GENE NUMBER AND SEQUENCE VARIATION AMONG FOOD ANIMAL SPECIES OF WC1 (AKA T19) THAT FUNCTIONS AS BOTH A UNIQUE ¿D T CELL PATTERN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1009067
• Grant No.: 2016-67015-24913
• Cumulative Award Amt.: $495,000.00
• Proposal No.: 2015-06970
• Project Start Date: Feb 1, 2016
• Project End Date: Jan 31, 2021
• Grant Year: 2016
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Veterinary Animal Sciences

Non Technical Summary
Animal health is of great importance, in agricultural, food security, general economic and public health terms. The diseases that this grant proposes to investigate (tuberculosis, anaplasmosis, Johne's disease, and porcine reproductive and respiratory syndrome virus (PRRSV)) cause billions of dollars in losses to U.S. agricultural producers. In addition, tuberculosis, anaplasmosis, and Johne's disease are zoonotic diseases, in which animals can serve as reservoirs and vectors of disease for humans. For example, Mycobacterium tuberculosis and Mycobacterium bovis, the bacteria that cause tuberculosis, infect one-third of the world's human population, and are responsible for 1.5 million human deaths worldwide per year. While Mycobacterium bovis infection of livestock and humans has been nearly eliminated in the U.S. by decades of culling infected animals and pasteurizing milk, importation of infected livestock and dairy products from Mexico and infected deer reservoirs have resulted in cattle and human infection and deaths in the U.S. The rise in consumer demand for "raw" unpasteurized milk also increases the danger of a Mycobacterium bovis outbreak. History has shown that the best way to treat disease is to prevent it through vaccination, with the long-term goal of driving it into extinction. Smallpox and rinderpest virus are two examples of pathogens that are now extinct because of vaccination. This is especially true in agriculture, in which diseases may not be fatal to all animals, but severely impact producers because animals fail to reach market weight in a timely fashion, incur veterinary medical bills, and fail to deliver viable young. Chronically infected animals can infect the whole herd or flock. Thus, it is critical to develop effective vaccines to prevent animal pathogens from gaining a foothold. Most classic vaccines rely on activating a white blood cell called a "B cell", which produce antibody proteins that bind specifically to the pathogen and lead to its elimination. These B cells produce only one type of antibody per cell and display a memory response in that they act faster the second time they encounter the pathogen and are thus able to prevent infection. However, many pathogens are able to evade these antibody proteins through mutation because the antibodies are so specific. For these pathogens, it has proven beneficial to design vaccines that recruit a second type of immune cell capable of a memory response, the T cell. There are two types of T cells: alpha beta T cells and gamma delta T cells. Much more is known about alpha beta than gamma delta T cells, but we do know that gamma delta T cell respond much more quickly than B cells or alpha beta T cells, that they can recognize pathogen molecules that the pathogens can't mutate because they are integral to pathogen survival, and that, unlike B cells, they make the potent protective chemical interferon-gamma. We have shown that the efficacy of a vaccine to the bacteria Leptospira in cattle is correlated with the development of an early-acting gamma delta T cell memory response, and that a gamma delta T cell protein called WC1-3, which binds directly to Leptospira, is involved. WC1-3 is only one of 13 WC1 genes in cattle that have been conserved over millions of years of evolution. Because the normal tendency of nucleotide sequences in genes that fulfill no function is to accumulate mutations that render them non-functional "pseudogenes", it is likely that each WC1 gene has been preserved through recognition of a different class of pathogens. Consistent with this hypothesis, we have found that gamma delta T cells respond to Mycobacterium, and that two other WC1 proteins bind to Mycobacterium. In this grant proposal, we propose to obtain the nucleotide sequence of previously unknown WC1 genes in sheep, cattle, and pigs and to investigate the mechanism of these proteins interaction with, and activation by, pathogens. Identification of the molecules bound by WC1 will allow to create more effective designer vaccines that recruit multiple arms of the immune response, including the early and potent gamma delta T cell response.

Animal Health Component

0%

Research Effort Categories

Basic

95%

Applied

0%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3910	1040	50%
311	3910	1090	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species;

Field Of Science
1040 - Molecular biology; 1090 - Immunology;

Keywords

cattle

co-receptor

gamma delta t cell

goat

pattern recognition receptor

sheep

swine

wc1

Goals / Objectives
Major long-term goal: To define the molecular recognition structures and mechanisms involved in γδ T cell interactions with pathogens in food animal species, so that activation of these cells may be targeted in next generation vaccines. γδ T cells are unusual T cells, in that they respond much faster than conventional T cells and respond more broadly to pathogen molecules than antibody-producing B cells, which predicts that pathogens will be less likely to evade the γδ T cell response by mutation. Designing vaccines enriched in the pathogen molecules that specifically activate γδ T cells will recruit these cells to the immune response and potentially result in more effective, more broadly reactive vaccines. In addition, the characterization of variation in this host:pathogen interaction is of potentially high impact for selective breeding or genetic modification for disease resistance in food animals. We have focused on the WC1 gene family in cattle, which is expressed on γδ T cell, and which we have shown contributes to the immune response to pathogens. This study extends this work to WC1 genes and pathogens with significant impact in pig, sheep and goats.Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats.Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats.Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation.

Project Methods
The general scientific methods for this project will be the molecular characterization of sheep, goat and pig WC1 nucleotide sequences, the subsequent production of sheep, goat and pig WC1 proteins, the characterization of their ligands on pathogens such as Mycobacteria, Anaplasma and PRRSV using recombinant proteins and engineered cells and the elucidation of WC1 signaling. We will deliver this science-based information to target audiences via deposition of nucleotide sequences in GenBank. The nucleotide sequence information will also be used in a cellular and molecular biology class at UMass Amherst to teach the concept of genome evolution to 95 students. Undergraduate students will also participate in this project through authentic discovery-based research experiences. Information on the pathogen recognition profiles ofsheep, goat and pig WC1, any further evidence supporting our working model of WC1 as a multi-genic array of hybrid pathogen recognition receptors and co-receptors for the gamma delta TCR, and the involvement of WC1 in the response to pathogens such as Mycobacteria, Anaplasma and PRRSV will be disseminated through public presentations and publication in peer-reviewed literature.The project will be evaluated by our achievement of milestones such as characterizing the WC1 gene numbers in sheep, goat and pigs and working with others to map those onto the sheep, goat and pig genome. Other milestones include screening sheep, goat and pig WC1 proteins for binding to pathogens such as Mycobacteria, Anaplasma and PRRSV and characterizing the signaling capacity of WC1 cytoplasmic domains.

Progress 02/01/20 to 01/31/21

Outputs
Target Audience:a cellular and molecular biology class taught to approximately 100 undergraduate students 2 undergraduates trained in molecular and cellular biology, genomics 3 graduate students working on this project Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?3 graduate students and 2 undergraduate studentshave been trained under this project during this reporting period. One of the graduate students has successfully defended his Ph.D. thesis, 2 other graduate students will defend their Ph.D. theses by the end of the summer. How have the results been disseminated to communities of interest?I meet with prospective undergraduate students and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the researchwe do that is is funded by the USDA, why that is important, and how they can get involved if they come to UMass Amherst. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals. In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. • In 2020, we made significant progress in verifying caprine gene models obtained through annotation of the PacBio sequenced San Clemente goat genome, by comparison to the Yunan goat genome and cloning of corresponding full-length WC1 cDNA from the UMass Boer goat herd. We found evidence for up to30 goat WC1 genes, which is more than twice that of cattle. Moreover, goats had seven different WC1 gene structures, of which 4 are unique to goats. Caprine WC1 genes also had multiple splice variants of their transcripts coding for the intracytoplasmic domains that in some cases eliminated tyrosines shown previously to be important for signal transduction. The N-terminal WC1 SRCR domains known as SRCR a1, based on sequence and position, were highly conserved among goat breeds, but fewer were conserved between goats and cattle (manuscript in submission). In the ovine Rambouillet sheep assembly, we found 15 complete genes and 42 partial genes, with 6 different predicted structures. We were able to confirm transcription of all but five of the annotated ovine WC1 genes in the UMass Amherst Dorset sheep flock. Of the five annotated a1 domains for which we were not able to obtain cDNA evidence, three had frame shift mutations, or are truncated, indicating they may be pseudogenes. We were also able to amplify the full-length transcript comprising the 11 SRCR-domain gene WC1-10, verifying that gene structure as it appears in the genome assembly. Along with the ruminant species goat, sheep, and cattle, non-ruminant swine belong to the orderArtiodactyla. In swine, cDNA evidence shows that porcine WC1 is also expressed as a multigenic array consisting of 9 genes (WC1-1 to WC1-9), each encoding 6 SRCR domains. We annotatedSscrofa11.1for sequence derived from full-length cDNA transcripts representing the 9 hypothetical porcine WC1 genes. We were able to map 7 of the 9 genes, leaving two (WC1-5 and WC1-8) unplaced in the current assembly (manuscript in submission). Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiple newly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis and tuberculosis (causes tuberculosis in pigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. We have evaluated the splice variants of the bovine WC1 genes using the system outlined. The results indicate that the for some of the WC1 genes there is constitutive splicing even in resting cells while for others, they may be spliced in activated cells. Thus, the data is more complex than we initially hypothesized. We plan to use the next generation sequencing and PacBio approach to get a more comprehensive picture of this.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yirsaw AW, Gillespie A, Britton E, Doerle A, Johnson L, Marston S, Telfer J, Baldwin CL. Goat gamma delta T cell subpopulations defined by WC1 expression, responses to pathogens and cytokine production. Dev. Comp. Immunol 2021 May; 118:103984. Doi:10.1016/j.dci.2020.103984. PMID:33352199
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Gillespie A, Yirsaw A, Kim S, Wilson K, McLaughlin J, Madigan M, Loonie K, Britton E, Zhang F, Damani-Yokota P, Gunasekaran KP, Telfer J, Baldwin CL. Gene characterization and expression of the gamma delta T cell co-receptor in sheep. Dev. Comp. Immunol. 2021 Mar;116:103911. doi: 10.1016/j.dci.2020.103911. PMID 33137393
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Gillespie A, Yirsaw A, Gunasekaran KP, Smith TP, Bickhart DM, Turley M, Connelley T, Telfer JC, Baldwin CL. Characterization of the domestic goat gamma delta T cell receptor gene loci and gene usage. Immunogenetics 2021 Apr;73(2):187-201. Doi:10.1007/s00251-021-01203-y. PMID:33479855.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Baldwin, C.L., A. Yirsaw, A. Gillespie, L. LePage, F. Zhang, P. Damani-Yokota, and J.C. Telfer. 2020. ?? T cells in livestock: Responses to pathogens and vaccine potential. Transboundary and Emerging Diseases 67(Suppl2) 119-1128.
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Yirsaw, A., A. Gillespie, F. Zhang, T.P.L. Smith, D Bickhart, K.P. Gunasekaran, M. Amir, H. Park, J. Telfer, and C.L. Baldwin. Defining the caprine gamma delta T cell WC1 multigenic array and evaluation of its expressed sequences and gene structure conservation among goat breeds and relative to cattle.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Lauren Le Page*, Jessica Buck, Natalie Boisvert, Alexandria Gillespie, Edward Hudgens, Alehegne Yirsaw, Haoting Hsu, Cynthia L. Baldwin and Janice C. Telfer, Genomic organization and expression of the swine WC1 multigenic array of hybrid corruptor/PRR molecules. CRWAD meeting, Chicago, IL, USA December 202o

Progress 02/01/16 to 01/31/21

Outputs
Target Audience:a cellular and molecular biology class taught to approximately 100 undergraduate students undergraduates trained in molecular and cellular biology, genomics 3 graduate students working on this project Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?3 graduate students and 2 undergraduate students have been trained under this project during this reporting period. One of the graduate students has successfully defended his Ph.D. thesis, 2 other graduate students will defend their Ph.D. theses by the end of the summer. How have the results been disseminated to communities of interest?I meet with prospective undergraduate students and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the research we do that is is funded by the USDA, why that is important, and how they can get involved if they come to UMass Amherst. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals. In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. • In 2020, we made significant progress in verifying caprine gene models obtained through annotation of the PacBio sequenced San Clemente goat genome, by comparison to the Yunan goat genome and cloning of corresponding full-length WC1 cDNA from the UMass Boer goat herd. We found evidence for up to 30 goat WC1 genes, which is more than twice that of cattle. Moreover, goats had seven different WC1 gene structures, of which 4 are unique to goats. Caprine WC1 genes also had multiple splice variants of their transcripts coding for the intracytoplasmic domains that in some cases eliminated tyrosines shown previously to be important for signal transduction. The N-terminal WC1 SRCR domains known as SRCR a1, based on sequence and position, were highly conserved among goat breeds, but fewer were conserved between goats and cattle (manuscript in submission). In the ovine Rambouillet sheep assembly, we found 15 complete genes and 42 partial genes, with 6 different predicted structures. We were able to confirm transcription of all but five of the annotated ovine WC1 genes in the UMass Amherst Dorset sheep flock. Of the five annotated a1 domains for which we were not able to obtain cDNA evidence, three had frame shift mutations, or are truncated, indicating they may be pseudogenes. We were also able to amplify the full-length transcript comprising the 11 SRCR-domain gene WC1-10, verifying that gene structure as it appears in the genome assembly. Along with the ruminant species goat, sheep, and cattle, non-ruminant swine belong to the order Artiodactyla. In swine, cDNA evidence shows that porcine WC1 is also expressed as a multigenic array consisting of 9 genes (WC1-1 to WC1-9), each encoding 6 SRCR domains. We annotated Sscrofa11.1 for sequence derived from full-length cDNA transcripts representing the 9 hypothetical porcine WC1 genes. We were able to map 7 of the 9 genes, leaving two (WC1-5 and WC1-8) unplaced in the current assembly (manuscript in submission). Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiple newly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis and tuberculosis (causes tuberculosis in pigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. We have evaluated the splice variants of the bovine WC1 genes using the system outlined. The results indicate that the for some of the WC1 genes there is constitutive splicing even in resting cells while for others, they may be spliced in activated cells. Thus, the data is more complex than we initially hypothesized. We plan to use the next generation sequencing and PacBio approach to get a more comprehensive picture of this.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Yirsaw AW, Gillespie A, Britton E, Doerle A, Johnson L, Marston S, Telfer J, Baldwin CL. Goat gamma delta T cell subpopulations defined by WC1 expression, responses to pathogens and cytokine production. Dev. Comp. Immunol 2021 May; 118:103984. Doi:10.1016/j.dci.2020.103984. PMID:33352199
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Gillespie A, Yirsaw A, Kim S, Wilson K, McLaughlin J, Madigan M, Loonie K, Britton E, Zhang F, Damani-Yokota P, Gunasekaran KP, Telfer J, Baldwin CL. Gene characterization and expression of the gamma delta T cell co-receptor in sheep. Dev. Comp. Immunol. 2021 Mar;116:103911. doi:10.1016/j.dci.2020.103911. PMID 33137393
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Gillespie A, Yirsaw A, Gunasekaran KP, Smith TP, Bickhart DM, Turley M, Connelley T, Telfer JC, Baldwin CL. Characterization of the domestic goat gamma delta T cell receptor gene loci and gene usage. Immunogenetics 2021 Apr;73(2):187-201. Doi:10.1007/s00251-021-01203-y. PMID:33479855.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Baldwin, C.L., A. Yirsaw, A. Gillespie, L. LePage, F. Zhang, P. Damani-Yokota, and J.C. Telfer. 2020. Gamma delta T cells in livestock: Responses to pathogens and vaccine potential. Transboundary and Emerging Diseases 67(Suppl2) 119-1128.
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Yirsaw, A., A. Gillespie, F. Zhang, T.P.L. Smith, D Bickhart, K.P. Gunasekaran, M. Amir, H. Park, J. Telfer, and C.L. Baldwin. Defining the caprine gamma delta T cell WC1 multigenic array and evaluation of its expressed sequences andgene structure conservation among goat breeds and relative to cattle.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Lauren Le Page*, Jessica Buck, Natalie Boisvert, Alexandria Gillespie, Edward Hudgens, Alehegne Yirsaw, Haoting Hsu, Cynthia L. Baldwin and Janice C. Telfer, Genomic organization and expression of the swine WC1 multigenic array of hybrid corruptor/PRR molecules. CRWAD meeting, Chicago, IL, USA December 2020

Progress 02/01/19 to 01/31/20

Outputs
Target Audience:The target audiences reached by our efforts during this reporting period included stakeholders in agricultural research via presentations at conferences and publications, and undergraduates at UMass Amherst via lectures and research experiences. Changes/Problems:We have completed, or are nearing completion, on many of the grant's specific aims. Major problems that have delayed the completion of all the specific aims include: Experiment 2.1 WC1 binding/gamma delta T hybridoma activation by Anaplasma. This experiment has been hampered by the retirement of our collaborator Professor Wendy Brown two years ago. We are still looking for a new source of this pathogen. Experiment 2.3 Porcine WC1 and CD163A binding/infection/activation by PRRSV . This sub-aim has been delayed due to the sad illness and death of our collaborator Professor Michael Murtaugh, University of Minnesota. Currently we are seeking a new collaborator and source of PRRSV. Experiment 3.2 Determine the occurrence of splice variants coding for the extracellular region of WC1 following activation of the gamma deltaT cell with a pathogen. We have evaluated the splice variants of the bovine WC1 genes using the system outlined. The results indicate that for some of the WC1 genes there is constitutive splicing even in resting cells while for others, they may be spliced in activated cells. Thus, the data is more complex than we initially hypothesized. We plan to use the next generation sequencing and PacBio approach to get a more comprehensive picture of this. What opportunities for training and professional development has the project provided?3 graduate students and 4 undergraduate students have been trained under this project during this reporting period. How have the results been disseminated to communities of interest?I meet with prospective undergraduate students and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the research we do that is is funded by the USDA, why that is important, and how they can get involved if they come to UMass Amherst. What do you plan to do during the next reporting period to accomplish the goals?Continued characterization of swine WC1 genes and RNA to obtain the last 2 full-length WC1 genes. • Continued characterization of ovine and caprine WC1 genes and RNA • Annotation of new Pac-Bio sequenced genomes and deposition of sequence into GenBank database • Continue WC1-pathogen binding studies, identify ligands • Start PRSSV and Anaplasma studies • WC1 alternative splicing and signalling studies

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals. In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. • 17 WC1 genes annotated in San Clemente goat genome. In confirmation that these genes are expressed, 6 WC1 cDNAs were cloned from goat, 1 of which was not found in San Clemente goat genome, indicating that the sequenced genome is not complete. There is evidence for as many as 28 caprine WC1 genes, but it is not clear how many of these may be pseudogenes or soluble forms with potential antibiotic activity. • cDNA and genomic evidence for 12 ovine WC1 genes; PacBio sequence of a1 domains shows additional sheep-specific WC1 genes. • 8 fulll length and 2 partial WC1 cDNAs cloned from pig. Only two of these have been previously annotated in the swine genome, 4 are novel. Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiple newly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis and tuberculosis (causes tuberculosis in pigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. Ongoing- Chimeric constructs under construction

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Lauren Le Page*, Jessica Buck, Natalie Boisvert, Alexandria Gillespie, Edward Hudgens, Alehegne Yirsaw, Haoting Hsu, Cynthia L. Baldwin and Janice C. Telfer, Classification of WC1 gene family in Sus scrofa and evaluation of individual SRCR domain affinity for Mycobacterium bovis and Leptospira spp. American Association of Immunologists (AAI) meeting. San Diego, California, USA. May 9-13, 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Lauren Le Page*, Jessica Buck, Natalie Boisvert, Alexandria Gillespie, Edward Hudgens, Alehegne Yirsaw, Haoting Hsu, Cynthia L. Baldwin and Janice C. Telfer, Genomic organization and expression of the swine WC1 multigenic array of hybrid corruptor/PRR molecules. CRWAD meeting, Chicago, IL, USA December 2019.

Progress 02/01/18 to 01/31/19

Outputs
Target Audience: a cellular and molecular biology class taught to approximately 96 undergraduate students 1 undergraduatetrained in molecular and cellular biology, genomics 3 graduate students working on this project Changes/Problems:We have completed, or are nearing completion, on many of the grant's specific aims.Major problems that have delayed the completion of all the specific aims include: Experiment 2.1 WC1 binding/gamma deltaT hybridoma activation by Anaplasma. This experiment has been hampered by the retirement of our collaborator Professor Wendy Brown two years ago. We are still looking for a new source of this pathogen. Experiment 2.3 Porcine WC1 and CD163A binding/infection/activation by PRRSV. This sub-aim has been delayed due to the sad illness and death of our collaborator Professor Michael Murtaugh, University of Minnesota. Currently we are seeking a new collaborator and source of PRRSV. Experiment 3.2 Determine the occurrence of splice variants coding for the extracellular region of WC1 following activation of thegamma deltaT cell with a pathogen. We have evaluated the splice variants of the bovine WC1 genes using the system outlined. The results indicate that the for some of the WC1 genes there is constitutive splicing even in resting cells while for others, they may be spliced in activated cells. Thus, the data is more complex than we initially hypothesized.we plan to use the next generation sequencing and PacBio approach to get a more comprehensive picture of this. What opportunities for training and professional development has the project provided? 3 graduate students and 4 undergraduate students have been trained under this project during this reporting period. Le Page, L., Yirsaw, A., Gillespie, A., Hudgeons, E., Baldwin, C.L. and Telfer, J.C*, Multigenic SRCR receptor arrays determine the gamma delta T cell response to antigen. (talk)Non-conventional T cells in health and disease,BSI Comparative Veterinary Immunology Group (CVIG) and Biotechnology and Biological Sciences Research Council (BBSRC) UK Veterinary Vaccinology Network (UK VVN), January 2019. Le Page, L., Yirsaw, A., Gillespie, A., Hudgeons, E., Baldwin, C.L. and Telfer, J.C*.Swine WC1 genes are a multigenic array with bacterial binding capacity.(talk)NIFA-USDA Animal Health Program Director meetingand CRWAD meeting, Chicago, IL, USA. December 2018. LePage, L.*, Hsu, H., Nandi, D., Buck, J., Boisvert, N., Damani-Yokota, P., Yirsaw, A., Gillespie, A., Hudgeons, E., Amir, M., Park, H., Baldwin, C.L. and Telfer, J.C. "Molecularand Functional Variationofthe Gamma Delta T Cell PRR/Co-Receptor WC1 Gene Family Among Livestock" (talk and poster)American Association of Immunologists (AAI) meeting. Austin, Texas, USA. May 4-8,2018. How have the results been disseminated to communities of interest? I meet with prospective undergraduate students and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the research we do that is is funded by the USDA, why that is important, and how they can get involved if they come to UMass Amherst. What do you plan to do during the next reporting period to accomplish the goals? Continued characterization of swine WC1 genes and RNA to obtain the last 2 full-length WC1 genes. Continued characterization of ovine and caprine WC1 genes and RNA Annotation of new Pac-Bio sequenced genomes and deposition of sequence into GenBank database Continue WC1-pathogen binding studies, identify ligands Start PRSSV and Anaplasma studies WC1 alternative splicing and signalling studies

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals. In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. • 17 WC1 genes annotated in San Clemente goat genome. In confirmation that these genes are expressed, 6 WC1 cDNAs were cloned from goat, 1 of which was not found in San Clemente goat genome, indicating that the sequenced genome is not complete. There is evidence for as many as 28 caprine WC1 genes, but it is not clear how many of these may be pseudogenes or soluble forms with potential antibiotic activity. • cDNA and genomic evidence for 12 ovine WC1 genes; PacBio sequence of a1 domains shows additional sheep-specific WC1 genes. • 8 fulll length and 2 partial WC1 cDNAs cloned from pig. Only two of these have been previously annotated in the swine genome, 4 are novel. Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiple newly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis andtuberculosis (causes tuberculosis in pigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. Ongoing- Chimeric constructs under construction

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: CL Baldwin, AW Yirsaw, A. Gillespie, L LePage, F. Zhang, P Damani-Yokota and JC Telfer. Cellular immune responses of livestock WC1+ gamma delta T cells to pathogens, Transboundary and Emerging Diseases, Sep 12, 2019, doi: 10.1111/tbed.13328; PMID: 31515956.

Progress 02/01/17 to 01/31/18

Outputs
Target Audience: a cellular and molecular biology class taught to approximately 94 undergraduate students 1 undergraduate students working on this project during the summer of 2017 and supported by a competitive UMass Amherst Center for Agriculture research fellowship 8 undergraduates trained in melecular and cellular biology, genomics 4 graduate students working on this project Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 4 graduate students and 8 undergraduate students have been trained under this project during this reporting period. LePage, L.*,Hsu, H., Nandi, D., Buck, J., Boisvert, N., Damani-Yokota, P., Yirsaw, A., Gillespie, A., Hudgeons, E., Amir, M., Park, H., Baldwin, C.L. and Telfer, J.C.*"Molecular and Functional Variation of the Gamma Delta T Cell PRR/Co-Receptor WC1 Gene Family Among Livestock" (poster)NIFA-USDA Animal Genomics Program Director meeting and 2017 International Plant and Animal Genome Conference,San Diego, CA,USA. January 2018 Telfer, J.C.* and Baldwin, C.L. (invited talk) "WC1 hybrid pathogen recognition receptors and signaling co-receptors direct immune responses by bovine γδ T cells to pathogens"CRWAD meeting, Chicago, IL, USA. December 2017. LePage, L.*,Hsu, H., Nandi, D., Buck, J., Boisvert, N., Damani-Yokota, P., Yirsaw, A.,Gillespie, A., Hudgeons, E., Amir, M., Park, H., Baldwin, C.L. and Telfer, J.C.* "Molecular and functional variation of the γδ T cell pattern recognition receptor/co-receptor WC1 gene family among livestock." (poster and talk)NIFA-USDA Animal Health Program Director meeting and CRWAD meeting, Chicago, IL, USA. December 2017. Le Page, L.,Hsu, H., Nandi, D., Buck, J., Boisvert, N., Damani-Yokota, P., Yirsaw, A., Gillespie, A., Hudgeons, E., Amir, M., Park, H., Baldwin, C.L. andTelfer, J.C.*"WC1 is a hybrid co-receptor and a pathogen-associated molecular pattern receptor and co-receptor for the gamma delta TCR." (talk and poster)American Association of Immunologists (AAI) meeting. Washington, D.C., USA. May 12-16, 2017. Gillespie A., Connelley T., Telfer J.C., Baldwin C.L. "Interaction of γδ TCR with the WC1 hybrid coreceptor/pathogen recognition receptor in cattle" (poster)American Association of Immunologists (AAI) meeting. Washington, D.C., USA. May 12-16, 2017. Damani-Yokota, P.*,Telfer, J.C., and Baldwin, C.L. "Variegated gene expression and Sox13-mediated regulation of WC1 molecules, hybrid PRR/Co-receptor exclusive to γδ T cells" (talk and poster.American Association of Immunologists (AAI) meeting.Washington, D.C., USA. May 12-16, 2017. How have the results been disseminated to communities of interest? I meet with prospective undergraduate students and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the research we do that is is funded by the USDA,why that is important, and how thwy can get involved if they come to UMass. As part of the AAI meeting in Washington DC, I met with the staff ofMassachusetts representatives and senators and shared with them the impact of USDA-funded research. What do you plan to do during the next reporting period to accomplish the goals? Continued characterization of swine WC1 genes and RNA to obtain the last 2 full-length WC1 genes.. Continued characterization of ovine and caprine WC1 genes and RNA Annotation of new Pac-Bio sequenced genomes and deposition of sequence into GenBank database Continue WC1-pathogen binding studies, identify ligands WC1 alternative splicing and signalling studies

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals. In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1: Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. 17 WC1 genes annotated in San Clemente goat genome. In confirmation that these genes are expressed, 6 WC1 cDNAs were cloned from goat, 1 of which was not found in San Clemente goat genome, indicating that the sequenced genome is not complete. There is evidence for as many as 28 caprine WC1 genes, but it is not clear how many of these may be pseudogenes or soluble forms with potential antibiotic activity. cDNA and genomic evidence for 12 ovine WC1 genes; PacBio sequence of a1 domains shows additional sheep-specific WC1 genes. 8 fulll length and 2 partial WC1 cDNAs cloned from pig. Only two of these have been previously annotated in the swine genome, 4 are novel. Objective 2: Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiple newly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis and tuberculosis (causes tuberculosis in pigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. Ongoing- Chimeric constructs under construction

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Damani-Yokota, P., Telfer, J.C., and Baldwin, C.L. 2018. Variegated transcription of the WC1 hybrid PRR/co-receptor genes by individual gamma delta T cells and correlation with pathogen responsiveness. Frontiers in Immunology. 07 May 2018. https://doi.org/10.3389/fimmu.2018.00717.

Progress 02/01/16 to 01/31/17

Outputs
Target Audience:Target audiences reached during ths reporting periodincluded: a cellular and molecular biology class taught to approximately 94 undergraduate students 1 undergraduate student in the NSF program Louis Stokes Alliances for Minority Participation who is training in my lab 2 undergraduate students working on this project in my lab during the summer of 2016 and supported by a competitive UMass Amherst Center for Agriculture research fellowship 7 undergraduates working on this project in my lab during the spring semester of 2016 2undergraduates working on this project in my lab during the fall semester of 2016 2 graduate students working on this project in my lab Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 2 graduate students and 8 undergraduate students have been trained under this project during this reporting period. Janice C Telfer (speaker), Lauren LePage, Alexandria Gillespie, Payal Damani-Yokota, Cynthia L Baldwin. WC1 is a Hybrid gamma delta TCR Coreceptor and Pattern Recognition Receptor for Pathogenic Bacteria. (#35) International Veterinary Immunology Symposium (IVIS) 2016. Gold Coast, Queensland, Australia. August 16-19, 2016. Cynthia L Baldwin (speaker), Janice C Telfer, Payal Damani-Yokota, Alexandria Gillespie. Variegated expression of members of the WC1 pattern recognition receptor and co-receptor multi-gene family on bovine gamma delta T cells direct their response to pathogens (#25) International Veterinary Immunology Symposium (IVIS) 2016. Gold Coast, Queensland, Australia. August 16-19, 2016. Participated in the Bill and Melinda Gates Foundation Grand Challenge Workshop at the International Veterinary Immunology Symposium (IVIS) 2016, Gold Coast, Queensland, Australia. August 17, 2016. "The Bill and Melinda Gates Foundation Grand Challenge workshop will explore the key challenges for the development of safe and effective vaccines for Livestock. Working in small groups participants will identify their top 3 areas of interest and recommend work that could be done to find solutions to these problems. Based on the feedback from this workshop the BMGF will make a call for proposals in the most promising areas." How have the results been disseminated to communities of interest?I meet with prospective undergraduatestudents and their parents who are are interested in our program at UMass. As part of those meetings, I tell them about the research we do that is is funded by the USDA. What do you plan to do during the next reporting period to accomplish the goals? Continued characterization of swine WC1 genes and RNA using 3' RACE and sequencing of PCR-amplified full-length sequences. Continued characterization of ovine and caprine WC1 genes and RNA Annotation of new Pac-Bio sequenced genomes and deposition of sequence into GenBank database Try Pac-Bio sequencing of PCR-amplified WC1 genes Continue WC1-pathogen binding studies WC1 alternative splicing and signalling

Impacts
What was accomplished under these goals? The impact of our accomplishments thus far under these goals is that we are closer to knowing the previously unknown DNA sequences of these important genes in pigs, sheep and goats and have been able to start screening the proteins that these genes encode for their binding to pathogens. This has the potential to lead to better vaccines against these pathogens or better breeding for resistance, which will lead to healthier animals.In the case of pathogens that also infect humans, healthier animals means a safer food supply and healthier humans. Objective 1:Molecular characterization of the WC1 genes and proteins in pigs, sheep, and goats. 16 WC1 genes annotated in San Clemente goat genome. In confirmation that these genes are expressed,6 WC1 cDNAs werecloned from goat, 1 of which was not found in San Clemente goat genome, indicating that the sequenced genome is not complete 2 WC1 cDNAs cloned from sheep. 6 WC1 cDNAs cloned from pig. Only two of these have been previously annotated in the swine genome, 4 are novel. Objective 2:Evaluate the ability of WC1 proteins to bind pathogens relevant to pigs, sheep and goats. Multiplenewly isolated swine WC1 proteins bind to Mycobacterium species such as Mycobacterium bovis and tuberculosis(causes tuberculosis inpigs, sheep, goats and humans) and Mycobacterium avium subsp. paratuberculosis (causes Johne's disease, a wasting disease seen in cattle, sheep, goats and pigs, and has been isolated from patients with Crohn's disease in humans). Objective 3: Characterization of the potential role of WC1 extracellular and intracellular domain splice variants in pathogen binding and signaling for γδ T cell activation. Ongoing

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mercy PrabhuDas, Cynthia L. Baldwin, Paul L. Bollyky, Dawn M.E. Bowdish, Kurt Drickamer, Maria Febbraio, Joachim Herz, Lester Kobzik, Monty Krieger, John Loike, Benita McVicker, Terry K. Means, Soren Moestrup, Steven R. Post, Tatsuya Sawamura, Samuel Silverstein, Robert C. Speth, Janice C. Telfer, Geoffrey M. Thiele, Xiang-Yang Wang, Samuel D. Wright, and Joseph El Khoury. A Consensus Definitive Classification of Scavenger receptors. Journal of Immunology 198 (10) 3775-3789 DOI: https://doi.org/10.4049/jimmunol.1700373.