Dendritic Cells Regulate Exposure of MHC Class II at Their Plasma Membrane by Oligoubiquitination Joost Snijder Vera Verhage Supervisor: Prof. W. Stoorvogel

Proteolytic processing of MHC-II

Research Aim To establish what role ubiquitination has in sorting of MHC-II complexes in DC’s

MHCII-ß is oligo-ubiquitinated

Ubiquitination takes place in the MVB after peptide loading

Proteolytic processing of Ii is required for Ubiquitination, DC maturation interferes

Summary MHCII-ß is oligo-ubiquitinated Ubiquitination takes place in the MVB after peptide loading Proteolytic processing of Ii is required for ubiquitination LPS-induced maturation interferes with ubiquitination

Target residue 225 Conserved Lysine residue 225 A k - β K225A = mutant Lysine  Alanine 2 MHCII Haplotypes – A b - α/β = Wild type – A k - α / β = Transduced haplotype

Fig 4B Wild-type A k - β was ubiquitinated – Not mutant A k - β K225A

Fig 4A Distribution of A b - α in A b - β -KO + A k - β -(wt) BMDC equal to WT BMDC In A b- β KO no MHCII expression A k -b- K225 accumulates at PM – Does not get ubiquitinated

Fig 4C Exogenous mixes haplotypes are properly assembled in transduced WT – ubiquitination-deficient A k - β K225A located at plasma membrane

Fig 5 Endogenous MHC II in MVB was associated 74% with LV (74%) In A k -b K225A, 16% was found located in LV – Sorting is drastically changed

Sorting steps Where is ubiquitination involved or required? – Is it endocytosis or recycling?

Fig 6A Transfer from EEA1 to DM positive structure – EEA1, early endosomal marker – DM class II compartment Delay in Mutant A k -b K225A 20°C, endocytosis occurs, but no recycling and transport to lysosomes

Less A k -b K225A was detected intracellularly (vs Ab- b) (ratio 5.5 ± 1.4) Due to reduced uptake or accelerated recyling?

Conclusion Ubiquitination of MHC II-b – occurs at lysine 225 and is required for intracellular retention of MHC II in immature DCs. – is required for sorting at MVB, failure result in transfer by default to the plasma membrane – Is required for efficient endocytosis