Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells
Abstract
:1. Introduction
2. Results
2.1. PKC Isozyme Expression in CB CD3+ T Cells
2.2. PKC Isozyme Expression in CB CD4+ and CD8+ T Cells
2.3. Transient Nature of the PKC Isozyme Deficiencies in CBTC during Maturation
2.4. Relationship between CBTC PKC Isozyme Levels and Cytokine Production in the Matured Cells
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Ethics Statement
4.3. Preparation of Mononuclear Cells (MC) from CB and Peripheral Blood of Adult Donors
4.4. Isolation of T Cells
4.5. CBTC Maturation
4.6. Flow Cytometric Detection of PKC Isozymes
4.7. Measurement of Intracellular Cytokines
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Ono, Y.; Fujii, T.; Ogita, K.; Kikkawa, U.; Igarashi, K.; Nishizuka, Y. The structure, expression, and properties of additional members of the protein kinase C family. J. Biol. Chem. 1988, 263, 6927–6932. [Google Scholar] [CrossRef]
- Ono, Y.; Fujii, T.; Igarashi, K.; Kuno, T.; Tanaka, C.; Kikkawa, U.; Nishizuka, Y. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc. Natl. Acad. Sci. USA 1989, 86, 4868–4871. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reina-Campos, M.; Diaz-Meco, M.T.; Moscat, J. The Dual Roles of the Atypical Protein Kinase Cs in Cancer. Cancer Cell 2019, 36, 218–235. [Google Scholar] [CrossRef] [PubMed]
- Duran, A.; Diaz-Meco, M.T.; Moscat, J. Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation. EMBO J. 2003, 22, 3910–3918. [Google Scholar] [CrossRef]
- Harb, H.; Irvine, J.; Amarasekera, M.; Hii, C.S.; Kesper, D.A.; Ma, Y.; Ferrante, A. The role of PKCζ in cord blood T-cell maturation towards Th1 cytokine profile and its epigenetic regulation by fish oil. Biosci. Rep. 2017, 37, BSR20160485. [Google Scholar] [CrossRef] [Green Version]
- Martin, P.; Moscat, J. Th1/Th2 Differentiation and B Cell Function by the Atypical PKCs and Their Regulators. Front. Immunol. 2012, 3, 241. [Google Scholar] [CrossRef] [Green Version]
- Metz, P.J.; Arsenio, J.; Kakaradov, B.; Kim, S.H.; Remedios, K.A.; Oakley, K.; Chang, J.T. Regulation of Asymmetric Division and CD8+T Lymphocyte Fate Specification by Protein Kinase Cζ and Protein Kinase Cλ/ι. J. Immunol. 2015, 194, 2249–2259. [Google Scholar] [CrossRef] [Green Version]
- Prescott, S.L.; Irvine, J.; Dunstan, J.A.; Hii, C.; Ferrante, A. Protein kinase C zeta: A novel protective neonatal T-cell marker that can be upregulated by allergy prevention strategies. J. Allergy Clin. Immunol. 2007, 120, 200–206. [Google Scholar] [CrossRef]
- D’Vaz, N.; Ma, Y.; Dunstan, J.A.; Lee-Pullen, T.F.; Hii, C.; Meldrum, S.; Prescott, S.L. Neonatal protein kinase C zeta expression determines the neonatal T-Cell cytokine phenotype and predicts the development and severity of infant allergic disease. Allergy 2012, 67, 1511–1518. [Google Scholar] [CrossRef]
- Hii, C.S.; Costabile, M.; Mayne, G.C.; Der, C.J.; Murray, A.W.; Ferrante, A. Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells. Biochem. J. 2003, 370 Pt 2, 497–503. [Google Scholar] [CrossRef] [Green Version]
- Acevedo, N.; Alashkar Alhamwe, B.; Caraballo, L.; Ding, M.; Ferrante, A.; Garn, H.; van Esch, B.C. Perinatal and Early-Life Nutrition, Epigenetics, and Allergy. Nutrients 2021, 13, 724. [Google Scholar] [CrossRef]
- Perveen, K.; Quach, A.; McPhee, A.; Prescott, S.L.; Barry, S.C.; Hii, C.S.; Ferrante, A. Validation of monoclonal anti-PKC isozyme antibodies for flow cytometry analyses in human T cell subsets and expression in cord blood T cells. Sci. Rep. 2019, 9, 9263. [Google Scholar] [CrossRef]
- Prescott, S.L.; Macaubas, C.; Smallacombe, T.; Holt, B.J.; Sly, P.D.; Loh, R.; Holt, P.G. Reciprocal age-related patterns of allergen-specific T-cell immunity in normal vs. atopic infants. Clin. Exp. Allergy 1998, 28 (Suppl. 5), 39–44, discussion 50-1. [Google Scholar] [CrossRef]
- Perveen, K.; Quach, A.; McPhee, A.; Prescott, S.L.; Barry, S.C.; Hii, C.S.; Ferrante, A. Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively. Int. J. Mol. Sci. 2021, 22, 4907. [Google Scholar] [CrossRef]
- Ferrante, A.; Prescott, S. Immunological Immaturity of the Neonate, Protein Kinase C Zeta and Allergy. J. Neonatal Biol. 2014, 3, 106e. [Google Scholar] [CrossRef]
- Durgeau, A.; Virk, Y.; Corgnac, S.; Mami-Chouaib, F. Recent Advances in Targeting CD8 T-Cell Immunity for More Effective Cancer Immunotherapy. Front. Immunol. 2018, 9, 14. [Google Scholar] [CrossRef]
- Yu, Y.; Ma, X.; Gong, R.; Zhu, J.; Wei, L.; Yao, J. Recent advances in CD8+ regulatory T cell research. Oncol. Lett. 2018, 15, 8187–8194. [Google Scholar] [CrossRef]
- Hinks, T.S.C.; Hoyle, R.D.; Gelfand, E.W. CD8+Tc2 cells: Underappreciated contributors to severe asthma. Eur. Respir. Rev. 2019, 28, 190092. [Google Scholar] [CrossRef] [Green Version]
- Yu, W.; Zhou, X.; Dunham, D.; Lyu, S.C.; Manohar, M.; Zhang, W.; Nadeau, K. Allergen-specific CD8+ T cells in peanut-allergic individuals. J. Allergy Clin. Immunol. 2019, 143, 1948–1952. [Google Scholar] [CrossRef] [Green Version]
- Hinks, T.S.C.; Zhou, X.; Staples, K.J.; Dimitrov, B.D.; Manta, A.; Petrossian, T.; Djukanović, R. Innate and adaptive T cells in asthmatic patients: Relationship to severity and disease mechanisms. J. Allergy Clin. Immunol. 2015, 136, 323–333. [Google Scholar] [CrossRef] [Green Version]
- Wang, W.; Cheng, Z.S.; Chen, Y.F.; Lin, Y.H. Increased circulating IL-9-producing CD8+ T cells are associated with eosinophilia and high FeNO in allergic asthmatics. Exp. Ther. Med. 2016, 12, 4055–4060. [Google Scholar] [CrossRef] [PubMed]
- Stone, K.D.; Prussin, C.; Metcalfe, D.D. IgE, mast cells, basophils, and eosinophils. J. Allergy Clin. Immunol. 2010, 125 (Suppl. 2), S73–S80. [Google Scholar] [CrossRef] [PubMed]
- Gour, N.; Wills-Karp, M. IL-4 and IL-13 signaling in allergic airway disease. Cytokine 2015, 75, 68–78. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kumar, R.K.; Herbert, C.; Yang, M.; Koskinen, A.M.; McKenzie, A.N.; Foster, P.S. Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin. Exp. Allergy 2002, 32, 1104–1111. [Google Scholar] [CrossRef] [PubMed]
- Micossé, C.; von Meyenn, L.; Steck, O.; Kipfer, E.; Adam, C.; Simillion, C.; Schlapbach, C. Human “TH9” cells are a subpopulation of PPAR-γ+ TH2 cells. Sci. Immunol. 2019, 4, eaat5943. [Google Scholar] [CrossRef]
- Schmitt, E.; Germann, T.; Goedert, S.; Hoehn, P.; Huels, C.; Koelsch, S.; Rüde, E. IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma. J. Immunol. 1994, 153, 3989–3996. [Google Scholar]
- Dardalhon, V.; Awasthi, A.; Kwon, H.; Galileos, G.; Gao, W.; Sobel, R.A.; Kuchroo, V.K. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3− effector T cells. Nat. Immunol. 2008, 9, 1347–1355. [Google Scholar] [CrossRef] [Green Version]
- Veldhoen, M.; Uyttenhove, C.; van Snick, J.; Helmby, H.; Westendorf, A.; Buer, J.; Stockinger, B. Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat. Immunol. 2008, 9, 1341–1346. [Google Scholar] [CrossRef]
- Goswami, R.; Kaplan, M.H. A Brief History of IL-9. J. Immunol. 2011, 186, 3283–3288. [Google Scholar] [CrossRef] [Green Version]
- Renauld, J.-C. New insights into the role of cytokines in asthma. J. Clin. Pathol. 2001, 54, 577–589. [Google Scholar] [CrossRef] [Green Version]
- Hauber, H.P.; Bergeron, C.; Hamid, Q. IL-9 in allergic inflammation. Int. Arch. Allergy Immunol. 2004, 134, 79–87. [Google Scholar] [CrossRef]
- Soussi-Gounni, A.; Kontolemos, M.; Hamid, Q. Role of IL-9 in the pathophysiology of allergic diseases. J. Allergy Clin. Immunol. 2001, 107, 575–582. [Google Scholar] [CrossRef]
- Liu, G.; Qian, L.; Xu, T.; Yu, J.; Li, M.; Cui, Y. Changes in the Th9 cell population and related cytokines in the peripheral blood of infants with recurrent wheezing. Cent. Eur. J. Immunol. 2020, 45, 60–68. [Google Scholar] [CrossRef]
- Petit-Frere, C.; Dugas, B.; Braquet, P.; Mencia-Huerta, J. Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes. Immunology 1993, 79, 146. [Google Scholar]
- Temann, U.-A.; Geba, G.P.; Rankin, J.A.; Flavell, R.A. Expression of Interleukin 9 in the Lungs of Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness. J. Exp. Med. 1998, 188, 1307–1320. [Google Scholar] [CrossRef]
- Matsuzawa, S.; Sakashita, K.; Kinoshita, T.; Ito, S.; Yamashita, T.; Koike, K. IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor. J. Immunol. 2003, 170, 3461–3467. [Google Scholar] [CrossRef] [Green Version]
- Sehra, S.; Yao, W.; Nguyen, E.T.; Glosson-Byers, N.L.; Akhtar, N.; Zhou, B.; Kaplan, M.H. TH9 cells are required for tissue mast cell accumulation during allergic inflammation. J. Allergy Clin. Immunol. 2015, 136, 433–440.e1. [Google Scholar] [CrossRef] [Green Version]
- Louahed, J.; Kermouni, A.; Van Snick, J.; Renauld, J.-C. IL-9 induces expression of granzymes and high-affinity IgE receptor in murine T helper clones. J. Immunol. 1995, 154, 5061–5070. [Google Scholar]
- Gounni, A.S.; Gregory, B.; Nutku, E.; Aris, F.; Latifa, K.; Minshall, E.; Hamid, Q. Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils. Blood J. Am. Soc. Hematol. 2000, 96, 2163–2171. [Google Scholar]
- Dong, Q.; Louahed, J.; Vink, A.; Sullivan, C.D.; Messler, C.J.; Zhou, Y.; Nicolaides, N.C. IL-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice. Eur. J. Immunol. 1999, 29, 2130–2139. [Google Scholar] [CrossRef]
- Alaskhar Alhamwe, B.; Khalaila, R.; Wolf, J.; von Bülow, V.; Harb, H.; Alhamdan, F.; Potaczek, D.P. Histone modifications and their role in epigenetics of atopy and allergic diseases. Allergy Asthma Clin. Immunol. 2018, 14, 39. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Cytokines | PKCα | PKCβ1 | PKCβ2 | PKCδ | PKCε | PKCη | PKCθ | PKCμ | PKCζ | PKCλ/ι |
---|---|---|---|---|---|---|---|---|---|---|
IFN-γ (%) | 0.5604 | 0.1782 | 0.4377 | 0.7085 * | 0.4995 | 0.5377 | 0.1967 | 0.3937 | 0.7019 * | 0.0939 |
IFN-γ (MFI) | 0.2106 | 0.1976 | 0.1796 | 0.3579 | 0.3686 | 0.1659 | 0.5463 | −0.0134 | 0.6768 * | −0.1691 |
IL-2 (%) | 0.2204 | 0.3329 | 0.1008 | 0.3092 | 0.2727 | 0.1757 | 0.3761 | −0.0376 | 0.5745 | −0.2421 |
IL-2 (MFI) | 0.2446 | 0.1459 | 0.2505 | 0.4598 | 0.2192 | 0.2446 | 0.4263 | 0.2310 | 0.7079 * | −0.0340 |
TNF (%) | 0.3748 | 0.2478 | 0.2076 | 0.4679 | 0.3332 | 0.2666 | −0.0105 | 0.0986 | 0.5599 | −0.0989 |
TNF (MFI) | 0.3008 | 0.3182 | 0.1975 | 0.4791 | 0.3996 | 0.2009 | 0.3994 | 0.0351 | 0.7659 ** | −0.1134 |
LT-α (%) | 0.3346 | 0.0347 | 0.2112 | 0.3929 | 0.3334 | 0.1884 | −0.2018 | 0.1209 | 0.3434 | 0.0537 |
LT-α (MFI) | −0.0937 | 0.0765 | −0.2068 | −0.0380 | −0.0044 | −0.1691 | −0.2348 | −0.3628 | 0.2329 | −0.4143 |
IL-4 (%) | 0.4928 | 0.2634 | 0.2714 | 0.6645 * | 0.4175 | 0.3514 | −0.0338 | 0.2853 | 0.5840 | 0.0918 |
IL-4 (MFI) | 0.2515 | 0.2140 | 0.0579 | 0.4423 | 0.1989 | 0.0969 | −0.1759 | 0.0935 | 0.4623 | −0.1301 |
IL-5 (%) | 0.2875 | 0.5194 | 0.1459 | 0.0834 | 0.5722 | 0.1974 | 0.4155 | −0.2627 | 0.2405 | 0.0372 |
IL-5 (MFI) | −0.0224 | 0.1130 | −0.1156 | −0.0436 | 0.3052 | −0.1455 | 0.1579 | −0.3850 | 0.0816 | −0.1837 |
IL-9 (%) | −0.4542 | −0.4489 | −0.3473 | −0.6409 * | −0.4878 | −0.3095 | −0.4324 | −0.1984 | −0.8345 ** | −0.03258 |
IL-9 (MFI) | −0.5337 | −0.6502 | −0.4375 | −0.6236 | −0.5397 | −0.4115 | −0.4924 | −0.1557 | −0.881 *** | −0.1508 |
IL-10 (%) | −0.2731 | −0.3727 | −0.1408 | −0.4348 | −0.2676 | −0.0802 | −0.0477 | −0.0500 | −0.6230 | 0.1204 |
IL-10 (MFI) | −0.4495 | −0.3071 | −0.2806 | −0.5754 | −0.4010 | −0.3320 | −0.1614 | −0.2134 | −0.6725 * | 0.0095 |
IL-13 (%) | −0.1708 | −0.1348 | −0.1926 | −0.1691 | −0.0013 | −0.0055 | 0.4277 | −0.1217 | −0.2527 | −0.1113 |
IL-13 (MFI) | −0.3522 | −0.3389 | −0.3298 | −0.4816 | −0.3356 | −0.3757 | 0.0836 | −0.2117 | −0.5320 | −0.0616 |
IL-17 (%) | −0.083 | −0.5377 | 0.0448 | −0.2234 | −0.0655 | 0.1499 | 0.0774 | 0.1560 | −0.5995 | 0.2036 |
IL-17 (MFI) | 0.2200 | 0.6979 * | 0.1645 | −0.0019 | 0.3411 | 0.0338 | 0.1120 | −0.3631 | 0.4925 | −0.0400 |
L-21 (%) | 0.5002 | 0.3347 | 0.2669 | 0.4598 | 0.5308 | 0.3461 | −0.0795 | 0.1010 | 0.5185 | 0.1458 |
IL-21 (MFI) | 0.0205 | −0.2084 | −0.0434 | 0.04250 | −0.1240 | −0.0438 | −0.3547 | 0.1681 | 0.0046 | −0.0509 |
IL-22 (%) | 0.0561 | −0.0125 | 0.0720 | 0.2144 | 0.2900 | 0.08116 | 0.7090 * | −0.03731 | 0.4114 | −0.2888 |
IL-22 (MFI) | 0.2119 | 0.4719 | −0.0041 | 0.3309 | 0.3908 | 0.04281 | 0.0788 | −0.2496 | 0.4745 | −0.2407 |
TGF-β (%) | 0.2417 | −0.6425 | 0.3993 | 0.1699 | 0.2979 | 0.5000 | 0.5319 | 0.5087 | −0.291 | 0.3904 |
TGF-β (MFI) | 0.1783 | −0.0354 | 0.2476 | −0.0299 | 0.4619 | 0.1653 | 0.5937 | −0.1791 | 0.3515 | 0.0906 |
Antibody (Clone) | Fluorochrome | Cat/Company |
---|---|---|
Anti-PKCα (H-7) | AF647 | sc-8393/Santa Cruz |
Anti-PKCβII (F-7) | AF647 | sc-13149/Santa Cruz |
Anti-PKCβ1 (EPR18512) | AF647 | ab223452/Abcam |
Anti-PKCζ (H-1) | PE | sc-17781/Santa Cruz |
Anti-PKCθ (E-7) | PE | sc-1680/Santa Cruz |
Anti-PKCλ/ι (H-12) | PE | sc-17837/Santa Cruz |
Anti-PKCη (EPR18513) | AF488 | ab179524/Abcam |
Anti-PKCδ (EPR17075) | AF488 | ab206282/Abcam |
Anti-PKCε (EPR1482) | AF488 | ab217980/Abcam |
Anti-PKCμ (EP1493Y) | AF647 | ab51246/Abcam |
Mouse IgG1k Isotype control (MOPC-31C) | AF647 | 566011/BD |
Rabbit mAb IgG Isotype Control | AF488 | 2975/CST |
Rabbit mAb IgG Isotype Control | AF647 | 2985/CST |
Mouse mAb IgG2ak (X39) | PE | 340459/BD |
Anti-CD3 (SK7) | APC-H7 | 560176/BD |
Anti-CD8 (RPA-T8) | PE-Cy7 | 557746/BD |
Antibody (Clone) | Fluorochrome | Cat/Company |
---|---|---|
Anti-IL-2 (5344.111) | BV421 | 562914/BD |
Anti-IL-10 (JES3-9D7) | AF488 | 501413/BioLegend |
Anti-LT-α (359-81-11) | PE | 554556/BD |
Anti-IL-17A (N49-653) | PerCP-Cy 5.5 | 560799/BD |
Anti-TGF-βI (TW4-2F8) | PE/Cy7 | 349610/BioLegend |
Anti-TNF (MAb11) | APC | 554514/BD |
Anti-IFN-γ (4S.B3) | APC/Cy7 | 502530/BioLegend |
BV510 (viability stain) | 564406/BD |
Antibody (Clone) | Fluorochrome | Cat/Company |
---|---|---|
Anti-IL-13 (JES10-5A2) | BV421 | 563580/BD |
Anti-IL-4 (MP4-25D2) | FITC | 562047/BD |
Anti-IL-21 (3A3-N2.1) | PE | 562042/BD |
Anti-IL-9 (MH9A3) | PerCP-Cy 5.5 | 561461/BD |
Anti-IL-5 (TRFK5) | APC | 562048/BD |
Anti-IL-22 (2G12A41) | PE/Cy7 | 366707/BioLegend |
Anti-IFN-γ (4S.B3) | APC/Cy7 | 502530/BioLegend |
BV510 (viability stain) | 564406/BD |
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Perveen, K.; Quach, A.; Stark, M.J.; Prescott, S.L.; Barry, S.C.; Hii, C.S.; Ferrante, A. Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells. Int. J. Mol. Sci. 2021, 22, 12650. https://doi.org/10.3390/ijms222312650
Perveen K, Quach A, Stark MJ, Prescott SL, Barry SC, Hii CS, Ferrante A. Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells. International Journal of Molecular Sciences. 2021; 22(23):12650. https://doi.org/10.3390/ijms222312650
Chicago/Turabian StylePerveen, Khalida, Alex Quach, Michael J. Stark, Susan L. Prescott, Simon C. Barry, Charles S. Hii, and Antonio Ferrante. 2021. "Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells" International Journal of Molecular Sciences 22, no. 23: 12650. https://doi.org/10.3390/ijms222312650