Downregulation Of PD-L1 Research Articles

Immune Checkpoint-Modulating Photosensitizer That Targets BRD4 for Cancer Photoimmunotherapy.

Photodynamic therapy is an efficient approach to promote cytotoxic T lymphocyte tumor infiltration to convert immunologically cold tumors into hot tumors through the induction of immunogenic cell death . However, tumors usually overexpress immune checkpoints such as PD-L1 to suppress T lymphocyte antitumor activity and evade immune surveillance. Therefore, the design of efficient photosensitizers to overcome checkpoint-mediated immune evasion is highly necessary. In this work, we report the design of BRD-PS, a BRD4-targeting photosensitizer, as a new class of immunomodulatory photosensitizer termed an immune checkpoint-modulating photosensitizer, to solve this issue. On one hand, BRD-PS induces immunogenic pyroptosis and ferroptosis to promote the activation and tumor infiltration of cytotoxic T cells. On the other hand, BRD-PS suppresses the expression of PD-L1 to avoid immune evasion. This work demonstrated the feasibility of utilizing a single photosensitizer to simultaneously induce immunogenic cell death and PD-L1 downregulation for synergistic cancer photoimmunotherapy.

PDL1 targeting by miR-138-5p amplifies anti-tumor immunity and Jurkat cells survival in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) has constituted over 80% of the lung cancer population with a poor prognosis. Over the past decade, immunotherapy has been constructed in the enlargement of immune checkpoint inhibitors as a promising approach for NSCLC treatment. Evading the immune system using the PD-1/PD-L1 axis is an intelligent way for cancers, and T cells cannot respond fully and confront cancer. Recently, the miR-138 was reported as a PD-L1 regulator in NSCLC. However, its inhibitory impact on T-cell exhaustion has not been characterized. The present study aims to impair PD-L1 (B7-H1) expression in Adenocarcinoma cell lines using miR-138-5p and determines how it prevents Jurak cell exhaustion. To gain the purpose, first, 18 highly significant dysregulated miRNAs containing hsa-miR-138 and CD274-mRNA network were detected in NSCLC based on bioinformatics analysis. Moreover, our study revealed a high level of miR-138-5p could make significant changes like PDL1 downregulation, proliferation, and mortality rate in A549/Calu6 cells. We also simulate cancer environmental conditions by culturing Jurak cells and NSCLC cell lines under the influence of stimulator cytokines to show how miR-138-5p survives Jurak cells by targeting PD-L1/PD-1pathway.

Effect of 1-L-MT on immunotherapy of diffuse large B-cell lymphoma via downregulation of PD-L1.

e19066 Background: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, accounting for more than 30% of all cases. Although with the development of treatment, the 5 years overall survival rates remain low, range from 30%-50% for all DLBCL patients. In addition to R-CHOP, immunotherapy has attracted much attention. However, the single-agent activity of most immune checkpoint inhibitors (ICI) in DLBCL has been overwhelmingly disappointing. It is urgent to develop new methods to enhance the efficacy of DLBCL treatment. Our previous study demonstrated that 1-L-MT, an IDO1 inhibitor, can inhibit tumor growth of DLBCL. Here, we investigate whether 1-L-MT promotes antitumor immunity of ICI in DLBCL. Methods: The role of 1-L-MT on cell proliferation in vitro and tumor growth in NOD/SCID mice were investigated. RNA-seq analysis was performed in 1-L-MT treated and control OCI-Ly10 cells. ChIP assay was performed using AhR-precipitated DNA samples of OCI-Ly10 cells (treated with or without 1-L-MT) and primers for PD-L1, and he expression of PD-L1 was evaluated by RT-qPCR. The combination of 1-L-MT and PD-1 blockade therapy were performed in C57BL/6 mouse bearing tumor cells to evaluate the antitumor activity. Results: In our study, IDO1 inhibitor 1-L-MT sensitized diffuse large B cell lymphoma to immunotherapy via downregulation of PD-L1. Further study confirmed that 1-L-MT could reduce the expression of PD-L1 and upregulate the expression of MHC-I antigen presentation factors. In addition, RNA-seq and bioinformatics analysis revealed that the transcription factor AhR may involved in the downregulation of PD-L1 transcription level induced by 1-L-MT. Mechanistically, ChIP and RT-qPCR assays were conducted to test AhR-PD-L1 interaction, and the results showed that AhR directly bound the promoter of PD-L1. 1-L-MT-mediated PD-L1 reduction promoted T cell activation and T cell killing of tumor cells. The combination of 1-L-MT and anti PD-1 antibody significantly inhibited DLBCL growth and extends the lifespan of tumor-bearing mice. Conclusions: Our study unveiled a mechanism by which 1-L-MT induces PD-L1 downregulation via AhR and provides methods to facilitate immunotherapy in diffuse large B-cell lymphoma. The present study may provide a theoretical foundation for overcoming the challenge of low-sensitivity to immunotherapy in DLBCL.

Molecular profiling after short-term preoperative endocrine therapy to identify oestrogen receptor positive (ER+)/HER2+ early breast tumours with favourable prognosis.

560 Background: The impeded anti-proliferative response of Estrogen Receptor positive (ER+) HER2+ breast cancer (BC) to endocrine therapy (ET) has been considered negated by treatment with anti-HER2 therapies but residual disease after anti-HER2 therapy remains at risk of recurrence. Studying the molecular changes (MolC) of ER+HER2+ BC in response to ET will provide clinical insights to treatment options. Methods: POETIC was a phase III trial of post-menopausal patients with ER+ BC randomized 2:1 to 2-weeks of peri-operative aromatase inhibitors (POAI) vs control, followed by standard-of-care. Paired pre-treatment (B) and on-treatment (2wk) samples from 313 ER+HER2+ BC (213 POAI/100 controls) were gene expression profiled (BC 360 codeset; NanoString). Association of MolC with Early biological response to AI was assessed by residual Ki672wk (low ≤10%; high >10%) by T-test, multiple testing corrected by Benjamini & Hochberg (FDR); with time to recurrence (TTR) was estimated using multivariable Cox regression models adjusted for post-surgery clinicopathological variables and age as adjuvant treatment surrogate. Results: In POAI tumours, immunity-related signatures and mammary stemness were significantly upregulated in responders while proliferation, DNA-damage repair (DDR), TP53mutational status and ER-signaling were downregulated (FDR<0.05). Controls had exclusive downregulation of PDL1 and upregulation of hypoxia (FDR < 0.05). We previously identified 5 new molecular subgroups based on baseline gene expression (1- Immune high, ESR1 low; 2- ECM, ESR1 low, highest ERBB2; 3- DDR deficiency, 4- Endocrine signalling high and 5- Endocrine and PI3K/MAPK/RAS signalling high) which were associated with different response to AI and differential outcome. Endocrine-signalling, PI3K/MAPK/RAS signalling, tumour-immunity and chemokines were upregulated at a higher magnitude in AI sensitive subgroups (3 and 4) while the molecular subgroup with early resistance to AI and poorer outcome at baseline (2) did not show significant changes. Intrinsic subtype (IS) shifting was significantly more prevalent in treated (37%, 79/213) than in controls (14%, 14/100) (p <0.001). In POAI most Luminal B (LumB) shifted to Luminal A (LumA; 79%, 59/75), driven by a reduction in proliferation. LumA2wk was associated with better outcome compared to LumB2wk (HR 0.2; CI95% 0.06-0.72, p=0.01). IS2wk provides additional information predicting TTR, than ISB (AIC value = 217.2 vs 221.6). Conclusions: We provide a comprehensive picture on how ER+HER2+ BC gene expression profile changes in response to ET. Most LumB BC shift to LumA, being LumA2wk IS significantly associated with better outcome. There is a clinical utility of peri-operative ET for ER+HER2+ BC, guiding who would have good prognosis for adjuvant ET and who may need additional therapy. Clinical trial information: NCT02338310 .

Enhancement of anti-PD-L1 antibody plus anlotinib efficacy due to downregulation of PD-L1 in the micro-conduit endothelium within the tumor: a randomized double-blind trial.

The possible enhancing effect of anlotinib on programmed death receptor ligand (PD-L1) antibody and the efficacy-predicting power of PD-L1 in micro-conduit endothelium, including lymphatic endothelial cells (LECs) and blood endothelial cells (BECs), were determined to identify patients who would benefit from this treatment. PD-L1 positivity in LECs, BECs, and tumor cells (TCs) was assessed using paraffin sections with multicolor immunofluorescence in an investigator's brochure clinical trial of TQB2450 (PD-L1 antibody) alone or in combination with anlotinib in patients with non-small cell lung cancer. Progression-free survival (PFS) with different levels of PD-L1 expression was compared between the two groups. Among 75 patients, the median PFS (mPFS) was longer in patients who received TQB2450 with anlotinib [10 and 12 mg (161 and 194 days, respectively)] than patients receiving TQB2450 alone (61 days) [hazard ratio (HR)10 mg = 0.390 (95% confidence interval {CI}, 0.201-0.756), P = 0.005; HR12 mg = 0.397 (0.208-0.756), P = 0.005]. The results were similar among 58 patients with high PD-L1 expression in LECs and TCs [159 and 209 vs. 82 days, HR10 mg = 0.445 (0.210-0.939), P = 0.034; HR12 mg = 0.369 (0.174-0.784), P = 0.009], and 53 patients with high PD-L1 expression in BECs and TCs [161 and 209 vs. 41 days, HR10 mg = 0.340 (0.156-0.742), P = 0.007; HR12 mg = 0.340 (0.159-0.727), P = 0.005]. No differences were detected in the mPFS between the TQB2450 and combination therapy groups in 13 low/no LEC-expressing and 18 low/no BEC-expressing PD-L1 cases. Mono-immunotherapy is not effective in patients with high PD-L1 expression in LECs and/or BECs. Anlotinib may increase efficacy by downregulating PD-L1 expression in LECs and/or BECs, which is presumed to be a feasible marker for screening the optimal immune patient population undergoing anti-angiogenic therapy.

A biomimetic spore nanoplatform for boosting chemodynamic therapy and bacteria-mediated antitumor immunity for synergistic cancer treatment

Bacterial-based antitumor immunity has become a promising strategy to activate the immune system for fighting cancer. However, the potential application of bacterial therapy is hindered by the presence of instability and susceptibility to infections within bacterial populations. Furthermore, monotherapy is ineffective in completely eliminating complex cancer with multiple contributing factors. In this study, based on our discovery that spore shell (SS) of Bacillus coagulans exhibits excellent tumor-targeting ability and adjuvant activity, we develop a biomimetic spore nanoplatform to boost bacteria-mediated antitumor therapy, chemodynamic therapy and antitumor immunity for synergistic cancer treatment. In detail, SS is separated from probiotic spores and then attached to the surface of liposome (Lipo) that was loaded with hemoglobin (Hb), glucose oxidase (GOx) and JQ1 to construct SS@Lipo/Hb/GOx/JQ1. In tumor tissue, highly toxic hydroxyl radicals (•OH) are generated via sequential catalytic reactions: GOx catalyzing glucose into H2O2 and Fe2+ in Hb decomposing H2O2 into •OH. The combination of •OH and SS adjuvant can improve tumor immunogenicity and activate immune system. Meanwhile, JQ1-mediated down-regulation of PD-L1 and Hb-induced hypoxia alleviation synergistically reshape immunosuppressive tumor microenvironment and potentiate immune response. In this manner, SS@Lipo/Hb/GOx/JQ1 significantly suppresses tumor growth and metastasis. To summarize, the nanoplatform represents an optimum strategy to potentiate bacteria-based cancer immunotherapy.

Bioresponsive nanocomplex integrating cancer-associated fibroblast deactivation and immunogenic chemotherapy for rebuilding immune-excluded tumors

Cancer-associated fibroblasts (CAFs) play a crucial role in creating an immunosuppressive environment and remodeling the extracellular matrix within tumors, leading to chemotherapy resistance and limited immune cell infiltration. To address these challenges, integrating CAFs deactivation into immunogenic chemotherapy may represent a promising approach to the reversal of immune-excluded tumor. We developed a tumor-targeted nanomedicine called the glutathione-responsive nanocomplex (GNC). The GNC co-loaded dasatinib, a CAF inhibitor, and paclitaxel, a chemotherapeutic agent, to deactivate CAFs and enhance the effects of immunogenic chemotherapy. Due to the modification with hyaluronic acid, the GNC preferentially accumulated in the tumor periphery and responsively released cargos, mitigating the tumor stroma as well as overcoming chemoresistance. Moreover, GNC treatment exhibited remarkable immunostimulatory efficacy, including CD8+ T cell expansion and PD-L1 downregulation, facilitating immune checkpoint blockade therapy. In summary, the integration of CAF deactivation and immunogenic chemotherapy using the GNC nanoplatform holds promise for rebuilding immune-excluded tumors.

Abstract PR-010: Dual targeting of IDO1/TDO2 inhibits tumor progression and attenuates the immune suppressive tumor microenvironment

Abstract Introduction: High-grade serous carcinoma (HGSC) tumors of the ovary, fallopian tube, or peritoneum are predicted to be immunogenic since the presence of tumor-infiltrating lymphocytes conveys a better prognosis. However, the efficacy of immunotherapies has been limited due to the immune-suppressed tumor immune microenvironment (TIME), which is established through tumor metabolism and immune-suppressive metabolites that directly affect immune cell function by depleting nutrients and activating immune-suppressive transcriptional programs. The tryptophan (TRP) catabolism pathway is a critical regulator of immune-suppressed TIME; however, clinical trials targeting the pathway to date have demonstrated limited response. Note there are two structurally distinct rate-limiting TRP catabolizing enzymes, Indoleamine 2,3-Dioxygenase (IDO1) and Tryptophan 2,3-Dioxygenase (TDO2), that evolved separately to catabolize tryptophan into Kynurenine (KYN). Most TRP catabolism inhibitors (e.g., Epacadostat) have primarily targeted IDO1, which may explain the mild anti-tumor effects of Epacadostat. Thus, we hypothesize that targeting both enzymes involved in TRP catabolism will alleviate the immune-suppressed TIME. Methods: We assessed primary human ascites fluid (IRB #07-0395) with global untargeted metabolomics. Using a panel of HGSC cell lines, we assessed genetic (small hairpin-mediated knockdown and overexpression) and pharmacologic (Epacadostat [IDO1 inhibitor], AT-0174 [IDO1/TDO2 inhibitor], 66cl4 [TDO2 inhibitor], StemRegenin 1 [AhR inhibitor]) modulation of IDO1, TDO2, and aryl hydrocarbon receptor (AhR, signal transducer of KYN). Proliferation assays in 2D and 3D. We examined the response of IDO1/TDO2 inhibition in two syngeneic murine models (ID8 Tp53-/- and BR-Luc Tp53-/-, Brca1-/-, myr-AKT, MYC). Multispectral immunohistochemistry (mIHC) and flow cytometry defined the TIME. Results: Elevated KYN levels are correlated to an inflamed TIME. The depletion of TRP and increased KYN negatively affect the anti-tumor immune response. HGSC cell lines and clinical outcomes depend more on TDO2 than IDO1. In contrast to knocking down IDO1 and AhR, TDO2 knockdown inhibited HGSC cell growth, and overexpression of TDO2 induced cancer cell proliferation. Targeting both IDO1 and TDO2 significantly inhibited TRP catabolism. Notably, only the combined IDO1/TDO2 inhibition resulted in the downregulation of PD-L1. In a syngeneic HGSC murine model, a first-in-class orally available dual IDO1/TDO2 inhibitor (AT-0174) significantly inhibited tumor progression, reduced tumor-associated macrophages, and reduced expression of immune-suppressive proteins, including PD-L1, on immune and tumor cells. Conclusions: These studies demonstrate the importance of TDO2 and the potential of AT-0174 to overcome an immune-suppressed TIME. The findings represent a novel approach to targeting TRP catabolism that may improve tumor response rates to immune therapy. Citation Format: Lyndsey Crump, Jessica Floyd, Elizabeth Woodruff, Mike Bickerdike, Li-Wei Kuo, Miriam Post, Silviu Itescu, Jennifer Richer, Benjamin G. Bitler. Dual targeting of IDO1/TDO2 inhibits tumor progression and attenuates the immune suppressive tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-010.

Metformin-Mediated Fast Charge-Reversal Nanohybrid for Deep Penetration Piezocatalysis-Augmented Chemodynamic Immunotherapy of Cancer.

Immune checkpoint blockade (ICB) therapy still suffers from insufficient immune response and adverse effect of ICB antibodies. Chemodynamic therapy (CDT) has been demonstrated to be an effective way to synergize with ICB therapy. However, a low generation rate of reactive oxygen species and poor tumor penetration of CDT platforms still decline the immune effects. Herein, a charge-reversal nanohybrid Met@BF containing both Fe3O4 and BaTiO3 nanoparticles in the core and Metformin (Met) on the surface was fabricated for tumor microenvironment (TME)- and ultrasound (US)-activated piezocatalysis-chemodynamic immunotherapy of cancer. Interestingly, Met@BF had a negative charge in blood circulation, which was rapidly changed into positive when exposed to acidic TME attributed to quaternization of tertiary amine in Met, facilitating deep tumor penetration. Subsequently, with US irradiation, Met@BF produced H2O2 based on piezocatalysis of BaTiO3, which greatly enhanced the Fenton reaction of Fe3O4, thus boosting robust antitumor immune response. Furthermore, PD-L1 expression was inhibited by the local released Met to further augment the antitumor immune effect, achieving effective inhibitions for both primary and metastatic tumors. Such a combination of piezocatalysis-enhanced chemodynamic therapy and Met-mediated deep tumor penetration and downregulation of PD-L1 provides a promising strategy to augment cancer immunotherapy.

Anoikis regulator GLI2 promotes NC cell immunity escape by TGF-β-mediated non-classic hedgehog signaling in colorectal cancer: based on artificial intelligence and big data analysis.

Anoikis is a speed-limited procedure to inhibit tumor metastasis during epithelial-mesenchymal transition (EMT). Previous studies have explored anoikis-related genes (ARG) in predicting prognosis and distinguishing tumoral immunity in many types of cancer. However, the role of ARGs in regulating NK cell exhaustion (NKE) and in predicting chemotherapy sensitivity is not clear. Therefore, it is necessary to work on it. Gene expression profiles and clinical features are collected from TCGA and GEO, and data analysis is performed in R4.2.0. The ARGs-based no-supervised learning algorithm identifies three ARG subgroups, amongst which the prognosis is different. WCGNA and Artificial intelligence (AI) are applied to construct an NKE-related drug sensitivity stratification and prognosis identification model in digestive system cancer. Pathways association analysis screens out GLI2 is a key gene in regulating NKE by non-classic Hedgehog signaling (GLI2/TGF-β/IL6). In vitro experiments show that down-regulation of GLI2 enhances the CAPE-mediated cell toxicity and accompanies with down-regulation of PD-L1, tumor-derive IL6, and snial1 whereas the expression of cleaved caspas3, cleaved caspase4, cleaved PARP, and E-cadherin are up-regulated in colorectal cancer. Co-culture experiments show that GLI2- decreased colorectal tumor cells lead to down-regulation of TIM-3 and PD1 in NK cells, which are restored by TGF-bate active protein powder. Besides, the Elisa assay shows that GLI2-decreased colorectal tumor cells lead to up-regulation of IFN-gamma in NK cells.

JAK1/2 Inhibition Modulates Key Markers of Lymphomagenesis in 9p24.1 Amplified Lymphoma, Priming for Enhanced Antigenicity

Background Primary Mediastinal B-cell Lymphoma (PMBCL) and Hodgkin Lymphoma (HL) are frequently defined by chromosome 9p24.1 amplification, leading to upregulation of PDL-1/-2 and JAK2. JAK2, in addition to its known role in the JAK/STAT pathway, has also been shown to act as an epigenetic modifier by phosphorylation of H3Y41, enabling an euchromatin state and expression of MYC. When these epigenetic modifications are coupled with upregulation of PDL-1/-2, the 9p amplicon leads to immune escape, unchecked cell proliferation, and tumorigenesis. We hypothesize that if deranged histone modification and amplified expression of PD-1 ligands cooperate to drive cell growth in 9p amplified PMBCL and HL, then dual targeting with JAK inhibitor and immune checkpoint blockade will lead to altered gene expression and enhanced immunogenicity inducing synergistic cell death. The following abstract demonstrates preliminary data using ruxolintib monotherapy in cell lines, with goal of combining immunotherapy in mouse models. Methods In order to understand how ruxolitinib modulates key drivers of lymphomagenesis in 9p24.1 amplified lymphoma, human lymphoma cell lines with 9p amplification, (n=2): Karpas1106p (PMBCL) and L540 (HL)were compared to cell lines lacking 9p24.1 amplification, (n=2): Farage (PMBCL) and U2940 (PMBCL). All samples were treated for 3- and 6-day intervals. The IC50 concentrations were established for ruxolintib using Cell TiterGlo assay: Karpas1106p 5.72 µM, L540 22 µM, Farage 29.46 µM, and U2940 32.99 µM. All cells were treated for 48 hours with control or ruxolitnib and probed by western blot, flow cytometry, and RNA-sequencing. The expression of key determinants of survival including JAK/STAT, pro- and anti-apoptotic proteins (SOCS1 and MYC, respectively) and MHCII were measured by western blot. PD-1/-2 ligands were measured by flow cytometry. Transcriptomic changes were evaluated by RNA-seq and data was presented as Log 2 fold change compared to vehicle controls. Results Pharmacodynamic effects were most prominent in the 9p amplified Karpas1106p cell line demonstrating increased markers of immunogenicity by protein quantification: increased CIITA by 3.5-fold (SEM ± 1.15) and increased MHCII by 2.67-fold (SEM ± 0.33). Expression of tumor suppressor SOCS1 was increased by 2.67-fold (SEM ± 0.68) and expression of oncogene MYC was decreased by 6.06-fold (SEM ± 4.66). The 9p amplified L540 line similarly demonstrated increased CIITA by 3.22-fold (SEM ± 1.59) and decreased MYC by 2.69-fold (SEM ± 0.65), however no significant change in MHCII or SOCS1 was observed. Significant reduction of STAT3p and/or STAT6p was demonstrated across all cell lines (p < 0.01). Cell lines without 9p amplification, Farage and U2940, did not demonstrate consistent increase nor decrease in markers of immunogenicity or oncogenes, suggesting ruxolitinib preferentially modulates downstream markers of survival and immune recognition driven by the 9p amplicon. Flow cytometry demonstrates significantly reduced expression of PDL-1 (p = 0.01) and PDL-2 (p = 0.001) in 9p amplified cell lines (Karpas1106p and L540), and non-amplified cell lines (Farage and U2940), as shown in Figure 1. Down-regulation of PDL-1/-2 was more prominent in the 9p amplified cell lines compared to those without 9p amplification (p = 0.055 and p = 0.18, respectively), suggesting that ruxolitinib may prime for increased immunogenicity by reducing PDL-1/-2, especially in 9p amplified cell lines. RNA-seq of the 9p amplified Karpas1106p cell line demonstrated increased MHCII: HLA-DQ (1.12, p < 0.001), HLA-DO (0.70, p < 0.001), HLA DM (0.14, p = 0.001), and decreased PDL-1 (-1.04, p < 0.009), PDL-2 (-1.70, p < 0.001), C-MYC (-0.95, p < 0.001), and STAT1/3/4 (all p < 0.001). Gene-ontology analysis demonstrated increased lymphocyte differentiation (p < 0.001) and immune response activating pathways (p = 0.003) ( Fig. 2). Conclusion Altogether, data suggests that ruxolitinib enhances immunogenicity primarily in 9p amplified cell lines and supports the hypothesis that ruxolitinib may prime for lymphoma cell death when combined with immunotherapy. These findings provide a foundation for in vivo assessment in humanized mouse models treated with combination anti-PDL-1 and JAK1/2 inhibitor, targeting tumorigenesis driven by 9p amplification. *This work has been funded by the ASH RTAF grant.

TRIM29 promotes antitumor immunity through enhancing IGF2BP1 ubiquitination and subsequent PD-L1 downregulation in gastric cancer

Tripartite motif-containing protein 29 (TRIM29) is a member of TRIM family protein which has been reported to play a role in the progress of inflammatory and cancer diseases. However, its specific role in gastric cancer (GC) has yet to be fully understood. Here, we investigated the expression of TRIM29 in gastric cancer and its functions in the antitumor immunity. TRIM29 expression was lower in tumor tissues than that in paired normal tissues. Lower expression of TRIM29 was related to aberrant hypermethylation of CpG islands in TRIM29 gene. Comprehensive proteomics and immunoprecipitation analyses identified IGF2BP1 as TRIM29 interactors. TRIM29 interacted with IGF2BP1 and induced its ubiquitination at Lys440 and Lys450 site by K48-mediated linkage for protein degradation. IGF2BP1 promoted PD-L1 mRNA stability and expression in a 3′UTR and m6A-dependent manner. Functionally, TRIM29 enhanced antitumor T-cell immunity in gastric cancer dependent on the IGF2BP1/PD-L1 axis in vivo and in vitro. Clinical correlation analysis revealed that TRIM29 expression in patient samples was associated with CD8+ immune cell infiltration in the GC microenvironment and the overall survival rates of GC patients. Our findings revealed a crucial role of TRIM29 in regulating the antitumor T-cell immunity in GC. We also suggested that the TRIM29/IGF2BP1/PD-L1 axis could be used as a diagnostic and prognostic marker of gastric cancer and a promising target for GC immunotherapy.

Canagliflozin enhances endogenous eliminatiton of senescent cells through downregulation of PD-L1

Abstract Sodium-glucose co-transporter 2 (SGLT2) inhibitors have been developed as a new class of anti-diabetic drug, and shown cardio- and renal-protective effects in patients with diabetes, heart failure, and chronic kidney diseases. Also, there's evidence that canagliflozin, one of the SGLT2 inhibitors has expanded lifespan in aged mice, but their detailed protective machinery is still to be clarified. Here we demonstrate that canagliflozin has a effect to eliminate senescent cells from pathological tissues, so-called "senolysis" in some murine disease models and alleviates their pathology. We treated canagliflozin mixed in food at 0.03% (w/w) to high-fat-diet(HFD)-fed obese mice and found that canagliflozin reduced senescent cells in visceral adipose tissue within 1 week. Also, we found that senolytic effect of canagliflozin was provided through enhancing endogenous senolytic function by NK or T cells. This machinery was mediated by enhanced AMPK signaling, which leads to the downregulation of PD-L1 expression on senescent cells. Furthermore, we treated canagliflozin to ApoE-knockout atherosclerotic mice with western diet feeding, progeroid mice and chronological aged mice. Canagliflozin reduced their senescent cell burden, resulting in alleviation of atherosclerosis, extended lifespan of progeroid, and altered physical function due to chronological aging. Our results would be promising evidence that SGLT2 inhibitors can be used as not only cardio- or renal-protective drugs but also anti-aging drugs.

Usp22 Deficiency Leads to Downregulation of PD-L1 and Pathological Activation of CD8+ T Cells and Causes Immunopathology in Response to Acute LCMV Infection.

Ubiquitin-specific peptidase 22 (Usp22) cleaves ubiquitin moieties from numerous proteins, including histone H2B and transcription factors. Recently, it was reported that Usp22 acts as a negative regulator of interferon-dependent responses. In the current study, we investigated the role of Usp22 deficiency in acute viral infection with lymphocytic choriomeningitis virus (LCMV). We found that the lack of Usp22 on bone marrow-derived cells (Usp22fl/fl Vav1-Cre mice) reduced the induction of type I and II interferons. A limited type I interferon response did not influence virus replication. However, restricted expression of PD-L1 led to increased frequencies of functional virus-specific CD8+ T cells and rapid death of Usp22-deficient mice. CD8+ T cell depletion experiments revealed that accelerated CD8+ T cells were responsible for enhanced lethality in Usp22 deficient mice. In conclusion, we found that the lack of Usp22 generated a pathological CD8+ T cell response, which gave rise to severe disease in mice.

Maltol has anti-cancer effects via modulating PD-L1 signaling pathway in B16F10 cells.

Introduction: Among skin cancers, melanoma has a high mortality rate. Recent advances in immunotherapy, particularly through immune checkpoint modulation, have improved the clinical treatment of melanoma. Maltol has various bioactivities, including anti-oxidant and anti-inflammatory properties, but the anti-melanoma property of maltol remains underexplored. The aim of this work is to explore the anti-melanoma potential of maltol through regulating immune checkpoints. Methods: The immune checkpoint PD-L1 was analyzed using qPCR, immunoblots, and immunofluorescence. Melanoma sensitivity towards T cells was investigated via cytotoxicity, cell viability, and IL-2 assays employing CTLL-2 cells. Results: Maltol was found to reduce melanin contents, tyrosinase activity, and expression levels of tyrosinase and tyrosinase-related protein 1. Additionally, maltol suppressed the proliferative capacity of B16F10 and induced cell cycle arrest. Maltol increased apoptotic rates by elevating cleaved caspase-3 and PARP. The co-treatment with maltol and cisplatin revealed a synergistic effect on inhibiting growth and promoting apoptosis. Maltol suppressed IFN-γ-induced PD-L1 and cisplatin-upregulated PD-L1 by attenuating STAT1 phosphorylation, thereby enhancing cisplatin's cytotoxicity against B16F10. Maltol augmented sensitivity to CTLL-2 cell-regulated melanoma destruction, leading to an increase in IL-2 production. Discussion: These findings demonstrate that maltol restricts melanoma growth through the downregulation of PD-L1 and elicits T cell-mediated anti-cancer responses, overcoming PD-L1-mediated immunotherapy resistance of cisplatin. Therefore, maltol can be considered as an effective therapeutic agent against melanoma.

Near-Infrared Triggered Self-Accelerating Nanozyme Camouflaged with a Cancer Cell Membrane for Precise Targeted Imaging and Enhanced Cancer Immunotherapy.

Although cancer immunotherapy has made encouraging progress, clinical therapeutic efficiency is often modest due to inadequate immunogenicity and immune resistance. Developing promising nanoagents for simultaneously activating tumor-specific immunity and suppressing immune resistance to achieve efficient immunotherapy is still challenging. Herein, we developed a biomimetic nanozyme consisting of a gold nanorod@mesoporous ceria core-shell scaffold with gold nanoparticle deposition and cancer cell membrane camouflage. The nanozyme exhibited near-infrared (NIR)-enhanced GOx-mimicking activity at high temperatures and performed well under hypoxic environments due to an increased in situ oxygen supply. In cancer cells, the nanozyme induced and amplified hyperthermia by triggering self-accelerating cascade reactions to deplete glucose and inhibiting the expression of heat shock protein under NIR irradiation, which can cause mitochondrial dysfunction and redox balance disruption to activate pyroptosis and elicit a robust immune response. Additionally, the immune checkpoint blockade caused by encapsulated JQ1-mediated PD-L1 downregulation synergistically contributed to excellent immune therapeutic effects. Besides, we demonstrated that cancer cell membrane coating endows the nanozyme targeting ability to tumor. The proposed nanozyme will broaden the application of GOx and have the potential as the nanoplatform for imaging-guided and O2-consuming combined treatments.

PD-L1 Downregulation and DNA Methylation Inhibition for Molecular Therapy against Cancer Stem Cells in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a heterogeneous cancer characterized by various cellular subtypes. This study investigates the potential of a combination strategy using immunotherapy and epigenetic reprogramming against HCC. We used a transgenic HCC mouse C57BL/6J-TG(ALB1HBV)44BRI/J to assess the dynamics of the programmed death receptor and its ligand (PD-1/PD-L1) and DNA methylation markers. In parallel, PD-L1 RNA silencing was performed in various human HCC cell lines, while combination therapy was performed in a co-culture system using long-term exposure of 5-Azacytidine (5-AZA) and an anti-PD-L1. Data from the mouse model showed that the expressions of Pdcd1, Pdcd1l1, and DNA methyltransferase 1 (Dnmt1) were significantly higher in HCC as compared to the wild-type mice (p < 0.01), supported by the high presence of PD-L1 methylated DNA. In HCC cell lines, PD-L1 silencing was accompanied by DNMT1 reduction, mostly noted in aggressive HCC cell lines, followed by the dysregulation of the cancer stem cell marker EpCAM. In combination therapy, the growth of HCC cells and lymphocytes was limited by the PD-L1 antibody, further reduced in the presence of 5-AZA by up to 20% (p < 0.001). The data demonstrated that combination therapy might be an option as a potential treatment for heterogeneous HCC.

Abstract P2165: Endothelial Leptin Resistance Exacerbates Vascular Dysfunction Through Attenuating PD-L1 Expression

Background: Obesity and its vascular complications are escalating globally that threaten millions of people and incurring tremendous financial costs. Fundamentally, endothelial dysfunction is the culprit among these cardiovascular diseases due to the presence of risk factors such as hyperglycemia and hyperlipidemia, which enhance the inflammatory responses and leucocyte infiltration. Persistent endotheliopathy is closely associated to severe cardiovascular events, ultimately leading to mortality. Leptin is a pleiotropic adipokine that is significantly expressed in obese people, resulting in leptin resistance. Programmed cell death ligand 1 (PD-L1; encoded by gene Cd274 ) is the inhibitory immune checkpoint that limits immunogenic responses of lymphocytes by causing functional exhaustion and apoptosis in lymphocytes. The relationship between endothelial PD-L1 expression and progression of vasculopathy has not yet been elucidated. Methods: Providing that circulating leptin level is closely associated with body adiposity and feeding behaviors, both hypoleptinemia and hyperleptinemia were generated on C57BL/6 mice by nutritional modulation or genetic knockout on leptin receptor. Results: Findings across multiple models suggested, the expression of PD-L1, an immune-coinhibitory molecule, was regulated by leptin, the impairment in leptin signaling resulted in vascular PD-L1 downregulation. By employing the mice with obesogenic hyperleptinemia, we showed that the vascular tissue failed to respond to exogenous leptin stimuli, thereby hindering PD-L1 expression in the inflammatory microenvironment. In fact, PD-L1 expression is crucial in maintaining tissue homeostasis by suppressing the activated PD-1 + leukocytes, as such hindered PD-L1 expression may favor the vascular inflammation by failing to inhibit immunogenic attack, especially when other risk factors are present. Beyond acting as a ligand to leukocytes, our findings also revealed PD-L1 reverse signaling in endothelium plays a role in resolving inflammation and rescuing endothelial function. Summary: These findings propose a new explanation for why obesity results in vasculopathy in relationship with PD-L1.

The role of WTX212, an erythrocyte-anti-PD1 antibody conjugate, in anti-tumor activities in tumor models and cancer patients with acquired resistance to immunotherapy.

29 Background: Despite the success of immune-checkpoint blockade (ICB) treatments, a considerable proportion of patients cannot benefit from ICB therapy due to acquired resistance. Recent studies have revealed that successful immunotherapy drives new immune responses arising from peripheral immune system instead of reinvigorating the pre-existing responses in the tumor. Here we designed WTX212, autologous engineered red blood cells (RBCs) conjugated with anti-PD1 antibodies capable of activating the peripheral immune system. We also evaluated its anti-cancer activities in anti-PD1 non-responsive tumor models and a first-in-human (FIH) trial. Methods: The anti-tumor efficacy of WTX212 was evaluated in both PD1 responsive (MC38 colorectal cancer) and non-responsive (KP lung cancer and pB3 breast cancer) mouse tumor models (n=7 / group). Mice were monitored twice a week for clinical signs and tumor sizes. The mechanism of action was investigated in splenectomized mice and by analyzing the biodistribution of WTX212 using a radiolabeling method. The immune landscape was also profiled through cytof mass and scRNA-seq analyses in the spleen, tumor and peripheral blood. The FIH trial (NCT05707325) explores safety, pharmacokinetics and mechanism in cancer patients with acquired resistance to ICB. WTX212 was manufactured by covalently engineering anti-PD1 antibody with RBCs isolated from patients. By 11th April 2023, 3 patients have been enrolled and received at least one dose of WTX212 (20*1010 cells, IV, Q3W). Results: Treatment of WTX212 significantly suppressed tumor growth up to 100% in anti-PD1 responsive and non-responsive tumor models. Unlike antibodies, WTX212 primarily distributed to the spleen and vascular system. The anti-tumor effect of WTX212 was abrogated in corresponding splenectomized mice. Compared to anti-PD1 antibody, single-cell analysis identified the immune landscape remodeling in the spleen from WTX212-treated mice, with down-regulation of PD-L1+ myeloid-derived suppressor cells (MDSCs) and up-regulation of effector CD8+ T cells. In the FIH trial, 3 patients have been administered with total thirteen cycles of WTX212. No treatment-related AEs greater than Grade 2 were observed. WTX212 remained in the peripheral blood day 21 post transfusion. WTX212 rapidly activates peripheral immune systems with decreased MDSCs, Tregs and increased effector T cells in peripheral blood from all patients. The first patient who received six cycles of WTX212 treatment demonstrated stable disease with a 20% reduction at the target lesion. Conclusions: WTX212 demonstrated anti-tumor activities by activating the peripheral immune system to overcome resistance to immunotherapy in both preclinical models and human patients. WTX212 is well-tolerated supporting the future clinical trials. Clinical trial information: NCT05707325 .

Multi-omics analysis of human mesenchymal stem cells shows cell aging that alters immunomodulatory activity through the downregulation of PD-L1

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.