Genetically engineered chicken models: novel tools to investigate lymphocyte development and function
Final Report Abstract
Within this project the overall aim was to enhance our knowledge on lymphocyte development and function in birds using genetically engineered chicken models. By using existing JH -/- birds with a knockout of peripheral B cells and deficient in antibody production it was possible to establish an adoptive transfer model showing that CXCR4/CXCL12 interaction is crucial for the immigration of B lymphocytes into the bursa of Fabricius during embryonic development. Also, we were able to show that signaling up on CXCR4 stimulation is dependent on the presence of surface B cell receptor expression. By further utilizing the antibody deficient JH -/- chickens it was for the first time possible to raise chickens deficient of maternal antibodies without performing chemical or surgical bursectomy. Surprisingly maternal antibody deficient chickens developed total immunoglobulin titers and antigen specific antibodies comparable to maternal antibody positive chickens. Interestingly maternal antibody negative chickens gained significantly less body weight compared to the control group pointing towards an important role of maternal antibody during development. In addition, it was possible to generate birds with a knockout of the immunoglobulin light chain. To our surprise we were able to show that chickens are able to produce single heavy chain antibodies and that a single heavy chain B cell receptor is supporting all steps of B cell development. Crossing of immunoglobulin light and heavy chain knockout chickens resulted in a chicken line completely deficient of gene rearrangement of the immunoglobulin loci. By analyzing these double immunoglobulin chickens, it was possible to show that gene rearrangement of the immunoglobulin loci is not involved in triggering the exclusive migration of B lymphocytes towards the bursa of Fabricius during embryonic development. Unexpectedly it was not possible to generate genetic modified inbred chicken lines using injection of primordial germ cells from inbred chicken lines into developing embryos. In order to be able to generate genetic modified inbred chicken lines we developed a new method transplanting primordial germ cells into the testes of roosters sterilized by irradiation. Transplanted PGCs were able to restore spermatogenesis and led to the generation of genetically modified inbred chickens. In addition, we succeeded in generating chickens with ubiquitous mCherry expression creating a useful chicken line for adoptive transfer experiments. Beside the focus on B lymphocytes we aimed to unravel the role of γ/δ T cells in chickens. Like other livestock species chicken belong to the so called γ/δ T cell high-species with up to 50% of the peripheral lymphocytes being γ/δ T cells. In order to analyze the role of this T cell population we generated a knockout of the constant region of the T cell receptor γ chain by using CRISPR/Cas9 in combination with homology directed repair. Using CRISPR/Cas9 it was for the first time possible to not use isogenic homology arms and to shorten the length of the homology regions making targeting in PGCs easier and more efficient compared to classical homologoues recombination. Resulting TCR Cγ-/- chickens showed a complete depletion of γ/δ T cells. To our surprise the TCR Cγ-/- chickens developed completely normal and did not show any immunosuppression nor any other phenotype caused by the depletion of the γ/δ T cells. Composition of the remaining peripheral lymphocytes populations was not influenced by the missing γ/δ T cells. Also, no effect on immunoglobulin titers was seen. Because of the large proportion of γ/δ T cells especially in the intestinal tract we speculated on a possible role of γ/δ T cells on the microbiome and gut integrity. Extensive histological analysis also showed no difference between γ/δ T cell deficient and control animals. 16s rRNA sequencing results also do no reveal a role of γ/δ T cells in the control of the gut microbiome. Taken together results obtained during this project increase our understanding of B cell development and function in birds and provide new useful tools to further analyze B as well as T lymphocytes in chickens.
Publications
- (2015). A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation. Front Immunol 6, 126
Leighton, P.A., Schusser, B., Yi, H., Glanville, J., and Harriman, W.
(See online at https://doi.org/10.3389/fimmu.2015.00126) - (2016). Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens. Eur J Immunol 46, 2137-2148
Schusser, B., Collarini, E.J., Pedersen, D., Yi, H., Ching, K., Izquierdo, S., Thoma, T., Lettmann, S., Kaspers, B., Etches, R.J., Van De Lavoir, M.C., Harriman, W., and Leighton, P.A.
(See online at https://doi.org/10.1002/eji.201546171) - (2017). Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken. Sci Rep 7, 14246
Trefil, P., Aumann, D., Koslova, A., Mucksova, J., Benesova, B., Kalina, J., Wurmser, C., Fries, R., Elleder, D., Schusser, B., and Hejnar, J.
(See online at https://doi.org/10.1038/s41598-017-14475-w) - (2018). "Restoration of fertility-new ways towards efficient transgenesis in chicken", Transgenic Research, 472-472
Trefil, P., Aumann, D., Lounkova, A., Mucksova, J., Benesova, B., Kalina, J., Wurmser, C., Fries, R., Elleder, D., and Hejnar, J., Schusser, B.
(See online at https://doi.org/10.1007/s11248-018-0086-x) - (2018). Applications of Gene Editing in Chickens: A New Era Is on the Horizon. Front Genet 9, 456
Sid, H., and Schusser, B.
(See online at https://doi.org/10.3389/fgene.2018.00456) - (2018). Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus. Proc Natl Acad Sci USA 115, 11603-11607
Bertzbach, L.D., Laparidou, M., Hartle, S., Etches, R.J., Kaspers, B., Schusser, B., and Kaufer, B.B.
(See online at https://doi.org/10.1073/pnas.1813964115) - (2019). Blocking of the CXCR4-CXCL12 Interaction Inhibits the Migration of Chicken B Cells Into the Bursa of Fabricius. Front Immunol 10, 3057
Laparidou, M., Schlickenrieder, A., Thoma, T., Lengyel, K., and Schusser, B.
(See online at https://doi.org/10.3389/fimmu.2019.03057) - (2020). "Analyzing the role of gamma delta T-cells in chickens by reverse genetics", Transgenic Research, 478-478
Hellmich, R., Aumann, D., Sid, H., Zenner, C., Kaspers, B., and Schusser, B.
