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. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-024-01009-6.

Table S1.The plasmids and their characteristics used in the current study.a The plasmid is used as a backbone vector therefore there is no inserted DNA fragment.
b DAPI is a commercial product for nuclear staining.
c Species is not applicable to this antibody in such an application.

Figure S1 .
Figure S1.The expression of PD-1 and Rab37 exhibited positive correlation in T cells.The correlation of Pdcd1 mRNA expression with Rab37, Rab3a, Rab8a or Rab11b in patients with lung adenocarcinoma (n=483) and lung squamous cell carcinoma (n=486) (A-D) or in normal tissues (n=109) (E-H) from the GEPIA dataset.R square and P value by Pearson correlation coefficient are shown.(I) Jurkat T cells were transfected with Rab37, Rab3a, Rab8a, or Rab11b plasmid for 24h, respectively, and protein expression were detected by immunoblotting.OE: overexpression.

Figure S3 .
Figure S3.Glycosylation on PD-1 promotes protein stability and transport to the PM.(A) Schematic diagram of full-length PD-1.SP, signal peptide; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain.Four putative Asn-X-Ser/Thr motifs in the ECD domain are labeled in red.The numbers indicate the amino acid positions.We established

Figure S5 .
Figure S5.The serum biochemical parameters and major organ histology in LLC tumor-bearing Rab37 KO and WT mice.(A-B) Serum biochemical markers (A) and major organ histology (B) of treated mice examinations revealed no significant adverse effects between Rab37 KO and WT mice.Scale bar: 200 μm.The data are shown as the mean ± S.D. P values were determined by two-tailed Student's t test.* P<0.05, ** P<0.01, *** P<0.001.

Figure S6 .
Figure S6.The relationship between Rab37 expression and population of PD-1/TIM3/CD8 cells derived from PBMCs treated with CD3 and CD28 antibodies.Blue dot represents PBMCs from healthy donor (N=3).Black dot represents PBMCs from lung cancer patients (N=9).

Table S3 . Characteristics of NSCLC patients and normal individuals for ex vivo assays in the current study.
a Not applicable.