1. Cellular lipids and immunity: characterisation of glycosphingolipids binding the antigen presenting molecule CD1 Karen Muindi OGBI seminar 30 th May 2007

2. CD1: an introduction MHC class 1- like glycoprotein Associates with  2m 5 CD1 genes (CD1a-e) expressed in humans 4 of these (CD1a-d) present self and foreign lipid antigens to T cells Brigl and Brenner. Annual Reviews in Immunology, 2004

3. CD1a intracellular trafficking Adapted from Hava et al. Curr Opin Immunol. 2005

4. CD1b,c and d intracellular trafficking Hava et al. Curr Opin Immunol. 2005

5. CD1 antigens

6. CD1 presentation of self GSLs implicated in etiology of several autoimmune diseases MS patients have elevated numbers of T cells responding to a variety of GSLs including sulfatide and GM1 sera from Guillain-Barré patients has elevated levels of Abs reactive to GM1, GA1 and GD1b The most potent activators of CD1d-restricted T cells (  -galactosylceramide and iGb3) are GSLs Endogenous glycosphingolipids

7. Wt mCD1d or mCD1d-tail deleted (TD) expressing cells assayed for their ability to activate CD1d restricted NKT cells CD1d-TD presentation greatly reduced. Suggests that lysosomal processing is required for activation of NKT cells CD1 lysosomal trafficking and T cell activation Chiu et al. JEM. 1999, 103-10.

8. 2 construct system to resolve CD1 pre- and post- lysosomal GSL load Adapted from Brigl and Brenner. Annual Reviews in Immunology, 2004 hCD1b-Fc hCD1b-TEV

9. hCD1b-Fc is expressed and maintains native conformation

10. TEV protease cleaves hCD1b-TEV: cleaved protein maintains native conformation

11. Lysosomal trafficking of hCD1b-TEV B2mExtracellular Domain linkerTMCYTEV

12. hCD1b-TEV steady state localisation hCD1b-TEV (151-150)hCD1b-TEV (2b4)

13. Lysosomal trafficking of recycling surface cleavable hCD1b constructs B2mExtracellular Domain linkerTMCYTEV

14. Lysosomal trafficking of recycling surface cleavable hCD1b constructs B2mExtracellular Domain linkerTMCYTEV

15. hCD1b-TEV (3:2-2) traffics to the lysosome … wt CD1b hCD1b-TEV (3:2-2)

16. …and recycles back to the cell surface Presentation of glucose monomycolate (GMM) with long alkyl chains (C80) to T cells requires lysosomal loading of lipid to CD1b (Moody et al. Nat Imm 2002) Not required for GMM with short alkyl chains (C32)

17. hCD1b-bound lipid extraction and characterisation

18. hCD1b-Fc expressed in CHO cells binds lactosylceramide and GM3.

19. hCD1b-TEV expressed in CHO cells binds lactosylceramide and GM3.

20. Transfer of the 2 construct system to other cellular models

21. Lysosomal processing of GSLs bound to mCD1d expressed in RAW cells GD1a GM1a

22. Conclusions We have successfully designed a 2 construct system to study lysosomal processing of CD1 GSL antigens Studies of mCD1d in RAW cells suggest that trimming of GSL headgroups from the non reducing end occurs in the lysosome (GD1a to GM1a)

23. Future Studies Using the U937 system, determine whether exchange or processing of hCD1b-bound GSLs occurs in the lysosomes Determine which of pre- and post- lysosomal GSL antigens is more antigenic Determine whether the GSL tails are modified in the lysosome Determine whether activation of APCs results in a change in GSL profile and CD1 GSL antigen load.

24. Acknowledgements HMS/BWH (Boston) Manuela Cernadas Gerald Watts Duarte Barral Michael Brenner UCD (Ireland) Louise Royle Pauline Rudd OGBI David Neville Terry Butters Raymond Dwek Work supported by; NIH grants to Michael Brenner OGBI studentship DPhil scholarship awards from ORS and Clarendon Fund schemes