Thrombospondins 1 and 4 undergo coordinated transport to multicore cytotoxic granules to regulate SMAP biogenesis and function in CTL-mediatedcytotoxicity

Cassioli, C.; Capitani, N.; Staton, C. C.; Schirra, C.; Finetti, F.; Onnis, A.; Alawar, N.; Tu, S.-M.; Lopresti, L.; Tatangelo, V.; Tangredi, C.; Valvo, S.; Chang, H.-F.; Miccoli, A.; Compeer, E. B.; Nicholls, J.; Blazar, B.; Marotta, G.; Wood, M. J.; Trentin, L.; Patrussi, L.; Dustin, M. L.; Becherer, U.; Baldari, C. T.

doi:10.1101/2024.04.25.590546

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bioRxiv · 10.1101/2024.04.25.590546

Thrombospondins 1 and 4 undergo coordinated transport to multicore cytotoxic granules to regulate SMAP biogenesis and function in CTL-mediatedcytotoxicity

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Abstract

Supramolecular Attack Particles (SMAPs) are particulate entities, characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a glycoproteic shell, released by CTLs and NK cells. Prior proteomic analysis identified thrombospondin-1 (TSP-1) and thrombospondin-4 (TSP-4) as putative components of SMAPs. While TSP-1 has been validated as a component of the SMAP shell and shown to contribute significantly to CTL-mediated killing, the expression and function of TSP-4 in CTLs, and its interplay with TSP-1 in SMAP biogenesis and function, has not been investigated as yet. Here we demonstrate that TSP-4 and TSP-1 have a complementary expression profile during in vitro human CD8+ T cell differentiation to CTLs and sequentially localize to lytic granules (LG), with TSP-4 being required for TSP-1 association with LGs. Correlative light microscopy identified the TSP-enriched LGs as the SMAP-containing multicore granules. We show by STED microscopy a heterogeneity among TSP-enriched LGs, the most abundant population being positive for both TSP-4 and TSP-1. We also show that TSP-1 and TSP-4 are co-released in association with SMAPs at immune synapses formed on planar supported lipid bilayers, as assessed by dSTORM imaging. Finally, we provide evidence that TSP-4 is required for CTL- and SMAP-mediated cell killing. Of note, we found that chronic lymphocytic leukemia (CLL) cell supernatants, which suppress CTL mediated killing, also suppress expression of TSP-4 as well as of cytolytic effectors and impair SMAP biogenesis. These results identify TSP-4 as a key player in SMAP structure and activity and suggest that SMAPs may be a new target for immune suppression by CLL.

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Cassioli, C., Capitani, N., Staton, C. C., Schirra, C., Finetti, F., Onnis, A., Alawar, N., Tu, S.-M., Lopresti, L., Tatangelo, V., Tangredi, C., Valvo, S., Chang, H.-F., Miccoli, A., Compeer, E. B., Nicholls, J., Blazar, B., Marotta, G., Wood, M. J., Trentin, L., Patrussi, L., Dustin, M. L., Becherer, U., Baldari, C. T.. 2024-04-28. Thrombospondins 1 and 4 undergo coordinated transport to multicore cytotoxic granules to regulate SMAP biogenesis and function in CTL-mediatedcytotoxicity. https://doi.org/10.1101/2024.04.25.590546

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