Progress 10/01/01 to 09/30/07
Outputs
OUTPUTS: The information from this project have been shared in a number of ways in the past year including the following: (1) participation as oral and poster presentations at the Conference for Research Workers on Animal Diseases in Chicago; the USDA-CSREES project directors workshop in Chicago; the International Veterinary Immunology Symposium in Brazil; (2) by seminars at universities and research institutions including University of Iowa, USDA-ARS Beltsville, USDA-ARS Ames Iowa, University of Pennsylvania college of veterinary medicine's annual faculty retreat; (3) interacting with undergraduate students in the Department of Veterinary and Animal Science through lecturing in Infection and Immunity and providing an honors symposium focusing on infectious diseases of livestock.
PARTICIPANTS: Project director was Cynthia Baldwin, University of Massachusetts. Collaborators included Drs Wendy Brown and Kevin Lahmers, Washington State University. Training and Profession Development included for graduate students working on the project Ms. Fei Wang and Ms. Chuang Chen, technicians Carolyn Herzig and Vanessa Mailloux and undergraduate students Ryan Powers and Vanessa Mailloux.
TARGET AUDIENCES: Formal classroom instruction, laboratory instruction, and practicum experiences were provided to undergraduate students in the Department of Veterinary and Animal Sciences and Dept of Microbiology at the University of Massachusetts. Information from this project was used to develop curriculum regarding animal diseases and defenses in an undergraduate course Infection and Immunity; it was also used in graduate education that included a Journal Club course reading and interpreting scientific papers and a graduate lecture course in Immunology; it was used in a Microbiology laboratory course in Immunology.
PROJECT MODIFICATIONS: none
Impacts
The results of this project have contributed to the general understanding of the immune response to infection in cattle. A brief report relevant to the grant objectives follows. WC1 molecules are transmembrane glycoproteins uniquely expressed on gamma delta T cells and have been implicated in both arresting and augmenting responses by ruminant γδ T cells. Two forms of WC1 can be serologically distinguished by monoclonal antibodies, i.e. WC1.1 and WC1.2. These serologically distinguishable forms are expressed by largely nonoverlapping subpopulations of γδ T cells from blood. Previously we showed that the form of the WC1 co-receptor expressed on γδ T cells divides the γδ T cells into functional subsets according to their ability to produce interferon-γ. However WC1 is known to be a large family of receptors based on genomic analysis by others and analyses of transcript sequences from peripheral blood γδ T cells by us
indicated at least 13 different intracytoplasmic tail sequences that could be loosely grouped into WC1.1, WC1.2 or WC1.3 families. Analysis of the bovine genome sequence indicated that the intracytoplasmic tail transcripts for the WC1.1 and WC1.2 families were coded for by 4 exons while the WC1.3 family has the analogous 4 exons with a 5th exon inserted between exons 2 and 3. Despite the difference in sequence, all tail transcripts coded for 5 tyrosines and mutations of the tyrosines for a cDNA clone representing the archetypal WC1.1 sequence and a second cDNA clone representing a WC1.2 sequence showed that the second tyrosine was the major tyrosine phosphorylated in both. Analysis of five T cells clones which were serologically WC1.2-positive/WC1.1-negative showed that a single clone may have two different WC1.2-family transcript sequences for the tail as well as extracellular regions. Yet among the WC1.2+ clones the transcripts were unique to particular T cell clones. We hypothesize
that the different WC1 co-receptors may increase the specificity of the WC1+ gamma delta T cells which otherwise rely on a very restricted set of genes to code for their antigen-specific T cell receptors.
Publications
- Herzig, C., S. Blumerman, M-P. Lefranc and C. Baldwin. (2006) Bovine T cell receptor gamma variable and constant genes: combinatorial usage by circulating gamma delta T cells. Immunogenetics 58:138-151.
- Rogers, A.N., D. Van Buren, B. Zou, K. Lahmers, C. Herzig, W.C. Brown, J.C. Telfer and C.L. Baldwin. (2006) Characterization of WC1 co-receptors on functionally distinct subpopulations of ruminant gamma delta T cells. Cellular Immunology 239:151-161.
- Blumerman, S., C. Herzig, A. Rogers and C.L. Baldwin. (2006) Differential TCR gene usage between WC1- and WC1+ ruminant gamma delta T cell subpopulations including those responding to bacterial antigen. Immunogenetics 58:680-692.
- Blumerman, S.., F. Wang, C. Herzig and C.L. Baldwin. (2007) Molecular cloning if bovine chemokine receptors and expression by WC1+ gamma delta T cells. Developmental and Compartive Immunology 31:87-102.
- Blumerman, S., C. Herzig, F. Wang, P. Coussens, and C. Baldwin. (2007) Comparison of gene expression by co-cultured WC1+ gamma delta and CD4+ alpha beta T cells exhibiting a recall response to bacterial antigen. Molecular Immunology 44:2033-2045.
- Blumerman, S., C. Herzig and C.L. Baldwin. (2007) WC1+ gamma delta T cell memory population is induced by killed bacterial vaccine. European Journal of Immunoogy 37:1204-1216.
- Sopp, P., D. Werling and C. Baldwin. (2007) Cross-reactivity of mAbs to human CD antigens with cells from cattle. Veterinary Immunology and Immunopathology 119:106-114.
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Progress 10/01/05 to 09/30/06
Outputs
WC1 molecules are transmembrane glycoproteins uniquely expressed on gamma delta T cells serving as cell type differentiation antigens. Two forms of WC1 previously identified by monoclonal antibodies, i.e. WC1.1 and WC1.2, are expressed by largely nonoverlapping subpopulations of gamma delta T cells in blood. Overall our results demonstrate that the form of the WC1 co-receptor expressed on gamma delta T cells divides them into functional subsets that respond to different types of antigen. Expression of the WC1.1 co-receptor was the main indicator of cell proliferation and IFN-gamma production in response to autologous and bacterial antigens and IL-12 suggesting these correspond to a Th1-type of cell. There was a steady decrease in proportion of WC1.1+ cells, but not WC1.2+ cells, within PBMC with animal aging suggesting the two subpopulations may have different roles in immune regulation although both had transcripts for regulatory cytokines IL-10 and TGF-beta. While
differences in the intracytoplasmic tail sequences of WC1.1 and WC1.2 molecules that may affect signaling have been found evaluation of transcripts for intracellular signaling molecules has shown only minor differences in expression between the subsets. To complicate the picture, gene cloning and protein studies have shown there are at least 16 different tail sequences for WC1 molecules associated with blood gamma delta T cells and about 20 biochemically distinct proteins. Since we found the tail sequences have different patterns of motifs that correlate with serine/threonine and tyrosine phosphorylation sites we are evaluating the association of the extracelluar domains with the various tail sequences. We postulate that in combination with the motifs in their tail sequences WC1 forms influence cell function as a result of the ligands they bind since similar molecules have bacterial ligands. Analysis of the bovine genome sequence indicated that the intracytoplasmic tail transcripts
for the WC1.1 and WC1.2 families were coded for by 4 exons while the WC1.3 family has the analogous 4 exons with a 5th exon inserted between exons 2 and 3. Despite the difference in sequence, all tail transcripts coded for 5 tyrosines and mutations of the tyrosines for a cDNA clone representing the archetypal WC1.1 sequence and a second cDNA clone representing a WC1.2 sequence showed that the second tyrosine was the major tyrosine phosphorylated in both. Analysis of five T cells clones which were serologically WC1.2-positive/WC1.1-negative showed that a single clone may have two different WC1.2-family transcript sequences for the tail as well as extracellular regions. Yet among the WC1.2+ clones the transcripts were unique to particular T cell clones. We hypothesize that the different WC1 co-receptors may increase the specificity of the WC1+ gamma delta T cells which otherwise rely on a very restricted set of genes to code for their antigen-specific T cell receptors.
Impacts
Understanding how to activate gamma delta T cells will allow us to exploit this knowledge to generate more effective vaccines or to induce innate immune responses when needed to combat unknown pathogens. This is particularly relevant with the current threat of biowarfare agents. We have found that the WC1+ gamma delta T cells are the first to respond to antigen following vaccination and thus may influence the development of the response by other T cell subpopulations such as CD4 T cells.
Publications
- Blumerman, S., C. Herzig, F. Wang, P. Coussens, and C. Baldwin. (2007)Comparison of gene expression by co-cultured WC1+ gamma delta and CD4+ gamma delta T cells exhibiting a recall response to bacterial antigen. Molecular Immunology 44:2033-2045.
- Rogers, A., D. G. VanBuren, E.E. Hedblom, M.E. Tilahun, J.C. Telfer and C.L. Baldwin. (2005) gamma delta T cell function varies with the expressed WC1 coreceptor. J. Immunol. 174:3386-3393.
- Herzig, C., S. Blumerman, M-P. Lefranc and C. Baldwin. (2006) Bovine T cell receptor gamma variable and constant genes: combinatorial usage by circulating γδ T cells. Immunogenetics 58:138-151.
- Rogers, A.N., D. Van Buren, B. Zou, K. Lahmers, C. Herzig, W.C. Brown, J.C. Telfer and C.L. Baldwin. (2006) Characterization of WC1 co-receptors on functionally distinct subpopulations of ruminant γδ T cells. Cell. Immunol. 239:151-161.
- Blumerman, S.., F. Wang, C. Herzig and C.L. Baldwin. (2007) Molecular cloning if bovine chemokine receptors and expression by WC1+ γδ T cells. Dev. Comp. Immunol., May2, 2006-EPUB.
- Blumerman, S., C. Herzig, A. Rogers and C.L. Baldwin. (2006) Differential TCR gene usage between WC1- and WC1+ ruminant γδ T cell subpopulations including those responding to bacterial antigen. Immunogenetics 58:680-692.
- Herzig, C.T.A., S.L. Blumerman and C.L. Baldwin. (2006) Identification of three new bovine T cell receptor delta variable gene subgroups expressed by peripheral blood T cells. Immunogenetics 58:746-757.
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Progress 10/01/04 to 09/30/05
Outputs
WC1 is a scavenger receptor cysteine rich type 1 transmembrane glycoprotein restricted to gamma delta T cells and implicated in augmenting cellular activation as well as induction of reversible cell cycle arrest. Conflicting roles for WC1 may be attributable to the response by different subpopulations of cells. Recent studies showed that the two major gd T cell populations, WC1.1+/WC1.2- and WC1.2+/WC1.1- cells, can be functionally segregated: WC1.1+ cells produced interferon-g and proliferated to bacterial and autologous antigens (Rogers et al., 2005) while the WC1.2+ cells proliferated more vigorously in response to stimulation with the mitogen concanavalin A and to peptides from the rickettsial parasite Anaplasma marginalis (Lahmers et al., 2005). The difference in functional responses is hypothesized to be a result of their expression of different WC1 molecules or isoforms. We have found 15 different transcript sequences for the WC1 intracellular tail suggesting
further diversity in signaling potential. We showed by 2-D gels and Western blotting that at least 20 biochemically distinguishable spots representing WC1 proteins are associated with peripheral blood gamma delta T cells with a slightly restricted portion of those associated with the plasma membranes and an even more limited number with the lipid rafts. The subpopulations defined as WC1.1+/WC1.2- and WC1.2+/WC1.1- cells each had a complex WC1 signature pattern although virtually all spots associated with WC1.2+/WC1.1- cells bore the WC1.2 epitope and were distinct from the WC1.1+ gamma delta T cell pattern of spots.
Impacts
Understanding how to activate gamma delta T cells will allow us to exploit this knowledge to generate more effective vaccines or to induce innate immune responses when needed to combat unknown pathogens. This is particularly relevant with the current threat of biowarfare agents. We have found that the WC1+ gamma delta T cells are the first to respond to antigen following vaccination and thus may influence the development of the response by other T cell subpopulations such as CD4 T cells.
Publications
- Baldwin, C.L., T. Sathiyaseelan, B. Naiman, A.M. White, R. Brown, S. Blumerman, A. Rogers and S.J. Black. (2002) Activation of bovine peripheral blood gamma delta T cells for cell division and IFN-g production. Veterinary Immunology & Immunopathology 87:251-259.
- Rogers, A.N., S. Welte, S.J. Black and C.L. Baldwin. (2002) Genetic identification of homologues of CD72 and CD166/ALCAM, ligands for SRCR family accessory molecules CD5 and CD6. Veterinary Immunology & Immunopathology 85:233-239.
- Naimam, B., S. Blumerman, D. Alt, C. Bolin, R. Brown, R. Zeurner and C.L.Baldwin. (2002) Evaluation of type 1 immune response in naive and vaccinated animals following challenge with Leptospira borgpetersenii serovar hardjo: involvement of WC1+ gamma delta and CD4 T cells. Infection and Immunity 70:6147-6157.
- Sathiyaseelan, T., A. Rogers and C.L. Baldwin. (2002) Response of bovine gamma delta T cells to activation through CD3. Veterinary Immunology and Immunopathology 90:155-168.
- Rogers, A., D. G. VanBuren, E.E. Hedblom, M.E. Tilahun, J.C. Telfer and C.L. Baldwin. (2005) Gamma deltaT cell function varies with the expressed WC1 coreceptor. J. Immunol. 174:3386-3393.
- Rogers, A., D. G. VanBuren, E.E. Hedblom, M.E. Tilahun, J.C. Telfer and C.L. Baldwin. (2005) Function of ruminant gamma delta T cells is defined by WC1.1 and WC1.2 isoform expression. Vet. Immunol. Immunopathol. 108:211-217
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Progress 10/01/03 to 09/30/04
Outputs
WC1 molecules are transmembrane glycoproteins belonging to the scavenger receptor cysteine rich family and uniquely expressed on gamma delta T cells. While participation of WC1+ gamma delta T cells in immune responses is well established, very little is understood regarding the significance of expressing different forms of the WC1 molecule. Two forms previously identified by monoclonal antibodies, i.e. WC1.1 and WC1.2, are expressed by largely nonoverlapping subpopulations of gamma delta T cells. Here, it was shown that expression of the WC1.1 co-receptor was the main indicator of proliferation, CD25 expression and interferon-gamma production in response to autologous and bacterial antigens, as well as for interferon-gamma production without proliferation in Th1-polarizing IL-12-containing cultures. However, after culture in either Th1-polarizing conditions or neutral conditions mRNA was present for both T-bet and GATA-3 as well as for IL-12Rbeta2 in WC1.1+ and WC1.2+
subpopulations and neither produced IL-4 under any conditions. Although the steady decrease in proportion of WC1.1+ cells, but not WC1.2+ cells, within PBMC with animal aging suggested the two subpopulations may have different roles in immune regulation, cells bearing either WC1.1 or WC1.2 expressed mRNA for regulatory cytokines IL-10 and TGF-beta, with TGF-beta being constitutively expressed by ex vivo cells. Overall the results demonstrate that the form of the WC1 co-receptor expressed on gamma delta T cells divides them into functional subsets according to IFN-gamma production and proliferative capacity to specific stimuli as well as with regard to representation within PBMC. Finally, evidence is provided for minor differences in the intracytoplasmic tail sequences of WC1.1 and WC1.2 that may affect signaling. We propose that it is the interaction with the natural ligand that directs these subsets down different functional pathways either as a result of signaling throught these
molecules or as a result of the cytokine mileau created by the cell expressing the ligand for the WC1 isoforms. To this end we have expressed a full-length sequence of WC1.1 to use to identify its ligand. A similar procedure is being followed for expression of WC1.2. Expression is done in mammalian cells and the recombinant molecule is reactive with anti-native WC1 molecules.
Impacts
Understanding how to activate gamma delta T cells will allow us to exploit this knowledge to generate more effective vaccines or to induce innate immune responses when needed to combat unknown pathogens. This is particularly relevant with the current threat of biowarfare agents.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Progress has been made in our endeavor to identify a ligand or ligands for WC1 in that we now have a vector capable of expressing the extracellular domain of WC1.1. The recombinant protein coded by the vector contains all 11 SRCR domains and contains a leader sequence that targets the resulting recombinant protein for secretion from transfected cells. It also contains a myc-His tag to facilitate isolation and screening of the recombinant protein. The region of the vector coding for the SRCR domains of WC1.1 was amplified using the highest fidelity polymerase currently available and recent sequencing has demonstrated that the vector coding for the recombinant protein does not contain errors. In addition to our ligand identification goals, we have made significant progress characterizing the WC1 homologs. It now appears that these different forms of WC1, which are expressed primarily in non-overlapping subsets of gamma delta T cells, react differently to the same
stimulus and play different roles with regard to cytokine production. WC1.1+ cells express interferon (IFN)-gamma when stimulated with IL-12 or when PBMC are obtained from leptospira-vaccinated animals and stimulated with leptospira sonicates. WC1.2+ cells do not appear to participate substantially under these conditions of stimulation with IFN-gamma production of their own. On the other hand, when PBMC are stimulated using Concanavalin A, WC1.2+ cells expand much more quickly than cells expressing WC1.1. Both projects are being carried out simultaneously and are well underway toward our overall goal of reducing what are currently disparate views in the literature regarding of the role of WC1 and of cells expressing this molecule.
Impacts
Understanding how to activate gamma delta T cells will allow us to exploit this knowledge to generate more effective vaccines or to induce innate immune responses when needed to combat unknown pathogens. This is particularly relevant with the current threat of biowarfare agents.
Publications
- White, A.M., S. Blumerman, B. Naiman and C.L. Baldwin. (2002) Expression of the bovine high affinity IL-12 receptor beta 2. Veterinary Immunology & Immunopathology 84:127-142.
- Baldwin, C.L., T. Sathiyaseelan, B. Naiman, A.M. White, R. Brown, S. Blumerman, A. Rogers and S.J. Black. (2002) Activation of bovine peripheral blood gamma delta T cells for cell division and IFN-gamma production. Veterinary Immunology & Immunopathology 87:251-259.
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Progress 10/01/01 to 09/30/02
Outputs
Preliminary data from our lab and others showed that monoclonal antibodies to the bovine gamma delta T cell lineage specific molecule WC1 induce or inhibit bovine T cell proliferation in vitro depending on the concentration of antibody used. Thus, we postulate that the natural ligand(s) for WC1 regulates bovine T cell function either through direct stimulation, or by acting as co-stimulatory molecules. We hypothesize that (i) the ligand for BoWC1 will enhance responses by bovine T cells if the level of expression is sufficient and (ii) expression of the ligand will be increased on cells at inflammatory sites due to their activation. The identification of this WC1 ligand will provide additional insight into the regulation of T cell function in cattle. The objectives are to clone and sequence the gene coding for the bovine WC1 ligand, make recombinant protein of this ligand and monoclonal antibodies reactive with the ligand; determine if the recombinant ligand (as
soluble molecule or transfected into cells) or monoclonal antibodies to the ligand affect responses by bovine gamma delta T cells; evaluate the level of expression of the WC1 ligand in resting and activated cells and in normal and inflamed tissue using the monoclonal antibodies and gene probes. We have obtained the cloned archetypal WC1 orginally described to have 1413 amino acids (Clevers et al., 1988) and a single transmembrane domain spanning amino acid 1251 to 1279. The large extracellular domain of WC1 comprises 3.75 kb of the 5' end of the gene. This region has been cloned from cDNA comprising the entire gene using PCR to produce soluble WC1. It has been ligated into a mammalian expression vector and sequencing of the vector with the ligated gene be sure the gene is in-frame is underway. The plasmid has been transfect by electroporation into Cos cells with variable success in obtaining sufficient secreted product suggesting stable transfectants are not easily selected for use in
large scale preparation of soluble WC1. The recombinant WC1 protein produced by the transfectants has been purified using anti-WC1 mAb IL-A29 on an affinity column and confirmed by SDS-PAGE. The WC1 will be biotinylated and used as a probe to identify cells in PBMC and tissue sections that express its putative ligand(s) using by a strepavidin-phycoerythrin immunofluorescence system. However we are currently investigating other expression systems before proceeding.
Impacts
Understanding how gamma delta T cells in cattle not only protect cattle from infection directly but influences the development of immune responses by other lymphocyte populations will be instrumental in the design of more effective vaccines and thus the improvement of bovine health.
Publications
- Rogers, A.N., S. Welte, S.J. Black and C.L. Baldwin. (2002) Genetic identification of homologues of CD72 and CD166/ALCAM, ligands for SRCR family accessory molecules CD5 and CD6. Veterinary Immunology and Immunopathology 85:233-239.
- Sathiyaseelan, T., A. Rogers and C.L. Baldwin. (2002) Response of bovine gamma delta T cells to activation through CD3. Veterinary Immunology and Immunopathology 90:155-168.
- Baldwin, C.L., T. Sathiyaseelan, B. Naiman, A.M. White, R. Brown, S. Blumerman, A. Rogers and S.J. Black. (2002) Activation of bovine peripheral blood gd T cells for cell division and IFN-g production. Veterinary Immunology & Immunopathology 87:251-259.
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Progress 10/01/00 to 09/30/01
Outputs
Funding received in October 2001. No results to report.
Impacts
(N/A)
Publications
- No publications reported this period
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