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. Krunkosky1, L.G. Beltran Garza1, R. M. Gogal Jr.2, D.J. Hurley3, M. García1

1Poultry Diagnostic Research Center, 2Department of Biosciences and Diagnostic Imaging, 3Food 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

– Epithelia cells of:

• Trachea
• Larynx
• Nares
• Sinus membranes
• Conjunctiva

Conjunctiva Trachea

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

• r 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 103 TCID 50 /chicken. A second group of chickens were mock inoculated with media

Post-Infection

CALT, Harderian gland, trachea, spleen

Day 1 Day 3 Day 5 Day 7 Day 9

Tissue disassociation, cells counts adjusted and viability determined

60um 70 microns 40 microns

Flow cytometry, cytospins, ILTV real time PCR

Cytospins

A B C D

lymphocyte lymphocyte red blood cell Glandular epithelial cell lymphocyte lymphocyte Ciliated epithelial cell

Results: Genome Viral Load

Flow Cytometry Procedure: How were cells gated?

• Based in peripheral blood lymphocytes:

P1: Leukocytes P2: Lymphocytes

Analysis @P2 Gate

CD8 CD4 IgM

CALT

C

low high

Day 3 mock/MHCII

low high

Day 3 ILTV- infected/MHCII Day 3 mock/MHCII

low high

Day 3 ILTV- infected/MHCII

low high

CALT SPLEEN

CALT

+Values are expressed as Mean ± SEM

*=p-value ≤0.05, **=p-value ≤0.01, ***=p-value ≤0.001, ****0.0001

% Expression Days Post-Infection Day 1 Day 3 Day 5 Mock+ Infected+ Mock+ Infected+ Mock+ Infected+ P2 gate cells 45±2 47±2 47±2 31±3 *** 45±2 31±2 **** MHCI 69±3 72±2 70±2 59±3** 60±4 58±2 MHCII low 14±1 15±1 17±1 9±1 **** 15±1 12±1 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 CD8 11±4 12±3 13±1 14±2 15±1 14±1 IgM 30±2 30±2 26±2 41±(2) **** 34±3 39±3

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 CD4 18±1 17±1 15±1 16±1 22±3 27±2 CD8 36±2 34±2 39±3 38±2 32±3 26±2 IgM 36±2 37±2 44±2 33±2 *** 42±3 32±2 **

+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 CD4 28±2 21±2* 31±7 32±17 26±8 32±12 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*** CD8 40±2 43±2 42±2 43±3 48±2 40±2** IgM 23±1 22±1 21±1 21±2 23±2 24±1

+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

• ILTV favorably controls the immune cells of

the HALT and trachea to promote viral replication Conclusion

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 5th 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 MHCIIhigh positive cells activated?

What type of cells are they?