技术资料

CD8(+) T cells have a central role in antitumour immunity,but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1,a key cholesterol esterification enzyme,led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells,which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe,which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile,to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1,an established target for atherosclerosis,is therefore also a potential target for cancer immunotherapy. View Publication

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases,which sulfonate proteins,glycoproteins,glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs),which sulfonate various small molecules including hormones,neurotransmitters,and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions,cell signaling,and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism,namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions,MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels,and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs,and through siRNA knockdown experiments,we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules,raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics. View Publication

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy,leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this,short-boosted Ag-specific CD8 T cells continue to contract gradually over time,which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant,functional Ag-specific CD8 T cells that are poised for immediate protection; however,this is at the expense of forming stable long-term memory. View Publication

Current combination antiretroviral therapies (cART) efficiently suppress HIV-1 reproduction in humans,but the virus persists as integrated proviral reservoirs in small numbers of cells. To generate an antiviral agent capable of eradicating the provirus from infected cells,we employed 145 cycles of substrate-linked directed evolution to evolve a recombinase (Brec1) that site-specifically recognizes a 34-bp sequence present in the long terminal repeats (LTRs) of the majority of the clinically relevant HIV-1 strains and subtypes. Brec1 efficiently,precisely and safely removes the integrated provirus from infected cells and is efficacious on clinical HIV-1 isolates in vitro and in vivo,including in mice humanized with patient-derived cells. Our data suggest that Brec1 has potential for clinical application as a curative HIV-1 therapy. View Publication

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function,the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore,under this stimulatory condition,CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10,which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation,which correlates with lower CD86 expression compared to patients with chronic rejection. Hence,the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production. View Publication

BACKGROUND Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore,the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence,we focus on optimizing hESCs for clinic application through gene modification. RESULTS Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-$\$ expression of HLA II. CONCLUSION We generated HLA II defected hESCs via deleting CIITA,a master regulator of constitutive and IFN-$\$ expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g.,T cells and DCs) and escape the attack of receptors' CD4(+) T cells,which are the main effector cells of cellular immunity in allograft. View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments,their presence is related to a poor prognosis,and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study,we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs,restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70,an increase in the Foxp3 methylation status and,ultimately,the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions,suggesting its possible use in the design of anticancer vaccines. View Publication

BACKGROUND: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK),activating transcription factor 6 (ATF6),and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways,leading to less protein production and activating genes that encode important proteins in folding processes,including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However,HIV infection's effect on the UPR has scarcely been investigated. METHODS: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered. RESULTS: In in vitro and in vivo analyses,our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile. CONCLUSIONS: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus,two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented. View Publication

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses,the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study,we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression,in this context CTLA-4 blocking Abs had no impact on the response. In contrast,in settings where CTLA-4(+) cells were present as regulators� View Publication

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However,limited information is available regarding the immunologic features of iPSCs. In this study,expression of MHC and T cell co-stimulatory molecules in hiPSCs,and the effects on activation,proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation,hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ,TNF-α and IL-17,hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10,and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity,which may result from their induction of IL-10-secreting Treg. View Publication