Fig 2: LAMTOR1 peptide administration promotes anti-PD-1 therapy sensitivity in lung tumors. (A) The flowchart depicts the utilization of LAMTOR1 peptide and anti-PD-1 in the treatment of lung tumors. Starting from day 6, injections of LAMTOR1 peptide (50 mg/kg) and anti-PD-1 (200 μg) were administered every three and six days, respectively, until day 24. The injections were given via intravenous administration to the Control group, anti-PD-1 group, LAMTOR1 peptide group, and LAMTOR1 peptide + anti-PD-1 group. (B, C) Using the IVIS imaging system, it was observed that the combination of LAMTOR1 peptide and anti-PD-1 hindered the growth of lung tumors (n = 6/group). (D, E) Additionally, the inhibitory effect on lung tumor growth due to the combined treatment of LAMTOR1 peptide and anti-PD-1 was validated through HE staining, with scale bars representing 200 μm. (F) The expression levels of PD-L1 in lung tumors were evaluated through immunohistochemistry. The scale bars represent 200 μm. (G) Flow cytometry analysis indicated that the synergy between LAMTOR1 peptide and anti-PD-1 enhanced the activity of CD8+ T cells, proliferation (%CFSE−), and cytotoxicity (GzmB+). (H) The survival of mice within tumors (n = 6/group) was evaluated. (I) Examine the potential adverse effects of co-administering the LAMTOR1 peptide with immunotherapy, employing H&E staining to observe the treatment-induced damage to the lungs, liver, heart, kidney, and spleen, with scale bars representing 50 μm. The results are depicted as mean ± SEM from 3 assays, with statistical significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on 1-way ANOVA or log-rank test

Fig 3: The dynamic transcriptional characteristics of T cell subpopulations during the progression of GO.(A) Major T cell subtypes in the scRNA-Seq dataset. (B and C) All subtypes of CD4 T (B) and CD8 T cells (C) in both scRNA-Seq and scATAC-Seq datasets. (D and E) The relative abundance of clonotypes and distribution of the GO top 10 unique clonotype among CD4 T cell subtype (D) and CD8 T cell subtype (E). (F) The expression levels for the cytotoxicity functional genes in CD4 CTL. Data are represented as the median IQR. ****Padj < 0.00001 by Mann-Whitney U test. (G) Chromatin accessibility peak of GZMB and FGFBP2 in CD4 CTL. (H) qPCR showed the gene expression levels of GZMB, FGFBP2, CTSW, and PRF1 in CD4 CTL (Healthy, n = 6; GO, n = 6). Data are represented as the median IQR. **P < 0.001 by Mann-Whitney U test. (I and J) The scores of exhaustion score (I) and aTreg functional gene score (J) in aTreg; heatmap displayed the expression levels of representative functional genes. Data are represented as the median IQR. ****Padj < 0.00001 by Mann-Whitney U test. (K) The expression levels of the top 10 GO-specific TRAV and TRBV genes in aTreg. (L and M) The expression of TGF-β1 (L) and CTLA4 (M) from 3 groups; dot plots indicate the mean expression intensity and target cell proportions (Healthy, n = 9; GH, n = 9; GO, n = 9). Data are represented as the median IQR. ***Padj < 0.0001 by Mann-Whitney U test. *Padj < 0.05, **Padj < 0.001. (N–P) The proliferation (N), inhibition (O) and IFN-γ (P) production of CD8+ T cells co-cultured with Treg (Healthy, n = 5; GH, n = 5; GO, n = 5). Data are represented as the median IQR. *Padj < 0.05 by Mann-Whitney U test. The experiments were repeated 3 times.

Fig 4: Rationally designed LAMTOR1 peptide targets PD-L1 to lysosomal degradation. (A) The study generated a GFP fusion protein based on LAMTOR1 sequences. (B-D) Western blot analysis identified the expression of different GFP fusion proteins (GFP-S1, GFP-S2, and GFP-S3) in NSCLC cells (H1975 and H358). Additionally, Western blot results showed that GFP-S1 underwent degradation, which was inhibited by treatment with Bafilomycin A1 (Baf-A1). (E-H) Furthermore, western blot analysis demonstrated that the degradation of exosomal PD-L1 could be prevented by Baf-A1 treatment. Subsequently, NSCLC cells (H1975 and H358) were exposed to the LAMTOR1 peptide with or without 50 μM Baf-A1 treatment. (I, J) In a separate experiment, NSCLC cells (H1975 and H358) co-cultured with LAMTOR1 peptide and CD8+ T cells in Transwell chambers underwent analysis of CD8+ T cell proliferation (%CFSE−) and cytotoxic (%GzmB+) function were evaluated through flow cytometry (I), while IL-2, IFN-γ, and TNF-α expression levels in CD8+ T cells using RT-PCR (J). (K) Immunoblots were performed to assess exosomal PD-L1 levels in NSCLC cells (H1975 and H358) after PD-L1 overexpression. (L) The study also involved injecting C57BL6 mice (n = 6/group) with LLC cells (1 × 10^6) to generate subcutaneous tumors. After 21 days, the animals were euthanized, and tumor tissues were collected for volume analysis. (M) Moreover, C57BL6 mice (n = 10/group) received injections of LLC/PD-L1 cells (1 × 10^6) and were infected with either control or LAMTOR1 peptide on day 4. After 24 days, the animals were euthanized to collect tumor tissues for volume analysis. (N) The expression levels of PD-L1 in lung tumor was assessed using immunohistochemistry. Scale bars indicate 200 μm. The results are presented as mean ± SEM from six independent experiments, with statistical significance denoted by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) based on Student’s t-test