Effect of IFNα-2a on Fas expression and apoptosis rate of peripheral blood cytotoxic T cells in patients with hepatitis B

Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.

Blockade of Arf1-mediated lipid metabolism in cancers promotes tumor infiltration of cytotoxic T cells via the LPE-PPARγ-NF-κB-CCL5 pathway

Tumor immunotherapy has achieved breakthroughs in a variety of tumors. However, the systemic absence of T cells in tumors and immunosuppressive tumor microenvironment so far limits the efficacy of immunotherapy to a small population of patients. Therefore, novel agents to increase T-cell tumor infiltration are urgently needed in the clinic. We recently found that inhibition of the ADP-ribosylation factor 1 (Arf1)-mediated lipid metabolism not only kills cancer stem cells (CSCs) but also elicits an anti-tumor immune response. In this study, we revealed a mechanism that targeting Arf1 promotes the infiltration of cytotoxic T lymphocytes (CTLs) into tumors through the C-C chemokine ligand 5 (CCL5)- C-C chemokine receptor type 5 (CCR5) pathway. We found that blockage of Arf1 induces the production of the unsaturated fatty acid (PE 18:1) that binds and sequestrates peroxisome proliferator- activated receptor-γ (PPARγ) from the PPARγ-nuclear factor-κB (NF-κB) cytoplasmic complex. The released NF-κB was then phospho-rylated and translocated into the nucleus to regulate the transcription of chemokine CCL5. CCL5 promoted infiltration of CTLs for tumor regression. Furthermore, the combination of the Arf1 inhibitor and programmed cell death protein 1 (PD-1) blockade induced an even stronger anti-tumor immunity. Therefore, targeting Arf1 represents a novel anti-tumor immune approach by provoking T-cell tumor infiltration and may provide a new strategy for tumor immunotherapy.

Recombinant E.coli LLO/OVA Vaccination Effectively Inhibits Murine Melanoma Metastasis to Lung by CD8^+T Cells Immunity

Objective： To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods： Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O （LLO） and ovalbumin （OVA） of the vaccine was determined by coomassie brilliant blue staining and western blotting, After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T, CD3^CD8^＋T and OVA257-264 SIINFEKL specific CD8^＋T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed. Results： Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3^＋CD4^＋T, CD4^＋CD25^＋T and CD3^＋CD8^＋T cells were equivalent in the two groups of mice. However, there were significantly more OVA257-264 SIINFEKL specific CD8^＋T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8^＋T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B I6 OVA challenged mice too. Conclusion： Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8^＋T cell immunity.

Adaptive immune response during hepatitis C virus infection

Hepatitis C virus(HCV)infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma.HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control.Liver damage and disease progression during HCV infection are driven by both viral and host factors.Specifically,adaptive immune response carries out an essential task in controllingnon-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery.HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion.To impair HCV-specific T cell reactivity,HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro-and antiapoptotic proteins.In this review,the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4^(+)T cells

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency(PAD),analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects.CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation.Indeed,while circulating CD57^(+)CD4^(+)T cells are normally rare,we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade.We performed single-cell RNA-seq analysis of matched CD57^(+)CD4^(+)T cells from blood and tonsil samples.Circulating CD57^(+)CD4^(+)T cells(CD4cyt)exhibited a cytotoxic transcriptome similar to that of CD8^(+)effector cells,could kill B cells,and inhibited B-cell responses.CTLA4 restrained the formation of CD4cyt.While CD57 also marked an abundant subset of follicular helper T cells,which is consistent with their antigen-driven differentiation,this subset had a preexhaustion transcriptomic signature marked by TCF7,TOX,and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition.Thus,CD57^(+)CD4^(+)T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers.CTLA4 is a key modifier of CD4^(+)T-cell cytotoxicity,and the pathological CD4cyt phenotype is accentuated by infection.

Dendritic cell-mimicking scaffolds for ex vivo T cell expansion

We propose an ex vivo T cell expansion system that mimics natural antigen-presenting cells(APCs)for adoptive cell therapy(ACT).Microfiber scaffolds coated with dendritic cell(DC)membrane replicate physicochemical properties of dendritic cells specific for T cell activation such as rapid recognition by T cells,long duration of T cell tethering,and DC-specific co-stimulatory cues.The DC membrane-coated scaffold is first surface-immobilized with T cell stimulatory ligands,anti-CD3(αCD3)and anti-CD28(αCD28)antibodies,followed by adsorption of releasable interleukin-2(IL-2).The scaffolds present both surface and soluble cues to T cells ex vivo in the same way that these cues are presented by natural APCs in vivo.We demonstrate that the DC-mimicking scaffold promotes greater polyclonal expansion of primary human T cells as compared toαCD3/αCD28-func-tionalized Dynabead.More importantly,major histocompatibility complex molecules derived from the DC membrane of the scaffold allow antigen-specific T cell expansion with target cell-specific killing ability.In addition,most of the expanded T cells(~97%)can be harvested from the scaffold by density gradient centri-fugation.Overall,the DC-mimicking scaffold offers a scalable,modular,and customizable platform for rapid expansion of highly functional T cells for ACT.

Graft-versus-leukemia effects of Wilms＇ tumor 1 protein-specific cytotoxic T lymphocytes in patients with chronic myeloid leukemia after allogeneic hematopoietic stem cell transplantation

Background The role of Wilms＇ tumor 1 protein （WT1）-specific cytotoxic T cells （CTL） in eradicating chronic myeloid leukemia （CML） cells is to be established. The aim of this study was to determine whether WT1 contributed to the graft-versus-leukemia effects （GVLE） for CML following allogeneic hematopoietic stem cell transplantation （HSCT）. Methods High-resolution human leukocyte antigen （HLA） class I genotyping was performed by sequence-specific polymerase chain reaction （PCR）. Fifteen HLA-A~＊2402 patients with CML who underwent allogeneic HSCT were enrolled in this study. We monitored the frequency of WT1-specific CTL by pentamer assay and the molecular minimal residual disease by real-time quantitative PCR.Results A CD8＋ T-cell response to WT1 was observed in 14 of 15 patients after HSCT. The median frequencies of WT1-CTL were 0.54%, 0.62%, 0.81% and 1.28% （%CD8） on days 30, 60, 90 and 180, respectively. The median frequency of WT1-CTL （1.38%） in patients with molecular remission （MoR） was significantly higher than that in those without MoR （0.38%） on day 30, while no significant differences between them were detected on days 60, 90 and 180. The increase of WT1-CTL was associated with a decrease in bcr-abl expression and MoR; and the decrease of WT1-CTL was associated with an increase in bcr-abl expression, suggesting a WT1 -driven GVL effect. WT1-CTL had a predominant effector-memory phenotype （CD45RO＋CD27-CD57＋）.Conclusions The emergence of WT1-CTL with an effector-memory phenotype is associated with GVLE in CML patients after HSCT. This will pave the way for the WT1 vaccines to enhance GVLE after HSCT in CML.

Human cancergermlineantigen-specific cytotoxicTcell-what canwelearnfrompatient

In this review,we will highlight the importance of cancer germline antigen-specific cytotoxic CD8^(+) T lymphocytes(CTL)and the factors affecting antitumor CTL responses.In light of cancer immunotherapy,we will emphasis the need to further understand the features,characteristics,and actions of modulatory receptors of human cancer germline-specific CTLs,in order to determine the optimal conditions for antitumor CTL responses.

Immune cells in term and preterm labor

Labor resembles an inflammatory response that includes secretion of cytokines/chemokines by resident and infiltrating immune cells into reproductive tissues and the maternal/fetal interface. Untimely activation of these inflammatory pathways leads to preterm labor, which can result in preterm birth. Preterm birth is a major determinant of neonatal mortality and morbidity; therefore, the elucidation of the process of labor at a cellular and molecular level is essential for understanding the pathophysiology of preterm labor. Here, we summarize the role of innate and adaptive immune cells in the physiological or pathological activation of labor. We review published literature regarding the role of innate and adaptive immune cells in the cervix, myometrium, fetal membranes, decidua and the fetus in late pregnancy and labor at term and preterm. Accumulating evidence suggests that innate immune cells （neutrophils, macrophages and mast cells） mediate the process of labor by releasing pro-inflammatory factors such as cytokines, chemokines and matrix metalloproteinases. Adaptive immune cells （T-cell subsets and B cells） participate in the maintenance of fetomaternal tolerance during pregnancy, and an alteration in their function or abundance may lead to labor at term or preterm. Also, immune cells that bridge the innate and adaptive immune systems （natural killer T （NKT） cells and dendritic cells （DCs）） seem to participate in the pathophysiology of preterm labor. In conclusion, a balance between innate and adaptive immune cells is required in order to sustain pregnancy; an alteration of this balance will lead to labor at term or preterm.

Induced regulatory T cells suppress Tel cells through TGF-β signaling to ameliorate STZ-induced type 1 diabetes mellitus

Type 1 diabetes mellitus(T1D)is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells.In addition to well-established pathogenic effector T cells,regulatory T cells(Tregs)have also been shown to be defective in T1D.Thus,an increasing number of therapeutic approaches are being developed to target Tregs.However,the role and mechanisms of TGF-β-induced Tregs(iTregs)in T1D remain poorly understood.Here,using a streptozotocin(STZ)-induced preclinical T1D mouse model,we found that iTregs could ameliorate the development of T1D and preserve β cell function.The preventive effect was associated with the inhibition of type 1 cytotoxic T(Tel)cell function and rebalancing the Treg/Tc1 cell ratio in recipients.Furthermore,we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1.In addition to the preventive role,the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic(NOD)mouse models were tested,which revealed improved β cell function.Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.

A mouse model of vitiligo based on endogenous auto-reactive CD8+T cell targeting skin melanocyte

Vitiligo is the most common human skin depigmenting disorder.It is mediated by endogenous autoreactive CD8+T cells that destruct skin melanocytes.This disease has an estimated prevalence of 1%of the global population and cur-rently has no cure.Animal models are indispensable tools for understanding vitiligo pathogenesis and for developing new therapies.Here,we describe a vitiligo mouse model which recapitulates key clinical features of vitiligo,including epidermis depigmentation,CD8+T cell infiltration in skin,and melanocyte loss.To activate endogenous autoreactive cytotoxic CD8+T cells targeting melanocytes,this model relies on transient inoculation of B16F10 melanoma cells and depletion of CD4+regulatory T cells.At cellular level,epidermal CD8+T cell infiltration and melanocyte loss start as early as Day 19 after treatment.Visually apparent epidermis depigmentation occurs 2 months later.This protocol can efficiently induce vitiligo in any C57BL/6 background mouse strain,using only commercially available reagents.This enables researchers to carry out in-depth in vivo vitiligo studies utilizing mouse genetics tools,and provides a powerful platform for drug discovery.

IFN-γ^(+) cytotoxic CD4^(+) T lymphocytes are involved in thepathogenesis of colitis induced by IL-23 and the foodcolorant Red 40

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin(IL)-23.This immune response is mediated by CD4^(+)T cells,but mechanistic insights into how these CD4^(+)T cells trigger andperpetuate colitis have remained elusive.Here,using single-cell transcriptomic analysis,we found that several CD4^(+)T-cell subsetsare present in the intestines of colitic mice,including an interferon(IFN)-γ-producing subset.In vivo challenge of primed mice withRed 40 promoted rapid activation of CD4^(+)T cells and caused marked intestinal epithelial cell(IEC)apoptosis that was attenuated bydepletion of CD4^(+)cells and blockade of IFN-γ.Ex vivo experiments showed that intestinal CD4^(+)T cells from colitic mice directlypromoted apoptosis of IECs and intestinal enteroids.CD4^(+)T cell-mediated cytotoxicity was contact-dependent and required FasL,which promoted caspase-dependent cell death in target IECs.Genetic ablation of IFN-γconstrained IL-23-and Red 40-inducedcolitis development,and blockade of IFN-γinhibited epithelial cell death in vivo.These results advance the understanding of themechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ^(+)cytotoxic CD4^(+)T cells as a newpotential therapeutic target for colitis.

Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

The minigenes encoding Plasmodium falciparum CTL epitopesrestricted to human MHC class I molecular HLA-A2 and HLA-B51, which were both at high frequency among Chinese population, were constructed as mono-epitope CTL vaccines named pcDNA3.1/tr and pcDNA3.1/ sh. The minigenes of the two epitopes were then tandem linked to form a dimeric CTL epitope minigene recombinant vaccine. After DNA transfection, the epitope minigenes were expressed respectively in two human cell lines, each bearing one MHC class I molecule named CIR/HLA-A2.1 and K562/HLA-B51. The intracellular expression of the CTL epitope minigenes not only enhanced the stability of HLA-A2.1 and HLA-B51 molecules but also increased the assemblage of MHC class I molecules on cell surfaces, which testified the specific process and presentation of those endogenous expressed epitopes. For the cells transfected with the dimeric minigene encoding two tandem linked epitopes, the expression and presentation of each epitope were also detected on cell membranes that bore different MHC class I molecules. It meant that the adjacency of the two CTL epitopes did not interfere with the specific process and presentation of each epitope. Compared with the ordinary CTL studies that inoculated synthesized epitope peptides with peripheral blood cells, this work aimed to process the epitopes directly inside HLA class I allele specific human cells, and thus theoretically imitated the same procedure in vivo. It was also an economical way to predict the immunogenicity of CTL epitopes at an early stage especially in laboratories with limited financial resource.

Paradox-driven adventures in the development of cancer immunology and immunotherapy

After more than one hundred years of documented trials,immunotherapy has become a standard of care in the treatment of human cancer.Much of the knowledge that led to recent breakthroughs seems quite logical from today’s point of view.However,what we now cite as facts were originally considered paradoxes,meaning something contrary to expectations or perceived opinion at the time.In order to make gains in the field of immunotherapy,one had to be willing to confront ideas and concepts that seemed to contradict one another,and reconcile how each could be true.This is what led to new knowledge and advances.Here,we highlight some of these paradoxes and the milestone discoveries that followed,each one critical for our understanding of immune checkpoint pathways.By outlining some of the steps that we took and the challenges that we overcame,we hope to inspire and encourage future generations of researchers to confront the paradoxes that still permeate the field.