技术资料

Soybeans are a rich source of isoflavones that have been linked with anti-inflammatory processes and various health benefits. However,specific mechanisms whereby soy bioactives impact immune cell subsets are unclear. Isoflavones,such as genistein and daidzein,are metabolized by microbes to bioactive metabolites as O-desmethylangolensin (O-DMA) and equol,whose presence has been linked to health benefits. We examined how soy isoflavones and metabolites impact natural killer (NK) cell signaling and function. We observe no impact of isoflavones on viability of healthy donor peripheral blood mononuclear cells (PBMCs) or NK cells,even at high (25 µM) concentrations. However,pre-treatment of PBMCs with physiologically-relevant concentrations of genistein (p = 0.0023) and equol (p = 0.006) decreases interleukin (IL)-12/IL-18-induced interferon-gamma (IFN-gamma) production versus controls. Detailed cellular analyses indicate genistein and equol decrease IL-12/IL-18-induced IFN-gamma production by human NK cell subsets,but do not consistently alter cytotoxicity. At the level of signal transduction,genistein decreases IL-12/IL-18-induced total phosphorylated tyrosine,and phosphorylation MAPK pathway components. Further,genistein limits IL-12/IL-18-mediated upregulation of IL-18Ralpha expression on NK cells (p = 0.0109). Finally,in vivo studies revealed that C57BL/6 mice fed a soy-enriched diet produce less plasma IFN-gamma following administration of IL-12/IL-18 versus control-fed animals (p {\textless} 0.0001). This study provides insight into how dietary soy modulates NK cell functions. View Publication

Mucosal-associated invariant T (MAIT) cells exhibit different characteristics from those of TCRalpha7.2- conventional T cells. They play important roles in various inflammatory diseases,including rheumatoid arthritis and inflammatory bowel disease. MAIT cells express a single T cell receptor alpha chain,TCRalpha7.2 segment associated with Jalpha33 and CDR3 with fixed length,which recognizes bacteria-derived vitamin B metabolites. However,the characteristics of MAIT cells and TCRalpha7.2+ CD161- T cells have never been compared. Here,we performed RNA sequencing to compare the properties of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. Genome-wide transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells,and TCRalpha7.2+ CD161- T cells were compared and analyzed using causal network analysis. This is the first report comparing the transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. We also identified the predominant signaling pathways of MAIT cells,which differed from those of TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells,through a gene set enrichment test and upstream regulator analysis and identified the genes responsible for the characteristic MAIT cell phenotypes. Our study advances the complete understanding of MAIT biology. View Publication

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics,we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo,LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells,solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation,enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally,enhancement of STAT5 activation,as a result of LAG-3 deficiency,contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. View Publication

The persistence of latent HIV proviruses in long-lived CD4(+) T cells despite antiretroviral therapy (ART) is a major obstacle to viral eradication. Because current candidate latency-reversing agents (LRAs) induce HIV transcription,but fail to clear these cellular reservoirs,new approaches for killing these reactivated latent HIV reservoir cells are urgently needed. HIV latency depends upon the transcriptional quiescence of the integrated provirus and the circumvention of immune defense mechanisms. These defenses include cell-intrinsic innate responses that use pattern-recognition receptors (PRRs) to detect viral pathogens,and that subsequently induce apoptosis of the infected cell. Retinoic acid (RA)-inducible gene I (RIG-I,encoded by DDX58) forms one class of PRRs that mediates apoptosis and the elimination of infected cells after recognition of viral RNA. Here we show that acitretin,an RA derivative approved by the US Food and Drug Administration (FDA),enhances RIG-I signaling ex vivo,increases HIV transcription,and induces preferential apoptosis of HIV-infected cells. These effects are abrogated by DDX58 knockdown. Acitretin also decreases proviral DNA levels in CD4(+) T cells from HIV-positive subjects on suppressive ART,an effect that is amplified when combined with suberoylanilide hydroxamic acid (SAHA),a histone deacetylase inhibitor. Pharmacological enhancement of an innate cellular-defense network could provide a means by which to eliminate reactivated cells in the latent HIV reservoir. View Publication

Binding of ICAM-1 (intercellular adhesion molecule-1) to the β2-integrin LFA-1 (leukocyte function associated antigen-1) is known to induce crosstalk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies we have been able to study the requirement and mechanism of action for the crosstalk in considerable detail. LFA-1 activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain,which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand VCAM-1 (vascular cell adhesion molecule-1). The results show that with LFA-1 antibodies,we can either 1) activate LFA-1 and inhibit α4β1,2) inhibit both LFA-1 and α4β1,3) inhibit LFA-1 but not α4β1 or 4) not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications. View Publication

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

Type 1 diabetes (T1D) is characterized by a chronic,progressive autoimmune attack against pancreas-specific antigens,effecting the destruction of insulin-producing β-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies,as well as T cells specific for a single orthologous epitope of IL-2,are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice,the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset. In T1D patients,circulating IgG memory B cells specific for IL-2 or insulin are present at similar frequencies. Anti-IL-2 autoantibodies cloned from T1D patients demonstrate clonality,a high degree of somatic hypermutation and nanomolar affinities,indicating a germinal centre origin and underscoring the synergy between cognate autoreactive T and B cells leading to defective immune tolerance. View Publication

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here,we adapted this methodology to a high-throughput platform for the efficient,arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner,whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously,enabling studies of interactions among multiple host and viral factors. Finally,in an arrayed screen of 45 genes associated with HIV integrase,we identified several candidate dependency/restriction factors,demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication

BACKGROUND/AIM Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies,suggesting the contribution of cell-mediated immunity (CMI). However,PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end,the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates. METHODS An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983) and another strain (type-2 PRRSVVR2332) with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection. RESULTS At 72 days post infection,T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates,while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore,five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes,including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes,neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate. CONCLUSION These results demonstrated that T-lymphocytes recognizing antigenically and genetically diverse isolates were induced by infection with a type 2 PRRSV strain (SD23983). If these reponses have cytotoxic or other protective functions,they may help overcome the suboptimal heterologous protection conferred by conventional vaccines. View Publication

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses,host defense mechanisms,and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system,DCs are very rare in blood,accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore,alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity,affordability,high purity,and high yield of cells is imperative to consider. In the current study,two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability,proliferation,and phenotype were assessed using viability dyes,MTT assay,and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method,the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded textgreater 70% CD11c+ MDDCs. Therefore,our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs. View Publication

Autologous platelet rich plasma (PRP) is clinically used to induce repair of different tissues through the release of bioactive molecules. In some patients,the production of an efficient autologous PRP is unfeasible due to their compromised health. We developed an allogeneic PRP mismatched for AB0 and Rh antigens. To broadcast its clinical applications avoiding side effects the outcome of allogeneic PRP on immune response should be defined. Thus,we investigated whether PRP affected the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4. Indeed,these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a(+) dendritic cells and favored the expansion of phagocytic CD163(+) CD206(+) fibrocyte-like cells. These cells produced inteleukin-10 and prostaglandin-E2,but not interferon-γ,upon stimulation with lipopolysaccharides. Moreover,they promoted the expansion of regulatory CD4(+) CD25(+) FoxP3(+) T cells upon allostimulation or antigen specific priming. Finally,the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population possibly favoring wound healing. View Publication