C\ ( 3 .i t I '( CHARACTERISATION OF THE IMMUNE RESPONSES OF THE KOALA RAY WILKTNSON BSc (Hons) Immuno-Virology VETLAB Primary Industries of South Australia Adelaide, South Australia Department of Microbiology and Immunology University of Adelaide Adelaide, South Australia A thesis submitted to the University of Adelaide in fulfilment of the requirement for the degree of Doctor of Philosophy. September 1996 Abstract The koala (Phascolarctos cinereøs) is an arboreal marsupial dear to the hearts of most Australians and is a symbol of Australian wildlife throughout the world. It has a total dependence on a rapidly shrinking food and habitat resource, is known to suffer from a fatal stress-related syndrome and appears to have higher disease susceptibility compared to other marsupials. These factors have contributed to the suggestion that the very survival of this species is under threat. Despite this, until very recently little was known of the physiology of this unique animal and its immune system and immunological capability in particular had received minimal scientific attention. This study was undertaken to characterise both the humoral and cell-mediated immune responses of the koala to provide information upon which future disease investigations and immunological research could be based. Koala immunoglobulins (Igs) were purified using the highly specific technique of afhnity chromatography and analysed using precipitation and chromatographic techniques. Antisera was raised against both the purified molecules and whole koala serum and used to further characterise these proteins in koala serum. The production of such antisera also enabled the development of highly sensitive immunoenzyme assays which were used to monitor the kinetics and dynamics of antibody production to a range of soluble and particulate antigens. The results from these studies indicate that koalas produce Igs analogous to those found in eutherian mammals. The major Ig isotype, IgG, is composed of two heavy and two light chains resulting in molecules with a molecular weight of approximately 156 kDa. These molecules have a higher net negative charge than that seen in most other mammals and they localise in the beta region when serum is subjected to agarose electrophoresis. Koalas produce at least two subclasses of IgG and an IgM-like molecule which are all present in normal koala sortlttt; Quantification of koala serum Ig using radial immunodiffirsion indicated a mean IgG level of 5.18 mg/ml, which is lower than that reported in many other mammalian species. Both the kinetics and dynamics of Ab production to all protein antigens used in this study were somewhat slower compared to those reported in other mammals. Ab levels to some soluble proteins such as BSA did not reach detectable levels until three to four weeks post exposure and required fifteen to twenty weeks to reach levels produced in rabbits within three weeks of exposure. Methods were developed to identiff and quantitate circulating immune effector cells in an attempt to define the reasons for the retarded responses to antigens. B lymphocytes were identified using an antiserum directed against SmIg. They represented 23.8% of the lymphoid cell population in the peripheral circulation. T lymphocytes were identified using a cross-reactive polyclonal antiserum directed against an intracytoplasmic portion of the epsilon chain of the CD3 Ag. These cells represented 65%o of the lymphoid cell population in the peripheral circulation. Koala macrophages were identified using a cross-reactive monoclonal Ab raised against a murine MHC class II Ag; la-H-2d. Immunohistochemical studies with these Abs enabled the identification of these immune cells in formalin fixed lymphoid tissue. The results obtained indicated that the secondary lymphoid organs of koalas contain fewer lymphoid cells than the more extensively studied mammals. They also display less organisational structure than equivalent eutherian mammal organs, providing one possible explanation for the retarded humoral response observed in this species. Ag specifi c in vitro proliferative responses of lymphocytes were demonstrated for the first time in this animal by sensitising koalas in vivo with Bacillus Calmette-Guerin (BCG). The level and timing of this cell-mediated immune (CMI) response were comparable with those seen in non-metatherian mammals. Blocking studies with the cross-reactive anti-murine MHC class II antibody indicate that these proliferation responses to BCG utilise similar induction mechanisms to those reported in other mammals. Attempts to generate long term T cell clones in this species were unsuccessful although it was demonstrated that supernatants from mitogen activated kangaroo mononuclear *IL-2 cells contained an co-factor like" activity capable of maintaining the proliferation of lectin-activated koala lymphoid cells. Studies designed to further dissect CMI responses in this species by inducing in vitro allogeneic responses using MLR assays, were also unsuccessful despite extensive optimisation of experimental conditions. The finding that MLR responses could not be generated using lymphoid cells obtained from South Australian and Queensland animals suggested that the lack of such responses was ii not due to a lack of genetic diversity within the South Australian population, but was a species wide phenomenon. This supports the findings of others that marsupials as a group display minimal or no MLR reactivity. Finally, koala responses to a hapten, 4-hydroxy-3-nitrophenyl acetyl (NP) were analysed to provide further information on the immune capability of this species. Interestingly, koala humoral responses to this hapten were much more rapid than those seen to more complex protein Ags, including the carrier proteins used to render NP immunogenic. In addition, the use of this hapten enabled the identification and quantification of NP- specific antibody secreting cells suggesting that further work with this and other haptens may prove useful in clarifring the apparent immune deficiencies of this animal. PUBLICATIONS AND PRESENTATIONS ARISING PUBLICATIONS Wilkinson R, M Allanson, V Kolega, I) Lawrence and S Neville 1991. Purification and initial characterisation of koala immunoglobulins. Vet Immunol Immunopathol29: I 89-l 95. Wilkinson R,I Kotlarski, M Barton and P Phillips Lg92.Isolation of koala lymphoid cells and their in vitro rcsponses to Mitogen. Vet Immunol Immunopathol 3l:21-33. Wilkinson R,I Kotlarski and M Barton 1992. Antigenic responses of koala lymphoid cells. Vet Immunol Immunopathol 33: 237-247 ' Wilkinson R,I Kotlarski and M Barton 1994. Further characterisation of the immune response of the koala. Vet Immunol Immunopathol 40:325-339' \ililkinson R, I Kotlarski and M Barton 1995. Identification of koala T lymphocytes using an anti-human CD3 antibody. Develop Comp Immunol 19(6): 537-545. Bertram E, I Kotlarski and R Wilkinson 1996. Identification of duck T lymphocytes using an anti-human CD3 antibody. Vet Immunol lmmunopathol. In Press' PRESENTATIONS Wilkinson R 1991. The kinetics and dynamics of immunoglobulin production in the koala. Oral presentation at the Annual Conference of the Australian Society for Microbiology, Gold Coast, Queensland. \ililkinson R 1993. How much caî a koala bear? Oral presentation at the Annual Conference of the Australian Society of Laboratory Animal Science, Adelaide, South Australia. 'Wilkinson R 1994. Would the koala make an interesting immunosuppressed animal model? Poster presentation at the 8th International Workshop of Immunosuppressed Animal Models, Utrecht, Netherlands. Wilkinson R, M Barton and I Kotlarski 1995. Identification and characterisation of a T cell specific antigen in the koala using an anti-human CD3 antibody. Poster presentation at the 4th International Veterinary Immunology Symposium, Davis, USA. Wilkinson R, M Barton and I Kotlarski 1995. Characterisation of the immune response of the koala. Poster presentation at the 9th International Congress of Immunology, San Francisco, USA' IV ACKNOWLEDGMENTS The work reported in this thesis was preformed in the Immuno-Virology section of VETLAB, Primary Industries, South Australia and received financial assistance from the Australian Koala Foundation. I am grateful to these organisations for their support. I wish to express my sincere thanks to my supervisors Professor leva Kotlarski and Doctor Mary Barton for their continued support, guidance and encouragement throughout this study. I also wish to thank the following people for the contributions they have made to this thesis: Staff of VETLAB and in particular those of the Immuno-Virology Section; Margaret Allanson, Peter Durham, Carolyn Coles, Vicki Katsos, David Lawtence, Bob Stevenson, Sandy Wright and especially Bridget V/right for their continued physical, emotional and cerebral support during this study. Edward Bertram and Garry Penney of the Microbiology and Immunology Department, University of Adelaide for their assistance, encouragement and fruitful discussions. Alan Bishop and Tracey Dowse of the Flow Cytometry Laboratory, IMVS for their hours of assistance and entertainment. The students and staff of Associate Professor Leonie Ashman's Laboratory, IMVS, for 'Western their advice and assistance, in the areas of cellular identification, blotting and immunoprecipitation. Frank Stomski of the Human Immunology Section, Hanson Centre IMVS for provision of equipment, reagents, advice and computer expertise. VI Helen Wainwright and Penny Leaney of the Neuropathology Laboratory