THE ROLE OF INNATE-LIKE T CELLS IN INFLUENZA IMMUNITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028674
• Grant No.: 2021-67015-37521
• Cumulative Award Amt.: $495,296.65
• Proposal No.: 2022-04775
• Project Start Date: Jul 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
(N/A)

Non Technical Summary
Present knowledge about the immune responses that protect pigs from influenza virus infections is largely based on our understanding of a group of immune cells called conventional T cells which have been well described for decades. However, much less is known about the role of more recently discovered populations of unconventional T cells called innate-like T cells (ITC). These ITCs specialize in producing large quantities of immune signaling molecules in barrier organs like the lung, and probably make pivotal contributions to influenza immunity. Although ITCs probably make up a substantial portion of the swine influenza response, most of how they contribute to pig immunity can only be speculated about from mouse models which have significant drawbacks, including that mice completely lack an entire category of ITCs called group I CD1-restricted T-cells. We hypothesize that ITCs play important roles in early immune responses during influenza infections as well as in shaping immune memory against subsequent infections. We will test this theory using genetically modified swine individually lacking three different types of ITCs. Firstly, ITC-deficient and -intact pigs will be compared for their susceptibility to different influenza A virus strains using a range of innovative tools. Secondly, we will determine the resistance of ITC-deficient and intact pigs to subsequent challenge with distant and closely related influenza virus strains. These experiments will provide the first comprehensive examination of ITCs to the health of pigs, which has the potential to transform current understanding of how swine protect themselves against respiratory viruses.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology;

Keywords

cd1

host immunity

influenza

innate-like t cells

mr1

swine

Goals / Objectives
While present understanding of swine influenza A virus (IAV) immunity is focused on conventional MHC-restricted T-cells that recognize peptide antigens, unconventional innate-like T cell (ITC) subsets, which include CD1- and MR1-restricted T-cells, are emerging as critical players in the respiratory system where they can play both a protective and pathogenic role in lung disease. This is largely through the production large amounts of cytokines in barrier organs like the lung where ITCs preferentially accumulate. Although ITCs probably make up a substantial portion of the swine influenza response, most of how they contribute to pig immunity can only be speculated about from mouse models which have significant drawbacks, including that mice completely lack an entire category of ITCs called group I CD1-restricted T-cells. We hypothesize that ITCs play important roles in early innate responses during an IAV infection as well as in shaping immune memory and cross-protection against subsequent infections. We will test this theory using genetically modified swine individually lacking CD1- and MR1-restricted ITCs.-Firstly, ITC-deficient and intact pigs will be compared for their susceptibility to different IAV subtypes using a range of innovative tools.-Secondly, we will compare virus-specific cellular and humoral immune responses in different CD1- and MR1-deficient pigs and determine their resistance to subsequent challenge with homologous and heterosubtypic IAV strains.These experiments will provide the first comprehensive examination of CD1- and MR1-restricted T cells to the health of pigs which has the potential to transform current understanding of the swine pulmonary immune response.

Project Methods
The CRISPR/Cas9 system will be used to produce lines of pigs that lack the antigen presenting molecules MR-1 and CD1A/B that are respectively required for the development of mucosal associated innate T (MAIT) cells and CD1A/B- restricted T cells. We already have pigs that lack CD1d that is required for the production of natural killer T (NKT) cells.Each stock of genetically modified pig will be infected with two strains of influenza A virus (IAV) to test whether they are more or less resistant to IAV infections.Each stock of genetically modified pig will be rechallenged after infection with two strains of influenza A virus (IAV) to test whether MAIT cells, NKT cells and CD1A/B-restricted T cells make important contributions to immune memory against IAV infection.Pigs will be evaluated for virus load and shedding, immune cytokines, frequency of immune cells in lung and lymphoid organs, and histopathology.We will isolate lung leukocytes to compare how IAV-induced immune responses and pulmonary immune cell concentrations vary between pigs that express and lack MAIT cells, NKT cells, and CD1A/B-restricted T cells.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:T cell biologists Swine immunologists Vaccine researchers Industry specialists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, Alyssa May and Laurie Touchard, presented research related to this project at two University of Missouri Research symposia: ShowMeResearch Week 2023 and the Animal Sciences Graduate Forum 2023. How have the results been disseminated to communities of interest?Through the additional publication: Ma W, Loving CL, Driver JP. From Snoot to Tail: A Brief Review of Influenza Virus Infection and Immunity in Pigs. J Immunol. 2023 Oct 15;211(8):1187-1194. doi: 10.4049/jimmunol.2300385. PMID: 37782856. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue analyzing samples taken from the 6 influenza challenge studies completed as part of this project. We are currently performing another study to determine their contribution of CD1d-resticted T cells to acquired immunity against subsequent influenza infection. In June, we will perform a study to determine their contribution of MR1-resticted T cells to acquired immunity against subsequent influenza infection. We plan to perform additional studies with CD1D and CD1A/B knockout pigs if they become available. We plan to continue analyzing single cell RNA sequencing data.

Impacts
What was accomplished under these goals? For the CD1A/B knockout pigs, we performed 2 influenza virus challenge studies. One study to test the role of CD1a/b-restricted cells in early innate T cell responses (Aim 1) and another study to determine their contribution to acquired immunity against subsequent influenza infection (Aim 2). For the MR1 knockout pigs, we performed 2 influenza virus challenge studies. Both studies were to test the role of MR1-restricted cells in early innate T cell responses (Aim 1). For the CD1D knockout pigs, we performed 2 influenza virus challenge studies. One study to test the role of CD1d-restricted cells in early innate T cell responses (Aim 1) and another study to determine their contribution to acquired immunity against subsequent influenza infection (Aim 2). We are currently analyzing results we obtained from our different studies, including histology, flow cytometry, and viral titers. Single cell RNA sequencing has been performed for lung immune cells from the CD1D KO pigs and we are currently analyzing results through our bioinformatics pipeline.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Ma W, Loving CL, Driver JP. From Snoot to Tail: A Brief Review of Influenza Virus Infection and Immunity in Pigs. J Immunol. 2023 Oct 15;211(8):1187-1194. doi: 10.4049/jimmunol.2300385. PMID: 37782856.

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:T cell biologists Swine immunologists Vaccine researchers Industry specialists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students were chosen to present their research at the 2023 CRWAD meeting in Chicago IL Jan 22-24: L. Touchard, A. May, D.M. De Carvalho Madrid, J.P. Driver; Multicolor flow cytometry panels for immunophenotyping pig leukocytes D.M. Madrid, W. Gu, J. Driver; A single cell analysis of pulmonary leukocytes in pigs How have the results been disseminated to communities of interest?Through the following publications: 1. W. Gu, D. M. C. Madrid, S. Joyce, J. P. Driver, A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs. Cell Rep 40, 111050 (2022). 2. D. M. C. Madrid et al., Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus. Front Vet Sci 9, 999507 (2022). 3. S. Joyce, G. D. Okoye, J. P. Driver, Die Kämpfe únd schláchten-the struggles and battles of innate-like effector T lymphocytes with microbes. Front Immunol 14, 1117825 (2023). What do you plan to do during the next reporting period to accomplish the goals? We plan to complete analyzing samples taken from out three influenza challenge studies completed this last year. We plan to commence analyzing single cell RNA sequencing data. We plan to commence studies in Aim 2 that involve investigating the role played by innate-like T cells in acquired immune responses against influenza.

Impacts
What was accomplished under these goals? We successfully targeted and deleted CD1A, CD1B, and MR1 genes and established breeding herds of genetically edited pigs that harbor these mutations, along with our already established CD1D knockout breeding herd. We produced pigs from all of our breeding herds that were enrolled in three separate influenza challenge studies. We are currently analyzing results we obtained from histology, flow cytometry, and viral titers. Tissues and cells collected from recipient pigs were cryopreserved in preparation for single cell sequencing.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: W. Gu, D. M. C. Madrid, S. Joyce, J. P. Driver, A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs. Cell Rep 40, 111050 (2022).
• Type: Journal Articles Status: Published Year Published: 2022 Citation: D. M. C. Madrid et al., Comparison of oseltamivir and ?-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus. Front Vet Sci 9, 999507 (2022).
• Type: Journal Articles Status: Published Year Published: 2023 Citation: S. Joyce, G. D. Okoye, J. P. Driver, Die Kÿ¤mpfe ÿºnd schlÿ¡chten-the struggles and battles of innate-like effector T lymphocytes with microbes. Front Immunol 14, 1117825 (2023).