TLR9 Activation as Immunotherapy in a Murine Model of Metastatic Lymphangioleiomyomatosis

Abstract

Pulmonary Lymphangioleiomyomatosis (LAM) is a slow progressing, metastasizing neoplasm primarily affecting women of reproductive age, marked by abnormal growth of smooth muscle-like cells leading to cystic lung destruction. Rapamycin, the only approved treatment for LAM, slows disease progression but ~40% of patients have partial or no response to treatment. There is an urgent need for new treatments. Research shows that LAM has hallmarks of cancer, like expression of immune checkpoint receptors, and is responsive to immune checkpoint inhibition in mouse models. This suggests that other anti-cancer strategies could be effective in treating LAM. In this thesis, we investigated toll like receptor (TLR) activation using intranasal administration of CpG, a TLR9 agonist, as LAM immunotherapy. We used a mouse model of metastatic LAM to determine survival after biweekly intranasal CpG therapy (10µg/ 5µg) with and without systemic α-PD-1, rapamycin, or α-CD317 therapy. We used ELISA to measure the cytokine profile and flow cytometry to quantify cell populations and characterize differences in the immune response between CpG-treated and untreated LAM lungs. We found that CpG treatment enhanced median survival from 32 to 60 days in murine LAM. Survival benefit of CpG treatment was inversely dose-dependent and more effective during early stages of disease. CpG-treatment was synergistic with both α-PD-1 checkpoint inhibition and rapamycin, with survival increasing from 60 days (CpG) to 71 days (CpG + α-PD-1) and 100 days (CpG + Rapamycin). Histological analysis showed that CpG treatment decreased the LAM nodule burden but inevitably caused tissue inflammation. Efficacy of CpG treatment in LAM is facilitated in part by plasmacytoid dendritic cells through decreased regulatory T cell numbers, priming of Th17 cells, and increased secretion of inflammatory and cytotoxic cytokines by CD8 T cells. Our findings suggest that adjuvant immunotherapy, like CpG, may offer new treatment strategies for LAM that are compatible with the current standard of care, rapamycin.