was monitored by analysis of melting curves. Gene expression data were analyzed using the Comparative CT (ddCT) Method. Samples for which the amplification of the housekeeping genes was outside of the expected range were discarded, so as to not misinterpret a change in the expression of a target gene as an inadequate PCR reaction.
Lymphocyte proliferation: Lymphocyte proliferation was evaluated by the incorporation of 5-bromo-2’-deoxyuridine (BrdU), a thymidine analogue, detected with a monoclonal antibody and colorimetric enzymatic reaction (Cell Proliferation ELISA BrdU (colorimetric), Roche Diagnostics GmbH, Mannheim, Germany) as per manufacturer’s instructions using an ELISA plate reader (Multiskan EX v.1.0) at 450 nm with a reference wavelength of 690 nm. Results were expressed as optical density (OD). Cells from mice (one mouse for each experimental day) were assayed concurrently with dolphin samples for quality control. After field sampling, mouse data were assessed for the presence of outliers using the SPSS software (IBM SPSS Statistics version 21, Armonk, NY 10504, USA). If outliers were detected, it was assumed that normal daily variability for the assay was exceeded, and the corresponding dolphin data for that assay on that day were eliminated from the dataset.
Some bottlenose dolphins living in Sarasota Bay were captured, sampled and released after blood and serum samples were taken.
Details on the medium used: Dolphin, human, porcine, bovine, and ovine whole blood was diluted 1:1 with phosphate buffered saline (PBS) with 2 mM EDTA (Miltenyi, Auburn, CA 95602, USA), layered on top of an equal volume of Ficoll-Paque Plus 1.077 (GE, Pittsburgh, PA 15264, USA), and centrifuged for 40 minutes at 400 g. The peripheral blood mononuclear cell (PBMC) layer was collected, washed twice with Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Grand Island, NY 14072, USA) supplemented with 1mM sodium pyruvate, 100 mM non-essential amino acids, 25 mM HEPES, 2 mM L-glutamine, 100 U/mL, penicillin, 100 mg/mL streptomycin and 0.25 mg/mL Fungizone (all from Thermo Fisher Scientific, Grand Island, NY 14072, USA), along with 10% fetal bovine serum (Hyclone, Logan, UT 84321, USA), hereafter referred to as complete DMEM, and cells were enumerated with their viability assessed using the exclusion dye trypan blue (Life Technologies, Grand Island, NY 14072, USA). Cell viability was typically greater than 90%. PBMC isolation after Ficoll-Paque centrifugation was confirmed using a BD FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) using side forward scatter (relative cell size) and side scatters (relative cell complexity) settings to assess the proportion of PBMCs in the sample. Starting concentrations of PBMCs were as follows: 1 x 106 cells for CD4 labeling, intracellular FOXP3 labeling; 2 x 105cells/well for CD25 labeling, BioPlex assays, and lymphocyte stimulation assays; 1 x 104 cells for FACs fluorescence.
Instruments:
FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) and the automated CellQuest software (Becton Dickinson Immunocytometry System, San Jose, CA 95131, USA).
BD Biosciences LSRFortessa X-20 Cell Analyzer (Becton Dickinson, Franklin Lakes, NJ 07417, USA) and the FACSDiva software (Becton Dickinson Immunocytometry System, San Jose, CA 95131, USA).
Bio-Plex 200 system (Bio-Rad, Hercules, CA 94547, USA), and analyzed using Bio-Rad Manager 5.0. CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA 94547, USA).
ELISA plate reader (Multiskan EX v.1.0) and SPSS software (IBM SPSS Statistics version 21, Armonk, NY 10504, USA).
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