技术资料

Adaptive immunity to self-antigens causes autoimmune disorders,such as multiple sclerosis,psoriasis and type 1 diabetes; paradoxically,T- and B-cell responses to amyloid-$\$(A$\$) reduce Alzheimer's disease (AD)-associated pathology and cognitive impairment in mouse models of the disease. The manipulation of adaptive immunity has been a promising therapeutic approach for the treatment of AD,although vaccine and anti-A$\$ approaches have proven difficult in patients,thus far. CD4(+) T cells have a central role in regulating adaptive immune responses to antigens,and A$\$-specific CD4(+) T cells have been shown to reduce AD pathology in mouse models. As these cells may facilitate endogenous mechanisms that counter AD,an evaluation of their abundance before and during AD could provide important insights. A$\$-CD4see is a new assay developed to quantify A$\$-specific CD4(+) T cells in human blood,using dendritic cells derived from human pluripotent stem cells. In tests of textgreater50 human subjects A$\$-CD4see showed an age-dependent decline of A$\$-specific CD4(+) T cells,which occurs earlier in women than men. In aggregate,men showed a 50% decline in these cells by the age of 70 years,but women reached the same level before the age of 60 years. Notably,women who carried the AD risk marker apolipoproteinE-ɛ4 (ApoE4) showed the earliest decline,with a precipitous drop between 45 and 52 years,when menopause typically begins. A$\$-CD4see requires a standard blood draw and provides a minimally invasive approach for assessing changes in A$\$ that may reveal AD-related changes in physiology by a decade. Furthermore,CD4see probes can be modified to target any peptide,providing a powerful new tool to isolate antigen-specific CD4(+) T cells from human subjects. View Publication

NK cells are innate immune cells known for their cytolytic activities toward tumors and infections. They are capable of expressing diverse killer Ig-like receptors (KIRs),and KIRs are implicated in susceptibility to Crohn's disease (CD),a chronic intestinal inflammatory disease. However,the cellular mechanism of this genetic contribution is unknown. In this study,we show that the licensing" of NK cells� View Publication

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs),thus preventing the development of durable antitumor immunity. Therefore,the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however,those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast,we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+),CD8(+),Tregs),B cells,and NK cells,which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells,B cells,and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4,TLR5L:flagellin,TLR4L:LPS,and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation,but not B cell proliferation. Besides CL097,TLR2L:PGN,CL075,and TLR9L:CpG-A,CpG-B,and CpG-C) were strong activators of NK cells. Importantly,we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation,2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg),and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients. 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

HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms,the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells,monocytes,and neutrophils as effector cells,to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio,NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets,but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination. View Publication

The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic,it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study,we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary,the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies. View Publication

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However,the activation requirements,the temporal aspects of the suppressive activity,and mode of action of human Tregs are subjects of controversy. In this study,we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously,whereas in the remaining donors,anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore,anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs,but could be fully obliterated by trypsin treatment,indicating that a cell surface protein is directly involved. By add-back of active,fixed Tregs at different time points after activation of responding T cells,the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly,we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation,indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells. View Publication

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

HIV elite controllers (EC) are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment. Adaptive immunity and host genetic factors,although implicated,do not entirely explain this phenomenon. On the other hand,plasmacytoid dendritic cells (pDCs) are the principal type I interferon (IFN) producers in response to viral infection,and it is unknown whether pDCs are involved in the control of HIV infection in EC. In our study,we analyzed peripheral pDC levels and IFN-α production by peripheral blood mononuclear cells (PBMCs) in EC compared to other groups of HIV-infected patients,the ability of pDCs to reduce HIV production in vitro,and the mechanisms potentially involved. We showed preserved pDC counts and IFN-α production in EC. We also observed a higher capacity of pDCs from EC to reduce HIV production and to induce T cell apoptosis,whereas pDCs from viremic patients barely responded without previous Toll-like receptor 9 (TLR-9) stimulus. The preserved functionality of pDCs from EC to reduce viral production may be one of the mechanisms involved in the control of HIV viremia in these subjects. These results demonstrate the importance of innate immunity in HIV pathogenesis,and an understanding of pDC mechanisms would be helpful for the design of new therapies. View Publication

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many,but not all,HICs may contribute to long-lasting viral control. However,other earlier defense mechanisms may be involved. Here,we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge,monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1),which has been involved in the control of HIV-1 replication,than cells from control subjects. However,HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts,leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula,suggesting the action of a saturable mechanism. Importantly,cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs. View Publication

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice,N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity,cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis,spontaneous inflammatory demyelination and neurodegeneration,the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3),including genetic variants in interleukin-7 receptor-α (IL7RA*C),interleukin-2 receptor-α (IL2RA*T),MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3),optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway,namely N-glycosylation. View Publication