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Background and Objectives : Sepsis involves a dysregulated host response, characterized by simultaneous immunosuppression and hyperinflammation. Initially, there is the release of pro-inflammatory factors and immune system dysfunction, followed by persistent immune paralysis leading to apoptosis. This study investigates sepsis-induced apoptosis and its pathways, by assessing changes in PD-1 and PD-L1 serum levels, CD4+ and CD8+ T cells, and Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE II) severity scores. Materials and Methods : This prospective, observational, single-centre study enrolled 87 sepsis patients admitted to the intensive care unit at the County Emergency Clinical Hospital in Târgu Mureș, Romania. We monitored the parameters on day 1 (the day sepsis or septic shock was diagnosed as per the Sepsis-3 Consensus) and day 5. Results : Our study found a statistically significant variation in the SOFA score for the entirety of the patients between the studied days ( p = 0.001), as well as for the studied patient groups: sepsis, septic shock, survivors, and non-survivors ( p = 0.001, p = 0.003, p = 0.01, p = 0.03). On day 1, we found statistically significant correlations between CD8+ cells and PD-1 ( p = 0.02) and PD-L1 ( p = 0.04), CD4+ and CD8+ cells ( p < 0.0001), SOFA and APACHE II scores ( p < 0.0001), and SOFA and APACHE II scores and PD-L1 ( p = 0.001 and p = 0.01). On day 5, we found statistically significant correlations between CD4+ and CD8+ cells and PD-L1 ( p = 0.03 and p = 0.0099), CD4+ and CD8+ cells ( p < 0.0001), and SOFA and APACHE II scores ( p < 0.0001). Conclusions : The reduction in Th CD4+ and Tc CD8+ lymphocyte subpopulations were evident from day 1, indicating that apoptosis is a crucial factor in the progression of sepsis and septic shock. The increased expression of the PD-1/PD-L1 axis impairs costimulatory signalling, leading to diminished T cell responses and lymphopenia, thereby increasing the susceptibility to nosocomial infections.

Introduction: This study aims to explore Programmed Death Receptor-1 (PD-1) and Programmed Death Ligand-1 (PD-L1) variations in Lung Cancer (LC) tissues and Peripheral Blood (PPB) and their association with immunotherapy efficacy and prognosis., Method: 72 patients with LC were included in the LC group and 39 patients with concurrent benign lung disease were included in the benign group. PD-1/PDL-1 was compared in PPB and lung tissue. All LC patients were treated with immunotherapy. The relationship between PD-1/PDL-1 in LC tissue and PPB and immunotherapy efficacy was analyzed. Patients were divided into death and survival groups, and PD-1/PDL-1 in tumor tissues and PPB were compared., Results: The authors found that PD-1 and PDL-1 positive expression in lung tissue and PPB in LC patients was elevated. Combined detection of PD-1 and PDL-1 was effective in diagnosing LC and evaluating the prognosis of LC patients. PD-1 and PDL-1 positive expression was reduced after disease remission while elevated in dead patients. The 3-year survival rate of patients with PD-1 positive expression was 45.45 % (25/55), which was lower (82.35 %, 14/17) than those with PD-1 negative expression. The 3-year survival rate of patients with positive and negative expression of PDL-1 was 48.78 % (20/41) and 61.29 % (19/31), respectively., Discussion: The present results demonstrated that PD-1 and PDL-1 are abnormal in cancer tissue and PPB of LC patients. The combined detection of PD-1 and PDL-1 has diagnostic value for LC and evaluation value for the efficacy and prognosis of immunotherapy., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Background/aim: Meningiomas are the most common primary brain tumors of the central nervous system. Immunotherapy is a promising treatment method applied in many types of cancer. There is no standard and effective medical treatment to reduce recurrence and mortality in cases of incomplete resection of meningiomas and in high-grade cases. In order to investigate medical treatments in addition to surgery and radiotherapy, in this study, the status of immune checkpoint molecules (PD-L1/PD-1), which are the target of immunotherapy, in meningiomas was investigated., Materials and Methods: Four hundred two cases of meningioma diagnosed between 2007 and 2020 at our institution were used. New blocks were prepared from the appropriate blocks of the cases using the tissue microarray method. Sections obtained from these blocks were immunohistochemically stained with PD-1 and PD-L1 antibodies. Obtained data were interpreted with statistical analysis., Results: Expression of PD-L1 was observed in 28.4% of meningiomas. Staining rates are higher in high-grade tumors. The staining rate of PD-L1 in the tumor increased significantly with pattern loss. PD-L1 expression in immune cells is 19.9%. Immune cell expression and the number of expressing immune cells correlate with spontaneous necrosis. Immune cell expression and the number of expressing immune cells are increased in high-grade meningiomas. PD-1 expression in immune cells is 9.0%, and this correlates with brain invasion., Conclusions: With these data, it was observed that the expression of immune checkpoint molecules PD-L1 and PD-1 increased especially in high-grade meningiomas. It may be the subject of research that these molecules may be targets of immunotherapy in the treatment of meningiomas., Competing Interests: Conflict of interest: The authors declare that there are no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© TÜBİTAK.)

Background: Oral lichen planus (OLP) and oral epithelial dysplasia (OED) present diagnostic challenges due to clinical and histologic overlap. This study explores the immune microenvironment in OED, hypothesizing that immune signatures could aid in diagnostic differentiation and predict malignant transformation., Methods: Tissue samples from OED and OLP cases were analyzed using immunofluorescence/immunohistochemistry (IF/IHC) for CD4, CD8, CD163/STAT1, and PD-1/PDL-1 expression. RNA-sequencing was performed on the samples, and data was subjected to CIBERSORTx analysis for immune cell composition. Gene Ontology analysis on the immune differentially expressed genes was also conducted., Results: In OED, CD8 + T-cells infiltrated dysplastic epithelium, correlating with dysplasia severity. CD4 + lymphocytes increased in the basal layer. STAT1/CD163 + macrophages correlated with CD4 + intraepithelial distribution. PD-1/PDL-1 expression varied. IF/IHC analysis revealed differential immune cell composition between OED and OLP. RNA-sequencing identified upregulated genes associated with cytotoxic response and immunosurveillance in OED. Downregulated genes were linked to signaling, immune cell recruitment, and tumor suppression., Conclusions: The immune microenvironment distinguishes OED and OLP, suggesting diagnostic potential. Upregulated genes indicate cytotoxic immune response in OED. Downregulation of TRADD, CX3CL1, and ILI24 implies dysregulation in TNFR1 signaling, immune recruitment, and tumor suppression. This study contributes to the foundation for understanding immune interactions in OED and OLP, offering insights into future objective diagnostic avenues., (© 2024. The Author(s).)

Colorectal cancer is the third most common cancer worldwide and the second cause of death from malignant tumors. Colorectal cancers are treated with surgery, chemotherapy, gene therapy and immunotherapy. PD-1 and PD-L1 proteins have recently been considered as potential targets of anticancer therapy in colorectal cancer. The aim of this study was to evaluate the association of immunohistochemical expression of PD-1 and PD-L1 proteins in colorectal cancer patients with selected clinical and morphological parameters and their survival. Ninety-eight cases of colorectal cancer were studied. Immunohistochemistry was used to evaluate the expression of PD-1 and PD-L1 proteins. Correlations were found between the expression of PD-L1 protein in lymphocytes and lack of lymph node metastases and a lower clinical stage. There was also a correlation between PD-L1 protein expression in cancer cells and a higher grade of histological malignancy.

Background: Preliminary studies have suggested that soluble programmed death-1 (sPD-1) and soluble programmed cell death ligand-1 (sPD-L1) have prognostic implications in many malignant tumors. However, the correlation between sPD-1/sPD-L1 level and prognosis of hepatocellular carcinoma (HCC) is still unclear., Methods: We searched several electronic databases from database inception to October 7, 2021. Meta-analyses were performed separately for overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS), time to progression (TTP), and tumor-free survival (TFS). Random effects were introduced to this meta-analysis. The correlation between sPD-1/sPD-L1 level and prognosis was evaluated using hazard ratios (HRs) with 95% confidence intervals (95%CIs)., Results: A total of 11 studies (1291 patients) were incorporated into this meta-analysis, including seven on sPD-L1, two on sPD-1, and two about both factors. The pooled results showed that high sPD-L1 level was associated with worse OS (HR = 2.46, 95%CI 1.74-3.49, P < 0.001; I 2  = 31.4, P = 0.177) and poorer DFS/RFS/TTP/TFS of patients with HCC (HR = 2.22, 95%CI 1.47-3.35, P < 0.001; I 2  = 66.1, P = 0.011), irrespective of method of detection, study type, treatment, cut-off value and follow-up time. In contrast, the level of sPD-1 was not correlated to the OS (HR = 1.19, 95%CI 0.55-2.56, P = 0.657) and DFS/TFS of patients with HCC (HR = 0.94, 95%CI 0.36-2.49, P = 0.906)., Conclusion: sPD-L1 rather than sPD-1 could be a good predictor for recurrence and survival after treatment for HCC. More high-quality prospective studies are warranted to assess the prognostic value of sPD-1 or sPD-L1 for HCC., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this era of personalized medicine, targeted immunotherapies like immune checkpoint inhibitors (ICI) blocking the programmed death-1 (PD-1)/program death ligand-1 (PD-L1) axis have become an integral part of treating advanced stage non-small cell lung carcinoma (NSCLC) and many other cancer types. Multiple monoclonal antibodies are available commercially to detect PD-L1 expression in tumor cells by immunohistochemistry (IHC). As most clinical trials initially required tumor biopsy for PD-L1 detection by IHC, many of the currently available PD-1/PD-L1 assays have been developed and validated on formalin fixed tissue specimens. The majority (>50%) of lung cancer cases do not have a surgical biopsy or resection specimen available for ancillary testing and instead must rely primarily on fine needle aspiration biopsy specimens for diagnosis, staging and ancillary tests. Review of the literature shows multiple studies exploring the feasibility of PD-L1 IHC on cytological samples. In addition, there are studies addressing various aspects of IHC validation on cytology preparations including pre-analytical (e.g., different fixatives), analytical (e.g., antibody clone, staining platforms, inter and intra-observer agreement, cytology-histology concordance) and post-analytical (e.g., clinical outcome) issues. Although promising results in this field have emerged utilizing cytology samples, many important questions still need to be addressed. This review summarizes the literature of PD-L1 IHC in lung cytology specimens and provides practical tips for optimizing analysis., (© 2022 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Less than 50% of patients with melanoma respond to anti-programmed cell death protein 1 (anti-PD-1), and this treatment can induce severe toxicity. Predictive markers are thus needed to improve the benefit/risk ratio of immune checkpoint inhibitors (ICI). Baseline tumor parameters such as programmed death ligand 1 (PD-L1) expression, CD8 + T-cell infiltration, mutational burden, and various transcriptomic signatures are associated with response to ICI, but their predictive values are not sufficient. Interaction between PD-1 and its main ligand, PD-L1, appears as a valuable target of anti-PD-1 therapy. Thus, instead of looking at PD-L1 expression only, we evaluated the predictive value of the proximity between PD-1 and its neighboring PD-L1 molecules in terms of response to anti-PD-1 therapy., Experimental Design: PD-1/PD-L1 proximity was assessed by proximity ligation assay (PLA) on 137 samples from two cohorts (exploratory n = 66 and validation n = 71) of samples from patients with melanoma treated with anti-PD-1±anti-CTLA-4. Additional predictive biomarkers, such as PD-L1 expression (MELscore), CD8 + cells density, and NanoString RNA signature, were also evaluated., Results: A PD-1/PD-L1 PLA model was developed to predict tumor response in an exploratory cohort and further evaluated in an independent validation cohort. This score showed higher predictive ability (AUC = 0.85 and 0.79 in the two cohorts, respectively) for PD-1/PD-L1 PLA as compared with other parameters (AUC = 0.71-0.77). Progression-free and overall survival were significantly longer in patients with high PLA values ( P = 0.00019 and P < 0.0001, respectively)., Conclusions: The proximity between PD-1 and PD-L1, easily assessed by this PLA on one formalin-fixed paraffin-embedded section, appears as a new biomarker of anti-PD-1 efficacy., (©2021 American Association for Cancer Research.)

Immune checkpoint inhibitors (ICIs) have shown unprecedented benefits in various adult cancers, and this success has prompted the exploration of ICI therapy even in childhood malignances. Although the use of ICIs as individual agents has achieved disappointing response rates, combinational therapies are likely to promise better results. However, only a subset of patients experienced prolonged clinical effects, thus suggesting the need to identify robust bio-markers that predict individual clinical response or resistance to ICI therapy as the main challenge. In this review, we focus on how the use of ICIs in adult cancers can be translated into pediatric malignances. We discuss the physiological mechanism of action of each IC, including PD-1, PD-L1 and CTLA-4 and the new emerging ones, LAG-3, TIM-3, TIGIT, B7-H3, BTLA and IDO-1, and evaluate their prognostic value in both adult and childhood tumors. Furthermore, we offer an overview of preclinical models and clinical trials currently under investigation to improve the effectiveness of cancer immunotherapies in these patients. Finally, we outline the main predictive factors that influence the efficacy of ICIs, in order to lay the basis for the development of a pan-cancer immunogenomic model, able to direct young patients towards more specific immunotherapy., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Local immunoglobulin hyperproduction is observed in nasal polyps (NPs) with and without ectopic lymphoid tissues (eLTs)., Objective: Our aim was to identify the T-cell subsets involved in local immunoglobulin production independent of eLTs in NPs., Methods: The localization, abundance, and phenotype of CD4 + T-cell subsets were studied by immunofluorescence, flow cytometry, and single-cell RNA sequencing. Purified nasal T-cell subsets were cultured with autologous peripheral naive B cells to explore their function. Programmed death ligand 1 and programmed death ligand 2 expression in NPs was investigated by immunofluorescence staining and flow cytometry., Results: Accumulation of PD-1 high CXCR5 - CD4 + T cells outside lymphoid aggregates was found in NPs. Nasal PD-1 high CXCR5 - CD4 + T cells were characterized by a unique phenotype that was related to B-cell help and tissue residency and distinct from PD-1 -/int CXCR5 - and CXCR5 + CD4 + T cells in NPs as well as PD-1 high CXCR5 high CD4 + follicular helper T cells in tonsils. Compared with the frequencies of PD-1 high CXCR5 - CD4 + T cells and their IFN-γ + , IL-17A + , and IL-21 + subsets in the control inferior turbinate tissues, the frequencies of these cells and their subsets were increased in both eosinophilic and noneosinophilic NPs, whereas the frequencies of the IL-4 + and IL-4 + IL-21 + subsets were increased only in eosinophilic NPs. Nasal PD-1 high CXCR5 - CD4 + T cells induced immunoglobulin production from B cells in a potency comparable to that induced by tonsillar follicular helper T cells. PD-1 high CXCR5 - CD4 + T-cell frequencies were correlated with IgE levels in eosinophilic NPs. PD-L1 and PD-L2 suppressed the function of PD-1 high CXCR5 - CD4 + T cells, and their levels were reduced in NPs. PD-1 high CXCR5 - CD4 + T-cell abundance was associated with the postsurgical relapse of NPs., Conclusion: PD-1 high CXCR5 - CD4 + T cells participate in local immunoglobulin production independent of eLTs in NPs., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Purpose: This pilot study aimed on generating insight on alterations in circulating immune cells during the use of FOLFIRINOX and gemcitabine/nab-paclitaxel in pancreatic ductal adenocarcinoma (PDAC)., Patients and Methods: Peripheral blood mononuclear cells were isolated before and 30 days after initiation of chemotherapy from 20 patients with advanced PDAC. Regulatory T cells (FoxP3+) and immune checkpoints (PD-1 and TIM-3) were analyzed by flow cytometry and immunological changes were correlated with clinical outcome., Results: Heterogeneous changes during chemotherapy were observed in circulating T-cell subpopulations with a pronounced effect on PD-1+ CD4+/CD8+ T cells. An increase in FoxP3+ or PD-1+ T cells had no significant effect on survival. An increase in TIM3+/CD8+ (but not TIM3+/CD4+) T cells was associated with a significant inferior outcome: median progression-free survival in the subgroup with an increase of TIM-3+/CD8+ T cells was 6.0 compared to 14.0 months in patients with a decrease/no change (p = 0.026); corresponding median overall survival was 13.0 and 20.0 months (p = 0.011), respectively., Conclusions: Chemotherapy with FOLFIRNOX or gemcitabine/nab-paclitaxel induces variable changes in circulating T-cell populations that may provide prognostic information in PDAC., (© 2021. The Author(s).)

Background: The progression to gastric cancer has been linked to chronic infection with Helicobacter pylori (H. pylori). Immune checkpoint inhibitors (programmed cell death -1, PD-1; programmed cell death -ligand 1, PD-L1) have a role in cancer immune escape. The relationship between H. pylori virulence factors with PD-1, PD-L1 T helper 1 (Th1), T helper 17 (Th17), and regulatory T cell (Treg) response genes, has not been thoroughly investigated in the development of gastric cancer. Therefore, we evaluated how H. pylori virulence factors influence the expression levels of immune-related genes in the development of gastric immunopathology., Methods: A total of 92 gastric tissues of normal controls and patients with gastritis, gastric ulcer, and gastric cancer were examined for the expression of immune-checkpoint inhibitor genes (PD-1 PD-L1), Th1 (interferon- γ, IFN-γ), Th17 (interleukin- 17, IL-17, Retinoic-acid-receptor- related orphan nuclear receptor gamma t, RORγ-t), and Treg (Forkhead box P3, FOXP3) response genes with quantitative real-time PCR (qRT-PCR). Furthermore, correlation of H. pylori virulence factors' (cytotoxin-associated gene A, cagA; vacuolating cytotoxin gene A, vacA (s1,s2,m1,m2); blood group antigen-binding adhesin gene A, babA, duodenal ulcer promoting gene A, dupA; the putative neuraminyllactose-binding hemagglutinin homolog, hpaA; neutrophil-activating protein A napA; outer inflammatory protein A, oipA; urease A, ureA; and urease B, ureB) genotypes with a degree of inflammation and density of H. pylori were investigated. Next, the relationship between H. pylori virulence factors and immune-checkpoint inhibitor genes, and T-cell response genes was evaluated. Eventually, a decision tree model was developed to determine the clinical outcome of patients using expression data., Results: The intensity of PD-1 and PD-L1 mRNA expression was increased significantly in gastric tissue of patients with gastric ulcer (PD-1: 2.3 fold, p=0.01; PD-L1: 2.1 fold, p=0.004), and gastric cancer (PD-1: 2 fold, p= 0.04; PD-L1: 1.8 fold, p=0.05) compared with control subjects. Also, PD-1: PD-L1 expression was significantly higher in patients with gastritis, who were infected with a marked density of H. pylori compared with its mildly infected counterparts. Furthermore, a novel negative correlation was found between PD-1 (r= -0.43) and PD-L1 (r= -0.42) with FOXP3 in patients with gastritis. CagA-positive H. pylori strain's negative association with PD-L1 expression (r=-0.34) was detected in patients with gastritis. Interestingly, PD-1 mRNA expression correlated positively with vacA s2/m2, in gastritis (r=0.43) and ulcer (r=0.43) patients. Furthermore, PD-1: PDL1 expression negatively correlated with vacA m1/m2 (r=-0.43 for PD-1; r=-0.38 for PD-L1) in gastritis patients. Moreover, an inverse correlation of PDL1 was present with vacA m1 (r=0.52) and vacA s1/m1 (r=0.46) versus vacA m2 (r=-0.44) and vacA m1 (r=0.52) and vacA s1/m2 (r=-0.14) in ulcer patients, respectively. Also, a correlation of vacA m2 (r=-0.47) and vacA s1/s2 (r= 0.45) with PD-1 was detected in ulcer patients. In addition, a novel negative correlation between FOXP3 mRNA levels and napA was shown in patients with gastritis and ulcer (r=-0.59). Finally, a computer-based model that was developed showed that knowing the expression levels of PD-L1, RORγ-t, and vacA s1/m2 would be useful to detect the clinical outcome of a patient., Conclusion: Our results suggested that PD-1:PD-L1 immune checkpoint inhibitors were increased in gastric pre-cancerous lesions that progress to gastric cancer. Herein, we report the relationship between H. pylori virulence factors and expression of host immune checkpoint inhibitors for diagnostic prediction of gastric malignancies using computer-based models., (Copyright © 2021. Published by Elsevier B.V.)

Background: Immune checkpoint proteins, especially PD-L1 and PD-1, play a crucial role in controlling the intensity and duration of the immune response, thus preventing the development of autoimmunity. These proteins play a vital role in enabling cancer cells to escape immunity, proliferate and progress., Methods: This brief review highlights essential points related to testing for immune checkpoint therapy that histopathologists need to know., Results: In recent years, several inhibitors of these proteins have been used to reactivate the immune system to fight cancer. Selection of patients for such therapy requires demonstration of PD-L1 activation on the tumor cells, best done by immunohistochemical staining of the tumor and immune cells using various antibodies with predetermined thresholds., Conclusions: Immune checkpoint therapy appears to be promising and is rapidly expanding to include a large variety of cancers., (© 2021. The Author(s).)

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β-dependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade.

In psoriasis, CD8 + CD103 + memory T cells residing in the epidermis represent an effector population capable of maintaining the condition and driving a recurrence of the disease. Tissue-infiltrating CD8 + T cells expressing PD-1 are regarded as antigen-primed effector cells in others chronic inflammatory diseases. However, the expression and significance of PD-1 on skin-infiltrating CD8 + T cells in human psoriasis is not known. By analyzing skin-infiltrating T cells from human psoriasis, we found that active psoriatic epidermis contained PD-1 expressing CD8 + CD103 + T cells that correlated with the disease severity and histopathology. PD-1 + CD8 + CD103 + T cells possessed a canonical psoriasis-specific resident memory phenotype with IL-23R expression and produced IL-17A, whereas PD-1 - CD8 + CD103 + T cells preferentially produced IFN-γ. The diversity of skin-infiltrating T cells was dominated by CD4 + T cells, while CD8 + T cells, especially CD8 + CD103 + T cells, represented an oligoclonal population in active psoriasis. In addition, PD-1 + CD8 + CD103 + T cells used different TCR Vβs from PD-1 - CD8 + CD103 + T cells counterpart. In the early resolved lesion, the composition and functional status of PD-1 + CD8 + CD103 + T cells were markedly altered, while PD-1 - CD8 + CD103 + T cells population was minimally changed. Collectively, PD-1 expression delineates a putative pathogenic subset of epidermal CD8 + CD103 + T cells, which possibly play a role in psoriasis pathogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8 + T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is a common malignancy with a worldwide distribution. Despite bowel cancer screening programmes, the management of patients with metastatic disease is still an important and challenging problem. Immune checkpoint inhibitor (ICI) therapy is a well-established treatment in several cancers, eg, malignant melanoma and non-small cell lung carcinoma and is used in metastatic disease. The principle of this treatment is to use monoclonal antibodies to block the immune tolerance that commonly develops to tumor cells, therefore allowing host T-cell immunity to recognise and lyse cancer cells. The cellular receptors most commonly targeted by ICI therapy are cytotoxic T-lymphocyte-associated protein-4 and the programmed death 1/programmed death ligand 1 system. This review provides a scientific background to current ICI therapy and discusses the factors that predict response to this treatment. This is followed by a description of the emerging evidence for the use of ICI therapy in CRC and the utility of cellular pathology in stratifying patients for this treatment, especially when the systemic disease is present.

Background: During the last decades, the management for metastatic colorectal cancer patients has improved due to novel therapeutic approaches. A mismatch-repair deficient status seems to favour a better response to checkpoint inhibitor therapy, but the question arises whether a specific subgroup of stage IV patients with mismatch-repair (MMR) proficient status should also be considered. RHAMM (Receptor for Hyaluronic Acid Mediated Motility/HAMMR/CD168) is characterized by tumor progression and immunogenicity. Therefore, the aim of this study is to determine whether RHAMM within the CRLM of MMR-proficient patients correlate with a more immunological microenvironment, represented by cytotoxic T-cells, PD-1 and PD-1., Methods: Two patient cohorts of liver metastases from MMR colorectal cancers were included into the study (n = 81 and 76) using ngTMA® technology and immunohistochemically analyzed for RHAMM, cytotoxic T-cells (CD8+), PD-1/PD-L1, intrametastatic budding (IMB) and perimetastatic budding (PMB)., Results: RHAMM-positive IMB was linked to a higher PD-L1 expression (r = 0.32; p = 0.233 and r = 0.28; p = 0.044) in the center and periphery of the metastasis and RHAMM-positive PMB was associated with a higher expression of PD-1 (r = 0.33; p = 0.0297), and especially PD-L1 (r = 0.604; p < 0.0001 and r = 0.43; p = 0.003) in the center and periphery of the metastasis. IMB and PMB were additionally associated with a higher count of CD8+ T-cells (p < 0.0001; r = 0.58; p < 0.0001; r = 0.53)., Conclusions: The RHAMM status can be assessed in IMB/PMB either in biopsies or in resections of colorectal cancer liver metastases. A positive RHAMM status in IMB and/or PMB may be a potential indicator for a checkpoint inhibitor therapy for stage IV colorectal cancer patients with MMR proficient status., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication of transplantation. In addition to reactivation of Epstein-Barr virus in immunocompromised patients, impaired tumor immunity is suggested to be a risk factor for PTLD. However, it remains unclear whether immune suppressive tumor-infiltrating lymphocytes (TILs) correlate with the occurrence or prognosis of PTLD. We analyzed TILs in 26 patients with PTLD to elucidate the clinicopathological significance of the expression of PD-1 and FoxP3, which are associated with exhausted T-cells and regulatory T-cells (Tregs), respectively. Numbers of PD-1 + TILs in the PTLD specimens were significantly higher in patients who developed PTLD early after transplantation (P = 0.0040), while numbers of FoxP3 + TILs were not (P = 0.184). There was no difference in overall response rate regardless of the expression of PD-1 or FoxP3. FoxP3 high patients tended to have a shorter time to progression compared with FoxP3 low patients, especially in the case of FoxP3 high patients with diffuse large B-cell lymphoma-subtype PTLD (P = 0.011), while PD-1 high patients did not. These results suggest that T-cell exhaustion may be mainly associated with PTLD development, while immune suppression by Tregs may be dominant in enhanced progression of PTLD following disease occurrence.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163 + PD-L1 - myeloid cells and CD8 + FoxP3 + PD-1 low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This work describes the modification of a gold electrode with the BMS-8 compound that interacts with the Programmed Death-Ligand 1 (PD-L1), an immune checkpoint protein. The results show that we can confirm the presence of the sPD-L1 in the concentration range of 10 -18 to 10 -8 M using electrochemical impedance spectroscopy (EIS) with a limit of detection (LOD) of 1.87 × 10 -14 M for PD-L1 (S/N = 3.3) and at a concentration of 10 -14 M via cyclic voltammetry (CV). Additionally, high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements were applied to confirm the functionalization of the electrode. We investigated the selectivity of the electrode for other proteins: Programmed Death-1 (PD-1), cluster of differentiation 160 (CD160), and B- and T-lymphocyte attenuator (BTLA) at concentrations of 10 -8 M. Differentiation between PD-L1 and PD-1 was achieved based on the analysis of the capacitance effect frequency dispersion at the surface of the modified Au electrode with BMS-8 after incubation at various concentrations of PD-L1 and PD-1 proteins in the range of 10 -18 to 10 -8 M. Significant differences were observed in the heterogeneity of PD-L1 and PD-1. The results of the quasi-capacitance studies demonstrate that BMS-8 strongly and specifically interacts with the PD-L1 protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Richter syndrome (RS) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. RS is known to be associated with a number of genetic alterations such as TP53 and NOTCH1 mutations. However, it is unclear what immune microenvironment changes are associated with RS. In this study, we analyzed expression of immune checkpoint molecules and infiltration of immune cells in nodal samples, and peripheral blood T-cell diversity in 33 CLL and 37 RS patients. Compared to CLL, RS nodal tissue had higher PD-L1 expression in histiocytes and dendritic cells (median 16.6% vs. 2.8%, P < 0.01) and PD1 expression in neoplastic B cells (median 26.0% vs. 6.2%, P < 0.01), and higher infiltration of FOXP3-positive T cells (median 1.7% vs. 0.4%, P < 0.01) and CD163-positive macrophages (median 23.4% vs. 9.1%, P < 0.01). In addition, peripheral blood T-cell receptor clonality was significantly lower in RS vs. CLL patients (median [25th-75th], 0.107 [0.070-0.209] vs. 0.233 [0.111-0.406], P = 0.046), suggesting that T cells in RS patients were significantly more diverse than in CLL patients. Collectively these data suggest that CLL and RS have distinct immune signatures. Better understanding of the immune microenvironment is essential to improve immunotherapy efficacy in CLL and RS.

Objectives: The aim of this study was to characterize the tumor microenvironment of patients with gastroenteropancreatic neuroendocrine tumors relative to progression-free survival (PFS)., Methods: Immune profiling for CD3, CD8, programmed death-1/programmed death-ligand 1, and indoleamine 2,3-dioxygenase expression in 2 cohorts of gastroenteropancreatic neuroendocrine tumors: patients with short PFS (<4 years, n = 12) versus long PFS (≥4 years, n = 14) after surgery. Immune infiltrates in the tumor and interface were quantified. Programmed death-ligand 1 expression was determined within the tumor, stroma, and interface., Results: Patients with shorter PFS had larger tumors (P = 0.02), mostly in the pancreas (P = 0.04). We observed a higher mean expression of CD3+, CD8+, programmed death-1+ cells, and indoleamine 2,3-dioxygenase at the interface compared with the tumor: log 10 mean differences 0.56 (95% confidence interval [CI], 0.43-0.68; P < 0.0001), 0.45 (95% CI, 0.32-0.59; P = 0.0002), 0.50 (95% CI, 0.40-0.61; P < 0.0001), and 0.24 (95% CI, 0.03-0.46; P = 0.046), respectively. Patients with longer PFS had higher intratumoral CD3+ T cells, log 10 mean difference 0.38 (95% CI, 0.19-0.57; P = 0.004). Programmed death-ligand 1 expression tended to be higher among patients with shortened PFS (odds ratio, 2.00; 95% CI, 0.68-5.91)., Conclusions: Higher intratumoral CD3+ T-cell infiltrate was associated with longer PFS after resection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Suppression of the immune system is intimately linked to the development and progression of malignancy, and immune modulating treatment options have shown promise in a variety of tumor types, including some triple-negative breast cancers (TNBC). The most dramatic therapeutic success has been seen with immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand, PD-L1. Difficulty remains, however, in appropriate patient selection for treatment, as many PD-L1-positive cancers fail to show durable responses to PD-1/PD-L1 inhibition. Checkpoint inhibitor targeting of the adaptive immune response relies on the presence of major histocompatibility complex (MHC) class I molecules on the tumor cell surface for tumor antigen presentation. MHC class I loss has been previously described in breast cancer and represents a putative mechanism of immunotherapeutic resistance in this tumor type. One hundred seventeen invasive primary breast carcinomas with a range of histologic subtypes were evaluated on tissue microarrays containing formalin-fixed paraffin-embedded tissue. Loss of MHC class I expression was common among breast cancers, with greater than half of cases demonstrating either subclonal or diffuse loss. Fifty-nine percent of TNBC demonstrated loss of MHC class I, including 46% of those meeting the Food and Drug Administration-approved threshold of 1% for tumor-associated immune cell PD-L1 expression. MHC class I loss was particularly common in the apocrine subtype of TNBC (78%). MHC class I's employment as a predictive biomarker should be considered, as its loss may represent a barrier to successful enhancement of the antitumor adaptive immune response by PD-1/PD-L1 inhibition., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: Laryngotracheal stenosis (LTS) is a fibrotic condition of the upper airway. Recent evidence suggests dysregulated host immunity plays a role in LTS development and progression. The programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis, targeted by paradigm-shifting immunotherapies for cancer treatment, has also recently been implicated in the pathogenesis of fibrotic pulmonary disease. However, a role for the PD-1/PD-L1 axis in the proximal airway fibrosis seen in LTS patients has not been explored., Study Design: Controlled ex vivo study., Methods: Expression of PD-1, PD-L1, CD4, and CD8 were evaluated using immunohistochemical staining of cricotracheal resection specimens from postintubation iatrogenic laryngotracheal stenosis (iLTS), idiopathic subglottic stenosis (iSGS) patients, and normal controls derived from rapid autopsy (n = 8 per group). Fibroblasts derived from iLTS scar were also treated with transforming growth factor beta 1 (TGFβ1) and analyzed for PD-L1 expression by quantitative real-time polymerase chain reaction (n = 6)., Results: iLTS specimens exhibited increased expression of PD-1, PD-L1, and CD4 (all P < .0167) compared to controls, whereas iSGS specimens exhibited increased expression of PD-1 and CD4 (P < .0167) compared to controls. PD-1, PD-L1, and CD4 showed periepithelial patterns of expression in both disease cohorts. TGFβ1 treatment of iLTS fibroblasts increased expression of PD-L1 (the cognate ligand for PD-1)., Conclusion: Expression of both PD-1 and its ligand PD-L1 are significantly greater in patients with iLTS compared to controls, and PD-1 expression is also elevated in patients with iSGS. Given published evidence implicating the PD-1/PD-L1 axis in pulmonary fibrosis, this suggests a possible role for checkpoint inhibitors targeting the PD-1/PD-L1 axis for the treatment of LTS., Level of Evidence: N/A Laryngoscope, 131:967-974, 2021., (© 2020 The American Laryngological, Rhinological and Otological Society, Inc.)

Introduction: The role of the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis is well established in classical Hodgkin lymphoma (HL), where PD-1 blockade demonstrated spectacular efficacy in relapsed/refractory disease. However, little is known about the frequency and cellular distribution of other immune checkpoints in HL samples., Patients and Methods: Using immunohistochemistry, we investigated, along with PD-L1 and PD-1, the expression of lymphocyte-activation gene 3 (LAG-3) and T-cell immunoglobulin and mucin-domain containing 3 (TIM-3) in 57 biopsy samples of patients with classical HL., Results: Hodgkin and Reed/Sternberg (HRS) cells were strongly positive for PD-L1 in nearly all cases. HRS cells were TIM-3 positive in 36% of samples, whereas LAG-3 was rarely expressed (5.2%). In the microenvironment, PD-1, LAG-3, and TIM-3 were expressed by ≥ 5% of cells in 65%, 98%, and 96% of cases, respectively. T-cell rosettes surrounding HRS cells consisted of CD4 + FoxP3- helper T cells expressing both PD-1 and LAG-3, with a variable expression of TIM-3., Conclusion: This study demonstrates for the first time that LAG-3 and TIM-3 are nearly always expressed in the microenvironment of classical HL. This may constitute the basis for targeting LAG-3 or TIM-3 in combination with anti-PD-1 antibodies in the treatment of relapsed/refractory HL., (Copyright © 2020 Elsevier Inc. All rights reserved.)

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types., (© 2021 by The American Society of Hematology.)

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti-programmed cell death protein 1 (PD-1) treatment is predominantly studied in tumor-infiltrating lymphocytes (TILs). Its impact on PD-1 expressing cells in tumor-draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD-1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD-1, CD69, and HLA-DR on CD4 + and CD8 + T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD-1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD-1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD-1 negative TILs expressed high levels of human leukocyte antigen - DR isotype and CD69. In contrast, PD-1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3 + PD-1 + cells in tumor-draining lymph nodes had significantly lower disease-free and overall survival rates (log-rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD-1 levels in TDLNs correspond with survival and potentially with response to anti-PD-1 therapy. Such knowledge may ultimately help guide anti-PD-1 treatment., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Purpose: Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients' sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients' overall survival (OS)., Methods: sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model., Results: We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62-1.01, P value = 0.062)., Conclusions: Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM.

Immunotherapy with checkpoint inhibitors is well established as an effective treatment for non-small cell lung cancer and melanoma. The list of approved indications for treatment with PD-1/PD-L1 checkpoint inhibitors is growing rapidly as clinical trials continue to show their efficacy in patients with a wide range of solid tumours. Clinical trials have used a variety of PD-L1 immunohistochemical assays to evaluate PD-L1 expression on tumour cells, immune cells or both as a potential biomarker to predict response to immunotherapy. Requests to pathologists for PD-L1 testing to guide choice of therapy are rapidly becoming commonplace. Thus, pathologists need to be aware of the different PD-L1 assays, methods of evaluation in different tumour types and the impact of the results on therapeutic decisions. This review discusses the key practical issues relating to the implementation of PD-L1 testing for solid tumours in a pathology laboratory, including evidence for PD-L1 testing, different assay types, the potential interchangeability of PD-L1 antibody clones and staining platforms, scoring criteria for PD-L1, validation, quality assurance, and pitfalls in PD-L1 assessment. This review also explores PD-L1 IHC in solid tumours including non-small cell lung carcinoma, head and neck carcinoma, triple negative breast carcinoma, melanoma, renal cell carcinoma, urothelial carcinoma, gastric and gastroesophageal carcinoma, colorectal carcinoma, hepatocellular carcinoma, and endometrial carcinoma. The review aims to provide pathologists with a practical guide to the implementation and interpretation of PD-L1 testing by immunohistochemistry., (Copyright © 2020 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4 + T cells, CD8 + T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8 + PD-1 + T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8 + PD-1 - T cells and non-Treg CD4 + FOXP3 - T cells; but increased exhausted CD8 + PD-1 + T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1 + CD8 + T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lim, Nguyen, Wasser, Kumar, Lee, Nasir, Chua, Lai, Hazirah, Loh, Khor, Yeong, Lim, Low, Albani, Chong and Chew.)

Introduction: The spleen plays an important role in regulating the immune response to infectious pathogens. T-cells dysfunction and exhaustion have been reported in patients with hepatitis B/C virus (HBV/HCV) infection, which contributes to persistent virus infection. The aims of this study were to investigate spleen-related evidence of immunosuppression and immune tolerance in HCV cirrhotic patients with portal hypertension (PH). Methods: The expression of programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) and its ligand PD-L1/2, and Galectin-9 in the spleens and livers of HCV cirrhotic patients ( n = 15) was analyzed using real-time PCR and immunohistochemistry. Flow cytometry was used to evaluate the expression of PD-1 and Tim-3 on splenic T-cells and the peripheral blood T-cells before and after splenectomy ( n = 8). Results: Spleens from patients with PH showed significantly increased mRNA levels of PD-L2, Tim-3, Galectin-9, CD80, and CD86, and decreased levels of CD28 compared to control spleens (spleens removed due to traumatic injury) (all p < 0.05). Additionally, protein expression of inhibitory signaling molecules was significantly increased in both the spleens and livers of cirrhotic patients compared with controls (all p < 0.05). Peripheral blood and splenic CD4 + and CD8 + T-cells also expressed higher protein levels of PD-1, Tim-3, and CTLA-4 in cirrhotic patients as compared with healthy controls (all p < 0.05). The proportion of PD-1 + CD4 + T lymphocytes (26.2% ± 7.12% vs. 21.0% ± 9.14%, p = 0.0293) and Tim-3 + CD8 + T lymphocytes (9.4% ± 3.04% vs. 6.0% ± 2.24%, p = 0.0175) in peripheral blood decreased followed splenectomy. Conclusion: The CD4 + and CD8 + T-cells in spleen and peripheral blood highly expressed PD-1 and Tim-3 in HCV-infected and cirrhotic patients with portal hypertension. Highly expressed PD-1 and Tim-3 in peripheral blood T-lymphocytes can be partly reversed following splenectomy.

Breast cancer is a common malignant tumor in women. Triple‑negative breast cancer (TNBC) is highly invasive with a high rate of metastasis and poor prognosis. Programmed death ligand 1 (PD‑L1) plays an important role in mediating the escape of tumor cells from immune surveillance. There have been significant advances in understanding the biology of TNBC. This review presents a detailed discourse on the available data on the expression of PD‑L1 in breast cancer and preliminary clinical outcome of PD‑L1/PD‑1 inhibitors in breast cancer patients. Early clinical trials involving PD‑L1/PD‑1 inhibitors have exhibited efficacy in tumor response and/or disease control in patients with refractory metastatic breast cancer, particularly TNBC. Furthermore, the mechanisms and factors that influence the immunoediting process are summarized and their functions in detail are analyzed.

Context: Programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin and ITIM domain (TIGIT) are considered major immune co-inhibitory receptors (CIRs) and the most promising immunotherapeutic targets in cancer treatment, but they are largely unexplored in medullary thyroid carcinoma (MTC)., Objective: We aimed to provide the first evidence regarding the expression profiles and clinical significance of CIRs in a large cohort of MTC patients., Design and Patients: In total, 200 MTC patients who received initial surgery in our hospital were included. Immunohistochemistry was performed to evaluate CIR expressions in tissue microarrays (TMAs). Combined with the results of our previous programmed cell death ligand-1 (PD-L1) study, clinicopathologic and prognostic correlations of these proteins were retrospectively analyzed., Results: TIM-3, PD-1, CTLA-4, LAG-3, and TIGIT positivity was detected in 96 (48.0%), 27 (13.5%), 25 (12.5%), 6 (3.0%), and 6 (3.0%) patients, respectively, in whom TIM-3, PD-1, and CTLA-4 expressions were positively correlated. Log-rank tests and multivariate Cox analyses both indicated that TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression were associated with worse structural recurrence-free survival. In addition, among 20 patients who developed advanced disease during follow-up, 12 (60%) showed TIM-3 positivity, among whom 6 cases also had concurrent moderate to strong PD-1, PD-L1, or CTLA-4 expression., Conclusions: Using the currently largest TMA cohort of this rare cancer, we delineated the CIR expression profiles in MTC, and identified TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression as promising biomarkers for tumor recurrence. Furthermore, a subset of advanced MTCs are probably immunogenic, for which single or combined immunotherapy including TIM-3, PD-1, PD-L1, or CTLA-4 blockade may be potential therapeutic approaches in the future., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Ovarian cancer is a heterogeneous disease, where chronic inflammation plays a key role in carcinogenesis. In this study, it is aimed to analyze the relationship with prognosis and chemotherapy response to clinicopathologicalnvariables in epithelial ovarian cancers such as proliferation of PD-1 +, CD8 +, CD4 +, CD3 + T-lymphocytes infiltrating the tumor and tumor stroma., Material and Methods: Seventy-six cases diagnosed with primary epithelial ovarian tumor from biopsy or surgical resection materials were included in the study. Immunreactivity of CD3, CD4, CD8, PD1 was evaluated immunohistochemically in lymphocytes in tumor infiltrating lymphocytes and stromal lymphocytes., Results: Seventeen (22.4%) of the cases were Type I, 59 (77.6%) of them were Type II ovarian carcinoma. PD-1 positivity was observed in stromal and intraepithelial lymphocytes in 22 (28.9%) of 76 cases. In the presence of PD-1 + T-lymphocytes that infiltrate tumor and stroma, disease-free survival are shorter (p = 0.037). The presence of stromal CD4 + and CD8 + T-lymphocytes was more common in late stage patients (p = 0.012, p = 0.036; respectively). The disease-free and overall survival rate was statistically significantly shorter in the presence of CD8 + T lymphocytes (p = 0.009, p = 0.003; respectively)., Conclusions: CD3, CD4 and CD8 may contribute to PD-1 mediated tumor control. Anti PD-1 therapy may be an alternative to chemotherapy in PD-1 positive patients. Identifying patients who do not respond to chemotherapy through PD-1 expression prior to immunotherapy will help develop potential personalized immunotherapy.

The role of immunotherapy in bladder urothelial cancers is rapidly expanding. Since the initial second-line therapy approval for patients who fail prior platinum-based chemotherapy, the use of immunotherapy with checkpoint inhibitors has been rapidly evolving. There are approved indications for first-line metastatic disease in the platinum-ineligible patients or the cisplatin-ineligible PD-L1 positive patients, and there is a label for high-risk non-muscle-invasive bladder cancer who are BCG-refractory. In addition, a trial on maintenance immunotherapy with avelumab showed positive findings with improvement in overall survival that has also changed standard of care for these patients. There are ongoing clinical trials evaluating its use in the neoadjuvant and adjuvant perioperative muscle-invasive bladder cancer setting. The pivotal trials that led to these FDA approvals and promising and ongoing trials are discussed herein., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The "inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies., Methods: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients., Results: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4 + T cells, whereas reductions in CD8 + T cells were more concentrated in memory subsets. Immature cytokine-producing CD56 bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56 dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4 + T cells and CD56 dim NK cells, and higher numbers of CD8 + effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20 + B cells in the tumor., Conclusions: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

Objectives: Aim of the study was to evaluate the prognostic impact of CD8-positive (CD8 + ) tumour-infiltrating lymphocytes (TILs) and PD-L1 expression on the outcome of patients with malignant salivary gland neoplasms., Materials and Methods: Formalin-fixed, paraffin-embedded tissue samples and clinicopathological data from patients treated for salivary gland carcinoma in a head and neck cancer centre were retrospectively retrieved. Immunohistochemical staining was applied on sections of 84 specimens of 12 different histological subtypes. Both CD8 and PD-L1 expression were rated by semi-automated cell counts by a digital image analysis programme. Survival analyses were performed by the log-rank test on the univariate level, and the Cox model was applied on the multivariate level. Associations between immunological markers and clinicopathological variables were estimated by the Pearson chi-squared test. Additionally, PD-1 was estimated as an exhaustion marker of CD8 + TILs., Results: Patients exceeding a tumour proportion score ≥5% regarding PD-L1 expression demonstrated a significantly decreased survival, as did individuals with an overall high CD8 + cell density. Particularly, high CD8 + cell counts in the invasive front of the respective tumour tissue significantly coincided with a poor outcome. Also, high numbers of CD8 + TILs significantly matched with a high quantity of PD-1 + TILs., Conclusion: CD8 + TILs abundance in the peritumoural microenvironment correlates with impaired outcome of patients with salivary gland carcinoma. The simultaneous negative prognostic impact of PD-L1 expression and presence of PD-1 + TILs advocates an immune checkpoint-controlled mechanism of CD8 + TILs exhaustion for these tumours and paves the way for future treatment strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: Persistent antigen exposure leads to the accumulation of lymphocytes and subsequent tertiary lymphoid structures (TLS). We investigated the relationship of tumor microenvironment (TME) with respect to programmed death ligand 1 (PD-L1), its receptor programmed death 1 (PD-1), and TLS in upper tract urothelial carcinoma (UTUC) cases and compared them with UTUC associated with urothelial bladder carcinoma (UTUC-BCa)., Methods: We retrospectively identified 72 patients with UTUC. Representative slides were reviewed, and TLS were counted. Immunohistochemical stains for PD-1 and PD-L1 were performed. PD-1-positive lymphocytes were counted and H-score for PD-L1-positive membranous staining was determined., Results: PD-L1 expression in the tumor was present in 55.1% of the UTUC cases. Higher stage was associated with increased PD-L1 expression (P = .035). TLS were present in 33.3% and their presence was significantly associated with PD-L1 positivity (P = .024). This association remained significant after adjustment for UTUC-BCa. TLS were also associated with a greater number of infiltrating PD-1-positive lymphocytes (P = .013)., Conclusions: This study is one of the first comparative studies of the TME in UTUC and UTUC-BCa. PD-L1 is expressed in a subset of UTUC and is associated with TLS. The presence of TLS is an inherent characteristic of UTUC and not secondary to the presence of BCa., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is a growing interest in immunotherapy in childhood cancers. Osteosarcoma is a compelling potential target as there are few targeted options available for this aggressive cancer. We provide a description of the landscape of programmed cell death-1 (PD-1), programmed cell death-ligand 1 (PD-L1) and relevant immune markers in serial samples from 15 osteosarcoma patients. PD-1 and PD-L1 expression was present in biopsy samples (47% and 53%, respectively), absent in resections, and present in metastases (40% and 47%). Both decalcified and nondecalcified specimens demonstrated expression of PD-1 and PD-L1. The results suggest that biopsy or metastatic specimens maybe most valuable in assessing expression of PD-1 and PD-L1.

mWasabi is a bright monomeric green fluorescent protein. It can be used as a fusion tag to monitor various biological events, e.g. protein localization. Here we report the selection of camelid-derived single-domain antibody fragments (nanobodies) against mWasabi. In this work, phage-display approach was employed to select the high affinity mWasabi-specific Nb (nanobodies). These nanobodies were able to recognize mWasabi or in a fused fashion with PD1. The interesting binding characteristics of these two mWasabi-specific nanobodies could be valuable for design new tools for cellular tracing or targeting based on the mWasabi-fusing protein in many different biological research fields., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: High tumor expression of programmed cell death protein (PD1) and programmed death-ligand 1 (PD-L1) is thought to be associated with positive clinical outcomes after treatment with anti-PD1 or anti-PD-L1 agents. Several sensitive methods based on immunohistochemistry, ligand binding assay (LBA), and liquid chromatography/mass spectrometry involving the measurement of PD1 and PD-L1 expression have been reported. Here, we expand on the characterization of different tumor types using a highly specific, sensitive, and robust immunoaffinity liquid chromatography/tandem mass spectrometry (IA-LC/MS/MS)-based method for the simultaneous quantitation of PD1 and PD-L1 in tumor tissues., Methods: Human tumor tissue samples were homogenized using a Precellys Evolution homogenizer. The samples were incubated with anti-PD1 and anti-PD-L1 capture polyclonal antibodies, which were bound to magnetic beads. Following enrichment, samples were digested with trypsin. A Waters iKEY HSS T3 1.8 um (150 μm × 100 mm) column with a gradient flow rate of 3 μL/min was used for chromatographic separation, and a Waters TQ-S triple quadrupole mass spectrometer was used for detection. Selected reaction monitoring (SRM) transitions with unit resolution for precursor/product ion masses were optimized for PD1 and PD-L1 surrogate peptides., Results: The surrogate peptides LAAFPEDR for PD1 and FTVTVPK for PD-L1 yielded the most intense SRM transitions at m/z 459.7 > 516.2 and m/z 396.2 > 543.3, respectively, and thus were selected for the quantitation of PD1 and PD-L1. The lower limit of quantitation for PD1 and PD-L1 was 0.062 ng/mL with an assay range up to 10 ng/mL. Using this method, human PD1 and PD-L1 were detected and quantified from four different types of tumor tissues. The data show that PD1 expression level was highly correlated with that of PD-L1 in all tumor tissues analyzed here., Conclusions: A highly specific and sensitive immunoaffinity microflow LC/MS/MS method for the simultaneous quantification of PD1 and PD-L1 in tumor tissues was developed and implemented. This method combines the advantage of immuno-capture for analyte enrichment with the high specificity of detection of multiple surrogate peptides by LC/MS/MS. The quantification of PD1 and PD-L1 co-expression in tumor could help evaluate their role in assessing tumor type selection and patient stratification., (© 2020 John Wiley & Sons, Ltd.)

Interaction of human programmed death factor-1 (hPD-1) of T cells and one of its ligands hPD-L1 which is expressed on cancer cells suppresses effector T cell functions. Studies showed that the hPD-1/hPD-L1 pathway is associated with killing mechanisms of tumor cells evading the immune system. Immunotherapy based on the checkpoint inhibitor on hPD-1 has been an important approach to treat cancer; however, not all cancer cells over-express hPD-L1. Detection of hPD-L1 over-expression in cancer cells may be a key factor for deciding on whether immunotherapy should be conducted. In the present study, we produced recombinant hPD-1 using Escherichia coli, and created a fluorescent probe termed quenched hPD-1 (QPD-1) for the detection of hPD-L1. We found that hPD-1 can quench fluorescence of carboxytetramethylrhodamine labeled on its N-terminal and QPD-1 is a convenient tool to rapidly detect hPD-L1 with a limit of detection of 10 nM and detectable range of 10 nM-1000 nM. QPD-1 may also function as a probe to screen for hPD-L1 over-expressing tumor cells and promote appropriate medical procedure through tumor immunotherapy., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

It is becoming increasingly important to obtain detailed diagnostic information on small-volume tissue biopsies, such as core needle biopsies. This is particularly crucial in the workup and diagnosis of classic Hodgkin lymphoma (CHL) and other morphologically similar lymphomas such as T-cell/histiocyte-rich large B-cell lymphoma (THRLBL), where small-volume lymph node biopsies often represent the frontline tissue source, and the differential diagnosis includes a reactive process. Immunohistochemical markers could be helpful to differentiate CHL from reactive lymph node changes (RLN) in this setting. The use of programmed cell death-1 (PD-1) and its ligand (PD-L1) immunohistochemistry has historically focused on prognostic and therapeutic value when evaluating CHL. However, the present study seeks to determine the diagnostic utility of these markers in core needle biopsies of CHL (25), THRLBL (3), and RLN (31). The cases of CHL and THRLBL were previously diagnosed and confirmed with standard immunohistochemistry, allowing the utility of PD-1 and PD-L1 to be tested in this setting. Different PD-1 and PD-L1 expression patterns were observed between the reactive process of RLN and the malignant lymphomas (CHL and THRLBL). CHL cases overall showed the greatest expression of PD-L1 within the malignant Reed-Sternberg cell population, with 40% of CHL cases exhibiting >50% PD-L1 expression. This degree of PD-L1 expression was not seen in the lymphocytic cell population of any RLN (P<0.001). Conversely, CHL cases showed an overall lower expression of PD-1, as 96% of CHLs had <5% PD-1 expression in Reed-Sternberg cells compared with only 10% expression within the lymphocytic population of RLN (P<0.001). THRLBL cases followed a similar trend to CHL. These results demonstrate that upfront PD-1 and PD-L1 immunohistochemistry can aid in the diagnosis of CHL in small-volume tissue biopsies.

Immunotherapy has revolutionized the management of cancers. At the end of 2018, 1,716 clinical trials assessed regimen that combine program death-1 (PD-1)/program death ligand-1 (PD-L1) blockers with other cancer therapies (tyrosine kinase inhibitor, chemotherapy and radiotherapy). There is a contrast between these clinical dynamics and the difficulty of identifying biomarkers to better select patients that could benefit from immunotherapy. In this context, different tumor classifications have been proposed to try to better stratify patients. They rely on the characteristics of the tumor microenvironment and led first to divide them into hot and cold tumors. In this review, we aim to demonstrate the limitations of this classification focusing on the differential significance of subpopulations of intratumor CD8 + T cells. We also underline novel mechanisms of resistance to anti-PD-1/PD-L1 blockade, focusing on myeloid cells, hypoxia and tumor immunoediting under treatment. Understanding the mechanisms of resistance to immune-checkpoint inhibitor is indeed a powerful research driver that allows further identification of novel biomarkers, drug development and bring a rational to innovative therapeutic combinations., (© 2020 UICC.)

Background: The prognosis of HPV - HNSCC was worse than that of HPV + HNSCC. Analysis of tumours and tumour-infiltrating lymphocytes (TILs) may provide insight into the progression of HPV - HNSCC., Methods: The tumour and TIL phenotypic characteristics of 134 HNSCC specimens (HPV - tumours were classified into "Infiltrating" and "Pushing" subtypes based on their different tumour nest configuration and prognosis) were retrospectively analysed. HNSCC data from the Cancer Genome Atlas (n = 263) were analysed for CD8α, HPV and overall survival (OS). A murine HNSCC model was used to verify the antitumour role of PD-1 + CD8 + TILs., Results: The "Infiltrating" HPV - subtype showed shorter OS than the "Pushing" subtype. Moreover, there is a tendency from "Pushing" to "Infiltrating" subtype from the primary to the recurrent lesion. Different from total CD8 + TILs, tumour-specific PD-1 + CD8 + TILs were fewer in invasive margin (IM) of "Infiltrating" HPV - tumours. PD-1 + CD8 + TILs recognised autologous HNSCC cells and showed stronger inhibition of tumour growth in a murine HNSCC model resistant to PD-1 blockade., Conclusions: Coevolution of HPV - HNSCC and TILs is characterised by an "Infiltrating" phenotype and less tumour-specific PD-1 + CD8 + TILs, which may provide a framework for further translational studies and patient stratification.

Background: Most tumors responding to immunotherapy with monoclonal antibodies targeting programmed cell death protein1 (PD1) and programmed death ligand-1 (PD-L1) show surface expression of PD-L1. Neuroblastoma has been reported to show low PD-L1 surface expression., Methods: The effect of immune checkpoint inhibitor on mouse neuroblastoma was investigated, and host immune cells were analyzed in the tumor microenvironment. Expression of co-stimulatory molecules by Neuro-2a mouse neuroblastoma cells was analyzed using flow cytometer. Neuro-2a cells were inoculated subcutaneously into A/J mice, followed by intraperitoneal injection of antibodies targeting PD-1 and PD-L1. Mice were sacrificed for the measurement of tumor weights on day 14 following tumor inoculation, and tumor-infiltrating cells were analyzed using a flow cytometer., Results: Dim expression of PD-L1 was observed on the cell surface of cultured Neuro-2a cells. Growth of subcutaneous tumors was significantly suppressed, and PD-L1-expressing tumor cells were depleted by the antibody treatment. We confirmed that Neuro-2a cells opsonized by the anti-PD-L1 antibody were phagocytosed in the in vitro setting. In the treated tumor microenvironments, CD8α + lymphocyte and CD11c + MHC II + cells were significantly accumulated in comparison with the control group. These CD11c + MHC II + cells expressed CD80, CD86, CD14, and CD40, but not CD205, PD-L1, or CTLA4. PD-1 expression was detected dimly. Immune suppressive effects of CD11b + Gr-1 + myeloid-derived suppressor cells by the administration of anti-PD-1 and PD-L1 antibodies were not observed in spleen, regional lymph nodes, or tumor microenvironment., Conclusions: Our findings raise the possibility that co-administration of anti-PD-1 and anti-PD-L1 antibodies have a synergistic effect on inhibition of tumor growth and could be an effective therapy against neuroblastoma with dim expression of PD-L1., (Copyright © 2020 Elsevier Inc. All rights reserved.)