Tracking immune cells phenotypic changes in Head associated lymphoid - PowerPoint PPT Presentation

Tracking immune cells phenotypic changes in Head associated lymphoid tissues (HALT), trachea, and spleen of chickens ocularly-infected with Infectious laryngotracheitis virus (ILTV) M. Krunkosky 1 , L.G. Beltran Garza 1 , R. M. Gogal Jr. 2 , D.J. Hurley 3 , M. García 1 1 Poultry Diagnostic Research Center, 2 Department of Biosciences and Diagnostic Imaging, 3 Food Animal Health and Management, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.

Clinical signs of the disease • conjunctivitis • swelling of the infraorbital sinuses, • nasal discharge • dyspnea and bloody mucus in the trachea

Sites of Lytic - Infection Trachea – Epithelia cells of: • Trachea • Larynx • Nares • Sinus membranes • Conjunctiva Conjunctiva

Mucosal tissues – First mucosal tissues to come in contact with ILTV during natural infection or vaccination are: • HALT (head associated lymphoid tissue) – CALT (conjunctiva associated lymphoid tissue) – NALT (nasal associated lymphoid tissue) – Harderian gland Maxilla cut between nostril and the eyes Nasal cavity and infra-orbital sinus

Immune response induced by ILT infection or vaccination? – Mucosal vaccination with live attenuated vaccines via eye-drop, drinking water or spray induce protective immunity – Resistance (adaptive immune response) is provided by cell mediated immunity – ILTV infections induces strong inflammation responses that influences: • Viral replication • Pathology of disease • Modulates adaptive immune response – Viral chemokine binding protein (gG) favors humoral immunity Therefore early local immune responses against ILTV will dictate adaptive immune responses and outcome of disease

• Objective : To identify changes of immune cells populations in CALT, Harderian gland, and trachea during ILTV lytic infection • Approach : Flow cytometry to quantify (%) – Cells expressing CD4+ or CD8+ (T lymphocytes) Cells expressing MHCI ( All Nucleated cells) – Cells expressing MHCII (Macrophages, Dendritic cells, B cells) – – Cells expressing IgM (B cell first isotype) • Hypothesis: During lytic infection ILTV favorably controls the immune cells of the HALT and trachea to promote viral replication

Experimental Design At 6 weeks of age specific pathogen free (SPF) chickens were inoculated via the ocular route (both eyes) with virulent isolate 63140 at a dose of 3.16 X 10 3 TCID 50 /chicken. A second group of chickens were mock inoculated with media Day 1 Day 3 Day 5 Day 7 Day 9 Post-Infection CALT, Harderian gland, trachea, spleen 60um Tissue disassociation, cells counts adjusted and viability determined Flow cytometry, cytospins, ILTV real time PCR 40 microns 70 microns

Cytospins A B lymphocyte red blood cell lymphocyte C D Glandular epithelial cell Ciliated lymphocyte epithelial cell lymphocyte

Results: Genome Viral Load

Flow Cytometry Procedure: How were cells gated? • Based in peripheral blood lymphocytes: P1: Leukocytes P2: Lymphocytes

Analysis @P2 Gate CALT CD8 CD4 IgM CALT SPLEEN Day 3 mock/MHCII Day 3 mock/MHCII high low high low C Day 3 ILTV- infected/MHCII Day 3 ILTV- infected/MHCII high low high low

CALT % Expression Days Post-Infection Day 1 Day 3 Day 5 Mock + Infected + Mock + Infected + Mock + Infected + 45 ± 2 47 ± 2 47 ± 2 31 ± 3 *** 45 ± 2 31 ± 2 **** P2 gate cells MHCI 69 ± 3 72 ± 2 70 ± 2 59 ± 3** 60 ± 4 58 ± 2 14 ± 1 15 ± 1 17 ± 1 9 ± 1 **** 15 ± 1 12 ± 1 MHCII low MHCII high 40 ± 3 38 ± 2 37 ± 2 52 ± 3 *** 41 ± 2 48 ± 2 CD4 44 ± 2 44 ± 2 45 ± 2 29 ± 2 **** 38 ± 2 35 ± 2 11 ± 4 12 ± 3 13 ± 1 14 ± 2 15 ± 1 14 ± 1 CD8 30 ± 2 30 ± 2 26 ± 2 41 ± (2) **** 34 ± 3 39 ± 3 IgM + Values are expressed as Mean ± SEM *=p- value ≤ 0.05, **=p- value ≤ 0.01, ***=p- value ≤ 0.001, ****0.0001

Harderian Gland % Expression Days Post-Infection Day 1 Day 3 Day 5 Mock + Infected + Mock + Infected + Mock + Infected + P2 gate cells 9 ± 2 7 ± 1 10 ± 2 10 ± 1 9 ± 1 22 ± 3 **** MHCI 82 ± 2 86 ± 2 80 ± 3 82 ± 2 79 ± 1 82 ± 1 MHCII low 8 ± 1 8 ± 1 9 ± 1 6 ± 1*** 7 ± 0 12 ± 2*** MHCII high 20 ± 2 14 ± 1** 16 ± 2 15 ± 2 17 ± 1 18 ± 1 18 ± 1 17 ± 1 15 ± 1 16 ± 1 22 ± 3 27 ± 2 CD4 CD8 36 ± 2 34 ± 2 39 ± 3 38 ± 2 32 ± 3 26 ± 2 36 ± 2 37 ± 2 44 ± 2 33 ± 2 *** 42 ± 3 32 ± 2 ** IgM + Values are expressed as Mean ± SEM *=p- value ≤ 0.05, **=p- value ≤ 0.01, ***=p- value ≤ 0.001, ****0.0001

Trachea % Expression Days Post-Infection Day 1 Day 3 Day 5 Mock + Infected + Mock + Infected + Mock + Infected + P2 gate cells 7 ± 2 5 ± 1 6 ± 1 7 ± 1 11 ± 2 17 ± 3 MHCI 89 ± 1 94 ± 1** 91 ± 1 91 ± 1 88 ± 1 92 ± 1 MHCII low 4 ± 1 3 ± 1 4 ± 2 5 ± 3 5 ± 2 6 ± 4 MHCII high 13 ± 4 8 ± 3** 10 ± 3 10 ± 5 10 ± 1 9 ± 1 28 ± 2 21 ± 2* 31 ± 7 32 ± 17 26 ± 8 32 ± 12 CD4 CD8 13 ± 1 13 ± 1 15 ± 1 18 ± 5 17 ± 1 18 ± 3 IgM 14 ± 1 11 ± 1 16 ± 5 12 ± 1 14 ± 1 11 ± 1 + Values are expressed as Mean ± SEM *=p- value ≤0.05, **=p- value ≤0.01, ***=p- value ≤0.001, ****0.0001

Spleen % Expression Days Post-Inoculation Day 1 Day 3 Day 5 Mock + Infected + Mock + Infected + Mock + Infected + P2 gate cells 48 ± 2 52 ± 2 50 ± 1 46 ± 1** 45 ± 2 42 ± 2 MHCI 76 ± 1 78 ± 1 77 ± 1 79 ± 1 73 ± 2 72 ± 1 MHCII low 27 ± 2 24 ± 2 24 ± 1 25 ± 2 27 ± 2 29 ± 2 MHCII high 30 ± 1 28 ± 2 26 ± 2 27 ± 2 28 ± 2 30 ± 1 CD4 23 ± 1 20 ± 1 22 ± 1 23 ± 1 18 ± 1 24 ± 1*** 40 ± 2 43 ± 2 42 ± 2 43 ± 3 48 ± 2 40 ± 2** CD8 23 ± 1 22 ± 1 21 ± 1 21 ± 2 23 ± 2 24 ± 1 IgM + Values are expressed as Mean ± SEM **=p- value ≤0.01, ***=p- value ≤0.001, ****0.0001

Summary • Immune cells changes or the lack-off that may favor viral replication – CALT: transient decrease of CD4+ and MHCI cells – HG: decrease of IgM+ cells – Trachea: static during lytic replication after ocular inoculation – Overall lack of CD8+ cells increase • Evidence of a developing immune response – CALT: increase of IgM+ cells – CALT & HG: increase in MHCII + cells (maybe APC’s) – Spleen: increase in CD4+ cells

Conclusion • ILTV favorably controls the immune cells of the HALT and trachea to promote viral replication

Objective 2.2.2: Characterization of LOCAL IMMUNE RESPONSES in chickens administered a live attenuated infectious laryngotracheitis virus (ILTV) chicken embryo origin (CEO) vaccine alone or combined with select viral respiratory vaccines Maricarmen García and Robert M. Gogal Jr. (University of Georgia)

• ✔ 1rst Year : Characterization of cell phenotypes in tissues infected with virulent ILTV isolate • 2nd Year : Characterization of cell phenotypes in tissues infected with ILTV CEO vaccine • 3rd Year : Characterization of cell phenotypes in tissues after sequential or co-administration of ILTV (CEO) and IBV vaccines • 4rd and 5 th Year : Characterization of cell phenotypes in tissues post-challenge of ILTV and IBV vaccinated chickens

Questions??

Questions to be Answered • In CALT Is the decrease in CD4+ cells due to apoptosis? • Are MHCI and MHCII high positive cells activated? What type of cells are they?