Expression and in vitro upregulation of MHCII in koala lymphocytes

Lau, Quintin; Canfield, P. J.; Higgins, Damien P.

doi:10.1016/j.vetimm.2012.04.010

Cited by 13 publications

(17 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Due to constant pathogen-driven selection, immune molecules are among the least conserved between species. Our ability to examine koala immune profiles has, therefore, been limited to detection of antibodies, lymphocyte proliferation assays, immunophenotyping by flow cytometry with a limited number of cross-reactive antibodies to conserved (mostly intra-cytoplasmic) domains (T cell, anti-human CD3; B cell, anti-human CD79b; MHCII, anti-human HLA-DP, DQ, DR; IFNg) (Higgins et al, 2004;Lau et al, 2012) and, very recently, qPCR for IL10, IFNg and TNFa (Mathew et al, 2013a;2013b). Our recent immunophenotypic studies on captive, KoRV-positive koalas (Lau et al, 2012) revealed some interesting features: elevated numbers of B cells relative to other species (1.0-4.9×10 6 cells/ml vs 0.17-0.56×10 6 cells/ml, respectively), and absence of MHCII expression on stimulated and nonstimulated T cells.…”

Section: Is Korv Immunomodulatory In Koalas?mentioning

confidence: 99%

Koala immunology and the koala retrovirus (KoRV)

Higgins¹,

Lau²,

Maher³

2014

Tech. Rep. Aust. Mus., Online

Self Cite

View full text Add to dashboard Cite

Abstract. Although koala retrovirus (KoRV) is widely termed a pathogen, direct evidence for causation of disease impacts in koalas (Phascolarctos cinereus) remains elusive. Examination of the immune system of koalas could provide a sharper tool to investigate this but progress has been slow due to a paucity of immunological reagents in this species, and historical contradictions in research findings in this area. Our work using cross reactive antibodies to examine behaviour of resting and stimulated koala T cells (anti-human CD3); B cells (anti-human CD79b); MHCII (anti-human HLA-DP, DQ, DR) and interferon gamma (anti-bovine IFNg) by flow cytometry have revealed some features consistent with a skew to a Th2 (B cell) immune focus. Assessing the role of KoRV in immunomodulation in koalas clearly requires more in-depth research. We have used recent advances in genomics of other marsupials to develop tools necessary to assess KoRV's effects on koala immune function in free-ranging, captive and in-vitro systems.Higgins, Damien P., Quintin Lau, and Iona Maher. 2014. Koala immunology and the koala retrovirus (KoRV). In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed.

show abstract

Section: Is Korv Immunomodulatory In Koalas?mentioning

confidence: 99%

Koala immunology and the koala retrovirus (KoRV)

Higgins¹,

Lau²,

Maher³

2014

Tech. Rep. Aust. Mus., Online

Self Cite

View full text Add to dashboard Cite

show abstract

“…Partial koala MHCII genes have been characterised (Jobbins et al, 2012; Lau et al, 2013), and variation across Australia described (Lau et al, 2014). Though MHCII is clearly functional in the koala immune system, based on its conservation of genetic structure relative to other species (Lau et al, 2013) and demonstration of expression and up-regulation in koala lymphocytes and tissues (Canfield, Hemsley & Connolly, 1996; Lau, Canfield & Higgins, 2012), associations between koala MHCII and disease have not yet been investigated, but the low genetic diversity of some koala populations (Lau et al, 2014) makes this an area worthy of study. To open investigation of the relevance of MHCII diversity to chlamydial disease in the koala, this study makes use of archived samples and data from hospital admissions from a wild Chlamydia -exposed koala population to identify MHCII DA and DB β (DAB and DBB, respectively) variants as candidates for more targeted investigation in prospective studies.…”

Section: Introductionmentioning

confidence: 99%

Identification of MHCII variants associated with chlamydial disease in the koala (Phascolarctos cinereus)

Lau

Griffith

Higgins

2014

PeerJ

Self Cite

View full text Add to dashboard Cite

Chlamydiosis, the most common infectious disease in koalas, can cause chronic urogenital tract fibrosis and infertility. High titres of serum immunoglobulin G against 10 kDa and 60 kDa chlamydial heat-shock proteins (c-hsp10 and c-hsp60) are associated with fibrous occlusion of the koala uterus and uterine tube. Murine and human studies have identified associations between specific major histocompatibility complex class II (MHCII) alleles or genotypes, and higher c-hsp 60 antibody levels or chlamydia-associated disease and infertility. In this study, we characterised partial MHCII DAB and DBB genes in female koalas (n = 94) from a single geographic population, and investigated associations among antibody responses to c-hsp60 quantified by ELISA, susceptibility to chlamydial infection, or age. The identification of three candidate MHCII variants provides additional support for the functional role of MHCII in the koala, and will inform more focused future studies. This is the first study to investigate an association between MHC genes with chlamydial pathogenesis in a non-model, free-ranging species.

show abstract

“…Cell cultures of 1 ml were incubated in plain media (unstimulated) or in media containing PMA 50 ng∕ml, Ionomycin 1 μ g∕ml (Higgins, Hemsley & Canfield, 2004); PMA 25 ng∕ml, PHA 2 μ g∕ml (Sullivan et al. , 2000); or Con A 5 μ g∕ml (Lau et al. , 2012) in a sealed tube at 37°C for 5 h. Following incubation, cells were washed twice in warm PBS, resuspended in 1 ml RNAlater (Applied Biosystems, Carlsbad, CA, USA), stored at room temperature overnight and then frozen at −20°C until RNA extraction.…”

Section: Methodsmentioning

confidence: 99%

“…, 2010; Kollipara et al. , 2012; Wilkinson, Kotlarski & Barton, 1992), and flow cytometric immunophenotyping of T and B lymphocytes via cross reactive antibodies to CD3, CD79b, MHCII and IFN γ at rest and following stimulation with PMA-ionomycin (Higgins, Hemsley & Canfield, 2004; Lau et al. , 2012).…”

Section: Introductionmentioning

confidence: 99%

Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR

Maher

Griffith

Lau

et al. 2014

PeerJ

Self Cite

View full text Add to dashboard Cite

Investigation of the immune response of the koala (Phascolarctos cinereus) is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV), which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1) and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNγ) along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A). Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not consistently up-regulated by any of the protocols. Expression of CD4 and CD8β was down-regulated by mitogen stimulation. We found that the reference genes GAPDH and 28s are valid for normalising cytokine expression by koala lymphocytes after mitogen stimulation.

show abstract

Expression and in vitro upregulation of MHCII in koala lymphocytes

Cited by 13 publications

References 33 publications

Koala immunology and the koala retrovirus (KoRV)

Koala immunology and the koala retrovirus (KoRV)

Identification of MHCII variants associated with chlamydial disease in the koala (Phascolarctos cinereus)

Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus) by qRT-PCR

Contact Info

Product

Resources

About