技术资料

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

The youngest peripheral T cells (recent thymic emigrants [RTEs]) are functionally distinct from naive T cells that have completed postthymic maturation. We assessed the RTE memory response and found that RTEs produced less granzyme B than their mature counterparts during infection but proliferated more and,therefore,generated equivalent target killing in vivo. Postinfection,RTE numbers contracted less dramatically than those of mature T cells,but RTEs were delayed in their transition to central memory,displaying impaired expression of CD62L,IL-2,Eomesodermin,and CXCR4,which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge,indicating that the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus,the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity,driving the efficacy of the RTE response to that of mature T cells. View Publication

The p110δ isoform of PI3K is known to play an important role in immunity,yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells,we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation,proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases,our study suggests caution in using these drugs in patients,as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Type I IFNs are key mediators of immune defense against viruses and bacteria. Type I IFNs were also previously implicated in protection against fungal infection,but their roles in antifungal immunity have not been thoroughly investigated. A recent study demonstrated that bacterial and fungal β-glucans stimulate IFN-β production by dendritic cells (DCs) following detection by the Dectin-1 receptor,but the effects of β-glucan-induced type I IFNs have not been defined. We investigated whether type I IFNs regulate CD8 T cell activation by fungal β-glucan particle-stimulated DCs. We demonstrate that β-glucan-stimulated DCs induce CD8 T cell proliferation,activation marker (CD44 and CD69) expression,and production of IFN-γ,IL-2,and granzyme B. Moreover,we show that type I IFNs support robust CD8 T cell activation (proliferation and IFN-γ and granzyme B production) by β-glucan-stimulated DCs in vitro and in vivo due to autocrine effects on the DCs. Specifically,type I IFNs promote Ag presentation on MHC I molecules,CD86 and CD40 expression,and the production of IL-12 p70,IL-2,IL-6,and TNF-α by β-glucan-stimulated DCs. We also demonstrate a role for autocrine type I IFN signaling in bacterial LPS-induced DC maturation,although,in the context of LPS stimulation,this mechanism is not so critical for CD8 T cell activation (promotes IFN-γ production but not proliferation or granzyme B production). This study provides insight into the mechanisms underlying CD8 T cell activation during infection,which may be useful in the rational design of vaccines directed against pathogens and tumors. View Publication

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However,in some instances,the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative,but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein,we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important,hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells,such as dendritic cells (DC). Indeed,a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore,compared to immature or mature DC,Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether,these findings indicate that allogeneic hAC inhibit,rather than trigger,immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting. View Publication

CD4(+) T cells develop distinct and often contrasting helper,regulatory,or cytotoxic activities. Typically a property of CD8(+) T cells,granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However,the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling,we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs,which distinguishes them from other CD4(+) T helper (Th) cells,including Th1 cells,and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study,and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions. View Publication