Upregulation of PD-L1 in Senescence and Aging

Abstract

Cellular senescence is a stable form of cell cycle arrest associated with proinflammatory responses. Senescent cells can be cleared by the immune system as a part of normal tissue homeostasis. However, senescent cells can also accumulate in aged and diseased tissues, contributing to inflammation and disease progression. The mechanisms mediating the impaired immune-mediated clearance of senescent cells are poorly understood. Here, we report that senescent cells upregulate the immune checkpoint molecule PD-L1, the ligand for PD-1 on immune cells, which drives immune cell inactivation. The induction of PD-L1 in senescence is dependent on the proinflammatory program. Furthermore, the secreted factors released by senescent cells are sufficient to upregulate PD-L1 in nonsenescent control cells, mediated by the JAK-STAT pathway. In addition, we show that prolongevity intervention rapamycin downregulates PD-L1 in senescent cells. Last, we found that PD-L1 is upregulated in several tissues in naturally aged mice and in the lungs of idiopathic pulmonary fibrosis patients. Together, our results report that senescence and aging are associated with upregulation of a major immune checkpoint molecule, PD-L1. Targeting PD-L1 may offer new therapeutic opportunities in treating senescence and age-associated diseases.

Keywords: PD-L1; SASP; aging; senescence.

FIG 1

Upregulation of PD-L1 in senescent IMR90 cells. (A) Primary IMR90 cells were left untreated or induced to senescence by various means. Replicative senescent cells were harvested 2 weeks after the cells reached cell cycle arrest. HRasV12 was expressed via retroviral vector and harvested 1 week postinfection. Etoposide (50 μM) was used to treat cells for 24 h, and the cells were harvested 1 week after the treatment. Ionizing irradiation (20 Gy)-treated cells were harvested 2 weeks after treatment. Verification of senescence is described under Materials and Methods. The cell lysates were analyzed by immunoblotting with indicated antibodies. Loss of lamin B1 and gain of p16 were used as markers for senescence. (B) Cells as in panel A were analyzed by reverse transcription-quantitative PCR (RT-qPCR) for PD-L1. The results were normalized to lamin A/C and presented as mean values with standard error of the mean (SEM; n = 3). The P values were calculated from two-tailed Student’s t test. (C) IMR90 cells were left untreated or induced to senescence by HRasV12 (1 week) or IR (2 weeks). The cells were fixed and stained with antibodies as indicated, followed by confocal microscopy imaging. The images were acquired under identical settings, under a 63× objective, and representative images are shown. β-Tubulin was used to label the cells. Bars, 20 μm. Arrows indicate PD-L1 at or in close proximity to the cell membrane. (Right) Quantification of cell membrane-localized PD-L1. The data are from four randomly selected fields with over 200 cells, quantified under a 10× objective. The results shown are mean values with standard deviation (SD). The P values were calculated from two-tailed Student’s t test. (D) Primary BJ fibroblasts were treated with etoposide (40 μM) and harvested at day 14 or were infected with HRasV12 and harvested at day 7. The lysates were analyzed by immunoblotting. (E) Low-passage-number proliferating IMR90 cells were left untreated or induced to quiescence by contact inhibition. Cells reaching 100% confluence were considered the start of contact inhibition and were harvested at indicated days. Lysates were subjected to immunoblotting. Phospho-Rb was used as a proliferation marker. (F, G) IMR90 cells were subjected to etoposide treatment (50 μM) (F) or HRasV12 infection (G). The cells were harvested at indicated time points and analyzed by immunoblotting. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IR, ionizing irradiation.

FIG 2

The senescence-associated secretory phenotype (SASP) program is required for the induction of PD-L1 in senescence. (A) IMR90 cells were subjected to short hairpin RNA (shRNA)-mediated gene inactivation with a nontargeting control (NTC) hairpin or hairpin targeting cGAS. (Left) Immunoblotting showing cGAS was successfully inactivated by shRNA. (Middle, right) Cells were subjected to ionizing irradiation (IR, 20 Gy) and analyzed at day 14. The conditional media were analyzed for interleukin-6 (IL-6) (middle), and cell lysates were analyzed for PD-L1 and lamin B1. (B) Cells as in panel A were subjected to immunofluorescence analyses of PD-L1, imaged under confocal microscopy. The images were acquired under identical settings, and representative images are shown. β-Tubulin was used to label the cells. Bars, 20 μm. Arrows indicate PD-L1 at or near the cell membrane. (C) Quantification of cell membrane-localized PD-L1. The data are from four randomly selected fields with over 200 cells. The results shown are mean values with SD. The P values were calculated from two-tailed Student’s t test. (D) Related to panel A, IMR90 cells stably expressing sh-NTC or sh-cGAS were subjected to etoposide treatment (50 μM) for 24 h. The cells were then cultured until day 10 and harvested for immunoblotting analyses. (E) IMR90 cells were subjected to shRNA-mediated gene inactivation of NF-κB p65 subunit and were treated with IR (20 Gy) for 2 weeks. Cell lysates were probed with indicated antibodies. (F, G) IMR90 cells were treated with IR (20 Gy) for 10 days and then treated with STING inhibitor H-151 (1 μM) for 4 days. The cells were analyzed by immunoblotting (F) or RT-qPCR analyses normalized to lamin A/C (G). The results shown are mean values with SEM (n = 3) The P values were calculated from two-tailed Student’s t test. (H) IMR90 cells were treated with p38 MAPK inhibitor SB203580 (20 μM) 4 days after IR, and the drug was replenished every other day. Cell lysates were subjected to immunoblotting with indicated antibodies.

FIG 3

The SASP upregulates PD-L1 through the JAK-STAT pathway. (A) The conditioned media from etoposide or HRasV12-induced senescent IMR90 cells were harvested and incubated with low-passage-number proliferating IMR90 cells. The protein lysates were analyzed for PD-L1. (B) The conditioned media were harvested from control cells, or IR-induced senescent cells with sh-NTC or cGAS knockdown, as described in the legend to Fig. 2A. These conditioned media were used to incubate low-passage-number proliferating IMR90 cells for 24 h. The cell lysates were probed for PD-L1. (C) IMR90 cells were induced to senescence with HRasV12 for 1 week. The cell lysates and the media were harvested and probed for indicated antibodies. (D) Low-passage-number proliferating IMR90 cells were stably inactivated with shRNA encoding NTC or two independent STAT3 hairpins. The cells were then incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were then analyzed with indicate antibodies. (E, F) Similar to panel D, low-passage-number proliferating IMR90 cells were stably inactivated with shRNA encoding NTC or two independent JAK2 hairpins. The cells were then incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were probed with indicated antibodies. (G) Cells as in panels D to F were analyzed by RT-qPCR. The results were normalized to lamin A/C and were presented as mean values with SEM (n = 3). The P values were calculated from two-tailed Student’s t test. (H, I) Low-passage-number proliferating IMR90 cells stably expressing shRNA against NTC, JAK1, or STAT1 hairpins were incubated with conditioned media from HRasV12-induced senescent IMR90 cells for 24 h. The cell lysates were probed for indicated antibodies. (J, K) IMR90 cells with indicated hairpins were subjected to IR (20 Gy) and cultured for 14 days. The cell lysates were analyzed as indicated.

FIG 4

Rapamycin downregulates PD-L1 of senescent cells. (A) Scheme of experimental plan. Low-passage-number IMR90 cells were infected with retroviral vector encoding HRasV12 to induce senescence. On days 7 and 8, the cells were treated with compounds and were harvested on day 9 for immunoblotting analysis of PD-L1. (B) Effects of compounds on PD-L1 analyzed by immunoblotting. The doses of compounds are indicated in the figure. (C, D) Rapamycin downregulates PD-L1 in senescent IMR90. (C) Scheme of experimental design. Rapamycin at 50 nM was used. (D) Immunoblotting of PD-L1. Phospho-S6 was shown to indicate that rapamycin successfully inhibited mTOR activity. (E) Cells were prepared as in panel C, fixed, and stained with antibodies as indicated, followed by confocal microscopy imaging. The images were acquired under identical settings, and representative images are shown. β-Tubulin was used to label the cells. Bars, 20 μm. Arrows indicate PD-L1 at or near the cell membrane. (F) Quantification of cell membrane-localized PD-L1. The data are from four randomly selected fields with over 200 cells. The results shown are the mean values with SD. The P values were calculated from two-tailed Student’s t test. (G) Cells as in panel C were analyzed by RT-qPCR with indicated primers. The results were normalized to lamin A/C and were presented as mean values with SEM (n = 3). The P values were calculated from two-tailed Student’s t test. (H) Scheme of experimental design. IMR90 cells were challenged with ionizing irradiation (IR, 20 Gy). On day 14, 50 nM rapamycin was added to the media and was replenished daily. The cells were then harvested at day 18 for analyses. (I, J) Cells treated as in panel H were analyzed for PD-L1 by immunoblotting (I) or by RT-qPCR (J). RT-qPCR results were normalized to lamin A/C and were presented as mean values with SEM (n = 3). The P values were calculated from two-tailed Student’s t test. (K) Quantification of cell membrane-localized PD-L1. The data are from four randomly selected fields with over 200 cells. The results shown are mean values with SD. The P values were calculated from two-tailed Student’s t test.

FIG 5

Upregulation of PD-L1 in aging. (A) PD-L1 (CD274) RNA levels in mouse tissues from the Tabula Muris Senis database. The normalized read counts (linear) were used to generate the results. The mean values with SEM are shown. (B) PD-L1 (CD274) RNA levels from Benayoun et al. (42). The transcriptome sequencing (RNA-seq) data were downloaded, and the number of cpm was used. The mean values with SEM are shown. (C) Mice that were 2 to 3 months or 24 to 27 months old were euthanized, and tissues were harvested. The RNA from these tissues were extracted and subjected to RT-qPCR analyses for PD-L1 (CD274) gene. The results were normalized to GAPDH. The mean values with SEM are shown. The P values were calculated from two-tailed Student’s t test.

FIG 6

Upregulation of PD-L1 in interstitial lung disease (ILD). (A) UMAP expression plots of PD-L1 (CD274), p16 (CDKN2A), p15 (CDKN2B), p21 (CDKN1A), and IL-8 (CXCL8) in merged healthy and ILD human lungs. The data were reanalyzed from Habermann et al. (49). Higher-magnification inset panels below each gene expression are shown to highlight PD-L1 high cells. (B) Bar plot showing relative cell number contributions of healthy control donors versus ILD patients to either cluster 1 or 2. (C) Bar plot showing relative contribution to the expression of PD-L1 from healthy control donors versus ILD patients in cluster 1 or 2. (D) GSEA of the cluster 1 (aberrant basal cells) and cluster 2 (AT2 cells) using MSigDB signatures demonstrating that both clusters 1 and 2 are enriched in inflammatory responses (Zhou_Inflammatory_Response_Live_Up), while only cluster 1 is enriched in senescence signatures (Friedman_Senescent_Up).