Progress 09/01/09 to 08/31/14
Outputs
Target Audience: The target audiences are thoseprimarily interested in the health of poultry andhow immune responses in chickens are generated.We have reached these individuals through journal publications and meeting presentations.Because poultry are raised for foodthis project isalso of interest to those promoting and protecting human health.This award has has brought the study ofYF molecules to the understanding that these unusualclass I-like molecules likely have arolein immune responses to microbes includingbacteria that sometimes contaminate poultry products andcausefoodborneillness. Changes/Problems: While we firstintended to focusdirectly on the role ofYF class I-like molecules in Marek's disease, our focus changed with the findingsshowing that similar humans molecules present microbial antigens and thus that YFlikely contributes indirectly in immune responses to Marek's disease.We have focused heavilyon defining the nature of YF bound ligands and how the differences in different YF isoforms affect the ligands bound. The data so far support the likelihood that the ligands are microbial rather than viral in origin.We are now considering the possibilitythat YF may act earlier and less specifically in the immuneresponses. A history of prior exposure tobacterial antigens presented by differentYFisoformsmight drive maturation of the immune systemin ways that indirectly confer resistance or susceptibility to Marek's disease. Additional funding from a local fundingsource,the Caltech-COH Biomedical Research Initiativewill allow this project to continue. What opportunities for training and professional development has the project provided? Training activities.As part of thisproject a post-doctoral fellow Dr. Lei Zhangdeveloped a very useful cell-mediated cytotoxicity assays based in flow cytometry to test NK cell recognition of BF and YF classI-like moleculesDr. Zhang greatlyincreasedskills and confidence as a scientist duringduring her training. Two undergraduates gained laboratory and analytical skills while contributingsubstantially to the understanding of the variabilityin YF genes intwochicken lines (Lines N and P)differing in susceptibility to Marek's disease. A CIRMintern with a Master's degree, Melissa Hamilton,also received laboratory training andbecame highly skilled at modeling the structure of the YF binding groove, while developing methods now used daily in our lab to prepare and analyze YF1*7.1 protein for ligand binding analyses. Professional development. Thisproject allowed the PI to develop additional skills in data analysis and communicating findings in writing and in oral presentations. This included a significant number of presentations to local high school students interested in science increasing her capacity to convey the excitment of scientific investigation. How have the results been disseminated to communities of interest? Results were distributed to the scientific communities interested in the health of chickens through oral presentations and journal publications. The PI also participated in science presentations for the local community including tours of elementary and high school students and a community science fair. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments: As part of thiswork we addressed the hypothesis that YF class I molecules provide chickens with a type of antigen presentation not found in mammals. Interestingly, this hypothesis is NOT fully supported. The first evidence that mammals have something similar to YF was reported in 2012 by Reantragoon et al. (J Exp Med).In this study the high resolution structure of YF1*7.1 (the YF1*7.1 structure is from our publications,see Hee et al. 2009,2010) was used for the final structural analysis of MR1. MR1 isa MHC class I-like molecule recognized in humans by a subset of T lymphocytes called mucosal-associated invariant T (MAIT) cells.This study established a structural link betweenchicken YF class I-like molecules and MR1 in mammals. In a second paper by the same group in 2012 (Kjer-Nielsen et al., Nature), MR1 molecules wereshown topresent bacterially derivedvitamin B metabolites to a special class of T cells called mucosal-associated invariant T (MAIT) cells. In our own recent work (not yet published) we have found evidencethat YF1*7.1 binds a component in the supernatantfrom bacterial cultures.YF1*7.1 also binds a component within a defined mixture of vitamins, some of which are of microbial origin. These findings suggest that YF1*7.1 may, like MR1,bind microbial ligands.We are concentrating now on defining the nature of the YF1*7.1 bound ligand. We have some data helping to define theYF residues controlling recognition of YF by cells with natural killing activity. Structural analyses allowed us to define the residues candidate residues for YF recognition. We have some data for swapped residuesaltering recognition.More work will be required to define the role of individual residues. We evaluated sequence variability among YF allotypes found within the experimental lines N and P, lines selected for MD resistance and susceptibility, respectively. There are four MHC-Y haplotypes segregating in Lines N and P.We determined by extensive sampling of cDNA that some haplotypes are simple with evidence for only one transcribed YF gene (Y7 only in Line N and Y9 onlyin Line P). Interestingly theY7 and Y9 isoforms have identical ligand binding grooves and are nearly identical across their entire sequences. The other twohaplotypes (Y5 and Y8), found in both Lines N and P but at different frequencies,have more complex restriction fragment patterns andyielded cDNA for multiple transcripts indicating that these two haplotypeshave multiple expressed YF genes. When all the predicted YF sequences from cDNA clones are aligned it is apparent that most sequence variabilityamong the clonesoccurwithin the region encoding theantigen-binding groove. YF gene number and sequence variability both contribute to MHC-Y polymorphism. It may be that this variability is selected forwith different isoforms providing selective advantages under different conditions. The variability observed within the antigen-binding groove is predicted to affect the shape and surface charge within the binding groove.It may be that different isoforms bind different ligands. We do not know yet how YF variability might affect the incidence of Marek's disease, but YF-mediated interactions with the microbiome could influence theimmune function. For exampledirecting the priming ofinnate immunity mightresult later in different degrees ofresponsiveness to the challenge of Marek's disesase virus. A manuscript describing the YF variability observed in Lines N and Pis nearly ready for submission. We will continue to facilitate the exchange of existing and new information on MHC-B and MHC-Y using the context of our growing understanding of how MHC-Y and MHC-B genetics influences Marek’s lymphoma and other diseases. This work is continuing with the first draft of a wikipedia page under review by colleagues. We are finishing up the sequencing ofBAC clones that defiine the YF gene region in the Red Jungle Fowl (RJF). The MHC-Y region is poorly represented in the assemblies from theRJF genome project andassembly into a singleregion has not been possible. To find the nature of MHC-Y gene regionYF-positive BACclones available in theRJF CHORIandTAMU BAClibraries were isolated and sent to Takashi Shiina andKazuyoshi Hosomichi at Tokai University in Kanagawa, Japan for sequencing using the Sangersequencing method. This sequence, which was very, very difficult to assemble,revealed the repetitive nature of the MHC-Y regionwhere many copies of essentially only two types of genes (YF and c-type lectin-like genes) are packed together. Despite considerable effort it hasnot be possible to coalesce theMHC-Y sequences into a single contig.We think it likely that theBACsreflect the presence of three distinct regions separatedfrom one another by disimilar genomic regions.The repetitive nature made it difficult to be fully confident that assembly of the BAC clones sequenceswere fully correct. To verify that the earlier sequence assemblywas correct, we recentlyresequencedthe two most informative BAC clonesusing high through-put methods. These datawere aligned with theearlier sequence as a test for accuracy. The alignments showed that theearlier Sanger sequences were indeed excellent.A third non-overlapping BAC was also sequenced recently. Asis typical of the MHC-Y region this too proved very difficult to assemble, but providedseveral contigs that will be analyzed forgene content.To testthe hypothesis first suggested by Solinhac et al 2010 (BMC Genomics)that MHC-Y is segmented (dispersed) on GGA 16,FISH studies are underway tolocalizeMHC-Ygenes at high resolution in interphase nuclei. It may be that segmentation is why we are not able to coalesce the BAC sequences into a single contig. A manuscript for reporting this work is in preparation. We have mapped additional genes onGGA 16that are inherited in linkage with the YF genes. We combined array comparative genomic hybridzation with trisomy mapping to screen unassigned genomic scaffolds (that remain assigned to chrUn_random in the latest Gallus gallus genome assembly, galGal3.0) for sequences originating from GGA16. We wanted to do this to define genes within the MHC-Y neighborhood on GGA16. We identified annotated 33 chrUn_random RefSeq as candidates for assignment to GGA 16. These contain a variety of interesting genes including a total of 80 olfactory receptor (OR) genes and multiple cysteine-rich domain savenger receptor(SRCR) genes. In addition, there area number of genes encoding putative immunoglobulin-like receptors and toher molecules. We used high-resolution cytogenomic analyses to confirm the assigenment of the OR and SRCR genes to GGA 16, pinpointing them to the q-arm in a region between the centromere and the nucleolar organizaing region (NOR). This study was published in 2014 in Journal of Heredity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Miller MM, Robinson CM, Abernathy J, Goto RM, Hamilton MK, Zhou H, Delany ME. 2014. Mapping Genes to Chicken Microchromosome 16 and Discovery of Olfactory and Scavenger Receptor Genes Near the Major Histocompatibility Complex. Journal of Heredity 105(2):203-215. PMID: 24336927
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Bauer MM, Miller MM, Briles WE, Reed KM. 2013. Genetic variation at the MHC in a population of introduced wild turkeys. Animal Biotechnology 24(3):210-228. PMID: 23777350
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Resolving genetic differences at the MHC that influence the incidence of Mareks disease in chickens presented at a symposium entitled, Applying Next-Generation Sequencing to Solve Poultry Problems at the 2013 Poultry Science Association Annual Meeting, San Diego, CA, July 22-25, 2013
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Trisomy Mapping of Olfactory Receptor and CD163 Genes to GGA16 Confirmed by FISH, Plant and Animal Genome Meeting, Jan 12-16, 2013
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Using trisomy mapping and array comparative genomic hybridization to assign candidate genes to chicken chromosome 16, Plant and Animal Genome Meeting, Jan 13-18, 2012
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Platform Report at NC-1170 - Advanced Technologies for the Genetic Improvement of Poultry and the NSRP-8, National Animal Genome Research Program held at XXII Plant and Animal Genome Meeting, San Diego, CA, Jan
11-14, 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Platform Report at NC-1170 - Advanced Technologies for the Genetic Improvement of Poultry and the NSRP-8, National Animal Genome Research Program held at Plant and Animal Genome Meeting, San Diego, CA, Jan 12-16, 2013
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Platform Project Report at NC-1170 - Advanced Technologies for the Genetic Improvement of Poultry and the NSRP-8, National Animal Genome Research Program held at Plant and Animal Genome Meeting, San Diego, CA, January 13-18, 2012
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Invited Participant, Agriculture and Food Research Initiative Joint Animal Systems Project Director Meeting, Arlington, Virginia, Apr 19-21, 2011
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2010
Citation:
Invited Speaker, Harvey Mudd College Summer Lecture Series, Jul 14, 2010
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2009
Citation:
Invited Participant, NIFA Animal Health and Well-being PD Workshop, Chicago, IL, Dec 6, 2009
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2009
Citation:
Invited Speaker, Cancer Biology Research Program Retreat, City of Hope Comprehensive Cancer Center, Mar 21, 2009
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Invited Participant, USDA-NIFA-Animal Health Program Director Workshop, Chicago, Illinois, Dec 01, 2012
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Platform Project Report at USDA NE-1034: Genetic bases for resistance and immunity to avian diseases, East Lansing, MI, Oct 02, 2012
|
Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The target audience for this reporting period are other scientists and poultry breeders. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided a CIRM Bridges Intern (10 month tenure) the opportunity to develop a new aspect of the project and to gain considerable laboratory skills. We were able to develop collaborative links with two different laboratories, one interested in the basis of immune responses to bacteria in farm animals and one with a laboratory interested in structures of antigen presenting molecules. We also used this project as a launching point for a segment of the graduate course here in the Irell and Manella Graduate School of Biological Sciences. We taught theliterature review devoted to Immunology in a courseentitled to Fundamental Skills for Research. How have the results been disseminated to communities of interest? We have begun to disseminate in one-on-one conversations (email and in person)with other interested investigators at meetings and otherformats.Findings were reported in publications, meeting abstracts and presentations.The meetings have included the October 2012 USDA NE-1034 Meeting in East Lansing, MI, the December 2012 USDA NIFA Animal Health Meeting in Chicago, IL, the January 2013 PAG Meeting in San Diego, CA and the July 2013Poultry Science Meeting inSan Diego. Illustration of similarity of YF1 isoforms with MR1 and the structural variability among YF isoforms in the binding groove (circled in green). At the outset of this project it was not known whether chicken MHC-Y class I genes contribute to innate and adaptive immune responses as do other class I genes located nearby in the MHC-B cluster. These studies are focused on defining MHC-Y class I gene function and learning whether MHC-Y genetic variability contributes to the often observed variation between chickens in immunity to Marek’s disease. The reported work is divided into two parts with one focusing on innate and the other on adaptive immunity. Given the central role of MHC class I molecules in guiding the activities of natural killer (NK) cells one set of experiments used cell-mediated cytotoxicity assays to define the basis of MHC-Y class I recognition by NK cells. A second set of experiments are being conducted to investigate, through cDNA cloning and sequencing, the number and sequence variability of MHC-Y class I transcripts produced from five well-defined MHC-Y haplotypes and how this variability affects function. To gain insight into the likelihood that differences in the MHC-Y region affects adaptive immune responses, we modeled the antigen binding grooves of the isoforms identified to learn whether sequence variability among the isoforms resulted in changes in the shape, surface charge and size of the interior volume where antigen in classical class I molecules is held for T lymphocyte recognition. The recent findings showing that mammalian MR1 protein, the closest mammalian structural counterpart to YF1*7.1 (Kjer-Nielsen et al. Nature 2012), binds small metabolites from microbes further encourages us to think that ligands for YF may be of microbial origin. Another goal is a detailed guide to the chicken MHC-B and MHC-Y regions to be published in reviews and on the internet. Findings were reported in publications, meeting abstracts and presentations. What do you plan to do during the next reporting period to accomplish the goals? We have nearly completed a rough draft of the material that will form the wiki site we intend to publish on the internet. We will soon be sending this draft to colleagues for comments and additions. Further experiments are planned to define natural ligands that bind the YF1*7.1 binding groove.
Impacts
What was accomplished under these goals?
Illustration of similarity of YF1 isoforms with MR1 and the structural variability among YF isoforms in the binding groove (circled in green). At the outset of this project it was not known whether chicken MHC-Y class I genes contribute to innate and adaptive immune responses as do other class I genes located nearby in the MHC-B cluster. These studies are focused on defining MHC-Y class I gene function and learning whether MHC-Y genetic variability contributes to the often observed variation between chickens in immunity to Marek’s disease. The reported work is divided into two parts with one focusing on innate and the other on adaptive immunity. Given the central role of MHC class I molecules in guiding the activities of natural killer (NK) cells one set of experiments used cell-mediated cytotoxicity assays to define the basis of MHC-Y class I recognition by NK cells. A second set of experiments are being conducted to investigate, through cDNA cloning and sequencing, the number and sequence variability of MHC-Y class I transcripts produced from five well-defined MHC-Y haplotypes and how this variability affects function. To gain insight into the likelihood that differences in the MHC-Y region affects adaptive immune responses, we modeled the antigen binding grooves of the isoforms identified to learn whether sequence variability among the isoforms resulted in changes in the shape, surface charge and size of the interior volume where antigen in classical class I molecules is held for T lymphocyte recognition. The recent findings showing that mammalian MR1 protein, the closest mammalian structural counterpart to YF1*7.1 (Kjer-Nielsen et al. Nature 2012), binds small metabolites from microbes further encourages us to think that ligands for YF may be of microbial origin. Another goal is a detailed guide to the chicken MHC-B and MHC-Y regions to be published in reviews and on the internet. Findings were reported in publications, meeting abstracts and presentations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Hee et al. 2010, PloS Biol 8:e1000557
Zhang et al. 2012 Dev Comp Immunol. 37:446.
|
Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: It is not yet known whether chicken MHC-Y class I genes located on GGA16 in the MHC-Y gene cluster contribute to innate and adaptive immune responses as do other class I genes located nearby in the classically MHC-B cluster. These studies are focused on defining MHC-Y class I gene function and learning whether MHC-Y genetic variability contributes to the often observed variation between chickens in immunity to Mareks disease. The reported work is divided into two parts with one focusing on innate and the other on adaptive immunity. Given the central role of MHC class I molecules in guiding the activities of natural killer (NK) cells one set of experiments used cell-mediated cytotoxicity assays to define the basis of MHC-Y class I recognition by NK cells. A second set of experiments were conducted to investigate, through cDNA cloning and sequencing, the number and sequence variability of MHC-Y class I transcripts produced from five well-defined MHC-Y haplotypes. To gain insight into the likelihood that differences in the MHC-Y region affects adaptive immune responses, we modeled the antigen binding grooves of the isoforms identified to learn whether sequence variability among the isoforms resulted in changes in the the interior volume where antigen in classical class I molecules is held for T lymphocyte recognition. PARTICIPANTS: 1. Dr. Lei Zhang, Research Fellow was trained and moved on. 2. Jennifer Dalton, Undergraduate, completed a second summer internship working on this project 3. Vaish Balendiran, high school student, completed a summer internship on this project. 4. Dr. Nagarajan Vaidehi and Dr. Supriyo Bhattacharya, Department of Immunology, COH helped us establish PyMol-based structural analysis in our lab. 5. Dr. Chee Seng Hee, University of Basel, for taught us how to generate binding groove images. TARGET AUDIENCES: This information is of interest primarily to avian immunologists but it will likely have applications in genetic selection for disease resistance in the future. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We developed a flow cytometry-based assay for observing NK cell killing of target cells expressing different chicken class I cDNA clones from MHC-B and MHC-Y genes. This is a 3 hr flow cytometry assay for cell-mediated cytotoxicity in which CFSE-labeled transduced RP9 cells are targets and 7-AAD is used to reveal the targets killed by NK cell activity. We found that the NK cells obtained from the spleens of 14-day embryos (a source of NK cells free from T cells) specifically recognize MHC class I from both MHC-B and MHC-Y. Some isoforms enhanced killing compared to non-transduced control cells, while other inhibited killing. This was true for both MHC-B and MHC-Y isoforms. We conducted epitope-swap experiments focused on defining the residues determining NK cell inhibition and activation. Early results indicate that different regions on the surface of the MHC-Y and MHC-B molecules are involved. It may be that there are different sub-populations of NK cells with one sub-population recognizing class I molecules encoded by MHC-B and another recognizing class I encoded MHC-Y. Thus it may be that allelic differences at MHC-Y class I loci contribute to differences among individuals in response to infection and to differences in early responses to Mareks disease virus, a virus known to modulate MHC class I on the cell surface. To gain insights into MHC-Y genetic variability likely influencing adaptive immune responses, we cloned and sequenced MHC-Y class I cDNA from five MHC-Y haplotypes. These included the four haplotypes well-characterized MHC-Y haplotypes segregating in Cornell Lines N and P (Y5, Y7, Y8 and Y9) and the single MHC-Y haplotype carried in the highly inbred Line UCD001 (RJF). All five haplotypes were previously established on the basis of distinctive restriction fragment patterns (RFPs) in Southern hybridizations. Two haplotypes with simple RFPs (Y7 and Y9) have a single transcribed Y class I locus. For two haplotypes (Y5 and Y8) with slightly more complex RFPs with 2 or 3 transcripts identified. For the RJF haplotype four distinct transcripts were found. We then modeled the binding grooves using the YF1*7.1 structure as the starting point. We found evidence that the YF binding grooves are lined with polymorphic residues that result in the binding grooves in different isoforms being quite different in shape, size and surface charge. This finding suggests that different isoforms may bind and present quite different ligands for T cell recognition. A further implication is that different isoforms may contribute differently in adaptive immune responses to infection with Mareks disease virus and other poultry pathogens. In the past year, we trained a Research Fellow, Dr. Lei Zhang who has gone on to another position at the City of Hope, and two summer interns, Vaish Balendiran (Youngstown State University BS/MD Program) and Jennifer Dalton (St. Johns College).
Publications
- Zhang, L., Katselis, G.S., Moore, R.E., Lekpor, K., Goto, R.M., Hunt, H.D., Lee, T.D., Miller, M.M. (2012). MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos, Developmental and Comparative Immunology 37:446-456.
|
Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Activities. Objective 1. To more fully understand the basis of MHC-Y class I recognition by NK cells conducted cell mediated cytotoxicity assays in which we tested NK cell recognition of YF isoforms using BF isoforms as controls. We conducted the first candidate domain swap experiments designed to define what portion of the YF (and BF control) molecules are recognized by natural killer cells. We prepared chick embryo fibroblasts from genetically defined lines to use in assays to determine whether infection with MD virus in vitro affects the expression of YF at the cell surface. We screened the CHORI-261 library for additional clones mapping to the MHC. Objective 2. We completed the YF structural study with our collaborators in Berlin. We cloned and sequenced YF cDNA from genetically defined Line N and Line P and other lines with which we are evaluating the expressed polymorphism of the YF loci. Objective 3. We facilitated exchange of information and immunogenetic reagents again this year through email and telephone exchanges with interested individuals and through discussions at workshops and meetings. We shared a hybridoma line. Events. We presented at the 5th International Workshop on the Molecular Pathogenesis of Marek's Disease Virus and 1st Symposium on Avian Herpesviruses, in Athens, Georgia, October 19, 2010, the Poultry Workshop at the PAG XIX Meeting, January 15-19, 2011 in San Diego, CA and at the NIFA Animal Health and Well-being PD Workshop, Arlington, VA, April 12-15, 2011. Our research fellow and our summer interns presented at posters at City of Hope research events. We presented a talk at Harvey Mudd College, Pomona, California, July 14, 2010 to an audience of undergraduates and their professors. Services. We guided the scientific community by reviewing manuscripts and grant proposal applications. Products. We trained one Pomona College intern and two summer students. For Dissemination. This project has allowed us to continue collaboration with C.S. Hee and A. Ziegler. A spinoff is the development of this structural work is an ongoing conversation with Dirk Zajonc, La Jolla Institute for Allergy and Immunology on how to identify the contents of the YF binding groove. The publication of the YF structure drew international attention and comments at two sites that review current literature (see below). This project allows continued collaboration with T. Shiina and K, Hosomichi at Tokai University and with H. Zhou at Texas A&M. PARTICIPANTS: Individuals Marcia M. Miller, PhD, Principal Investigator Lei Zhang, PhD, Research Fellow Ronald M. Goto, Senior Research Associate Partner Organizations Insititut fur Immunogenetik, Charitie-Universitatmedizin Berlin, Freie Universitat Berlin, Berlin, Germany (Chee Seng Hee, Barbara Unchanska-Ziegler, Andreas Ziegler) Max-Delbruck-Centrum fur Molekulare Medizin, Berlin, Germany (Song Gao, Oliver Daumke Institut fur Chemie und Biochemie, Abteilung Strukturbiochemie, Freie Universtitat Berlin, Berlin, Germany (Bernhard Loll) Tokai University, Kanagawa, Japan (Takashi Shiina, Kazuyoshi Hosomichi, Hidetoshi Inoko) Texas A&M, College Station, Texas (Huaijun Zhou) La Jolla Institute for Allergy and Immunology, La Jolla, California (Dirk Zajonc) Collaborators George Katselis (now at U. Saskatchewan), Terry D. Lee and Roger Moore, Department of Immunology, City of Hope Beckman Research Institute Training Lei Zhang, Research Fellow Shaira Bhanji, now at Harvard University Vaishvavi Balendiran, Arcadia High School Jennifer Dalton, St John's College David DiTullio, Pomona College TARGET AUDIENCES: This project is targeted to those interested in the molecule mechanisms of disease resistance. Included in this group are immunologists, geneticists, poultry breeders and students interested in immunology, genetics and poultry science. We are targeted high school and undergraduate students interested in biology in the training portion of this effort by providing opportunities to do hands-on lab research at the cutting edge of this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Change in Knowledge. This study shows that in crystals grown from YF1*7.1 and beta2-microglobulin in the absence of added ligand the YF binding groove contains a linear chain of 17 atoms and a tetragonal head (a compound apparently gleaned during sample preparation and suspected to be cetrimonium). Addition of PEG200 during crystallization leads to the presence of two additional densities within the groove. These findings support the hypothesis that YF1 ligands are hydrophobic compounds. Additional refold experiments analyzed by isoelectric focusing showed interactions with LPS and mycolic acid. How this interaction occurs is not yet defined. The findings clearly demonstrate the hydrophobic nature of the YF binding groove. These findings reinforce the conclusion that YF molecules represent a so far unique type of molecule more like classical antigen presenting molecules in structure except for the binding groove. Others quickly commented on the uniqueness of YF See Nature Reviews Immunology 11, 3 (January 2011) and Protein Crystallography Newsletter 2, 12, (December 2010). The cloning of YF cDNA has so far provided data indicating that perhaps few YF genes are expressed. Our continued work on mapping genes to the MHC has revealed new genes in the YF region that will also likely be important in the genetics of disease resistance. Change in actions. We have learned to test NK cell cytotoxicity in a non-radioactive flow cytometry assay. We are learning ways in which to make the domain swaps more quickly. This will allow more constructs to be tested. Change in conditions. The data obtained are supportive of new ways of thinking about the MHC genetics in chickens. Our data indicate that different YF and BF isoforms have the capacity to either stimulate or inhibit NK cell killing when tested in killing assays in vitro. These data suggest that YF and BF isoforms encoded by different YF and BF haplotypes may affect early immune responses in vivo. If found to have a significant influence in vivo, future in selective breeding might include consideration of the affect of YF and BF alleles on early immune responses.
Publications
- Zhang, L., Katselis, G., Moore, R.E., Lekpor, K., Goto, R.M., Lee, T.D., Miller, M.M. (2011). Proteomic analysis of surface and endosomal membrane proteins from the avian LMH epithelial cell line. J. Proteome Res. 10(9):3973-82. Hee, C.S., Gao, S., Loll, B., Miller, M.M., Uchanska-Ziegler, B., Daumke, O., Ziegler, A. (2010). Structure of a classical MHC class I molecule that binds "non-classical" ligands. PloS Biology 8(e1000557):1-12. Gendzekhadze, K., Goto, R.M., Miller, M.M., Senitzer, D. (2010). The novel HLA-B*15:180 allele appears to be a recombinant B*08/B*15 allele. Tissue Antigens 76:334-335. Zhang, L., Katselis, G., Moore, R.E., Lekpor, K., Goto, R.M., Lee, T., Miller, M.M. (2010). Strategies for comprehensive profiling cell surface/membrane proteins by mass spectrometry. Collaboration: Road to the Cure: Annual City of Hope Poster Session, Duarte, CA, July 9, 2010, abstract and poster presentation. Bhanji, S., Goto, R.M., Porter-Kelley, J., Ehsani, J., Miller, M.M. (2010). The effect of microRNA (miRNA) on gene expression and tumor incidence in Marek's Disease (MD). Eugene & Ruth Roberts Summer Student Academy Poster Session, Duarte, CA, August 4, 2010, abstract and poster presentation.
- Miller, M.M., Goto, R.M., Ehsani, A., Wang, Y. (2010). MicroRNA and BG1 in MHC-linked resistance to Marek's disease tumors. The 5th International Workshop on the Molecular Pathogenesis of Marek's Disease Virus and 1st Symposium on Avian Herpesviruses, in Athens, Georgia, October 19, 2010, abstract and oral presentation.
- Miller, M.M. (2009). Genetic factors contributing to chicken MHC-B haplotype diversity. Poultry Workshop. Plant and Animal Genome XVIII Meeting, San Diego, CA January 9 - 13, 2010, oral presentation. Shiina, T., Goto, R.M., Hosomichi, K., Suzuki, S., Inoko, H., Miller, M.M. (2011). Genomic sequence analysis of the chicken major histocompatibility complex (MHC) regions. Poultry Workshop. Plant and Animal Genome XIX Meeting, San Diego, CA January 15 - 19, 2011, abstract and oral presentation by Dr. Shiina.
- Hosomichi, K., Goto, R.M., Suzuki, S., Inoko, H., Inoue, I., Miller, M.M., Shiina, T. (2011). Application of targeted resequencing methods to the MHC regions on chicken chromosome 16 (GGA16). Poultry Workshop. Plant and Animal Genome XIX Meeting, San Diego, CA January 15 - 19, 2011, abstract and oral presentation by Dr. Hosomichi.
- Miller, M.M. (2011). Chicken MHC-Y class I genes -- Clues to Function. Poultry Workshop. Plant and Animal Genome XIX Meeting, San Diego, CA January 15 - 19, 2011, abstract and oral presentation.
- Miller, M.M. (2011). MHC-Y class I determinants in innate and adaptive immune responses to Marek's disease. Agriculture and Food Research Initiative Joint Animal Systems Project Director Meeting, Arlington, VA, April 19-21, 2011, abstract and poster presentation.
- Balendiran, V., Dalton, J., Goto, R.M., Miller, M.M.. (2011). CD8 and Binding Groove Interaction Affected by Polymorphisms in the YF1 Locus of Chickens (Gallus gallus). Eugene and Ruth Roberts Summer Student Academy Poster Session, City of Hope, Duarte CA, August 4, 2011, abstract and poster.
- Dalton, J., Balendiran, V.,. Miller, M.M., Goto, R.M. (2011). Understanding Alternative Splicing in the Context of YF1 Alleles in Chickens. Eugene and Ruth Roberts Summer Student Academy Poster Session, City of Hope, Duarte CA, August 4, 2011, abstract and poster.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Activities. To more fully understand the basis of MHC-Y class I recognition by NK cells (Objective 1) we have worked on defining the YF MHC class I loci present within the chicken genome. We have isolated YF positive BAC clones from four Red Jungle Fowl BAC libraries analyzing clones to verify their origin from the MHC-Y region. The YF-positive BAC clones have been sent to Takashi Shiina at Tokai University for sequence analysis. The much of the sequence data are collected and a preliminary map contains three contigs. Assembly of the sequence data is extremely difficult because of many repetitive sequences. We established the position of MHC-Y relative to MHC-B and the nucleolus organizer region (NOR). Contrary to our hypothesis that MHC-Y and MHC-B were separated by the NOR we found, in collaboration with Mary Delany and Charmaine Robinson at UC Davis, that MHC-Y and MHC-B are on the same side of the NOR and separated by a relatively GC-rich region previously unknown. The order of the three markers and this new intervening region between MHC-Y and MHC-B were clearly resolved by high-resolution cytogenomic analysis of meiotic (prophase I pachytene chromosomes). These chromosomes are in an extended or decondensed state that allows greater resolution of closely spaced loci. We investigated the nature of the ligands bound within the YF binding groove (Objective 2). In a series of experiments we attempted to define the contents of the YF binding groove by eluting the contents from the groove of YF first by pH mediated denaturation and then chloroform/methanol extraction. Since structural studies have indicated that the binding groove of YF1*7.1 is narrow and hydrophobic we anticipated that the antigen by be found in the chloroform/methanol extractions. Mass spectrometry did indeed reveal a number of phospholipids but it was not possible clearly identify a component present within the experimental sample versus the control as a specific candidate for an eluted antigen. Other approaches, such as analysis of the binding of candidate ligands to YF1*7.1 and beta2-microglobulin complexes by isoelectric focusing under non-denaturing conditions, may be better suited for defining bound ligands. We facilitated exchange of information and immunogenetic reagents (Objective 3). This has been accomplished during the first year of this award through email and telephone exchanges with interested individuals and through discussions at workshops and meetings. Events. We presented at the NIFA Animal Health and Well-being PD Workshop, 12/6/2009 Chicago, IL. Services. We guided the scientific community by reviewing manuscripts and grant proposal applications. Products. This project provided a preliminary map of the MHC-Y region. For Dissemination. This project fostered continuing collaboration with T. Shiina and K, Hosomichi at Tokai University. This project has allowed us to continue collaboration with C. S. Hee and A. Ziegler. A spinoff is the development of collaboration with H. Zhou at Texas A&M. We sponsored a high school and undergraduate interns in the lab as the result of this award. PARTICIPANTS: Principal Investigator - M.M. Miller, PhD; Research Fellow - Lei Zhang, PhD; Senior Research Associate - R.M. Goto TARGET AUDIENCES: Tokai University, Japan; Freie Universitat Berlin, Berlin, Germany; University of California, Davis PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Change in Knowledge The result of work in the first year of this award we have changed the way the participants (the scientists involved) think about the function of the YF gene region. The work places YF class I molecules into a so far unique category of molecules that have a structure most similar to MHC class I molecules that present peptide antigen, but which have another function, perhaps that of presenting hydrophobic ligands such as lipids, glycolipids or lipopeptides. The YF molecules appear to be recently evolved in chickens. There are no equivalents found in mammals. The only other antigen presenting molecules that present such molecules are the ancient CD1 molecules. CD1 molecules are found in both mammals and chickens. The finding that YF have may a role in presenting some sort of hydrophobic antigens leads to the question of why such specialized molecules should evolved in chickens. While there may be something similar to MHC-Y region in turkeys gene sequence alignments suggest that chicken YF and BF are more similar to each other than, for examples chicken BF and BF-like genes in quail. Change in actions The results of the first year's work have led us to pursue alternative methods for identification of the ligands bound bye YF1*7.1. Change in conditions The data obtained are supportive of a new way of thinking about the MHC genetics in chickens that may have practical applications in selective breeding in the future.
Publications
- Hee, C.S., Gao, S., Miller, M.M., Goto, R.M., Ziegler, A., Daumke, O., Uchanska-Ziegler, B. (2009). Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1. Acta Cystallographica F65:422-425.
- Delany, M.E., Robinson, C.M., Goto, R.M., Miller, M.M. (2009). Architecture and organization of chicken micro-chromosome 16: Order of the NOR, MHC-Y and MHC-B subregions. J. Heredity 100:507-514.
- Goto, R.M., Wang, Y., Taylor, Jr., R.L., Wakenell, P.S., Hosomichi, K., Shiina, T., Blackmore, C.S., Briles, W.E., Miller, M.M. (2009). BG1 has a major role in MHC-linked resistance to malignant lymphoma in the chicken. Proc Natl Acad Sci U S A. 106:16740-16745.
- Miller, M.M. (2009). Report for NE-1034 Project - Genetic bases for resistance and immunity to avian diseases. Sent by email to all project members and presented at meeting, University of Arkansas, Fayetteville, Arkansas, October 3-5, 2009.
- Miller, M.M. (2009). MHC-Y class I determinants in innate and adaptive immune responses to Marek's disease. Abstract for poster presented at the AFRI Animal Health & Well-being PD Workshop, Chicago, Illinois December 6, 2009
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