Cannabis suppresses antitumor immunity by inhibiting JAK/STAT signaling in T cells through CNR2

THC suppresses T-cell immunity against cancer

Since cannabis has anti-inflammation properties, and THC is the key ingredient of cannabis,18 we hypothesized that it might affect the therapeutic efficacy of immunotherapy. Consistent with previous reports,19 we found that THC could inhibit the growth of tumor cells highly expressing CNR2 (Supplementary Fig. S1a, b). To avoid the effect of THC on tumor cells, we used tumor cell lines expressing low levels of CNR2 for the following experiments.

Mice bearing MC38 colon carcinoma or B16 melanoma were treated with PD-1 antibody, THC, or the combination of these two drugs. Tumor growth was measured every other day. Similar to other reports,20 MC38 tumors demonstrated a better response to PD-1 blockade than B16 tumors (Fig. 1a, b). Compared to DMSO controls, THC treatment significantly accelerated tumor growth in these two tumor models. Moreover, the therapeutic effect of PD-1 antibody was dramatically reduced in the combination groups, suggesting that the administration of THC might impair the antitumor immune response triggered by the PD-1 blockade. We then analyzed T-cell immune response in these groups. Consistent with the significant therapeutic effect in the PD-1 antibody group, an increased ratio of CD8+ T cells was observed in these tumors (Fig. 1c). However, the combination with THC diminished the effect of PD-1 blockade on both CD4+ T and CD8+ T cells while leading to a significant decrease of CD8+ T cells. The activity of tumor-infiltrating T cells was evaluated by in vitro activation using PMA plus Ionomycin for 4 h. While the highest production of IFN-γ was observed in T cells from tumors treated with PD-1 antibody, a significant reduction in the combination group (Fig. 1d). These data indicated that THC suppressed T-cell-mediated antitumor immunity decrease the effect of PD-1 blockade. Moreover, the therapeutic effect of PD-1 blockade was still suppressed by THC in mice depleted macrophages or B cells (Supplementary Fig. S1c, d), suggesting that THC mainly impaired T-cell immunity against cancer.

Fig. 1 THC suppresses T-cell immunity against cancer. Mice bearing MC38 (a) or B16 (b) tumors were treated with DMSO, THC, PD-1 antibody, or THC plus PD-1 antibody on day 10 after tumor inoculation. Tumor volumes were measured every other day (two-way ANOVA, mean ± SEM; *P < 0.05, and **P < 0.01). c The percentages of CD3+ T cells, CD4+ T cells and CD8+ T cells were analyzed by flow cytometry in the B16 tumors on day 17. d The expression of INF-γ in T cells isolated from tumor was detected by intracellular staining after in vitro re-stimulation with PMA and Ionomycin for 4 h. Statistical analysis was performed on biological replicates, ordinary one-way ANOVA, mean ± SD; *P < 0.05, and **P < 0.01. e, f Wild-type CD8+ T cells were pretreated with different concentrations of THC and anti-CD3 (5 μg/ml) plus anti-CD28 (5 μg/ml) simultaneously for 48 h. Proliferation was determined by CFSE dilution assay (e) and the production of IFN-γ and TNF-α was detected by intracellular staining (f). one-way ANOVA, mean ± SD. **P < 0.01. Data are representative of three independent experiments. g, h 6–10 weeks old C57BL/6J mice were subcutaneously engrafted with 105 B16-OVA tumor cells in 200 μl PBS. 10 days later, 1 × 106 OT-I (CD45.1+) T cells were transferred intravenously through tail veins, and THC was intraperitoneally injected on day 12, 14 and 16. Tumor growth was measured every other day (g), and the frequencies and numbers of OT-I T cells and the production of IFN-γ in OT-I T cells after in vitro activation were measured by flow cytometry (h). Two-tailed unpaired Student’s t-test, mean ± SD; *P < 0.05, **P < 0.01. Data are representative of three independent experiments. Full size image

We further examined the impact of THC on T-cell activation in vitro. CD8+ T cells isolated from wild-type C57BL/6J mice were pretreated with THC for 12 h in medium and then activated with CD3/CD28 antibodies for 48 h. Consistent with the previous study, we found that THC significantly inhibited the proliferation of CD8+ T cells, as detected by the CFSE dilution assay (Fig. 1e). Reduced expression of TNF-α and IFN-γ was also observed in T cells pretreated with THC (Fig. 1f). These data showed that THC inhibited both proliferation and function of T cells during activation in vitro.

Next, we studied the effect of THC on tumor-specific T cells by using the OT-I/B16-OVA mouse model, in which OT-I T cells specifically recognize the surrogate tumor antigen ovalbumin (OVA) expressed in B16 melanoma cells. CD45.2+ C57BL/6J mice bearing B16-OVA melanoma were adoptively transferred with CD45.1+ OT-I T cells via tail vein injection and then treated with THC or DMSO as control. Similar to its effect in the PD-1 antibody treatment, administration of THC significantly diminished the therapeutic effect of the adaptively transferred OT-I T cells (Fig. 1g). A dramatic reduction in the number and function of OT-I T cells was observed in THC-treated tumors (Fig. 1h). These data further demonstrated that THC suppressed T-cell immunity against cancer.

Endocannabinoid AEA inhibits function and proliferation of CD8+ T cells

ECS has been shown to be involved in the control of chronic inflammatory injury21,22 suggesting that it may suppress T-cell immunity as well. Indeed, we found that AEA (an endocannabinoid) treatment significantly promoted tumor growth and diminished the therapeutic effect of PD-1 antibody both in MC38 (Fig. 2a) and B16 (Fig. 2b) mouse models. CD8+ T cells pretreated with AEA also showed decreased proliferation and reduced production of IFN-γ and TNF-α during in vitro activation by CD3/CD28 antibodies (Fig. 2c–e). We further investigated the impact of AEA on T-cell-mediated antitumor immunity by using the OT-I/B16-OVA model. CD45.1+ OT-I T cells were intravenously injected into CD45.2+ mice bearing B16-OVA tumors of similar size and then treated with AEA or DMSO as control. Similar to the results of the THC treatment above, significantly accelerated tumor growth was observed in mice treated with AEA (Fig. 2f). Dramatic reduction in the percentage and activities of OT-I T cells were also found in AEA-treated tumors (Fig. 2g, h). These data indicated that both cannabis-derived and endogenous cannabinoids could suppress T-cell-mediated antitumor immunity.

Fig. 2 Endocannabinoid AEA inhibits function and expansion of CD8+ T cells. Mice bearing MC38 (a) or B16 (b) tumors were treated with DMSO, AEA, PD-1 antibody, or AEA plus PD-1 antibody on day 10 after tumor inoculation. Tumor volumes were measured every other day (two-way ANOVA, mean ± SEM, *P < 0.05, and **P < 0.01). Wild-type CD8+ T cells were stimulated with 5 μg/ml plate-bound anti-CD3 and anti-CD28 antibodies, and were incubated with different concentrations of endocannabinoid AEA simultaneously for 48 h. c The proliferation of CD8+ T cells was measured by CFSE dilution. d, e The production of IFN-γ and TNF-α cytokines in CD8+ T cells were detected by intracellular staining (mean ± SD, *P < 0.05, **P < 0.01). Statistical significance was assessed by ordinary one-way ANOVA. Data are representative of three independent experiments. B16-OVA tumors were established subcutaneously in 6–10 weeks old C57BL/6J mice 10 days before adoptive cell transfer of 1× 106 OT-I T cells (CD45.1+) and AEA was intraperitoneally injected on day 12, 14, and 16. f Tumors were measured every 2 days and the volume was calculated. Data in bar graphs represent mean ± SEM, three independent experiments were performed. g, h The frequencies and numbers of OT-I T cells in tumors and the production of IFN-γ in OT-I T cells from tumor after in vitro activation with PMA and Ionomycin were shown. i Kaplan–Meier estimates of overall survival comparing high to low levels of AEA in serum of lung cancer patients measured by ELISA. Data are shown as mean ± SEM; log-rank test. j Representative IHC images of CNR2high and CNR2low tumor sections stained with CNR2 (left). Scale bars correspond to 100 μm. Kaplan–Meier estimates of patients’ overall survival comparing high to low expression of CNR2 (right). Statistical significance was assessed by the log-rank (Mantel–Cox) test of survival curve. Full size image

Having found that AEA could impair the antitumor immunity, we further checked if the AEA levels would affect disease progression in cancer patients. The AEA levels in the sera of 170 lung cancer patients were measured by ELISA. The patients were divided into high and low groups by the median level of AEA. Compared to the low-level group, patients with high levels of AEA showed worse overall survival (Fig. 2i, Table 1). We then examined if the expression of CNR2, a receptor of AEA, also affects disease progression in these patients. The CNR2 expression was measured by IHC (Fig. 2j, left). We found that high expression of CNR2 was associated with worse overall survival in these patients (Fig. 2j, right). These data suggested that the ECS could promote tumor progression through the inhibition of antitumor immunity.

Table 1 Clinical characteristics for the SYSUCC lung cancer cohort Full size table

Cannabinoids impair T-cell-mediated antitumor immunity through CNR2

CNR2 is thought to be the receptor mediating the immune function of ECS due to its primary expression in immune cells.23 Moreover, THC and AEA are selective agonists of CNR2.24 We suspected that these two cannabinoids suppress the antitumor immunity through CNR2. To investigate the role of CNR2 in the antitumor immune response, we generated a Cnr2-2×Flag-IRES-Egfpflox/flox knock-in mouse line that expresses FLAG-tagged Cnr2 with an EGFP reporter, and the second exon of the Cnr2 gene was floxed (termed as Cnr2GFP). These mice were crossed with CD4Cre mice to generate mice with conditional knockout of Cnr2 in T cells (termed as Cnr2CKO, Fig. 3a, and Supplementary Fig. S2) and Cnr2GFP mice were served as littermate control.

Fig. 3 Cannabinoids impair T-cell-mediated antitumor immunity through CNR2. a Schematic diagram depicting the strategy used to generate Cnr2 condition knockout (Cnr2CKO, CNR2flox/floxCD4cre) mice (E: exon). LoxP sites flanking exon 2 of Cnr2 are indicated. Cnr2-2xFlag-IRES-Egfpflox/flox mice (Cnr2GFP) were crossed with CD4cre mice to delete the second exon of Cnr2. b Flow cytometry analysis of CNR2 expression from CNR2-GFP reporter mice in immune cell subsets, showing B cell (CD45+CD3−CD19+), T-cell (CD45+CD3+CD4+/CD8+), macrophage (CD45+CD3-F4-80+) and NK cell (CD45+CD3−NK1.1+). Cells from wild-type C57BL/6J mice were served as the negative control. Cnr2GFP and Cnr2CKO CD8+ T cells were treated with AEA or THC and stimulated by anti-CD3 plus anti-CD28 for 48 h. DMSO was used as the negative control. c Proliferation of CD8+ T cells was determined by CFSE dilution assay. d, e The production of IFN-γ and TNF-α in Cnr2GFP and Cnr2CKO CD8+ T cells were measured by intracellular staining (two-way ANOVA, mean ± SD, **P < 0.01). Full size image

We first checked the expression of CNR2 by the EGFP reporter in different lineages of immune cells. As shown in Fig. 3B, majority of the CD8+ T cells and B cells expressed high levels of CNR2 while low expression was observed in CD4+ T cells and NK cells. Macrophages had variable levels of CNR2.

We then examined if Cnr2 deficiency affected T-cell development. Compared to the Cnr2GFP controls, increased percentages and numbers of CD4 and CD8 single-positive subsets were observed in the thymus of Cnr2CKO mice, while the double-positive cells were slightly decreased. Similar phenotypes were observed in the spleen. In the mesenteric lymph node, only CD8+ T cells showed increased numbers in Cnr2CKO mice. These data suggested that Cnr2 ablation promoted T-cell development (Supplementary Fig. S3). We first checked the early development of T cells in the thymus. The numbers of DN1 (CD44+CD25−), DN2 (CD44+CD25+), and DN3 (CD44−CD25+) cells were not altered while DN4 cells (CD44−CD25−) were slightly reduced in the thymus of Cnr2CKO mice compared to Cnr2GFP mice (Supplementary Fig. S4a), which indicated that Cnr2 was not critical for the early development of T cells in the thymus. We further examined the four distinct developmental stages defined by the expression of TCRβ and the activation marker CD69. Although the portions of TCR-CD69- cells were slightly decreased, more TCR+CD69+ cells and TCR+CD69− cells were observed in Cnr2CKO mice compared to Cnr2GFP mice (Supplementary Fig. S4a), indicating that Cnr2 deficiency promoted the positive selection of T cells in the thymus and exported more T cells to the periphery. Expression of activation markers such as CD69 and CD25 was comparable on CD8+ T cells in aged Cnr2CKO and Cnr2GFP mice (Supplementary Fig. S4b). Histological analysis showed no inflammation in the organs of these aged mice in either group (Supplementary Fig. S4c). These data indicated that the negative selection in the thymus of Cnr2CKO mice was normal.

Since increased T-cell numbers were observed in the periphery, we also examined the homeostasis of Cnr2CKO and Cnr2GFP T cells. Cnr2GFP (CD45.1+CD45.2+) and Cnr2CKO (CD45.2+) CD8+ T cells were mixed in a ratio of 1:1, and then transferred into Rag2−/− recipient mice (CD45.1+) (Supplementary Fig. S5a). The frequencies of these two kinds of T cells in the lymph node (LN) and spleen (SP) were measured in 7 days. We found that the ratios of Cnr2CKO to Cnr2GFP T cells were slightly increased, which indicated that Cnr2 ablation increased T-cell homeostasis (Supplementary Fig. S5b).

Next, we investigated if THC and AEA would suppress T-cell activation through CNR2. CD8+ T cells isolated from Cnr2GFP or Cnr2CKO mice were pretreated with THC or AEA for 12 h and then stimulated with CD3/CD28 antibodies for 48 h. Although pretreatment of THC or AEA both significantly inhibited the proliferation and function of wild-type Cnr2GFP CD8+ T cells, these two cannabinoids did not affect the proliferation and the IFN-γ and TNF-α production of Cnr2CKO CD8+ T cells, indicating that cannabinoids inhibited T-cell function through CNR2 (Fig. 3c–e).

Surprisingly, in the DMSO treated groups, significantly increased proliferation and higher levels of IFN-γ and TNF-α were observed in the Cnr2CKO T cells, compared to Cnr2GFP T cells, suggesting that CNR2 suppressed T-cell function. Similar results were observed when these T cells were activated in vitro without DMSO (Supplementary Fig. S6). These data supported that CNR2 itself could directly inhibit T-cell proliferation and function.

Cnr2 deficiency promotes T-cell-mediated antitumor immunity

Given that CNR2 inhibited T-cell activation in vitro, we wondered whether it would affect antitumor immunity in vivo. To this end, we employed three tumor models, MC38, B16, and LLC. Compared to wild-type mice, all of these three kinds of tumors showed slower growth and prolonged survival in Cnr2CKOmice (Fig. 4a–c). The frequency of CD8+ T cells was significantly increased in the tumors from Cnr2CKO mice (Fig. 4d). Moreover, T cells isolated from tumors of Cnr2CKO mice produced more IFN-γ during the activation in vitro (Fig. 4e). The expression of exhaustion markers such as PD-1, LAG3, and CD39 was also reduced in T cells from tumors of Cnr2CKO mice compared to wild-type mice (Fig. 4f). These data indicated that Cnr2 deficiency enhanced the antitumor function of T cells, thus inhibiting tumor growth.

Fig. 4 CNR2 facilitates tumor development by suppressing immune response. Tumor growth and survival were assessed in B16 (a), MC38 (b) and LLC (c) models in Cnr2GFP and Cnr2CKO mice. Data are representative of three independent experiments. Statistical significance was assessed by ordinary one-way ANOVA or log-rank (Mantel–Cox) test of survival curve, mean ± SEM. **P < 0.01. d Flow cytometric analysis of CD4+ and CD8+ T cells of Cnr2GFP and Cnr2CKO mice bearing B16 tumor. e The production of IFN‐γ in CD8+ T cells from B16 tumors was assessed by flow cytometric analysis. Data are representative of three independent experiments. Statistical significance was assessed by two-way ANOVA (d) or two-tailed unpaired Student’s t-test (e), mean ± SD, *P < 0.05, **P < 0.01. f Flow cytometry (left) and quantification (right) of PD-1, LAG3, and CD39 positive cells in Cnr2GFP and Cnr2CKO OT-I cells from B16-OVA tumors. Two-tailed unpaired Student’s t-test, mean ± SD, *P < 0.05, **P < 0.01. Full size image

We then examined the effect of CNR2 on tumor-specific T cells in the OT-I/B16-OVA model. Cnr2GFP OT-I (CD45.1+CD45.2+) and Cnr2CKO OT-I (CD45.2+) T cells were mixed in a ratio of 1:1, and then adoptively transferred into C57BL/6J recipient mice (CD45.1+) with established subcutaneous B16-OVA tumors (Fig. 5a). The frequencies of these two kinds of OT-I T cells in B16-OVA tumors were measured 5 days after transfer. More Cnr2CKO OT-I cells were found in the tumors of recipient mice. The ratio of Cnr2CKO to Cnr2GFP OT-I cells was also significantly increased (Fig. 5a). In addition, Cnr2-deficient OT-I T cells produced more IFN-γ than their Cnr2GFP counterparts (Fig. 5b). These data demonstrated that Cnr2 deficiency in tumor-specific T cells enhanced their expansion and function in the tumor microenvironment.

Fig. 5 Cnr2 deficiency promotes T-cell-mediated antitumor immunity. a Cnr2GFP (CD45.1+ CD45.2+) and Cnr2CKO (CD45.2+) OT-I CD8+ T cells were 1:1 mixed and intravenously injected into mice (CD45.1+) with B16-OVA tumor. Flow cytometric analysis shows the frequencies of Cnr2GFP and Cnr2CKO OT-I T cells from TILs in tumors (left). The representative ratio of wild-type to Cnr2CKO OT-I T cells in the tumor were evaluated on day 5 (right). b Flow cytometry analysis of the production of IFN‐γ in Cnr2GFP and Cnr2CKO OT-I T cells isolated from tumors stimulated with PMA and Ionomycin in vitro. Data are representative of three independent experiments. Statistical significance was assessed by two-tailed unpaired Student’s t-test, mean ± SD. *P < 0.05 and **P < 0.01. c Mice bearing B16-OVA tumors were treated with 1 × 106 Cnr2GFP or Cnr2CKO OT-I T cells. PBS was used as control. Tumor volume was measured every other day (mean ± SEM, **P < 0.01) (Left). The survival curves of the three groups were compared (right). Statistical significance was assessed by the two-way ANOVA (left), or log-rank (Mantel–Cox) test of survival curve (right). d Mice bearing B16-OVA tumors were treated with 1 × 106 Cnr2GFP, Cnr2CKO OT-I T cells, Cnr2GFP OT-I T cells plus THC and Cnr2CKO OT-I T cells plus THC. Tumor volume were measured every other day (mean ± SEM, **P < 0.01) (left). The survival curves of the three groups were compared (right). Statistical significance was assessed by the two-way ANOVA (left), or log-rank (Mantel–Cox) test of survival curve (right). e T cells were transduced for 48 h by lentivirus and positive cells were sorted by flow cytometry. The expression of CNR2 was validated by qPCR and Western blotting. f Mice bearing B16-OVA tumors were treated with 1 × 106 shLacZ or shCnr2 OT-I T cells. PBS was used as control. Tumor volume was measured every other day (mean ± SEM, **P < 0.01). The survival curves of the three groups were compared. Statistical significance was assessed by the two-way ANOVA, or log-rank (Mantel–Cox) test of survival curve. g The frequencies of OT-I T cells in tumors were shown. h Flow cytometry analysis of the production of IFN-γ in shLacZ and shCnr2 OT-I T cells isolated from tumors stimulated with PMA and Ionomycin in vitro. Statistical significance was assessed by two-tailed unpaired Student’s t-test, mean ± SD, **P < 0.01. Full size image

Having found that Cnr2 deficiency enhanced the activities of tumor-specific T cells, we questioned whether these T cells could have superior therapeutic efficacy than wild-type T cells in tumor treatment. Mice bearing B16-OVA tumors were treated with Cnr2CKO or Cnr2GFP OT-I T cells through adoptive transfer. As expected, mice treated with Cnr2CKO OT-I T cells showed much slower tumor growth and longer survival than the Cnr2GFP OT-I T-cell-treated group (Fig. 5c). Moreover, THC administration impaired the efficacy of the Cnr2GFP OT-I cells but did not affect the efficacy of the Cnr2CKO OT-I T cells in treating B16-OVA tumors (Fig. 5d). These data suggested that targeting Cnr2 in T cells could be a potential approach to improve the efficacy of T-cell adoptive transfer therapy.

Since Cnr2 deficiency slightly affects T-cell development, we further used shRNA to knockdown Cnr2 in WT OT-I T cells, and then examined their function in B16-OVA tumors. Both qPCR and Western blotting showed that the Cnr2 shRNA-2 efficiently knocked down the expression of CNR2 in T cells (Fig. 5e). Mice bearing B16-OVA tumors were then treated with OT-I T cells traduced with Cnr2 shRNA-2 or control LacZ shRNA. Similar to Cnr2-deficient T cells, Cnr2 knockdown OT-I T cells significantly inhibited the growth of B16-OVA tumors and improved survival of tumor-bearing mice compared to the OT-I T cells transduced with LacZ shRNA (Fig. 5f). In addition, Cnr2 knockdown increased OT-I T cell numbers in tumors (Fig. 5g), and enhance the expression of IFN-γ in these T cells (Fig. 5h). These data indicated that Cnr2 knockdown enhanced the function of tumor-specific T cells in the tumor microenvironment.

Taken together, our results revealed that the ECS attenuated T-cell-mediated antitumor immunity through CNR2.

CNR2 binds to JAK1 and inhibits the STATs signaling

Finally, we addressed how CNR2 impaired T-cell activities. To identify the signaling pathways regulated by CNR2 in T cells, we performed an RNA-seq of Cnr2CKO and Cnr2GFP CD8+ T cells activated by anti-CD3/28 antibody for 24 h. Gene Ontology (GO) category analysis showed that T-cell differentiation and activation were the two most differentiated biological processes (Fig. 6a), consistent with the enhanced proliferation and effector function of Cnr2-deficient T cells. We further performed the Heatmap GO analysis on the Metascape website (http://metascape.org) to find out the key regulators. Interestingly, we found that two of the mainly affected genes were Stat1 and Stat3 (Fig. 6b). Gene Set Enrichment Analysis (GSEA) further indicated a strong activation of the JAK-STAT signaling pathway in Cnr2-deficient T cells (Fig. 6c). Additionally, the target genes of the JAK-STAT signaling including Ifnγ, Ifngr1, Il2, Akt2, Ccnd1, Ccnd2, and Socs1 were upregulated and Il6, Il10, or Socs3 were downregulated, validating the activation of the JAK-STAT signaling pathway in Cnr2-deficient T cells (Fig. 6d). Consistently, the expression of downstream genes such as Ifnγ, Il2, Ccnd2, and Socs1 was also suppressed by THC in Cnr2GFP T cells but not in Cnr2CKO T cells (Fig. 6d).

Fig. 6: CNR2 binds to JAK1 and inhibits the STATs signaling. CNR2 binds to JAK1 and inhibits the STATs signaling. a, b Gene Ontology (GO) category analysis and Heatmap GO analysis of RNA-seq data of Cnr2CKO and Cnr2GFP CD8+ T cells treated with anti-CD3 plus (5 μg/ml) anti-CD28 (5 μg/ml) for 24 h by using Metascape website (http://metascape.org). c GSEA analysis of the differentially expressed genes (RNA-seq datasets) of the JAK-STAT signaling pathway in Cnr2-deficient CD8+ T cells versus wild-type CD8+ T cells. d qPCR validation of the expression of genes downstream JAK-STAT signaling pathway in Cnr2-deficient and wild-type CD8+ T cells (Left), and in THC-treated Cnr2-deficient and wild-type CD8+ T cells (Right). Data are presented as the mean ± SD. of three biological replicates. **P < 0.01. e Mass spectrum analysis of CNR2 associated proteins in CD8+ T cells from Cnr2-2xFlag-IRES-Egfpflox/flox reporter mice after Flag pull-down assay. Six protein bands detected by silver-staining in the FLAG group but not in the IgG group were cut and performed mass spectrum analysis. f The top ten identified peptides were shown in the list. g Cnr2-2xFlag-IRES-Egfpflox/flox CD8+ T cells were treated with THC or DMSO as a control for 24 h. Cell lysates were then immunoprecipitated with Flag antibody and analyzed by immunoblot with anti-JAK1 and anti-Flag. h Cnr2GFP and Cnr2CKO CD8+ T cells were stimulated with anti-CD3 plus anti-CD28 for 10–30 min. Cell lysates were analyzed by immunoblot with anti-phosphorylated JAK1, anti-phosphorylated STAT1, anti-phosphorylated STAT3, anti-total STAT1, and anti-total STAT3. i Cnr2GFP and Cnr2CKO CD8+ T cells were pretreated with THC or DMSO for 24 h and then stimulated by anti-CD3 plus anti-CD28 for 30 min. Cell lysates were analyzed by immunoblot with anti-phosphorylated JAK1, anti-phosphorylated STAT1, anti-phosphorylated STAT3, anti-total STAT1, and anti-total STAT3. Full size image

To further explore the signaling pathways regulated by CNR2 in T cells, we employed immunoprecipitation followed by mass spectrometry to identify proteins interacting with CNR2. As mentioned above, the CNR2 protein was tagged by FLAG in the knock-in mouse line. We sorted CD8+GFP+ T cells from the spleen and lymph nodes of the knock-in mice and performed immunoprecipitation using FLAG antibody or control IgG (Fig. 6e). Differential bands were collected and mass spectrometry was performed. Consistent with the findings in RNA-seq, we found that JAK1 was one of the top proteins binding to CNR2, while the others belonged to unspecific cell adhesion proteins (Fig. 6f). We further confirmed the binding between CNR2 and JAK1 by co-immunoprecipitation assay (Fig. 6g).

Next, we compared the expression of pJAK1 or pSTAT1 and pSTAT3 in Cnr2CKO and Cnr2GFP control T cells during the activation in vitro. Compared to Cnr2GFP T cells, increased levels of phosphorylated JAK1, STAT1, and STAT3 proteins were observed in Cnr2CKO T cells in a time-dependent manner after the activation, while the levels of total JAK1 or STAT1 and STAT3 proteins remained unchanged (Fig. 6h).

We further checked if THC suppressed the JAK-STAT signaling in T cells through CNR2. Cnr2GFP cells and Cnr2CKO T cells were pretreated with or without THC and then activated by CD3 and CD28 antibodies for 30 min. THC treatment decreased the phosphorylation of STAT1 and STAT3 in Cnr2GFP T cells but not in Cnr2CKO T cells during activation (Fig. 6i). These data suggested that THC inhibited the JAK1-STAT1/3 signaling in T cells through CNR2.

Overall, our results indicated that the ECS impaired T-cell activity through the inhibition of the JAK1-STATs signaling in T cells.