Influence of PD‐L1 cross‐linking on cell death in PD‐L1‐expressing cell lines and bovine lymphocytes

Programmed death‐ligand 1 (PD‐L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death‐1 (PD‐1). However, the mechanism of PD‐L1‐mediated inhibitory signalling after PD‐L1 cross‐linking by anti‐PD‐L1 monoclonal antibody (mAb) or PD‐1–immunogloblin fusion protein (PD‐1‐Ig) is still unknown, although it may induce cell death of PD‐L1⁺ cells required for regular immune reactions. In this study, PD‐1‐Ig or anti‐PD‐L1 mAb treatment was tested in cell lines that expressed PD‐L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD‐L1‐mediated cell death. PD‐L1 cross‐linking by PD‐1‐Ig or anti‐PD‐L1 mAb primarily increased the number of dead cells in PD‐L1^high cells, but not in PD‐L1^low cells; these cells were prepared from Cos‐7 cells in which bovine PD‐L1 expression was induced by transfection. The PD‐L1‐mediated cell death also occurred in Cos‐7 and HeLa cells transfected with vectors only encoding the extracellular region of PD‐L1. In bovine lymphocytes, the anti‐PD‐L1 mAb treatment up‐regulated interferon‐γ (IFN‐γ) production, whereas PD‐1‐Ig treatment decreased this cytokine production and cell proliferation. The IFN‐γ production in B‐cell‐depleted peripheral blood mononuclear cells was not reduced by PD‐1‐Ig treatment and the percentages of dead cells in PD‐L1⁺ B cells were increased by PD‐1‐Ig treatment, indicating that PD‐1‐Ig‐induced immunosuppression in bovine lymphocytes could be caused by PD‐L1‐mediated B‐cell death. This study provides novel information for the understanding of signalling through PD‐L1.     

Expression and prognostic significance of programmed death protein 1 and programmed death ligand‐1, and cytotoxic T lymphocyte‐associated molecule‐4 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies and causes of death worldwide. In this study, we assessed the correlation between clinicopathologic factors with programmed cell death protein 1 (PD‐1) and programmed cell death ligand‐1 (PD‐L1), and cytotoxic T lymphocyte‐associated molecule‐4 (CTLA‐4) expressions. Furthermore, we analyzed the prognostic significance of these proteins in a subgroup of patients. We retrospectively evaluated the PD‐1, PD‐L1, and CTLA‐4 expressions in 294 HCC tissue microarray samples using immunohistochemistry. PD‐1 and PD‐L1 expressions were significant related to high CD8+ tumor‐infiltrating lymphocytes (TILs) (r = 0.664, p < 0.001 and r = 0.149, p = 0.012). Only high Edmondson–Steiner grade was statistically related to high PD‐1 expression. High PD‐L1 expression was demonstrated as an independent poor prognostic factor for disease‐free survival in addition to previous known factors, size >5 cm and serum albumin ≤3.5 g/dL in high CD8+ TILs group. We have demonstrated that the combined high expression of PD‐L1 and CD8+ TIL is an important prognostic factor related to the immune checkpoint pathway in HCC and furthermore, there is a possibility that it could be used as a predictor of therapeutic response. Also, this result would be helpful in evaluating the applicable group of PD‐1/PD‐L1 blocking agent for HCC patients.     

MMP‐9 expression increases according to the grade of squamous intraepithelial lesion in cervical smears

Studies about cervical carcinogenesis have demonstrated the increased expression of matrix‐metalloproteinase (MMP) according to the grade of cervical intraepithelial lesions. Considering the importance of innovative techniques to introduce noninvasive and rapid diagnoses for patients, this study aimed to perform MMP‐9 immunocytochemistry in cervical smears according to the cytopathological diagnoses, in order to monitor MMP activity in cervical smears. This cross‐sectional study investigated the expression of MMP‐9 in normal cervical smears, inflammatory cervical smears, squamous intraepithelial lesions, and cervical carcinoma. Cervical smears from 630 women were collected for cytopathological diagnoses and immunocytochemistry. Women with squamous intraepithelial lesions showed an increase in MMP‐9 expression, with moderate to intense staining occurring with increasing cervical lesion grade. The prevalence of moderate to intense MMP‐9 staining was 9% in normal cervical smears, 12% in cervical inflammation, 24% in low‐grade squamous intraepithelial lesion (LSIL), 92% in high‐grade squamous intraepithelial lesions (HSIL) and 100% in cervical carcinoma cases. In the specific case of LSIL, we found that association with MMP‐9 is more evident when there is the simultaneous presence of an infectious agent. Thus, the expression of MMP‐9 in cervical smears increases according to the grade of cervical lesion and LSIL in the presence of infectious agents showed higher MMP‐9 expression than women with LSIL without infectious agents. Diagn. Cytopathol. 2014;42:827–833. © 2014 Wiley Periodicals, Inc.     

PD‐L1 testing for lung cancer in the UK: recognizing the challenges for implementation

A new approach to the management of non‐small‐cell lung cancer (NSCLC) has recently emerged that works by manipulating the immune checkpoint controlled by programmed death receptor 1 (PD‐1) and its ligand programmed death ligand 1 (PD‐L1). Several drugs targeting PD‐1 (pembrolizumab and nivolumab) or PD‐L1 (atezolizumab, durvalumab, and avelumab) have been approved or are in the late stages of development. Inevitably, the introduction of these drugs will put pressure on healthcare systems, and there is a need to stratify patients to identify those who are most likely to benefit from such treatment. There is evidence that responsiveness to PD‐1 inhibitors may be predicted by expression of PD‐L1 on neoplastic cells. Hence, there is considerable interest in using PD‐L1 immunohistochemical staining to guide the use of PD‐1‐targeted treatments in patients with NSCLC. This article reviews the current knowledge about PD‐L1 testing, and identifies current research requirements. Key factors to consider include the source and timing of sample collection, pre‐analytical steps (sample tracking, fixation, tissue processing, sectioning, and tissue prioritization), analytical decisions (choice of biomarker assay/kit and automated staining platform, with verification of standardized assays or validation of laboratory‐devised techniques, internal and external quality assurance, and audit), and reporting and interpretation of the results. This review addresses the need for integration of PD‐L1 immunohistochemistry with other tests as part of locally agreed pathways and protocols. There remain areas of uncertainty, and guidance should be updated regularly as new information becomes available.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

A study of PD‐L1 expression in intravascular large B cell lymphoma: correlation with clinical and pathological features

Intravascular large B cell lymphoma (IVLBCL) is a rare, aggressive, extranodal large B cell lymphoma characterised by growth of tumour cells within the lumen of vessels, particularly capillaries. Programmed cell death ligand 1 (PD‐L1) is a cell surface glycoprotein that interacts with programmed death 1 (PD‐1) on the T cell surface, leading to modulation of the immune response. PD‐L1 is a targetable immune check‐point molecule that is expressed on neoplastic cells in various cancers, including a subset of lymphomas. We correlated the expression of PD‐L1 with clinical and pathological findings in this rare disease. Eleven cases of IVLBCL were identified in the archives of Laboratory of Pathology at the National Cancer Institute, NIH. A panel of immunostains (CD20, CD3, CD5, PD‐L1) was performed. The cases were classified as the classic form or the variant associated with haemophagocytic syndrome (HPS) based on published 2017 WHO criteria. Three cases (27.3%) were HPS variant and eight cases (72.7%) were the classic form. Five (45.5%) of 11 cases were CD5‐positive; two of three (66%) were HPS variants and three of eight (37.5%) were classic form. Overall, four of nine evaluable cases (44.4%) were positive for PD‐L1, three of which were classic. Only one CD5‐positive case was PD‐L1‐positive, a classic variant. In summary, a subset of IVLBCL express PD‐L1. Although limited, these data suggest that PD‐L1 is expressed in both the so‐called classic form as well as the HPS variant. PD‐L1 is expressed irrespective of CD5 expression. Finally, detection of PD‐L1 expression in a subset of IVLBCL lymphoma cases may identify patients who might benefit from targeted immunotherapy.     

Oestrogen treatment of experimental autoimmune encephalomyelitis requires 17β‐oestradiol‐receptor‐positive B cells that up‐regulate PD‐1 on CD4+ Foxp3+ regulatory T cells

It is now well accepted that sex hormones have immunoregulatory activity and may prevent exacerbations in multiple sclerosis during pregnancy. Our previous studies demonstrated that oestrogen (17β-oestradiol; E₂) protection against experimental autoimmune encephalomyelitis (EAE) is mediated mainly through oestrogen receptor-α (ERα) and the membrane receptor G-protein-coupled receptor 30 (GPR30) and is abrogated in the absence of B cells and the co-inhibitory receptor, Programmed Death-1 (PD-1). To critically evaluate the cell source of the E₂ and PD-1 co-inhibitory pathways in EAE regulation, we assessed the requirement for ERs on transferred B cells and downstream effects on expression of PD-1/PD-ligand on CD4⁺ Foxp3⁺ regulatory T (Treg) cells in B-cell-replenished, E₂-treated B-cell-deficient (μMT^−/−) mice with EAE. The results clearly demonstrated involvement of ERα and GPR30 on transferred B cells that mediated the protective E₂ treatment effect on EAE and further showed an E₂-mediated B-cell-dependent up-regulation of PD-1 on CD4⁺ Foxp3⁺ Treg cells. These findings identify regulatory B-cell populations as key players in potentiating Treg-cell activity during E₂-mediated protection against EAE.