Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer.

BACKGROUND: Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of T cells. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). However, the mechanism of intracellular trafficking and plasma membrane presentation of PD-1 remains unclear. METHODS: Multiple databases of lung cancer patients were integratively analyzed to screen Rab proteins and potential immune-related signaling pathways. Imaging and various biochemical assays were performed in Jurkat T cells, splenocytes, and human peripheral blood mononuclear cells (PBMCs). Rab37 knockout mice and specimens of lung cancer patients were used to validate the concept. RESULTS: Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37 small GTPase to sustain T cell exhaustion, thereby leading to poor patient outcome. PD-1 colocalized with Rab37-specific vesicles of T cells in a GTP-dependent manner whereby Rab37 mediated dynamic trafficking and membrane presentation of PD-1. However, glycosylation mutant PD-1 delayed cargo recruitment to the Rab37 vesicles, thus stalling membrane presentation. Notably, T cell proliferation and activity were upregulated in tumor-infiltrating T cells from the tumor-bearing Rab37 knockout mice compared to those from wild type. Clinically, the multiplex immunofluorescence-immunohistochemical assay indicated that patients with high Rab37+/PD-1+/TIM3+/CD8+ tumor infiltrating T cell profile correlated with advanced tumor stages and poor overall survival. Moreover, human PBMCs from patients demonstrated high expression of Rab37, which positively correlated with elevated levels of PD-1+ and TIM3+ in CD8+ T cells exhibiting reduced tumoricidal activity. CONCLUSIONS: Our results provide the first evidence that Rab37 small GTPase mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion, and the tumor promoting function of Rab37/PD-1 axis in T cells of TME in lung cancer. The expression profile of Rab37high/PD-1high/TIM3high in tumor-infiltrating CD8+ T cells is a biomarker for poor prognosis in lung cancer patients.

Assessment of cellular responses in three-dimensional cell cultures through chemical exchange saturation transfer and 1 H MRS.

The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 9, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:9 on Wiley Online Library: https://doi.org/10.1002/nbm.4757.

Assessment of cellular responses in three-dimensional cell cultures through chemical exchange saturation transfer and 1 H MRS.

Metabolic responses to physiological changes have been detected using chemical exchange saturation transfer (CEST) imaging in clinical settings. Similarly to other MRI techniques, the CEST technique was based originally on phantoms from buffer solutions and was then further developed through animal experiments. However, CEST imaging can capture certain dynamics of metabolism that solution phantoms cannot model. Cell culture phantoms can fill the gap between buffer phantoms and animal models. In this study, we used 1 H NMR and CEST in a B0 field of 9.4 T to investigate HEK293T cells from two-dimensional (2D) cultures, three-dimensional (3D) cultures, and 3D cultures seeded with cell spheroids. Two CEST dips were observed: the magnitude of the amine dip at 2.8 ppm increased during the incubation period, whereas the hydroxyl dip at 1.2 ppm remained approximately the same or modestly increased. We also observed a CEST dip at 2.8 ppm from the 2D culture responding dramatically to doxorubicin treatment. By cross-validating with pH values and the concentrations of amine and hydroxyl protons extracted through 1 H NMR, we observed that they did not correspond to an increase in the amine pool. We believe that the denaturation or degradation of proteins from the fetal bovine serum increased the size of the amine pool. Although 3D culture conditions can be further improved, our study suggests that 3D cultures have the potential to bridge studies of solution phantoms and those on animals.