Progress 09/01/11 to 08/31/17
Outputs
Target Audience:Our target audiences were those attending scientific meetings. The following talks or posters were presented: Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have provided extensive training to undergraduate animal science and pre-veterinary science studentsin our laboratories as well as graduate students. How have the results been disseminated to communities of interest?Through presentations of oral talks and posters at scientific meetings and workshops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Overall Impact (layman's terms) The goal of this project was to use cattle to better understand the diseases leptospirosis and tuberculosis, both global emergingdiseases that can be rapidly fatal and have enormous negative economic impact in agriculture and in human health. Since cattle and humans have similar immune systems, we studied the cattle immune response to the bacteria Leptospira andMycobacteria.Working with cattleallowed us to look at what happens inside the animal after vaccination and infection in a waythat isimpossible with humans. This project allowed us to discover exactlyhow these WC1 receptors bindto the Leptospira bacteria and Mycobacteria to activate bovine gamma delta T cells andwhich WC1 proteins of the thirteen possible are potentially involved. We hypothesized that someof the WC1 receptors bind to molecules made by Mycobacteria and that they are critical for the cattle's immune response to thebacteria, just as in the case of Leptospira.Activation means those cells can react to an infection and aid the infected animal in defeating the infection. We propose thatvaccines could be designed to activate gamma delta T cells through engagement of the WC1 co-receptor with the T cellreceptor to stimulate rapid responses by these cells providing a new paradigm for developing vaccines. Specific Aim 1: Determine the functional differences of the 3 WC1 molecules expressed by the cells within thegamma delta T cell subset that responds to Leptospira. 1.1 How do the different intracytoplasmic tail sequences affect gamma delta T cell responsiveness? Complete and reported in Year 2. 1.2 Do the 2 types of intracytoplasmic tail sequences differ in aspects of signaling? Complete and reported in Year 2. 1.3 Are all 3 WC1 molecules needed for the gamma delta T cells to respond to Leptospira? See Aim 1.4. 1.4 Does SRCR domain 'a' of all 3 WC1 molecules expressed by the responding gamma delta T cells bind andrespond to Leptospira? Completed and reported in Year 3 and/or in a publication that was submitted to Frontier in Immunology. The major conclusion of that report answers the questions posed by the specific aims and subaims indicating that a responsive gamma delta T cell withonly one leptospira-binding molecule responds to the bacteria. We generated 79 gamma delta T cell clones. Our qRT-PCR evaluation showed that approximately 75% of the clones had one to three WC1 genes transcribed per cell but up to six per cell occurred. We also report that the co-transcription of WC1 genes by clones did not have a set pattern although there were differences in the predominant WC1 genestranscribed by cells belonging tothe two subpopulations (WC1.1+ and WC1.2+). Finally, Leptospira-responsive WC1+ memory gamma delta T cell clones were shown to have a high propensity (80 percent) to express WC1 molecules that bind to the pathogen but for most only one of theirWC1s was pathogen-binding. Specific Aim 2: Determine the role of WC1 molecules in responses to mycobacteria and whether the response is restricted to a subset of WC1.1+ cells different from that responding to leptospira. 2.1 Which subset of WC1+ gamma delta T cells respond to M. bovis or M. tuberculosis? Completed and reported in Year 3. 2.2 Does WC1 have a role in the response of bovine gamma delta T cells to Mycobacteria? See subaim 2.3. 2.3 Do the WC1 molecules expressed by the responding cells bind mycobacterial components directly? This work has been completed but not yet published. It was presented as part of a keynote talk by Dr Telfer at the Keystone Conference on Veterinary Immunology in January 2015 in Colorado. We have found that WC1 SRCR domains bindto Mycobacterium spp. However, SRCR domains from different WC1 molecules than those that bind to thespirochetes Leptospira and Borrelia bind to Mycobacterium spp. Thus far, wehave found that WC1 binds to Mycobacterium spp. ligands that we have shown to activate bovine γδ T cellsfrom Mycobacterium bovis vaccinated and infected cattle (this latteris published and reported in Year 3). Unlike the caseof the currently unknown non-peptidic, non-LPS, Leptospira ligand for WC1, our results suggest that while WC1 SRCRdomains bind several Mycobacterium proteins, non-peptidic ligands also exist. Further elucidation of which WC1 SRCRdomains bind to known peptidic ligands and identification of unknown non-peptidic ligands will lead to the identification of thebest molecular candidates to use in designing vaccines targeted to activate the underexploited gamma delta T cell response. 2.4 Is the TCR restricted to a specific set of V genes of either the TCR gamma or TCR delta chain or CDR3 length? We have one publication towards this aim using spectratyping and reported in Year 4 butit is not completed andour new approach using Next Generation Seqeuencing (reported in Year 5) will provide a much finer and thorough analysis. Considerableprogress has been made in the technique that allows us to track each individual transcript in responding cells and sequencethem. We have successfully constructed libraries that have individual unique molecular identifiers for each individual mRNAtranscript that was converted to cDNA and we can amplify T cell receptor genes from those libraries. Specific Aim 3: Evaluate cells and tissue from mycobacteria and leptospira vaccinated cattle for recruitment of WC1 subsets. 3.1 What are the kinetics of responding gamma delta T cells in animals receiving a protective Leptospira serovar hardjo vaccine vs those receiving BCG? Completed for mycobacteria response and reported in Year 3; not completed for Leptospira. 3.2 Which gamma delta T cell subsets are localized/expanded with in the tissues of mycobacteria challenged animals and how does it correlate with protection? Completed and reported in Year 3. 3.3 What is the TCR gene usage by gamma delta T cells? This aim is not completed and our new approach is describedin Year 4. See Aim 2.4 above for further explanation. Here we proposed to use Q-RT-PCR in the grant proposalbut the new next gen sequencing will be much more informative as it will provide us with the entire transcript sequencesincluding the CDR3 of the gamma and delta chains. We have been able to establish the baseline of gamma and delta TCR gene transcription as well as thatof WC1 in newborns and adults.Using next generation sequencing a previous report showed a rapid and global shift in transcription of immunoglobulin genes by neonatal calves during the first month after birth. In contrast,transcription of genes that encode the TCR chains and the gamma delta co-receptor family WC1 hadfew changes during that time period. Also differences between calves and adults was minimal for TCR gene transcription and for most WC1 genes although large differences in transcription frequencies for some WC1 genes occurred suggesting aging or immunological experience changes the baseline repertoire. The transcription frequency of TRAV genes correlated with the number of members in a subgroup, indicating that the choice is stochastic. In contrast, this did not occur for the TCR beta, gamma or delta genes or the WC1 multigenic family indicating their gene expression is deterministic. For example, transcripts for genes in two TRDV1 clades of six TRDV subgroups and eleven TRDV1 clades available, those in one of 23 TRBV subgroups, and those in one of six TRGV cluster predominated. This has been prepared for publication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
PrabhuDas M, Baldwin CL, Bollyky PL, Bowdish DME, Drickamer K, Febbraio M, Herz J, Kobzik L, Krieger M, Loike J, McVicker B, Means TK, Moestrup S, Post SR, Tatsuya Sawamura T, Silverstein S, Speth RC, Telfer JC, Thiele GM, Wang X-Y, Wright SD, El Khoury J. A Consensus Definitive Classification of Scavenger receptors. Journal of Immunology 2017; 198(10):3775-3789. doi: 10.4049/jimmunol.1700373. PMID: 28483986
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Damani-Yokota P, Telfer JC and Baldwin CL. Variegated expression of WC1 hybrid PRR/Co-receptor genes by individual gamma delta T cell clones and correlation with pathogen responsiveness.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2018
Citation:
Damani-Yokota P, Gillespie A, Pasman Y, Merico D, Connelley TK, Kaushik A and Baldwin CL. Transcript analysis of bovine T cell receptor genes as well as the gamma delta WC1 co-receptor/pattern recognition receptor genes during the first month of life.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2018
Citation:
Payal Damani-Yokota. Regulation of expression o f the gamma delta T cell co-recptor and pattern recognition receptor multi-gene family WC1. Program in Cellular and Molecular Biology. University of Massachusetts Amherst.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gillespie A., Connelley T., Telfer J.C., Baldwin C.L. "Interaction of ?? 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 (poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
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 ?? T cells" (talk and poster. American Association of Immunologists (AAI) meeting. Washington, D.C., USA. May 12-16, 2017 (oral and poster presentations).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
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. "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 (poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
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 ?? T cell pattern recognition receptor/co-receptor WC1 gene family among livestock." (poster and talk) NIFA-USDA Program Director meeting and CRWAD meeting, Chicago, IL, USA. December 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Damani-Yokota P, Telfer JC and Baldwin CL. Variegated expression of members of a pathogen recognition receptor and co-receptor multi-gene family on gd T cells known as WC1. New England Immunology Conference: Woodshole, MA. October-2016. (Poster presentation).
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Our target audiences were those who attend scientific meetings. We presented the following papers: Maggioli MF, MV Palmer, TC Thacker, HM Vordermeier, JL McGill, MH Larsen,WR Jacobs Jr, and WR Waters. 2016. Polyfunctional cytokine production bycentral memory T cells from cattle in response to Mycobacterium bovisinfection and BCG vaccination. American Association of Immunologist,Annual Conference. Seattle, WA (abstract), May 2016. Maggioli MF, MV Palmer, TC Thacker, HM Vordermeier, JL McGill, MH Larsen,WR Jacobs Jr, and WR Waters. 2015. Polyfunctional cytokine production bycentral memory T cells from cattle in response to Mycobacterium bovisinfection and BCG vaccination. Autumn Immunology Conference. Chicago, IL(abstract), 2015. 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. Payal Yokota (speaker), Janice Telfer, Cynthia Baldwin. Variegated gene expression by the bovine gamma delta WC1 co-receptor. 11th Gamma Delta T cell conference, London, England, June 2016. Payal Yokota (speaker), Janice Telfer, Alie Gillespie, Cynthia Baldwin. Variegated gene expression of WC1 co-receptor by gamma delta T cell clones. Annual meeting of the American Association of Immunologists, Seattle, WA, May 2016. Rusk, RA; Palmer, MV; Waters, WR; McGill, JL. 2016. Determining bovinegamma delta T cell function at the site of Mycobacterium bovis infection.Conference for Research Workers in Animal Diseases, Chicago, IL. December2016, accepted for presentation. Guerra-Maupome, M; Larsen, MH; Palmer, MV; Waters, WR; McGill, JL. 2016.Neonatal gamma delta T cell responses to mucosal TB vaccination.Conference for Research Workers in Animal Diseases, Chicago, IL. December2016, accepted for persentation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training has been offered to graduate students, of which 5of 6are female (A. Gillespie, P. Yokota, L. LePage, A. Yirsaw, M. Amir, Mayara Maggioli) in this past year, to a female laboratory technician who is in a temporary position (Shelly Zimmerman,and to sevenundergraduate students in animal science, all of whom are female (Natalie Boisvert and Jessica Bach who were Center for Agriculture and Food and the Environment scholars and awarded summer fellowships by UMass; Katherine Wilson and Ariel Nenninger who are now veterinary students at Tufts University; Julie MacLaughlin and Haeree Park; and Lauren Holz who is now in a MS in Public Health program). They were able to work one-on-one with research scientists during the academic year as well as during summer-long projects. Training was offered to veterinarians from developing countries, from Pakistan (Maria Amir who came on a USAID fellowship) and Ethiopia (Al Yirsaw who is on release from the National Diagnostic Center in Ethiopia to complete a PhD). They are being trained in the most modern molecular biology techniques and next generation sequencing on projects relevant to increasing food animal health. They will be a great asset to their institutions upon returning home. Also Dr. Jodi McGill was a post-doctoral fellow on this project and she is now an assistant professor in the College of Veterinary Medicine at Kansas State University. She continues to collaborte with our labs. How have the results been disseminated to communities of interest? Through scientific publications and presentations at major conferences nationally and internationally as described in a separate section. There were 2platform presentations at the International Veterinary Immunology Symposium, 1 at the AAI general meeting and 1 at the winter meeting, 1 at the Gamma Delta T cell meeting in London.Dr Baldwin was also awarded the Chancellor's Medal for excellence in research and presented a public address to the University of Massachusetts and the public on our work and its importance to food security and human nutrition.Also discussions were held in a workshop at the International Veterinary Immunology Symposium were relevant and to which Drs Baldwin and Telfer contributed: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 explored 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. In addition we had 4 publications and links are provided to those published:http://journal.frontiersin.org/article/10.3389/fimmu.2016.00421/full?utm_so urce=newsletter&utm_medium=email&utm_campaign=Immunology-w43-2016 and http://www.cabi.org/bookshop/book/9781780643960 What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Determine the functional differences of the 3 WC1 molecules expressed by the cells within the gamma delta T cell subset that responds to Leptospira. 1.1 How do the different intracytoplasmic tail sequences affect gamma delta T cell responsiveness? None, complete. 1.2 Do the 2 types of intracytoplasmic tail sequences differ in aspects of signaling? None, complete. 1.3 Are all 3 WC1 molecules needed for the gamma delta T cells to respond to Leptospira? None, complete. 1.4 Does SRCR domain 'a' of all 3 WC1 molecules expressed by the responding gamma delta T cells bind and respond to Leptospira? None, completed but must be prepared for publication. Specific Aim 2: Determine the role of WC1 molecules in responses to mycobacteria and whether the response is restricted to a subset of WC1.1+ cells different from that responding to leptospira. 2.1 Which subset of WC1+ gamma delta T cells respond to M. bovis or M. tuberculosis? None, complete. 2.2 Does WC1 have a role in the response of bovine gamma delta T cells to Mycobacteria? This will be completed in the coming year. Previously we addressed for responses to leptopsiraby using shRNA to silence WC1 genes in primary gamma delta T cells and can do that in the coming year for the response to Mycobacteria. However it may now be possible to use a CRISPR-Cas9 approach although because this is a large gene family that may not be feasible. 2.3 Do the WC1 molecules expressed by the responding cells bind mycobacterial components directly? Further elucidation of which WC1 SRCR domains bind to known peptidic ligands and identification of unknown non-peptidic ligands of mycobacteriawill lead to the identification of the best molecular candidates to use in designing vaccines targeted to activate the underexploited gamma delta T cell response. The WC1 proteins are being expressed for further binding assays. 2.4 Is the TCR restricted to a specific set of V genes of either the TCR gamma or TCR delta chain or CDR3 length? We are currently preparing the libraries needed for gamma delta T cells responsive to various components of mycobacteria and leptospira as well as non-responsive control populations of cells and cell populations activated nonspecifically. and will subject them to next generation sequencing. We are working with scientists, statisticians and bioinformaticists at Roslin Institute in Edinburgh, UK to perfect the analysis. Specific Aim 3: Evaluate cells and tissue from mycobacteria and leptospira vaccinated cattle for recruitment of WC1 subsets. 3.1 What are the kinetics of responding gamma delta T cells in animals receiving a protective Leptospira serovar hardjo vaccine vs those receiving BCG? Completed for BCGresponse and reported in Year 3. The kinetic response to Leptospira is being evaluated in heifer calves following vaccination by collecting peripheral blood mononuclear cells and evaluating the responding gamma delta T cells. Using our next generation sequecing we will be able to evaluate both their WC1 gene usage and their T cell receptor sequences and monitor changes over time. 3.2 Which gamma delta T cell subsets are localized/expanded with in the tissues of mycobacteria challenged animals and how does it correlate with protection? We are currently analyzing expression of select genes by gamma delta T cells both in vitro in response to various mycobacterial antigens or live M. bovis, and in vivo at the site of M. bovis infection, as they may relate to the role of gamma delta T cells in TB granuloma formation and maintenance and thus protective immunity. We are also currently involved in determining the memory phenotype and expression of lung-homing receptors by gamma delta T cells responding to mucosal TB vaccination or virulent M. bovis infection. We are assessing expression of lung-homing moleculesby M. bovis-responsive gamma delta T cells which have been isolated from the peripheral blood or airways of calves vaccinated via aerosol with M. bovis BCG, or from cattle that have been infected via aerosol with virulent M. bovis. 3.3 What is the TCR gene usage by gamma delta T cells? See Aim 2.4 above for further explanation of our new approach to this subaim. ?
Impacts
What was accomplished under these goals?
Specific Aim 1: Determine the functional differences of the 3 WC1 molecules expressed by the cells within the gamma delta T cell subset that responds to Leptospira. 1.1 How do the different intracytoplasmic tail sequences affect gamma delta T cell responsiveness? Complete and reported in Year 2. 1.2 Do the 2 types of intracytoplasmic tail sequences differ in aspects of signaling? Complete and reported in Year 2. 1.3 Are all 3 WC1 molecules needed for the gamma delta T cells to respond to Leptospira? Results were reported in an oral and poster presentation at the American Association of Immunologists' meeting in Seattle in May 2016. It was selected as an oral presentation in London at the international Gamma Delta T cell 2016 conference. When the proposal was written 6 years ago our data using flow cytometrically sorted populations of leptospira responsive cells suggested that individual gamma delta T cells expressed 2 or 3 WC1 molecules. We have since made gamma delta T cells clones responsive to Leptospira and developed q-RT-PCR assays using Taqman probes to more closely assess their WC1 gene transcript expression. We found that the T cell clones express either a single WC1 gene or 2WC1 genes.These results indicate that a single type of WC1 molecule is sufficient for the gamma delta T cell to respond. 1.4 Does SRCR domain 'a' of all 3 WC1 molecules expressed by the responding gamma delta T cells bind and respond to Leptospira? Completed and reported in Year 3 and here by our unpublished results evaluating the gamma delta leptospira-responsiveT cell clones. We found that those clones while differing in the single WC1 gene or multiple WC1 genes they express, that the various WC1 geneproducts express bind leptospira. We are completing this work for publication. Specific Aim 2: Determine the role of WC1 molecules in responses to mycobacteria and whether the response is restricted to a subset of WC1.1+ cells different from that responding to leptospira. 2.1 Which subset of WC1+ gamma delta T cells respond to M. bovis or M. tuberculosis? Completed and reported in Year 3. 2.2 Does WC1 have a role in the response of bovine gamma delta T cells to Mycobacteria? Not started yet. 2.3 Do the WC1 molecules expressed by the responding cells bind mycobacterial components directly? This work has been partially completed but not yet published. It was presented as part of a keynote talk by Dr Telfer at the Keystone Conference on Veterinary Immunology in January 2015 in Colorado. We have found thatWC1 SRCR domains bind toMycobacterium spp.However, SRCR domains from different WC1 molecules than those that bind to the spirochetesLeptospiraandBorreliabind toMycobacteriumspp. This is consistent with our hypothesis that multi-genic genes WC1 arrays were conserved over millions of years of evolution by selective pressure from diverse pathogens. Thus far, we have found that WC1 binds toMycobacteriumspp. ligands that we have shown to activate bovine γδ T cells fromMycobacterium bovisvaccinated and infected cattle (thislatter part is published and reported in Year 3). Unlike the case of the currently unknown non-peptidic, non-LPS,Leptospiraligand for WC1, our results suggest that while WC1 SRCR domains bind severalMycobacteriumproteins, non-peptidic ligands also exist. Further elucidation of which WC1 SRCR domains bind to known peptidic ligands and identification of unknown non-peptidic ligands will lead to the identification of the best molecular candidates to use in designing vaccines targeted to activate the underexploited gamma delta T cell response. 2.4 Is the TCR restricted to a specific set of V genes of either the TCR gamma or TCR delta chain or CDR3 length? While we have one publication towards this aim using spectratyping and reported in Year 4 we feel it is not completed and that our new approach using Next Generation Seqeuencingwill provide amuch finer and thorough analysis. Considerable progress has been made in the technique that allows us to track each individual transcript in responding cells and sequence them. We have successfully constructed libraries that have individual unique molecular identifiers for each individual mRNA transcript that was converted to cDNA and we can amplify T cell receptor genes from those libraries. We are currently preparing the libraries needed for gamma delta T cells responsive to various components of mycobacteria and leptospira as well as non-responsive control populations of cells and cell populations activated nonspecifically. and will subject them to next generation sequencing. We are working with scientists, statisticians and bioinformaticists at Roslin Institute in Edinburgh, UK to perfect the analysis. Specific Aim 3: Evaluate cells and tissue from mycobacteria and leptospira vaccinated cattle for recruitment of WC1 subsets. 3.1 What are the kinetics of responding gamma delta T cells in animals receiving a protective Leptospira serovar hardjo vaccine vs those receiving BCG? Completed for mycobacteria response and reported in Year 3. The kinetic response to Leptospira is being evaluated in heifer calves following vaccination by collecting peripheral blood mononuclear cells and evaluating the responding gamma delta T cells. Using our next generation sequecing we will be able to evaluate both their WC1 gene usage and their T cell receptor sequences and monitor changes over time. We recently completed a trial project through a collaboration with Professor Azad Kaushik, Guelph, Ontario, evaluating the WC1 gene transcripts and T cell receptor genes in normal calves over the first month of life to establish a baseline. 3.2 Which gamma delta T cell subsets are localized/expanded with in the tissues of mycobacteria challenged animals and how does it correlate with protection? Paritally completed and reported in Year 3 and now we are currently analyzing expression of select genes by gamma delta T cells both in vitro in response to various mycobacterial antigens or live M. bovis, and in vivo at the site of M. bovis infection, as they may relate to the role of gamma delta T cells in TB granuloma formation and maintenance and thus protective immunity. We are also currently involved in determining the memory phenotype and expression of lung-homing receptors by gamma delta T cells responding to mucosal TB vaccination or virulent M. bovis infection. We are assessing expression of lung-homing moleculesby M. bovis-responsive gamma delta T cells which have been isolated from the peripheral blood or airways of calves vaccinated via aerosol with M. bovis BCG, or from cattle that have been infected via aerosol with virulent M. bovis. 3.3 What is the TCR gene usage by gamma delta T cells? This aim is not completed and our new approach is described in the report and also was recorded in the last 'REEPORT' progress report Year 4. See Aim 2.4 above for further explanation. Here we proposed to us Q-RT-PCR in the grant proposal but the new next gen sequencing will be much more informative as it will provide us with the entire transcript sequences including the CDR3 of the gamma and delta chains. Overall Impact (layman's terms) Because we have shown that molecules known as WC1, and found on the survaceof a particular type of cell in the immune system known as a gamma delta T cell, bind infectious disease agents specifically resulting in activation of those cells. Activation means those cells can reactto an infection and aid the infected animal in defeating the infection. We propose that vaccines could be designed to activate gamma delta T cells through engagement of the WC1 co-receptor with the T cell receptor to stimulate rapid responses by these cells providing a new paradigm for developing vaccines for diseases that to date we have been unsuccessful with regard to.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
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, Joseph El Khoury. A Consensus Definitive Classification of Scavenger receptors. Journal of Immunology 2016 (in press).
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Vordermeier H.M., W.R. Waters, and B.M. Buddle. 2015. (editors: H
Mukundan, MA Chambers, WR Waters, and MH Larsen) Vaccines and biomarkers
for Mycobacterium bovis infection in cattle. In: Many Hosts of
Mycobacteria: Tuberculosis, Leprosy, and other Mycobacterial Diseases of
Man and Animals, 10:185-201.
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Waters W.R., J.C. Hope, C.A. Hamilton, M.V. Palmer, J. McNair, R.A. Skuce,A.A. Allen, B.M. Buddle, B. Villarreal-Ramos, and H.M. Vordermeier. 2015.
(editors: H Mukundan, MA Chambers, WR Waters, and MH Larsen)
Immunopathogenesis of Mycobacterium bovis infection of cattle. In: Many
Hosts of Mycobacteria: Tuberculosis, Leprosy, and other Mycobacterial
Diseases of Man and Animals, 8:136-167.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Maggioli M.F., M.V. Palmer, T.C. Thacker, H.M. Vordermeier, J.L. McGill,
A.O. Whelan, M.H. Larsen, W.R. Jacobs Jr., and W.R. Waters. Increased
TNF-?/IFN-?/IL-2 and decreased TNF-?/IFN-? production by central memory
polyfunctional T cells are associated with protective responses against
bovine tuberculosis following BCG vaccination. Frontiers in Immunology, in
press and published online [doi: 10.3389/fimmu.2016.00421]
|
Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Our results were presented at a variety of scientific meetings as follows: Haoting Hsu, Chuang Chen, Ariel Nenninger, Lauren Holz, Cynthia L. Baldwin and Janice C. Telfer* "WC1 is a hybrid gamma delta TCR coreceptor and Pattern Recognition Receptor for pathogenic bacteria." (invited talk) Keystone Symposia on Molecular and Cellular Biology: Immunity to Veterinary Pathogens:Informing Vaccine Development. Keystone, Colorado, USA January 20-25, 2015. Haoting Hsu, Chuang Chen, Ariel Nenninger, Lauren Holz, Cynthia L. Baldwin and Janice C. Telfer* "WC1 is a hybrid gamma delta TCR coreceptor and Pattern Recognition Receptor for pathogenic bacteria." (invited talk) Sixth North American Comparative Immunology Workshop. Toronto, Ontario, Canada. June 13-15, 2015. Cynthia L. Baldwin* and Janice C. Telfer "Bovine gamma delta T cells' unique WC1 pattern recognition receptors (PRR) direct cellular immune responses to bacterial pathogens." Brucellosis 2014 International Research Conference, Berlin, Germany, September 9-12, 2014. Rusk, RA, MV Palmer, WR Waters, JL McGill. 2015. Measuring gamma delta T cell function at the site of Mycobacterium bovis infection. Conference for Research Workers in Animal Diseases, Chicago, IL. December 6-8 2015. McGill, JL. 2015 The role of gamma delta T cells in immunity to respiratory infections in the bovine. 14th Annual Great Plains Infectious Disease Meeting, Lawrence, KS. November 6-7 2015. Waters W.R. 2015.Vaccine Approaches for Bovine Tuberculosis: Correlates of Protection and Relevance to Human Tuberculosis. Vaccines for the Modern Era: Implications of Human andAnimal Health II. American Association of Immunologists Annual Meeting, New Orleans, Louisiana, May 8 - 12, 2015. Waters W.R. 2015.Vaccine Approaches for Bovine Tuberculosis: Correlates of Protection and Relevance to Human Tuberculosis. Immunity to Veterinary Pathogens: Informing Vaccine Development. Keystone Symposia, Keystone Colorado, January 20-25, 2015. Maggioli MF, MV Palmer, TC Thacker, HM Vordermeier, JL McGill, MH Larsen, WR Jacobs Jr, and WR Waters. 2015. Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination. Autumn Immunology Conference. Chicago, IL Maggioli M, M. Palmer, A. Whelan, H. Vordermeier, W. Waters. 2015. Polyfunctional cytokine responses by central memory CD4+ T cells in response to bovine tuberculosis. Host Response to Tuberculosis and Granulomas in Infectious and Non-Infectious Disease, Keystone Symposia,Santa Fe, New Mexico, January 22-27, 2015. Changes/Problems:The development of bioinformatics programs to use efficiently the Next Gen Sequencing methods for evaluation of immune response in livestock has proven challenging for a number of reasons including incomplete and incorrectly assembled genomes particularly in the ares of immune response genes and pattern recognition receptors. On 26-27thOctober 2015 the BBSRC Veterinary Vaccinology Network hosted a bioinformatics workshop focussing on functional genomics at The Roslin Institute, Edinburgh. The workshop was conceived at a joint US-UK Funders and Research workshop hosted in Washington earlier in the year. Discussions there among US and UK participants validated the fundamental importance and enormous potential of bioinformatics for informing veterinary vaccinology research and the value of US-UK collaborations for moving this field forward.Nearly 100 delegates from a variety of UK and USA organisations came together. The aim of the workshop was to understand the current research focus of different disciplines and the bioinformatic and biological resources currently available, focusing on the unique problems in analysing sequencing data from non-human/non-model species particularly when studying immune responses. It was affirmed that there is a pressing need for further collaborative discussion between veterinary vaccinologists and bioinformaticians to realise the potential of high-throughput data and for the need to use a unified approach to generating and analysing the data given the relatively small size of the veterinary vaccinology groups worldwide. What opportunities for training and professional development has the project provided?Training has been offered to graduate students, of which 4 of 5 are female, toa female laboratory technician who is in a temporary position,to a female veterinary student and to undergraduate students in animal science, both of whom are female. How have the results been disseminated to communities of interest?Through scientific publications and presentations at major conferences nationally and internationally. What do you plan to do during the next reporting period to accomplish the goals?We plan to repeat the study with the BCG deletion mutants in calves using a higher vaccine dose and improved aerosol delivery method and evaluate the gamma delta T cell response for WC1 gene usage and the TCR using Next Gen Sequencing methods. Scientists at both University of Massachusetts Amherst and NADC in Ames, Iowa who are PIs on the grantare collaborating with other scientists at NADC and at the USDA-ARS MARC center in Nebraska as well as those at the Roslin Institute of Genetics in Edinburgh, Scotland and Pirbright Institute in England to solve these problems. We can then use these new methods more efficiently to analyze populations of gamma delta T cells that respond to both mycobacteria and leptospira to understand the diversity of receptors that are used by such cells than the methods proposed originally in the grant application (spectratyping, etc).
Impacts
What was accomplished under these goals?
Th17-associated cytokines are integral in the immune response to tuberculosis, initiating both protective and harmful inflammatory responses. We have shown in our previous studies that both CD4 and WC1+ gamma delta T cells produce IL-17 and IFN-gamma in response to mycobacterial antigens, although CD4 T cells were the major producer of IL-17 in cattle, contrary to observations in mice challenged with mycobacteria. We evaluate applied aspects of IL-17 biology in the context of Mycobacterium bovis infection of cattle using RNA-seq to show that numerous Th17-associated cytokine genes (including IL-17A, IL-17F, IL-22, IL-19, and IL-27) were up-regulated > 9 fold in response to purified protein derivative stimulation of peripheral blood mononuclear cells from experimentally M. bovis-infected cattle. Protective vaccines elicited IL-17A, IL-17F, IL-22, and IL-27 responses. As compared to non-vaccinated animals, reduced IL-17A responses by vaccinate recipients at 2.5 weeks after M. bovis challenge correlated with reduced disease burden. Additionally, IL-17A and IFN-gamma?responses were highly correlated and exhibited similar diagnostic capacity. Present findings support the use of Th17-associated cytokines as biomarkers of infection and protection in the immune response to bovine tuberculosis (Waters et al. 2015 Clin Vac Immunol). We have also completed a calf study to analyze the systemic and mucosal gamma delta T cell response in neonatal calves vaccinated with a cocktail of the attenuated BCG deletion strains referred to above. Our collaborators at Albert Einstein College of Medicine have constructed deletion derivatives of BCG Danish that, compared to the parental BCG Danish strain, have (1) an improved safety profile by additional attenuation and (2) comparable or superior vaccine efficacy. The vaccine preparation we tested in this study was a cocktail of four BCG Danish deletion strains including BCG fdrA K/O,BCG leuCD K/O pks16 K/O, BCG metA K/O, BCG mmaA4 K/O. We had demonstrated that this cocktail had similar efficacy to BCG when administered parenterally (Waters et al. 2015 Clin Vac Immunol). Therefore, the objective of our study was to determine (1) the safety and overall immunogenicity of the BCG mutant cocktail delivered via aerosol vaccination; and (2) determine the gamma delta T cell response to aerosol BCG mutant vaccination. Neonatal calves were vaccinated or not via aerosol inoculation with 10^6 CFU total of the BCG mutant cocktail (2.5x10^5) of each mutant. Two and seven weeks after vaccination, animals were sacrificed and the gamma delta T cell response analyzed in the lungs and lymphoid tissues of the upper respiratory tract. Analysis on this project is currently underway.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Telfer JC, Baldwin CL. Bovine gamma delta T cells and the function of gamma delta T cell specific WC1 co-receptors. Cellular Immunology. 2015; 296(1):76-86. PubMed PMID: 26008759
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Waters WR, Maggioli MF, MV Palmer, TC Thacker, JL McGill, HM Vordermeier, L Berney-Meyer, WR Jacobs Jr, MH Larsen. 2016. Interleukin-17A as a biomarker for bovine tuberculosis. Clinical and Vaccine Immunology, 2015 Dec 16. pii: CVI.00637-15. [Epub ahead of print]
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: Scientists interested in basic immunology as well as those interested in vaccines. Potentially pharmaceutical companies interested in vaccine design. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students in pre-veterinary science and animal science have been trained as well as graduate students and post-doctoral fellows. Most of these have been women. How have the results been disseminated to communities of interest? The results of the project were presented at a Keystone Conference on Immunity to Veterinary Pathogens: Informing Vaccine Development with principal invited talks by Dr Telfer and a second by Dr. Waters. Results were also presented at the American Association of Immunologists annual general meeting with a tallk by Dr Baldwin in the Veterinary and Comparative Immunology section. What do you plan to do during the next reporting period to accomplish the goals? Work is planned to evaluate the role of the T cell receptor in the responses by the WC1-bearing gamma delta T cells using cattle infected with Mycobacterium bovis or vaccinated with BCG. In addition we will continue to evaluate the variegated gene expression of the WC1 multigenic family on gamma delta T cell subpopulations.
Impacts
What was accomplished under these goals?
The following is exerted from our peer-reviewed publications. WC1 coreceptors are scavenger receptor cysteine-rich (SRCR) family members, related to T19 in sheep, SCART in mice, and CD163c-α in humans, and form a 13-member subfamily in cattle exclusively expressed on γδ T cells. Subpopulations of γδ T cells are defined by anti-WC1 mAbs and respond to different pathogen species accordingly. In this study, variegated WC1 gene expression within subpopulations and differences in signaling and cell activation due to endodomain sequences are described. The endodomains designated types I to III differ by a 15- or 18-aa insert in type II and an additional 80 aa containing an additional eight tyrosines for type III. Anti-WC1 mAbs enhanced cell proliferation of γδ T cells when cross-linked with the TCR regardless of the endodomain sequences. Chimeric molecules of human CD4 ectodomain with WC1 endodomains transfected into Jurkat cells showed that the tyrosine phosphorylation of the type II was the same as that of the previously reported archetypal sequence (type I) with only Y24EEL phosphorylated, whereas for type III only Y199DDV and Y56TGD were phosphorylated despite conservation of the Y24EEL/Y24QEI and Y199DDV/I tyrosine motifs among the three types. Time to maximal phosphorylation was more rapid with type III endodomains and sustained longer. Differences in tyrosine phosphorylation were associated with differences in function in that cross-linking of type III chimeras with TCR resulted in significantly greater IL-2 production. Identification of differences in the signal transduction through the endodomains of WC1 contributes to understanding the functional role of the WC1 coreceptors in the γδ T cell responses. The WC1 proteins uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily are present in variable, and sometimes high, numbers in the genomes of mammals and birds. In cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials. The WC1 cytoplasmic domains contain multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens. Promoting effective immunity to Mycobacterium bovis infection is a challenge that is of interest to the fields of human and animal medicine alike. We report that γδ T cells from virulent M. bovis-infected cattle respond specifically and directly to complex, protein, and nonprotein mycobacterial Ags. Importantly, to our knowledge, we demonstrate for the first time that bovine γδ T cells specifically recognize peptide Ags derived from the mycobacterial protein complex ESAT6:CFP10 and that this recognition requires direct contact with APCs and signaling through the T cell Ag receptor but is independent of MHC class I or II. Furthermore, we show that M. bovis infection in cattle induces robust IL-17A protein responses. Interestingly, in contrast to results from mice, bovine CD4 T cells, and not γδ T cells, are the predominant source of this critical proinflammatory mediator. Bovine γδ T cells are divided into subsets based upon their expression of Workshop Cluster 1 (WC1), and we demonstrate that the M. bovis-specific γδ T cell response is composed of a heterogeneous mix of WC1-expressing populations, with the serologically defined WC1.1(+) and WC1.2(+) subsets responding in vitro to mycobacterial Ags and accumulating in the lesions of M. bovis-infected animals. The results enhance our understanding of γδ T cell biology and, because virulent M. bovis infection of cattle represents an excellent model of tuberculosis in humans, contribute to our overall understanding of the role of γδ T cells in the mycobacterial-specific immune response.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
McGill, J.L., R.E. Sacco, C.L. Baldwin, J. C. Telfer, M. V. Palmer and W. R. Waters. (2014) Specific recognition of mycobacterial protein and peptide antigens by gamma delta T cell subsets following infection with virulent Mycobacterium bovis. Journal of Immunology 192:27560-27569.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Baldwin, C.L., H. Hsu, C. Chen, M.Palmer, J. McGill, W.R. Waters and J. Telfer. (2014) The role of bovine gamma delta T cells and their WC1 co-receptor in response to bacterial pathogens and promoting vaccine efficacy: a model for cattle and humans. Veterinary Immunology and Immunopathoogy 159:144-155.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
McGill, J.L., R.E. Sacco, C.L. Baldwin, J.C. Telfer, M.V. Palmer, and W.R. Waters. (2014) The role of gamma delta T cells in immunity to Mycobacterium bovis infection in cattle. Veterinary Immunology and Immunopathoogy 159:133-143.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Chen, C., H. Hsu, E. Hudgens, J. Telfer, and C.L. Baldwin. (2014) Signal transduction by different forms of the ?? T cell-specific pattern recognition receptor WC1. Journal of Immunology 193:379-390.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hsu, H, C.L. Baldwin and J.C. Telfer. (2015) The endocytosis and signaling of the gamma delta T cell coreceptor WC1 is regulated by a dileucine motif. Journal of Immunology PMID: 25632004.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hsu, H, C. Chen, A. Nenninger, L. Holz, C.L. Baldwin and J.C. Telfer. (2015) WC1 is a hybrid gammadelta TCR coreceptor and Pattern Recognition Receptor for pathogenic bacteria. Journal of Immunology, PMID: 25632007.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Baldwin, C.L. and J.C. Telfer. (2015). The bovine model for elucidating the role of ?? T cells in controlling infectious diseases of importance to cattle and humans. Molecular Immunology, PMID: 25547715.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Herzig, C.T.A.H. and C.L. Baldwin. (2015) Characterizing the T cell receptor ? chain CDR3 region by spectratyping of bovine gamma delta T cells responding to leptospira. Immunogenetics 67:95-109.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Waters, W. R., M.F. Maggioli, J L. McGill, K.P. Lyashchenko, M.V. Palmer. (2014) Relevance of bovine tuberculosis research to the understanding of human disease: Historical perspectives, approaches, and immunologic mechanisms. Veterinary Immunology and Immunopathology 159:113-132.
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: Scientists interested in basic immunology as well as those interested in vaccines. Potentially pharmaceutical companies interested in vaccine design. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students and post-doctoral fellows along with undergraduate students and technicians have been trained. How have the results been disseminated to communities of interest? Presented at several scientific meetings and publications produced. What do you plan to do during the next reporting period to accomplish the goals? We are continuing our work to determine the distribution of WC1 molecules among subpopulations of gamma delta T cells. We will be making gamma delta T cells clones by using cytokine activation but also with transformation with the protozoan parasite Theileria parva. We will evaluate direct interaction between WC1 domains and mycobacterial components and products and also that of anaplasma.
Impacts
What was accomplished under these goals?
Promoting effective immunity to Mycobacterium bovis infection is a challenge that is of interest to the fields of human and veterinary medicine alike. We report that γδ T cells from virulent M. bovis infected cattle respond specifically and directly to protein and non-protein mycobacterial antigens. Importantly, we demonstrate for the first time that bovine γδ T cells specifically recognize peptides of the mycobacterial protein ESAT6:CFP10 and that this recognition requires direct contact with APC and signaling through the T cell antigen receptor but is independent of MHC class I or II. The responding γδ T cells produce interferon-γ and IL-17 but interestingly, in contrast to results from mice bovine CD4 T cells and not γδ T cells are the predominant source of the critical pro-inflammatory mediator IL-17A. Bovine γδ T cells are divided into subsets based upon their expression of Workshop Cluster 1 (WC1) and we demonstrate that the M. bovis-specific γδ T cell response is composed of a heterogeneous mix of WC1-expressing populations, with the serologically defined WC1.1+ and WC1.2+ subsets responding in vitro to mycobacterial antigens and accumulating in the lesions of M. bovis infected animals. The results described herein enhance our understanding of γδ T cell biology and, as virulent M. bovis infection of cattle represents an excellent model of tuberculosis in humans, contribute to our overall understanding of the role of γδ T cells in the mycobacterial-specific immune response.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
McGill, J.L., R.E. Sacco, C.L. Baldwin, J. C. Telfer, M. V. Palmer, W. R. Waters. 2014. Specific recognition of mycobacterial protein and peptide antigens by gamma delta T cell subsets following infection with virulent Mycobacterium bovis. J. Immunology, accepted
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Chen, C., H. Hsu, E. Hudgens, J. Telfer, and C.L. Baldwin. Signal transduction by different forms of the ?? T cell-specific pattern recognition receptor WC1.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: Gamma delta T cells are deemed critical to immune surveillance and protection since they are found as resident cells in many organs and tissues including in humans, cattle and mice, and circulate at substantial numbers ranging from 5 to 30 percent in the peripheral blood mononuclear cells of cattle and comprise up to 15 percent in human pre-adolescents. It is known that they contribute to cellular immunity and protection against important pathogens, organizing granulomas in response to Mycobacteria and producing interferon-gamma following vaccination to serovar Hardjo strains of Leptospira. However, it is not clear how best to prime gamma delta T cells for recall responses and few efforts have directly addressed the contribution of co-receptor stimulation. We are contributing to filling this gap by addressing the overarching hypothesis that the WC1 co-receptors on gamma delta T cells, as an example of PRRs on nonconventional T cells, can be exploited to fully activate these cells when combined with TCR activation. Annotation of the bovine genome showed there are multiple WC1 molecules coded for by individual genes. The WC1 molecules are distributed among cells such that they result in division of the cells into a number of gamma delta T cell subsets. Using RNA silencing we have shown that the WC1 co-receptor contributes to the ability of gamma delta T cells to respond to leptospira. The leptospira-responsive gamma delta T cells are found within a subset of the serologically-defined WC1.1 gamma delta T cell. Previous studies also suggest that M. bovis responsive gamma delta T cells are contained within the WC1.1 subset; however, this evidence is primarily correlative and it remains unknown which of these subsets are directly responding to M. bovis. The specific aims are to determine the functional differences of the WC1 molecules expressed by the cells within the gamma delta T cell subset that responds to Leptopsira and mycobacteria and determine what restricts the responses to a subset of cells within the WC1.1 population; and evaluate cells and tissue from mycobacteria (vaccinated and challenged) and leptospira vaccinated cattle for recruitment of WC1 subsets in vivo as defined by their WC1 gene expression. PARTICIPANTS: This proposal represents a collaboration between the University of Massachusetts Amherst (UMA) with Drs Janice Telfer and Cynthia Baldwin as PDs with graduate students Chuang Chen, HaoTing Hsu and Payal Yokota and several undergraduate science majors participating. At the National Animal Disease Center, Ames, Iowa, which is part of the United States Department of Agriculture's Agricultural Research Service (USDA-ARS), Drs Ray Waters and Mitchel Palmer are the PDs and post-doctoral fellow Jodi McGill participates. TARGET AUDIENCES: Basic and applied scientists and vaccine and pharmaceutical companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Completion of the annotation of the bovine genome and sequencing of full length cDNA transcripts of WC1 molecules show there are 13 WC1 molecules coded for by individual genes, that the WC1 molecules are distributed among cells to form a number of gamma delta T cell subsets, that this number of WC1 molecules is conserved among breeds and individuals as are the gene sequences. Using RNA silencing we have shown that the WC1 co-receptor contributes to the ability of gamma delta T cells to respond to leptospira. The leptospira-responsive gamma delta T cells are found within a subset of the serologically-defined WC1.1 gamma delta T cell subpopulation and our data indicate that the WC1 molecules expressed act as pattern recognition receptors interacting directly with bacterial components. In a comparison of a representative of a WC1.1 type of WC1 molecule (i.e. WC1-3) to a representative of a WC1.2 type (i.e. WC1-4), we found that five out of eleven WC1-3 SRCR domains and none of the eleven WC1-4 SRCR domains bound to two Leptospira species, L. borgpetersenii and L. interrogans. Binding for two of the five leptospire-binding WC1-3 SRCR domains is dependent on temperature at which the leptospires are cultured, suggesting that the WC1-3 SRCR domains do not all bind to the same ligand. Proteinase K digestion or polymyxin B treatment of the leptospires does not negatively impact binding, indicating that the ligands are not proteins or lipopolysaccharide (LPS). In contrast, treatment of leptospires with alkaline phosphatase decreased WC1-3 SRCR binding, indicating that the ligand is phosphorylated and may be similar to isopentenyl pyrophosphate (IPP), a non-peptidic T cell ligand and bacterial product whose binding is decreased with dephosphorylation. With regard to mycobacteria, experimental infection and vaccine studies with cattle have demonstrated a robust gamma delta T cell responses to mycobacterial antigens. We found the gamma delta T cells respond robustly to both protein and non-protein mycobacterial antigens following M. bovis infection. We observed notable responses to the protein antigens PPD-B and recombinant ESAT6, a target known to be highly antigenic for CD4 T cells. Further, we observed significant IFN gamma production to the non-protein antigens mycolylarabinogalactan peptidoglycan (mAGP) and the glycolipid mannosylated lipoarabinomannan (LAM), both major components of the mycobacterial cell wall. This response was both specific and direct, as gamma delta T cell IFN gamma production was observed in the presence of mixed PBMC and when purified gamma delta T cells were cultured with antigen presenting cells alone
Publications
- Wang, F., C. T. A. Herzig, C. Chen, H. Hsu, C. L. Baldwin, and J. C. Telfer. 2011. Scavenger receptor WC1 contributes to the gamma delta T cell response to Leptospira. Molecular Immunology 48:801-809.
- Chen, C., C.T.A. Herzig, L.J. Alexander, T. McDaneld, J. Keele, J.C. Telfer and C.L. Baldwin. 2012. Genomic copy number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes. BMC Genetics 13:86 (17 pages).
- Zeng, X., Y-L Wei, J. Huang, E.W. Newell, H. Yu, B.A. Kidd, M.S. Kuhns, R.W. Waters, M.M. Davis, C.T. Weaver, and Y. Chien. 2012. Gamma delta T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response. Immunity 37:524-534.
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