Immune Checkpoint Protein Expression Research Articles

Expression patterns of immune checkpoint proteins and Plasmodium falciparum-induced cytokines in chronic hepatitis B virus-infected and uninfected individuals: A cross-sectional study.

Chronic hepatitis B virus (CHB) infection remains a major public health problem. The American Association for the Study of Liver Diseases (AASLD) 2018 Hepatitis B Guidelines provide that CHB individuals not requiring antiviral therapy yet are monitored to determine the need for antiviral therapy in the future; however, these tests do not include measurement of cytokines and immune cell characterization. This case-control study compared the cytokine and immune checkpoint protein expression profiles between CHB individuals not yet on antiviral treatment and hepatitis B virus (HBV)-negative individuals. CD4 and CD8 T cells from CHB and HBV-negative individuals were characterized for immune checkpoint proteins programmed cell death-1 (PD1), T cell Immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (CD152), and a memory marker CXCR3 (CD183) using flow cytometry. Malaria-induced cytokine expression levels were determined by stimulating their blood cells with Plasmodium falciparum 3D7 strain antigens (CSP, AMA-1, and TRAP) in whole blood assays, and cytokine levels were measured using a 13-plex Luminex kit. HBV-negative and CHB individuals had comparable levels of CD4+ and CD8+ T cells. However, a proportion of the CD4+ and CD8+ populations from both groups, which were CXCR3+, expressed PD-1 and CD152. The ability to produce cytokines in response to malaria antigen stimulation was not significantly different between the groups. These findings support excluding CHB individuals from antiviral therapy at this stage of infection. However, CHB individuals require regular monitoring to determine the need for later antiviral treatment.

Multiomic factor analysis for pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched (HER2-E) early breast cancer (eBC) in the Keyriched-1 trial.

590 Background: pCR successfully identified candidates for (chemotherapy) de-escalation in the ADAPT HER2+ trial program. Here, we report biomarker analysis for pCR in the single arm, hypothesis-generating Keyriched-1 trial (NCT03988036) in HER2-E eBC treated with pembrolizumab and dual HER2 blockade. The trial rationale is based on high efficacy of anti-HER2 agents and potentially high immunogenicity and immune checkpoint protein expression in HER2-E eBC. Methods: 48 patients with HER2 2+ (ISH+) or 3+ eBC (stage I-III) and HER2-E subtype received pembrolizumab (200 mg), trastuzumab biosimilar (loading dose, LD 8 mg/kg, maintenance dose, MD 6 mg/kg), and pertuzumab (LD 840 mg/kg, MD 420 mg/kg) q3w for 12 weeks. Primary outcome was pCR (ypT0/is ypN0). Multiomic factor analysis was performed using different combinations of biomarker datasets: baseline BC360 single genes and gene signatures, multiplexed immunohistochemistry (mIHC), stromal tumor infiltrating lymphocytes (sTILs), somatic mutations and tumor mutational burden, and changes in circulating tumor DNA status under therapy. Factors representing combined markers developed from these pooled datasets were identified based on eigenvalue >1 criteria. Logistic regression with factors dichotomized by median score was conducted to analyze their association with pCR. Furthermore, logistic regression with elastic net regularization was performed using all single markers, factors from multiomic factor analysis, and clinical characteristics. Results: Factor involving gene expression (mainly related to immune response and tumorigenesis), mIHC markers (immune and HER2) in stroma, and sTILs datasets was significantly associated with pCR. Using the median as cutoff, higher scores increased the odds of pCR (unadj. OR 5.00, 95%CI 1.35, 18.56; p=.016). pCR rates were 66.7% and 28.6% in the high and low score groups, respectively. Effect of the factor (high vs low; OR 2.19) was also important in a model that additionally included clinical markers: grade (3 vs 2; OR 2.57), ER status (ER+ vs ER-; OR 0.89), cT (cT 2/3 vs 1; OR 0.69), cN (cN 1/2 vs 0; OR 0.51), and PR status (PR+ vs PR-; OR 0.34). When standardized values for all individual markers from gene expression, mIHC, and sTIL datasets were analyzed, markers with greatest impact on pCR vs. non-pCR were genes FNBP1, CD36, MYCN, and SIX1 (OR 1.43-1.62) and PR status (OR 0.65), with stronger impact than grade (OR 1.22) or nodal status (OR 0.90). Conclusions: Our multiomic analysis uncovered potential biomarkers for pCR in HER2-E eBC treated with immune therapy and dual-HER2 blockade alone. These results together with the results from the ADAPT translational research program support validation in larger trials investigating chemotherapy-free regimens in selected patients with HER2+ eBC. Clinical trial information: NCT03988036 .

Abstract A050: CD8+ T cell exhaustion and their DNA damage in BRCA1 mutated TNBC

Abstract PARP Poly (ADP-ribose) polymerase inhibitors (PARPi) are treatment options in TNBC patients with inherited BRCA1 mutations. Recent studies have shown that immune checkpoint therapies (ICT) in combination with PARPi are effective in other solid cancer types such as melanoma, pancreatic and BRCA deficient ovarian cancer. In early and advanced BRCA1 TNBC, ICT studies are limited and not fully elucidated especially for new ICT that target LAG-3, CTLA-4, ICOS. There is a need for identifying potential prognostic markers suggesting which patients could benefit most from ICT. Therefore, we conducted a detailed single cell analysis of the tumor microenvironment (TME) mainly focused on immune checkpoint expression, CD8 T cell exhaustion and DNA damage associated with BRCA1 TNBC compared to WT TNBC. Combining single cell spatial tissue multiplexing (PhenoCycler) (BRCA1 TNBC n=77, WT TNBC n=55), scRNA seq (BRCA2 n=1, WT n=5), FLOW (PBMCs: BRCA1 n=13, BRCA2 n=17, WT n=62) as well as bulk RNA seq (BRCA1 TNBC n=35, WT TNBC n=11), we performed a detailed analysis of the TME associated with BRCA1 and WT breast cancer. We developed a 32-plex antibody panel that evaluated T-cells and other immune cells with markers of exhaustion, DNA damage and immune checkpoint expression. CD8+ T cell frequency was significantly increased in BRCA1 TNBC compared to WT TNBC (p=0.0015). A detailed CD8 T cell characterization revealed increased dysfunctional/exhausted cells (e.g., TOX+/LAG-3+/PD-1+), T cell DNA damage (CD3e+/CD8+/RAD51+, p=0.0047) and LAG-3 transcript levels in BRCA1 TNBC compared to WT TNBC (p=0.0625). Furthermore, immune checkpoint protein expression in tumor- (PD-L1) and immune cells (PD-1, LAG-3, ICOS) was increased in the TME associated with BRCA1 TNBC compared to WT TNBC. Of note, we also found that PBMCs of healthy tumor-free BRCA mutation carriers contained significantly increased CD8+/PD+1 T cells compared to WT (p=0.012). Our data indicates that BRCA1 haploinsufficiency leads to DNA damaged and dysfunctional CD8+ T cells within the TME compared to WT. Thus, the TME of BRCA1 TNBC patients is more immune suppressive due to increased exhausted CD8+ T cell frequency, their DNA damage as well as immune checkpoint expression of both tumor-/and immune cells compared to WT TNBC. The effect of BRCA1 haploinsufficiency on immune cells is important to consider when trials of ICT options are designed. Citation Format: Dana Pueschl, Anupma Nayak, Derek A. Oldridge, William Chandler, Bria Fulmer, Sokratis Apostolidis, Danielle Bragen, Bradley Wubbenhorst, John Pluta, kConFab Investigator, Antonis Antoniou, Georgia Chenevix-Trench, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. CD8+ T cell exhaustion and their DNA damage in BRCA1 mutated TNBC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A050.

Inhibitory effect of adenosine on adaptive antitumor immunity and intervention strategies.

The tumors with a microenvironment characterized by a lack of immune cell infiltration, referred as "cold tumors", typically exhibit low responsiveness to immune therapy. Targeting the factors contributing to "cold tumors" formation and converting them into "hot tumors" would be a novel strategy for improving the efficacy of immunotherapy. Adenosine, a hydrolysis product of ATP, accumulates with a significantly higher concentration in the tumor microenvironments compared to normal tissue and exerts inhibitory effects on tumor-specific adaptive immunity. Tumor cells, dendritic cells, macrophages, and T cells express abundant adenosine receptors on their surfaces. The binding of adenosine to these receptors initiates downstream signaling pathways that suppress tumor antigen presentation and immune cell activation, consequently dampening adaptive immune responses against tumors. Adenosine downregulates the expression of major histocompatibility complex class Ⅱ and co-stimulatory factors on dendritic cells and macrophages, thereby inhibiting antigen presentation to T cells. Adenosine also inhibits ligand-receptor binding and transmembrane signaling on T cells, concomitantly suppressing the secretion of anti-tumor cytokines and impairing T cell activation. Furthermore, adenosine hinders effector T cell trafficking to tumor sites and infiltration by inhibiting chemokine secretion and KCa3.1 channels. Additionally, adenosine promotes the secretion of immunosuppressive cytokines, increases immune checkpoint protein expression, and enhances the activity of immunosuppressive cells, collectively curbing cytotoxic T cell-mediated tumor cell killing. Given the immunosuppressive role of adenosine in adaptive antitumor immunity, several inhibitors targeting adenosine generation or adenosine receptor blockade are currently in preclinical or clinical development with the aim of enhancing the effectiveness of other immunotherapies. This review provides an overview of inhibitory effects of adenosine on adaptive antitumor immunity, elucidating the molecular mechanisms involved, and summarizes the latest advances in the application of adenosine inhibition strategies in anticancer immunotherapy.

PPARγ agonist inhibits c-Myc-mediated colorectal cancer tumor immune escape.

As a master transcription factor, c-Myc plays an important role in promoting tumor immune escape. In addition, PPARγ (peroxisome proliferator-activated receptor γ) regulates cell metabolism, inflammation, and tumor progression, while the effect of PPARγ on c-Myc-mediated tumor immune escape is still unclear. Here we found that cells treated with PPARγ agonist pioglitazone (PIOG) reduced c-Myc protein expression in a PPARγ-dependent manner. qPCR analysis showed that PIOG had no significant effect on c-Myc gene levels. Further analysis showed that PIOG decreased c-Myc protein half-life. Moreover, PIOG increased the binding of c-Myc to PPARγ, and induced c-Myc ubiquitination and degradation. Importantly, c-Myc increased PD-L1 and CD47 immune checkpoint protein expression and promoted tumor immune escape, while PIOG inhibited this event. These findings suggest that PPARγ agonist inhibited c-Myc-mediated tumor immune escape by inducing its ubiquitination and degradation.

Vulnerability to immune therapy in BRAF- and MYB-altered pediatric gliomas.

2066 Background: The frequency and expression of predictive immune biomarkers in pediatric gliomas is relatively unknown. Here, we sought to define predictive signatures of immunotherapy responders by examining immune checkpoint expression, immune cell population signatures, and gene amplifications in a variety of pediatric gliomas. Methods: We profiled a cohort of pediatric, adolescent, and young adult glioma samples submitted to Caris Life Sciences (Phoenix, AZ) for analysis (N = 207). The cohort was further stratified by driver mutations: IDH mutant (N = 103), H3-3A mutant (N = 36), MYB-altered (N = 4), BRAF-altered (N = 39), and IDH wild type [IDH WT] (N = 25). De-identified next generation DNA sequencing (592-gene or whole exome) and RNA (whole transcriptome) sequencing were used to determine tumor-infiltrating immune cell signatures and immune checkpoint protein expression. Transcriptomic signatures predictive of response to immunotherapy (T cell inflamed score) and replication stress response defect (RSRD) score were calculated on transcripts per million (TPM) values. Immune cell fractions were estimated using RNA deconvolution (quanTIseq). Results: BRAF-altered tumors showed the highest IFN gamma signature (a clinically relevant inflammatory biomarker), a high pro-inflammatory M1 macrophage signature, and very high CD4+/CD8+ T cell signatures compared to other tumor groups. IDH mutant, but not WT gliomas, were relatively deficient in T cell infiltration but revealed an increase in dendritic cell signatures. IDH WT gliomas showed more T cell enrichment but higher immune suppressive checkpoint expression. H3-3A mutant tumors showed relatively low immune cell infiltration and immune checkpoint expression. Pro-inflammatory M1 macrophages were lower in the high-grade glioma microenvironment compared to low-grade. Immune suppressive M2 macrophages were elevated in the IDH WT microenvironment relative to other gliomas. There was no apparent difference in the frequency of PD-1+ T cells and tumor-expressed PD-L1 among the various molecularly defined pediatric glioma groups. Conclusions: In general, BRAF- and MYB-altered gliomas displayed high immune activation relative to other tumors. IDH WT tumors have an immune suppressive microenvironment with relatively high immune checkpoint expression. Based on predictive markers, BRAF- and MYB-driven gliomas show signatures suggesting the possibility of a greater response to immunotherapies than IDH WT/mutant gliomas or H3-3A gliomas.

5-Azacytidine-Mediated Modulation of the Immune Microenvironment in Murine Acute Myeloid Leukemia.

Cancer cells accumulate epigenetic modifications that allow escape from intrinsic and extrinsic surveillance mechanisms. In the case of acute myeloid leukemias (AML) and myelodysplastic syndromes, agents that disrupt chromatin structure, namely hypomethylating agents (HMAs), have shown tremendous promise as an alternate, milder treatment option for older, clinically non-fit patients. HMAs reprogram the epigenetic landscape in tumor cells through the reversal of DNA hypermethylation. Therapeutic effects resulting from these epigenetic changes are incredibly effective, sometimes resulting in complete remissions, but are frequently lost due to primary or acquired resistance. In this study, we describe syngeneic murine leukemias that are responsive to the HMA 5-azacytidine (5-Aza), as determined by augmented expression of a transduced luciferase reporter. We also found that 5-Aza treatment re-established immune-related transcript expression, suppressed leukemic burden and extended survival in leukemia-challenged mice. The effects of 5-Aza treatment were short-lived, and analysis of the immune microenvironment reveals possible mechanisms of resistance, such as simultaneous increase in immune checkpoint protein expression. This represents a model system that is highly responsive to HMAs and recapitulates major therapeutic outcomes observed in human leukemia (relapse) and may serve as a pre-clinical tool for studying acquired resistance and novel treatment combinations.

Glioblastoma Associated Natural Killer Cell EVs Generating Tumour-Specific Signatures: Noninvasive GBM Liquid Biopsy with Self-Functionalized Quantum Probes.

Diagnosis of glioblastoma (GBM) poses a recurring struggle due to many factors, including the presence of the blood-brain barrier (BBB) in addition to the significant tumor heterogeneity. Natural killer (NK) cells of the innate immune system are the primary immune surveillance mechanism for GBM and identify GBM tumors without any previous sensitization. The metabolic reprogramming of NK cells during GBM association is expected to be reflected in its extracellular vesicles. Therefore, tracking the activity of NK cell vesicles in circulation (circulating immune vesicles, CIVs) has great potential for accurate GBM diagnosis. However, identification GBM associated CIVs in circulation is immensely challenging as there is no availability of clinically validated GBM-specific circulating biomarkers. Here, we present GBM associated CIV profiling for noninvasive GBM diagnosis. We investigated the feasibility of using the signals derived from GBM associated CIVs as a de novo methodology for GBM diagnosis. An ultrasensitive sensor and a marker-free approach were essential for the detection of rare signals of GBM associated CIVs. For this purpose, we designed GBM ImmunoProfiler platform using scalable ultrafast laser multiphoton ionization mechanism and adopted surface enhanced Raman spectroscopy (SERS) ensuring simultaneous detection of multiple CIV signals to identify GBM. We experimentally demonstrated that GBM associated CIVs carry unique, tumor-specific signals. The features of GBM associated CIVs were explored through machine learning identifying its similarity with GBM patient blood (without cell isolation) using a very small amount of peripheral blood (5 μL) with 96.82% sensitivity and 100% specificity. In addition, we demonstrated that a tumor associated CIV profile can classify between multiple brain cancer types (astrocytoma, oligodendroglioma, and glioblastoma). We also experimentally demonstrated significant variation in the immune checkpoint protein expression (PDL-1 and CTLA-4) between GBM associated CIVs and uninteracted CIVs. Preclinical analysis with serum specimens of GBM patients showed the possibility of using our technology for minimally invasive GBM diagnosis. With clinical validation, our technology has potential to improve GBM diagnostics with a useful, minimally invasive GBM liquid biopsy.

The CHST11‐CP‐metal ionic axis contributes to NSCLC immune escape and metastasis

Escaping of immune surveillance is essential for tumor metastasis. Recently, intratumoral metal ionic balance plays an important role in controlling cancer immune checkpoint protein expression and tumor immune escape. Copper (Cu) promotes the expression of PD‐L1. In contrast to Cu, heme biosynthesis disturbs PD‐L1 expression. Heme protein is a kind of metalloproteins with ferrous iron (Fe2+)‐porphyrin core. Here, we found CHST11 served as a pro‐metastatic gene in non‐small cell lung cancer (NSCLC). We further identified the expression of ceruloplasmin (CP) was regulated by CHST11. CP is not only the major blood Cu carrier protein but also a Cu‐dependent ferroxidase in iron metabolism. It may hint a crosstalk mechanism between lung cancer metastasis and tumor immune surveillance through the CHST11‐CP axis. CP was also associated with poor prognosis in patients with NSCLC. Both ectopic CP expression and high intracellular ionic stimulations, like ferric iron and copper, promoted NSCLC progression, such as in vitro migration and invasion, as well as in vivo tumorigenesis abilities in Nod‐SCID mice. The ionic chelator treatments repressed in vitro metastatic potential and, interestingly, regulated the gene expressions of immune checkpoints in NSCLC cells. By using microarray analysis, we found CHST11 controlled CP expression through interferon‐gamma (IFNγ), Janus kinase (JAK), and signal transducer and activator of transcription (STAT) pathways. Treatment with ruxolitinib, a JAK inhibitor, suppressed the expressions of CP and immune checkpoint proteins. In conclusion, our findings prove that the CHST11‐CP axis plays an important role in both NSCLC immune escape and metastasis through IFNγ‐JAK‐STAT pathway. The combination of an iron chelator and ruxolitinib may provide a potential translation application that protects patients with NSCLC from cancer immune escape and metastasis.

Apoptosis-Related Signature Predicts Prognosis and Immune Microenvironment Infiltration in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD), a malignancy with high incidence and mortality rates worldwide, contains multiple genomic and epigenomic abnormalities. And the useful tumor markers associated with these abnormalities need further investigation. Whereas apoptosis is a form of programmed cell death, the expression of apoptosis-related genes in LUAD and its relationship with prognosis is unclear. In the present study, we identified 64 differentially expressed apoptosis-related genes (DEARGs) that were differentially expressed between LUAD tissue and normal lung tissue. Based on these DEARGs, all LUAD cases were classified into two subtypes using The Cancer Genome Atlas (TCGA) cohort to assess the prognostic value of apoptosis-related genes for survival. An 11-gene signature was established by applying the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression method to construct a multigene prediction model and classify all LUAD patients in the TCGA cohort into high or low AS-score groups. Patients in the low AS-score group had significantly higher survival and prognosis than those in the high AS-score group. Taking the median risk score of the AS-score, LUAD patients in the GSE68465 cohort were divided into two risk groups, low and high. The overall survival (OS) time was longer in the low AS-score group. Combined with clinical characteristics, the AS-score was an independent predictor of LUAD patients. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that the differential genes between the two groups were mainly enriched in cellular immunity. Further analysis revealed higher immune checkpoint protein expression and higher tumor mutational burden (TMB) in the high AS-score group, suggesting better efficacy of immunotherapy in the high AS-score group than the low AS-score group. And the high AS-score group was better in chemotherapy and targeted therapy efficiency. In conclusion, the AS-score constructed based on apoptosis-related genes can predict the prognosis of LUAD patients and provide some guidance for the antitumor treatment of LUAD patients.

Immune Checkpoint Protein Expression Defines the Prognosis of Advanced Thyroid Carcinoma.

BackgroundPatients with advanced thyroid carcinoma (TC), such as anaplastic thyroid carcinoma (ATC), poorly differentiated thyroid carcinoma (PDTC), and locally advanced papillary thyroid carcinoma (PTC), have poor prognoses and require novel treatments. Immune checkpoint (ICP) inhibitors have demonstrated encouraging and good results; nevertheless, their effect in advanced TCs remains largely unclear. Thus, we demonstrated ICP profiles and investigated their potential clinical significance.MethodsA total of 234 TC patients were involved, with 22 ATCs, 44 PDTCs, and 168 PTCs, including 58 advanced PTCs. Immunohistochemistry was performed to evaluate nine ICPs [programmed cell death ligand 1 (PDL1), Programmed cell death 1 (PD1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), B and T lymphocyte attenuator (BTLA), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), lymphocyte activation gene 3 (LAG3), V-domain immunoglobulin suppressor of T-cell activation (VISTA), B7 homolog 3 (B7-H3), and T-cell immunoglobulin and mucin domain- 3 protein (TIM3)] expression via tissue microarrays (TMAs), and clinical correlations were analyzed simultaneously.ResultsATC had the highest positive rate of ICPs among the three pathological types, as well as relatively high ICP co-expression. ATC with high expression of PDL1 positivity had a poor prognosis. Shorter survival was associated with VISTA, B7H3, TIM3, and TIGIT expression in PDTC. The greater the co-expression of these four ICPs, the poorer the prognosis in PDTC patients. VISTA and B7H3 were the two most commonly expressed ICPs in advanced PTC, both of which were linked to a poor prognosis.ConclusionsPDL1 is linked to the overall survival (OS) of ATC. A subset of PDTC is likely immunogenic with poor prognosis and co-expression of VISTA, B7H3, TIM3, and TIGIT. Furthermore, VISTA and B7H3 are prognostic biomarkers in advanced PTC. Single or combined blockade targeting these ICPs might be effective for advanced TCs in the future.

A Nicotinamide Phosphoribosyltransferase Inhibitor, FK866, Suppresses the Growth of Anaplastic Meningiomas and Inhibits Immune Checkpoint Expression by Regulating STAT1.

Anaplastic meningioma is classified as a World Health Organization (WHO) grade III tumor and shows a strong tendency to recur. Although the incidence of anaplastic meningioma is low, the high rate of recurrence and death still makes treatment a challenge. A proteomics analysis was performed to investigate the differentially expressed proteins between anaplastic meningiomas and fibrous meningiomas by micro-LC-MS/MS. The key metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT) showed upregulated expression in anaplastic meningiomas. However, targeting NAMPT to treat anaplastic meningiomas has not been reported. In vitro, NAMPT inhibitor -FK866 reduced the viability of anaplastic meningiomas by inducing cell cycle arrest at the G2/M phase. Intriguingly, the NAMPT inhibitor -FK866 decreased the protein expression of immune checkpoints PD-L1 and B7-H3 by down-regulating the STAT1 and p-STAT1 expression in vitro. Furthermore, FK866 suppressed the growth of anaplastic meningiomas in an in vivo xenograft model. The expression of Ki-67 and immune checkpoint proteins (PD-L1 and B7-H3) showed significant differences between the group treated with FK866 and the control group treated with DMSO. In conclusion, the expression of NAMPT, which plays a crucial role in energy metabolism, was upregulated in anaplastic meningiomas. The NAMPT inhibitor -FK866 significantly suppressed the growth of anaplastic meningiomas in vitro and in vivo. More strikingly, FK866 potently inhibited immune checkpoint protein (PD-L1 and B7-H3) expression by regulating STAT1 in vitro and in vivo. Our results demonstrated that NAMPT inhibitors could potentially be an effective treatment method for patients suffering from anaplastic meningiomas.

Inhibition of the Na+/K+-ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2′,3′-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.

Prognostic implication of CTLA-4, PD-1, and PD-L1 expression in aggressive adult T-cell leukemia–lymphoma

The prognosis of patients with aggressive adult T cell leukemia-lymphoma (ATLL) is dismal even with intensive chemotherapy. Allogeneic hematopoietic stem cell transplantation (HSCT) is a promising option for patients with aggressive ATLL, but the posttransplant outcome remains unsatisfactory. Hence, to further improve clinical outcomes, novel therapeutic approaches are needed. The clinical significance of immune checkpoint protein expression has not been well-established in aggressive ATLL. This study aims to identify the association between the expression profile of immune checkpoint proteins on ATLL cells and clinical outcomes. This retrospective study cohort included 65 patients with aggressive ATLL diagnosed between 2001 and 2015 at the National Cancer Center Hospital, Tokyo, Japan. Formalin-fixed paraffin-embedded tissue was used to immunohistochemically determine the expression of immune checkpoint proteins and assess the impact of expression profile on the probability of overall survival from diagnosis or HSCT. The current analysis shows that cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) expressions were adverse prognostic factors in patients with aggressive ATLL. Experiments that assess the efficacy of immune checkpoint inhibitors are warranted to alleviate the adverse impacts associated with negative immune checkpoints.

Integrative analysis of the molecular mechanisms, immunological features and immunotherapy response of ferroptosis regulators across 33 cancer types

Ferroptosis is a recently described mode of cell death caused by the accumulation of intracellular iron and lipid reactive oxygen species (ROS), which play critical roles in tumorigenesis and cancer progression. However, the underlying molecular mechanisms and promising biomarkers of ferroptosis among cancers remain to be elucidated. In this study, 30 ferroptosis regulators in ferroptosis-related signaling pathways were identified and analyzed in 33 cancer types. We found transcriptomic aberrations and evaluated the prognostic value of ferroptosis regulators across 33 cancer types. Then, we predicted and validated potential transcription factors (including E2F7, KLF5 and FOXM1) and therapeutic drugs (such as cyclophosphamide, vinblastine, and gefitinib) that target ferroptosis regulators in cancer. Moreover, we explored the molecular mechanisms of ferroptosis and found that signaling pathways such as the IL-1 and IL-2 pathways are closely associated with ferroptosis. Additionally, we found that ferroptosis regulators have a close relationship with immunity-related parameters, including the immune score, immune cell infiltration level, and immune checkpoint protein level. Finally, we determined a ferroptosis score using GSVA method. We found that the ferroptosis score effectively predicted ferroptotic cell death in tumor samples. And ferroptosis score is served as an independent prognostic indicator for the incidence and recurrence of cancers. More importantly, patients with high ferroptosis scores received greater benefit from immunotherapy. We aslo created an online webserver based on the nomogram prognostic model to predict the survival in immunotherapy cohort. The reason for this outcome is partially the result of patients with a high ferroptosis rate also having high immune scores, HLA-related gene expression and immune checkpoint protein expression, such as PDL2 and TIM3. Moreover, patients with high ferroptosis scores exhibited CD8 T cell and TIL infiltration and immune-related signaling pathway enrichment. In summary, we systematically summarize the molecular characteristics, clinical relevance and immune features of ferroptosis across cancers and show that the ferroptosis score can be used as a prognostic factor and for the evaluation of immunotherapy effects.

Improving Immunotherapy Efficacy in Soft-Tissue Sarcomas: A Biomarker Driven and Histotype Tailored Review.

Anti-PD-(L)1 therapies yield a disappointing response rate of 15% across soft-tissue sarcomas, even if some subtypes benefit more than others. The proportions of TAMs and TILs in their tumor microenvironment are variable, and this heterogeneity correlates to histotype. Tumors with a richer CD8+ T cell, M1 macrophage, and CD20+ cells infiltrate have a better prognosis than those infiltrated by M0/M2 macrophages and a high immune checkpoint protein expression. PD-L1 and CD8+ infiltrate seem correlated to response to immune checkpoint inhibitors (ICI), but tertiary lymphoid structures have the best predictive value and have been validated prospectively. Trials for combination therapies are ongoing and focus on the association of ICI with chemotherapy, achieving encouraging results especially with pembrolizumab and doxorubicin at an early stage, or ICI with antiangiogenics. A synergy with oncolytic viruses is seen and intratumoral talimogene laherpavec yields an impressive 35% ORR when associated to pembrolizumab. Adoptive cellular therapies are also of great interest in tumors with a high expression of cancer-testis antigens (CTA), such as synovial sarcomas or myxoid round cell liposarcomas with an ORR ranging from 20 to 50%. It seems crucial to adapt the design of clinical trials to histology. Leiomyosarcomas are characterized by complex genomics but are poorly infiltrated by immune cells and do not benefit from ICI. They should be tested with PIK3CA/AKT inhibition, IDO blockade, or treatments aiming at increasing antigenicity (radiotherapy, PARP inhibitors). DDLPS are more infiltrated and have higher PD-L1 expression, but responses to ICI remain variable across clinical studies. Combinations with MDM2 antagonists or CDK4/6 inhibitors may improve responses for DDLPS. UPS harbor the highest copy number alterations (CNA) and mutation rates, with a rich immune infiltrate containing TLS. They have a promising 15-40% ORR to ICI. Trials for ICB should focus on immune-high UPS. Association of ICI with FGFR inhibitors warrants further exploration in the immune-low group of UPS. Finally translocation-related sarcomas are heterogeneous, and although synovial sarcomas a poorly infiltrated and have a poor response rate to ICI, ASPS largely benefit from ICB monotherapy or its association with antiangiogenics agents. Targeting specific neoantigens through vaccine or adoptive cellular therapies is probably the most promising approach in synovial sarcomas.

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects.

SUMMARYDevelopmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

Prognostic Significance of Tumor Infiltrating Lymphocytes (TILs) and Programmed Cell Death-Ligand 1 (PD-L1) in Nasopharyngeal Carcinoma

Purpose/Objective(s)The role of PD-L1 and different subtypes of TILs in nasopharyngeal carcinoma (NPC) are till poorly studied. The purpose of this study is to evaluate the prognostic significance of TILs and immune checkpoint protein expression in NPC.Materials/MethodsA total of 125 NPC patients consisting of 68 patients with favorable prognosis (Group 1) and 57 patients with unfavorable prognosis (Group 2) treated between 2010 to 2014 were included. Archival tumor tissues of selected patients were retrieved and a tissue microarray (TMA) was conducted. The TMA was stained with CD3, CD4, CD8, Foxp3, CK, PDL1 and DAPI by using multiplexed quantitative fluorescence. The marker CD3 was used to defined T cells, subtypes of T cells were defined by the combination of different markers: cytotoxic T lymphocytes (CTLs, CD3+CD8+), CD4+ effector T cells (CD4+ Teff, CD3+CD4+Foxp3-), regulatory T cells (Treg, CD3+CD4+Foxp3+). The counts and densities of every marker and labelled cells were calculated. High density was defined as higher than the median value for each marker. The density differences and association with survival of PD-L1 and TILs were analyzed between two groups.ResultsPD-L1 positive tumor cells (TCs-PDL1) were observed in 96% of patients and PD-L1 positive immune cells (ICs-PDL1) were observed in 90.4% of patients. ICs-PDL1 were significantly correlated with high infiltration of CD3+ TILs, CD4+ Teff, CTLs, and Treg. Patients with high PDL1+ Treg had unfavorable overall survival (OS, HR 2.093, 95% CI 1.038-4.222, P = 0.035) and progression-free survival (PFS, HR 2.501, 95% CI 1.421-4.403, P = 0.001). High TCs-PDL1 expression was associated with a trend approaching significance for improved OS (P = 0.055) and PFS (P = 0.078). High density of Treg, Treg/CTL ratio and Treg/CD4+ Teff ratio were significantly associated with poor survival of PFS (P = 0.027, P = 0.001 and P = 0.004 respectively). Patients with lower Treg/CTL ratio presented better OS, but did not reach statistical significance (P = 0.082).ConclusionHigher density of Treg, Treg/ CTL and Treg/CD4+ Teff ratio were associated with worse survival of NPC which may contribute to the formation of immunosuppressive microenvironment. The role of PD-L1 and different subtypes of TILs in nasopharyngeal carcinoma (NPC) are till poorly studied. The purpose of this study is to evaluate the prognostic significance of TILs and immune checkpoint protein expression in NPC. A total of 125 NPC patients consisting of 68 patients with favorable prognosis (Group 1) and 57 patients with unfavorable prognosis (Group 2) treated between 2010 to 2014 were included. Archival tumor tissues of selected patients were retrieved and a tissue microarray (TMA) was conducted. The TMA was stained with CD3, CD4, CD8, Foxp3, CK, PDL1 and DAPI by using multiplexed quantitative fluorescence. The marker CD3 was used to defined T cells, subtypes of T cells were defined by the combination of different markers: cytotoxic T lymphocytes (CTLs, CD3+CD8+), CD4+ effector T cells (CD4+ Teff, CD3+CD4+Foxp3-), regulatory T cells (Treg, CD3+CD4+Foxp3+). The counts and densities of every marker and labelled cells were calculated. High density was defined as higher than the median value for each marker. The density differences and association with survival of PD-L1 and TILs were analyzed between two groups. PD-L1 positive tumor cells (TCs-PDL1) were observed in 96% of patients and PD-L1 positive immune cells (ICs-PDL1) were observed in 90.4% of patients. ICs-PDL1 were significantly correlated with high infiltration of CD3+ TILs, CD4+ Teff, CTLs, and Treg. Patients with high PDL1+ Treg had unfavorable overall survival (OS, HR 2.093, 95% CI 1.038-4.222, P = 0.035) and progression-free survival (PFS, HR 2.501, 95% CI 1.421-4.403, P = 0.001). High TCs-PDL1 expression was associated with a trend approaching significance for improved OS (P = 0.055) and PFS (P = 0.078). High density of Treg, Treg/CTL ratio and Treg/CD4+ Teff ratio were significantly associated with poor survival of PFS (P = 0.027, P = 0.001 and P = 0.004 respectively). Patients with lower Treg/CTL ratio presented better OS, but did not reach statistical significance (P = 0.082). Higher density of Treg, Treg/ CTL and Treg/CD4+ Teff ratio were associated with worse survival of NPC which may contribute to the formation of immunosuppressive microenvironment.

Immunohistochemical Detection of Bacteria in the Resected Valves was Associated with Stromal Immune Checkpoint Protein Expression that may Contribute to Calcific Aortic Stenosis

Aortic stenosis (AS) is a disease characterized by narrowing of the aortic valve (AV) orifice. The purpose of this study was to clarify the significance of bacterial detection and clinicopathological factors, including valve-infiltrating immune cells and disease severity, in relation to AS. After obtaining the written informed consent form from 50 patients with AS, we performed immunohistochemical analysis of lipopolysaccharide (LPS) for gram-negative bacteria and lipoteichoic acid (LTA) for gram-positive bacteria on surgically resected-AVs. Moreover, we evaluated the relationships among the presence of bacteria, immune checkpoint protein PD-L1 expression, and immune cell infiltrations such as CD8-positive T lymphocytes, CD163-positive macrophages, and FOXP3-positive regulatory T cell (Treg) in resected-aortic valves. LPS detection in the resected-aortic valve tissues was significantly associated with stromal PD-L1 expression, valve calcification, and LTA existence in resected samples. We showed that the presence of LPS was significantly related to high PD-L1 expression only in calcified-AVs, not in non-calcified-AVs. Moreover, the high expression of PD-L1 in AS samples without LPS was significantly associated with positive infiltration of CD163-positive macrophages and FOXP3-positive Tregs. Immunohistochemical bacterial detection in resected-aortic valves was associated with PD-L1 accumulation and valve calcification. PD-L1 significantly accumulated only in calcified valves with LPS existence.

Clinical Impact of X-Ray Repair Cross-Complementary 1 (XRCC1) and the Immune Environment in Colorectal Adenoma-Carcinoma Pathway Progression.

PurposeColorectal cancer (CRC) can develop via a hypermutagenic pathway characterized by frequent somatic DNA base-pair mutations. Alternatively, the immunogenicity of tumor cells themselves may influence the anticancer activity of the immune effector cells. Impaired DNA repair mechanisms drive mutagenicity, which then increase the neoantigen load and immunogenicity. However, no studies have analyzed immune checkpoint protein expression, particularly programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1), in adenoma–carcinoma progression and its relationship with the emergence of other DNA repair gene mutation.Materials and MethodsWe investigated mutations of 10 genes involved in DNA repair function: XRCC1, TP53, MLH1, MSH, KRAS, GSTP, UMP, MTHF, DPYD, and ABCC2. We performed sequencing to determine mutations and immunohistochemistry of immune checkpoints in clinical samples and determined changes in XRCC1 expression during progression through the adenoma–carcinoma pathway. We further investigated the prognostic associations of gene XRCC1 according to the expression, mutational profile, and immune profile using The Cancer Genome Atlas-colon adenocarcinoma (TCGA-COAD) dataset.ResultsFrom clinical samples, XRCC1 mutation demonstrated the strongest association with adenomas with a mutation frequency of 56.2% in adenomas and 34% in CRCs (p =0.016). XRCC1 was abnormally expressed and altered by mutations contributing to adenoma carcinogenesis. High expression of XRCC1, CD4, FOXP3, and PD-1/PD-L1 showed an overall upward trend with increased lesion severity (all p < 0.01). PD-1/PD-L1 expression and CD4+ intraepithelial lymphocytes (IELs) correlated with cytological dysplasia progression, specifically in patients with wild-type XRCC1 (all p < 0.01), whereas FOXP3 expression was independently associated with adenoma–carcinoma progression. From TCGA-COAD analysis, XRCC1 expression was associated with patients survival, tumor-infiltrating lymphocytes and immune marker expression.ConclusionIncreased IEL density and PD-1/PD-L1 expression correlate with cytological dysplasia progression and specifically with the XRCC1 mutation status in CRC. Our findings support a stepwise dysplasia-carcinoma sequence of adenoma carcinogenesis and an XRCC1 hypermutated phenotypic mechanism of lesions.