{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T19:03:12Z","timestamp":1791226992703,"version":"4.1.0"},"reference-count":52,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T00:00:00Z","timestamp":1628121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100005825","name":"National Institute of Food and Agriculture","doi-asserted-by":"publisher","award":["2015-68004-23131"],"award-info":[{"award-number":["2015-68004-23131"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>While the protective efficacy of the infectious laryngotracheitis virus (ILTV) vaccines is well established, little is known about which components of the immune response are associated with effective resistance and vaccine protection. Early studies have pointed to the importance of the T cell-mediated immune responses. This study aimed to evaluate the activation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and to quantify the presence of regulatory T cells (Tregs) in the larynx\u2013trachea of chickens vaccinated with chicken embryo origin (CEO), tissue culture origin (TCO) and recombinant Herpesvirus of Turkey-laryngotracheitis (rHVT-LT) vaccines after challenge. Our results indicated that CEO vaccine protection was characterized by early CTLs and activated CTLs enhanced responses. TCO and rHVT-LT protection were associated with a moderate increase in resting and activated CTLs followed by an enhanced NK cell response. Tregs increase was only detected in the non-vaccinated challenged group, probably to support healing of the severe trachea epithelial damage. Taken together, our results revealed main differences in the cellular immune responses elicited by CEO, TCO, and rHVT-LT vaccination in the upper respiratory tract after challenge, and that activated CTLs rather than NK cells play a main role in vaccine protection.<\/jats:p>","DOI":"10.3390\/vaccines9080865","type":"journal-article","created":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T09:35:32Z","timestamp":1628156132000},"page":"865","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Activation of Cytotoxic Lymphocytes and Presence of Regulatory T Cells in the Trachea of Non-Vaccinated and Vaccinated Chickens as a Recall to an Infectious Laryngotracheitis Virus (ILTV) Challenge"],"prefix":"10.3390","volume":"9","author":[{"given":"Daniel","family":"Maekawa","sequence":"first","affiliation":[{"name":"Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylva M.","family":"Riblet","sequence":"additional","affiliation":[{"name":"Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick","family":"Whang","sequence":"additional","affiliation":[{"name":"Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2108-4496","authenticated-orcid":false,"given":"David J.","family":"Hurley","sequence":"additional","affiliation":[{"name":"Food Animal Health and Management Program, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2175-0287","authenticated-orcid":false,"given":"Maricarmen","family":"Garcia","sequence":"additional","affiliation":[{"name":"Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Swayne, D.E., Boulianne, M., Logue, C.M., McDougald, L.R., Nair, V., and Suarez, D.L. (2020). Infectious laryngotracheitis. Diseases of Poultry, Wiley-Blackwell. [14th ed.].","DOI":"10.1002\/9781119371199"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1080\/03079457.2019.1644449","article-title":"Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination","volume":"48","author":"Maekawa","year":"2019","journal-title":"Avian Pathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1080\/03079457.2011.631983","article-title":"Protection induced by commercially available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler chickens","volume":"41","author":"Vagnozzi","year":"2012","journal-title":"Avian Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1637\/10383-092412-Reg.1","article-title":"Safety and efficacy of a turkey herpesvirus vector laryngotracheitis vaccine for chickens","volume":"57","author":"Esaki","year":"2013","journal-title":"Avian Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"348","DOI":"10.2307\/1591188","article-title":"Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage","volume":"35","author":"Guy","year":"1991","journal-title":"Avian Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1637\/8011-041907-REGR.1","article-title":"Replication and transmission of live attenuated infectious laryngotracheitis virus (ILTV) vaccines","volume":"51","author":"Oldoni","year":"2007","journal-title":"Avian Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2352","DOI":"10.1099\/jgv.0.000521","article-title":"Attenuation and protection efficacy of ORF C gene-deleted recombinant of infectious laryngotracheitis virus","volume":"97","author":"Spatz","year":"2016","journal-title":"J. Gen. Virol."},{"key":"ref_8","first-page":"1","article-title":"The old and new landscapes of infectious laryngotracheitis","volume":"118","author":"Zavala","year":"2011","journal-title":"Poult. Inf. Prof."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1637\/12029-011119-Reg.1","article-title":"Protection Efficacy of a recombinant herpesvirus of turkey vaccine against Infectious Laryngotracheitis virus administered in ovo to broilers at three standardized doses","volume":"6","author":"Maekawa","year":"2019","journal-title":"Avian Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1080\/03079458308436195","article-title":"Laryngotracheitis herpesvirus infection in the chicken: The role of humoral antibody in immunity to a graded challenge infection","volume":"12","author":"Fahey","year":"1983","journal-title":"Avian Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1080\/03079458408418530","article-title":"Laryngotracheitis herpesvirus infection in the chicken. II. The adoptive transfer of resistance with immune spleen cells","volume":"13","author":"Fahey","year":"1984","journal-title":"Avian Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1051","DOI":"10.1292\/jvms.56.1051","article-title":"The role of cell-mediated immunity in chickens inoculated with the cell-associated vaccine of attenuated infectious laryngotracheitis virus","volume":"56","author":"Honda","year":"1994","journal-title":"J. Vet. Med. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1080\/03079458908418639","article-title":"The appearance of viral antigen and antibody in the trachea of na\u00efve and vaccinated chickens infected with infectious laryngotracheitis virus","volume":"18","author":"York","year":"1989","journal-title":"Avian Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1016\/j.dci.2013.03.022","article-title":"Immune responses to infectious laryngotracheitis virus","volume":"41","author":"Coppo","year":"2013","journal-title":"Dev. Comp. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5173","DOI":"10.1128\/jvi.71.7.5173-5177.1997","article-title":"Specific cytotoxic T lymphocytes are involved in in vivo clearance of infectious bronchitis virus","volume":"1","author":"Seo","year":"1997","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Bertzbach, L.D., van Haarlem, D.A., H\u00e4rtle, S., Kaufer, B.B., and Jansen, C.A. (2019). Marek\u2019s Disease virus infection of natural killer cells. Microorganisms, 7.","DOI":"10.3390\/microorganisms7120588"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Umthong, S., Dunn, J.R., and Cheng, H.H. (2020). Depletion of CD8\u03b1\u03b2 + T cells in chickens demonstrates their involvement in protective immunity towards Marek\u2019s Disease with respect to tumor incidence and vaccinal protection. Vaccines, 24.","DOI":"10.3390\/vaccines8040557"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3103","DOI":"10.1007\/s00018-020-03477-z","article-title":"Revisiting cellular immune response to oncogenic Marek\u2019s disease virus: The rising of avian T-cell immunity","volume":"77","author":"Yang","year":"2020","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2478","DOI":"10.1038\/srep02478","article-title":"Differential lung NK cell responses in avian influenza virus infected chickens correlate with pathogenicity","volume":"3","author":"Jansen","year":"2013","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4886","DOI":"10.1128\/JVI.76.10.4886-4890.2002","article-title":"Protective cross-reactive cellular immunity to lethal A\/Goose\/Guangdong\/1\/96-like H5N1 influenza virus is correlated with the proportion of pulmonary CD8(+) T cells expressing gamma interferon","volume":"76","author":"Seo","year":"2002","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1906","DOI":"10.3389\/fimmu.2019.01906","article-title":"CD8(+) T Lymphocyte and NK cell network: Circuitry in the cytotoxic domain of immunity","volume":"10","author":"Uzhachenko","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1146\/annurev.immunol.23.021704.115526","article-title":"NK cell recognition","volume":"23","author":"Lanier","year":"2005","journal-title":"Annu. Rev. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1016\/j.immuni.2006.09.010","article-title":"Perforin and granzymes have distinct roles in defensive immunity and immunopathology","volume":"25","author":"Sumaria","year":"2006","journal-title":"Immunity"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1685","DOI":"10.1002\/eji.1830240734","article-title":"Characterization of avian natural killer cells and their intracellular CD3 protein complex","volume":"24","author":"Chen","year":"1994","journal-title":"Eur. J. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"759","DOI":"10.1016\/j.dci.2010.02.009","article-title":"Identification of new populations of chicken natural killer (NK) cells","volume":"34","author":"Jansen","year":"2010","journal-title":"Dev. Comp. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4485","DOI":"10.4049\/jimmunol.154.9.4485","article-title":"Identification and analysis of the expression of CD8 alpha beta and CD8 alpha alpha isoforms in chickens reveals a major TCR-gamma delta CD8 alpha beta subset of intestinal intraepithelial lymphocytes","volume":"15","author":"Tregaskes","year":"1995","journal-title":"J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0091-679X(04)75020-7","article-title":"Detection of T-cell degranulation: CD107a and b","volume":"75","author":"Betts","year":"2004","journal-title":"Methods Cell. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.vetimm.2012.11.012","article-title":"Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41","volume":"151","author":"Vervelde","year":"2013","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.jim.2015.02.011","article-title":"CD107a as a marker of activation in chicken cytotoxic T cells","volume":"419","author":"Wattrang","year":"2015","journal-title":"J. Immunol. Methods."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"925135","DOI":"10.1155\/2012\/925135","article-title":"CD4\u207aT cells: Differentiation and functions","volume":"2012","author":"Luckheeram","year":"2012","journal-title":"Clin. Dev. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1093\/ps\/77.12.1858","article-title":"Chicken CD4, CD8alphabeta, and CD8alphaalpha T cell co-receptor molecules","volume":"77","author":"Luhtala","year":"1998","journal-title":"Poult. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.dci.2013.04.018","article-title":"Avian CD4(+)CD25(+) regulatory T cells: Properties and therapeutic applications","volume":"41","author":"Selvaraj","year":"2013","journal-title":"Dev. Comp. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1997","DOI":"10.4049\/jimmunol.1002040","article-title":"Regulatory T cell properties of chicken CD4+CD25+ cells","volume":"186","author":"Shanmugasundaram","year":"2011","journal-title":"J. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Gurung, A., Kamble, N., Kaufer, B.B., Pathan, A., and Behboudi, S. (2017). Association of Marek\u2019s Disease induced immunosuppression with activation of a novel regulatory T-cells in chickens. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006745"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1111\/imr.12085","article-title":"Role of regulatory T-cells during virus infection","volume":"255","author":"Sehrawat","year":"2013","journal-title":"Immunol. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"883","DOI":"10.3389\/fimmu.2018.00883","article-title":"Emerging Functions of Regulatory T Cells in Tissue Homeostasis","volume":"9","author":"Sharma","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty percent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1080\/03079450802632031","article-title":"Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States","volume":"38","author":"Oldoni","year":"2009","journal-title":"Avian Pathol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1080\/03079450802043742","article-title":"Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States","volume":"37","author":"Oldoni","year":"2008","journal-title":"Avian Pathol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"223","DOI":"10.3382\/ps.2010-00759","article-title":"Field evaluation of the accuracy of vaccine deposition by two different available in ovo injection systems","volume":"90","author":"Williams","year":"2011","journal-title":"Poult. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"106","DOI":"10.2307\/1591340","article-title":"Virulence of infectious laryngotracheitis viruses: Comparison of modified-live vaccine viruses and North Carolina field isolates","volume":"34","author":"Guy","year":"1990","journal-title":"Avian Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1038\/nri778","article-title":"Effector and memory T-cell differentiation: Implications for vaccine development","volume":"2","author":"Kaech","year":"2002","journal-title":"Nat. Rev. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Maekawa, D., Whang, P., Riblet, S.M., Hurley, D., and Garc\u00eda, M. (2021). Assessing the infiltration of T lymphocytes and macrophages in the upper trachea of non-vaccinated and vaccinated chickens after an Infectious laryngotracheitis virus (ILTV) challenge. Avian Pathol., submitted.","DOI":"10.3390\/vaccines9080865"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"886","DOI":"10.1016\/j.immuni.2014.12.007","article-title":"Tissue-resident memory T-cells","volume":"41","author":"Schenkel","year":"2014","journal-title":"Immunity"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1080\/03079457.2011.588196","article-title":"Replication of recombinant herpesvirus of turkey expressing genes of infectious laryngotracheitis virus in specific pathogen free and broiler chickens following in ovo and subcutaneous vaccination","volume":"40","author":"Gimeno","year":"2011","journal-title":"Avian Pathol."},{"key":"ref_47","first-page":"585","article-title":"Latent infections with Marek\u2019s disease virus and turkey herpesvirus","volume":"66","author":"Calnek","year":"1981","journal-title":"J. Natl. Cancer. Inst."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"257","DOI":"10.2307\/1590094","article-title":"The early pathogenesis of turkey herpesvirus infection in chickens and turkeys","volume":"26","author":"Fabricant","year":"1982","journal-title":"Avian Dis."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1099\/0022-1317-53-1-133","article-title":"Latency of herpesvirus of turkey and Marek\u2019s disease virus genomes in a chicken T-lymphoblastoid cell line","volume":"53","author":"Hirai","year":"1981","journal-title":"J. Gen. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Koyanagi, N., and Kawaguchi, Y. (2020). Evasion of the cell-mediated immune response by Alphaherpesviruses. Viruses, 12.","DOI":"10.3390\/v12121354"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1007\/s00251-018-1066-2","article-title":"Two class I genes of the chicken MHC have different functions: BF1 is recognized by NK cells while BF2 is recognized by CTLs","volume":"70","author":"Kim","year":"2018","journal-title":"Immunogenetics"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"645426","DOI":"10.3389\/fimmu.2021.645426","article-title":"An anti-tumor vaccine against Marek\u2019s Disease virus induces differential activation and memory response of \u03b3\u03b4 T cells and CD8 T cells in chickens","volume":"12","author":"Hao","year":"2021","journal-title":"Front. Immunol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/9\/8\/865\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:40:53Z","timestamp":1760164853000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/9\/8\/865"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,5]]},"references-count":52,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["vaccines9080865"],"URL":"https:\/\/doi.org\/10.3390\/vaccines9080865","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,5]]}}}