Cover Page The handle

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Cover Page

The handle

http://hdl.handle.net/1887/136519

holds various files of this Leiden University

dissertation.

Author: Benne, N.

Title: Vaccination and targeted therapy using liposomes : opportunities for treatment of

atherosclerosis and cancer

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ϰ

ŶŝŽŶŝĐϭ͕ϮͲŝƐƚĞĂƌŽǇůͲƐŶͲŐůǇĐĞƌŽͲϯͲ

ƉŚŽƐƉŚŽŐůǇĐĞƌŽů;^W'ͿůŝƉŽƐŽŵĞƐŝŶĚƵĐĞ

ĂŶƟŐĞŶͲƐƉĞĐŝĮĐƌĞŐƵůĂƚŽƌǇdĐĞůůƐĂŶĚƉƌĞǀĞŶƚ

ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶŵŝĐĞ

ƵƚŚŽƌŶĂŵĞƐĂŶĚĂĸůŝĂƟŽŶƐ

Naomi Benne1_{͕:ĂŶŝŶĞǀĂŶƵŝũŶ}1_{͕&ĞƌŶĂŶĚŽ>ŽǌĂŶŽsŝŐĂƌŝŽ}1_{͕ZŽŵĂŝŶ:͘d͘>ĞďŽƵǆ}1_͕WĞƚĞƌ

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4

'ƌĂƉŚŝĐĂůĂďƐƚƌĂĐƚ

ďƐƚƌĂĐƚ

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/ŶƚƌŽĚƵĐƟŽŶ

Atherosclerosis is a disease involving large and medium-sized arteries, which ĂīĞĐƚƐ ŵŝůůŝŽŶƐ ŽĨ ƉĞŽƉůĞ ǁŽƌůĚǁŝĚĞ1_{͘ /ƚ ŝƐ ŝŶŝƟĂƚĞĚ ďǇ ƚŚĞ ƌĞƚĞŶƟŽŶ ŽĨ >> ŝŶ ƚŚĞ}

ƐƵďĞŶĚŽƚŚĞůŝĂůƐƉĂĐĞŽĨĂƌƚĞƌŝĞƐ͕ĂŶĚƐƵďƐĞƋƵĞŶƚŽǆŝĚĂƟŽŶĂŶĚƵƉƚĂŬĞŽĨ>>;Žǆ>>Ϳ ďǇ ŝŶĮůƚƌĂƟŶŐ ŵĂĐƌŽƉŚĂŐĞƐ ůĞĂĚŝŶŐ ƚŽ ĨŽĂŵ ĐĞůů ĨŽƌŵĂƟŽŶ2_{. In recent years, it has}

ďĞĐŽŵĞ ĐůĞĂƌ ƚŚĂƚ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ŝƐ Ă ĐŚƌŽŶŝĐ ŝŶŇĂŵŵĂƚŽƌǇ ĚŝƐĞĂƐĞ͘ ĞůůƐ ŽĨ ďŽƚŚ ƚŚĞ ŝŶŶĂƚĞ ĂŶĚ ĂĚĂƉƟǀĞ ŝŵŵƵŶĞ ƐǇƐƚĞŵ͕ ƐƵĐŚ ĂƐ ŵĂĐƌŽƉŚĂŐĞƐ͕ ĚĞŶĚƌŝƟĐ ĐĞůůƐ ;ƐͿ͕ d ĂŶĚ ůǇŵƉŚŽĐǇƚĞƐ͕ ĂƌĞ ƉƌĞƐĞŶƚ ŝŶ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞƐ ĂŶĚ ĂƌĞ ŝŶǀŽůǀĞĚ ŝŶ ƚŚĞ progression of the disease3,4_{͘tŚŝůĞƚŚĞƌĞŝƐƐƟůůĚĞďĂƚĞĂďŽƵƚƚŚĞƐƉĞĐŝĮĐƌŽůĞŽĨĐĞƌƚĂŝŶ}

ĐĞůůƚǇƉĞƐŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͕ƚŚĞŐĞŶĞƌĂůĐŽŶƐĞŶƐƵƐŝƐƚŚĂƚdŚĞůƉĞƌϭ;dŚϭͿϰ+_{T cells} ĂƌĞ ƉƌŽͲĂƚŚĞƌŽŐĞŶŝĐ͕ ǁŚŝůĞ ƌĞŐƵůĂƚŽƌǇ d ĂŶĚ ĐĞůůƐ ;dƌĞŐƐ ĂŶĚ ƌĞŐƐ͕ ƌĞƐƉĞĐƟǀĞůǇͿ ĂƌĞ ƉƌŽƚĞĐƟǀĞ5,6_{͘ dƌĞŐƐ ĂƌĞ ŬŶŽǁŶ ƚŽ ďĞ ǀŝƚĂů ĨŽƌ ŝŵŵƵŶĞ ƐƵƉƉƌĞƐƐŝŽŶ͕ ƌĞŐƵůĂƟŽŶ͕} ĂŶĚ ƌĞƐŽůƵƟŽŶ ŽĨ ŝŶŇĂŵŵĂƟŽŶ ĂŌĞƌ ŝŶĨĞĐƟŽŶ7_{͘ dŚĞǇ ĨŽƌŵ Ă ƐƵďƐĞƚ ŽĨ ϰ}+_{T cells} ƚŚĂƚ ĞǆƉƌĞƐƐ ƚŚĞ ƚƌĂŶƐĐƌŝƉƟŽŶ ĨĂĐƚŽƌ ĨŽƌŬŚĞĂĚ ďŽǆ Wϯ ;&KyWϯͿ ĂŶĚ ƚŚĞ />ͲϮ ƌĞĐĞƉƚŽƌ ɲ ĐŚĂŝŶ ;ϮϱͿ͘ dƌĞŐƐ ŵĞĚŝĂƚĞ ƚŽůĞƌĂŶĐĞ ďǇ ƚŚĞ ƉƌŽĚƵĐƟŽŶ ŽĨ ƚŚĞ ĂŶƟͲŝŶŇĂŵŵĂƚŽƌǇ ĐǇƚŽŬŝŶĞƐŝŶƚĞƌůĞƵŬŝŶ;/>ͿͲϭϬĂŶĚƚƌĂŶƐĨŽƌŵŝŶŐŐƌŽǁƚŚĨĂĐƚŽƌ;d'&ͿͲɴ8_{, consuming IL-2,}

ŝŶƚĞƌƌƵƉƟŶŐĞīĞĐƚŽƌdĐĞůůŵĞƚĂďŽůŝƐŵŽƌĞǀĞŶůǇƐŝŶŐĞīĞĐƚŽƌdĐĞůůƐ9_{. In many diseases,} including atherosclerosis10-12_{͕ dƌĞŐƐ ŚĂǀĞ Ă ƌĞĚƵĐĞĚ ĨƵŶĐƟŽŶ Žƌ ĂďŝůŝƚǇ ƚŽ ƉƌŽůŝĨĞƌĂƚĞ͘} ƵƌƌĞŶƚ ƐƚƌĂƚĞŐŝĞƐ ĨŽƌ ƚƌĞĂƚŵĞŶƚ ŽĨ ƐƵĐŚ ĚŝƐŽƌĚĞƌƐ ŝŶǀŽůǀĞ ƐǇƐƚĞŵŝĐ ƐƵƉƉƌĞƐƐŝŽŶ ŽĨ ŝŶŇĂŵŵĂƟŽŶ ǁŝƚŚ ĚƌƵŐƐ Žƌ ďǇ ƐĞůĞĐƟǀĞ ĐĞůů ĚĞƉůĞƟŽŶ͘ ,ŽǁĞǀĞƌ͕ ƚŚĞƐĞ ƚŚĞƌĂƉŝĞƐ ĐĂŶ ƌĞƐƵůƚŝŶƐĞǀĞƌĞƐŝĚĞĞīĞĐƚƐ͕ĞƐƉĞĐŝĂůůǇƵƉŽŶůŽŶŐͲƚĞƌŵƚƌĞĂƚŵĞŶƚ13-15_{͘ŵŽƌĞƐƉĞĐŝĮĐ} ƐƚƌĂƚĞŐǇǁŽƵůĚďĞƚŽĚĞƐŝŐŶĂǀĂĐĐŝŶĞƚŚĂƚŝŶĚƵĐĞƐƐƉĞĐŝĮĐƚŽůĞƌĂŶĐĞƚŚƌŽƵŐŚŝŶĚƵĐƟŽŶ ŽĨ dƌĞŐƐ ƚŚĂƚ ƌĞĐŽŐŶŝǌĞ ƚŚĞ ĂƵƚŽĂŶƟŐĞŶƐ ŝŶǀŽůǀĞĚ ŝŶ ŝŶŇĂŵŵĂƚŽƌǇ ĚŝƐĞĂƐĞƐ͘ >> ŚĂƐ ďĞĞŶŝĚĞŶƟĮĞĚĂƐƚŚĞŵŽƐƚƌĞůĞǀĂŶƚĂŶƟŐĞŶŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͕ĂƐŝƚŝƐŝŵƉŽƌƚĂŶƚŝŶƚŚĞ ŝŶŝƟĂƟŽŶŽĨĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ2,16_{͘/ŶĚĞĞĚ͕Žǆ>>ͲƐƉĞĐŝĮĐdĐĞůůƐŚĂǀĞďĞĞŶĨŽƵŶĚŝŶŚƵŵĂŶ} ĂƚŚĞƌŽƐĐůĞƌŽƟĐƉůĂƋƵĞƐ17_{͕ĂŶĚĂŶƟďŽĚŝĞƐĂŐĂŝŶƐƚƉŽϭϬϬ;ƚŚĞĂƉŽůŝƉŽƉƌŽƚĞŝŶŽĨ>>Ϳ} ŚĂǀĞďĞĞŶŝĚĞŶƟĮĞĚŝŶƉĂƟĞŶƚƐǁŝƚŚĐĂƌĚŝŽǀĂƐĐƵůĂƌĚŝƐĞĂƐĞ18-20_{. LDL, ApoB100,} ApoB100-ĚĞƌŝǀĞĚ ƉĞƉƟĚĞƐ ĂŶĚ ĂŶƟďŽĚŝĞƐ ĂŐĂŝŶƐƚ ƉŽϭϬϬͲApoB100-ĚĞƌŝǀĞĚ ƉĞƉƟĚĞƐ ŚĂǀĞ ƐƵĐĐĞƐƐĨƵůůǇ been used as vaccine targets in mice21-28_.

tŚŝůĞ ƚŚĞƐĞ ƐƚƵĚŝĞƐ ƐŚŽǁ Ă ƌĞĚƵĐƟŽŶ ŝŶ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞ ĨŽƌŵĂƟŽŶ ŝŶ ŵŝĐĞ͕ŽŶůǇĂĨĞǁŚĂǀĞďĞĞŶĚĞƐŝŐŶĞĚǁŝƚŚƚŚĞŐŽĂůŽĨŝŶĚƵĐŝŶŐĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƐ29_or

ƵƐŝŶŐĐŽŵƉůĞǆĨŽƌŵƵůĂƟŽŶƐ24_{. To our knowledge, there are no reports about the use of}

ĂƚŚĞƌŽͲƉƌŽƚĞĐƟǀĞĂŶƟŐĞŶƐǁŝƚŚĂĚǀĂŶĐĞĚĚƌƵŐĚĞůŝǀĞƌǇĨŽƌŵƵůĂƟŽŶƐ͘/ŵŵƵŶŽƚŚĞƌĂƉǇ ǁŝƚŚƐŽͲĐĂůůĞĚƚŽůĞƌŽŐĞŶŝĐƐ;ƐƉƵůƐĞĚex vivoǁŝƚŚĂŶƟŐĞŶƐĂŶĚ/>ͲϭϬŽƌd'&ͲɴͿŽƌ Žǆ>>ͲƉƵůƐĞĚ;ĂƉŽƉƚŽƟĐͿƐŚĂǀĞďĞĞŶƐƵĐĐĞƐƐĨƵůĂƚŝŶĚƵĐŝŶŐdƌĞŐƐ͘,ŽǁĞǀĞƌ͕ƚŚĞƐĞ ƚƌĞĂƚŵĞŶƚƐĂƌĞĞǆƉĞŶƐŝǀĞ͕ĂƐƚŚĞǇƌĞƋƵŝƌĞex vivoŝƐŽůĂƟŽŶĂŶĚůŽŶŐͲƚĞƌŵĐƵůƚƵƌŝŶŐŽĨ Ɛ ƵŶĚĞƌ 'DW ĐŽŶĚŝƟŽŶƐ30-32_{͘ DŽƌĞŽǀĞƌ͕ ƚŚĞƌĞ ŝƐ ĞǀŝĚĞŶĐĞ ƚŚĂƚ Ɛ ŝŶĚƵĐĞĚ ŝŶ ƚŚŝƐ} ǁĂǇĐĂŶůŽƐĞƚŚĞŝƌŵŝŐƌĂƚŽƌǇĂŶĚdĐĞůůĂĐƟǀĂƟŽŶƉŽƚĞŶƟĂůŽƌĐĂŶĞǀĞŶƌĞǀĞƌƚƚŽĂƉƌŽͲ ŝŶŇĂŵŵĂƚŽƌǇƉŚĞŶŽƚǇƉĞin vivo33_{͘dŚĞƌĞĨŽƌĞ͕ƚŚĞƌĞŝƐĂŶĞĞĚĨŽƌǀĂĐĐŝŶĞĨŽƌŵƵůĂƟŽŶƐ} ƚŚĂƚŝŶĚƵĐĞƚŽůĞƌŽŐĞŶŝĐƐĂŶĚƉŽϭϬϬͲƐƉĞĐŝĮĐdƌĞŐƐin vivo. >ŝƉŽƐŽŵĞƐĂƌĞĞƐƚĂďůŝƐŚĞĚĚĞůŝǀĞƌǇǀĞŚŝĐůĞƐǁŚŝĐŚĐĂŶĚĞůŝǀĞƌĂĚƌƵŐŽƌĂŶƟŐĞŶ directly into the cell34_{͘ DŽƌĞŽǀĞƌ͕ ƚŚĞŝƌ ƉƌŽƉĞƌƟĞƐ ĂŶĚ ĐŽŶƚĞŶƚƐ ĐĂŶ ďĞ ĮŶĞͲƚƵŶĞĚ ƚŽ}

the therapy. For instance, it has been shown that anionic liposomes composed of ƉŚŽƐƉŚĂƟĚǇůƐĞƌŝŶĞ;W^ͿƌĞƐĞŵďůĞĂƉŽƉƚŽƟĐĐĞůůƐ;W^ďĞĐŽŵĞƐĞǆƉŽƐĞĚŽŶƚŚĞƐƵƌĨĂĐĞ ŽĨ ĂƉŽƉƚŽƟĐ ĐĞůůƐ35_{Ϳ͘ dŚƌŽƵŐŚ ^ZͲŵĞĚŝĂƚĞĚ ƵƉƚĂŬĞ ďǇ ĂŶƟŐĞŶͲƉƌĞƐĞŶƟŶŐ ĐĞůůƐ͕ W^Ͳ}

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&ŝŶĞƚĞŬ ;͕ h^Ϳ͘ &Žƌ ŝŵŵƵŶŽŚŝƐƚŽĐŚĞŵŝĐĂů ƐƚĂŝŶŝŶŐ͕ ,ĞŵĂƚŽǆǇůŝŶ͕ KŝůͲZĞĚͲK͕ ^ŝƌŝƵƐ ZĞĚ͕ĂŶĚĂŶƟͲƌĂƚ/Ő';ǁŚŽůĞŵŽůĞĐƵůĞͿʹĂůŬĂůŝŶĞƉŚŽƐƉŚĂƚĂƐĞĂŶƟďŽĚǇƉƌŽĚƵĐĞĚŝŶŐŽĂƚ ǁĞƌĞ ƉƵƌĐŚĂƐĞĚ ĨƌŽŵ ^ŝŐŵĂͲůĚƌŝĐŚ ;ǁŝũŶĚƌĞĐŚƚ͕ ƚŚĞ EĞƚŚĞƌůĂŶĚƐͿ͕ ƌĂƚ ĂŶƟͲŵŽƵƐĞ ŵĂĐƌŽƉŚĂŐĞƐͬŵŽŶŽĐǇƚĞƐĂŶƟďŽĚǇ;DKDϮͿǁĂƐƉƵƌĐŚĂƐĞĚĨƌŽŵŝŽͲZĂĚ;sĞĞŶĞŶĚĂĂů͕ ƚŚĞEĞƚŚĞƌůĂŶĚƐͿ͘ϱͲďƌŽŵŽͲϰͲĐŚůŽƌŽͲϯͲŝŶĚŽůǇůƉŚŽƐƉŚĂƚĞ;/WͿͬŶŝƚƌŽďůƵĞƚĞƚƌĂǌŽůŝƵŵ ;EdͿƐƵďƐƚƌĂƚĞƐǇƐƚĞŵďƵīĞƌǁĂƐƉƵƌĐŚĂƐĞĚĨƌŽŵ<K;ŐŝůĞŶƚ͕͕h^Ϳ͘ ŶŝŵĂůƐ ϱϳ>ͬϲ͕ KdͲ// ƚƌĂŶƐŐĞŶŝĐ͕ dͲĐĞůů ŝŵŵƵŶŽŐůŽďƵůŝŶͲ ĂŶĚ ŵƵĐŝŶͲĚŽŵĂŝŶͲĐŽŶƚĂŝŶŝŶŐ ŵŽůĞĐƵůĞ ϰ ;d/DϰͿͲͬͲ_{and LDLr}ͲͬͲ_{ŵŝĐĞ ŽŶ Ă ϱϳ>ͬϲ ďĂĐŬŐƌŽƵŶĚ ǁĞƌĞ ƉƵƌĐŚĂƐĞĚ ĨƌŽŵ} :ĂĐŬƐŽŶ>ĂďŽƌĂƚŽƌǇ;͕h^Ϳ͕ďƌĞĚŝŶͲŚŽƵƐĞƵŶĚĞƌƐƚĂŶĚĂƌĚůĂďŽƌĂƚŽƌǇĐŽŶĚŝƟŽŶƐ͕ĂŶĚ provided with food and water ad libitum. LDLrͲͬͲ_{ŵŝĐĞǁĞƌĞĨĞĚĂtĞƐƚĞƌŶͲƚǇƉĞĚŝĞƚ;tdͿ}

ĐŽŶƚĂŝŶŝŶŐϬ͘ϮϱйĐŚŽůĞƐƚĞƌŽůĂŶĚϭϱйĐŽĐŽĂďƵƩĞƌ;^ƉĞĐŝĂůŝĞƚ^ĞƌǀŝĐĞƐ͕ƐƐĞǆ͕h<Ϳ͘ůů animal work was performed in compliance with the Dutch government guidelines and ƚŚĞŝƌĞĐƟǀĞϮϬϭϬͬϲϯͬhŽĨƚŚĞƵƌŽƉĞĂŶWĂƌůŝĂŵĞŶƚ͘ǆƉĞƌŝŵĞŶƚƐǁĞƌĞĂƉƉƌŽǀĞĚďǇ ƚŚĞƚŚŝĐƐŽŵŵŝƩĞĞĨŽƌŶŝŵĂůǆƉĞƌŝŵĞŶƚƐŽĨ>ĞŝĚĞŶhŶŝǀĞƌƐŝƚǇ͘ ŽŶĞŵĂƌƌŽǁͲĚĞƌŝǀĞĚĚĞŶĚƌŝƟĐĐĞůůƐ;DƐͿ ŽŶĞ ŵĂƌƌŽǁ ǁĂƐ ŝƐŽůĂƚĞĚ ĨƌŽŵ ƚŚĞ ƟďŝĂƐ ĂŶĚ ĨĞŵƵƌƐ ŽĨ ϱϳ>ͬϲ Žƌ d/DϰͲͬͲ_{mice. A} ƐŝŶŐůĞͲĐĞůůƐƵƐƉĞŶƐŝŽŶŽĨďŽŶĞŵĂƌƌŽǁĐĞůůƐǁĂƐŽďƚĂŝŶĞĚďǇƵƐŝŶŐĂϳϬͲђŵĐĞůůƐƚƌĂŝŶĞƌ ;'ƌĞŝŶĞƌŝŽͲKŶĞ͘s͕͘ůƉŚĞŶĂĂŶĚĞŶZŝũŶ͕E>Ϳ͘dŚĞĐĞůůƐǁĞƌĞĐƵůƚƵƌĞĚŝŶ/DD;>ŽŶǌĂͿ ƐƵƉƉůĞŵĞŶƚĞĚǁŝƚŚϮŵD>ͲŐůƵƚĂŵŝŶĞ͕ϴй;ǀͬǀͿ&^͕ϭϬϬhͬŵ>ƉĞŶŝĐŝůůŝŶͬƐƚƌĞƉƚŽŵǇĐŝŶ ;>ŽŶǌĂͿ͕ĂŶĚϱϬђDɴͲŵĞƌĐĂƉƚŽĞƚŚĂŶŽů;^ŝŐŵĂͿĂƚϯϳȗĂŶĚϱйK₂ŝŶϵϱŵŵWĞƚƌŝĚŝƐŚĞƐ ;'ƌĞŝŶĞƌŝŽͲKŶĞ͘s͕͘ůƉŚĞŶĂĂŶĚĞŶZŝũŶ͕E>ͿĂŶĚϮϬŶŐͬŵ>'DͲ^&;WĞƉƌŽdĞĐŚͿĨŽƌ 10 days. Medium was refreshed every other day.

/ŵŵƵŶŽƉƌĞĐŝƉŝƚĂƟŽŶ DƐ ǁĞƌĞ ŝŶĐƵďĂƚĞĚ ǁŝƚŚ ϭϬй ƐĞƌƵŵ ĨƌŽŵ >>ƌͲͬͲ_{ŵŝĐĞ ŽŶ Ă td ĂĐƟǀĂƚĞĚ ǁŝƚŚ} Ϭ͘ϭ ђŐͬŵ> >W^͘ ĸŶŝƚǇͲƉƵƌŝĮĐĂƟŽŶ ŽĨ D,Ͳ// ;/ͲďͿ ŵŽůĞĐƵůĞƐ ĨƌŽŵ DƐ ĂŶĚ ƐƵďƐĞƋƵĞŶƚ ƉĞƉƟĚĞ ĞůƵƟŽŶ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ĂƐ ĚĞƐĐƌŝďĞĚ ƉƌĞǀŝŽƵƐůǇ41_{͘ ƉƉƌŽǆŝŵĂƚĞůǇ} ϱϬǆϭϬ6_{DƐǁĞƌĞůǇƐĞĚŝŶϬ͘ϱŵ>ůǇƐŝƐďƵīĞƌ;ϱϬŵDdƌŝƐͲůƉ,ϴ͘Ϭ͕ϭϱϬŵDEĂů͕} ϱŵDƚŚǇůĞŶĞĚŝĂŵŝŶĞƚĞƚƌĂĂĐĞƟĐĂĐŝĚ;dͿ͕Ϭ͘ϱйǁŝƩĞƌŐĞŶƚϯͲϭϮ;EͲĚŽĚĞĐǇůͲE͕EͲ ĚŝŵĞƚŚǇůͲϯͲĂŵŵŽŶŝŽͲϭͲƉƌŽƉĂŶĞƐƵůĨŽŶĂƚĞͿ ĂŶĚ ƉƌŽƚĞĂƐĞ ŝŶŚŝďŝƚŽƌ ;ŽŵƉůĞƚĞ͕ ZŽĐŚĞ ƉƉůŝĞĚ^ĐŝĞŶĐĞͿͿĨŽƌϮŚŽƵƌƐĂƚϬΣ41_{. Lysates were centrifuged for 10 min at 2500 × g}

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4

ĞǆƉĞƌŝŵĞŶƚƐǁŝƚŚŝŶϮǁĞĞŬƐ͘ >ŝƉŽƐŽŵĞĐŚĂƌĂĐƚĞƌŝǌĂƟŽŶ dŚĞ ͲĂǀĞƌĂŐĞ ĚŝĂŵĞƚĞƌ ;_aveͿ ĂŶĚ ƉŽůǇĚŝƐƉĞƌƐŝƚǇ ŝŶĚĞǆ ;W/Ϳ ŽĨ ƚŚĞ ůŝƉŽƐŽŵĞƐ ǁĞƌĞ ŵĞĂƐƵƌĞĚ ďǇ ĚǇŶĂŵŝĐ ůŝŐŚƚ ƐĐĂƩĞƌŝŶŐ ;>^Ϳ ƵƐŝŶŐ Ă EĂŶŽ^ ĞƚĂƐŝǌĞƌ ;DĂůǀĞƌŶ >ƚĚ͕͘ DĂůǀĞƌŶ͕ h<Ϳ͘ ĞƚĂͲƉŽƚĞŶƟĂů ǁĂƐ ĚĞƚĞƌŵŝŶĞĚ ďǇ ƵƐŝŶŐ ůĂƐĞƌ ŽƉƉůĞƌ ĞůĞĐƚƌŽƉŚŽƌĞƐŝƐ using the same instrument. For measurements, the liposomes were diluted 100-fold ŝŶ ϭϬ ŵD ƉŚŽƐƉŚĂƚĞ ďƵīĞƌ Ăƚ Ɖ, ϳ͘ϰ ƚŽ Ă ƚŽƚĂů ǀŽůƵŵĞ ŽĨ ϭ ŵ>͘ dŽ ĚĞƚĞƌŵŝŶĞ ƚŚĞ ĐŽŶĐĞŶƚƌĂƟŽŶŽĨůŽĂĚĞĚKsϯϮϯ͕ƚŚĞƉĞƉƟĚĞǁĂƐƐĞƉĂƌĂƚĞĚĨƌŽŵůŝƉŽƐŽŵĞƐďǇƵƐŝŶŐ Ă ŵŽĚŝĮĞĚ ůŝŐŚͲǇĞƌ ŵĞƚŚŽĚ͕ ĂƐ ĚĞƐĐƌŝďĞĚ ƉƌĞǀŝŽƵƐůǇ42_{͘ ƌŝĞŇǇ͕ ϭϬϬ ђ> ŽĨ ĂƋƵĞŽƵƐ} ůŝƉŽƐŽŵĂů ĚŝƐƉĞƌƐŝŽŶ Žƌ Ă ŬŶŽǁŶ ĐŽŶĐĞŶƚƌĂƟŽŶ ŽĨ ĨƌĞĞ ƉĞƉƟĚĞ ĂƐ ĐŽŶƚƌŽů ǁĂƐ ŵŝǆĞĚ ǁŝƚŚ ϮϱϬ ђ> ŵĞƚŚĂŶŽů ĂŶĚ ϭϮϱ ђ> ĐŚůŽƌŽĨŽƌŵ ĂŶĚ ǀŽƌƚĞǆĞĚ ďƌŝĞŇǇ͘ ϮϱϬ ђ> ŽĨ Ϭ͘ϭ D ,ůĂŶĚϭϮϱĐŚůŽƌŽĨŽƌŵǁĂƐĂĚĚĞĚĂŶĚƚŚĞŵŝǆƚƵƌĞǁĂƐǀŽƌƚĞǆĞĚĂŐĂŝŶ͘dŚŝƐǁĂƐƚŚĞŶ ĐĞŶƚƌŝĨƵŐĞĚĨŽƌϱŵŝŶĂƚϭϱϬϬƌƉŵƚŽƐĞƉĂƌĂƚĞƚŚĞǁĂƚĞƌͲŵĞƚŚĂŶŽůƉŚĂƐĞ;ĐŽŶƚĂŝŶŝŶŐƚŚĞ ƉĞƉƟĚĞͿ ĨƌŽŵ ƚŚĞ ĐŚůŽƌŽĨŽƌŵ ƉŚĂƐĞ͘ dŚĞ ƵƉƉĞƌ ǁĂƚĞƌͲŵĞƚŚĂŶŽů ƉŚĂƐĞ ǁĂƐ ĐŽůůĞĐƚĞĚ ĂŶĚĂŶĂůǇǌĞĚďǇƌĞǀĞƌƐĞĚƉŚĂƐĞhW>;tĂƚĞƌƐYh/dzhW>͕tĂƚĞƌƐ͕D͕h^Ϳ͘&Žƌ ƚŚŝƐ͕ϱђ>ŽĨƚŚĞƐĂŵƉůĞǁĂƐŝŶũĞĐƚĞĚŝŶƚŽĂϭ͘ϳђŵ,ϭϴĐŽůƵŵŶ;Ϯ͘ϭǆϱϬŵŵ͕tĂƚĞƌƐ Yh/dzhW>͕tĂƚĞƌƐ͕D͕h^Ϳ͘dŚĞĐŽůƵŵŶƚĞŵƉĞƌĂƚƵƌĞĂŶĚƚŚĞƚĞŵƉĞƌĂƚƵƌĞŽĨƚŚĞ ƐĂŵƉůĞ ǁĞƌĞ ƐĞƚ Ăƚ ϰϬΣ ĂŶĚ ϰΣ ƌĞƐƉĞĐƟǀĞůǇ͘ dŚĞ ŵŽďŝůĞ ƉŚĂƐĞƐ ǁĞƌĞ DŝůůŝͲY ǁĂƚĞƌ ǁŝƚŚϬ͘ϭйd&;ƐŽůǀĞŶƚͿĂŶĚĂĐĞƚŽŶŝƚƌŝůĞǁŝƚŚϬ͘ϭйd&;ƐŽůǀĞŶƚͿ͘&ŽƌĚĞƚĞĐƟŽŶ͕ƚŚĞ ŵŽďŝůĞƉŚĂƐĞƐǁĞƌĞĂƉƉůŝĞĚŝŶĂůŝŶĞĂƌŐƌĂĚŝĞŶƚĨƌŽŵϱйƚŽϵϱйƐŽůǀĞŶƚŽǀĞƌϱŵŝŶƵƚĞƐ ĂƚĂŇŽǁƌĂƚĞŽĨϬ͘ϯϳϬŵ>ͬŵŝŶ͘WĞƉƟĚĞƐǁĞƌĞĚĞƚĞĐƚĞĚďǇĂďƐŽƌďĂŶĐĞĂƚϮϮϬŶŵƵƐŝŶŐ ĂŶYh/dzhW>dhsĚĞƚĞĐƚŽƌ;tĂƚĞƌƐYh/dzhW>͕tĂƚĞƌƐ͕D͕h^Ϳ͘ WƌŽƚĞŝŶĐŽƌŽŶĂĂŶĂůǇƐŝƐ dŽĐŚĂƌĂĐƚĞƌŝǌĞƚŚĞĨŽƌŵĂƟŽŶŽĨĂƉƌŽƚĞŝŶĐŽƌŽŶĂŽŶůŝƉŽƐŽŵĞƐ͕ůŝƉŽƐŽŵĞƐǁĞƌĞĚŝůƵƚĞĚ ƚŽĂůŝƉŝĚĐŽŶĐĞŶƚƌĂƟŽŶŽĨϬ͘ϭŵŐͬŵ>ĂŶĚŝŶĐƵďĂƚĞĚĨŽƌϭŚŽƵƌĂƚϯϳΣǁŝƚŚ&^ŽƌϭϬђŐͬ ŵ>ϭƋŝŶW͘>ŝƉŽƐŽŵĞƐǁĞƌĞǁĂƐŚĞĚƚŚƌĞĞƟŵĞƐĂŶĚĐŽŶĐĞŶƚƌĂƚĞĚǁŝƚŚĂsŝǀĂƐƉŝŶϱϬϬ ĐĞŶƚƌŝĨƵŐĞŵĞŵďƌĂŶĞĐŽŶĐĞŶƚƌĂƚŽƌ;DtKϭ͕ϬϬϬŬĂ͕^ĂƌƚŽƌŝƵƐ͕'ŽĞƫŶŐĞŶ͕'ĞƌŵĂŶǇͿ to remove unbound proteins, leaving the ‘hard’ protein corona47_{͘ ^ŝǌĞ͕ W/͕ ĂŶĚ ǌĞƚĂͲ}

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ŶĂůǇƐŝƐŽĨĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶŵŝĐĞ

ŝŐŚƚͲƚŽϭϰͲǁĞĞŬͲŽůĚŵĂůĞ>>ƌͲͬͲ_{mice were randomized into 3 groups of 9 mice. Mice}

ǁĞƌĞ ĨĞĚ Ă td ĨŽƌ ϭϬ ǁĞĞŬƐ ƚŽ ŝŶĚƵĐĞ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ ƵƌŝŶŐ ƚŚŝƐ ƟŵĞ͕ ŵŝĐĞ ǁĞƌĞ ŝŵŵƵŶŝǌĞĚĂƚǁĞĞŬϬ͕ϯ͕ϲĂŶĚϵǀŝĂŝ͘Ɖ͘ŝŶũĞĐƟŽŶǁŝƚŚĞŝƚŚĞƌW^͕ϭϬŶŵŽůŽĨĨƌĞĞƉϯϱϬϬ ƉĞƉƟĚĞŝŶW^͕Ϭ͘ϱŵŐ^W'ͲůŝƉŽƐŽŵĞƐŝŶW^͕ŽƌϬ͘ϱŵŐ^W'ͲůŝƉŽƐŽŵĞƐĐŽŶƚĂŝŶŝŶŐϭϬ ŶŵŽůŽĨƉϯϱϬϬŝŶW^͕ŝŶĂƚŽƚĂůǀŽůƵŵĞŽĨϮϬϬђ>͘ŌĞƌϭϬǁĞĞŬƐ͕ŵŝĐĞǁĞƌĞĞƵƚŚĂŶŝǌĞĚ ďǇ Ă ƐƵďĐƵƚĂŶĞŽƵƐ ŝŶũĞĐƟŽŶ ;ϭϮϬ ʅ>Ϳ ŽĨ Ă ĐŽĐŬƚĂŝů ĐŽŶƚĂŝŶŝŶŐ ŬĞƚĂŵŝŶĞ ;ϰϬ ŵŐͬŵ>Ϳ͕ ĂƚƌŽƉŝŶĞ ;ϱϬ ʅŐͬŵ>Ϳ͕ ĂŶĚ ƐĞĚĂǌŝŶĞ ;ϲ͘Ϯϱ ŵŐͬŵ>Ϳ͘ DŝĐĞ ǁĞƌĞ ĞǆƐĂŶŐƵŝŶĂƚĞĚ ĂŶĚ ƉĞƌĨƵƐĞĚǁŝƚŚW^͘&ŽƌŇŽǁĐǇƚŽŵĞƚƌǇ͕ĂŽƌƚĂƐǁĞƌĞŚĂƌǀĞƐƚĞĚĂŶĚĐƵƚŝŶƚŽƐŵĂůůƉŝĞĐĞƐ͘ dŚĞƐĞǁĞƌĞƚŚĞŶŝŶĐƵďĂƚĞĚĨŽƌϯϬŵŝŶĂƚϯϳΣǁŝƚŚϰϱϬhͬŵ>ĐŽůůĂŐĞŶĂƐĞ/͕ϮϱϬhͬŵ> ĐŽůůĂŐĞŶĂƐĞy/͕ϭϮϬhͬŵ>EƐĞ͕ĂŶĚϭϮϬhͬŵ>ŚǇĂůƵƌŽŶŝĚĂƐĞ͕ĂŶĚƐƚƌĂŝŶĞĚƚŚƌŽƵŐŚĂ ϳϬͲђŵĐĞůůƐƚƌĂŝŶĞƌƚŽŽďƚĂŝŶĂƐŝŶŐůĞͲĐĞůůƐƵƐƉĞŶƐŝŽŶ͘ĞůůƐǁĞƌĞƐƚĂŝŶĞĚĨŽƌdŚǇϭ͘Ϯ͕ϰ͕ ϴĂŶĚǀŝĂďŝůŝƚǇ͘,ĞĂƌƚƐǁĞƌĞŚĂƌǀĞƐƚĞĚĂŶĚĮǆĞĚĨƌŽǌĞŶŝŶKdĨŽƌŵƵůĂƟŽŶĂƚͲϴϬΣ͘ ,ĞĂƌƚƐǁĞƌĞƐƵďƐĞƋƵĞŶƚůǇĐƌǇŽƐĞĐƟŽŶĞĚŚŽƌŝǌŽŶƚĂůůǇƚŽƚŚĞĂŽƌƟĐĂǆŝƐĂŶĚƚŽǁĂƌĚƐƚŚĞ ĂŽƌƟĐĂƌĐŚ͘hƉŽŶŝĚĞŶƟĮĐĂƟŽŶŽĨƚŚĞĂŽƌƟĐƌŽŽƚ͕ĚĞĮŶĞĚďǇƚŚĞƚƌŝǀĂůǀĞůĞĂŇĞƚƐ͕ϭϬʅŵ ƐĞĐƟŽŶƐǁĞƌĞĐŽůůĞĐƚĞĚ͘^ĞĐƟŽŶƐǁĞƌĞƐƚĂŝŶĞĚĨŽƌKŝůͲZĞĚͲKĂƐƉƌĞǀŝŽƵƐůǇĚĞƐĐƌŝďĞĚ48 ƚŽǀŝƐƵĂůŝǌĞůŝƉŝĚͲƌŝĐŚƉůĂƋƵĞƐ͘DĂĐƌŽƉŚĂŐĞƐŝŶƚŚĞƉůĂƋƵĞƐǁĞƌĞƐƚĂŝŶĞĚƵƐŝŶŐDKDϮ staining as previously described49_{͘ŽůůĂŐĞŶŝŶƚŚĞƉůĂƋƵĞƐǁĂƐƐƚĂŝŶĞĚƵƐŝŶŐ^ŝƌŝƵƐZĞĚ}

staining, as previously described50_{. All stainings were analyzed by microscopy using Leica}

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^W'ͲůŝƉŽƐŽŵĞƐŝŶĚƵĐĞĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƐin vivo

dŽƵŶĐŽǀĞƌƚŚĞƚǇƉĞŽĨKsϯϮϯͲƐƉĞĐŝĮĐϰ+_{T cells induced by the liposomes}

ŝŶ ŵŝĐĞ͕ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ ;ϰϱ͘ϭ+_ϰ+_Ϯϱ+_&KyWϯ+_{Ϳ dƌĞŐƐ ǁĞƌĞ ŵĞĂƐƵƌĞĚ ďǇ ŇŽǁ} ĐǇƚŽŵĞƚƌǇĂŌĞƌŝŵŵƵŶŝǌĂƟŽŶ;&ŝŐƵƌĞϮ͕͕ĂŶĚͿ͘tĞĨŽƵŶĚĐŽŵƉĂƌĂďůĞƉĞƌĐĞŶƚĂŐĞƐ ŽĨŶŽŶͲƐƉĞĐŝĮĐdƌĞŐƉŽƉƵůĂƟŽŶƐ;ϰϱ͘ϭ-_ϰ+_Ϯϱ+_&KyWϯ+_{ͿŝŶĂůůŵŝĐĞ;&ŝŐƵƌĞ^ϯͿ͘^W'Ͳ} ůŝƉŽƐŽŵĞƐ ĞŶĐĂƉƐƵůĂƟŶŐ KsϯϮϯ ŝŶĚƵĐĞĚ ƚŚĞ ŚŝŐŚĞƐƚ ƉĞƌĐĞŶƚĂŐĞ ŽĨ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ dƌĞŐƐ͕ǁŚŝĐŚǁĂƐƐŝŐŶŝĮĐĂŶƚůǇŚŝŐŚĞƌƚŚĂŶƚŚĞďĂĐŬŐƌŽƵŶĚdƌĞŐƌĞƐƉŽŶƐĞĂŌĞƌŝŶũĞĐƟŽŶ ŽĨĨƌĞĞKsϯϮϯ͘^ƵƌƉƌŝƐŝŶŐůǇ͕WW^ͲůŝƉŽƐŽŵĞƐǁĞƌĞŶŽƚĂƐĞĸĐŝĞŶƚĂƚdƌĞŐŝŶĚƵĐƟŽŶĂƐ ^W'ͲůŝƉŽƐŽŵĞƐ͕ďƵƚƚŚĞǇĚŝĚƐŚŽǁĂŶŽŶͲƐŝŐŶŝĮĐĂŶƚŝŶĐƌĞĂƐĞŝŶdƌĞŐƐĐŽŵƉĂƌĞĚƚŽĨƌĞĞ KsϯϮϯ͘WdWͲůŝƉŽƐŽŵĞƐĚŝĚŶŽƚĂůƚĞƌdƌĞŐƌĞƐƉŽŶƐĞƐĂƐĐŽŵƉĂƌĞĚƚŽƚŚĞďĂĐŬŐƌŽƵŶĚ͘ tĞĂůƐŽŵĞĂƐƵƌĞĚĂŶƟŐĞŶͲƐƉĞĐŝĮĐŝŶƚƌĂĐĞůůƵůĂƌĐǇƚŽŬŝŶĞƌĞƐƉŽŶƐĞƐďǇŇŽǁĐǇƚŽŵĞƚƌǇ ĂŌĞƌƌĞƐƟŵƵůĂƟŽŶǁŝƚŚWDĂŶĚŝŽŶŽŵǇĐŝŶ;&ŝŐƵƌĞϮ͕͕&͕ĂŶĚ'Ϳ͘WdWͲůŝƉŽƐŽŵĞƐ ŐƌĞĂƚůǇ ŝŶĐƌĞĂƐĞĚ ƉƌŽͲŝŶŇĂŵŵĂƚŽƌǇ ĐǇƚŽŬŝŶĞ ƉƌŽĚƵĐƟŽŶ ;ŝŶƚĞƌĨĞƌŽŶ ;/&EͿͲɶ ĂŶĚ />ͲϭϳͿ ŽĨ ƚŚĞ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ ϰ+_{T cells, while both anionic liposomes induced almost no}

ƉƌŽĚƵĐƟŽŶŽĨƚŚĞƐĞĐǇƚŽŬŝŶĞƐ͘dŚĞƌĞǁĞƌĞŶŽĚŝīĞƌĞŶĐĞƐŝŶ/>ͲϰŽƌ/>ͲϭϬƉƌŽĚƵĐƟŽŶŝŶ any of the groups.

&ŝŐƵƌĞϮ͗/ŶĚƵĐƟŽŶŽĨKsϯϮϯͲƐƉĞĐŝĮĐŝŵŵƵŶĞƌĞƐƉŽŶƐĞƐŝŶŝ>EƐĂŶĚƐƉůĞĞŶƐŽĨŵŝĐĞϴĚĂǇƐ ĂŌĞƌŝ͘ǀ͘ŝŶũĞĐƟŽŶŽĨKsϯϮϯͲĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐ͘;ͿϮϱ+_&KyWϯ+_ϰ+_{dĐĞůůƐĨƌŽŵĂĚŽƉƟǀĞ}

ƚƌĂŶƐĨĞƌŽĨϰ+_{dĐĞůůƐĨƌŽŵĂŶKdͲ//ŵŽƵƐĞǁĞƌĞĚĞƚĞĐƚĞĚŝŶŝ>EƐŽĨtdŵŝĐĞĂŌĞƌŝŵŵƵŶŝǌĂƟŽŶ}

ǁŝƚŚ ĨƌĞĞ KsϯϮϯ͕ Žƌ ^W'Ͳ͕ WW^Ͳ Žƌ WdWͲůŝƉŽƐŽŵĞƐ ĐŽŶƚĂŝŶŝŶŐ KsϯϮϯ ƉĞƉƟĚĞ͘

ZĞƉƌĞƐĞŶƚĂƟǀĞŇŽǁĐǇƚŽŵĞƚƌǇƉůŽƚƐŽĨƉƌĞͲŐĂƚĞĚϰϱ͘ϭ+_ϰ+_{T-cells in the iLNs of a mouse 8}

ĚĂǇƐĂŌĞƌŝŵŵƵŶŝǌĂƟŽŶǁŝƚŚ;ͿWdW͕Žƌ;Ϳ^W'ůŝƉŽƐŽŵĞƐ͘^ƉůĞŶŽĐǇƚĞƐǁĞƌĞŝŶĐƵďĂƚĞĚĨŽƌϲ ŚŽƵƌƐǁŝƚŚWDнŝŽŶŽŵǇĐŝŶĂŶĚďƌĞĨĞůĚŝŶ͕ĂŶĚƐƵďƐĞƋƵĞŶƚůǇ͕;Ϳ/&Eɶ͕;Ϳ/>Ͳϭϳ͕;&Ϳ/>ͲϰĂŶĚ;'Ϳ

/>ͲϭϬƉƌŽĚƵĐƟŽŶďǇĂŶƟŐĞŶͲƐƉĞĐŝĮĐϰϱ͘ϭ+_ϰ+_{dĐĞůůƐǁĂƐĂŶĂůǇǌĞĚďǇŇŽǁĐǇƚŽŵĞƚƌǇ͘'ƌĂƉŚƐ}

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4

ŶŝŽŶŝĐůŝƉŽƐŽŵĞƐĂƩƌĂĐƚϭƋĨƌŽŵƐĞƌƵŵĂŶĚĂƌĞƚĂŬĞŶƵƉďǇ^ZƐ

tŚŝůĞ ďŽƚŚ ĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĂů ĨŽƌŵƵůĂƟŽŶƐ ŚĂĚ ƐŝŵŝůĂƌ ƉŚǇƐŝĐŽĐŚĞŵŝĐĂů ƉƌŽƉĞƌƟĞƐĂŶĚĐŽƵůĚŝŶĚƵĐĞdƌĞŐƐ͕^W'ͲĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐǁĞƌĞĐůĞĂƌůǇŵŽƌĞƉŽƚĞŶƚ͘ dŚĞĐĂƟŽŶŝĐWdWůŝƉŽƐŽŵĞƐŝŶĚƵĐĞĚŶŽdƌĞŐƐ͘tĞŚǇƉŽƚŚĞƐŝǌĞĚƚŚĂƚĂƉƌŽƚĞŝŶĐŽƌŽŶĂ ĂƌŽƵŶĚƚŚĞůŝƉŽƐŽŵĞƐĐŽƵůĚďĞƌĞƐƉŽŶƐŝďůĞĨŽƌƚŚĞĚŝīĞƌĞŶĐĞƐŝŶin vivo immunological ƌĞƐƉŽŶƐĞƐ͘ dŽ ĂƐƐĞƐƐ ƚŚĞ ĨŽƌŵĂƟŽŶ ŽĨ Ă ƉƌŽƚĞŝŶ ĐŽƌŽŶĂ͕ ǁĞ ŝŶĐƵďĂƚĞĚ ƚŚĞ ĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĞƐŝŶ&^ĨŽƌϭŚŽƵƌĂƚϯϳΣĂŶĚǁĂƐŚĞĚƚŚĞŵƚŽƌĞŵŽǀĞĂŶǇƵŶďŽƵŶĚƉƌŽƚĞŝŶƐ͕ ůĞĂǀŝŶŐƚŚĞ͚ŚĂƌĚ͛ƉƌŽƚĞŝŶĐŽƌŽŶĂ͘^^ͲW'ĂŶĂůǇƐŝƐƐŚŽǁĞĚƚŚĂƚƚŚĞůŝƉŽƐŽŵĞƐĂƩƌĂĐƚ ƉƌŽƚĞŝŶƐĨƌŽŵ&^;&ŝŐƵƌĞ^ϰͿ͘^ĞǀĞƌĂůƐĞƌƵŵƉƌŽƚĞŝŶƐĐĂŶďŝŶĚƚŽŶĂŶŽƉĂƌƟĐůĞƐ͕ŝŶĐůƵĚŝŶŐ complement proteins52_{͘KĨƚŚĞƐĞĐŽŵƉůĞŵĞŶƚƉƌŽƚĞŝŶƐ͕ϭƋŝƐĞƐƉĞĐŝĂůůǇŝŶƚĞƌĞƐƟŶŐƐŝŶĐĞ}

it can bind to various receptors53_{͘EŽƚĂďůǇ͕ďŝŶĚŝŶŐŽĨϭƋƚŽW^ŽŶĂƉŽƉƚŽƟĐĐĞůůƐůĞĂĚƐ}

ƚŽƌĞĐŽŐŶŝƟŽŶĂŶĚĐůĞĂƌĂŶĐĞďǇƉŚĂŐŽĐǇƟĐĐĞůůƐǀŝĂ^ZƐ54-56_{. To test whether the protein}

ĐŽƌŽŶĂŽƌϭƋŝƐŝŵƉŽƌƚĂŶƚĨŽƌƵƉƚĂŬĞŽĨůŝƉŽƐŽŵĞƐďǇDƐĂŶĚdƌĞŐŝŶĚƵĐƟŽŶ͕ǁĞ ŵĞĂƐƵƌĞĚƵƉƚĂŬĞŽĨŇƵŽƌĞƐĐĞŶƚůǇůĂďĞůĞĚůŝƉŽƐŽŵĞƐďǇDƐŝŶƚŚĞĂďƐĞŶĐĞŽĨƐĞƌƵŵ͕ ŽƌŝŶŵĞĚŝƵŵĐŽŶƚĂŝŶŝŶŐĞŝƚŚĞƌϴй&^ŽƌϭƋ͘tĞĂůƐŽĂƐƐĞƐƐĞĚKdͲ//&KyWϯ+_<ŝϲϳ+_ϰ+ dĐĞůůŝŶĚƵĐƟŽŶďǇƚŚĞƐĞDƐ͘ŽƚŚ^W'ͲĂŶĚWW^ͲůŝƉŽƐŽŵĞƐƐŚŽǁĞĚĂŶƵƉƚĂŬĞŽĨ ůĞƐƐƚŚĂŶϭϬйŝŶƚŚĞĂďƐĞŶĐĞŽĨ&^ŽƌϭƋ͕ŚŽǁĞǀĞƌ͕ƚŚĞƵƉƚĂŬĞŽĨWW^ͲůŝƉŽƐŽŵĞƐǁĂƐ ĂƌŽƵŶĚϭϬͲĨŽůĚůŽǁĞƌŝŶƚŚŝƐĐŽŶĚŝƟŽŶ;&ŝŐƵƌĞϯͿ͘ĚĚŝƟŽŶŽĨϭƋŽƌ&^ƐŝŐŶŝĮĐĂŶƚůǇ ŝŶĐƌĞĂƐĞĚƵƉƚĂŬĞŽĨďŽƚŚůŝƉŽƐŽŵĞƐ͕ǁŝƚŚ^W'ͲůŝƉŽƐŽŵĞƐƐŚŽǁŝŶŐƐŝŐŶŝĮĐĂŶƚůǇŚŝŐŚĞƌ ƵƉƚĂŬĞƚŚĂŶWW^ͲůŝƉŽƐŽŵĞƐŝŶŶŽƌŵĂůƐĞƌƵŵĐŽŶĚŝƟŽŶƐĂƚƚŚĞĐŽŶĐĞŶƚƌĂƟŽŶŽĨůŝƉŝĚƐ ǁĞĞǆĂŵŝŶĞĚ;&ŝŐƵƌĞϯͿ͘&Žƌ^W'ͲůŝƉŽƐŽŵĞƐ͕ĚĞƉůĞƟŽŶŽĨϭƋĨƌŽŵƐĞƌƵŵƐŝŐŶŝĮĐĂŶƚůǇ ƌĞĚƵĐĞĚƵƉƚĂŬĞ͕ǁŚŝĐŚǁĂƐƌĞŵĞĚŝĞĚďǇƌĞĐŽŶƐƟƚƵƟŶŐϭƋ;&ŝŐƵƌĞ^ϱͿ͘>ŝƉŽƐŽŵĞƐŝŶ ƚŚĞƉƌĞƐĞŶĐĞŽĨƐĞƌƵŵƐŝŐŶŝĮĐĂŶƚůǇŝŶĐƌĞĂƐĞĚdƌĞŐŝŶĚƵĐƟŽŶĐŽŵƉĂƌĞĚƚŽƐĞƌƵŵͲĨƌĞĞ ĐŽŶĚŝƟŽŶƐ;&ŝŐƵƌĞϯͿ͘dŚĞin vitro data supports the in vivoĮŶĚŝŶŐƐ͕ŝ͘Ğ͕͘ŝŶƚŚĞƉƌĞƐĞŶĐĞ ŽĨƐĞƌƵŵ͕ďŽƚŚůŝƉŽƐŽŵĞƐĞŶŚĂŶĐĞĚdƌĞŐŝŶĚƵĐƟŽŶĐŽŵƉĂƌĞĚƚŽĨƌĞĞKsϯϮϯ͕ĂŶĚ^W' ůŝƉŽƐŽŵĞƐǁĞƌĞŵŽƌĞƉŽƚĞŶƚ͘/ƚƐŚŽƵůĚďĞŶŽƚĞĚƚŚĂƚ͕ĂůƚŚŽƵŐŚƐĐĂŶƉƌŽĚƵĐĞϭƋ57_,

ƚŚĞ ƐŚŽƌƚ ŝŶĐƵďĂƟŽŶ ƟŵĞ ŽĨ ϰ ŚŽƵƌƐ ƐŚŽƵůĚ ŶŽƚ ĂůůŽǁ ĨŽƌ Ă ƐŝŐŶŝĮĐĂŶƚ ƉƌŽĚƵĐƟŽŶ ŽĨ ϭƋ͘tĞĂůƐŽĂƐƐĞƐƐĞĚǁŚĞƚŚĞƌd/Dϰ͕ĂŬŶŽǁŶƌĞĐĞƉƚŽƌĨŽƌĂƉŽƉƚŽƟĐĐĞůůƐƚŚĂƚƉůĂǇƐĂŶ important role in atherosclerosis58_{͕ƉůĂǇƐĂƌŽůĞŝŶŵĞĚŝĂƟŶŐƚŚĞƵƉƚĂŬĞŽĨŽƵƌůŝƉŽƐŽŵĞƐ}

ĂŶĚ ŝŶĚƵĐƟŽŶ ŽĨ dƌĞŐƐ͘ ,ŽǁĞǀĞƌ͕ d/DϰͲͬͲ_{DƐ ƐŚŽǁĞĚ ŶŽ ĚŝīĞƌĞŶĐĞƐ ŝŶ ůŝƉŽƐŽŵĞ}

(17)

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ŽĨ͞ͲƐĞƌƵŵ͕͟ŝŶĚŝĐĂƟŶŐƚŚĂƚ^ZƐĂƌĞƌĞƐƉŽŶƐŝďůĞĨŽƌƚŚĞƵƉƚĂŬĞŽĨŵŽƐƚŽĨƚŚĞůŝƉŽƐŽŵĞƐ ďǇDƐƵŶĚĞƌŶŽƌŵĂůƐĞƌƵŵĐŽŶĚŝƟŽŶƐ;&ŝŐƵƌĞϱͿ͘dŚĞĂĚĚŝƟŽŶŽĨƉŽůǇ/ĚŝĚŶŽƚĂůƚĞƌ ƚŚĞƵƉƚĂŬĞŽĨůŝƉŽƐŽŵĞƐŝŶƐĞƌƵŵͲĨƌĞĞĐŽŶĚŝƟŽŶƐ͕ƐƵŐŐĞƐƟŶŐƚŚĂƚďĂƌĞůŝƉŽƐŽŵĞƐĚŝĚ ŶŽƚŝŶƚĞƌĂĐƚǁŝƚŚ^ZƐ;&ŝŐƵƌĞϱͿ͘ &ŝŐƵƌĞ ϱ͗ īĞĐƚ ŽĨ ^Z ďůŽĐŬŝŶŐ ŽŶ in vitro ƵƉƚĂŬĞ ŽĨ ŇƵŽƌĞƐĐĞŶƚůǇ ůĂďĞůĞĚ ĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĞƐ ĞŶĐĂƉƐƵůĂƟŶŐKsϯϮϯďǇDƐ͘WĞƌĐĞŶƚĂŐĞŽĨƐǁŚŝĐŚŚĂǀĞƚĂŬĞŶƵƉŇƵŽƌĞƐĐĞŶƚůǇůĂďĞůĞĚ KsϯϮϯͲůŽĂĚĞĚ^W'ͲŽƌWW^ͲĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐĂŌĞƌϰŚŽƵƌƐŝŶĐƵďĂƟŽŶ͕ĂƐŵĞĂƐƵƌĞĚďǇ ŇŽǁĐǇƚŽŵĞƚƌǇ͘ĞůůƐǁĞƌĞŝŶĐƵďĂƚĞĚǁŝƚŚůŝƉŽƐŽŵĞƐŝŶ/DDƐƵƉƉůĞŵĞŶƚĞĚǁŝƚŚ&^ǁŝƚŚŽƵƚ ^ZďůŽĐŬŝŶŐ;ďůĂĐŬďĂƌƐͿŽƌǁŝƚŚ^ZďůŽĐŬŝŶŐƵƐŝŶŐϮϱϬђŐͬŵ>ƉŽůǇ/;ďůĂĐŬͬŐƌĂǇďĂƌƐͿ͕ŽƌŝŶƐĞƌƵŵͲ ĨƌĞĞ/DDǁŝƚŚŽƵƚƉŽůǇ/;ǁŚŝƚĞďĂƌƐͿŽƌǁŝƚŚƉŽůǇ/;ǁŚŝƚĞͬŐƌĂǇďĂƌƐͿ͘'ƌĂƉŚƐŚŽǁƐŵĞĂŶц^ ;ŶсϯͿ͕ΎΎΎΎƉфϬ͘ϬϬϬϭ͕ĐŽŵƉĂƌĞĚƚŽнƐĞƌƵŵǁŝƚŚŝŶůŝƉŽƐŽŵĞŐƌŽƵƉ͕ĚĞƚĞƌŵŝŶĞĚďǇƚǁŽͲǁĂǇ EKsĂŶĚŽŶĨĞƌƌŽŶŝ͛ƐŵƵůƟƉůĞĐŽŵƉĂƌŝƐŽŶƐƚĞƐƚƐ͘ ^W'ͲůŝƉŽƐŽŵĞƐĞŶĐĂƉƐƵůĂƟŶŐĂŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐͲƐƉĞĐŝĮĐƉĞƉƟĚĞƐŝŐŶŝĮĐĂŶƚůǇƌĞĚƵĐĞ ƉůĂƋƵĞĨŽƌŵĂƟŽŶĂŶĚŝŶĐƌĞĂƐĞƉůĂƋƵĞƐƚĂďŝůŝƚǇŝŶĂƚŚĞƌŽƐĐůĞƌŽƟĐŵŝĐĞ dŽůĞƌĂŶĐĞŝŶĚƵĐƟŽŶĂŐĂŝŶƐƚĂƚŚĞƌŽƐĐůĞƌŽƐŝƐƵƐŝŶŐƉĞƉƟĚĞƐƚĂƌŐĞƚĞĚĂŐĂŝŶƐƚƚŚĞ ŵĂŝŶ ĂŶƟŐĞŶ ŝŶ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͕ >>͕ ŚĂƐ ǇŝĞůĚĞĚ ƐŽŵĞ ƐƵĐĐĞƐƐ23-28_{͘ tĞ ŚǇƉŽƚŚĞƐŝǌĞĚ} ƚŚĂƚ ĞŶĐĂƉƐƵůĂƟŽŶ ŽĨ ĂŶ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐͲƐƉĞĐŝĮĐ ƉĞƉƟĚĞ ŝŶ ^W'ͲůŝƉŽƐŽŵĞƐ ǁŽƵůĚ ƌĞĚƵĐĞĂƚŚĞƌŽƐĐůĞƌŽƐŝƐƉƌŽŐƌĞƐƐŝŽŶŵŽƌĞĞĸĐŝĞŶƚůǇƚŚĂŶƚŚĞĨƌĞĞƉĞƉƟĚĞ͕ǀŝĂŝŶĚƵĐƟŽŶ ŽĨĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƐ͘dŚĞƉƌŽƚĞŝŶƐƵƌƌŽƵŶĚŝŶŐ>>͕ƉŽϭϬϬ͕ŝƐĂůĂƌŐĞƉƌŽƚĞŝŶ;ϱϭϱ ŬĂŝŶŚƵŵĂŶƐ͕ϱϬϵŬĂŝŶŵŝĐĞͿĐŽŶƚĂŝŶŝŶŐƐĞǀĞƌĂůƉŽƚĞŶƟĂůϰ+_{T cell epitopes. To} ŝĚĞŶƟĨǇ Ă ƌĞůĞǀĂŶƚ ƉŽϭϬϬ ƉĞƉƟĚĞ ĨŽƌ ŝŵŵƵŶŝǌĂƟŽŶ͕ ǁĞ ĞůƵƚĞĚ D,Ͳ// ƌĞƐƚƌŝĐƚĞĚ ƉĞƉƟĚĞƐ ĨƌŽŵ DƐ ĞǆƉŽƐĞĚ ƚŽ ŚǇƉĞƌĐŚŽůĞƐƚĞƌŽůĞŵŝĐ ƐĞƌƵŵ͘ tĞ ŝĚĞŶƟĮĞĚ ƐĞǀĞƌĂů ƉŽϭϬϬͲĚĞƌŝǀĞĚ ƉĞƉƟĚĞƐ ƵƐŝŶŐ ŽƵƌ ƉĞƉƟĚŽŵŝĐƐ ƐƚƌĂƚĞŐǇ ;dĂďůĞ ^ϭͿ͘ ĂƐĞĚ ŽŶ ƚŚĞ ƉƌĞĚŝĐƚĞĚ D,Ͳ// ďŝŶĚŝŶŐ ;dĂďůĞ ^ϮͿ ǁĞ ƐĞůĞĐƚĞĚ ƚŚĞ ƉĞƉƟĚĞ ƉŽϭϬϬ_3500-3514 ;ƉϯϱϬϬͿ ĂŶĚƐƵĐĐĞƐƐĨƵůůǇůŽĂĚĞĚŝƚŝŶƚŽ^W'ͲůŝƉŽƐŽŵĞƐ;dĂďůĞ^ϰͿ͘>>ƌͲͬͲ_{ŵŝĐĞŽŶĂtdǁĞƌĞ}

selected as a model for diet-induced atherosclerosis12_{͘dŚĞŵŝĐĞǁĞƌĞĨĞĚĂtdĨŽƌϭϬ}

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4

ůŝƉŽƐŽŵĞƐƐŝŐŶŝĮĐĂŶƚůǇƌĞĚƵĐĞĚƚŚĞůĞƐŝŽŶĂƌĞĂďǇϱϬй;&ŝŐƵƌĞϲĂŶĚͿ͘ƐĞǆƉĞĐƚĞĚ͕Ăůů ŵŝĐĞŐĂŝŶĞĚǁĞŝŐŚƚĚƵĞƚŽƚŚĞtd͕ďƵƚƚŚĞƌĞǁĞƌĞŶŽĚŝīĞƌĞŶĐĞƐďĞƚǁĞĞŶƚŚĞŐƌŽƵƉƐ ;&ŝŐƵƌĞϲͿ͘^ŝŵŝůĂƌůǇ͕ƐĞƌƵŵĐŚŽůĞƐƚĞƌŽůůĞǀĞůƐǁĞƌĞĞůĞǀĂƚĞĚŝŶĂůůŐƌŽƵƉƐďĞĐĂƵƐĞŽĨ ƚŚĞtd͘/ŶƚĞƌĞƐƟŶŐůǇ͕ŽŶůǇƚŚĞŐƌŽƵƉŽĨŵŝĐĞƚŚĂƚƌĞĐĞŝǀĞĚƚŚĞůŝƉŽƐŽŵĂůƚƌĞĂƚŵĞŶƚ ŚĂĚƐŝŐŶŝĮĐĂŶƚůǇůŽǁĞƌůĞǀĞůƐŽĨƐĞƌƵŵĐŚŽůĞƐƚĞƌŽůĐŽŵƉĂƌĞĚƚŽƚŚĞW^ĐŽŶƚƌŽůŐƌŽƵƉ ;&ŝŐƵƌĞϲͿ͘dŚĞĂŽƌƟĐƐĞĐƟŽŶƐǁĞƌĞĨƵƌƚŚĞƌƐƚĂŝŶĞĚĨŽƌŵĂĐƌŽƉŚĂŐĞĐŽŶƚĞŶƚ͕ǁŚŝĐŚŝƐĂŶ ŝŶĚŝĐĂƚŽƌŽĨŝŵŵƵŶĞĂĐƟǀĂƟŽŶ60_{͘ŝīĞƌĞŶĐĞƐŝŶŵĂĐƌŽƉŚĂŐĞĐŽŶƚĞŶƚďĞƚǁĞĞŶƚŚĞŐƌŽƵƉƐ} ǁĞƌĞ ŶŽƚ ƐŝŐŶŝĮĐĂŶƚ͕ ĂůƚŚŽƵŐŚ ƚŚĞƌĞ ǁĂƐ Ă ƚƌĞŶĚ ƚŽǁĂƌĚƐ ůŽǁĞƌ ŵĂĐƌŽƉŚĂŐĞ ĐŽŶƚĞŶƚ ŝŶ ƚŚĞ ŵŝĐĞ ŝŵŵƵŶŝǌĞĚ ǁŝƚŚ ůŝƉŽƐŽŵĞƐ ;&ŝŐƵƌĞ ϲ ĂŶĚ Ϳ͕ ǁŚŝĐŚ ĐŽƵůĚ ďĞ ;ƉĂƌƟĂůůǇͿ ƌĞƐƉŽŶƐŝďůĞĨŽƌƚŚĞƌĞĚƵĐƟŽŶŝŶƉůĂƋƵĞƐŝǌĞ͘&ƵƌƚŚĞƌŵŽƌĞ͕ƚŚĞƌĞǁĞƌĞƐŝŐŶŝĮĐĂŶƚůǇĨĞǁĞƌ ϴ+_{dĐĞůůƐƉƌĞƐĞŶƚŝŶƚŚĞĂŽƌƚĂŽĨŵŝĐĞŝŶũĞĐƚĞĚǁŝƚŚůŝƉŽƐŽŵĞƐ;&ŝŐƵƌĞ^ϳͿ͘>ĞǀĞůƐŽĨ}

ƚŚĞŝŶŇĂŵŵĂƚŽƌǇZϮ+_>ǇͲϲhi_{monocytes were unchanged in the blood of mice in all}

ŐƌŽƵƉƐ;&ŝŐƵƌĞ^ϴͿ͕ĨƵƌƚŚĞƌŝŶĚŝĐĂƟŶŐƚŚĂƚƚŚĞƌĞǁĂƐŶŽŝŶĐƌĞĂƐĞĚŝŶŇĂŵŵĂƟŽŶ͘&ŝŶĂůůǇ͕ the collagen content in the lesions was assessed, which is a measure of lesion stability51_.

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4

ŝƐĐƵƐƐŝŽŶΘŽŶĐůƵƐŝŽŶ

Atherosclerosis is the main underlying pathology for cardiovascular disease and is one of the leading causes of death worldwide1_{͘ tŚŝůĞ ǀĂĐĐŝŶĂƟŽŶ ĂŐĂŝŶƐƚ}

atherosclerosis has been successful in murine models23-28_{, a major challenge is the}

ŝŶĚƵĐƟŽŶŽĨĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƐŝŶĂƐĂĨĞĂŶĚĞīĞĐƟǀĞǁĂǇ͘,ĞƌĞǁĞŝŶƚƌŽĚƵĐĞ^W'Ͳ ůŝƉŽƐŽŵĞƐ ĂƐ Ă ƉĞƉƟĚĞ ĂŶƟŐĞŶ ĐĂƌƌŝĞƌ ƚŽ ŝŶĚƵĐĞ ƌĞŐƵůĂƚŽƌǇ dͲĐĞůůƐ ĂŶĚ ĂƐ Ă ƉŽƚĞŶƟĂů ǀĂĐĐŝŶĞ ĂŐĂŝŶƐƚ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ tŚĞƌĞĂƐ WdWͲůŝƉŽƐŽŵĞƐ ĐĂŶ ŝŶĚƵĐĞ ƐƚƌŽŶŐ ƉƌŽͲ ŝŶŇĂŵŵĂƚŽƌǇ ƌĞƐƉŽŶƐĞƐ͕ ǁĞ ŚǇƉŽƚŚĞƐŝǌĞĚ ƚŚĂƚ ^W'Ͳ ĂŶĚ WW^ͲůŝƉŽƐŽŵĞƐ ůĞĂĚ ƚŽ ŝŵŵƵŶĞƐƵƉƉƌĞƐƐŝŽŶďĞĐĂƵƐĞŽĨƚŚĞŝƌƐŝŵŝůĂƌŝƚǇƚŽĂƉŽƉƚŽƟĐĐĞůůƐ͘KsϯϮϯͲĐŽŶƚĂŝŶŝŶŐ liposomes were prepared with high-T_m lipids, since rigid liposomes have been shown ƚŽ ĞŶŚĂŶĐĞ W ƵƉƚĂŬĞ61_{ĂŶĚ ĂĐƟǀĂƟŽŶ}62_{, and would, therefore, be more potent at}

ŝŶĚƵĐŝŶŐdĐĞůůƌĞƐƉŽŶƐĞƐĐŽŵƉĂƌĞĚƚŽŇƵŝĚͲƐƚĂƚĞůŝƉŽƐŽŵĞƐ͘tĞƐŚŽǁƚŚĂƚĂůůůŝƉŽƐŽŵĞƐ ŝŶĚƵĐĞĚ ĞǆƉĂŶƐŝŽŶ ŽĨ KsϯϮϯͲƐƉĞĐŝĮĐ d ĐĞůůƐ in vivo͘ dŚĞ ĐĂƟŽŶŝĐ ůŝƉŽƐŽŵĞƐ ŝŶĚƵĐĞĚ ƉƌŽͲŝŶŇĂŵŵĂƚŽƌǇ ĐǇƚŽŬŝŶĞƐ͕ ǁŚŝĐŚ ǁĞ ŚĂǀĞ ĂůƐŽ ƉƌĞǀŝŽƵƐůǇ ŽďƐĞƌǀĞĚ42_{͘ KŶůǇ ^W'Ͳ} ĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐŝŶĚƵĐĞĚƐŝŐŶŝĮĐĂŶƚůǇŚŝŐŚĞƌŶƵŵďĞƌƐŽĨϮϱ+_&KyWϯ+_ϰ+_Tregs ĐŽŵƉĂƌĞĚ ƚŽ ĨƌĞĞ KsϯϮϯ ŝŶ ŵŝĐĞ͘ dŚŝƐ ǁĂƐ ƐƵƌƉƌŝƐŝŶŐ ƐŝŶĐĞ W^ͲĐŽŶƚĂŝŶŝŶŐ ůŝƉŽƐŽŵĞƐ ŚĂǀĞ ďĞĞŶ ƌĞƉŽƌƚĞĚ ƚŽ ŝŶĚƵĐĞ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ dƌĞŐƐ ŝŶ Ă ƚǇƉĞ / ĚŝĂďĞƚĞƐ ŵŽĚĞů36_{. In}

accordance with our study, however, IL-10 and IL-4 responses were also unchanged in the diabetes model36_{͘ŚĞĂĚͲƚŽͲŚĞĂĚĐŽŵƉĂƌŝƐŽŶŽĨƚŚĞĞīĞĐƚŽĨW^ŽƌW'ůŝƉŽƐŽŵĞƐ}

ĐŽŵƉůĞǆĞĚǁŝƚŚ&ĂĐƚŽƌs///;&s///Ϳin vitroƐŚŽǁĞĚƚŚĂƚW^ůŝƉŽƐŽŵĞƐƐŝŐŶŝĮĐĂŶƚůǇƌĞĚƵĐĞĚ ϴϲĂŶĚϰϬĞǆƉƌĞƐƐŝŽŶ͕ŝŵƉŽƌƚĂŶƚĐŽͲĂĐƟǀĂƟŶŐŵŽůĞĐƵůĞƐ͕ŝŶƐĂƐĐŽŵƉĂƌĞĚƚŽ ĨƌĞĞ&s///͕ǁŚŝůĞW'ůŝƉŽƐŽŵĞƐĚŝĚŶŽƚ63_{͘dŚŝƐƐƵƉƉŽƌƚƐŽƵƌĮŶĚŝŶŐƚŚĂƚ^W'ůŝƉŽƐŽŵĞƐ} ŚĂǀĞ Ă ŚŝŐŚĞƌ ƉŽƚĞŶĐǇ ƚŽ ĞǆƉĂŶĚ d ĐĞůůƐ͘ hŶĨŽƌƚƵŶĂƚĞůǇ͕ d ĐĞůů ƉƌŽůŝĨĞƌĂƟŽŶ ǁĂƐ ŽŶůǇ ŵĞĂƐƵƌĞĚĨŽƌW^ůŝƉŽƐŽŵĞƐ͕ĂŶĚdƌĞŐůĞǀĞůƐǁĞƌĞŶŽƚŵĞĂƐƵƌĞĚŝŶƚŚĞĂĨŽƌĞŵĞŶƟŽŶĞĚ study63_{͘ tĞ ƐŚŽǁ ƚŚĂƚ ^W'ͲůŝƉŽƐŽŵĞƐ ĂƌĞ ŵŽƌĞ ĞīĞĐƟǀĞůǇ ƚĂŬĞŶ ƵƉ ďǇ DƐ} in vitro ƚŚĂŶ WW^ͲůŝƉŽƐŽŵĞƐ͕ ǁŚŝĐŚ ĐŽƵůĚ ĞǆƉůĂŝŶ ƚŚĞŝƌ ŚŝŐŚĞƌ ƉŽƚĞŶĐǇ ƚŽ ŝŶĚƵĐĞ dƌĞŐ͘ ZĞŐĂƌĚůĞƐƐ͕ ƵƉƚĂŬĞ ŽĨ ďŽƚŚ ůŝƉŽƐŽŵĞƐ ǁĂƐ ůŽǁ ;ůĞƐƐ ƚŚĂŶ ϯϬйͿ͕ ǁŚŝĐŚ ŚĂƐ ďĞĞŶ ŽďƐĞƌǀĞĚƉƌĞǀŝŽƵƐůǇ͕ŵŽƐƚůŝŬĞůǇĚƵĞƚŽƵŶĨĂǀŽƌĂďůĞĞůĞĐƚƌŽƐƚĂƟĐŝŶƚĞƌĂĐƟŽŶƐǁŝƚŚƚŚĞ ŶĞŐĂƟǀĞůǇ ĐŚĂƌŐĞĚ ĐĞůů ƐƵƌĨĂĐ64_{. The mechanism of uptake could also be responsible}

ĨŽƌ ƚŚĞ ƉŽƚĞŶĐǇ ŽĨ ƚŚĞ ^W'ͲůŝƉŽƐŽŵĞƐ͘ In vitro and in vivo, liposomes interact with ƉƌŽƚĞŝŶƐ ŝŶ ƚŚĞ ƉŚǇƐŝŽůŽŐŝĐĂů ŵĞĚŝƵŵ͕ ƌĞƐƵůƟŶŐ ŝŶ ƚŚĞ ĨŽƌŵĂƟŽŶ ŽĨ Ă ƉƌŽƚĞŝŶ ĐŽƌŽŶĂ ĂƌŽƵŶĚ ƚŚĞ ůŝƉŽƐŽŵĞƐ͘ ĐĐŽƌĚŝŶŐůǇ͕ ǁĞ ŽďƐĞƌǀĞĚ ƚŚĂƚ ƉƌŽƚĞŝŶƐ ĨƌŽŵ ƐĞƌƵŵ ĂƩĂĐŚĞĚ ƚŽ ƚŚĞ ůŝƉŽƐŽŵĞƐ ĂŶĚ ƚŚĂƚ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ƐĞƌƵŵ ǁĂƐ ƌĞƋƵŝƌĞĚ ĨŽƌ ĞĸĐŝĞŶƚ ƵƉƚĂŬĞ ďǇDƐĂŶĚƐƵďƐĞƋƵĞŶƚdƌĞŐŝŶĚƵĐƟŽŶ͘dŚŝƐŝƐŝŶůŝŶĞǁŝƚŚŽƚŚĞƌƐƚƵĚŝĞƐƚŚĂƚŚĂǀĞ ƐŚŽǁŶ ƚŚĞ ƉƌŽƚĞŝŶ ĐŽƌŽŶĂ ƚŽ ďĞ ĞƐƐĞŶƟĂů ĨŽƌ ƚŚĞ ďŝŽůŽŐŝĐĂů ĨƵŶĐƟŽŶ ŽĨ ƉĂƌƟĐůĞƐ65_{. As} ŵĞŶƟŽŶĞĚĂďŽǀĞ͕ƐĞǀĞƌĂů^ZƐĐŽƵůĚďĞƌĞƐƉŽŶƐŝďůĞĨŽƌĂŶŝŽŶŝĐůŝƉŽƐŽŵĞƵƉƚĂŬĞ͕ĂŶĚ^ZͲ mediated uptake may lead to immune suppression66_{͘tĞĨŽƵŶĚĂƐŝŐŶŝĮĐĂŶƚƌĞĚƵĐƟŽŶ}

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via SRs54-56,72_{͕ ǁĞ ƚĞƐƚĞĚ ǁŚĞƚŚĞƌ ϭƋ ƉƌĞƐĞŶƚ ŝŶ ƚŚĞ ƉƌŽƚĞŝŶ ĐŽƌŽŶĂ ŽĨ ƚŚĞ ůŝƉŽƐŽŵĞƐ}

ǁĂƐƌĞƐƉŽŶƐŝďůĞĨŽƌ^ZͲŵĞĚŝĂƚĞĚƵƉƚĂŬĞĂŶĚdƌĞŐŝŶĚƵĐƟŽŶ͘ϭƋĨŽƌŵƐƉĂƌƚŽĨƚŚĞϭ ĐŽŵƉůĞǆƚŚĂƚŝƐƌĞƋƵŝƌĞĚĨŽƌƚƌŝŐŐĞƌŝŶŐŽĨƚŚĞĐůĂƐƐŝĐĂůĐŽŵƉůĞŵĞŶƚƉĂƚŚǁĂǇďƵƚĐĂŶĂůƐŽ regulate immunity53,73_{͘ŽŵƉůĞŵĞŶƚĂĐƟǀĂƟŽŶƐĞĞŵƐƚŽďĞĚĞƉĞŶĚĞŶƚŽŶƚŚĞƐƚƌƵĐƚƵƌĞ}

ŽĨ ϭƋ͖ ǁŚĞŶ ŝƚ ďŝŶĚƐ ƚŽ /Ő'ϭ͕ ϭƋ ŚĂƐ Ă ĚŝīĞƌĞŶƚ ĐŽŶĨŽƌŵĂƟŽŶ ƚŚĂŶ ǁŚĞŶ ŝƚ ďŝŶĚƐ ĚŝƌĞĐƚůǇƚŽW^ĞǆƉŽƐĞĚŽŶƚŚĞĐĞůůƐƵƌĨĂĐĞŽƌůŝƉŽƐŽŵĞƐ74_{. Moreover, there is evidence of}

ǀŝƌƵƐĞƐďŝŶĚŝŶŐϭƋĂƐĂďƌŝĚŐŝŶŐŵŽůĞĐƵůĞƚŽĞǀĂĚĞƚŚĞŝŵŵƵŶĞƐǇƐƚĞŵĂŶĚĞŶŚĂŶĐĞ ŝŶĨĞĐƟŽŶ75-77_{͘ϭƋĚĞĮĐŝĞŶĐǇ͕ĞŝƚŚĞƌŐĞŶĞƟĐ}78_{ŽƌǀŝĂĂŶƟͲϭƋĂƵƚŽĂŶƟďŽĚŝĞƐ}72_{, can lead}

ƚŽ ƐǇŵƉƚŽŵƐ ĂůŵŽƐƚ ŝĚĞŶƟĐĂů ƚŽ ƐǇƐƚĞŵŝĐ ůƵƉƵƐ ĞƌǇƚŚĞŵĂƚŽƐƵƐ ;^>Ϳ͘ ^ĞǀĞƌĂů ŽƚŚĞƌ ĂƵƚŽŝŵŵƵŶĞĚŝƐŽƌĚĞƌƐŚĂǀĞďĞĞŶĂƐƐŽĐŝĂƚĞĚǁŝƚŚĂĚǇƐƌĞŐƵůĂƟŽŶŽĨƚŚĞĐŽŵƉůĞŵĞŶƚ ƐǇƐƚĞŵĂŶĚƐƉĞĐŝĮĐĂůůǇϭƋĚĞĮĐŝĞŶĐŝĞƐ͕ŝŶĐůƵĚŝŶŐĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ79_{͘tĞƐŚŽǁƚŚĂƚϭƋŝƐ}

present in the protein corona and binds to anionic liposomes. This is in accordance with ŽƚŚĞƌƌĞƉŽƌƚƐŽĨϭƋďŝŶĚŝŶŐƚŽW^Ͳ70_{ĂŶĚW'ͲĐŽŶƚĂŝŶŝŶŐůŝƉŽƐŽŵĞƐ}80_{͘dŚĞĂĚĚŝƟŽŶŽĨϭƋ} ŝŶƐĞƌƵŵͲĨƌĞĞĐŽŶĚŝƟŽŶƐĐŽŵƉůĞƚĞůǇƌĞƐƚŽƌĞĚƚŚĞƵƉƚĂŬĞŽĨďŽƚŚW'ͲĂŶĚW^ͲůŝƉŽƐŽŵĞƐ͘ &ƵƌƚŚĞƌŵŽƌĞ͕ĚĞƉůĞƟŽŶŽĨϭƋƐŝŐŶŝĮĐĂŶƚůǇƌĞĚƵĐĞĚƵƉƚĂŬĞŽĨW'ͲůŝƉŽƐŽŵĞƐ͘dŚĞƌĞĨŽƌĞ͕ ĂŶŽƚŚĞƌĞǆƉůĂŶĂƟŽŶĨŽƌƚŚĞŚŝŐŚĞƌƉŽƚĞŶĐǇŽĨ^W'ͲůŝƉŽƐŽŵĞƐĐŽƵůĚďĞƚŚĂƚƚŚĞǇĂƩƌĂĐƚ ϭƋĨƌŽŵƚŚĞĐŝƌĐƵůĂƟŽŶŵŽƌĞĞĸĐŝĞŶƚůǇƚŚĂŶWW^ͲůŝƉŽƐŽŵĞƐ͘^ŝŶĐĞϭƋĐĂŶŶŽƚďŝŶĚ ƚŽĨƌĞĞW^ŽƌW'70_{͕ƚŚĞĚĞŶƐŝƚǇĂŶĚƌĞƉĞƟƟǀĞŶĞƐƐŽĨĂŶŝŽŶŝĐŚĞĂĚŐƌŽƵƉƐŽŶůŝƉŽƐŽŵĞƐ} ŵĂǇďĞĂŶŝŵƉŽƌƚĂŶƚƉĂƌĂŵĞƚĞƌƚŚĂƚĂīĞĐƚƐϭƋďŝŶĚŝŶŐ81_{͘dŚĞŵŽůĂƌƌĂƟŽƐŽĨƚŚĞůŝƉŝĚƐ} ƵƐĞĚŝŶƚŚŝƐƐƚƵĚǇǁĞƌĞŝĚĞŶƟĐĂů͕ƐŽƚŚŝƐǁŽƵůĚŶŽƚĂīĞĐƚďŝŶĚŝŶŐŽĨϭƋ͘/ƚŚĂƐĂůƐŽďĞĞŶ ƐƵŐŐĞƐƚĞĚƚŚĂƚƚŚĞĞůĞĐƚƌŽƐƚĂƟĐĐŚĂƌŐĞŽĨƚŚĞůŝƉŽƐŽŵĞƐŝƐĂŶŝŵƉŽƌƚĂŶƚƉĂƌĂŵĞƚĞƌĨŽƌ binding80,82_{, or that the chemical structure of the lipids is crucial}83_{. In this work, the}

zeta-ƉŽƚĞŶƟĂů ǁĂƐ ƚŚĞ ƐĂŵĞ ĨŽƌ ďŽƚŚ ůŝƉŽƐŽŵĂů ĨŽƌŵƵůĂƟŽŶƐ͕ ůĞĂǀŝŶŐ ƚŚĞ ƐƚƌƵĐƚƵƌĞ ŽĨ ƚŚĞ ůŝƉŝĚƐĂƐƚŚĞŽŶůǇĚŝīĞƌŝŶŐĨĂĐƚŽƌ͘ ^ŝŶĐĞƚŚĞƌĞǁĂƐĂĐůĞĂƌƌŽůĞĨŽƌϭƋŝŶƚŚĞƵƉƚĂŬĞŽĨƚŚĞůŝƉŽƐŽŵĞƐ͕ǁĞŚǇƉŽƚŚĞƐŝǌĞĚ ƚŚĂƚƚŚŝƐŵĂǇĂůƐŽŝŶŇƵĞŶĐĞdƌĞŐƐŬĞǁŝŶŐ͘tŚŝůĞǁĞĚŝĚŽďƐĞƌǀĞƚŚĂƚƚŚĞĂĚĚŝƟŽŶŽĨϭƋ ŝŶĐƌĞĂƐĞƐdƌĞŐƌĞƐƉŽŶƐĞƐĐŽŵƉĂƌĞĚƚŽƐĞƌƵŵͲĨƌĞĞĐŽŶĚŝƟŽŶƐ͕ƚŚŝƐǁĂƐŶŽƚƐŝŐŶŝĮĐĂŶƚ͘ ^ŝŵŝůĂƌůǇ͕ ůĂƌŬĞ et al. ƐŚŽǁĞĚ ƚŚĂƚ͕ ǁŚŝůĞ ϭƋ ƚŽůĞƌŝǌĞƐ ŵĂĐƌŽƉŚĂŐĞƐ ;ŝŶĐƌĞĂƐĞĚ WͲ >ϭĂŶĚWͲ>Ϯ͕ĚĞĐƌĞĂƐĞĚϰϬͿĂŶĚƐ;ŝŶĐƌĞĂƐĞĚWͲ>ϮĂŶĚĚĞĐƌĞĂƐĞĚϴϲͿ͕ƚŚĞƌĞ was only a trend towards higher Treg responses84_{͘ dŚŝƐ ƐƵŐŐĞƐƚƐ ƚŚĂƚ ϭƋ ŝƐ ƉĂƌƟĂůůǇ}

ƌĞƐƉŽŶƐŝďůĞĨŽƌƚŚĞdƌĞŐŝŶĚƵĐƟŽŶŽĨďŽƚŚ^W'ͲĂŶĚWW^ͲůŝƉŽƐŽŵĞƐ͕ďƵƚƚŚĞƉƌŽƚĞŝŶ corona likely contains more components that help to induce Tregs.

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4

ĚŽƐĞƚŽŝŶĚƵĐĞƚŚĞƐĂŵĞĂƚŚĞƌŽƉƌŽƚĞĐƟǀĞĞīĞĐƚǁĞƌĞƉŽƌƚ͘DŽƌĞŽǀĞƌ͕ďĞĐĂƵƐĞŽĨƚŚĞŝƌ ŚŝŐŚƚŽǆŝĐŝƚǇ͕ƵƐĞŽĨ&Žƌ/&ŝŶŚƵŵĂŶƐŝƐƐƚƌŽŶŐůǇĚŝƐĐŽƵƌĂŐĞĚ85_{. Unfortunately, we} ǁĞƌĞƵŶĂďůĞƚŽŵĞĂƐƵƌĞƉϯϱϬϬͲƐƉĞĐŝĮĐdƌĞŐƐŝŶƚŚŝƐĂƚŚĞƌŽƐĐůĞƌŽƐŝƐƐƚƵĚǇ͕ĚƵĞƚŽůĂĐŬŽĨ ƐƉĞĐŝĮĐƚĞƚƌĂŵĞƌƐ͘ tĞĨƵƌƚŚĞƌŽďƐĞƌǀĞĚĂƐŝŐŶŝĮĐĂŶƚĚĞĐƌĞĂƐĞŝŶƐĞƌƵŵĐŚŽůĞƐƚĞƌŽůůĞǀĞůƐŝŶŵŝĐĞ ƌĞĐĞŝǀŝŶŐ^W'ͬƉϯϱϬϬƚƌĞĂƚŵĞŶƚ͘/ƚŚĂƐďĞĞŶƐŚŽǁŶƚŚĂƚĚĞƉůĞƟŽŶŽĨdƌĞŐƐŝŶĐƌĞĂƐĞĚ cholesterol levels in LDLrͲͬͲ_mice86_{, while in vivodƌĞŐĞǆƉĂŶƐŝŽŶŝŶ>>ƌ}ͲͬͲ_{mice reduced}

cholesterol levels87_{, so this decrease in cholesterol levels could be caused by an increase}

ŝŶƚŚĞŶƵŵďĞƌŽĨĂŶƟŐĞŶͲƐƉĞĐŝĮĐdƌĞŐƐ͘&ŝŶĂůůǇ͕ĂŶŝŶĐƌĞĂƐĞŝŶĐŽůůĂŐĞŶĐŽŶƚĞŶƚŝŶƚŚĞ lesions of mice that received liposomes indicated more stable lesions. In accordance with ƚŚŝƐ͕ƚƌĞĂƚŵĞŶƚŽĨƉŽͲͬͲ_{mice with Tregs decreased lesion size and lesion macrophage}

content while increasing lesion collagen content88_.

ŽůůĞĐƟǀĞůǇ͕ ƚŚĞƐĞ ĚĂƚĂ ƐŚŽǁ ƚŚĂƚ ǁĞ ǁĞƌĞ ĂďůĞ ƚŽ ŝŶĚƵĐĞ ŚŝŐŚ ŶƵŵďĞƌƐ ŽĨ ĂŶƟŐĞŶͲƐƉĞĐŝĮĐ Ϯϱ+_&KyWϯ+_ϰ+_{dƌĞŐƐ ŝŶ ŵŝĐĞ ĂŌĞƌ Ă ƐŝŶŐůĞ ŝŶũĞĐƟŽŶ ŽĨ ^W'Ͳ}

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ZĞĨĞƌĞŶĐĞƐ

ϭ tĂŶŐ͕,͘ et al.'ůŽďĂů͕ƌĞŐŝŽŶĂů͕ĂŶĚŶĂƟŽŶĂůůŝĨĞĞǆƉĞĐƚĂŶĐǇ͕ĂůůͲĐĂƵƐĞŵŽƌƚĂůŝƚǇ͕ ĂŶĚĐĂƵƐĞͲƐƉĞĐŝĮĐŵŽƌƚĂůŝƚǇĨŽƌϮϰϵĐĂƵƐĞƐŽĨĚĞĂƚŚ͕ϭϵϴϬʹϮϬϭϱ͗ĂƐǇƐƚĞŵĂƟĐ analysis for the Global Burden of Disease Study 2015. The Lancet ϯϴϴ, 1459-ϭϱϰϰ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϭϰϬͲϲϳϯϲ;ϭϲͿϯϭϬϭϮͲϭ;ϮϬϭϲͿ͘

Ϯ WŝƌŝůůŽ͕ ͕͘ EŽƌĂƚĂ͕ '͘ ͘ Θ ĂƚĂƉĂŶŽ͕ ͘ >͘ >KyͲϭ͕ Kǆ>>͕ ĂŶĚ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ DĞĚŝĂƚŽƌƐ/ŶŇĂŵŵ ϮϬϭϯ͕ϭϱϮϳϴϲ͕ĚŽŝ͗ϭϬ͘ϭϭϱϱͬϮϬϭϯͬϭϱϮϳϴϲ;ϮϬϭϯͿ͘

ϯ >ŝďďǇ͕W͘/ŶŇĂŵŵĂƟŽŶŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘Arterioscler Thromb Vasc Biol ϯϮ, 2045-ϮϬϱϭ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬds,͘ϭϬϴ͘ϭϳϵϳϬϱ;ϮϬϭϮͿ͘

ϰ dĂďĂƐ͕/͘Θ>ŝĐŚƚŵĂŶ͕͘,͘DŽŶŽĐǇƚĞͲDĂĐƌŽƉŚĂŐĞƐĂŶĚdĞůůƐŝŶƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ Immunity ϰϳ͕ϲϮϭͲϲϯϰ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŝŵŵƵŶŝ͘ϮϬϭϳ͘Ϭϵ͘ϬϬϴ;ϮϬϭϳͿ͘

ϱ &ŽŬƐ͕ ͘ ͕͘ >ŝĐŚƚŵĂŶ͕ ͘ ,͘ Θ <ƵŝƉĞƌ͕ :͘ dƌĞĂƟŶŐ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ǁŝƚŚ regulatory T cells. Arterioscler Thromb Vasc Biol ϯϱ͕ ϮϴϬͲϮϴϳ͕ ĚŽŝ͗ϭϬ͘ϭϭϲϭͬ ds,͘ϭϭϰ͘ϯϬϯϱϲϴ;ϮϬϭϱͿ͘

ϲ ŽƵŶĂ͕,͘Θ<ƵŝƉĞƌ͕:͘EŽǀĞůͲĐĞůůƐƵďƐĞƚƐŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘Curr Opin Lipidol Ϯϳ͕ϰϵϯͲϰϵϴ͕ĚŽŝ͗ϭϬ͘ϭϬϵϳͬDK>͘ϬϬϬϬϬϬϬϬϬϬϬϬϬϯϯϱ;ϮϬϭϲͿ͘

ϳ ^ĂŬĂŐƵĐŚŝ͕ ^͕͘ zĂŵĂŐƵĐŚŝ͕ d͕͘ EŽŵƵƌĂ͕ d͘ Θ KŶŽ͕ D͘ ZĞŐƵůĂƚŽƌǇ d ĐĞůůƐ ĂŶĚ immune tolerance. Cell ϭϯϯ͕ϳϳϱͲϳϴϳ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐĞůů͘ϮϬϬϴ͘Ϭϱ͘ϬϬϵ;ϮϬϬϴͿ͘ ϴ DĂůůĂƚ͕͕͘ŝƚͲKƵĨĞůůĂ͕,͘ΘdĞĚŐƵŝ͕͘ZĞŐƵůĂƚŽƌǇdͲĐĞůůŝŵŵƵŶŝƚǇŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘

Trends Cardiovasc Med ϭϳ͕ϭϭϯͲϭϭϴ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ƚĐŵ͘ϮϬϬϳ͘Ϭϯ͘ϬϬϭ;ϮϬϬϳͿ͘ ϵ <ĞŝũǌĞƌ͕͕͘ǀĂŶĚĞƌĞĞ͕Z͕͘ǀĂŶĚĞŶ͕t͘ΘƌŽĞƌĞ͕&͘dƌĞŐŝŶĚƵĐŝŶŐĂĚũƵǀĂŶƚƐĨŽƌ ƚŚĞƌĂƉĞƵƟĐǀĂĐĐŝŶĂƟŽŶĂŐĂŝŶƐƚĐŚƌŽŶŝĐŝŶŇĂŵŵĂƚŽƌǇĚŝƐĞĂƐĞƐ͘Front Immunol ϰ͕Ϯϰϱ͕ĚŽŝ͗ϭϬ͘ϯϯϴϵͬĮŵŵƵ͘ϮϬϭϯ͘ϬϬϮϰϱ;ϮϬϭϯͿ͘ ϭϬ ƚŬŝŶƐŽŶ͕D͕͘͘ŝƐĞŶďĂƌƚŚ͕'͘^͘ΘDŝĐŚĞůƐ͕͘t͘dǇƉĞϭĚŝĂďĞƚĞƐ͘Lancet ϯϴϯ, ϲϵͲϴϮ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬ^ϬϭϰϬͲϲϳϯϲ;ϭϯͿϲϬϱϵϭͲϳ;ϮϬϭϰͿ͘ ϭϭ DĐ&ĂƌůĂŶĚ͕ ,͘ &͘ Θ DĂƌƟŶ͕ Z͘ DƵůƟƉůĞ ƐĐůĞƌŽƐŝƐ͗ Ă ĐŽŵƉůŝĐĂƚĞĚ ƉŝĐƚƵƌĞ ŽĨ autoimmunity. Nat Immunol ϴ͕ϵϭϯͲϵϭϵ͕ĚŽŝ͗ϭϬ͘ϭϬϯϴͬŶŝϭϱϬϳ;ϮϬϬϳͿ͘

ϭϮ ,ĂŶƐƐŽŶ͕'͘<͘Θ,ĞƌŵĂŶƐƐŽŶ͕͘dŚĞŝŵŵƵŶĞƐǇƐƚĞŵŝŶĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘Nat Immunol ϭϮ͕ϮϬϰͲϮϭϮ͕ĚŽŝ͗ϭϬ͘ϭϬϯϴͬŶŝ͘ϮϬϬϭ;ϮϬϭϭͿ͘ ϭϯ ŽƵƟŶŚŽ͕͘͘ΘŚĂƉŵĂŶ͕<͘͘dŚĞĂŶƟͲŝŶŇĂŵŵĂƚŽƌǇĂŶĚŝŵŵƵŶŽƐƵƉƉƌĞƐƐŝǀĞ ĞīĞĐƚƐŽĨŐůƵĐŽĐŽƌƟĐŽŝĚƐ͕ƌĞĐĞŶƚĚĞǀĞůŽƉŵĞŶƚƐĂŶĚŵĞĐŚĂŶŝƐƟĐŝŶƐŝŐŚƚƐ͘Mol Cell Endocrinol ϯϯϱ͕ϮͲϭϯ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ŵĐĞ͘ϮϬϭϬ͘Ϭϰ͘ϬϬϱ;ϮϬϭϭͿ͘ 14 Barr, T. A. et al. ĐĞůůĚĞƉůĞƟŽŶƚŚĞƌĂƉǇĂŵĞůŝŽƌĂƚĞƐĂƵƚŽŝŵŵƵŶĞĚŝƐĞĂƐĞƚŚƌŽƵŐŚ ĂďůĂƟŽŶ ŽĨ />ͲϲͲƉƌŽĚƵĐŝŶŐ ĐĞůůƐ͘ J Exp Med ϮϬϵ͕ ϭϬϬϭͲϭϬϭϬ͕ ĚŽŝ͗ϭϬ͘ϭϬϴϰͬ ũĞŵ͘ϮϬϭϭϭϲϳϱ;ϮϬϭϮͿ͘ ϭϱ ŚĂƚĞŶŽƵĚ͕ >͘ Θ ůƵĞƐƚŽŶĞ͕ :͘ ͘ ϯͲƐƉĞĐŝĮĐ ĂŶƟďŽĚŝĞƐ͗ Ă ƉŽƌƚĂů ƚŽ ƚŚĞ treatment of autoimmunity. Nat Rev Immunol ϳ͕ϲϮϮͲϲϯϮ͕ĚŽŝ͗ϭϬ͘ϭϬϯϴͬŶƌŝϮϭϯϰ ;ϮϬϬϳͿ͘

16 Shoji, T. et al. /ŶǀĞƌƐĞ ƌĞůĂƟŽŶƐŚŝƉ ďĞƚǁĞĞŶ ĐŝƌĐƵůĂƟŶŐ ŽǆŝĚŝǌĞĚ ůŽǁ ĚĞŶƐŝƚǇ ůŝƉŽƉƌŽƚĞŝŶ ;Žǆ>>Ϳ ĂŶĚ ĂŶƟͲŽǆ>> ĂŶƟďŽĚǇ ůĞǀĞůƐ ŝŶ ŚĞĂůƚŚǇ ƐƵďũĞĐƚƐ͘ Atherosclerosis ϭϰϴ͕ϭϳϭͲϭϳϳ͕ĚŽŝ͗ŽŝϭϬ͘ϭϬϭϲͬ^ϬϬϮϭͲϵϭϱϬ;ϵϵͿϬϬϮϭϴͲy;ϮϬϬϬͿ͘ 17 Stemme, S. et al.důǇŵƉŚŽĐǇƚĞƐĨƌŽŵŚƵŵĂŶĂƚŚĞƌŽƐĐůĞƌŽƟĐƉůĂƋƵĞƐƌĞĐŽŐŶŝǌĞ

ŽǆŝĚŝǌĞĚ ůŽǁ ĚĞŶƐŝƚǇ ůŝƉŽƉƌŽƚĞŝŶ͘ Proc Natl Acad Sci U S A ϵϮ, 3893-3897, ĚŽŝ͗ϭϬ͘ϭϬϳϯͬƉŶĂƐ͘ϵϮ͘ϵ͘ϯϴϵϯ;ϭϵϵϱͿ͘

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ŵŽĚŝĮĞĚ ƉĞƉƟĚĞ ƐĞƋƵĞŶĐĞƐ ŝŶ ĂƉŽ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ĐĂƌĚŝŽǀĂƐĐƵůĂƌ disease. Arterioscler Thromb Vasc Biol Ϯϯ͕ ϴϳϮͲϴϳϴ͕ ĚŽŝ͗ϭϬ͘ϭϭϲϭͬϬϭ͘ ds͘ϬϬϬϬϬϲϳϵϯϱ͘ϬϮϲϳϵ͘Ϭ;ϮϬϬϯͿ͘

ϭϵ ^ũŽŐƌĞŶ͕W͘ et al.,ŝŐŚƉůĂƐŵĂĐŽŶĐĞŶƚƌĂƟŽŶƐŽĨĂƵƚŽĂŶƟďŽĚŝĞƐĂŐĂŝŶƐƚŶĂƟǀĞ ƉĞƉƟĚĞϮϭϬŽĨĂƉŽͲϭϬϬĂƌĞƌĞůĂƚĞĚƚŽůĞƐƐĐŽƌŽŶĂƌǇĂƚŚĞƌŽƐĐůĞƌŽƐŝƐĂŶĚůŽǁĞƌ ƌŝƐŬŽĨŵǇŽĐĂƌĚŝĂůŝŶĨĂƌĐƟŽŶ͘Eur Heart J Ϯϵ͕ϮϮϭϴͲϮϮϮϲ͕ĚŽŝ͗ϭϬ͘ϭϬϵϯͬĞƵƌŚĞĂƌƚũͬ ĞŚŶϯϯϲ;ϮϬϬϴͿ͘

ϮϬ &ĂŐĞƌďĞƌŐ͕ ͕͘ WƌĂŚů 'ƵůůďĞƌŐ͕ h͕͘ ůŵ͕ Z͕͘ EŝůƐƐŽŶ͕ :͘ Θ &ƌĞĚƌŝŬƐŽŶ͕ '͘ E͘ ŝƌĐƵůĂƟŶŐĂƵƚŽĂŶƟďŽĚŝĞƐĂŐĂŝŶƐƚƚŚĞĂƉŽůŝƉŽƉƌŽƚĞŝŶͲϭϬϬƉĞƉƟĚĞƐƉϰϱĂŶĚ ƉϮϭϬ ŝŶ ƌĞůĂƟŽŶ ƚŽ ƚŚĞ ŽĐĐƵƌƌĞŶĐĞ ŽĨ ĐĂƌŽƟĚ ƉůĂƋƵĞƐ ŝŶ ϲϰͲǇĞĂƌͲŽůĚ ǁŽŵĞŶ͘ PLoS One ϭϬ͕ĞϬϭϮϬϳϰϰ͕ĚŽŝ͗ϭϬ͘ϭϯϳϭͬũŽƵƌŶĂů͘ƉŽŶĞ͘ϬϭϮϬϳϰϰ;ϮϬϭϱͿ͘

Ϯϭ ŚŽƵ͕ y͕͘ ĂůŝŐŝƵƌŝ͕ '͕͘ ,ĂŵƐƚĞŶ͕ ͕͘ >ĞĨǀĞƌƚ͕ ͘ <͘ Θ ,ĂŶƐƐŽŶ͕ '͘ <͘ >> /ŵŵƵŶŝǌĂƟŽŶ /ŶĚƵĐĞƐ dͲĞůůʹĞƉĞŶĚĞŶƚ ŶƟďŽĚǇ &ŽƌŵĂƟŽŶ ĂŶĚ WƌŽƚĞĐƟŽŶ Against Atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology Ϯϭ, ϭϬϴ;ϮϬϬϭͿ͘

22 Schiopu, A. et al. ZĞĐŽŵďŝŶĂŶƚ ŚƵŵĂŶ ĂŶƟďŽĚŝĞƐ ĂŐĂŝŶƐƚ ĂůĚĞŚǇĚĞͲŵŽĚŝĮĞĚ ĂƉŽůŝƉŽƉƌŽƚĞŝŶ ͲϭϬϬ ƉĞƉƟĚĞ ƐĞƋƵĞŶĐĞƐ ŝŶŚŝďŝƚ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ ŝƌĐƵůĂƟŽŶ ϭϭϬ͕ϮϬϰϳͲϮϬϱϮ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬϬϭ͘/Z͘ϬϬϬϬϭϰϯϭϲϮ͘ϱϲϬϱϳ͘ϱ;ϮϬϬϰͿ͘

Ϯϯ dƐĞ͕<͘ et al.ƚŚĞƌŽƉƌŽƚĞĐƟǀĞsĂĐĐŝŶĂƟŽŶǁŝƚŚD,Ͳ//ZĞƐƚƌŝĐƚĞĚWĞƉƟĚĞƐĨƌŽŵ ApoB-100. Front Immunol ϰ͕ϰϵϯ͕ĚŽŝ͗ϭϬ͘ϯϯϴϵͬĮŵŵƵ͘ϮϬϭϯ͘ϬϬϰϵϯ;ϮϬϭϯͿ͘ Ϯϰ <ůŝŶŐĞŶďĞƌŐ͕Z͘ et al./ŶƚƌĂŶĂƐĂůŝŵŵƵŶŝǌĂƟŽŶǁŝƚŚĂŶĂƉŽůŝƉŽƉƌŽƚĞŝŶͲϭϬϬĨƵƐŝŽŶ

ƉƌŽƚĞŝŶŝŶĚƵĐĞƐĂŶƟŐĞŶͲƐƉĞĐŝĮĐƌĞŐƵůĂƚŽƌǇdĐĞůůƐĂŶĚƌĞĚƵĐĞƐĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ Arterioscler Thromb Vasc Biol ϯϬ͕ϵϰϲͲϵϱϮ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬds,͘ϭϬϵ͘ϮϬϮϲϳϭ ;ϮϬϭϬͿ͘

25 Herbin, O. et al. Regulatory T-cell response to apolipoprotein B100-derived ƉĞƉƟĚĞƐƌĞĚƵĐĞƐƚŚĞĚĞǀĞůŽƉŵĞŶƚĂŶĚƉƌŽŐƌĞƐƐŝŽŶŽĨĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶŵŝĐĞ͘ Arterioscler Thromb Vasc Biol ϯϮ͕ϲϬϱͲϲϭϮ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬds,͘ϭϭϭ͘ϮϰϮϴϬϬ ;ϮϬϭϮͿ͘

26 Fredrikson, G. N. et al. /ŶŚŝďŝƟŽŶ ŽĨ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ŝŶ ĂƉŽͲŶƵůů ŵŝĐĞ ďǇ ŝŵŵƵŶŝǌĂƟŽŶǁŝƚŚĂƉŽͲϭϬϬƉĞƉƟĚĞƐĞƋƵĞŶĐĞƐ͘Arterioscler Thromb Vasc Biol Ϯϯ͕ϴϳϵͲϴϴϰ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬϬϭ͘ds͘ϬϬϬϬϬϲϳϵϯϳ͘ϵϯϳϭϲ͘;ϮϬϬϯͿ͘

Ϯϳ &ƌĞĚƌŝŬƐŽŶ͕ '͘ E͕͘ ũŽƌŬďĂĐŬĂ͕ ,͕͘ ^ŽĚĞƌďĞƌŐ͕ /͕͘ >ũƵŶŐĐƌĂŶƚǌ͕ /͘ Θ EŝůƐƐŽŶ͕ :͘ dƌĞĂƚŵĞŶƚ ǁŝƚŚ ĂƉŽ ƉĞƉƟĚĞ ǀĂĐĐŝŶĞƐ ŝŶŚŝďŝƚƐ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ŝŶ ŚƵŵĂŶ ĂƉŽ ͲϭϬϬ ƚƌĂŶƐŐĞŶŝĐ ŵŝĐĞ ǁŝƚŚŽƵƚ ŝŶĚƵĐŝŶŐ ĂŶ ŝŶĐƌĞĂƐĞ ŝŶ ƉĞƉƟĚĞͲƐƉĞĐŝĮĐ ĂŶƟďŽĚŝĞƐ͘J Intern Med Ϯϲϰ͕ϱϲϯͲϱϳϬ͕ĚŽŝ͗ϭϬ͘ϭϭϭϭͬũ͘ϭϯϲϱͲϮϳϵϲ͘ϮϬϬϴ͘Ϭϭϵϵϱ͘ǆ ;ϮϬϬϴͿ͘ Ϯϴ <ŝŵƵƌĂ͕d͘ et al.ZĞŐƵůĂƚŽƌǇϰ;нͿdĞůůƐZĞĐŽŐŶŝǌĞDĂũŽƌ,ŝƐƚŽĐŽŵƉĂƟďŝůŝƚǇ ŽŵƉůĞǆ ůĂƐƐ // DŽůĞĐƵůĞͲZĞƐƚƌŝĐƚĞĚ WĞƉƟĚĞ ƉŝƚŽƉĞƐ ŽĨ ƉŽůŝƉŽƉƌŽƚĞŝŶ ͘ ŝƌĐƵůĂƟŽŶ ϭϯϴ͕ϭϭϯϬͲϭϭϰϯ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬ/Zh>d/KE,͘ϭϭϳ͘ϬϯϭϰϮϬ;ϮϬϭϴͿ͘ Ϯϵ ǀĂŶ WƵŝũǀĞůĚĞ͕ '͘ ,͘ et al. /ŶĚƵĐƟŽŶ ŽĨ ŽƌĂů ƚŽůĞƌĂŶĐĞ ƚŽ ŽǆŝĚŝǌĞĚ ůŽǁͲ density lipoprotein ameliorates atherosclerosis. ŝƌĐƵůĂƟŽŶ ϭϭϰ, 1968-1976, ĚŽŝ͗ϭϬ͘ϭϭϲϭͬ/Zh>d/KE,͘ϭϬϲ͘ϲϭϱϲϬϵ;ϮϬϬϲͿ͘

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ůŽĂĚĞĚĚĞŶĚƌŝƟĐĐĞůůƐĂƩĞŶƵĂƚĞƐĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶŚǇƉĞƌĐŚŽůĞƐƚĞƌŽůĞŵŝĐŵŝĐĞ͘ ŝƌĐƵůĂƟŽŶ ϭϮϯ͕ϭϬϴϯͲϭϬϵϭ͕ĚŽŝ͗ϭϬ͘ϭϭϲϭͬ/Zh>d/KE,͘ϭϭϬ͘ϵϳϯϮϮϮ;ϮϬϭϭͿ͘ ϯϮ ,ĂďĞƚƐ͕ <͘ >͘ et al. sĂĐĐŝŶĂƟŽŶ ƵƐŝŶŐ ŽǆŝĚŝǌĞĚ ůŽǁͲĚĞŶƐŝƚǇ ůŝƉŽƉƌŽƚĞŝŶͲƉƵůƐĞĚ ĚĞŶĚƌŝƟĐĐĞůůƐƌĞĚƵĐĞƐĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶ>>ƌĞĐĞƉƚŽƌͲĚĞĮĐŝĞŶƚŵŝĐĞ͘Cardiovasc Res ϴϱ͕ϲϮϮͲϲϯϬ͕ĚŽŝ͗ϭϬ͘ϭϬϵϯͬĐǀƌͬĐǀƉϯϯϴ;ϮϬϭϬͿ͘ ϯϯ ZĂŬĞƌ͕ s͘ <͕͘ ŽŵŽŐĂůůĂ͕ D͘ W͘ Θ ^ƚĞŝŶďƌŝŶŬ͕ <͘ dŽůĞƌŽŐĞŶŝĐ ĞŶĚƌŝƟĐ ĞůůƐ ĨŽƌ ZĞŐƵůĂƚŽƌǇ d Ğůů /ŶĚƵĐƟŽŶ ŝŶ DĂŶ͘ Front Immunol ϲ͕ ϱϲϵ͕ ĚŽŝ͗ϭϬ͘ϯϯϴϵͬ ĮŵŵƵ͘ϮϬϭϱ͘ϬϬϱϲϵ;ϮϬϭϱͿ͘ ϯϰ WĂƩŶŝ͕ ͘ ^͕͘ ŚƵƉŝŶ͕ s͘ s͘ Θ dŽƌĐŚŝůŝŶ͕ s͘ W͘ EĞǁ ĞǀĞůŽƉŵĞŶƚƐ ŝŶ >ŝƉŽƐŽŵĂů Drug Delivery. Chem Rev ϭϭϱ͕ϭϬϵϯϴͲϭϬϵϲϲ͕ĚŽŝ͗ϭϬ͘ϭϬϮϭͬĂĐƐ͘ĐŚĞŵƌĞǀ͘ϱďϬϬϬϰϲ ;ϮϬϭϱͿ͘

ϯϱ EĂŐĂƚĂ͕^͕͘,ĂŶĂǇĂŵĂ͕Z͘Θ<ĂǁĂŶĞ͕<͘ƵƚŽŝŵŵƵŶŝƚǇĂŶĚƚŚĞĐůĞĂƌĂŶĐĞŽĨĚĞĂĚ cells. Cell ϭϰϬ͕ϲϭϵͲϲϯϬ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĐĞůů͘ϮϬϭϬ͘ϬϮ͘Ϭϭϰ;ϮϬϭϬͿ͘

ϯϲ WƵũŽůͲƵƚŽŶĞůů͕/͘ et al.hƐĞŽĨĂƵƚŽĂŶƟŐĞŶͲůŽĂĚĞĚƉŚŽƐƉŚĂƟĚǇůƐĞƌŝŶĞͲůŝƉŽƐŽŵĞƐ to arrest autoimmunity in type 1 diabetes. PLoS One ϭϬ͕ĞϬϭϮϳϬϱϳ͕ĚŽŝ͗ϭϬ͘ϭϯϳϭͬ ũŽƵƌŶĂů͘ƉŽŶĞ͘ϬϭϮϳϬϱϳ;ϮϬϭϱͿ͘ ϯϳ ZĂŵŽƐ͕ '͘ ͘ et al. ƉŽƉƚŽƟĐ ŵŝŵŝĐƌǇ͗ ƉŚŽƐƉŚĂƟĚǇůƐĞƌŝŶĞ ůŝƉŽƐŽŵĞƐ ƌĞĚƵĐĞ ŝŶŇĂŵŵĂƟŽŶƚŚƌŽƵŐŚĂĐƟǀĂƟŽŶŽĨƉĞƌŽǆŝƐŽŵĞƉƌŽůŝĨĞƌĂƚŽƌͲĂĐƟǀĂƚĞĚƌĞĐĞƉƚŽƌƐ ;WWZƐͿ ŝŶ ǀŝǀŽ͘ Br J Pharmacol ϭϱϭ͕ ϴϰϰͲϴϱϬ͕ ĚŽŝ͗ϭϬ͘ϭϬϯϴͬƐũ͘ďũƉ͘ϬϳϬϳϯϬϮ ;ϮϬϬϳͿ͘ 38 Hosseini, H. et al. WŚŽƐƉŚĂƟĚǇůƐĞƌŝŶĞ ůŝƉŽƐŽŵĞƐ ŵŝŵŝĐ ĂƉŽƉƚŽƟĐ ĐĞůůƐ ƚŽ ĂƩĞŶƵĂƚĞ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ ďǇ ĞǆƉĂŶĚŝŶŐ ƉŽůǇƌĞĂĐƟǀĞ /ŐD ƉƌŽĚƵĐŝŶŐ ϭĂ lymphocytes. Cardiovasc Res ϭϬϲ͕ϰϰϯͲϰϱϮ͕ĚŽŝ͗ϭϬ͘ϭϬϵϯͬĐǀƌͬĐǀǀϬϯϳ;ϮϬϭϱͿ͘ ϯϵ sĂƌǇƉĂƚĂŬŝ͕͘D͕͘ĞŶŶĞ͕E͕͘ŽƵǁƐƚƌĂ͕:͕͘:ŝƐŬŽŽƚ͕t͘ΘKƐƐĞŶĚŽƌƉ͕&͘ĸĐŝĞŶƚ

ƌĂĚŝĐĂƟŽŶŽĨƐƚĂďůŝƐŚĞĚdƵŵŽƌƐŝŶDŝĐĞǁŝƚŚĂƟŽŶŝĐ>ŝƉŽƐŽŵĞͲĂƐĞĚ^ǇŶƚŚĞƟĐ >ŽŶŐͲWĞƉƟĚĞ sĂĐĐŝŶĞƐ͘ Cancer Immunol Res ϱ͕ ϮϮϮͲϮϯϯ͕ ĚŽŝ͗ϭϬ͘ϭϭϱϴͬϮϯϮϲͲ ϲϬϲϲ͘/ZͲϭϲͲϬϮϴϯ;ϮϬϭϳͿ͘

ϰϬ ĂƌŶŝĞƌ YƵĞƌ͕ ͕͘ ůƐŚĂƌŬĂǁǇ͕ ͕͘ ZŽŵĞŝũŶ͕ ^͕͘ <ƌŽƐ͕ ͘ Θ :ŝƐŬŽŽƚ͕ t͘ ĂƟŽŶŝĐ ůŝƉŽƐŽŵĞƐ ĂƐ ĂĚũƵǀĂŶƚƐ ĨŽƌ ŝŶŇƵĞŶǌĂ ŚĞŵĂŐŐůƵƟŶŝŶ͗ ŵŽƌĞ ƚŚĂŶ ĐŚĂƌŐĞ ĂůŽŶĞ͘ Eur J Pharm Biopharm ϴϭ͕ϮϵϰͲϯϬϮ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ĞũƉď͘ϮϬϭϮ͘Ϭϯ͘Ϭϭϯ;ϮϬϭϮͿ͘ ϰϭ ,ĂƐƐĂŶ͕ ͘ et al. dŚĞ ŚƵŵĂŶ ůĞƵŬŽĐǇƚĞ ĂŶƟŐĞŶͲƉƌĞƐĞŶƚĞĚ ůŝŐĂŶĚŽŵĞ ŽĨ 

lymphocytes. Molecular & cellular proteomics : MCP ϭϮ͕ϭϴϮϵͲϭϴϰϯ͕ĚŽŝ͗ϭϬ͘ϭϬϳϰͬ ŵĐƉ͘DϭϭϮ͘ϬϮϰϴϭϬ;ϮϬϭϯͿ͘ ϰϮ sĂƌǇƉĂƚĂŬŝ͕͘D͕͘ǀĂŶĚĞƌDĂĂĚĞŶ͕<͕͘ŽƵǁƐƚƌĂ͕:͕͘KƐƐĞŶĚŽƌƉ͕&͘Θ:ŝƐŬŽŽƚ͕t͘ ĂƟŽŶŝĐůŝƉŽƐŽŵĞƐůŽĂĚĞĚǁŝƚŚĂƐǇŶƚŚĞƟĐůŽŶŐƉĞƉƟĚĞĂŶĚƉŽůǇ;/͗Ϳ͗ĂĚĞĮŶĞĚ ĂĚũƵǀĂŶƚĞĚǀĂĐĐŝŶĞĨŽƌŝŶĚƵĐƟŽŶŽĨĂŶƟŐĞŶͲƐƉĞĐŝĮĐdĐĞůůĐǇƚŽƚŽǆŝĐŝƚǇ͘AAPS J ϭϳ͕ϮϭϲͲϮϮϲ͕ĚŽŝ͗ϭϬ͘ϭϮϬϴͬƐϭϮϮϰϴͲϬϭϰͲϵϲϴϲͲϰ;ϮϬϭϱͿ͘ ϰϯ DĂďƌĞǇ͕ ^͘ Θ ^ƚƵƌƚĞǀĂŶƚ͕ :͘ D͘ /ŶǀĞƐƟŐĂƟŽŶ ŽĨ ƉŚĂƐĞ ƚƌĂŶƐŝƟŽŶƐ ŽĨ ůŝƉŝĚƐ ĂŶĚ ůŝƉŝĚŵŝǆƚƵƌĞƐďǇƐĞŶƐŝƟǀŝƚǇĚŝīĞƌĞŶƟĂůƐĐĂŶŶŝŶŐĐĂůŽƌŝŵĞƚƌǇ͘Proc Natl Acad Sci U S A ϳϯ͕ϯϴϲϮͲϯϴϲϲ͕ĚŽŝ͗ϭϬ͘ϭϬϳϯͬƉŶĂƐ͘ϳϯ͘ϭϭ͘ϯϴϲϮ;ϭϵϳϲͿ͘

ϰϰ ŚĂŶŐ͕ z͘ W͕͘ >ĞǁŝƐ͕ Z͘ E͘ Θ DĐůŚĂŶĞǇ͕ Z͘ E͘ ĂůŽƌŝŵĞƚƌŝĐ ĂŶĚ ƐƉĞĐƚƌŽƐĐŽƉŝĐ studies of the thermotropic phase behavior of the n-saturated ϭ͕ϮͲĚŝĂĐǇůƉŚŽƐƉŚĂƟĚǇůŐůǇĐĞƌŽůƐ͘ Biophys J ϳϮ͕ ϳϳϵͲϳϵϯ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϬϬϲͲ ϯϰϵϱ;ϵϳͿϳϴϳϭϮͲϱ;ϭϵϵϳͿ͘

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4

Biochim Biophys Acta ϳϯϮ͕ϰϬϱͲϰϭϭ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬϬϬϬϱͲϮϳϯϲ;ϴϯͿϵϬϬϱϳͲϯ;ϭϵϴϯͿ͘ ϰϲ ZĞŐĞůŝŶ͕͘͘ et al.ŝŽƉŚǇƐŝĐĂůĂŶĚůŝƉŽĨĞĐƟŽŶƐƚƵĚŝĞƐŽĨKdWĂŶĂůŽŐƐ͘Biochim

Biophys Acta ϭϰϲϰ͕ϭϱϭͲϭϲϰ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϬϬϱͲϮϳϯϲ;ϬϬͿϬϬϭϮϲͲϳ;ϮϬϬϬͿ͘ ϰϳ tĞŝƐƐ͕͘͘'͕͘<ĞŵƉĞ͕<͕͘&ŽƌƐƚĞƌ͕^͘ΘĂƌƵƐŽ͕&͘DŝĐƌŽŇƵŝĚŝĐǆĂŵŝŶĂƟŽŶŽĨ

ƚŚĞ ͞,ĂƌĚ͟ ŝŽŵŽůĞĐƵůĂƌ ŽƌŽŶĂ &ŽƌŵĞĚ ŽŶ ŶŐŝŶĞĞƌĞĚ WĂƌƟĐůĞƐ ŝŶ ŝīĞƌĞŶƚ Biological Milieu. Biomacromolecules ϭϵ͕ ϮϱϴϬͲϮϱϵϰ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬĂĐƐ͘ ďŝŽŵĂĐ͘ϴďϬϬϭϵϲ;ϮϬϭϴͿ͘

ϰϴ DĞŚůĞŵ͕͕͘,ĂŐďĞƌŐ͕͕͘͘DƵŚů͕>͕͘ƌŝŬƐƐŽŶ͕h͘Θ&ĂůŬĞǀĂůů͕͘/ŵĂŐŝŶŐŽĨŶĞƵƚƌĂů lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc ϴ͕ϭϭϰϵͲϭϭϱϰ͕ĚŽŝ͗ϭϬ͘ϭϬϯϴͬŶƉƌŽƚ͘ϮϬϭϯ͘Ϭϱϱ;ϮϬϭϯͿ͘

ϰϵ <ƌŝƟŬŽƵ͕͘ et al./ŶŚŝďŝƟŽŶŽĨůǇƐŽƉŚŽƐƉŚĂƟĚŝĐĂĐŝĚƌĞĐĞƉƚŽƌƐϭĂŶĚϯĂƩĞŶƵĂƚĞƐ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐĚĞǀĞůŽƉŵĞŶƚŝŶ>>ͲƌĞĐĞƉƚŽƌĚĞĮĐŝĞŶƚŵŝĐĞ͘Sci Rep ϲ, 37585, ĚŽŝ͗ϭϬ͘ϭϬϯϴͬƐƌĞƉϯϳϱϴϱ;ϮϬϭϲͿ͘

ϱϬ tĞǌĞů͕ ͘ et al. ŽŵƉůĞŵĞŶƚ ĨĂĐƚŽƌ ϱĂ ŝŶĚƵĐĞƐ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞ ĚŝƐƌƵƉƟŽŶƐ͘J Cell Mol Med ϭϴ͕ϮϬϮϬͲϮϬϯϬ͕ĚŽŝ͗ϭϬ͘ϭϭϭϭͬũĐŵŵ͘ϭϮϯϱϳ;ϮϬϭϰͿ͘ ϱϭ EĂĚŬĂƌŶŝ͕^͘<͘ et al. Measurement of collagen and smooth muscle cell content

ŝŶ ĂƚŚĞƌŽƐĐůĞƌŽƟĐ ƉůĂƋƵĞƐ ƵƐŝŶŐ ƉŽůĂƌŝǌĂƟŽŶͲƐĞŶƐŝƟǀĞ ŽƉƟĐĂů ĐŽŚĞƌĞŶĐĞ tomography. J Am Coll Cardiol ϰϵ͕ ϭϰϳϰͲϭϰϴϭ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ũĂĐĐ͘ϮϬϬϲ͘ϭϭ͘ϬϰϬ ;ϮϬϬϳͿ͘

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ĂŶƟďŽĚǇͲĚĞƉĞŶĚĞŶƚ ĞŶŚĂŶĐĞŵĞŶƚ ŽĨ ,/sͲϭ ŝŶĨĞĐƟŽŶ͘ AIDS ϭϭ, 949-958, ĚŽŝ͗ϭϬ͘ϭϬϵϳͬϬϬϬϬϮϬϯϬͲϭϵϵϳϬϴϬϬϬͲϬϬϬϬϮ;ϭϵϵϳͿ͘ ϳϳ dĂŬĂĚĂ͕ ͕͘ &ĞůĚŵĂŶŶ͕ ,͕͘ <ƐŝĂǌĞŬ͕ d͘ '͘ Θ <ĂǁĂŽŬĂ͕ z͘ ŶƟďŽĚǇͲĚĞƉĞŶĚĞŶƚ ĞŶŚĂŶĐĞŵĞŶƚ ŽĨ ďŽůĂ ǀŝƌƵƐ ŝŶĨĞĐƟŽŶ͘ J Virol ϳϳ͕ ϳϱϯϵͲϳϱϰϰ͕ ĚŽŝ͗ϭϬ͘ϭϭϮϴͬ ũǀŝ͘ϳϳ͘ϭϯ͘ϳϱϯϵͲϳϱϰϰ͘ϮϬϬϯ;ϮϬϬϯͿ͘ ϳϴ tĂůƉŽƌƚ͕D͘:͕͘ĂǀŝĞƐ͕<͘͘ΘŽƩŽ͕D͘ϭƋĂŶĚƐǇƐƚĞŵŝĐůƵƉƵƐĞƌǇƚŚĞŵĂƚŽƐƵƐ͘ Immunobiology ϭϵϵ͕ϮϲϱͲϮϴϱ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬ^ϬϭϳϭͲϮϵϴϱ;ϵϴͿϴϬϬϯϮͲϲ;ϭϵϵϴͿ͘ ϳϵ ŚĂƟĂ͕s͘<͘ et al.ŽŵƉůĞŵĞŶƚϭƋƌĞĚƵĐĞƐĞĂƌůǇĂƚŚĞƌŽƐĐůĞƌŽƐŝƐŝŶůŽǁͲĚĞŶƐŝƚǇ ůŝƉŽƉƌŽƚĞŝŶ ƌĞĐĞƉƚŽƌͲĚĞĮĐŝĞŶƚ ŵŝĐĞ͘ Am J Pathol ϭϳϬ͕ ϰϭϲͲϰϮϲ͕ ĚŽŝ͗ϭϬ͘Ϯϯϱϯͬ ĂũƉĂƚŚ͘ϮϬϬϳ͘ϬϲϬϰϬϲ;ϮϬϬϳͿ͘ ϴϬ ƌĂĚůĞǇ͕͘:͕͘DĂƵƌĞƌͲ^ƉƵƌĞũ͕͕͘ƌŽŽŬƐ͕͘͘ΘĞǀŝŶĞ͕͘s͘hŶƵƐƵĂůĞůĞĐƚƌŽƐƚĂƟĐ ĞīĞĐƚƐ ŽŶ ďŝŶĚŝŶŐ ŽĨ ϭƋ ƚŽ ĂŶŝŽŶŝĐ ůŝƉŽƐŽŵĞƐ͗ ƌŽůĞ ŽĨ ĂŶŝŽŶŝĐ ƉŚŽƐƉŚŽůŝƉŝĚ domains and their line tension. Biochemistry ϯϴ͕ ϴϭϭϮͲϴϭϮϯ͕ ĚŽŝ͗ϭϬ͘ϭϬϮϭͬ ďŝϵϵϬϰϴϬĂ;ϭϵϵϵͿ͘

ϴϭ ĂĐŚŵĂŶŶ͕D͘&͘Θ:ĞŶŶŝŶŐƐ͕'͘d͘sĂĐĐŝŶĞĚĞůŝǀĞƌǇ͗ĂŵĂƩĞƌŽĨƐŝǌĞ͕ŐĞŽŵĞƚƌǇ͕ ŬŝŶĞƟĐƐ ĂŶĚ ŵŽůĞĐƵůĂƌ ƉĂƩĞƌŶƐ͘ Nat Rev Immunol ϭϬ͕ ϳϴϳͲϳϵϲ͕ ĚŽŝ͗ϭϬ͘ϭϬϯϴͬ ŶƌŝϮϴϲϴ;ϮϬϭϬͿ͘

ϴϮ ƌĂĚůĞǇ͕ ͘ :͕͘ ƌŽŽŬƐ͕ ͘ ͕͘ EŽƌƌŝƐͲ:ŽŶĞƐ͕ Z͘ Θ ĞǀŝŶĞ͕ ͘ s͘ ϭƋ ďŝŶĚŝŶŐ ƚŽ ůŝƉŽƐŽŵĞƐŝƐƐƵƌĨĂĐĞĐŚĂƌŐĞĚĞƉĞŶĚĞŶƚĂŶĚŝƐŝŶŚŝďŝƚĞĚďǇƉĞƉƟĚĞƐĐŽŶƐŝƐƟŶŐ ŽĨƌĞƐŝĚƵĞƐϭϰͲϮϲŽĨƚŚĞŚƵŵĂŶϭƋĐŚĂŝŶŝŶĂƐĞƋƵĞŶĐĞŝŶĚĞƉĞŶĚĞŶƚŵĂŶŶĞƌ͘ Biochim Biophys Acta ϭϰϭϴ͕ϭϵͲϯϬ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬƐϬϬϬϱͲϮϳϯϲ;ϵϵͿϬϬϬϭϯͲϵ;ϭϵϵϵͿ͘ ϴϯ ^ŽƵ͕ <͘ Θ dƐƵĐŚŝĚĂ͕ ͘ ůĞĐƚƌŽƐƚĂƟĐ ŝŶƚĞƌĂĐƟŽŶƐ ĂŶĚ ĐŽŵƉůĞŵĞŶƚ ĂĐƟǀĂƟŽŶ

ŽŶ ƚŚĞ ƐƵƌĨĂĐĞ ŽĨ ƉŚŽƐƉŚŽůŝƉŝĚ ǀĞƐŝĐůĞ ĐŽŶƚĂŝŶŝŶŐ ĂĐŝĚŝĐ ůŝƉŝĚƐ͗ ĞīĞĐƚ ŽĨ ƚŚĞ structure of acidic groups. Biochim Biophys Acta ϭϳϳϴ͕ϭϬϯϱͲϭϬϰϭ͕ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ ďďĂŵĞŵ͘ϮϬϬϴ͘Ϭϭ͘ϬϬϲ;ϮϬϬϴͿ͘ ϴϰ ůĂƌŬĞ͕ ͘ s͕͘ tĞŝƐƚ͕ ͘ D͕͘ tĂůƐŚ͕ ͘ D͘ Θ dĞŶŶĞƌ͕ ͘ :͘ ŽŵƉůĞŵĞŶƚ ƉƌŽƚĞŝŶ ϭƋďŽƵŶĚƚŽĂƉŽƉƚŽƟĐĐĞůůƐƐƵƉƉƌĞƐƐĞƐŚƵŵĂŶŵĂĐƌŽƉŚĂŐĞĂŶĚĚĞŶĚƌŝƟĐĐĞůůͲ ŵĞĚŝĂƚĞĚdŚϭϳĂŶĚdŚϭdĐĞůůƐƵďƐĞƚƉƌŽůŝĨĞƌĂƟŽŶ͘J Leukoc Biol ϵϳ, 147-160, ĚŽŝ͗ϭϬ͘ϭϭϴϵͬũůď͘ϯϬϲϭϰͲϮϳϴZ;ϮϬϭϱͿ͘ ϴϱ ^ƟůůƐ͕,͘&͕͘:ƌ͘ĚũƵǀĂŶƚƐĂŶĚĂŶƟďŽĚǇƉƌŽĚƵĐƟŽŶ͗ĚŝƐƉĞůůŝŶŐƚŚĞŵǇƚŚƐĂƐƐŽĐŝĂƚĞĚ with Freund’s complete and other adjuvants. ILAR J ϰϲ͕ϮϴϬͲϮϵϯ͕ĚŽŝ͗ϭϬ͘ϭϬϵϯͬ ŝůĂƌ͘ϰϲ͘ϯ͘ϮϴϬ;ϮϬϬϱͿ͘

ϴϲ <ůŝŶŐĞŶďĞƌŐ͕ Z͘ et al. ĞƉůĞƟŽŶ ŽĨ &KyWϯн ƌĞŐƵůĂƚŽƌǇ d ĐĞůůƐ ƉƌŽŵŽƚĞƐ hypercholesterolemia and atherosclerosis. J Clin Invest ϭϮϯ, 1323-1334, ĚŽŝ͗ϭϬ͘ϭϭϳϮͬ:/ϲϯϴϵϭ;ϮϬϭϯͿ͘

ϴϳ &ŽŬƐ͕͘͘ et al. ŝīĞƌĞŶƟĂůĞīĞĐƚƐŽĨƌĞŐƵůĂƚŽƌǇdĐĞůůƐŽŶƚŚĞŝŶŝƟĂƟŽŶĂŶĚ regression of atherosclerosis. Atherosclerosis Ϯϭϴ͕ ϱϯͲϲϬ͕ ĚŽŝ͗ϭϬ͘ϭϬϭϲͬũ͘ ĂƚŚĞƌŽƐĐůĞƌŽƐŝƐ͘ϮϬϭϭ͘Ϭϰ͘ϬϮϵ;ϮϬϭϭͿ͘

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