PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8⁺ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4⁺ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4⁺ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4⁺ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4⁺ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4⁺ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.     

Mechanisms of immune checkpoint inhibitor-mediated liver injury

The immune checkpoints, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein-1/ligand-1 (PD-1/PD-L1) are vital contributors to immune regulation and tolerance. Recently immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy; however, they come with the cost of immune related adverse events involving multiple organs such as the liver. Due to its constant exposure to foreign antigens, the liver has evolved a high capacity for immune tolerance, therefore, blockade of the immune checkpoints can result in aberrant immune activation affecting the liver in up to 20% of patients depending on the agent(s) used and underlying factors. This type of hepatotoxicity is termed immune mediated liver injury from checkpoint inhibitors (ILICI) and is more common when CTLA4 and PD-1/PD-L1 are used in combination. The underlying mechanisms of this unique type of hepatotoxicity are not fully understood; however, the contribution of CD8⁺ cytotoxic T lymphocytes, various CD4⁺ T cells populations, cytokines, and the secondary activation of the innate immune system leading to liver injury have all been suggested. This review summarizes our current understanding of the underlying mechanisms of liver injury in immunotherapy using animal models of ILICI and available patient data from clinical studies.     

CXCR5+ CD8+ T cells could induce the death of tumor cells in HBV-related hepatocellular carcinoma

The follicular CXCR5⁺ CD8⁺ T cells have recently emerged as a critical cell type in mediating peripheral tolerance as well as antiviral immune responses during chronic infections. In this study, we investigated the function of CXCR5⁺ CD8⁺ T cells in HBV-related hepatocellular carcinoma patients. Compared to CXCR5⁻ CD8⁺ T cells, CXCR5⁺ CD8⁺ T cells presented elevated PD-1 expression but reduced Tim-3 and CTLA-4 expression. Upon anti-CD3/CD28 stimulation, CXCR5⁺ CD8⁺ T cells demonstrated higher proliferation potency than CXCR5⁻ CD8⁺ T cells, especially after PD-1 blockade. CXCR5⁺ CD8⁺ T cells also demonstrated significantly higher granzyme B synthesis and release, as well as higher level of degranulation. Tumor cells were more readily eliminated by CXCR5⁺ CD8⁺ T cells than by CXCR5⁻ CD8⁺ T cells. Interestingly, we found that B cells were more resistant to CXCR5⁺ CD8⁺ T cell-mediated killing than tumor cells, possibly through IL-10-mediated protection. In addition, the CXCR5⁺ CD8⁺ T cell-mediated cytotoxic effects on tumor cells could be significantly enhanced by PD-L1 blockade. Together, we presented that in patients with in HBV-related hepatocellular carcinoma, CXCR5⁺ CD8⁺ T cells could mediate tumor cell death more potently than the CXCR5⁻ CD8⁺ T cells in vitro while the autologous B cells were protected.     

Naloxone can improve the anti-tumor immunity by reducing the CD4CD25Foxp3 regulatory T cells in BALB/c mice

New strategies that stimulate cell-mediated immunity (CMI) against tumors and inhibit regulatory T cells are needed to improve the outcome of cancer immunotherapy. The aim of this study was to enhance the anti-tumor immunity of gp96 vaccine through naloxone administration. Therefore, we used BALB/c mouse model of fibrosarcoma tumor and analyzed the tumor size, splenocyte proliferation, spleen and tumor-infiltrated lymphocytes. Tumor and spleen CD4⁺CD25⁺Foxp3⁺ regulatory T lymphocytes, cytotoxic activity of the splenocytes, IFN-γ and IL-4 secretion were assessed to describe the anti-tumor immune response. Our findings showed that co-administration of gp96 and naloxone has resulted in a significant reduction in CD4⁺CD25⁺Foxp3⁺ regulatory T cells in the spleen. The results indicated that on days 27 and 32 the tumors in the gp96 + Nal group grew significantly slower. Moreover, co-administration of gp96 and naloxone significantly increased the intra-tumor CD8⁺ T cells and cytotoxic activity. In addition the results indicated a significant increase in the proliferation of splenocytes and IFN-γ production. Our results demonstrate that naloxone is an effective immunoadjuvant in cancer immunotherapy.     

Enhanced chemo-immunotherapy against melanoma by inhibition of cholesterol esterification in CD8+ T cells

Cholesterol facilitated the formation of T cell receptor on cytotoxic CD8⁺ T lymphocytes (CTLs). However, the activation of CD8⁺ T cells always resulted in the upregulation of acetyl-CoA acetyltransferase-1 (ACAT-1) and enhanced the esterification of cholesterol. To relieve the suppression on CD8⁺ T cells, an ACAT-1 inhibitor avasimibe was combined with chemo-immunotherapy. Paclitaxel and immunoadjuvant αGC were co-encapsulated in liposomes modified with pH sensitive TH peptide (PTX/αGC-TH-Lip). After intravenous injections, the combination of avasimibe significantly elevated the free cholesterol level and relieved the inhibition of CD8⁺ T cells caused by PTX/αGC-TH-Lip, leading to enhanced CTL responses and anti-tumor effects of PTX/αGC-TH-Lip in B16F10 melanoma xenograft and lung metastasis models. The adoptive immunotherapy further confirmed the enhanced anti-tumor immune responses of the combined strategy. The combination of avasimibe and PTX/αGC-TH-Lip was proven as a feasible approach to enhance the antitumor effects of chemo-immunotherapy by relieving the inhibition of CD8⁺ T cells.     

Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35–40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4⁺Fop3⁺ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8⁺ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.     

Cationic Liposomes Loaded with a Synthetic Long Peptide and Poly(I:C): a Defined Adjuvanted Vaccine for Induction of Antigen-Specific T Cell Cytotoxicity

For effective cancer immunotherapy by vaccination, co-delivery of tumour antigens and adjuvants to dendritic cells and subsequent activation of antigen-specific cytotoxic T cells (CTLs) is crucial. In this study, a synthetic long peptide (SLP) harbouring the model CTL epitope SIINFEKL was encapsulated with the TLR3 ligand poly(inosinic-polycytidylic acid) (poly(I:C)) in cationic liposomes consisting of DOTAP and DOPC. The obtained particles were down-sized to about 140 nm (measured by dynamic light scattering) and had a positive zeta-potential of about 26 mV (according to laser Doppler electrophoresis). SLP loading efficiency was about 40% as determined by HPLC. Poly(I:C) loading efficiency was about 50%, as assessed from the fluorescence intensity of fluorescently labelled poly(I:C). Immunogenicity of the liposomal SLP vaccine was evaluated in vitro by its capacity to activate dendritic cells (DCs) and present the processed SLP to SIINFEKL-specific T cells. The effectiveness of the vaccine to activate CD8⁺ T cells was analysed in vivo after intradermal and subcutaneous immunisation in mice, by measuring antigen-specific T cells in blood and spleens and assessing their functionality by cytokine production and in vivo cytotoxicity. The liposomal formulation efficiently delivered the SLP to DCs in vitro and induced a functional CD8⁺ T cell immune response in vivo to the CTL epitope present in the SLP. The SLP-specific CD8⁺ T cell frequency induced by the poly(I:C)-adjuvanted liposomal SLP formulation showed an at least 25 fold increase over the T cell frequency induced by the poly(I:C)-adjuvanted soluble SLP. In conclusion, cationic liposomes loaded with SLP and poly(I:C) have potential as a powerful therapeutic cancer vaccine formulation.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9686-4) contains supplementary material, which is available to authorized users.KEY WORDS: cancer immunotherapy, cationic liposomes, CTL epitope, peptide antigen, Poly(I:C)     

Functional tumor specific CD8 + T cells in spleen express a high level of PD-1

The inhibitory effects of programmed cell death 1 (PD-1) receptor on tumor specific T cells were mainly investigated at tumor site. While PD-1 expression can be rapidly unregulated upon T cell activation at lymphoid tissues, little is known about where PD-1 signal exerts its inhibitory function in tumor-bearing host. To address this issue, we assessed the effects of PD-1 on vaccine induced activation of splenic CD8 + T cells in mice. The vaccine consisted of mice CD8 + T cell epitope peptide and poly IC. After vaccination, spleen or tumor tissues were dissociated, IFN-γ synthesis and PD-1 expression by CD8 + T cells were detected by flow cytometry. We found that CD8 + T cells could be successfully activated in spleen after immunization, characterized by the capability of producing IFN-γ when encountering relevant peptide. These activated splenic CD8 + T cells also expressed a high level of PD-1. Although PD-L1 expression in spleen parenchyma was also increased after vaccination, PD-1 blockade did not affect the activation of splenic CD8 + T cells, but enhanced the anti-tumor effects of peptide vaccine. This synergetic effect of peptide vaccine plus PD-1 blockade was coupled with increased aggregation of IFN-γ + CD8 + tumor infiltrated lymphocytes (TILs), rather than CD4 + TILs. The results indicated that for tumor-bearing host, PD-1 pathway exerted its inhibitory function at tumor site and PD-1 expression on the splenic CD8 + T cells correlated positively with IFN-γ synthesis.     

PD-1 regulates CXCR5+ CD4 T cell-mediated proinflammatory functions in non-small cell lung cancer patients

PD-1 inhibitors have been used to revive exhausted T cell responses in non-small cell lung cancer (NSCLC) and other malignancies. CXCR5⁺ T follicular helper (Tfh) cells are characterized by constitutive high PD-1 expression and have been associated with the formation of tertiary lymphoid structures and implicated in antitumor immunity. In this study, we investigated the effect of PD-1 and PD-1 inhibition on CXCR5⁺ CD4 T cells. Data showed that CXCR5⁺ CD4 T cells in both healthy subjects and NSCLC patients presented markedly higher PD-1 expression than CXCR5⁻ CD4 T cells. Both CXCR5⁻ and CXCR5⁺ CD4 T cells from NSCLC patients presented higher PD-1 expression than their counterparts in healthy subjects. PD-1⁺ CXCR5⁺ CD4 T cells were functional, could express IL-21, IL-10, and CXCL13 upon stimulation, demonstrated auxiliary effects toward CD8 T cell-mediated IFN-γ production and proliferation, and promoted IgM and IgG production. However, the potency of PD-1⁺ CXCR5⁺ CD4 T cells was lower than the potency of PD-1⁻ CXCR5⁺ CD4 T cells. PD-1 blocking could significantly enhance the effector functions of PD-1⁺ CXCR5⁺ CD4 T cells. Overall, this study demonstrated that PD-1⁺ CXCR5⁺ CD4 T cells could promote CD8 T cell and B cell inflammation and could be modulated by PD-1 inhibition.     

Anti-PD-L1 mediating tumor-targeted codelivery of liposomal irinotecan/JQ1 for chemo-immunotherapy

Immune checkpoint blockade therapy has become a first-line treatment in various cancers. But there are only a small percent of colorectal patients responding to PD-1/PD-L1 blockage immunotherapy. How to increase their treatment efficacy is an urgent and clinically unmet need. It is acknowledged that immunogenic cell death (ICD) induced by some specific chemotherapy can enhance antitumor immunity. Chemo-based combination therapy can yield improved outcomes by activating the immune system to eliminate the tumor, compared with monotherapy. Here, we develop a PD-L1-targeting immune liposome (P-Lipo) for co-delivering irinotecan (IRI) and JQ1, and this system can successfully elicit antitumor immunity in colorectal cancer through inducing ICD by IRI and interfering in the immunosuppressive PD-1/PD-L1 pathway by JQ1. P-Lipo increases intratumoral drug accumulation and promotes DC maturation, and thereby facilitates adaptive immune responses against tumor growth. The remodeling tumor immune microenvironment was reflected by the increased amount of CD8⁺ T cells and the release of IFN-γ, and the reduced CD4⁺Foxp3⁺ regulatory T cells (Tregs). Collectively, the P-Lipo codelivery system provides a chemo-immunotherapy strategy that can effectively remodel the tumor immune microenvironment and activate the host immune system and arrest tumor growth.     

Changes of phenotype and function of human CD4+CD25-T cells induced by transfection of Foxp3

The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4⁺ T cells. The pMSCV-Foxp3 retroviral vector encoding Foxp3 gene was transduced into the PT67 packaging cell line. Virus-containing supernatant was applied to differentiate CD4⁺CD25⁻ T cells. The resulting cells were sorted with flow cytometry. The expressions of CD25, CD127, CTLA-4 and the proliferation of transfected T cells were examined. The effect of transfected CD4⁺ T cells on the proliferation and cytokine production of CD4⁺CD25⁻ T cells was examined. Foxp3-gene transfected CD4⁺ T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4, but down-regulated CD127 expression. After transfection, the proliferation of CD4⁺ T cells was eliminated. Transfected T cells inhibited the proliferation of CD4⁺CD25⁻ T cells. CD4⁺CD25⁻ T cells acquired a regulatory phenotype and function after it was transduced with the Foxp3 gene. This suggested a key role of Foxp3 in the generation of CD4⁺CD25⁺ regulatory T cells. __________ Translated from Acta Academiae Medicinae Militaris Tertiae, 2008, 30(3): 186–188 [译自: 第三军医大学学报]     

CD163+ M2-type tumor-associated macrophage support the suppression of tumor-infiltrating T cells in osteosarcoma

Osteosarcoma is one of the most common childhood cancers with high numbers of cancer-related deaths. Progress in conventional therapies is showing limited improvement. An adaptive T cell-based immunotherapy represents a promising new therapeutic option, but to improve its efficacy, regulatory mechanisms in osteosarcoma need further elucidation. Here, to evaluate the regulatory effect of tumor microenvironment of T cells in osteosarcoma, we examined the peripheral blood (PB) and tumor infiltrating (TI) T cells, and their correlations with PB and tumor immune characteristics. We found that TI T cells contained significantly higher levels of TIM-3⁺ PD-1⁻ and TIM-3⁺ PD-1⁺ cells than their PB counterparts. Similar to that in chronic HIV and HCV infections, these TIM-3⁺ PD-1⁻ and TIM-3⁺ PD-1⁺ T cells presented reduced proliferation and proinflammatory cytokine secretion in response to stimulation. Presence of M2-type (CD163⁺) macrophages exacerbated T cell immunosuppression, since frequencies of CD163⁺ tumor-associated macrophages were directly correlated with the frequencies of suppressed TIM-3⁺ PD-1⁺ T cells. Moreover, depletion of CD163⁺ macrophages significantly improved T cell proliferation and proinflammatory cytokine production. Overall, our data presented an intratumoral T cell-specific immunosuppression that was amplified by M2-type tumor-associated macrophages.     

基于生物信息学的CD39对肺腺癌免疫微环境及其免疫治疗的影响分析

目的 采用生物信息学方法探讨CD39基因在肺腺癌组织中的表达情况,并分析CD39与肺腺癌免疫微环境及免疫治疗响应的关系。方法 通过The Cancer Genome Atlas （TCGA）数据库分析CD39在肺腺癌组织和正常肺组织中的表达差异。采用基因集富集分析（GSEA）研究CD39可能富集的免疫相关通路;通过肿瘤免疫数据库（TIMER）分析肺腺癌患者中CD39基因表达情况与肿瘤微环境免疫细胞浸润的关系;同时分析CD39和其他免疫检查点基因PD-1、PD-L1、CTLA-4、IDO1的相关性,采用药物敏感性基因组学数据库（GDSC）和肿瘤免疫功能障碍和排斥（TIDE）数据库,预测CD39基因表达与化疗药物敏感性以及免疫治疗的响应。结果 CD39在肺腺癌组织中的表达低于在正常组织中的表达,CD39富集了免疫相关通路如炎症反应、干扰素α反应、干扰素γ反应。TIMER数据库均显示在肺腺癌组织中,CD39的表达与B细胞、CD8⁺T细胞、CD4⁺T细胞、巨噬细胞、中性粒细胞以及树突状细胞呈正相关性。同时CD39与其他的免疫检查点PD-1、PD-L1、CTLA-4、IDO1均呈一定的正相关性,化疗敏感性显示在肺腺癌组织中,对于博来霉素、顺铂、多西他赛、长春花碱等化疗药物,高表达CD39组对其敏感。但对于免疫治疗,其响应率却低于CD39低表达组。结论 CD39在肺腺癌组织中低表达,能激活免疫相关通路,对免疫治疗响应率高,可作为肺腺癌新型分子标志物。     

Co-immunizing with PD-L1 induces CD8+ DCs-mediated anti-tumor immunity in multiple myeloma

Tumor therapeutic vaccines have faced a challenge for effective protection against malignant tumors by inducing tumor-specific CD8⁺ T cell responses. Here, we designed a DNA vaccine containing a tumor-specific antigen of Dickkopf-1 (DKK-1) and an immune checkpoint of programmed death ligand 1 (PD-L1) delivered by PLGA/PEI nanoparticle-mediated delivery system for multiple myeloma therapy. Murine subcutaneous tumor model established with human DKK1 (hDKK-1)-SP2/0 cells were intramuscularly immunized with PLGA/PEI-pPD-L1/pDDK-1 vaccine and equal amount of control 3 times at 10 day-intervals. Compared with PLGA/PEI-pDKK1 immunization group, PLGA/PEI-pPD-L1/pDKK-1 co-immunization enhanced the induction and mature of CD11c⁺ DCs and CD8⁺CD11c⁺ DCs, and promoted antigen-specific Th1 responses and cytotoxic T lymphocyte (CTL) responses. The reduced tumor volume and weight as well as increased tumor inhibition rate were observed in PLGA/PEI-pPD-L1/pDKK-1 vaccine co-immunization group, indicated that the vaccine could effectively inhibit the tumor growth of multiple myeloma. The anti-tumor activity of PLGA/PEI-pPD-L1/pDKK-1 vaccine was abrogated by CD8 cell depletion accompanied with the reduced percentages of CD8⁺CD11c⁺ DCs and CD8⁺ T cells in the spleen and TILs. These results indicated that the anti-tumor efficacy of PLGA/PEI-pPD-L1/pDKK-1 vaccine was required for CD8⁺CD11c⁺ DCs-mediated CD8⁺ T cell immunity responses. This vaccine strategy may represent a potential and promising approach for hematological malignancy treatment.     

Comparative phenotypes of peripheral blood and spleen cells from cancer patients

Patients with resectable tumor, either in the body or the tail of the pancreas, and cancer patients with a primary tumor adjacent to the splenic vasculature frequently undergo a splenectomy as standard of care during resection. The spleen provides an unutilized source of lymphocytes with potential utility for adoptive cellular therapy (ACT). In this report, spleen and peripheral blood (PB) cells from cancer patients were compared to one another and normal PB by flow cytometry with a focus on CD8⁺ T-cells, memory phenotype, and their relative expression of checkpoint proteins including program death ligand-1 (PD1). PD1 is both an activation marker for T-cells including antigen (Ag) specific responses, as well as a marker of T-cell exhaustion associated with co-expression of other checkpoint molecules such as lymphocyte activating gene-3 (LAG-3) and T-cell immunoglobulin and mucin domain containing-3 (TIM-3). In summary, the spleen is a rich source of CD8⁺PD1⁺ T-cells, with an 8-fold higher frequency compared to the PB. These CD8+ T-cells are predominantly central and transitional memory T-cells with associated effector phenotypes and low expression of TIM-3 and LAG-3 with potential utility for ACT”.     

Gemcitabine and checkpoint blockade exhibit synergistic anti-tumor effects in a model of murine lung carcinoma

Lung cancer is leading cause of cancer death in the world. Chemotherapy is currently one of the standard treatments for lung cancer. Gemcitabine is a pyrimidine nucleoside drug which has been approved by FDA to treat lung cancer. However, acquired resistance inevitable develops after Gemcitabine treatment, limiting clinical efficacy. Lewis lung carcinoma (LLC) cells were treated with Gemcitabine and cell apoptosis and programmed cell death-ligand 1 (PD-L1) expression were analyzed by flow cytometry. LLC mouse model was established to analysis the proportion and programmed cell death-1 (PD-1) expression of CD8 + T cells. Anti-tumor effect by treating with Gemcitabine and anti-PD-1 antibody was measured through in vivo LLC mouse model. Gemcitabine treatment induces tumor cell apoptosis and PD-L1 expression. Further study showed that Gemcitabine treatment also increases CD8⁺ and CD4⁺ T cells proportion, PD-1 and PD-L1 expression in LLC mouse model. Combination therapy with Gemcitabine and αPD-1 not only has strong anti-tumor effect, but also could inhibit postsurgical recurrence of LLC. Our findings demonstrated that the combination therapy of Gemcitabine and αPD-1 is an effective therapeutic strategy for lung cancer.     

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC₅₀ = 8.9 nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD₅₀ > 5000 mg/kg and showed significant in vivo antitumor activity through promoting CD8⁺ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.     

Upregulation of PD-1 on CD4+CD25+T cells is associated with immunosuppression in liver of mice infected with Echinococcus multilocularis

Alveolar echinococcosis is a zoonotic disease caused by Echinococcus multilocularis (E. multilocularis) infection. The relationship between PD-1/PD-L1 pathway and Tregs at different stages of E. multilocularis infection has rarely been reported. This study aims to investigate the role of PD-1/PD-L1 in immunosuppression of Tregs in E. multilocularis infection. Hematoxylin-eosin staining, flow cytometry, immunohistochemistry, quantitative RT-PCR analysis, cytometric bead array and MTT assay were used to analyze liver pathological changes, percentages of PD-1⁺ Tregs and PD-L1⁺ dendritic cells (DCs), expression levels of PD-1, PD-L1 and Foxp3, levels of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) and proliferation of lymphocytes. During middle-late stage (day 30 to day 330) the percentages of PD-1⁺ Tregs and PD-L1⁺ DCs together with levels of Foxp3, IL-10 and TGF-β increased significantly and maintained at high level. The expression of PD-1 and PD-L1 was increased with the enlarging erosion of E. multilocularis, and was mainly distributed in hepatic sinus, fibrous wall of alveolar hydatid and germinal layer around foci of infection. PD-1/PD-L1 promoted the secretion of IL-10 and TGF-β. Our results indicate that engagement of the PD-1 and PD-L1 correlates with inhibition of T-cell effector function, cytokine secretion and proliferation. High expression of PD-1/PD-L1 may play an important role in stimulating CD4⁺CD25⁺ T cells, and maintaining peripheral tolerance and immune evasion during chronic infection of E. multilocularis.     

基于生物信息数据库分析CD47对肺鳞癌免疫微环境及其免疫治疗的影响

目的 采用生物信息学方法分析基因CD47对肺鳞癌免疫微环境的影响并预测其免疫治疗响应。方法 从The Cancer Genome Atlas (TCGA)数据库下载基因CD47在肺鳞癌组织和正常组织中的测序数据并分析其表达差异。采用基因集富集分析研究CD47富集的免疫相关通路;通过肿瘤免疫数据库(Tumor Immune Estimation Resource,TIMER)和TISIDB数据库(http://cis.hku.hk/TISIDB/TISIDB)分析肺鳞癌患者中CD47基因表达情况与肿瘤微环境免疫细胞浸润的关系;同时也分析了CD47和其他免疫检查点基因PD-1、PD-L1、CTLA-4、IDO1的相关性;采用pRRophetic和TIDE算法,预测CD47基因表达与化疗药物敏感性以及免疫治疗的响应。结果 与正常组织相比,CD47在肺鳞癌组织中低表达;CD47富集了免疫相关通路如炎症反应、干扰素α反应、干扰素γ反应和JAK-STAT通路。2个数据库分析结果显示,在肺鳞癌组织中CD47表达水平与B细胞、CD8⁺T细胞、CD4⁺T细胞、巨噬细胞、中性粒细胞以及树突状细胞呈正相关性。同时CD47与其他的免疫检查点PD-1、PD-L1、CTLA-4、IDO1均呈一定的正相关性,化疗敏感性结果显示,在肺鳞癌组织中,对于舒尼替尼、达沙替尼、阿霉素、吉西他滨等化疗药物,高表达CD47组IC₅₀值低于低表达组。但对于免疫治疗,其响应率却低于低表达组。结论 CD47在肺鳞癌组织中低表达,能激活免疫相关通路,对免疫治疗响应率高,可作为肺鳞癌新型分子标志物。     

Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall Skeletons (Nr-CWS)

Nocardia rubra cell wall skeleton (Nr-CWS) has been reported to have innate immunostimulating and anti-tumor activities. However, the immunomodulatory effects of Nr-CWS on CD8+ T cells and their related mechanisms are still unknown. In this work, our team purified CD8⁺T cells from spleen cells and explored the phenotype and function of NR-CWS in vitro on CD8⁺T cells. We observed that Nr-CWS can significantly up-regulate the expression of CD69 and CD25 on CD8⁺T cells, with no significant effect on apoptosis or cell death of CD8⁺T cells that occurs in vitro during culture. In addition, the effect of perforin and granzyme B was increased after Nr-CWS treatment, but did not substantially alter the expression of TRAIL and FasL. A variety of cytokine analyses have shown that of the cytokines examined (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6 and IL-10), only IFN-γ and TNF The increase in -α was more pronounced, and the effect of Nr-CWS in CD8⁺T cell culture medium on CD8+ T cells was independent of Th cells. Our results demonstrated that Nr-CWS could up-regulate CD69 and CD25 expression on CD8⁺T cells, promoting IFN-γ and TNF-α secretion, and enhancing perforin and granzyme B production. Thus Nr-CWS may have Immunoaugmenting therapeutic activity via an increase in CD8⁺T cells response.