Partial restoration of T‐cell function in aged mice by in vitro blockade of the PD‐1/ PD‐L1 pathway

Programmed cell death‐1 (PD‐1) is a newly characterized negative regulator of immune responses. The interaction of PD‐1 with its ligands (PD‐L1 and PD‐L2) inhibits T‐cell proliferation and cytokine production in young mice. Increased PD‐1 expression has been described during chronic infections, inducing chronic activation of the immune system to control it. As aging is associated with chronic immune activation, PD‐1 may contribute to age‐associated T‐cell dysfunction. Our data showed the following results in aged mice: (i) the number of PD‐1‐expressing T cells and the level of expression of PD‐Ls was increased on dendritic cell subsets and T cells; (ii) PD‐1⁺ T cells were exhausted effector memory T cells, as shown by their lower level of CD127, CD25 and CD28, as well as their limited proliferative and cytokine‐producing capacity; (iii) the expression of PD‐1 was up‐regulated after T‐cell receptor‐mediated activation of CD8⁺ T cells, but not of CD4⁺ T cells; (iv) blockade of the PD‐1/PD‐L1 pathway moderately improved the cytokine production of T cells from old mice but did not restore their proliferation; and (v) blockade of the PD‐1/PD‐L1 pathway did not restore function of PD‐1⁺ T cells; its effect appeared to be exclusively mediated by increased functionality of the PD‐1^? T cells. Our data thus suggest that blockade of the PD‐1/PD‐L1 is not likely to be efficient at restoring exhausted T‐cell responses in aged hosts, although improving the responses of PD‐1^? T cells may prove to be a helpful strategy in enhancing primary responses.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

Inhibition of yes‐associated protein down‐regulates PD‐L1 (CD274) expression in human malignant pleural mesothelioma

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

Inhibition of NF‐κB is required for oleanolic acid to downregulate PD‐L1 by promoting DNA demethylation in gastric cancer cells

Gastric cancer is one of the most common causes of cancer‐related death worldwide. Immunotherapy via programmed cell death protein 1 (PD‐1)/programmed cell death‐ligand 1 (PD‐L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD‐L1 expression in gastric cancer cells. Interleukin‐1β (IL‐1β) at 20 ng/mL was used to stimulate human gastric cancer MKN‐45 cells. IL‐1β significantly increased PD‐L1 expression, which was abolished by OA. Next, OA‐treated MKN‐45 cells were co‐cultured with activated and PD‐1‐overexpressing Jurkat T cells. OA restored IL‐2 levels in the co‐culture system and increased T cell killing toward MKN‐45 cells. Overexpression of PD‐L1 eliminated OA‐enhanced T cell killing capacity; however, PD‐1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL‐1β‐increased DNA demethylase activity in MKN‐45 cells. DNA methyltransferase inhibitor 5‐azacytidine rescued OA‐reduced PD‐L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD‐L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL‐1β‐treated MKN‐45 cells, and overexpression of TET3 restored OA‐reduced PD‐L1 expression. Finally, OA disrupted nuclear factor κB (NF‐κB) signaling IL‐1β‐treated MKN‐45 cells, and overexpression of NF‐κB restored OA downregulation of TET3 and PD‐L1. The cytotoxicity of T cells toward MKN‐45 cells was also weakened by NF‐κB overexpression. Altogether, OA blocked the IL‐1β/NF‐κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD‐L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.     

Autocrine VEGF signalling on M2 macrophages regulates PD‐L1 expression for immunomodulation of T cells

M2‐polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death‐ligand 1 (PD‐L1) on M2 macrophages was significantly up‐regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD‐L1 expression on M2 macrophages was dramatically down‐regulated. Furthermore, transplantation of PD‐L1⁺ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4⁺/CD8⁺ T cells in the peripheral blood and increased CD4⁺CD25⁺ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD‐1 signalling blockade in cancer therapy.     

Indirubin modulates CD4+ T‐cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4⁺ T‐cell homeostasis with simultaneous decrease of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4⁺ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell‐axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose‐dependent manner. Indirubin was observed to restore the expression of programmed cell‐death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4⁺ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4⁺ T cells in a PD1/PTEN/AKT signalling pathway.     

An Evaluation of the Impact of PD‐1 Pathway Blockade on Reproductive Safety of Therapeutic PD‐1 Inhibitors

This report discusses the principles of reproductive toxicity risk assessment for biopharmaceuticals blocking the PD‐1/programmed cell death ligand 1 (PD‐L1) pathway, which have been developed for the treatment of patients with advanced malignancies. The PD‐1/PD‐L1 pathway is a T‐cell co‐inhibitory pathway that normally maintains immune tolerance to self. Its role in pregnancy is to maintain immune tolerance to the fetal allograft. In cancer patients, this signaling pathway is hijacked by some neoplasms to avoid immune destruction. PD‐1/PD‐L1‐blocking agents enhance functional activity of the target lymphocytes to eventually cause immune rejection of the tumor. A therapeutic blockade of PD‐1/PD‐L1 pathway that occurs at full target engagement provides a unique challenge to address the risk to pregnancy because disruption of the same pathway may also reduce or abrogate maternal immune tolerance to the fetal alloantigens inherited through the father. Typically, nonclinical reproductive and developmental toxicity (DART) studies in animals (rats and rabbits) with clinical drug candidates are conducted to identify potential risk in humans and to determine exposure margin for the effects on reproduction as part of the risk assessment. However, for biopharmaceuticals for which the desired mechanism of action cannot be separated from potential deleterious effects to the fetus and when the only relevant toxicology species is nonhuman primate (NHP), the risk to reproduction can be predicted by a mechanism‐based assessment using data generated from murine surrogate models as supportive information without conducting DART in NHPs. Such an approach has been used in the evaluation of pregnancy risk of anti‐PD‐1 agent, pembrolizumab, and has been demonstrated as an important alternative to performing DART studies in NHPs     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

MicroRNA‐93‐5p expression in tumor tissue and its tumor suppressor function via targeting programmed death ligand‐1 in colorectal cancer

This work aimed to investigate miR‐93‐5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand‐1 (PD‐L1). MiR‐93‐5p and PD‐L1 expression was detected in CRC and adjacent normal tissues by quantitative real‐time polymerase chain reaction and immunohistochemistry. The correlation between miR‐93‐5p and PD‐L1 was validated by a dual‐luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR‐NC, miR‐93‐5p mimics, miR‐93‐5p inhibitor, PD‐L1 small interfering RNA (siRNA) and miR‐93‐5p inhibitor + PD‐L1 siRNA groups, and wound‐healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR‐93‐5p was downregulated in CRC tissues with upregulated PD‐L1. In PD‐L1‐negative patients, miR‐93‐5p expression was increased compared with that in PD‐L1‐positive patients. MiR‐93‐5p and PD‐L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD‐L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase‐1 (MMP‐1), ‐2, and ‐9, and these effects were abolished by the miR‐93‐5p inhibitor. Additionally, anti‐PD‐L1 upregulated the expressions of interleukin‐2 (IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon γ (IFN‐γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL‐1β, IL‐10, and TGF‐β. However, these changes were partially reversed by miR‐93‐5p inhibition. miR‐93‐5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD‐L1.     

MiR‐16 regulates mouse peritoneal macrophage polarization and affects T‐cell activation

MiR‐16 is a tumour suppressor that is down‐regulated in certain human cancers. However, little is known on its activity in other cell types. In this study, we examined the biological significance and underlying mechanisms of miR‐16 on macrophage polarization and subsequent T‐cell activation. Mouse peritoneal macrophages were isolated and induced to undergo either M1 polarization with 100 ng/ml of interferon‐γ and 20 ng/ml of lipopolysaccharide, or M2 polarization with 20 ng/ml of interleukin (IL)‐4. The identity of polarized macrophages was determined by profiling cell‐surface markers by flow cytometry and cytokine production by ELISA. Macrophages were infected with lentivirus‐expressing miR‐16 to assess the effects of miR‐16. Effects on macrophage–T cell interactions were analysed by co‐culturing purified CD4⁺ T cells with miR‐16‐expressing peritoneal macrophages, and measuring activation marker CD69 by flow cytometry and cytokine secretion by ELISA. Bioinformatics analysis was applied to search for potential miR‐16 targets and understand its underlying mechanisms. MiR‐16‐induced M1 differentiation of mouse peritoneal macrophages from either the basal M0‐ or M2‐polarized state is indicated by the significant up‐regulation of M1 marker CD16/32, repression of M2 marker CD206 and Dectin‐1, and increased secretion of M1 cytokine IL‐12 and nitric oxide. Consistently, miR‐16‐expressing macrophages stimulate the activation of purified CD4⁺ T cells. Mechanistically, miR‐16 significantly down‐regulates the expression of PD‐L1, a critical immune suppressor that controls macrophage–T cell interaction and T‐cell activation. MiR‐16 plays an important role in shifting macrophage polarization from M2 to M1 status, and functionally activating CD4⁺ T cells. This effect is potentially mediated through the down‐regulation of immune suppressor PD‐L1.     

PD‐L1 expression in melanoma shows marked heterogeneity within and between patients: implications for anti‐PD‐1/PD‐L1 clinical trials

This study evaluated the expression of PD‐L1 in immunotherapy‐naïve metastatic melanoma patients to determine longitudinal intrapatient concordance and correlate PD‐L1 status with clinicopathologic characteristics and outcome. PD‐L1 expression was assessed by immunohistochemistry in 58 patients (43 primary tumors, 96 metastases). Seventy‐two percent of patients had at least one specimen expressing PD‐L1 in ≥1% of tumor cells. Median positive tumor cell count overall was low (8% in nonzero specimens). PD‐L1 expression was frequently discordant between primary tumors and metastases and between intrapatient metastases, such that 23/46 longitudinal patient specimens were discordant. PD‐L1 was associated with higher TIL grade but not with other known prognostic features. There was a positive univariate association between PD‐L1 expression in locoregional metastases and melanoma‐specific survival, but the effect was not observed for primary melanoma. In locoregional lymph node metastasis, PD‐L1+/TIL+ patients had the best outcome, and PD‐L1+/TIL? patients had poor outcome.     

IL‐12 stimulates the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) gene expression in CD4+ T cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (?1 to ?1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009. © 2009 Wiley‐Liss, Inc.     

Identification and characterization of bovine programmed death‐ligand 2

Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)‐1 receptor and its ligand, PD‐L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD‐L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD‐L2 was analyzed in vitro. Recombinant PD‐L2 (PD‐L2‐Ig), which comprises an extracellular domain of bovine PD‐L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD‐L2. Bovine PD‐L2‐Ig bound to bovine PD‐1‐expressing cells and addition of soluble bovine PD‐1‐Ig clearly inhibited the binding of PD‐L2‐Ig to membrane PD‐1 in a dose‐dependent manner. Cell proliferation and IFN‐γ production were significantly enhanced in the presence of PD‐L2‐Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD‐L2‐Ig significantly enhanced IFN‐γ production from virus envelope peptides‐stimulated PBMCs derived from bovine leukemia virus‐infected cattle. Interestingly, PD‐L2‐Ig‐induced IFN‐γ production was further enhanced by treatment with anti‐bovine PD‐1 antibody. These data suggest potential applications of bovine PD‐L2‐Ig as a therapy for bovine diseases.     

IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα⁺ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα⁺ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα⁺IFN‐γ⁺ cells and lower IL‐27Rα⁺IL‐10⁺ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα⁺ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.     

Association of programmed death ligand‐1 (PD‐L1) expression with treatment outcomes in patients with BRAF mutation‐positive melanoma treated with vemurafenib or cobimetinib combined with vemurafenib

The prognostic significance of programmed death ligand‐1 (PD ‐L1) on treatment outcomes in patients receiving BRAF with or without MEK inhibitors is not well understood. This retrospective exploratory analysis evaluated the association of tumour PD ‐L1 expression with progression‐free survival (PFS ) and overall survival (OS ) among 210 patients in the coBRIM trial treated with cobimetinib plus vemurafenib or placebo plus vemurafenib. In the vemurafenib cohort, there was a trend of increased PFS and OS in those with PD ‐L1⁺ melanoma, with hazard ratios (HR s; PD ‐L1⁺ vs. PD ‐L1^?) of 0.70 (95% CI , 0.46–1.07) and 0.69 (95% CI , 0.42–1.13) for PFS and OS , respectively. However, in patients treated with cobimetinib plus vemurafenib, a similar trend was not observed with HR s (PD ‐L1⁺ versus PD ‐L1^?) of 1.04 (95% CI , 0.66–1.68) and 0.94 (95% CI , 0.57–1.57) for PFS and OS , respectively. The combination cobimetinib plus vemurafenib appears to overcome the poor prognosis associated with low PD ‐L1 expression.     

Expression of PD‐1/LAG‐3 and cytokine production by CD4+ T cells during infection with Plasmodium parasites

CD4⁺ T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4⁺ T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4⁺ T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD‐1 and LAG‐3, as early as 6 days after infection, whereas those from either Listeria monocytogenes‐ or Leishmania major‐infected mice did not. In response to T‐cell receptor stimulation, CD4⁺ T cells from mice infected with all the pathogens under study produced high concentrations of IFN‐γ. IL‐2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD‐1 and its ligands resulted in increased IFN‐γ production in response to Plasmodium antigens, implying that PD‐1 expressed on activated CD4⁺ T cells actively inhibits T cell immune responses. Studies using Myd88^?/?, Trif^?/? and Irf3^?/? mice showed that induction of these CD4⁺ T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD‐1 and LAG‐3 on CD4⁺ T cells and their reduced IL‐2 production are common characteristic features of Plasmodium infection.

     

The miR‐3127‐5p/p‐STAT3 axis up‐regulates PD‐L1 inducing chemoresistance in non‐small‐cell lung cancer

It is less known about miRNA3127‐5p induced up‐regulation of PD‐L1, immune escape and drug resistance caused by increased PD‐L1 in lung cancer. In this study, lentivirus was transduced into lung cancer cells, and quantitative PCR and Western blot were used to detect the expression of PD‐L1. Then immunofluorescence assay was applied to detect autophagy, finally we explored the relationship between PD‐L1 expressions and chemoresistance in patients. As a result, we found that microRNA‐3127‐5p promotes pSTAT3 to induce the expression of PD‐L1; microRNA‐3127‐5p promotes STAT3 phosphorylation through suppressing autophagy, and autophagy could retaine pSTAT3 into the nucleus in miRNA‐3127‐5p knocked cells, and immune escape induced by elevated level of PD‐L1 results in chemoresistance of lung cancer. In conclusion, microRNA‐3127‐5p induces PD‐L1 elevation through regulating pSTAT3 expression. We also demonstrate that immune escape induced by PD‐L1 can be dismissed by corresponding monoclonal antibody.