Background Pancreatic ductal adenocarcinoma (PDAC) remains immunologically intractable, owing to a complicated tumor microenvironment (TME). We have previously demonstrated that PDAC secreted factors antagonize the activity of healthy γδ T cells, which are disproportionately present in human PDAC samples. Here, we sought to investigate the underlying mechanism of γδ T dysfunction, characterizing a role for decreased cholesterol uptake induced by PDAC secretome, while also identifying potentially useful modulators of this signaling that reverse PDAC triggered γδ T dysfunction through restoration of NKG2D expression.

Methods γδ T cells were obtained from healthy, consenting donors and exposed to conditioned media from patient-derived PDAC organoids. After 12 hours of PDAC secretome exposure, γδ Ts were pulsed with BODIPY labeled LDL and assessed via flow cytometry for LDL uptake. Separately, γδ Ts were co-cultured with patient-derived PDAC organoids at an effector:target (E:T) of 5:1 for 48 hours. After this co-culture, apoptosis induction was measured by assessing the percentage of organoid area positive for a cleaved caspase 3/7 dye. Organoids were either incubated with γδ T alone + DMSO vehicle control, or γδ T + SR9243 (a small molecule inverse agonist of the LXR transcription factor), with or without addition of an NKG2D blocking antibody.

Results Culture of γδ T cells in the presence of conditioned media from two separate patient derived PDAC organoid lines significantly diminishes γδT uptake of LDL in the microenvironment (p<0.005; n= 3 biological replicates), with a ~22% decrease (PDAC 1) and 60% decrease in LDL uptake (PDAC 2). γδT exhibit increased killing of PDAC organoids in the presence of SR9243, as measured by apoptotic induction (cleaved caspase 3/7), which was attenuated with the addition of an NKG2D blocking antibody. We demonstrate that SR9243 increases expression of NKG2D, which is essential for γδ T mediated killing of PDAC organoids.

Conclusions The harsh TME in PDAC includes secreted factors that signal through the LXR transcription factor axis, decreasing the fitness of infiltrating lymphocytes. γδT dysfunction is associated with impaired LDL uptake after exposure to PDAC secretome, which can be modulated through addition of SR9243, an LXR inverse agonist. In an ex vivo patient derived model system, γδT tumor killing is dependent on NKG2D expression, which is enhanced through modulation of LXR. These findings may suggest new approaches to overcome the challenging TME present within PDAC to advance next generation immunotherapies.

Ethics Approval Informed consent was recieved from donors for both blood draws and patient biopsies used to develop 3D cancer organoid models. IRB #: 2017-1070.