Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection
<p>mAb 5F12 specifically recognizes recombinant and natural porcine CD69 protein. (<b>A</b>) Recombinant porcine CD69, expressed in pcDNA3.1-poCD69-transfected HEK-293T cells, was detected by mAb 5F12 against porcine CD69. The CD69-positive cells (white arrows) and negative cells were magnified. Scale bars, 40 μm. (<b>B</b>) mAb 5F12 specifically recognizes recombinant porcine CD69 in the cell lysates of pcDNA3.1-poCD69-transfected HEK-293T cells but not in pcDNA3.1-transfected or un-transfected HEK-293T cells (Mock) by Western blotting. (<b>C</b>,<b>D</b>) The expression of CD69 on pig lymphocytes was detected by mAb 5F12 and polyclonal anti-poCD69 (pAb) antibodies, followed by Alexa Fluor 488-labeled goat anti-mouse-IgG antibody, via flow cytometry after stimulation with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h. These CD69<sup>+</sup> lymphocytes were gated on lymphocytes within the PBMCs of pigs. *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 2
<p>The identification of the epitope recognized by mAb 5F12 against CD69 by dot-ELISA. (<b>A</b>) Three overlapping peptides covering the CD69 polypeptide (aa 133–161) were synthesized and named Pep 1 (aa 133–147), Pep 2 (aa 140–154), Pep 3 (aa 147–161), an extra cysteine was added at the N terminus of each peptide for the conjugation of KLH. (<b>B</b>) These peptides were conjugated to KLH carrier and their reactivity with anti-poCD69 mAb 5F12 was determined by dot-ELISA.</p> "> Figure 3
<p>Anti-poCD69 mAb 5F12 shows weak or no cross-reactivity with bovine, mouse, and chicken CD69. (<b>A</b>) Homologous comparison of CD69 polypeptide (aa 133–161) in pig samples and human, mouse, and bovine samples. The percentage of homology was indicated. (<b>B</b>) The cross-reactivities of Dylight<sup>®</sup>755-conjugated anti-poCD69 mAb 5F12 with PBMCs from mice, cows, and chickens were examined by flow cytometry after the in vitro stimulation of PBMCs with or without PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h (Unstim vs. PMA/Iono). The CD69<sup>+</sup> lymphocytes were gated on lymphocytes within PBMCs. The data shown are representative of three independent experiments.</p> "> Figure 4
<p>Anti-poCD69 mAb 5F12 is valid for the detection of CD69 expression on different leukocyte subsets of pigs. PBMCs from healthy pigs were stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 6 h, then stained with a cocktail of antibodies containing Dylight<sup>®</sup>755-CD69, anti-porcine CD3, CD8α, CD4, and CD21 or a cocktail containing Dylight<sup>®</sup>755-CD69, SLA-II DR, CD163, and CD172a. The expression of CD69 on CD21<sup>+</sup> B cells (gated on lymphocytes) and T cell subsets (CD4<sup>+</sup>, CD8<sup>+</sup>, CD4<sup>+</sup>CD8<sup>+</sup>, gated on CD3<sup>+</sup> T lymphocytes), NK cells (gated on CD21<sup>−</sup>CD3<sup>−</sup>CD8α<sup>+</sup> cells), monocytes/macrophages (MHCII<sup>+</sup>CD163<sup>+</sup>), and neutrophils (MHCII<sup>−</sup>CD163<sup>−</sup>CD172<sup>+</sup>) was determined by flow cytometry. Monocytes/macrophages and neutrophils were gated on leukocytes, excluding lymphocytes.</p> "> Figure 5
<p>The detection of CD69 expression on T cells in different organs after PRRSV immunization and infection. PRRSV-free piglets were immunized intranasally and intramuscularly with the rJS-ORF2-6-CON vaccine or were administered RPMI-1640 (as the control), then challenged with a virulent PRRSV NADC30-like SD17-38 isolate on day 42 post-immunization. Peripheral blood, mediastinal lymph nodes (mLN), and spleens were harvested on days 7 and/or 14 post-challenge (dpc) and single-cell suspensions were prepared for the detection of CD69 expression on T cell subsets via flow cytometry. (<b>A</b>) Representative pseudocolor plots (left panel) and the dynamic changes (right panel) of total CD69<sup>+</sup> lymphocytes in the PBMCs of unvaccinated (<span class="html-italic">n</span> = 3) and immunized pigs (<span class="html-italic">n</span> = 3) after challenge. (<b>B</b>,<b>C</b>) Representative dot-plots (left panel) and the comparison (right panel) of CD69<sup>+</sup>CD4<sup>+</sup> and CD69<sup>+</sup>CD8<sup>+</sup> T cells in the mLNs and spleens of unvaccinated (<span class="html-italic">n</span> = 3) and immunized pigs (<span class="html-italic">n</span> = 3) at 14 dpc. Data shown are mean ± SEM. ns, no statistical significance. * <span class="html-italic">p</span> < 0.05.</p> "> Figure 6
<p>ASFV infection induces the early activation of different lymphocyte subsets in the blood and lymphoid organs of pigs. Healthy piglets were infected or not infected with the ASFV HLJ/18 strain by intramuscular injection. Peripheral blood, spleens, and submandibular lymph nodes were collected on day 5 after infection and single-cell suspensions were prepared for the detection of CD69 expression on different lymphocyte subsets, using flow cytometry. The percentages of CD69-positive NK cells, B cells, CD8, CD4, and CD4<sup>+</sup>CD8<sup>+</sup> T cells, as well as γδ T cells in the PBMCs (<b>A</b>), spleen (<b>B</b>), and submandibular lymph node cells (<b>C</b>) were compared between mock-infected and ASFV-infected pigs (<span class="html-italic">n</span> = 3). Data shown are mean ± SEM. ns = no statistical significance. ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals, Cells, and Virus Strains
2.2. Porcine CD69 Polypeptide Immunization and Cell Fusion
2.3. Hybridoma Screening, Ascites Production, Purification, and Fluorescence Labeling of CD69 mAb
2.4. Cloning and Construction of pcDNA3.1-poCD69 Plasmid
2.5. Transfection and Indirect Immunofluorescence Assay
2.6. Western Blotting
2.7. Dot-ELISA
2.8. Infection Experiments with PRRSV and ASFV
2.9. Single-Cell Suspension Preparation and Culture
2.10. Flow Cytometry
2.11. Statistical Analysis
3. Results
3.1. Generation and Characterization of Monoclonal Antibodies against Pig CD69
3.2. Anti-Pig CD69 mAb 5F12 Recognizes Epitope Residues 147–161 of Pig CD69
3.3. mAb 5F12 Shows Weak or No Cross-Reactivity with PBMCs from Bovine, Mice, and Chickens
3.4. Detection of CD69 Expression on Different Leukocyte Subsets by Flow Cytometry
3.5. Detection of CD69 Expression on Lymphocytes in Pigs after Challenge with PRRSV
3.6. Detection of CD69 Expression on Different Lymphocyte Subsets in Pigs after ASFV Infection
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Peptides | Sequence | Start | End | Length * |
---|---|---|---|---|
CD69-peptide | CLKQHVGRAEHWIGLKNEDGQTWKWSNGKE | 133 | 161 | 30 |
Pep 1 | CLKQHVGRAEHWIGLK | 133 | 147 | 16 |
Pep 2 | CAEHWIGLKNEDGQTW | 140 | 154 | 16 |
Pep 3 | CKNEDGQTWKWSNGKE | 147 | 161 | 16 |
Antigen | Clone | Isotype | Conjugate | Source |
---|---|---|---|---|
CD3 | BB23-8E6-8C8 | Mouse IgG2a | PerCP-cy5.5 | BD |
CD4 | 74-12-4 | Mouse IgG2b | PE-Cy™7 | BD Pharmingen |
CD8α | 76-2-11 | Mouse IgG2a | biotin | Southernbiotech |
CD21 | BB6-11C9.6 | Mouse IgG1 | AlexaFluor®488 | Southernbiotech |
γδTCR | MAC320 | Rat PVG IgG2a | PE | BD |
CD69 | 5F12 | Mouse IgG1 | Dylight®755 | in-house |
CD163 | 2A10/11 | Mouse IgG1 | RPE | Bio-rad |
CD172a | 74-22-15 | Mouse IgG1 | FITC | Southernbiotech |
SLA II DR | 2E9/13 | Mouse IgG2b | APC | Bio-rad |
Mouse IgG | Poly4053 | Goat polyclonal IgG | AlexaFluor®488 | Biolegend |
Streptavidin | — | — | BV 510™ | Biolegend |
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Tian, Y.; Hao, Y.; Dong, M.; Li, S.; Wang, D.; Jiang, F.; Wang, Q.; Hao, X.; Yang, Y.; Chen, N.; et al. Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection. Viruses 2022, 14, 1343. https://doi.org/10.3390/v14061343
Tian Y, Hao Y, Dong M, Li S, Wang D, Jiang F, Wang Q, Hao X, Yang Y, Chen N, et al. Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection. Viruses. 2022; 14(6):1343. https://doi.org/10.3390/v14061343
Chicago/Turabian StyleTian, Yunfei, Yuxin Hao, Maoli Dong, Shuai Li, Dongyue Wang, Fei Jiang, Qingqing Wang, Xiaoli Hao, Yi Yang, Nanhua Chen, and et al. 2022. "Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection" Viruses 14, no. 6: 1343. https://doi.org/10.3390/v14061343