Immune cells and checkpoints in pancreatic adenocarcinoma: Association with clinical and pathological characteristics.

INTRODUCTION: Pancreatic adenocarcinoma is an extremely aggressive neoplasm, with many challenges to be overcome in order to achieve a truly effective treatment. It is characterized by a mostly immunosuppressed environment, with dysfunctional immune cells and active immunoinhibitory pathways that favor tumor evasion and progression. Thus, the study and understanding of the tumor microenvironment and the various cells subtypes and their functional capacities are essential to achieve more effective treatments, especially with the use of new immunotherapeutics. METHODS: Seventy cases of pancreatic adenocarcinoma divided into two groups 43 with resectable disease and 27 with unresectable disease were analyzed using immunohistochemical methods regarding the expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and human leukocyte antigen G (HLA-G) molecules as well as the populations of CD4+ and CD8+ T lymphocytes, regulatory T cells (Tregs), and M2 macrophages (MM2). Several statistical tests, including multivariate analyses, were performed to examine how those immune cells and immunoinhibitory molecules impact the evolution and prognosis of pancreatic adenocarcinoma. RESULTS: CD8+ T lymphocytes and M2 macrophages predominated in the group operated on, and PD-L2 expression predominated in the unresectable group. PD-L2 was associated with T stage, lymph node metastasis, and clinical staging, while in survival analysis, PD-L2 and HLA-G were associated with a shorter survival. In the inoperable cases, Tregs cells, MM2, PD-L1, PD-L2, and HLA-G were positively correlated. CONCLUSIONS: PD-L2 and HLA-G expression correlated with worse survival in the cases studied. Tumor microenvironment was characterized by a tolerant and immunosuppressed pattern, mainly in unresectable lesions, where a broad positive influence was observed between immunoinhibitory cells and immune checkpoint proteins expressed by tumor cells.

Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity.

BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch. METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous ß-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed. RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo. CONCLUSION: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

PD-L2 Expression in Breast Cancer Promotes Tumor Development and Progression.

Background: This work focused on investigating the role of programmed death ligand 2 (PD-L2) in the progression of breast cancer by utilizing breast cancer specimens and cells. Materials and Methods: The serum levels of soluble PD-L2 (sPD-L2) in breast cancer patients and healthy individuals were analyzed by means of the enzyme-linked immunosorbent assay, and the PD-L2 levels within 416 resected breast cancer specimens were assessed through immunohistochemistry. Concurrently, in vitro cell experiments and in vivo animal experiments were carried out to analyze the relationship between PD-L2 and the invasion and migration of breast cancer. Results: The concentration of sPD-L2 in breast cancer patients significantly increased compared to that in the control groups. Additionally, breast cancer patients with high concentrations of sPD-L2 had higher Ki67 values (≥30%) and tumor grades. PD-L2 was expressed in 79.09% of the cancer samples, which exhibited a positive correlation with the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Furthermore, we discovered that knockdown of PD-L2 inhibited the migratory and invasive abilities of both MCF-7 and MDA-MB231 cells. Conclusion: Our findings demonstrated that knockdown of PD-L2 suppressed tumor growth, providing novel insights into important biological functions.

Differential Immune Checkpoint Protein Expression in HNSCC: The Role of HGF/MET Signaling.

Although inhibitors targeting the PD1/PD-L1 immune checkpoint are showing comparably good outcomes, a significant percentage of head and neck squamous cell carcinoma (HNSCC) patients do not respond to treatment. Apart from using different treatment strategies, another possibility would be to target other immune checkpoints operating in these non-responding tumors. To obtain an overview of which checkpoint ligands are expressed on HNSCC tumor cells and if these ligands are affected by HGF/MET signaling, we used mRNA sequencing and antibody-based techniques for identifying checkpoint ligands in six HNSCC tumor cell lines. Furthermore, we compared our results to mRNA sequencing data. From the checkpoint ligands we investigated, VISTA was expressed the highest at the RNA level and was also the most ubiquitously expressed. PD-L2 and B7-H3 were expressed comparably lower and were not present in all cell lines to the same extent. B7-H4, however, was only detectable in the Detroit 562 cell line. Concerning the effect of HGF on the ligand levels, PD-L2 expression was enhanced with HGF stimulation, whereas other checkpoint ligand levels decreased with stimulation. B7-H4 levels in the Detroit 562 cell line drastically decreased with HGF stimulation. This is of interest because both the checkpoint ligand and the growth factor are reported to be connected to epithelial-mesenchymal transition in the literature.

PD-L1 and PD-L2 Expression in Different Tumor Stages and Types of Malignant Salivary Gland Neoplasms: A Single-center Experience.

There is a limited amount of data on the role of programmed cell death ligand (PD-L) -1 and PD-L2 in salivary gland carcinomas. We aimed to evaluate the prognostic value of PD-L1 and PD-L2 expressions, which are closely related to immune mechanisms, with respect to salivary gland tumor types and stages. Data from patients with salivary gland masses surgically removed between 2006 and 2021, diagnosed with a malignant salivary gland neoplasm, were retrospectively analyzed. Immunoreactivity for PD-L1 and PD-L2 was performed on resection materials. The mean age of 90 patients was 52.1±18.8 and 46.7% were male. Overall, 55.6% of patients were diagnosed with adenoid cystic carcinoma (ACC), 23.3% with mucoepidermoid carcinoma (MEC), 16.7% with acinic cell carcinoma (AciCC), 3.3% with ductal carcinoma (DC), and 1 patient with pleomorphic adenoma ex carcinoma (PA-ex-CA). In all, 52% of ACC, 12% of AciCC, 24% of MEC, and 12% of DC cases were at stage IV. The tumor diameter, frequencies of lymphovascular invasion, metastasis, positive surgical margin, recurrence, and mortality rates of patients at stages III and IV were significantly larger than those at stages I and II ( P <0.05). The percentages of tumor cell score (TCS) and immune cell score (ICS) for PD-L1 were significantly higher among patients with MEC compared with those with other types of tumors ( P =0.0011). However, the percentages of combined score (CS) for PD-L1 and tumor cell score for PD-L2 were comparable among tumor types ( P >0.05). No significant difference was found in these scores for PD-L1 between tumor stages ( P >0.05), but for PD-L2, all patients at stage I had TCS <1% for PD-L2, while all patients at stages II and III, and 92% of patients at stage IV had TCS ≥1% ( P <0.0001). High expression of PD-L1 was mostly observed in MEC cases ( P =0.0016), while all patients with AciCC had a low PD-L1 expression level ( P =0.0206). The mean tumor diameter, rate of lymphovascular invasion, perineural invasion, metastasis, positive surgical margin, recurrence, type of treatment, mortality, and TILs ratio did not differ significantly according to PD-L1 expression level ( P >0.05). The percentage of tumor-infiltrating lymphocytes was comparable among negative and positive PD-L1 scores according to both 1% and 5% threshold values ( P >0.05). High PD-L1 expression is rare in AciCC, while PD-L1 expression is high in MEC. Our findings underline the importance of future screening for PD-L1 and PD-L2 before patients undergoing immunotherapies in all salivary gland tumors.

In Search of Better Peptide-(Derived from PD-L2)-Based Immune Checkpoint Inhibitors.

Current anti-cancer immune checkpoint therapy relies on antibodies that primarily target the PD-1/PD-L1(-L2) negative regulatory pathway. Although very successful in some cases for certain cancers, these antibodies do not help most patients who, presumably, should benefit from this type of therapy. Therefore, an unmet clinical need for novel, more effective drugs targeting immune checkpoints remains. We have developed a series of high-potency peptide inhibitors interfering with PD-1/PD-L1(-L2) protein-protein interaction. Our best peptide inhibitors are 12 and 14 amino acids long and show sub-micromolar IC50 inhibitory activity in the in vitro assay. The positioning of the peptides within the PD-1 binding site is explored by extensive modeling. It is further supported by 2D NMR studies of PD-1/peptide complexes. These results reflect substantial progress in the development of immune checkpoint inhibitors using peptidomimetics.

[Progress in PD-1/PD-L1, PD-L2 signaling pathway and its role in host anti-tuberculosis immunity].

Programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, expressed on a variety of immune cells, play multiple regulatory roles in the host immune response to Mycobacterium tuberculosis infection. In this study, we reviewed that the regulatory roles of PD-1/PD-L1, PD-L2 signaling in the host adaptive immune response, such as the innate response of macrophages, and the interaction between T cells and macrophages in response to MTB. In addition, during MTB infection, PD-1/PD-L1, PD-L2 signaling is also involved in the host inflammatory response, as well as the potential roles of PD-1/PD-L1, PD-L2 in the diagnosis and treatment of tuberculosis.

Upregulation of PD-1 and its ligands and expansion of T peripheral helper cells in the nephritic kidneys of lupus-prone BXSB-Yaa mice.

OBJECTIVE: This study aimed to investigate the role of the programmed cell death protein 1 (PD-1) pathway and T peripheral helper (Tph) cells in the pathogenesis of lupus nephritis using lupus-prone BXSB-Yaa mice. METHODS: Male BXSB-Yaa mice and age-matched male C57BL/6 mice were used. The expression of PD-1 and its ligands (programmed cell death 1 ligand-1, PD-L1 and programmed cell death 1 ligand-2, PD-L2) and the phenotypes of kidney-derived cells and splenocytes expressing these molecules were analyzed by immunofluorescence and flow cytometry. RESULTS: Nephritis spontaneously developed in 16-week-old but not in 8-week-old BXSB-Yaa or C57BL/6 mice. PD-1 was expressed on CD4+ mononuclear cells (MNCs) that infiltrated the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of CD4+PD-1+CXCR5-ICOS+ kidney-derived Tph cells was higher in 16-week-old than in 8-week-old BXSB-Yaa and C57BL/6 mice, whereas the frequency of CD4+PD-1+CXCR5+ICOS+ kidney-derived T follicular helper (Tfh) cells was not significantly different between the mice. PD-L1 was constitutively expressed in the renal tubules. PD-L2 was expressed in the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of PD-L1highCD11c+CD3-CD19- and PD-L2+CD11c+CD3-CD19- kidney-derived MNCs in 16-week-old BXSB-Yaa mice was significantly higher than that of the control mice. The percentage of kidney-derived Tph cells but not Tfh cells was correlated with the urinary protein levels in the nephritic mice. CONCLUSION: The results of this study suggest that kidney-infiltrating PD-1+ Tph cells expanded concomitantly with the upregulation of PD-L1 and PD-L2 in the kidneys and the progression of lupus nephritis.

Deciphering Cholesterol's Role in PD-L2 Stability: A Distinct Regulatory Mechanism From PD-L1.

Programmed cell death 1 ligand 2 (PD-L2), a member of the B7 immune checkpoint protein family, emerges as a crucial player in immune modulation. Despite its functional overlap with programmed cell death 1 ligand 1 (PD-L1) in binding to the programmed cell death protein 1 (PD-1) on T cells, PD-L2 exhibits a divergent expression pattern and a higher affinity for PD-1. However, the regulatory mechanisms of PD-L2 remain under-explored. Here, our investigations illustrate the pivotal role of cholesterol in modulating PD-L2 stability. Using advanced nuclear magnetic resonance (NMR) and biochemical analyses, we demonstrate a direct and specific binding between cholesterol and PD-L2, mediated by an F-xxx-V-xx-LR motif in its transmembrane domain, distinct from that in PD-L1. This interaction stabilizes PD-L2 and prevents its downstream degradation. Disruption of this binding motif compromises PD-L2's cellular stability, underscoring its potential significance in cancer biology. These findings not only deepen our understanding of PD-L2 regulation in the context of tumors, but also open avenues for potential therapeutic interventions.

Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines.

BACKGROUND: Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. METHODS: We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) cells. RESULTS: Increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK. CONCLUSIONS: The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.

An alternatively spliced PD-L1 isoform PD-L1∆3, and PD-L2 expression in breast cancers: implications for eligibility scoring and immunotherapy response.

Targeting PD-1/PD-L1 has shown substantial therapeutic response and unprecedented long-term durable responses in the clinic. However, several challenges persist, encompassing the prediction of treatment effectiveness and patient responses, the emergence of treatment resistance, and the necessity for additional biomarkers. Consequently, we comprehensively explored the often-overlooked isoforms of crucial immunotherapy players, leveraging transcriptomic analysis, structural modeling, and immunohistochemistry (IHC) data. Our investigation has led to the identification of an alternatively spliced isoform of PD-L1 that lacks exon 3 (PD-L1∆3) and the IgV domain required to interact with PD-1. PD-L1∆3 is expressed more than the canonical isoform in a subset of breast cancers and other TCGA tumors. Using the deep learning-based protein modeling tool AlphaFold2, we show the lack of a possible interaction between PD-L1∆3 and PD-1. In addition, we present data on the expression of an additional ligand for PD-1, PD-L2. PD-L2 expression is widespread and positively correlates with PD-L1 levels in breast and other tumors. We report enriched epithelial-mesenchymal transition (EMT) signature in high PD-L2 transcript expressing (PD-L2 > PD-L1) tumors in all breast cancer subtypes, highlighting potential crosstalk between EMT and immune evasion. Notably, the estrogen gene signature is downregulated in ER + breast tumors with high PD-L2. The data on PD-L2 IHC positivity but PD-L1 negativity in breast tumors, together with our results on PD-L1∆3, highlight the need to utilize PD-L2 and PD-L1 isoform-specific antibodies for staining patient tissue sections to offer a more precise prediction of the outcomes of PD-1/PD-L1 immunotherapy.

PD-L1 and PD-L2 expression in colorectal cancer.

Context: The programmed death-1 (PD-1) is an immune checkpoint molecule that suppresses T-cell response. The binding of PD-1 to PD-L1/PD-L2 results cytokine production, and T-cell proliferation are reduced. Tumors expressing PD-L1 and PD-L2 escape from cytotoxic T-cells and are exposed to tumor progression. For this reason, immunotherapy has become a new option in the treatment of cancer. Aims: In this study, we examined the PD-L1 and PD-L2 expression in colorectal carcinoma (CRC), and evaluated the relationship between clinicopathological parameters and CD8+ T cells. Methods and Material: We evaluated CD8 expression in tumor-infiltrating lymphocytes and surrounding tumor lymphocytes with PD-L1, PD-L2 staining in tumor cells and immune cells formalin-fixed paraffin embedded samples of 124 patient diagnosed with CRC. Statistical Analysis Used: Pearson Chi-Square, Fisher Exact Chi-Square, and Pearson Exact Chi-Square analyses were used in the analysis of the cross tables. Survival distributions predicted Kaplan--Meier method and it was evaluated using log-rank statistics. Results: In our study, a significant correlation was found between PD-L1 expression and female sex and tumors with medullary morphology. No expression of PD-L2 was observed in tumors containing medullary morphology, and a statistically inverse relationship was observed between PD-L2 and the medullary component. PD-L1 positive tumor-infiltrating lymphocytes were determined to be an important predictor for recurrence-free survival. Conclusions: We believe that the evaluation of these parameters may be useful in the selection of patients who will benefit from immunotherapy in CRC cases.

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

Programmed death receptor ligand-2 (PD-L2) bearing extracellular vesicles as a new biomarker to identify early triple-negative breast cancer patients at high risk for relapse.

PURPOSE: Based on the tumor-promoting features of extracellular vesicles (EV) and PD-L1/2-bearing EV subpopulations (PD-L1/2EV), we evaluated their potential as surrogate markers for disease progression or eligibility criteria for PD-1 immune checkpoint inhibition (ICI) approaches in early triple-negative breast cancer (TNBC). METHODS: After enrichment of EV from plasma samples of 56 patients before and 50 after chemotherapy (CT), we determined levels of EV particle number and PD-L1/2EV by nanoparticle tracking analysis or ELISA and associated the results with clinical status/outcome and the presence of distinct circulating tumor cells (CTC) subpopulations. RESULTS: Compared to healthy controls, patients had a tenfold higher EV concentration and significantly elevated PD L2EV but not PD L1EV levels. The most important clinical implications were found for PD-L2EV. High PD-L2EV levels were associated with a significantly reduced 3-year progression-free and overall survival (PFS and OS). A loss of PD-L2EV after CT was significantly more prominent in patients achieving pathological complete response (pCR). Increased pre-CT PD-L2EV levels were found in patients having NOTCH1-positive or ERBB3-positive CTC. The presence of ERBB3-positive CTC combined with high pre-CT PD-L2EV resulted in a shorter PFS. CONCLUSION: This study highlights PD L2EV as a promising biomarker for risk assessment of TNBC patients and represents the basic for additional studies introducing PD-L2EV as an eligibility criterion for PD-1 ICI approaches.

Clinical Value of the PD-1/PD-L1/PD-L2 Pathway in Patients Suffering from Endometriosis.

The interaction between dendritic cells (DCs) and T cells mediated by the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1)/programmed cell death ligand 2 (PD-L2) pathway is the most important point in regulating immunological tolerance and autoimmunity. Disturbances in the quantity, maturity, and activity of DCs may be involved in the implantation and growth of endometrial tissue outside the uterus in endometriosis (EMS). However, little is known about the role of the immune checkpoint pathways in EMS. In our study, we examined the expression of PD-L1/PD-L2 on myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the peripheral blood (PB) and peritoneal fluid (PF) of both EMS patients (n = 72) and healthy subjects (n = 20) via flow cytometry. The concentration of soluble PD-L1 and PD-L2 in the plasma and PF of EMS patients and the control group were determined using ELISA. We demonstrated an elevated percentage of mDCs, mDCs and pDCs with the PD-L1or PD-L2 expression, and a higher concentration of the soluble forms of PD-L1 and PD-L2 in the PF than in the plasma of EMS patients. We conclude that the peritoneal cavity environment and the PD-1/PD-L1/PD-L2 axis may play an important role in the modulation of immune response and the development and/or progression of EMS.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

Investigation of pd-l1 (cd274), pd-l2 (pdcd1lg2), and ctla-4 expressions in malignant pleural mesothelioma by immunohistochemistry and real-time polymerase chain reaction methods.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive malignant disease with a poor prognosis, which affects the surface mesothelium of the pleural cavity. Immune checkpoints are responsible for controlling the immune system to avoid autoimmunity and prevent tissue damage. In this study, we aimed to investigate the expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) immuno-control receptors in MPM patients and the relationship of the expression with tumour types and prognostic parameters. MATERIAL AND METHODS: In this study, we evaluated 50 MPM cases. Immunohistochemically CTLA-4, PD-L1, and PD-L2 were detected by using monoclonal anti-CTLA-4, anti-PD-L1, and anti-PD-L2. Real-time polymerase chain reaction (RT-PCR) analysis was performed with the primers CTLA-4, PD-L1, and PD-L2. RESULTS: Statistically, no significant relation was determined between the PD-L1, PD-L2, and CTLA-4 expressions (immunohistochemical and RT-PCR methods) and the MPM histological type. Interestingly significant correlation was observed between the mean survival time and immunohistochemical PD-L2 expression; thus, long-term survival was observed in cases with PD-L2 expression. CONCLUSIONS: Programmed death ligand 1, PD-L2, and CTLA-4 expression were observed in some MPM cases, suggesting that treatments targeting immune checkpoints may be effective. Because immunohistochemical expression of PD-L2 is associated with better prognosis, it may provide useful clues in the follow-up of patients.

An Extended PD-L2 Cytoplasmic Domain Results From Alternative Splicing in NSCLC Cells.

Antibody-based immunotherapy targeting the interaction between programmed cell death 1 (PD-1) and its ligand PD-L1 has shown impressive clinical outcomes in various cancer types, including nonsmall cell lung cancer (NSCLC). However, regulatory mechanisms in this immune checkpoint pathway still needs clarification. PD-L2 is structurally homologous to PD-L1 and is a second PD-1 ligand. Alternative mRNA splicing from the CD274 and PDCD1LG2 genes holds the potential to generate PD-L1 and PD-L2 isoforms, respectively, with novel functionality in regulation of the PD-1 immune checkpoint pathway. Here, we describe alternative splicing in NSCLC cells potentially generating eight different PD-L2 isoforms from the PDCD1LG2 gene. Extension of exon 6 by four nucleotides is the most prominent alternative splicing event and results in PD-L2 isoform V with a cytoplasmic domain containing a 10 amino acid extension. On average 13% of the PDCD1LG2 transcripts in NSCLC cell lines and 22% of the transcripts in NSCLC tumor biopsies encode PD-L2 isoform V. PD-L2 isoform V localizes to the cell surface membrane but less efficiently than the canonical PD-L2 isoform I. The cytoplasmic domains of PD-1 ligands can affect immune checkpoint pathways by conferring membrane localization and protein stability and thereby represent alternative targets for immunotherapy. In addition, cytoplasmic domains are involved in intracellular signalling cascades in cancer cells. The presented observations of different cytoplasmic domains of PD-L2 will be important in the future delineation of the PD-1 immune checkpoint pathway.

PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability.

Regulatory T (Treg) cells are central to limit immune responses to allergens. Here we show that PD-L2 deficiency prevents the induction of tolerance to ovalbumin and control of airway hyperreactivity, in particular by limiting pTreg numbers and function. In vitro, PD-1/PD-L2 interactions increase iTreg numbers and stability. In mice lacking PD-L2 we find lower numbers of splenic pTregs at steady state, producing less IL-10 upon activation and with reduced suppressive activity. Remarkably, the numbers of splenic pTregs are restored by adoptively transferring PD-L2high dendritic cells to PD-L2KO mice. Functionally, activated pTregs lacking PD-L2 show lower Foxp3 expression, higher methylation of the Treg-Specific Demethylation Region (TSDR) and a decreased Tricarboxylic Acid (TCA) cycle associated with a defect in mitochondrial function and ATP production. Consequently, pyruvate treatment of PD-L2KO mice partially restores IL-10 production and airway tolerance. Together, our study highlights the importance of the PD-1/PD-L2 axis in the control of metabolic pathways regulating pTreg Foxp3 stability and suppressive functions, opening up avenues to further improve mucosal immunotherapy.