1. Peripheral Immune System of Glycosphingolipid Storage Disorder Mouse Models Aruna Jeans

2. Plasma membrane Golgi– GSL synthesis Lysosome– GSL degradation Glycosphingolipid Storage Disorders –GSL degradation is blocked Cytosol Glycosphingolipids

3. Sandhoff Mouse Model Mutation in hexB, loss of  -hexosaminidase A and B Accumulation of GM2 and GA2 Neurodegenerative disease course with symptom onset at 8 weeks (head tremor) End stage 16-18 weeks (ataxic gate, hind limb paralysis) Characterised by CNS inflammation (contributes to disease progression)

4. Peripheral Immune System GSLs accumulate in haematopoietic cells. GSLs have a number of specific roles within the peripheral immune system, making particular lymphocyte subsets vulnerable to GSL accumulation. Human disease course is highly variable – peripheral inflammation is a potential influential factor. Common neurodegenerative disorders characterised by CNS inflammation are exacerbated by peripheral inflammation.

5. Aims… Phenotype the peripheral immune system Functionally characterise the immune system Determine whether peripheral inflammation can exacerbate disease progression

6. Phenotype Cell type CD4 T cellsReduction in splenic CD4 T cells from 6/8 weeks CD8 T cellsNo detected change in numbers, but double positive thymocytes show a decrease in CD8 expression NKT cellsLoss of NKT cells (defective selection) B cellsReduction in marginal zone B cells from 8 weeks. Elevation in peritoneal B1 cells. Reduction in splenic and circulating B cells at 16-18 weeks (end stage) Monocytes/ macrophages Elevation in monocytes 3-6 weeks. Decrease in CD11b expression in a subset of splenic macrophages NeutrophilsElevation in neutrophils at 16-18 weeks (end stage)

7. Immune response Innate response – recognition by pre-formed non- specific effectors, activation of adaptive response Adaptive response – generation of specific effectors and immunological memory Antigen presenting cell – process antigens and display them on cell surface in conjunction with molecules required to activate lymphocytes CD8+ killer T cells –kill infected cells (cytosolic pathogens ) CD4+ helper T cells – activate B cells in response to T-Dependent antigens B cell – produces antibodies (inactivate toxins, label pathogens for complement recognition or opsonisation)

8. Killer T cells Female mice primed and boosted with UTY peptide. Percentage of UTY specific CD8+ T cells determined using UTY-tetramer

9. Killer T cells – VITAL assay Mice injected with stained male splenocytes or female splenocytes loaded with UTY- peptide. After 24 hrs, mice bled and cell populations recorded – calculate percentage of specific lysis, relative to female non-pulsed YX XX Injected into mice (IV), bled at 24 hrs FL1 FL2 10uM 1uM

10. T-Dependent Response Antigen binds BCR, and is internalised and processed Antigen taken up, processed and presented by APC CD4+ T cells with specific TCR ‘recognises’ presented antigen. T cells are activated and divide. T cells secrete IL4 and IFN  influencing the immune response B cell is partly activated. Antigen-complexed with MHC II is presented at cell surface T cells recognise antigen presented by partially activated B cells in lymph node. Delivers second activation signal. Germinal Centre – Fully activated B cells undergo clonal expansion, affinity maturation and B cell maturation to produce plasma cells and memory cells T-dependent IL4 IFN IL4 IFN

11. T-Dependent Response in the Sandhoff Mouse Mice were injected with 100ug TNP-KLH, precipitated with alum. Mice were bled on day 7 and serum Ig levels measured by ELISA.

12. CD4+ T cells Antigen processing and presentation IL4 IFN IL4 IFN

13. Germinal Centre Formation Mice were injected with TNP-KLH or Sheep red blood cells On day 7, mice were culled and the percentage of germinal centre B cells (PNAhiCD19+) determined by flow cytometry.

14. Plasma Cell Formation Percentage of Plasma Cells (Ig+CD138+) MFI of CD138 expression Control0.9+/- 0.061141+/-49 Homozygote0.6 +/- 0.14876+/-33 Plasma cells – 0.87% Serum anti-TNP titre- 37139 ng/ml Plasma cells – 1.0% Serum anti-TNP titre – 23670 ng/ml HeterozygoteHomozygote

15. Conclusion GSL accumulation effects all major groups of myeloid cells and lymphocytes, altering the response to immune stimulus. Sandhoff mice are able to generate functional killer T cells. Sandhoff mice have a reduced response to T- dependent antigens, due to a defect in plasma cell production and/or function. Defect in plasma cell formation could be due to errors in cell signalling and/or interaction with follicular dendritic cells Changes in the way human patients respond to infection, may make them vulnerable to secondary infection.

16. Future Work Look at germinal centre architecture Look at secondary response to T- dependent antigens Determine whether peripheral inflammation exacerbates disease progression

17. Acknowledgements Fran Platt Prof. Raymond Dwek David Smith Gabriele Reinkensmeier All of the first floor Jon Silk (IMM) Prof. Crundolo (IMM)

18. T-Independent Response T- independent Germinal centre? Mice were injected with 25g TNP-Ficoll IP. On day 7, mice were bled and serum expressed. Anti-TNP antibodies were measured by Elisa.

19. Macrophages