Publications by authors named "Rushika C Wirasinha"

12 Publications

  • Page 1 of 1

c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

Eur J Immunol 2021 May 7. Epub 2021 May 7.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.
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http://dx.doi.org/10.1002/eji.202048900DOI Listing
May 2021

DOCK8 deficiency diminishes thymic T-regulatory cell development but not thymic deletion.

Clin Transl Immunology 2021 7;10(1):e1236. Epub 2021 Jan 7.

Infection and Immunity Program Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology Monash University Melbourne VIC Australia.

Objective: To define the effect of DOCK8 deficiency on thymic tolerance in mice.

Methods: Thymocytes from wild-type ( ) and DOCK8-deficient ( ) mice were examined by flow cytometry. Some mice had transgenic expression of the BCL2 anti-apoptotic protein in haemopoietic cells. Some mice expressed the transgenic 3A9 T-cell receptor (TCR), which triggers thymocyte deletion in mice also expressing hen egg lysozyme under the insulin promoter.

Results: In mice, the proportion of thymocytes induced to acquire tolerance at the immature CCR7 stage was normal. Deletion of strongly self-reactive CD4 thymocytes occurred efficiently in mice in a TCR-transgenic model that requires self-antigen transfer from epithelial cells to bone marrow (BM)-derived antigen-presenting cells. Thymic Foxp3 T-regulatory cells (T) and Helios Foxp3 T precursors were decreased in mice, including when apoptosis was inhibited by BCL2 transgene expression. thymic T expressed CD25 and CTLA-4 at normal levels. The results suggest that DOCK8 deficiency does not affect the function of BM-derived antigen-presenting cells in the thymus, the TCR self-reactivity threshold that activates tolerance mechanisms in thymocytes or the apoptotic deletion of these thymocytes. However, DOCK8 is required to prevent a subset of developing T cells from undergoing cell death via a mechanism that is distinct from apoptosis.

Conclusion: DOCK8 deficiency diminishes T development in the thymus without compromising thymocyte deletion.
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http://dx.doi.org/10.1002/cti2.1236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790591PMC
January 2021

Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility.

J Exp Med 2021 Feb;218(2)

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.
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http://dx.doi.org/10.1084/jem.20200476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595743PMC
February 2021

Deletion of self-reactive CCR7- thymocytes in the absence of MHC expression on thymic epithelial cells.

Cell Death Differ 2019 12 24;26(12):2727-2739. Epub 2019 Apr 24.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia.

The selection of αβ T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αβ T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRβ + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRβ chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.
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http://dx.doi.org/10.1038/s41418-019-0331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224288PMC
December 2019

αβ T-cell receptors with a central CDR3 cysteine are enriched in CD8αα intraepithelial lymphocytes and their thymic precursors.

Immunol Cell Biol 2018 07 3;96(6):553-561. Epub 2018 May 3.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia.

The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαβ CD8αα intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRβ chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3 regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.
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http://dx.doi.org/10.1111/imcb.12047DOI Listing
July 2018

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Immunology 2018 07 27;154(3):522-532. Epub 2018 Feb 27.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.
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http://dx.doi.org/10.1111/imm.12904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002238PMC
July 2018

GPR65 inhibits experimental autoimmune encephalomyelitis through CD4 T cell independent mechanisms that include effects on iNKT cells.

Immunol Cell Biol 2018 02 19;96(2):128-136. Epub 2017 Dec 19.

Immunology Division, Garvan Institute of Medical Research, Sydney, NSW, Australia.

The G protein-coupled receptor 65 (GPR65) gene has been genetically associated with several autoimmune diseases, including multiple sclerosis (MS). GPR65 is predominantly expressed in lymphoid organs and is activated by extracellular protons. In this study, we tested whether GPR65 plays a functional role in demyelinating autoimmune disease. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we found that Gpr65-deficient mice develop exacerbated disease. CD4 helper T cells are key drivers of EAE pathogenesis, however, Gpr65 deficiency in these cells did not contribute to the observed exacerbated disease. Instead, Gpr65 expression levels were found to be highest on invariant natural killer T (iNKT) cells. EAE severity in Gpr65-deficient mice was normalized in the absence of iNKT cells (CD1d-deficient mice), suggesting that GPR65 signals in iNKT cells are important for suppressing autoimmune disease. These findings provide functional support for the genetic association of GPR65 with MS and demonstrate GPR65 signals suppress autoimmune activity in EAE.
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http://dx.doi.org/10.1111/imcb.1031DOI Listing
February 2018

IL-2 prevents deletion of developing T-regulatory cells in the thymus.

Cell Death Differ 2017 06 12;24(6):1007-1016. Epub 2017 May 12.

Immunology Department, The John Curtin School of Medical Research, The Australian National University, Canberra 0200, Australia.

In the thymus, strongly self-reactive T cells may undergo apoptotic deletion or differentiate into Foxp3+ T-regulatory (T-reg) cells. Mechanisms that partition T cells into these two fates are unclear. Here, we show that IL-2 signalling is required to prevent deletion of CD4+ CD8- CCR7+ Helios+ thymocytes poised to upregulate Foxp3. The deletion prevented by IL-2 signalling is Foxp3 independent and occurs later in thymocyte development than the deletion that is prevented by Card11 signalling. Our results distinguish two bottlenecks at which strongly self-reactive thymocytes undergo deletion or progress to the next stage of T-reg differentiation; Card11 regulates the first bottleneck and IL-2 regulates the second.
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http://dx.doi.org/10.1038/cdd.2017.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442470PMC
June 2017

IL-27 Directly Enhances Germinal Center B Cell Activity and Potentiates Lupus in Sanroque Mice.

J Immunol 2016 10 12;197(8):3008-3017. Epub 2016 Sep 12.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia;

Germinal centers (GC) give rise to high-affinity and long-lived Abs and are critical in immunity and autoimmunity. IL-27 supports GCs by promoting survival and function of T follicular helper cells. We demonstrate that IL-27 also directly enhances GC B cell function. Exposure of naive human B cells to rIL-27 during in vitro activation enhanced their differentiation into CD20CD38CD27CD95CD10 cells, consistent with the surface marker phenotype of GC B cells. This effect was inhibited by loss-of-function mutations in STAT1 but not STAT3 To extend these findings, we studied the in vivo effects of IL-27 signals to B cells in the GC-driven Roquin lupus mouse model. Il27raRoquin mice exhibited significantly reduced GCs, IgG2a(c) autoantibodies, and nephritis. Mixed bone marrow chimeras confirmed that IL-27 acts through B cell- and CD4 T cell-intrinsic mechanisms to support GCs and alter the production of pathogenic Ig isotypes. To our knowledge, our data provide the first evidence that IL-27 signals directly to B cells promote GCs and support the role of IL-27 in lupus.
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http://dx.doi.org/10.4049/jimmunol.1600652DOI Listing
October 2016

A timeline demarcating two waves of clonal deletion and Foxp3 upregulation during thymocyte development.

Immunol Cell Biol 2016 Apr 29;94(4):357-66. Epub 2015 Oct 29.

Immunology Department, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.

Thymocytes that bind strongly to self-antigens are prevented from becoming naive T cells by several mechanisms. They undergo clonal deletion at two stages of development; wave 1 in immature thymocytes lacking the medulla-homing chemokine receptor, CCR7, or wave 2 in more mature CCR7(+) thymocytes. Alternatively, self-reactive thymocytes upregulate Foxp3 to become T-regulatory cells. Here, we describe the differential timing of the two waves of deletion and Foxp3 upregulation relative to the immature proliferating stage. Proliferating thymocytes were pulse-labeled in normal C57BL/6 mice with 5-ethynyl-2'-deoxyuridine (EdU). Thymocytes progressed into wave 1 (CCR7(-)) and wave 2 (CCR7(+)) of clonal deletion ~2 and 5 days after proliferation, respectively. Foxp3 upregulation occurred between 4 and 8 days after proliferation, predominantly in thymocytes with a Helios(+) CCR7(+) phenotype. These findings establish a timeline that suggests that wave 1 of clonal deletion occurs in the thymic cortex, whereas wave 2 and Foxp3 upregulation both occur in the thymic medulla.
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http://dx.doi.org/10.1038/icb.2015.95DOI Listing
April 2016

Interleukin-27 signaling promotes immunity against endogenously arising murine tumors.

PLoS One 2013 12;8(3):e57469. Epub 2013 Mar 12.

Immunological Diseases Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

Interleukin-27 (IL-27) is a pleiotropic cytokine but its immunosuppressive effects predominate during many in vivo immunological challenges. Despite this, evidence from tumor cell line transfer models suggested that IL-27 could promote immune responses in the tumor context. However, the role of IL-27 in immunity against tumors that develop in situ and in tumor immunosurveillance remain undefined. In this study, we demonstrate that tumor development and growth are accelerated in IL-27 receptor α (Il27ra)-deficient mice. Enhanced tumor growth in both carcinogen-induced fibrosarcoma and oncogene-driven mammary carcinoma was associated with decreased interferon-γ production by CD4 and CD8 T cells and increased numbers of regulatory T-cells (Treg). This is the first study to show that IL-27 promotes protective immune responses against endogenous tumors, which is critical as the basis for future development of an IL-27 based therapeutic agent.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595259PMC
September 2013