Data_Sheet_1_Toll-Like Receptor 4 Expression on Lymphoma Cells Is Critical for Therapeutic Activity of Intratumoral Therapy With Synthetic TLR4 Agonist Glucopyranosyl Lipid A.xlsx

Intratumoral (IT) injections of Glucopyranosyl lipid A (G100), a synthetic toll-like receptor 4 (TLR4) agonist formulated in a stable emulsion, resulted in T-cell inflammation of the tumor microenvironment (TME) and complete cure of 60% of mice with large established A20 lymphomas. Strong abscopal effects on un-injected lesions were observed in a bilateral tumor model and surviving mice resisted a secondary tumor challenge. Depletion of CD8 T-cells, but not CD4 or NK cells, abrogated the anti-tumor effect. Unexpectedly, TLR4 knock-out rendered A20 tumors completely non-responsive to G100. In vitro studies showed that GLA has direct effect on A20 cells, but not on A20 cells deficient for TLR4. As shown by genotyping and phenotyping analysis, G100 strongly activated antigen presentation functions in A20 cells in vitro and in vivo and induced their apoptosis in a dose dependent manner. Similarly, the TLR4 positive human mantle cell lymphoma line Mino showed in vitro activation with G100 that was blocked with an anti-TLR4 antibody. In the A20 model, direct activation of B-lymphoma cells with G100 is sufficient to induce protective CD8 T-cell responses and TLR4 expressing human B-cell lymphomas may be amenable to this therapy as well.

瘤内（Intratumoral, IT）注射的吡喃葡萄糖基脂质A（Glucopyranosyl lipid A, G100）是一种配制于稳定乳剂中的合成Toll样受体4（Toll-like receptor 4, TLR4）激动剂。该制剂可诱导肿瘤微环境（tumor microenvironment, TME）出现T细胞浸润性炎症，并使60%携带已形成大型A20淋巴瘤的小鼠获得完全治愈。在双侧肿瘤模型中，可观察到其对未注射瘤灶的显著远隔效应（abscopal effect）；存活小鼠可抵御继发肿瘤攻击。实验证实，清除CD8 T细胞（而非CD4 T细胞或自然杀伤细胞natural killer, NK）可完全抵消该抗肿瘤作用。令人意外的是，TLR4基因敲除可使A20肿瘤完全丧失对G100的应答能力。体外研究表明，GLA可对A20细胞产生直接作用，但对TLR4缺陷的A20细胞无此效应。经基因分型与表型分型分析证实，G100可在体内外显著激活A20细胞的抗原呈递功能，并以剂量依赖性方式诱导其凋亡。与之类似，TLR4阳性的人套细胞淋巴瘤细胞系Mino在体外可被G100激活，且该激活效应可被抗TLR4抗体阻断。在A20模型中，直接通过G100激活B细胞淋巴瘤细胞即可诱导保护性CD8 T细胞应答，而表达TLR4的人类B细胞淋巴瘤或许也可适用于该治疗方案。