1. RESEARCH FOCUS
The studies of my lab focus on basic and applied aspects of the fish immune system. Moreover, as teleost fish represent the most ancient living bony species with an immunoglobulin-based adaptive immune system, we use these species to study key aspects of the evolution of adaptive immunity.
Our main animal model is Rainbow trout. While earlier work focused on investigating the structure, function and evolution of fish complement (see below refs# 1-7), during the last 7 years our studies have mainly focused on B cells and mucosal immunity aspects of teleost fish.
In 2006 we reported for the first time that primary B cells from a vertebrate animal (a teleost fish) contained potent phagocytic and bactericidal capacities (ref# 6). This signified a paradigm shift since B cells were not recognized as professional phagocytes at that point. Later in 2012 we found for the first time subsets of mammalian B-1 B cells with phagocytic and bactericidal capacities (ref# 17). Critically, these cells were able to present particulate antigen to CD4+ T cells.
Another primary interest of our lab is the study of fish mucosal immune responses. In 2010 we reported the first mucosal immunoglobulin (IgT) in the gut of a teleost fish (ref# 17). In this study, IgT exemplified the most ancient mucosal immunoglobulin found in a vertebrate species. Thus, this finding represented a paradigm shift as the dogma at that time dictated that mucosal immunoglobulins had evolved first in tetrapod species. Later in 2013 we showed that IgT comprised the main immunoglobulin in the mucosa of the fish skin, thus supporting further the concept that IgT is a mucosal immunoglobulin (ref# 28). Both in the gut and the skin we have shown that IgT is the main immunoglobulin responding to mucosal pathogens (refs# 17 and 28). Conversely, we have demonstrated that IgM-specific responses are predominantly found in the serum of the same animals. Thus, teleost fish generate compartmentalized immunoglobulin responses against pathogens: while IgT plays a key role in mucosal areas, IgM is the principal immunoglobulin responding in systemic immunity. Critically, we have also found that IgT is the main immunoglobulin coating the microbiota of the fish gut and skin (refs# 17 and 28), similar to the immune exclusion role of IgA in mammals. Thus, a strong interest of our lab is to understand the regulation of fish microbiota by IgT, as well as to ascertain the role of microbiota in modulating fish immunity.
Our findings on IgT mucosal responses are not only of interest at the basic level but have important implications for the way that fish vaccines will be designed and tested, as a large number of fish pathogens enter through the skin and gut mucosal areas (ref# 19 and 29). In that regard, our laboratory is currently assessing the induction of IgT immune and protective responses upon vaccination through different routes (i.e., oral, bath, injection). Moreover, we are also developing immunostimulants that stimulate strong immune and protective mucosal immunity in farmed fish.
In addition to the above mentioned projects, we are currently analyzing the interactions of the different rainbow trout B cell subsets with T cells. To this end, we have recently generated mAb antibodies against rainbow trout CD4-1 and CD4-2 molecules. These mAbs have enabled us to identify and functionally characterize subpopulations of CD4-T cells in this species (manuscript in preparation). We are interested in assessing B-T cell interactions in mucosal and systemic lymphoid organs of rainbow trout, including the ability of trout APCs (i.e., phagocytic B cells, macrophages) in presenting antigen to the different subsets of systemic and mucosal trout CD4-T cells. Understanding B-T cell interactions will be crucial for the future design of more effective fish vaccines and immunostimulants.
From an evolutionary perspective, our studies are relevant to deciphering unresolved paradigms of the mammalian immune system, and thus, we are using rainbow trout as a model to address several aspects of mammalian phagocytic B cells and mucosal immunity (ref# 24).
It is important to mention that the antibodies generated in our lab that have already been reported in peer-reviewed journals, are being freely shared and distributed to other research groups. See below (Antibodies Distribution) for the availability of antibody reagents and how to obtain them. Alternatively, visit Dr. Sunyer's lab website at http://www.vet.upenn.edu/research/research-laboratories/research-laboratory/sunyer-laboratory
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2. ANTIBODIES DISTRIBUTION
2.1. Currently available antibodies:
Our laboratory has generated monoclonal and polyclonal antibodies against rainbow trout IgT. After the publication of these antibodies (see ref# 17), we started their free distribution to interested research groups. A full description of these polyclonal and monoclonal anti-IgT antibodies can be found in the datasheets shown below under the ANTIBODY DATASHEETS section. If you are interested in obtaining any of the antibodies displayed in the datasheets, please contact Dr. Sunyer (sunyer@vet.upenn.edu).
2.2. Antibodies under development:
We have recently produced polyclonal and monoclonal antibodies against rainbow trout IL-10, CD4-1 and CD4-2. We are in the process of characterizing their reactivity and specificity. These antibodies will be freely distributed after they have been fully characterized and after they are reported in a peer-reviewed journal. Peer review publication of a reagent prior to its distribution ensures a high degree of scrutiny by the scientific community which may point to pitfalls of the reagent not previously detected by the laboratory producing it.
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3. ANTIBODY DATASHEETS
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3.1. Antibody Datasheet #1: Anti-rainbow trout IgT monoclonal antibody
Clone Name: 41.8
Host/Isotype: MOUSE/IgG2b
Background: Three immunoglobulin classes (IgM, IgD and IgT) are present in teleost fish. IgT, also called IgZ in some fish species, was identified in 2005. In rainbow trout, there are two major B cell subsets expressing either IgT or IgM/IgD, but no B cells expressing both IgM and IgT. In contrast to IgM+ B cells, IgT+ B cells do no co-express IgD. Significant titers of rainbow trout IgT and accumulation ofs IgT+ B cells are detected in mucus and mucosal tissues (skin and gut) respectively from fish infected with mucosal pathogens. Moreover, bacterial microbiota in mucus from gut and skin are prevalently coated by IgT. Therefore, IgT and IgT+ B cells play specialized roles in rainbow trout mucosal immune responses and homeostasis. The above information regarding the production of IgT monoclonal antibodies and the detection of IgT and IgT+ B cells in rainbow trout gut and skin have been reported in the publications shown in the “references” section below.
Specificity: The clone 41.8 produces mouse IgG2b that recognizes refolded recombinant protein of the constant domains 2-4 of rainbow trout IgT heavy chain which was recombinantly produced in E. coli. This monoclonal antibody recognizes soluble IgT from rainbow trout fluids (plasma, serum, skin mucus, gut mucus) as well as membrane IgT from IgT+ B cells. The antibody specificity was demonstrated in publication #1 of the ”references” section.
Immunogen: Recombinant protein of heavy chain constant domain (CH2-4 domains) of rainbow trout produced in E. coli.
Applications:
-- Western blot: 2 µg/ml. Recognizes IgT from serum, plasma, whole cell lysates.
-- Perform SDS-PAGE under non-reducing conditions prior to blotting proteins.
-- FACS: 2 µg/ml for 100 µL cell (1x107 cells/mL)
Presentation: Protein G purified immunoglobulin in PBS.
Storage/Handling: Store antibody in aliquot, tightly sealed at +2-4 oC, although sodium azide or Bronidox (both at 0.05%) is recommended as preservative if stored at that temperature for more than 1 day. It is important to avoid multiple thaw and freezing cycles as well as warm and chill cycles. If product requires storage for longer than 1-2 weeks, it is recommended that it is stored at -20-80 oC.
References for Antibody Datasheet #1:
Zhang, Y. A., I. Salinas, J. Li, D. Parra, S. Bjork, Z. Xu, S. E. LaPatra, J. Bartholomew, and J. O. Sunyer. 2010. IgT, a primitive immunoglobulin class specialized in mucosal immunity. Nat. Immunol. 11:827-835.
Xu, Z., Parra, D., Gomez, D., Salinas, I., Zhang, Y.A., Jørgensen G.L., Skjødt, K., Rasmussen, K.J.,Buchmann K., LaPatra, S.L., J. O. Sunyer. 2013. Teleost skin, an ancient mucosal surface that elicits gut-like immune-responses. PNAS.110:13097-13102.
Important notification: It is possible that during shipment, small volumes of product may become entrapped in the seal of the enclosed vial. Therefore, for products with 250 µl or less, we suggest centrifuging the vial in a tabletop centrifuge in order to dislodge any product in the container’s cap.
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3.2. Antibody Datasheet #2: Anti-rainbow trout IgT polyclonal antibody
Host/Isotype: Rabbit/IgG
Background: Three immunoglobulin classes (IgM, IgD and IgT) are present in teleost fish. IgT, also called IgZ in some fish species, was identified in 2005. In rainbow trout, there are two major B cell subsets expressing either IgT or IgM/IgD, but no B cells expressing both IgM and IgT. In contrast to IgM+ B cells, IgT+ B cells do no co-express IgD. Significant titers of rainbow trout IgT and accumulation ofs IgT+ B cells are detected in mucus and mucosal tissues (skin and gut) respectively from fish infected with mucosal pathogens. Moreover, bacterial microbiota in mucus from gut and skin are prevalently coated by IgT. Therefore, IgT and IgT+ B cells play specialized roles in rainbow trout mucosal immune responses and homeostasis. The above information regarding the production of IgT polyclonal antibodies and the detection of IgT and IgT+ B cells in rainbow trout gut and skin have been reported in the publications shown in the “references” section below.
Specificity: This polyclonal antibody recognizes the refolded recombinant protein of the constant domains 2-4 of rainbow trout IgT heavy chain which was recombinantly produced in E. coli. This polyclonal antibody recognizes soluble IgT from rainbow trout fluids (plasma, serum, skin mucus, gut mucus) as well as membrane IgT from IgT+ B cells. The antibody specificity was demonstrated in publication #1 of the ”references” section.
Immunogen: Recombinant protein of heavy chain constant domain (CH2-4 domains) of rainbow trout produced in E. coli.
Applications:
-- Western blot: 1 µg/ml. Recognizes IgT from serum, plasma, whole cell lysates.
-- Perform SDS-PAGE under reducing or non-reducing conditions prior to blotting proteins.
-- FACS: 2 µg/ml for 100 µL cell (1x107 cells/mL)
Presentation: Rabbit IgG was first purified by protein G chromatography. Thereafter, specific IgG was affinity purified against a column containing the recombinant immunogen. The antibody is in PBS.
Storage/Handling: Store antibody in aliquot, tightly sealed at +2-4 oC, although sodium azide or Bronidox (both at 0.05%) is recommended as preservative if stored at that temperature for more than 1 day. It is important to avoid multiple thaw and freezing cycles as well as warm and chill cycles. If product requires storage for longer than 1-2 weeks, it is recommended that it is stored at -20-80 oC.
References for Antibody Datasheet #2:
Zhang, Y. A., I. Salinas, J. Li, D. Parra, S. Bjork, Z. Xu, S. E. LaPatra, J. Bartholomew, and J. O. Sunyer. 2010. IgT, a primitive immunoglobulin class specialized in mucosal immunity. Nat. Immunol. 11:827-835.
Xu, Z., Parra, D., Gomez, D., Salinas, I., Zhang, Y.A., Jørgensen G.L., Skjødt, K., Rasmussen, K.J.,Buchmann K., LaPatra, S.L., J. O. Sunyer. 2013. Teleost skin, an ancient mucosal surface that elicits gut-like immune-responses. PNAS.110:13097-13102.
Important notification: It is possible that during shipment, small volumes of product may become entrapped in the seal of the enclosed vial. Therefore, for products with 250 µl or less, we suggest centrifuging the vial in a tabletop centrifuge in order to dislodge any product in the container’s cap.
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4. LAB PUBLICATIONS
1. Boshra, H., J. Li, R. Peters, J. Hansen, A. Matlapudi, and J. O. Sunyer. 2004. Cloning, expression, cellular distribution and role in chemotaxis of a C5a receptor in rainbow trout: The first identification of a C5a receptor in a non-mammalian species. J. Immunol. 172:4381-90.
2. Boshra, H., A. Gelman, and J. O. Sunyer. 2004. Structural and functional characterization of complement C4 and C1s-like molecules in teleost fish: Insights into the evolution of classical and alternative pathways. J. Immunol. 173:349-59.
3. Li, J., R. Peters, S. LaPatra, M. Vazzana, and J. O. Sunyer. 2004. Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes. Dev. Comp. Immunol. 28:1005-21.
4. Boshra, H.., R. Peters, J. Li, and J. O. Sunyer. 2004. Production of recombinant C5a from rainbow trout (Oncorhynchus mykiss): Role in leukocyte chemotaxis and respiratory burst. Fish & Shellfish Immunol. 17:293-303.
5. Wang, L., J. O. Sunyer, L. J. Bello. 2005. Immunogenicity of a bovine viral diarrhea virus E2-C3d fusion protein containing a bovine homolog of C3d. Dev. Comp. Immunol. 29:907-15.
6. Boshra, H., T. Wang, L. Hove-Madsen, J. Hansen, J. Li, A. Matlapudi, C. J. Secombes, L. Tort, and J. O. Sunyer. 2005. Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species. J. Immunol. 175:2427-37.
7. Li, J., H. Boshra, and J. O. Sunyer. 2005. Evolution of anaphylatoxins, their diversity and novel roles in innate in immunity: Insights from the study of fish complement. Veterinary Immunol. Immunopathol. 108: 77-89.
8. Li, J., D. R. Barreda, Y. A. Zhang, H. Boshra, A. E. Gelman, S. LaPatra, L. Tort, and J. O. Sunyer. 2006. B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nat. Immunol. 7:1116-1124.
9. Lovoll, M., T. Kilvik, H. Boshra, J. Bogwald, J. O. Sunyer, and R. A. Dalmo. 2006. Maternal transfer of complement components C3-1, C3-3, C3-4, C4, C5, C7, Bf, and Df to offspring in rainbow trout (Oncorhynchus mykiss). Immunogenetics .58:168-179.*
10. Boshra, H., J. Li, and J. O. Sunyer. 2006. Recent advances on the complement system of Teleost Fish: Novel components and new functions. Fish & Shellfish Immunol. 20 (2): 239-262.
11. Gelman, A. E., D. F. LaRosa, J. Zhang, P. T. Walsh, Y. Choi, J. O. Sunyer, and L. A. Turka. 2006. The adaptor molecule MyD88 activates PI-3 kinase signaling in CD4+ T cells and enables CpG oligodeoxynucleotide-mediated costimulation. Immunity. 25:783-793.
12. Løvoll M, Johnsen H, Boshra H, Bøgwald J, J. O. Sunyer and R. A. Dalmo. 2007. The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar). Fish & Shellfish Immunol. 23:542-552.
13. Hodawadekar, S., D. Yu, D. Cozma, B. Freedman, J. O. Sunyer, M. L. Atchinson, and Thomas-Tikhonenko A. 2007. B-Lymphoma cells with epigenetic silencing of Pax5 trans-differentiate into macrophages, but not other hematopoietic lineages. Exp Cell Res. 15:331-40.
14. McCarthy, S. E., R. F. Johnson, Y. A. Zhang, J. O. Sunyer, and R. N. Harty. 2007. Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding. J. Virol. 81:11452-11460.
15. Zhang, Y. A., J. Hikima, J. Li, S. E. LaPatra, Y. P. Luo, and J. O. Sunyer. 2009. Conservation of structural and functional features in a primordial CD80/86 molecule from rainbow trout (Oncorhynchus mykiss), a primitive teleost fish. J. Immunol. 183:83-96.
16. Liu, Y., L. Cocka, A. Okumura, Y. A. Zhang, J. O. Sunyer, and R. N. Harty. 2010. Conserved motifs within Ebola and Marburg virus VP40 proteins are important for stability, localization, and subsequent budding of virus-like particles. J. Virol. 84:2294-2303.
17. Zhang, Y. A., I. Salinas, J. Li, D. Parra, S. Bjork, Z. Xu, S. E. LaPatra, J. Bartholomew, and J. O. Sunyer. 2010. IgT, a primitive immunoglobulin class specialized in mucosal immunity. Nat. Immunol. 11:827-835.
18. Zhang, Y.A., Salinas, I., and J. O. Sunyer. 2011. Recent findings on the structure and function of teleost IgT. Fish & Shellfish Immunol. 31:627-34.
19. Salinas, I., Zhang, Y.A., and J. O. Sunyer. 2011. Mucosal immunoglobulins and B cells of teleost fish. Dev. Comp. Immunol. 35:1346-65.
20. Parra, D., A. M. Rieger, J. Li, Y.A. Zhang, L. M. Randall, C.A. Hunter, D. R. Barreda and J. O. Sunyer. 2012. Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities, and present phagocytosed antigen to CD4+ T cells. J. Leuk. Biol. 91(4):526-36.
21. Rowley, A.F., Morgan, E.L., Taylor, G.W., Sunyer, J.O., Holland, J.W., Vogan C,L., C.J. Secombes. 2012. Interaction between eicosanoids and the complement system in salmonid fish. Dev. Comp. Immunol. 36:1-9.
22. J. O. Sunyer. 2012. Evolutionary and Functional Relationships of B Cells from Fish and Mammals: Insights into Their Novel Roles in Phagocytosis and Presentation of Particulate Antigen. Infect. Disor.-Drug Targ. 12:200-12.
23. Ordas, M.C., B. Dixon, J. O. Sunyer, S. Bjork, J. Barholomew, T. Korytar, T., B. koller, A. Cuesta, C. Tafalla. 2012. Identification of a novel CCR7 gene in rainbow trout with differential expression in the context of mucosal or systemic infection. Dev Comp Immunol. 38:302-11.
24. J. O. Sunyer. 2013. Perspective article: Fishing for new paradigms in teleost fish immune systems. Nat. Immunol. 14:320-6.
25. Parra, D., Takizawa F. & J. O. Sunyer. 2013. Evolution of B-cell Immunity. Ann. Rev. Ani. Vet. Biosc. 1:65-97.
26. Castro, R., L. Jouneau, H.P. Pham, O. Bouchez, V. Giudicelli, M-P. Lefranc, E. Quillet, A. Benmansour, A. Six, S. Fillatreau, O. Sunyer, P. Boudinot. 2013. Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection. PLoS Pathog. 2013 Jan;9(1):e1003098. doi: 10.1371/journal.ppat.1003098. Epub 2013 Jan 10.
27. S. Fillatreau, A. Six, S.Magadan, R. Castro, J. O. Sunyer, P. Boudinot. 2013. The astonishing diversity of Ig classes and B cell repertoires in teleost fish. Front. Immunol. 2013;4:28. doi: 10.3389/fimmu.2013.00028. Epub 2013 Feb 13.
28. Xu, Z., Parra, D., Gomez, D., Salinas, I., Zhang, Y.A., Jørgensen G.L., Skjødt, K., Rasmussen, K.J.,Buchmann K., LaPatra, S.L., J. O. Sunyer. 2013. Teleost skin, an ancient mucosal surface that elicits gut-like immune-responses. Proc. Natl. Acad. Sci. U S A.110:13097-13102.
29. Gomez, D., Sunyer, J.O., Salinas, I. The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. 2013. Fish & Shellfish Immunol. 35:1729-39.
30. Gomez, D., Bartholomew, J., Sunyer, J.O. 2014. Biology and mucosal immunity to myxozoans. Dev. Comp. Immunol. 43:243-56.
31. Bjork, S.J., Zhang, Y.A., Hurst, C.N., Alonso-Naveiro, M.E., Alexander, J.D., Sunyer, J.O., Bartholomew, J.L. 2014. Defenses of susceptible and resistant Chinook salmon (Onchorhynchus tshawytscha) against the myxozoan parasite Ceratomyxa shasta. Fish & Shellfish Immunol. 37:87-95.
32. Wu, N., LaPatra, S.E., Li, J., Sunyer, J.O**., Zhang, Y.A. 2014. Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen. Fish & Shellfish Immunol.40:616-23.
33. Reyes-Cerpa, S., Reyes-López, F., Toro-Ascuy, D., Montero, R., Maisey, K., Acuña-Castillo, C., Sunyer, J.O., Parra, D., Sandino, A.M., Imarai, M. 2014. Induction of anti-inflammatory cytokine expression by IPNV in persistent infection. Fish & Shellfish Immunol. 41:172-182.
*Dr. Sunyer and Dr. Dalmo are equal corresponding authors for manuscript # 12.
** Dr. Sunyer and Dr. Zhang are equal corresponding authors for manuscript# 32.