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Cancer Technologies

Cancer Technologies - Poster Session E

Poster H4 - TSC-2 deficient Lymphangioleiomyomatosis Cells Modulate PD-L1 and MHC-I Expression on Lymphatic Endothelial Cells

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Lymphangioleiomyomatosis (LAM) is defined as a progressive lung disease that primarily affects women of reproductive age. It is characterized by the progressive development of abnormal smooth muscle cells in the lung which resembles a close characterization of cancer. LAM arises from inhibiting mutations in the tumor suppressor genes TSC1 and TSC2, which lead to uncontrolled activation of the mammalian target of rapamycin complex 1 (mTORC1). Recent research has also found that another characteristic LAM shares with cancer is the development of an immunosuppressive environment in the lung, to evade the immune response. LAM is associated with inducing lymphangiogenesis and LAM nodules might interfere with lymphatic flow, all of which could lead to changes in immunity. Although the full mechanism of how LAM uses the lymphatic system to evade the immune system is unclear, recent findings have shown that lymphatic endothelial cells (LECs) play a large role in modulating immunity through transport and direct interactions with immune cells. Through the production of immuno-regulatory cytokines, LECs can control the activation of antigen-presenting cells and express immuno-suppressive factors to modulate immune function. LECs have been known to express various cytokines such as TGF-β, IL-7, IDO, and surface/intracellular markers such as Ki67, PD-L1 MHC-I, and MHC-II. Therefore, we hypothesize that LECs could play a major role in contributing to immunosuppression in LAM disease through secretion of cytokines and changes in surface marker expression. Investigating the effect that LAM cells have on LECs will help to uncover the mechanisms of LAM-induced lymphangiogenesis and proliferation.

Materials and

Methods:  We cultured human dermal lymphatic endothelial cells in vitro to model the lymphatic endothelial cells (LECS) found in the structure of lymphatic vessels. To model LAM disease, human smooth muscle-like cells deficient in the TSC2 gene were cultured. We collected the supernatant containing any secreted factors from these LAM cells as LAM-conditioned media (LAM-CM). The LECs were then exposed to this LAM-conditioned media and LAM growth media (LAM-GM) (void of any cytokines) diluted 1:5 with LEC basal media (endothelial cell media w/o growth factors) for 24 hours to assess the effect of LAM secretions on LEC activity. We used flow cytometry to measure the viability and expression of MHC-I, PD-L1, MHC-II, and Ki-67 in LAM-exposed LECs. ELISA was used to measure levels of VEGF-C/D, TGF-β, and IL-7 in the media. FlowJo and GraphPad Prism softwares were used for data analysis.

Results, Conclusions, and Discussions: 

Results:
We initially assessed whether the LAM growth and LAM conditioned media used for culturing lymphatic endothelial cells (LECs) exhibited any cytotoxic effects on the LECs. Our data suggest that neither the LAM-conditioned media (LAM-CM) nor the LAM-growth media (LAM-GM) significantly impact LEC viability (Figure 1). Our results indicate a < 10% difference in viability between the treatment groups. These findings suggest that exposure to LAM media via a dilution of basal LEC media did not impact LEC viability. However, whether LAM- conditioned media encouraged proliferation of LECs in these treatment groups remains to be explored.
We further examined the impact of LAM cell secretions on MHC-1 expression in LECs. We found decreased MHC-1 expression in LECs following exposure to LAM-CM. Although LECs from all treatment groups were found to express MHC-1 (Figure 2), analysis of median fluorescence intensity (MFI) revealed a 50% reduction in surface MHC-1 expression in LAM-CM compared to the basal control (Figure 1). This suggests that LAM-secreted factors present in LAM-CM may be responsible for the observed decrease in LEC MHC-1 expression. PD-L1 expression was also measured in these samples. The same trend was observed where PD-L1 expression decreased by approximately 50% in LAM-CM compared to the controls (Figure 3). This suggests that LAM cells may modulate immune responses or encourage prolonged inflammation through decreased LEC expression of PD-L1. Further studies need to be done to determine what LAM cell-secreted factors contribute to these observed changes in LECs.

Conclusion:
Our data suggests LECs indirectly exposed to LAM cells have decreased expression of MHC-1 & PD-L1. These findings have implications for understanding how LAM disease might affect LEC structure and function and elucidating a mechanism for the characteristic pathophysiology of lymphangiogenesis in LAM.

Acknowledgements (Optional):