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Analysis of DOCK2 and SENP2 mutations on the immune system and CD8 T cell survival

Mei, Yan (Angela)

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The discovery of genes essential for normal T cell numbers and T cell-mediated immune responses provides valuable insights into the adaptive immune system and targets for modulating disorders of immunity in cancer, transplantation, infection and autoimmunity. The studies presented in this thesis investigate two novel gene mutations that diminish the number of circulating T cells. The mutations were discovered by genome-wide ENU-mutagenesis in mice by flow cytometric screening of lymphocyte...[Show more]

dc.contributor.authorMei, Yan (Angela)
dc.date.accessioned2019-08-29T00:41:01Z
dc.date.available2019-08-29T00:41:01Z
dc.identifier.urihttp://hdl.handle.net/1885/165368
dc.description.abstractThe discovery of genes essential for normal T cell numbers and T cell-mediated immune responses provides valuable insights into the adaptive immune system and targets for modulating disorders of immunity in cancer, transplantation, infection and autoimmunity. The studies presented in this thesis investigate two novel gene mutations that diminish the number of circulating T cells. The mutations were discovered by genome-wide ENU-mutagenesis in mice by flow cytometric screening of lymphocyte subsets in splenocytes and in peripheral blood. Genetic mapping and sequencing identified the two mutations. The first was a premature stop-gain mutation in Dock2, encoding a GTP exchange factor for the Rac family of cytoplasmic small G proteins, which has previously been found to be critical for T cells and immunity in mice and humans. The second mutation was a synonymous exonic nucleotide substitution in Senp2, encoding a specific protease for cleaving the ubiquitin-like SUMO protein from its conjugates with other proteins, whose critical role in T cells had not previously been shown. Mice homozygous for the ‘dockland’ E775X mutation in DOCK2 (doc/doc) had 80% fewer naïve (CD44low) CD8 and CD4 T cells, 70% fewer NKT cells, 50% fewer B cells in the blood and 80% fewer B cells in the spleen, and absence of splenic marginal zone B cells. Mutant T cells nevertheless divided normally to anti-CD3 in vitro, consistent with intact TCR signaling, and peripheral CD44high activated memory T cells were present in normal or increased numbers consistent with homeostatic proliferation. Thymocytes showed evidence of developmental defects, including 50% decreased CD4+CD8+ double positive (DP) cells with lower CD5 expression and greatly decreased NKT cells. The percentage of CD4+ or CD8+ single positive (SP) thymocytes lacking CD69 was increased, consistent with delayed egress of fully mature cells. In competitive bone marrow transplantation experiments, mutant hematopoietic stem cells engrafted efficiently based on their contribution to granulocytes, but failed to contribute to peripheral T cells due to progressive under-representation at the DP and SP thymocyte stages of development. Mutant B cells contributed poorly to the follicular and germinal centre subsets, and were completely absent from the marginal zone subset. These results establish that DOCK2 is essential within T and B lymphocytes. Despite decreased circulating naïve T cells, Dock2 mutant doc/doc mice exposed sequentially to H3N2 and H1N1 influenza viruses formed normal numbers of memory CD8 T cells binding H2-Db MHC tetramers bearing the dominant influenza NP peptide epitope, NP366-374 (NP+), in contrast to Dock8 mutant mice analysed in parallel which had greatly diminished memory T cells. Dock2 mutant mice nevertheless formed fewer NP-binding effector CD8 cells than wildtype at the peak of the primary and recall response. During acute lung infection with PR8-strain H1N1 influenza virus, Dock2 mutant mice were protected from weight loss and morbidity occurring between days 5 and 7 in wild-type mice. Mice with combined homozygous mutations in Dock2 and Dock8 tended to have fewer circulating naïve T cells and NKT cells than either single mutant, although this did not achieve statistical significance in the cohort available. Collectively, these results extend earlier published findings in Dock2 knockout mice, and provide new insights into the effects of DOCK2 deficiency on the anti-viral immune response that are relevant to the fatal infections and T cell lymphopenia in DOCK2-deficient children. In the second mutant strain, duan, a 70% decrease selectively in circulating naïve CD8 T cells mapped as a recessive trait to a synonymous A>G substitution at Ch16:22,036,478 in exon 11 of Senp2. The mutation was +9 nucleotides from the exon 11 splice acceptor and diminished splicing of exon 10 to this acceptor. Of spliced mRNA that was produced, 55% skipped exon 11 to create an in-frame deletion of 15 amino acids. Upon expression of GFP-tagged SENP2, the internally deleted protein nevertheless exhibited normal intracellular localization to the nuclear membrane. While Senp2-/- null mutation in mice is homozygous lethal prior to blastocyst implantation, a complementation cross with the duan point mutant revealed that compound heterozygous Senp2dua/- mice were viable and recapitulated the CD8 T cell deficiency of Senp2dua/dua homozygotes, confirming that partial deficiency of SENP2 causes a selective T cell deficit. Thymic T cells subsets were unaffected in Senp2dua/dua mice, and the basis for the decreased number of naïve CD8 T cells was shown to be diminished survival in the periphery. This was most clearly shown by comparing the persistence of ovalbumin-specific OT-I CD8 T cells with normal or mutant Senp2 after the T cells were “parked” in wild-type mice without antigenic stimulation. In this setting, mutant OT-I T cells transduced with a retroviral vector expressing wildtype SENP2 preferentially survived, further confirming that SENP2 deficiency was responsible. Competitive bone marrow transplantation also showed a cell autonomous requirement for normal splicing of Senp2 to maintain peripheral CD8 T cell numbers but not for CD4 T cells or B cells. Upon sequential exposure to recombinant H3N2 and H1N1 influenza viruses expressing ovalbumin peptide, OT-I T cells bearing the Senp2dua/dua mutation formed an equally large population of effector CD8 T cells 7 days after primary or recall challenge, but persistence of memory CD8 T cells was decreased more than 90%. Senp2 mutant CD8 T cells responded normally to CD3 stimulation in vitro as measured by cell division. They also responded normally to IL-7 in vitro as measured by STAT5 phosphorylation, despite a cell autonomous 25% decrease in IL7Ra/CD127 on their cell surface. Collectively, these results define a key role for controlling SUMO conjugation by SENP2 in the survival of naïve and memory CD8 T cells, apparently involving a pathway independent of the established TCR and IL-7 signalling mechanisms.
dc.language.isoen_AU
dc.subjectENU
dc.subjectDOCK2
dc.subjectDOCK8
dc.subjectIL-7
dc.subjectSENP2
dc.titleAnalysis of DOCK2 and SENP2 mutations on the immune system and CD8 T cell survival
dc.typeThesis (PhD)
local.contributor.supervisorGoodnow, Christopher
local.contributor.supervisorcontactc.goodnow@garvan.org.au
dcterms.valid2019
local.description.notesthe author deposited 28/08/2019
local.type.degreeDoctor of Philosophy (PhD)
dc.date.issued2018
local.contributor.affiliationDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University
local.identifier.doi10.25911/5d7a2dbd3a1ba
local.mintdoimint
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