Abstract
Tumor-infiltrating lymphocyte (TIL) deficiency is the most conspicuous obstacle to limit the cancer immunotherapy. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 antibody, have achieved great success in clinical practice. However, due to the limitation of response rates of ICIs, some patients fail to benefit from monotherapy. Thus, novel combination therapy that could improve the response rates emerges as new strategies for cancer treatment. Here, we reported that the natural product rocaglamide (RocA) increased tumor-infiltrating T cells and promoted Th17 differentiation of CD4+ TILs. Despite RocA monotherapy upregulated PD-1 expression of TILs, which was considered as the consequence of T cell activation, combining RocA with anti-PD-1 antibody significantly downregulated the expression of PD-1 and promoted proliferation of TILs. Taken together, these findings demonstrated that RocA could fuel the T cell anti-tumor immunity and revealed the remarkable potential of RocA as a therapeutic candidate when combining with the ICIs.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
1. Materials and methods
1.1. Reagents
RocA (BBP00609) was purchased from BioBioPha Co., Ltd. (Yunnan, China). PE anti-mouse CD3(100205), FITC anti-mouse CD4(100405), PerCP/Cyanine5.5 anti-mouse CD8(100733), APC anti-mouse NKp46(137608), purified anti-mouse NK1.1 (PK136) (108702) were obtained from BioLegend Inc. Anti-mouse PD-1(BE0146-RMP1-14) and rat IgG2a(BE0089-2A3) were supplied by BioXcell. Anti-mouse CD45.2(560694), FITC anti-mouse CD3(553061), BV605 anti-mouse CD4(563151), BV510 anti-mouse CD8(563068), BV650 anti-mouse NKp46 (740627), BV421 anti-mouse PD-1 (562584), PE anti-mouse CD107a (558661), PE/Cy7 anti-mouse IFN-γ(557649), PE anti-mouse RORγt(562607), BV421 mouse anti-T-bet(563318), BB700 mouse anti-GATA3(566642), PE-CF594 anti-mouse IL-4(562450), AF647 mouse anti-Ki-67(558615), BV650 anti-mouse IL-17A(564170) were provided by BD Biosciences. APC mouse anti-FOXP3(17-5773-82) was purchased from Thermo Fisher Scientific. PMA(Abs9107), ionomycin calcium salt (Abs9108) were supplied by Absin. Collagenase I(40507ES60) and DNase I(10607ES15) were obtained from Yeasen.
1.2. Cell culture
Mouse Lewis lung cancer (LLC) cells, mouse B16F10 melanoma cells and mouse CT26 colon cancer cells were supplied by the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. LLC cells and B16F10 cells were incubated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, SH30022.01B), and CT26 cells were incubated in RPMI-1640 medium(HyClone, SH30809.01). Both media were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Yeasen, 60162ES76).
1.3. Subcutaneous tumor models
Male C57BL/6 or BALB/c mice were purchased from Vital River Laboratory Animal Technology Co. (Beijing, China), and maintained under a specific pathogen-free (SPF) environment. For subcutaneous tumor models, 1.5 × 106 LLC cells, 2 × 105 B16F10 cells or 5 × 105 CT26 cells were suspended in 100 μL serum-free medium and subcutaneously injected into the right flank of 6-week-old C57BL/6 or BALB/c mice on day 0 and then 1.0 mg/kg of RocA was administered via i.p. injection every 2 days from day 3, anti-PD-1 antibody was administered via i.p. injection every 3 days from day 9. For NK cell depletion, mice were administered with 100 μg of PK136 antibody per mouse via i.p. injection on day 0, 3, 7 10 and 13. The tumor length and width were measured every 2 days using a caliper and the tumor volume was calculated using the following formula: V = (π/8)a × b2, where V = tumor volume, a = maximum tumor diameter and b = minimum tumor diameter. All animal procedures, including tumor transplantation, tumor volume monitoring and euthanasia, were approved by the Institutional Animal Care and Use Committee at Shanghai University of Traditional Chinese Medicine.
1.4. Flow cytometry analysis
Cells were exposed to the appropriate fluorescence-conjugated antibodies at 4 °C for 30 min in the dark, whereas control cells were incubated with the corresponding IgG Fc antibodies under the same conditions, then washed and resuspended in PBS containing 1% FBS. The data were obtained by a BD Accuri C6(BD Biosciences) and analyzed using Flowjo software.
1.5. RNA sequencing
1.5 × 106 LLC cells per mouse were subcutaneously inoculated on the upper back of C57BL/6 mice and 1.0 mg/kg of RocA was administered via i.p. injection every 2 days. Mice were sacrificed on day 21, tumors were isolated and analyzed by RNA sequencing, which was performed by Shanghai Biotechnology Corporation. Genes with fold change ≥ 2 and P < 0.05 were identified as differentially expressed genes (DEGs).
1.6. CD4 depletion in vivo
1.5 × 106 LLC cells were subcutaneously injected into the right flank of C57BL/6 mice on day 0. Tumor-bearing mice were treated with 1.0 mg/kg of RocA via i.p. injection every 2 days from day 3. For CD4+ T cell depletion, purified anti-mouse CD4 antibody was intraperitoneally injected with a dose of 50 μg per mouse at 24 h before LLC cell injection and every 5 days until the end of experiments, IgG antibody was used as control for anti-CD4 antibody.
1.7. In vitro differentiation analysis of CD4+ T cells
Splenic lymphocytes were isolate from healthy C57BL/6 mice and were then cultured in vitro. The cultured lymphocytes were treated by 20 ng/mL of IL-6, 1 ng/mL of TGF-β and 2 μg/mL of anti-CD3 beads and 1 μg/mL of anti-CD28 beads. Intracellular staining of transcription factors and cytokines were conducted to analyze the CD4+ T cell subpopulation after 7-day culturing.
1.8. Statistical analysis
All data were analyzed using GraphPad Prism software 8.3 (GraphPad, San Diego, CA, USA) and expressed as mean ± standard deviation (S.D.). A two-tailed unpaired t-test or one-way analysis of variance (ANOVA) was applied to determine statistical significance (P < 0.05).
Introduction
Cancer immunotherapy has become a prominent strategy in recent years, such as immune checkpoint blockade (ICB) therapy. Programmed cell death protein 1(PD-1), an immune checkpoint molecule expressed on T cells as well as other cells including but not limited to B cells, natural killer (NK) cells and myeloid cells, has received much attention in the past decade [1]. The interaction of PD-1 with its ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2), can deliver an inhibitory signal and limit effector T cell responses to protect tissues from immune-mediated damage [2], whereas tumor cells take advantage of this signal as a mechanism for immune escape[3]. The high expression of PD-1 is considered to be a marker of functional T cells [4] or exhausted T cells [5], and T cell exhaustion can be reversed by blocking PD-1 signaling [6].
The ICB therapy that bases on blocking the PD-1/PD-L1 axis has demonstrated prominent antitumor activity in multiple cancer types [7,8,9]. However, the durable remission rates still remain low due to immune resistance, a majority of patients fail to benefit from the therapy as a single agent, thereby novel combination treatments that can overcome the resistance and increase the response rates have attracted extensive attention [10,11,12]. In patients with solid tumors, responders of ICB therapy exhibit an immune-hot phenotype, characterized by T lymphocyte infiltration, whereas nonresponders may exhibit an immune-cold phenotype, characterized by the absence or exclusion of T cells in the tumor parenchyma [13]. Therefore, promoting T cell infiltration to turn nonresponsive cold tumors into responsive hot ones may become a breakthrough in enhancing ICB therapeutic efficacy.
T cells play a central role in immune-hot tumors, where CD4+ T cell release various cytokines that recruit and regulate the activity of other immune cells [14]. Naïve CD4+ T cells, under diverse stimulation, differentiate into regulatory T cells (Tregs) and distinct T helper (Th) cell subsets, such as follicular helper T (Tfh), Th1, Th2, Th17, Th9 and Th22 cells. The production of signature cytokines defines Th cell subsets and functional capacities [15]. For instance, Th1 cells are induced by IL-12 [16], Th2 cells are induced by IL-4 [17], while the differentiation of Th17 cells is promoted by IL-1β, IL-6, IL-21, IL-23 and TGF-β [18]. The mature Th cells acquire well-defined functions to combat specific pathogens but are also equipped with plasticity in response to changing microenvironment [19].
As professional cytokine-producing cells, Th cells are an essential and complex component of the immune system. Briefly, Th1 cells can activate macrophages through IFN-γ production [20], Th2 cells have an excellent performance for orchestrating immune responses against extracellular parasites and are involved in the induction of asthma and other allergic diseases by producing IL-4, IL-5 and IL-13 [15]. Th17 cells, capable of producing IL-17, IL-21 and IL-22, mediate immune responses to extracellular bacteria and fungi and are also responsible for different forms of autoimmunity [21, 22]. However, there is a lack of consensus on the role of Th17 cells in cancer progression, due to the plasticity of this subtype, enabling Th17 cell transdifferentiate into other types including Th1, Th2, Treg and Tfh cells in distinct microenvironment [23].
Rocaglamide (RocA), a compound originally isolated from Aglaia elliptifolia, has exhibited significant anti-cancer properties [24]. In our previous studies, we have found that RocA boosts NK cell-based immunotherapy by promoting the tumor infiltration of NK cells via triggering cGAS-STING signaling pathway [25] and targeting autophagy initial gene ULK1 to enhance NK cell-mediated killing of cancer cells [26]. In this study, we observed that RocA facilitated the infiltration and differentiation of T cells, thereby transforming immune-cold tumor into hot ones. When combined with anti-PD-1 antibody, RocA promoted functional activation of CD4+ TILs and anti-PD-1 antibody downregulated the PD-1 expression that prevented T cell dysfunction. Furthermore, the depletion of CD4+ T cells partially abolished the antitumor activity of RocA in vivo. These findings demonstrated that RocA could promote T cell anti-tumor immunity and had a potential capability in enhancing the response rate of ICB therapy.
Results
RocA promotes the intra-tumoral infiltration of CD4+ and CD8+ T cells
In the previous studies, we have found that RocA could enhance antitumor activity of NK cell-based immunotherapy. In addition to NK cells, T cells play a critical role in cancer immunotherapy. The previous findings [25, 26] lead us to investigate whether RocA could increase the tumor infiltration of T cells. In this study, we found that RocA suppressed the growth of mouse Lewis lung cancer (LLC) cells in vivo (Fig. 1A), the tumor weight of mice treated with RocA were significantly lower than those of mice treated with vehicle control (Fig. 1B). And tumor growth was significantly suppressed by RocA (Fig. 1C) compared to vehicle control. Furthermore, flow cytometry analysis revealed that RocA significantly increased the tumor-infiltrating lymphocytes (TILs) (Fig. 1D and 1E), including both CD4+ and CD8+ T cells (Fig. 1D and 1F). The percentage of splenic CD4+ and CD8+ T cells was also increased by RocA treatment (Fig. 1G and 1H). These results demonstrated that RocA could promote T cells infiltrating to the tumor sites.
Tumors can be divided into immune-hot, -cold and -desert tumors according to the level of TILs. We further investigated the promoting effect of RocA on intra-tumoral T cells in B16F10 melanoma, a typical immune-cold tumor. RocA significantly decreased tumor weight and suppressed tumor growth of B16F10 melanoma (Fig.S1A, B). Similarly, RocA remarkably increased the percentage of tumor-infiltrating CD4+ and CD8+ T cells (Fig.S1C). Unexpectedly, RocA did not increase the percentage of splenic lymphocytes in tumor-bearing mice (Fig.S1D), which was not consistent with the results in Lewis lung cancer. Those results showed that RocA increased tumor-infiltrating T cells independent of the increasing of splenic T cells. Moreover, RocA inhibited the growth of immune-hot CT26 colon cancer in vivo, but did not affect the intratumoral infiltration of T cells (Fig. S2A-D), which could be attributed to the abundance of lymphocytes within immune-hot tumors.
Previously, we reported that RocA could promote the tumor infiltration of NK cells [25]. We next confirmed whether RocA-mediated intratumoral infiltration of T cells were dependent on NK cells. LLC tumor-bearing mice were treated with PK136 antibody to deplete NK cells in vivo (Fig.S3A, B). We found that NK cell depletion failed to abolish the RocA-mediated intratumoral infiltration of T cells (Fig.S3C). Similar results were observed in splenic T cells of tumor-bearing mice, RocA increased the proportion of T cells in the absence of NK cells (Fig.S3D). These results showed that RocA-mediated increase of TILs was independent of NK cells.
The combination therapy of RocA and anti-PD-1 antibody exhibited potent antitumor activity in checkpoint-resistant tumor models
Promoting T cell infiltration to turn nonresponsive cold tumors into immune-responsive hot ones has become a breakthrough in enhancing the ICB therapy. The above-mentioned results demonstrated that RocA could transform immune-cold tumors into hot ones by fueling intratumoral infiltration of T cells. We next explored whether RocA can promote antitumor activity of anti-PD-1 antibody. We found that despite monotherapy of PD-1 blockade or RocA treatment suppressed tumor growth in a certain extent, the combination therapy of RocA and anti-PD-1 antibody exhibited the most potent suppressive effect on tumor growth (Fig. 2A-C). Moreover, we found that B16F10 melanoma cells and CT26 colorectal cancer cells were more resistant to checkpoint blockade therapy. The monotherapy of anti-PD-1 antibody showed minimal antitumor activity, while the combination therapy of RocA and anti-PD-1 antibody significantly suppressed tumor growth in both melanoma and colorectal cancer mouse models (Fig. 2D-I).
The combination therapy of RocA and anti-PD-1 antibody overcomes checkpoint-resistant tumor via coordinated operations
We further explored the mechanism of action by which combination therapy suppressed tumor growth remarkably. As RocA increased tumor-infiltrating CD4+ and CD8+ T cells (Fig. 1D-F), we next investigated whether these TILs were functional in tumor microenvironment. Tumor tissues were collected from LLC tumor-bearing mice and tumor-infiltrating T cells were analyzed by flow cytometry. Indeed, RocA was found to significantly promote the degranulation and intracellular production of IFN-γ in CD4+ TILs but did not activate CD8+ TILs in LLC tumor tissues (Fig. 3A, 3B and 3D).
T cell activation is often associated with upregulation of PD-1. As expected, monotherapy of RocA significantly increased CD4+/PD-1+ T cells in LLC tumor tissues (Fig. 3C). We investigated whether anti-PD-1 antibody could prevent CD4+/PD-1+ TILs from checkpoint-mediated dysfunction. The results showed that CD4+/PD-1+ TILs were not downregulated in combination therapy group compared to those in RocA monotherapy group (Fig. 3C). However, the mean fluorescent intensity of PD-1 was significantly lower in combination therapy group compared to those in RocA monotherapy group (Fig. 3E). Moreover, the percentage of CD4+/PD-1+/Ki67+ and CD8+/PD-1+/Ki67+ cells was significantly increased by combination therapy compared to RocA monotherapy, which suggested that anti-PD-1 antibody promoted proliferation of TILs and helped RocA to overcome checkpoint-mediated inhibition of TILs (Fig. 3F). Taken together, these results suggested that RocA and anti-PD-1 antibody coordinated to promote immune response against checkpoint-resistant tumor.
RocA upregulated genes involved in T helper cells
To explore the effects that RocA imposed on the tumor microenvironment, the tumor tissues were isolated and underwent analysis through RNA Sequencing. Strikingly, the functional enrichment analysis of differential genes showed that 5 of top 30 enrichments were about T helper cell differentiation (Fig. 4A). Therefore, we further analyzed the gene expression of transcription factors, cytokines, effect factors and chemokines associated with Th1, Th2 and Th17 cells, the three most common subtypes of T helper cells. We found that RocA did have a significant effect on the expression of related genes (Fig. 4B). The results of RNA-Seq were validated through qRT-PCR (Fig. 4C). These results indicated that RocA had an ability to regulate Th cell differentiation and effector function.
RocA promoted CD4+ T cell differentiate into Th17 cells both in vivo and in vitro
We next investigated whether the antitumor activity of RocA is dependent on CD4+ T helper cells. Purified anti-mouse CD4 antibody was intraperitoneally injected for CD4+ T cell depletion (Fig. 5A), flow cytometry analysis showed that the CD4+ T cells of C57BL/6 mice were depleted by anti-CD4 antibody (Fig. 5B and C). We found that the antitumor activity of RocA was partially abolished by CD4+ T cell depletion (Fig. 5D-F), which indicated that CD4+ T cells play a pivotal role in mediating the antitumor activity of RocA.
The CD4+ T cells mainly consist of T helper cells that can differentiate into Th1, Th2 and Th17 subpopulations (Fig. 6A). As the results of bulk RNA sequencing suggested that RocA might promoted Th17 differentiation of TILs. We sought to validate this concept both in vivo and in vitro. We investigated the CD4+ T cell subsets in RocA-treated tissues from tumor-bearing mice via intracellular stain flow cytometry. And the results showed that CD4+/RORγt+ population and CD4+/IL-17+ population, which mainly represents Th17 cells, were significantly increased after RocA treatment, while Th1 and Th2 cells were not increased by RocA (Fig. 6B-D). Moreover, splenic lymphocytes were isolate from healthy C57BL/6 mice and were then cultured in vitro. The cultured lymphocytes were treated with IL-6, TGF-β and anti-CD3/CD28 beads. Intracellular staining of transcription factors and cytokines were conducted to analyze the CD4+ T cell subpopulation (Fig. 6E-H). The results showed that RocA significantly increased the percentage of CD4+/RORγt+/IL-17+ subset, despite the percentage of IL-17+ cells in parental gate were not altered by RocA, which might be attributed to the release of IL-17. These results demonstrated that RocA promoted CD4+ T cell differentiate into Th17 cells.
Discussion
The ICB therapy holds great promise as a revolutionary therapeutic approach, whereas the unsatisfactory response rates limited its application, which is mainly attributed to T cell dysfunction. In this study, we found that RocA transformed the immune-cold tumors into immune-hot ones through increasing the quantity and immunity of tumor-infiltrating T cells. On the other hand, the combination of RocA and anti-PD-1 antibody significantly downregulated the PD-1 expression and promote proliferation of TILs compared with those of RocA monotherapy. Moreover, we demonstrated that the antitumor activity of RocA depended in part on CD4+ T cells and RocA promoted the CD4+T cell differentiate into Th17 cells. These results revealed that RocA could be potentially applied in T cell-based cancer immunotherapy, especially in ICB therapy.
Immune checkpoint molecules regulate the immune system by stimulating and suppressing the immune response under physiological conditions, thus preventing excessive immune responses. PD-1 is expressed on the surface of activated T cells as an immune checkpoint molecule that negatively regulates T cell function to prevent inordinate killing [27]. PD-1 overexpression was initially thought to be a sign of T cell exhaustion [5]; however, recent studies have shown that PD-1 overexpression is actually a hallmark of high-functioning T cells [4]. In this study, we found that RocA upregulated the expression of PD-1, but we still observed increased effector function of these PD-1+T cells by detecting activation markers IFNγ and LAMP1, suggesting that RocA probably promoted the early killing of T cells, leading to the upregulation of PD-1 expression.
The interaction of PD-1 with PD-L1 and PD-L2 expressed by tumor cells has been suggested as a major mechanism of tumor immune evasion and is therefore an attractive target for cancer therapy [28]. Anti-PD-1 antibodies have been widely used in a variety of cancer types, including non-small-cell lung carcinoma, melanoma, colorectal cancer and renal cell carcinoma, and have shown significant antitumor activity over the past decade [29,30,31,32]. Nevertheless, some patients have demonstrated a lack of initial response to treatment (primary resistance) or patients with initial promising response to treatment can develop resistance overtime (acquired resistance), which necessitates improved treatment strategies [33]. Recently, many researches have been focused on the combination of anti-PD-1 antibody and other anti-tumor therapies such as anti-CTLA-4 agents, chemotherapy, radiotherapy and so on [34,35,36]. In this study, we demonstrated that the combination of RocA and anti-PD-1 antibody exerted potent antitumor activity even on the immune-cold B16F10 melanoma, a type of tumor with poor response to ICB therapy.
Indeed, RocA alone increased the quantity of intratumoral T cells while also upregulating tumor PD-L1 expression (data not shown), which to some extent increased the likelihood of T cell exhaustion. Although anti-PD-1 antibody alone can block the T cell inhibitory signaling, intratumoral infiltrating T cells remains insufficient to maximize the anti-tumor immune response, which is one of the major challenges facing ICB therapy. However, the combination of RocA and anti-PD-1 antibody increased intratumoral T cells and prevented T cell dysfunction, ultimately achieving a robust anti-tumor immune response.
Moreover, in the context of immune-hot CT26 colon cancer, RocA demonstrated a definite anti-tumor effect and promoted tumor suppressive activity of anti-PD-1 antibody, but RocA did not increase the quantity of T cells in the spleen and tumor microenvironment of mice. This discrepancy could be attributed to the abundance of lymphocytes within immune-hot tumors. Thus, lymphocyte function emerges as a critical factor influencing the anti-tumor effect of RocA, warranting further investigation.
Th17 cells, a subset of T helper cells, are originally identified and studied in detail in autoimmune diseases and have been associated with a variety of inflammatory contexts [37, 38]. The differentiation of Th17 cells is definitively orchestrated by the transcription factor orphan nuclear receptor RORgammat (RORγt) [39]. Th17 cells express the cytokines IL-17, IL-21, IL-22 and other cytokines such as IFNγ and TNFα in human tumor tissues [40]. The debate about whether tumor-infiltrating Th17 cells inhibit or promote cancer progression continues, the nature of these cells depends on the various cancer environment [41,42,43]. In fact, Th17 cells can be further categorized into various lineages based on the specificity of cytokines production. In this study, we observed that RocA promoted CD4+ T cells to differentiate into Th17 cells. However, the specific role of RocA in promoting tumor-infiltrating Th17 cells and the underlying mechanism still require further elucidation.
In summary, our study elucidated that RocA could facilitate T cell anti-tumor immunity and combine with anti-PD-1 antibody against tumors. Consequently, RocA holds promise for enhancing the efficacy of cancer immunotherapy, offering potential solutions to challenges posed by existing immunotherapies such as immune checkpoint blockade therapy.
Conclusion
This study reported the therapeutic potential of combination therapy of RocA and anti-PD-1 antibody. We found that RocA promoted the infiltration and differentiation of CD4+ TILs and coordinated with anti-PD-1 antibody to overcome checkpoint resistance in multiple tumor models. Indeed, RocA showed a capacity of fueling the T cell anti-tumor immunity and serving as a therapeutic candidate in enhancing the ICB therapy.
Data availability
The data are available from the corresponding author on reasonable request.
Abbreviations
- CD:
-
Clusters of differentiation
- cGAS:
-
Cyclic GMP-AMP synthase
- FOXP3:
-
Forkhead box protein P3
- GATA3:
-
GATA binding protein 3
- ICB:
-
Immune checkpoint blockade
- IFN-γ:
-
Interferon gamma
- IL:
-
Interleukin
- LAMP1:
-
Lysosomal-associated membrane protein 1
- NK:
-
Natural killer cell
- PD-1:
-
Programmed cell death protein 1
- PD-L1:
-
Programmed death-ligand 1
- PD-L2:
-
Programmed death-ligand 2
- RocA:
-
Rocaglamide
- RORγt:
-
RAR-related orphan receptor gamma
- RNA-Seq:
-
RNA sequencing
- STING:
-
Stimulator of interferon genes
- TBET(TBX21):
-
T-box transcription factor 21
- Tfh:
-
Follicular helper T cell
- TGF-β:
-
Transforming growth factor beta
- Th:
-
T helper cell
- TIL:
-
Tumor-infiltrating lymphocyte
- Treg:
-
Regulatory T cell
- ULK1:
-
Unc-51-like kinase 1
References
Morad G et al (2021) Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell 184(21):5309–5337
Keir ME et al (2008) PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26:677–704
Nam S et al (2019) Analysis of the expression and regulation of PD-1 protein on the surface of myeloid-derived suppressor cells (MDSCs). Biomol Ther 27(1):63–70
Patel SP et al (2015) PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther 14(4):847–856
Barber DL et al (2006) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439(7077):682–687
Day CL et al (2006) PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443(7109):350–354
Ninomiya K et al (2018) Pembrolizumab for the first-line treatment of non-small cell lung cancer. Expert Opin Biol Th 18(10):1015–1021
Adams S et al (2019) Pembrolizumab monotherapy for previously untreated, PD-L1-positive, metastatic triple-negative breast cancer: cohort B of the phase II KEYNOTE-086 study. Ann Oncol 30(3):405–411
El-Khoueiry AB et al (2017) Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet 389(10088):2492–2502
Yi CH et al (2021) Lenvatinib targets FGF receptor 4 to enhance antitumor immune response of anti-programmed cell death-1 in HCC. Hepatology 74(5):2544–2560
Huuhtanen J et al (2023) Single-cell characterization of anti-LAG-3 and anti- PD-1 combination treatment in patients with melanoma. J Clin Invest. https://doi.org/10.1172/JCI164809
Fang DD et al (2019) MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment. J Immunother Cancer. https://doi.org/10.1186/s40425-019-0750-6
Zhang JH et al (2022) Turning cold tumors hot: from molecular mechanisms to clinical applications. Trends Immunol 43(7):523–545
Kennedy R, Celis E (2008) Multiple roles for CD4 T cells in anti-tumor immune responses. Immunol Rev 222:129–144
Zhu JF (2018) T Helper Cell Differentiation, Heterogeneity, and Plasticity. Csh Perspect Biol 10(10):a030338
Hsieh CS et al (1993) Development of Th1 Cd4+ T-cells through Il-12 produced by listeria-induced macrophages. Science 260(5107):547–549
Seder RA, Paul WE et al (1994) Acquisition of lymphokine-producing phenotype by Cd4+ T-Cells. Annu Rev Immunol 12:635–673
Saravia J et al (2019) Helper T cell differentiation. Cell Mol Immunol 16(7):634–643
DuPage M, Bluestone JA (2016) Harnessing the plasticity of CD4 T cells to treat immune-mediated disease. Nat Rev Immuno 16(3):149–163
Szabo SJ et al (2003) Molecular mechanisms regulating Th1 immune responses. Annu Rev Immunol 21:713–758
Ouyang WJ, Kolls JK, Zheng Y (2008) The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28(4):454–467
Karpisheh V et al (2022) The role of Th17 cells in the pathogenesis and treatment of breast cancer. Cancer Cell Int 22(1):108
Guery L, Hugues S (2015) Th17 cell plasticity and functions in cancer immunity. Biomed Res Int 2015:314620
Li-Weber M (2015) Molecular mechanisms and anti-cancer aspects of the medicinal phytochemicals rocaglamides (=flavaglines). Int J Cancer 137(8):1791–1799
Yan X et al (2022) Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer. Int J Biol Sci 18(2):585–598
Yao C et al (2018) Rocaglamide enhances NK cell-mediated killing of non-small cell lung cancer cells by inhibiting autophagy. Autophagy 14(10):1831–1844
Latchman Y et al (2001) PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2(3):261–268
Yearley JH et al (2017) PD-L2 expression in human tumors: relevance to Anti-PD-1 therapy in cancer. Clin Cancer Res 23(12):3158–3167
Garon EB et al (2015) Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372(21):2018–2028
Topalian SL et al (2014) Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol 32(10):1020–1030
De Giorgi U et al (2019) Safety and efficacy of nivolumab for metastatic renal cell carcinoma: real-world results from an expanded access programme. BJU Int 123(1):98–105
Lenz HJ et al (2022) First-line nivolumab plus low-dose ipilimumab for microsatellite instability-high/mismatch repair-deficient metastatic colorectal cancer: the phase II CheckMate 142 study. J Clin Oncol 40(2):161–170
Kirchhammer N et al (2022) Combination cancer immunotherapies: Emerging treatment strategies adapted to the tumor microenvironment. Sci Transl Med 14(670):eabo605
Asrir A et al (2022) Tumor-associated high endothelial venules mediate lymphocyte entry into tumors and predict response to PD-1 plus CTLA-4 combination immunotherapy. Cancer Cell 40(3):318-334.e9
Ho TTB et al (2022) Combination of gemcitabine and anti-PD-1 antibody enhances the anticancer effect of M1 macrophages and the Th1 response in a murine model of pancreatic cancer liver metastasis. J Immunother Cancer 8(2):e001367
Chen D et al (2020) SHP-2 and PD-L1 inhibition combined with radiotherapy enhances systemic antitumor effects in an anti-PD-1-resistant model of non-small cell lung cancer. Cancer Immunol Res 8(7):883–894
Harrington LE et al (2005) Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6(11):1123–1132
Park H et al (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6(11):1133–1141
Ivanov et al (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126(6):1121–1133
Kryczek I et al (2009) Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114(6):1141–1149
Amicarella F et al (2017) Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer. Gut 66(4):692–704
Gamal W, Sahakian E, Pinilla-Ibarz J (2023) The role of Th17 cells in chronic lymphocytic leukemia: friend or foe? Blood Adv 7(11):2401–2417
Qian X et al (2017) Interleukin-17 acts as double-edged sword in anti-tumor immunity and tumorigenesis. Cytokine 89:34–44
Acknowledgements
We sincerely thank all the people who have provided helpful support.
Funding
This study was supported by the National Natural Science Foundation of China (82373908, 81803933), Natural Science Foundation of Shanghai (23ZR1460800, 22ZR1461600). The funding body had no role in the design of the study, collection, analysis, interpretation of data and writing the manuscript.
Author information
Authors and Affiliations
Contributions
CY and SZ conducted conception of the research and designed the experiment. WN performed the experiments and analyzed the data. JL wrote, edited and reviewed the paper. YL assisted with the analysis. WN, JL and YL contribute equal to this study. LW, CG, YZ and CF provided advice with the experiments. All authors read and corrected the final version of the paper. CY and SZ are responsible for the overall content as guarantor.
Corresponding authors
Ethics declarations
Conflict of interest
SZ has equity and consulting relationships with Base Therapeutics. All other authors have no conflict of interest to declare.
Ethical approval
This study was approved by the Local Ethics Committee (Shanghai University of Traditional Chinese Medicine).
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Luo, J., Ng, W., Liu, Y. et al. Rocaglamide promotes infiltration and differentiation of T cells and coordinates with PD-1 inhibitor to overcome checkpoint resistance in multiple tumor models. Cancer Immunol Immunother 73, 137 (2024). https://doi.org/10.1007/s00262-024-03706-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00262-024-03706-5