UNRAVELING THE ENDOCYTIC CONNECTIONS FOR ANTIGEN PRESENTATION AND TOXOPLASMA GONDII PROLIFERATION

FACUNDO GARRIDO; CRISTINA CROCE; SOFÍA DINAMARCA; FRANCO NIETO; NICOLAS BLANCHARD; LUIS MAYORGA; IGNACIO CEBRIÁN

San Luis

Congreso; LXXI Reunión Anual de la SAI; 2023

Sociedad Argentina de Inmunología (SAI)

Dendritic cells (DCs) are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway or with MHC-I molecules through the cross-presentation pathway to activate CD4+ or CD8+ T lymphocytes, respectively. Moreover, DCs represent one of the main cellular targets during infection by Toxoplasma gondii. This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, T. gondii modulates the host immune response through multiple interactions with proteins and lipids. In this study, we focus on the interaction between the PV and two intracellular compartments that are relevant for the efficient transport of MHC-I molecules in DCs, multivesicular bodies (MVBs) and the endocytic recycling compartment (ERC). In order to arrest MVBs biogenesis we used the inhibitor U18666A, a drug that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of T. gondii-associated antigens and parasite proliferation were hampered in treated cells. Since U18666A impairs the formation of MVBs, we analyzed in T. gondii-infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by T. gondii. Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. We used a different approach to impair the ERC functionality in DCs by silencing the expression of Rab22a. We have previously shown that this experimental strategy leads to a drastic disrupture of the main intracellular pool of MHC-I molecules distributed at the ERC, and consequently it affects the cross-presentantion of T. gondii-associated antigens. Now, we deppened into the replicative capacity of this parasite inside Rab22a KD DCs. We studied the kinetics of Rab22a recruitment towards the PV, and how this GTPase modulates the size and number of lipid droplets in T. gondii-infected DCs. Finally, we found that after the initial infection, the parasite is unable to replicate efficiently inside Rab22a silenced DCs. Altogether, our results highlight the importance of MVBs formation and the ERC integrity in DCs for optimal antigen presentation and T. gondii proliferation.