PD-1 Expression Research Articles

Nivolumab receptor occupancy on effector regulatory T cells predicts clinical benefit.

Immune checkpoint inhibitor discovery represents a turning point in cancer treatment. However, the response rates of solid tumors remain ~10%-30%; consequently, prognostic and immune-related adverse event (irAE) predictors are being explored. The programmed cell death protein 1 (PD-1) receptor occupancy (RO) of PD-1 inhibitors depends on the number of peripheral blood lymphocytes and their PD-1 expression levels, suggesting that the RO may be related to efficacy and adverse events. As PD-1 inhibition affects each T-cell subset differently, the RO of each cell population must be characterized. However, relevant data have not been reported, and the prognostic relevance of this parameter is not known. In this study, we aimed to clarify the association between the nivolumab RO in each T-cell population and patient prognosis and reveal the development of irAEs in nivolumab-treated patients. Thirty-two patients were included in the study, and the mean follow-up period was 364 days. The nivolumab RO on effector regulatory T cells (eTregs) was significantly lower in the group that presented clinical benefits, and a significant negative association was observed between PD-1 occupancy on eTregs and all-cause mortality. The results suggest that the nivolumab RO on eTregs may be a prognostic factor in PD-1 inhibitor therapy, implying that the inhibition of PD-1/PD-ligand 1 (PD-L1) signaling on eTregs may attenuate antitumor effects.

Serologic, radiographic, and tissue-based markers associated with major pathologic response after treatment with neoadjuvant immunotherapy in patients with resectable hepatocellular carcinoma.

561 Background: Neoadjuvant immunotherapy can induce a major pathologic response (MPR) in patients with resectable hepatocellular carcinoma (HCC), which may be associated with prolonged recurrence-free survival. This study aimed to understand which variables correlate with achieving an MPR. Methods: Patients with resectable HCC who received either neoadjuvant nivolumab plus ipilimumab or nivolumab alone and underwent surgery were included. 18 patients had baseline and post-treatment computed tomography, alpha-fetoprotein (AFP), and elastography. 17 of these patients had baseline and post-treatment tissue available for immunohistochemistry. Patients were classified into two groups: MPR, defined as necrosis of >70%, and no MPR. Data was summarized using descriptive statistics and compared using Wilcoxon rank sum test. P value <0.05 was considered statistically significant. Results: Statistically significant differences in objective response (OR) and changes in CD8, Granzyme B, and PD-1 expression were identified upon comparison of patients with an MPR to those without an MPR (Table 1). Additionally, numerical differences in baseline tumor size, change in AFP, and both non-tumor and tumor liver fibrosis (at baseline, after treatment, and interim change) were identified upon comparison of patients with an MPR to those without an MPR. Conclusions: Our data show that large baseline tumor size, greater OR, reduction in AFP, increase in liver and tumor fibrosis, and increase in CD8, Granzyme B, and PD-1 expression after receipt of neoadjuvant immunotherapy are associated with achieving an MPR in patients with resectable HCC. [Table: see text]

PD1 and CD39 expression on tumor infiltrating lymphocytes in patients with gastric cancer.

377 Background: Activation and clonal expansion of tumor antigen-specific T cells in cancer patients are crucial for immunotherapies. Recent reports indicate that CD39 is a marker to distinguish tumor-reactive CD8 T cell populations from bystander populations. In gastric cancer (GC), anti-CD39 antibody, anti-PD-1 antibody, and FOLFOX combination therapy may be effective therapy at AACR 2022. Methods: We retrospectively reviewed the data of 62 GC patients who underwent gastrectomy at our hospital (2017 - 2018) and examined their rejected specimen’s immune-microenvironment using immunohistochemistry. Then we prospectively enrolled 16 GC patients. Their tumor tissue and tumor-infiltrating lymphocytes (TILs) were extracted from surgical specimens, these fractions were subjected to the RNA-seq analysis ( n = 3), CD107a degranulation assay ( n = 7) and 3H-Thymidine incorporation assay ( n = 7). Results: The densities of CD39+CD8 TILs positively correlated with the densities of CD4 T cells and B-cells in GC tissues ( P = 0.01, < 0.01, respectively). Furthermore, higher densities of CD39+CD8 TILs in GC patients were associated with better overall survival ( P < 0.01). We classified TILs into four groups based on expression levels of the PD-1 and CD39. The proportion of CD39+CD8 T cells was significantly higher in GC tissues than their PBMCs ( n = 13) and non-cancerous tissues ( n = 4). CD39+PD-1+CD8 (DP) TILs in primary tumors were elevated expression of multiple inhibitory receptors such as LAG3, TIGIT and reduced expression of IL-2 by RNA-seq analysis. T cells expanded in vitro from DP-TILs ( n = 5) efficiently recognized autologous tumor cells in CD107a assay compared with other populations, and considered to be tumor-reactive T cells fraction. However, since these DP TILs in primary tumors show exhausted phenotypes, capacities to kill tumor are expected to be reduced in the tumor-microenvironment (TME). By adding ATP to the DP-TILs in vitro at the concentration reported in the TME, we observed reduction of cell proliferation by 3H-thymidene incorporation assay and of degranulation by CD107a assay, and we observed recovery of these changes by adding anti-CD39 inhibitory antibody. Conclusions: DP-TILs may have superior T cell receptors recognizing tumor-antigens and contribute to tumor eradication, especially when administrated anti-CD39 antibody.

Helios characterized circulating follicular helper T cells with enhanced functional phenotypes and was increased in patients with systemic lupus erythematosus

Helios was related to the immunosuppressive capacity and stability of regulatory T cells. However, the significance of Helios in follicular help T (TFH) and follicular regulatory T (TFR) cells is unclear. This research aimed to clarify the significance of Helios (IKZF2) in TFH and TFR cells and its clinical value in systemic lupus erythematosus (SLE). IKZF2 mRNA in different cell subsets was analyzed. Helios+ percentages in TFH and TFR cells were identified in the peripheral blood of 75 SLE patients and 62 HCs (healthy controls). PD-1 and ICOS expression were compared between Helios+ and Helios− cells. The capacity of TFH cells to secrete IL-21 and TFR cells to secrete IL-10 was measured. Correlation analysis and receiver operating characteristic (ROC) curve analysis were conducted to assess the clinical significance of Helios-related TFH and TFR cell subsets in SLE. There was Helios expression in TFH and TFR cells. PD-1 and ICOS were lower in Helios+ TFR than in Helios− TFR. ICOS was increased in Helios+ TFH cells compared with Helios− TFH cells, and ICOS in Helios+ TFH cells was downregulated in SLE. Helios+ TFH cells secreted more IL-21 than Helios− TFH cells, and Helios+ TFH cells from SLE patients had a stronger IL-21 secretion than HCs. Helios+ TFH percentages were negatively correlated with C3 and C4 and positively related to CRP and SLEDAI, and the AUC of Helios+ TFH to distinguish SLE from HC was 0.7959. Helios characterizes circulating TFH cells with enhanced function. Increased Helios+ TFH cells could reflect the autoimmune status of SLE.Graphical abstract

Role of thymosin α1 in restoring immune response in immunological nonresponders living with HIV

BackgroundImmunological nonresponders (INRs) living with HIV are at increased risk of co-infection and multiple tumors, with no effective strategy currently available to restore their T-cell immune response. This study aimed to explore the safety and efficacy of thymosin α1 in reconstituting the immune response in INRs.MethodsINRs with CD4 + T cell counts between 100 and 350 cells/μL were enrolled and received two-staged 1.6 mg thymosin α1 subcutaneous injections for 24 weeks (daily in the first 2 weeks and biweekly in the subsequent 22 weeks) while continuing antiretroviral therapy. T cell counts and subsets, the expression of PD-1 and TIM-3 on T cells, and signal joint T cell receptor excision circles (sjTREC) at week 24 were evaluated as endpoints.ResultsTwenty three INRs were screened for eligibility, and 20 received treatment. The majority were male (19/20), with a median age of 48.1 years (interquartile range: 40.5–57.0) and had received antiretroviral therapy for 5.0 (3.0, 7.3) years. Multiple comparisons indicated that CD4 + T cell count and sjTREC increased after initiation of treatment, although no significant differences were observed at week 24 compared to baseline. Greatly, levels of CD4 + T cell proportion (17.2% vs. 29.1%, P < 0.001), naïve CD4 + and CD8 + T cell proportion (17.2% vs. 41.1%, P < 0.001; 13.8% vs. 26.6%, P = 0.008) significantly increased. Meanwhile, the proportion of CD4 + central memory T cells of HIV latent hosts (42.7% vs. 10.3%, P < 0.001) significantly decreased. Moreover, the expression of PD-1 on CD4 + T cells (14.1% vs. 6.5%, P < 0.001) and CD8 + T cells (8.5% vs. 4.1%, P < 0.001) decreased, but the expression of TIM-3 on T cellsremained unaltered at week 24. No severe adverse events were reported and HIV viral loads kept stable throughout the study.ConclusionsThymosin α1 enhance CD4 + T cell count and thymic output albeit as a trend rather than an endpoint. Importantly, it improves immunosenescence and decreases immune exhaustion, warranting further investigation.Trial registrationThis single-arm prospective study was registered with ClinicalTrials.gov (NCT04963712) on July 15, 2021.

Negative prognostic behaviour of PD-L1 expression in tongue and larynx squamous cell carcinoma and its significant predictive power in combination with PD-1 expression on TILs

BackgroundBiomarkers that can predict outcome will improve the efficacy of treatment for HNSCC patients. In this regard, we retrospectively evaluated the prognostic effect of PD1, PD-L1, and CD45RO in tongue and larynx squamous cell carcinomas.MethodsFFPE tissue blocks of 63 larynx and 40 tongue squamous cell carcinoma samples were selected, cut into 3 µm sections, and immunohistochemically stained for PD1, PD-L1, and CD45RO. The slides were evaluated by an expert pathologist, and results were analysed using Chi-square, univariate, and multivariable Cox regression methods.ResultsTC-PD-L1 expression (P = 0.001) and its expression intensity (P = 0.002) were significantly correlated with a higher percentage of PD-1 + tumor infiltrating lymphocytes. In univariate survival analysis, TC-PD-L1 and its expression intensity had a significant impact on both DFS (HR: 0.203; P = 0.003 and HR: 0.320; P = 0.005) and OS (HR: 0.147; P = 0.002 and HR: 0.322; P = 0.005). Based on the multivariate analysis, PD1 (DFS: HR: 3.202; P = 0.011, OS: HR: 2.671; P = 0.027) and TC-PD-L1 (DFS: HR: 0.174; P = 0.006, OS: HR: 0.189; P = 0.009) were found to be independent prognostic markers. In the second part, scoring systems were defined based on the expression status of PD1 and PD-L1. Patients with higher scores were expected to have longer DFS and OS. In multivariate analysis, the PD1/TC-PD-L1 (DFS: P = 0.001, OS: P = 0.003) scoring systems showed superior prognostic effects. Interestingly, at the highest levels of this score, none of the patients experienced recurrence or cancer-caused death.ConclusionCollectively, this study suggests negative prognostic behaviour for TC-PD-L1 protein and introduces the PD-1/TC-PD-L1 scoring system as a strong prognostic marker in OS and DFS prediction of tongue and larynx HNSCC patients.

Abstract B014: Immuno-modulation of the tumor microenvironment of pancreatic adenocarcinoma following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT)

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors. Unlike other tumors, the progress of systemic therapies has been very slow, mainly due to the peculiar and resistant tumor microenvironment (TME) of PDAC. The addition of ablative stereotactic body radiation therapy (SBRT) in a total neoadjuvant strategy is promising for the treatment of localized PDAC and is currently being explored in several clinical trials. However, if radiation therapy possesses the ability to modulate the TME, the impact of high-dose SBRT is still poorly known in PDAC. Here, we aim to characterize by immunohistochemistry (IHC) and RNA sequencing (RNAseq) analysis the immuno-modulations of the TME following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT). MATERIAL: Paraffin-embedded residual tumoral tissues of 50 localized PDAC resected between 2011 and 2020 were used: seventeen patients had surgery first, seventeen received an induction chemotherapy with FOLFIRINOX (FFX) only and sixteen with FFX followed by an iHD-SBRT designed to individually maximize the dose prescribed to the tumor and vessels interfaces up to Dmax(0.5cc)&amp;lt;53Gy in 5 fractions. RESULTS: We found that although collagen deposition increases significantly after iHD-SBRT (COL1: 83.27 vs 78.60 vs 68.04%, p&amp;lt;0.001 for iHD-SBRT, FFX and treatment naïve cohort respectively), the intra-tumoral lymphocyte infiltration is globally not altered, including for cytotoxic CD8+ lymphocytes. Only the IHC expression of CD4+ T helper population is significantly decreased after iHD-SBRT. Using gene set enrichment analysis (GSEA) of the KEGG pathway, the IL-17 signaling pathway related to the T17 cells is particularly significantly increased after iHD-SBRT compared to FFX only. After iHD-SBRT, the IHC expression levels of FOXP3+ cells and PD-L1 are significantly increased. No difference is observed for CD68+ expression however, cell type enrichment analysis from our RNA-seq data using xCell algorithm shows that the M2-polarized CD68+ cells are increased after iHD-SBRT compared to FFX alone. PD-1 expression is significantly decreased after iHD-SBRT. Furthermore, RNAseq data revealed that iHD-SBRT is associated with significant enrichment in basaloid cells (p&amp;lt;0.0024), a subtype of basal–like cells associated with immunomodulatory stroma and better clinical outcomes. GSEA canonical pathway analysis further revealed metabolism-related pathways such as oxidative phosphorylation and gluthathione metabolism significantly enriched in iHD-SBRT treated group, suggesting the potential of mitochondrial inhibitors as candidates for combination therapy. CONCLUSIONS: iHD-SBRT is able to durably immuno-modulate the TME of PDAC. If the overall T-cell infiltration is preserved, we have identified some increased pro-tumoral cells and pathways after iHD-SBRT that could improve the development of better-oriented combination trials involving high-dose SBRT. Citation Format: Christelle Bouchart, Oier Azurmendi Senar, Julie Navez, Laurine Verset, Anaïs Boisson, Matthieu Hein, Nicky D’Haene, Dirk Van Gestel, Luigi Moretti, Pieter Demetter, Jean-Baptiste Bachet, Karen Willard-Gallo, Rémy Nicolle, Tatjana Arsenijevic, Jean-Luc Van Laethem. Immuno-modulation of the tumor microenvironment of pancreatic adenocarcinoma following isotoxic high-dose stereotactic body radiotherapy (iHD-SBRT) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B014.

Rechallenge of immune checkpoint inhibitors in advanced non-small cell lung cancer.

Immune checkpoint inhibitor (ICI) rechallenge in non-small cell lung cancer (NSCLC) is a promising therapeutic strategy. The situation for ICI rechallenge can be divided into three categories: adverse events (AEs); resistance to ICIs, and rechallenge becomes compulsive because of tumor relapse while the patients had completed a 2 year course of immunotherapy. However, these categories are still controversial and should be explored further. Through voting at the 6th Straits Summit Forum on Lung Cancer, in this study we summarize the consensus of 147 experts in ICI rechallenges. A total of 97.74% experts agreed to rechallenge; 48.87% experts rechallenge with the original drug, and the others rechallenge with a different drug; 40.3% agreed to rechallenge directly after progression; 88.06% experts agreed to ICI rechallenge with a combination regimen; and factors such as previous performance status score, PD-1 expression, and age should also be considered. Understanding the the clinical studies in ICI rechallenge could bring us one step closer to understanding the consensus. In patients with advanced NSCLC who have suffered recurrent or distant metastasis after immunotherapy, the option of rechallenge with ICIs is a promising treatment option.

T lymphocyte-derived immunogenic hybrid nanovesicles for immunosuppression reversion and boosted photothermal immunotherapy

Photothermal therapy (PTT) can mediate the immunogenic cell death (ICD) of tumors through photothermal transformation, thus releasing tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) to stimulate the maturation of dendritic cells (DCs) and elicit the immune response against tumors. However, PTT-mediated upregulation of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) will cause the immune evasion of tumor cells and thus increases the immunosuppression of cytotoxic T lymphocytes (CTLs), resulting in restricted photothermal immune activation. Taking advantage of the high expression of programmed cell death protein 1 (PD-1) in T lymphocytes, we develop an immunogenic photothermal hybrid nanovesicle to reverse immunosuppression and enhance PTT-induced ICD. The hybrid vesicle (TMVL-I) is constructed by phospholipid bilayer fusion of T cell-derived membrane vesicle (TMV) with high PD-1 expression and the indocyanine green (ICG)-loaded thermosensitive liposome (L-I). TMVL-Is bind to the tumor cells with PTT-induced upregulation of PD-L1, effectively blocking the PD-1/PD-L1 pathway between CTLs and tumor cells, thus reversing PD-L1-mediated tumor immunosuppression and intensifying photothermal-induced ICD effects. These processes significantly induce the maturation of DCs and enhance the infiltration of CTLs into both primary and distal tumors and the activation of spleen CTLs, strongly inhibiting the growth of B16 xenograft tumors and greatly improving the survival rate of B16 xenograft mice. This work provides a feasible strategy for immunosuppressive microenvironment reprogramming-based combination therapy.

Immunophenotyping with high-dimensional flow cytometry identifies Treg cell subsets associated with recurrence in papillary thyroid carcinoma.

The activation of Treg cell subsets is critical for the prognosis of tumor patients; however, their heterogeneity and disease association in papillary thyroid carcinoma (PTC) need further investigation. We performed high-dimensional flow cytometry for immunophenotyping on thyroid tissues and matched peripheral blood samples from patients with multinodular goiters or PTC. We analyzed CD4+ T cell and Treg cell phenotypes and compared the recurrence-free survival of PTC patients with different Treg cell subset characteristics using TCGA. Furthermore, PTC recurrent and non-recurrent group were compared by multiplex immunohistochemistry. High-dimensional flow cytometry and bioinformatics analysis revealed an enrichment of Tregs in tumors compared with multinodular goiters and peripheral blood specimens. Moreover, effector Tregs (e-Tregs) as well as FOXP3+ non-Tregs were enriched in tumor samples, and the expression of CD39, PD-1, and CD103 increased on tumor Tregs. TCGA data analysis showed that individuals with CD39hi PD-1loCD103loe-Treghi and CD39loPD-1loCD103hie-Treghi expression patterns had a high recurrence rate. According to the multiplex immunohistochemistry and analysis, compared with non-recurrent group, the proportion of high recurrence rate effector Treg clusters (CD39+PD-1-CD103- plus CD39-PD-1-CD103+) was increased in recurrent patients. Overall, our results highlight the potential of e-Treg subsets as future immunotherapy targets for PTC recurrence.

Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Glycolysis Induced by METTL14 Is Essential for Macrophage Phagocytosis and Phenotype in Cervical Cancer.

N 6-methyladenosine (m6A) is the most abundant mRNA modification in mammals and it plays a vital role in various biological processes. However, the roles of m6A on cervical cancer tumorigenesis, especially macrophages infiltrated in the tumor microenvironment of cervical cancer, are still unclear. We analyzed the abnormal m6A methylation in cervical cancer, using CaSki and THP-1 cell lines, that might influence macrophage polarization and/or function in the tumor microenvironment. In addition, C57BL/6J and BALB/c nude mice were used for validation invivo. In this study, m6A methylated RNA immunoprecipitation sequencing analysis revealed the m6A profiles in cervical cancer. Then, we discovered that the high expression of METTL14 (methyltransferase 14, N6-adenosine-methyltransferase subunit) in cervical cancer tissues can promote the proportion of programmed cell death protein 1 (PD-1)-positive tumor-associated macrophages, which have an obstacle to devour tumor cells. Functionally, changes of METTL14 in cervical cancer inhibit the recognition and phagocytosis of macrophages to tumor cells. Mechanistically, the abnormality of METTL14 could target the glycolysis of tumors invivo and vitro. Moreover, lactate acid produced by tumor glycolysis has an important role in the PD-1 expression of tumor-associated macrophages as a proinflammatory and immunosuppressive mediator. In this study, we revealed the effect of glycolysis regulated by METTL14 on the expression of PD-1 and phagocytosis of macrophages, which showed that METTL14 was a potential therapeutic target for treating advanced human cancers.

Antigen-presenting B cells promote TCF-1+ PD1- stem-like CD8+ T-cell proliferation in glioblastoma.

Understanding the spatial relationship and functional interaction of immune cells in glioblastoma (GBM) is critical for developing new therapeutics that overcome the highly immunosuppressive tumor microenvironment. Our study showed that B and T cells form clusters within the GBM microenvironment within a 15-μm radius, suggesting that B and T cells could form immune synapses within the GBM. However, GBM-infiltrating B cells suppress the activation of CD8+ T cells. To overcome this immunosuppression, we leveraged B-cell functions by activating them with CD40 agonism, IFNγ, and BAFF to generate a potent antigen-presenting B cells named BVax. BVax had improved antigen cross-presentation potential compared to naïve B cells and were primed to use the IL15-IL15Ra mechanism to enhance T cell activation. Compared to naïve B cells, BVax could improve CD8 T cell activation and proliferation. Compared to dendritic cells (DCs), which are the current gold standard professional antigen-presenting cell, BVax promoted highly proliferative T cells in-vitro that had a stem-like memory T cell phenotype characterized by CD62L+CD44- expression, high TCF-1 expression, and low PD-1 and granzyme B expression. Adoptive transfer of BVax-activated CD8+ T cells into tumor-bearing brains led to T cell reactivation with higher TCF-1 expression and elevated granzyme B production compared to DC-activated CD8+ T cells. Adoptive transfer of BVax into an irradiated immunocompetent tumor-bearing host promoted more CD8+ T cell proliferation than adoptive transfer of DCs. Moreover, highly proliferative CD8+ T cells in the BVax group had less PD-1 expression than those highly proliferative CD8+ T cells in the DC group. The findings of this study suggest that BVax and DC could generate distinctive CD8+ T cells, which potentially serve multiple purposes in cellular vaccine development.

Profiling of VEGF Receptors and Immune Checkpoints in Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is caused by human papilloma virus (HPV) infection of the aerodigestive tract that significantly impacts quality-of-life including the ability to communicate and breathe. Treatment was traditionally limited to serial ablative procedures in the O.R. with possible local adjuvant therapy, but new systemic therapies, such as Vascular endothelial growth factor (VEGF) inhibitors, are showing significant promise. This study aims to determine whether rationale exists for combination therapeutic approaches using VEGF inhibitors and/or immune checkpoint blockade. Using fresh specimens from the O.R., we performed flow cytometry on papilloma, normal adjacent tissue, and blood. Papilloma and surrounding tissue were examined for expression of PD-L1, PD-L2, Galectin-9, VEGFR2, and VEGFR3. CD8+ and CD4+ T cells were assayed for expression of PD-1, TIGIT, LAG3, and TIM3. Our data shows that papilloma tissue exhibits significantly higher levels of PD-L1 and PD-L2 compared to adjacent tissue. Elevated levels of the VEGF receptor VEGFR3 were also observed in papilloma tissue. When examining T cells within the papilloma, elevated PD-1 and TIGIT expression was observed on CD8+ T cells, while levels of PD-1, TIGIT, and TIM3 were elevated on CD4+ T cells compared to PBMCs. Heterogenous marker expression was observed between individuals. Our analysis shows that RRP tissue shows elevated levels of multiple immune check point targets and VEGFR3, with varied patterns unique to each papilloma patient. Some of these immune checkpoint markers already have novel immunotherapies available or in development, providing molecular rationale to offer these systemic treatments to selected patients affected by RRP alongside VEGF inhibitors. Laryngoscope, 134:2819-2825, 2024.

Could we use PD-1 and PD-L1 expression on lymphocytes and monocytes as predictive markers for prognosis of acute biliary pancreatitis?

PurposeThis study aimed to assess the significance of immunophenotyping and serum cytokines in predicting the clinical progression of acute biliary pancreatitis (ABP). Materials and methodsCytokine levels, T-helper, cytotoxic T, natural killer (NK) cells, monocytes, HLA-DR, and PD-1, as well as PDL-1 immune checkpoints, were measured in ABP patients at the time of diagnosis and compared with results from healthy volunteers. The study also compared leukocyte counts, hematocrit, immunophenotyping results, cytokine statuses, and PD-1, PDL-1 expression between healthy volunteers and ABP subgroups categorized by pancreatitis severity. ResultsThe study included 65 ABP patients and 20 healthy volunteers. Significant differences were observed between groups in hematocrit, leukocyte counts, total monocytes, lymphocytes, CD3+ total T cells, CD4+ Th cells, PD-1 expression on CD4+ and CD8+T lymphocytes, HLA-DR expression on CD14+ monocytes, NK cells, PD-L1 expression on CD14+ monocytes, classical and intermediate monocytes, as well as levels of IL-6, IL-8, IL-10, IL-18, and IL-33 cytokines. Moderate correlations were found with lymphocyte counts, PD-1+CD4+ cells, PD-L1+CD14+ cells, and strong correlations with HLA-DR+CD14+ cells. Hematocrit, CD3+ total T cells, NK cells, CD4+PD-1 + T cells, and CD8+PD-1 + T cells showed independent associations with the severity of ABP. Lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, CD4+PD-1 + T cells, classical, and intermediate monocytes exhibited the highest Area Under the Curve rates in determining organ failure. ConclusionsHematocrit, lymphocyte counts, CD14+HLA-DR+ cells, CD14+PD-L1+ cells, and intermediate monocytes emerged as parameters most closely associated with the severity and these parameters could be useful in predicting the severity of ABP.

Upregulation of PD-1/PD-L1 and downregulation of immune signaling pathways lead to more severe visceral leishmaniasis in undernutrition mice

BackgroundLeishmaniasis is mainly prevalent in tropical and subtropical developing countries, where chronic undernutrition often co-exists. Undernutrition is reported to promote the progression of leishmaniasis, but its immune mechanisms have not been fully elucidated.MethodsTo simulate chronic undernutrition of patients in epidemic areas and explore the immune mechanism of undernutrition promoting leishmaniasis, BALB/c mouse models with different nutritional imbalances were established, including undernutrition 75%, undernutrition 65% and obesity mouse models. After infection with Leishmania donovani in these model mice, we focused on evaluating the progress of leishmaniasis in the spleen and liver, the expression of important immunosuppressive and immunoactivation molecules, and changes of spleen transcriptome. The immune signaling pathways enriched by differentially expressed genes and hub genes were analyzed.ResultsThe results showed that among the mouse infection models, undernutrition 75% + infection group had the highest parasite load in the spleen and liver at the 8th week post-infection, possibly due to the continuous increase of PD-1, PD-L1 and TCR. Spleen RNA-seq results suggested that some immune signaling pathways were downregulated in undernutrition 75% + infection group, including neutrophil extracellular trap formation, IL-17 signaling pathway, natural killer cell-mediated cytotoxicity, etc. Among them, neutrophil extracellular trap formation pathway had the largest number of downregulated genes. This also explained why undernutrition 75% + infection group had the highest parasite load. Through PPI network analysis, hub genes such as Lcn2, Ltf, Mpo, Dnaja1, Hspa1a, Hspa1b and Hsph1 were screened out and might play important roles in the process of undernutrition promoting leishmaniasis.ConclusionsUndernutrition upregulated PD-1 and PD-L1 expression and downregulated immune signaling pathways in mice with visceral leishmaniasis. The signaling pathways and hub genes may serve as drug targets or intervention targets for the treatment of leishmaniasis patients with undernutrition.Graphical

Anti-PD-L1 therapy altered inflammation but not survival in a lethal murine hepatitis virus-1 pneumonia model.

Because prior immune checkpoint inhibitor (ICI) therapy in cancer patients presenting with COVID-19 may affect outcomes, we investigated the beta-coronavirus, murine hepatitis virus (MHV)-1, in a lethal pneumonia model in the absence (Study 1) or presence of prior programmed cell death ligand-1 (PD-L1) antibody (PD-L1mAb) treatment (Study 2). In Study 1, animals were inoculated intratracheally with MHV-1 or vehicle and evaluated at day 2, 5, and 10 after infection. In Study 2, uninfected or MHV-1-infected animals were pretreated intraperitoneally with control or PD-L1-blocking antibodies (PD-L1mAb) and evaluated at day 2 and 5 after infection. Each study examined survival, physiologic and histologic parameters, viral titers, lung immunophenotypes, and mediator production. Study 1 results recapitulated the pathogenesis of COVID-19 and revealed increased cell surface expression of checkpoint molecules (PD-L1, PD-1), higher expression of the immune activation marker angiotensin converting enzyme (ACE), but reduced detection of the MHV-1 receptor CD66a on immune cells in the lung, liver, and spleen. In addition to reduced detection of PD-L1 on all immune cells assayed, PD-L1 blockade was associated with increased cell surface expression of PD-1 and ACE, decreased cell surface detection of CD66a, and improved oxygen saturation despite reduced blood glucose levels and increased signs of tissue hypoxia. In the lung, PD-L1mAb promoted S100A9 but inhibited ACE2 production concomitantly with pAKT activation and reduced FOXO1 levels. PD-L1mAb promoted interferon-γ but inhibited IL-5 and granulocyte-macrophagecolony-stimulating factor (GM-CSF) production, contributing to reduced bronchoalveolar lavage levels of eosinophils and neutrophils. In the liver, PD-L1mAb increased viral clearance in association with increased macrophage and lymphocyte recruitment and liver injury. PD-L1mAb increased the production of virally induced mediators of injury, angiogenesis, and neuronal activity that may play role in COVID-19 and ICI-related neurotoxicity. PD-L1mAb did not affect survival in this murine model. In Study 1 and Study 2, ACE was upregulated and CD66a and ACE2 were downregulated by either MHV-1 or PD-L1mAb. CD66a is not only the MHV-1 receptor but also an identified immune checkpoint and a negative regulator of ACE. Crosstalk between CD66a and PD-L1 or ACE/ACE2 may provide insight into ICI therapies. These networks may also play role in the increased production of S100A9 and neurological mediators in response to MHV-1 and/or PD-L1mAb, which warrant further study. Overall, these findings support observational data suggesting that prior ICI treatment does not alter survival in patients presenting with COVID-19.

Spatial features of specific CD103+CD8+ tissue-resident memory T cell subsets define the prognosis in patients with non-small cell lung cancer

BackgroundTissue-resident memory T (TRM) cells can reside in the tumor microenvironment and are considered the primary response cells to immunotherapy. Heterogeneity in functional status and spatial distribution may contribute to the controversial role of TRM cells but we know little about it.MethodsThrough multiplex immunofluorescence (mIF) (CD8, CD103, PD-1, Tim-3, GZMB, CK), the quantity and spatial location of TRM cell subsets were recognized in the tissue from 274 patients with NSCLC after radical surgery. By integrating multiple machine learning methods, we constructed a TRM-based spatial immune signature (TRM-SIS) to predict the prognosis. Furthermore, we conducted a CD103-related gene set enrichment analysis (GSEA) and verified its finding by another mIF panel (CD8, CD103, CK, CD31, Hif-1α).ResultsThe density of TRM cells was significantly correlated with the expression of PD-1, Tim-3 and GZMB. Four types of TRM cell subsets was defined, including TRM1 (PD-1−Tim-3−TRM), TRM2 (PD-1+Tim-3−TRM), TRM3 (PD-1−Tim-3+TRM) and TRM4 (PD-1+Tim-3+TRM). The cytotoxicity of TRM2 was the strongest while that of TRM4 was the weakest. Compare with TRM1 and TRM2, TRM3 and TRM4 had better infiltration and stronger interaction with cancer cells. The TRM-SIS was an independent prognostic factor for disease-free survival [HR = 2.43, 95%CI (1.63–3.60), P < 0.001] and showed a better performance than the TNM staging system for recurrence prediction. Furthermore, by CD103-related GSEA and mIF validation, we found a negative association between tumor angiogenesis and infiltration of TRM cells.ConclusionsThese findings reveal a significant heterogeneity in the functional status and spatial distribution of TRM cells, and support it as a biomarker for the prognosis of NSCLC patients. Regulating TRM cells by targeting tumor angiogenesis may be a potential strategy to improve current immunotherapy.

Sulconazole inhibits PD-1 expression in immune cells and cancer cells malignant phenotype through NF-κB and calcium activity repression.

The overexpression of the immunoinhibitory receptor programmed death-1 (PD1) on T-cells is involved in immune evasion in cancer. The use of anti-PD-1/PDL-1 strategy has deeply changed the therapies of cancers and patient survival. However, their efficacy diverges greatly along with tumor type and patient populations. Thereby, novel treatments are needed to interfere with the anti-tumoral immune responses and propose an adjunct therapy. In the current study, we found that the antifungal drug Sulconazole (SCZ) inhibits PD-1 expression on activated PBMCs and T cells at the RNA and protein levels. SCZ repressed NF-κB and calcium signaling, both, involved in the induction of PD-1. Further analysis revealed cancer cells treatment with SCZ inhibited their proliferation, and migration and ability to mediate tumor growth in zebrafish embryos. SCZ found also to inhibit calcium mobilization in cancer cells. These results suggest the SCZ therapeutic potential used alone or as adjunct strategy to prevent T-cell exhaustion and promotes cancer cell malignant phenotype repression in order to improve tumor eradication.

Exploring the prognostic potential of m6A methylation regulators in low-grade glioma: implications for tumor microenvironment modulation

BackgroundThe biological behavior of low-grade glioma (LGG) is significantly affected by N6-methyladenosine (m6A) methylation, an essential epigenetic alteration. Therefore, it is crucial to create a prognostic model for LGG by utilizing genes that regulate m6A methylation.MethodsUsing TCGA and GTEx databases. We examined m6A modulator levels in LGG and normal tissues, and investigated PD-L1 and PD-1 expression, immune scores, immune cell infiltration, tumor immune microenvironment (TIME) and potential underlying mechanisms in different LGG clusters. We also performed immunohistochemistry and RT-qPCR to identify essential m6A adjustment factor.ResultsThe results showed that m6A regulatory element expression was significantly increased in LGG tissues and was significantly associated with TMIE. A substantial increase in PD-L1 and PD-1 levels in LGG tissues and high-risk cohorts was observed. PD-L1 expression was positively correlated with FTO, ZCCHC4, and HNRNPD, whereas PD-1 expression was negatively correlated with FTO, ZC3H7B, and HNRNPD. The prognostic signature created using regulators of m6A RNA methylation was shown to be strongly associated with the overall survival of LGG patients, and FTO and ZCCHC4 were confirmed as independent prognostic markers by clinical samples. Furthermore, the results revealed different TIME characteristics between the two groups of patients, indicating disrupted signaling pathways associated with LGG.ConclusionOur results present that the m6A regulators play vital role in regulating PD-L1/PD-1 expression and the infiltration of immune cells, thereby exerting a sizable impact on the TIME of LGG. Therefore, m6A regulators have precise predictive value in the prognosis of LGG.