Nicotine exhausts CD8+ T cells against tumor cells through increasing miR-629-5p to repress IL2RB-mediated granzyme B expression

Abstract

The mechanism exhausting CD8⁺ T cells is not completely clear against tumors. Literature has demonstrated that cigarette smoking disables the immunological activity, so we propose nicotine is able to exhaust CD8⁺ T cells. The CD8⁺ T cells from healthy volunteers with and without cigarette smoking and the capacity of CD8⁺ T cells against tumor cells were investigated. RNAseq was used to investigate the gene profiling expression in CD8⁺ T cells. Meanwhile, small RNAseq was also used to search novel microRNAs involved in the exhaustion of CD8⁺ T cells. The effect of nicotine exhausting CD8⁺ T cells was investigated in vitro and in the humanized tumor xenografts in vivo. We found that CD8⁺ T cells were able to reduce cell viability in lung cancer HCC827 and A549 cells, that secreted granzyme B, but CD8⁺ T cells from the healthy cigarette smokers lost anti-HCC827 effect. Moreover, nicotine suppressed the anti-HCC827 effect of CD8⁺ T cells. RNAseq revealed lower levels of IL2RB and GZMB in the exhausted CD8⁺ T cells. We identified that miR-629-5p was increased by nicotine, that targeted IL2RB. Transfection of miR-629-5p mimic reduced IL2RB and GZMB levels. We further validated that nicotine reduced granzyme B levels using a nuclear imaging technique, and demonstrated that nicotine exhausted peripheral blood mononuclear cells against HCC827 growth in the humanized tumor xenografts. This study demonstrated that nicotine exhausted CD8⁺ T cells against HCC827 cells through increasing miR-629-5p to suppress IL2RB.

Keywords: CD8+ T cells; Granzyme B; IL2RB; Nicotine.

Fig. 1

Reduction of anti-tumor capacity in the CD8⁺ T cells treated with nicotine and from healthy smokers. a The CD8⁺ T cell isolated from a healthy volunteer without smoking (NS#1, Table 1) significantly reduced the cell viability of EGFR-positive lung cancer HCC827 and A549 cells. b The same effects of anti-HCC827 and A549 cells were observed in the isolated natural killer (NK) cells. c There were granzyme B (GZMB) secretion in the cultured medium from HCC827 and A549 treated with CD8⁺ T cells detected by ELISA technique, but no detection in the treatments with NK cells. d The cell viability of H520 cells, an EGFR-negative cell line, was reduced in CD8⁺ T cells treatment, but 1975 cells, a EGFR T790M cell line, were resistant to CD8⁺ T cells. e PD-L1 expression: H1975 > A549 > HCC827 > 520. Moreover, IFNr-induced PD-L1 expression in EGFR-positive lung cancers. f CD8⁺ T cells isolated from healthy smokers (S#1, S#2, and S#3, Table 1) lost anti-HCC827 effect compared to healthy non-smokers (NS#1, NS#2, and NS#3, Table 1). g CD16, an activated marker of CD8⁺ T cells, decreased in smokers compared to non-smokers, h but, there is no significant for PD-1 expression. i Meanwhile, we found that CD8⁺ T cells treated with 1 μg/mL of nicotine for 24 h lost the anti-HCC827 effect compared to non-treated CD8⁺ T cells. j Moreover, nicotine diminished the expression of CD16 in CD8⁺ T cells (n = 9), but increased PD-1 expression (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

IL2RB and GZMB decreased in nicotine-treated CD8⁺ T cells. a To investigate the mechanism exhausting CD8⁺ T cells against HCC827, RNAseq was performed to discover the potential gene-regulating GZMB expression. The differential genes were shown in CD8⁺ T cells between a smoker and a non-smoker, and b there were 423 gene upregulated and 698 genes downregulated in CD8⁺ T cells from a smoker based on log² fold changes  > 1 and  < −1 with p < 0.05. c The differential genes were consequently analyzed using NetworkAnalyst for selecting the potential driver genes, that validated GZMB decreased in the CD8⁺ T cells with smoking. d We also figured out that IL2RB, a receptor mediating IL2 signaling pathway, decreased in the CD8⁺ T cells with smoking after classifying the differential genes according to PANTHER classification. e IL2RB and GZMB were validated in the enrolled volunteers, it revealed that IL2RB decreased in CD8⁺ T cells with smoking. f Nicotine treatment for 24 h significantly decreased IL2RB (CD122) expression. g CD122 expressions were correlated with CD16 expressions in CD8⁺ T cells. h In addition, nicotine treatment significantly decreased IL2RB and GZMB expression detected using RT-qPCR

Fig. 3

Nicotine reduced IL2-mediated downstream gene expression in CD8⁺ T cells. a RNAseq was used to discover the IL2-mediated genes in CD8⁺ T cells, that the differential genes with log² fold change  > 2 after IL2 treatment for 2 h were shown. b The 26 genes, including GZMB, were analyzed using NetworkAnalyst, that revealing GZMB was associating with apoptosis function according to PATHER BP. c The IL2-mediated 26 genes were compared and overlapped with the genes downregulating in smoking CD8⁺ T cells. The results demonstrated there were 11 genes, including CISH, GZMB, CCL4, S100A1, NCR3, ANXA2, GZMH, LTA, LTB, CLIC1, and NKG7. d Consequently, qPCR was used to validate the gene expression in IL2-treated and e nicotine-treated CD8⁺ T cells. The results revealed nicotine reduced the IL2-mediated gene expression in CD8⁺ T cells, including CISH, GZMB, CCL4, GZMH, LTA, and LTB. *p < 0.05

Fig. 4

Nicotine increases miR-629-5p to reduce IL2RB in CD8⁺ T cells. a Small RNAseq analysis revealed the differential expression of microRNAs in CD8⁺ T cells from a healthy smoker compared to a non-smoker. b Among the increased microRNAs, miR-146-3p and miR-629-5p are selected according to matching by TargetScan that targets IL2RB. The predicted target genes by these two microRNAs contained 48 overlapping genes compared with downregulation genes in smoking CD8⁺ T cells (c) that were consequently analyzed using NetworkAnalyst and figured out IL2RB was the common gene. d miR-629-5p was validated in CD8⁺ T cells from smokers, higher than non-smokers. e Meanwhile, A549 co-culture increased miR-629-5p, and f decreased IL2RB expression in CD8⁺ T cells. g Nicotine was validated to increase miR-629-5p, it decreased IL2RB in CD8⁺ T cells. h The transfection of the miR-629-5p-labeled FAM in CD8⁺ T cells was 20.6%, i that reduced IL2RB expression. j Moreover, miR-629-5p mimic diminished CISH, GZMB, CCL4, S100A1, LTB, NKG7. k Transfection of miR-629-5p mimic reduced anti-HCC827 effect for CD8⁺ T cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Nicotine reduced healthy PBMCs-mediated granzyme B activity in the HCC827-derived humanized tumor xenografts. a, b The GZMB-binding peptide (GZP) labeled with radioactive In-111 through DTPA chelating was completed and c injected via tail vein into the HCC827-derived humanized tumor xenografts. The HCC827 cells were injected subcutaneously for 14 days (para-tumor), and then injected with isolated PBMCs from a healthy non-smoker (intra-tumor). d, e The results from real-time radioactive nuclear imaging revealed that nicotine treatment in PBMCs for 24 h significantly reduced the PBMCs-mediated granzyme B activity against para-tumors. *p < 0.05

Fig. 6

Nicotine reduced PBMCs-mediated anti-HCC827 effect in vivo. a There were three groups to investigate the effect of nicotine on reducing the anti-HCC827 effect of healthy PBMCs, including the HCC827-derived tumor xenografts (ctrl as para-tumor, n = 4), para-tumor with injected with PBMCs and HCC827 cells in the opposite flank leg (intra-tumor, n = 4), and the PBMCs pretreated with 1 μg/mL of nicotine for 24 h (n = 3). b, c The results indicated that PBMCs caused remarkable reductions of HCC827-derived tumors, including intra- and para-tumors. Meanwhile, PBMC pretreated with nicotine lost the anti-HCC827 capacity, resulting in tumor growth. *p < 0.05

Fig. 7

Schematic diagram illustrates the mechanism of nicotine to exhaust CD8⁺ T cells. a IL2 binds IL2R complex to induce granzyme B expression in the active CD8⁺ T cells. b Nicotine increases miR-629-5p to repress IL2RB expression, resulting in a reduction of IL2-mediated granzyme B levels and exhaustion of CD8⁺ T cells against tumors