Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and ...activated states, mouse peripheral CD4(+)CD25(+) T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4(+)CD25(-) T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1beta and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2(-/-) mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.
Granulysin (GNLY), an antimicrobial protein present in the granules of human cytotoxic T lymphocytes and natural killer (NK) cells, is produced as an intact 15-kDa form that is cleaved to yield a ...9-kDa form. Alarmins are endogenous mediators that can induce recruitment and activation of antigen-presenting cells (APCs) and consequently promote the generation of immune response. We hypothesized that GNLY might function as an alarmin. Here, we report that both 9- and 15-kDa forms of recombinant GNLY-induced in vitro chemotaxis and activation of both human and mouse dendritic cells (DCs), recruited inflammatory leucocytes, including APCs in mice, and promoted antigen-specific immune responses upon coadministration with an antigen. GNLY-induced APC recruitment and activation required the presence of Toll-like receptor 4. The observed activity of recombinant GNLY was not due to endotoxin contamination. The capability of the supernatant of GNLY-expressing HuT78 cells to activate DC was blocked by anti-GNLY antibodies. Finally we present evidence that supernatants of degranulated human NK92 or primary NK cells also activated DCs in a GNLY- and Toll-like receptor 4–dependent manner, indicating the physiologic relevance of our findings. Thus, GNLY is the first identified lymphocyte-derived alarmin capable of promoting APC recruitment, activation, and antigen-specific immune response.
Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune ...modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into α and β families. Structures of several β-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys&4, and Cys3-Cys6. We found that human β-defensin 3 (hBD3), a recently described member of the growing β family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native β pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by α-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human β-defensins and the design of novel peptide antibiotics.
Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human ...cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.
Immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) promote tumor immune evasion and thus targeting of Tregs has become an strategy in cancer immunotherapy. Tumor necrosis factor receptor 2 ...(TNFR2) is highly expressed and important for the immunosuppressive function of Tregs in humans and mice. Thus, the benefit of targeting TNFR2 in cancer immunotherapy merits more investigation. A previous report identified a new murine monoclonal anti-TNFR2 antibody (designated TY101), which showed therapeutic efficacy in murine cancer models, but its mechanism of action was less understood. In this study, the capacity of a combination of immunostimulants to enhance the effect of this inhibitor of Tregs was investigated. We examined the efficacy of TY101 as an anti-tumor immune reagent combined with HMGN1 (N1, a dendritic cell activating TLR4 agonist) and R848 (a synthetic TLR7/8 agonist). This immunotherapeutic combination exerted synergistic antitumor effects as compared with any single treatment. The antitumor response was mainly mediated by the depletion of Tregs and stimulation of cytotoxic CD8 T cell activation. The result also suggested that the effect of TY101 was similar to that of anti-PD-L1 when used in combination with these immunostimulants. Therefore, we propose that treatment strategies of antagonizing TNFR2 on Tregs would behave as potent checkpoint inhibitors and can potentially be utilized to develop a novel antitumor immunotherapy.
Previous studies have demonstrated that β‐defensins exhibit chemotactic activity by sharing the chemokine receptor CCR6 with the CC chemokine ligand CCL20/macrophage‐inflammatory protein‐3α (MIP‐3α). ...Structural analysis of CCL20/MIP‐3α revealed that most of the positively charged residues are concentrated at one area of its topological surface, a characteristic considered to be important for the antimicrobial activity of defensins. Here, we report that similar to defensins, CCL20/MIP‐3α has antimicrobial effects on Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Streptococcus pyogenes, Enterococcus faecium, Staphylococcus aureus, and Candida albicans. Additionally, by screening a total of 30 human chemokines, we have identified an additional 17 human chemokines, which exhibit antimicrobial activity in vitro. Collectively, about two‐thirds of the chemokines investigated so far has the capacity to kill microorganisms in vitro, suggesting that antimicrobial activity may be another host‐defense function for certain chemokines. Comparison of the structural characteristics between antimicrobial and nonantimicrobial chemokines suggests that topological formation of a large, positively charged electrostatic patch on the surface of the molecule is likely to be a common structural feature of antimicrobial chemokines.
Dendritic cells play a pivotal role in host immune defense, such as elimination of foreign pathogen and inhibition of tumorigenesis. In this paper, we report that Gd@C82(OH)22 n could induce ...phenotypic maturation of dendritic cells by stimulating DC production of cytokines including IL-12p70, upregulating DC co-stimulatory (CD80, CD83, and CD86) and MHC (HLA-A,B,C and HLA-DR) molecules, and switching DCs from a CCL5-responsive to a CCL19-responsive phenotype. We found that Gd@C82(OH)22 n can induce dendritic cells to become functionally mature as illustrated by their capacity to activate allogeneic T cells. Mice immunized with ovalbumin in the presence of Gd@C82(OH)22 n exhibit enhanced ovalbumin-specific Th1-polarized immune response as evidenced by the predominantly increased production of IFNγ, IL-1β, and IL-2. The Gd@C82(OH)22 n nanoparticle is a potent activator of dendritic cells and Th1 immune responses. These new findings also provide a rational understanding of the potent anticancer activities of Gd@C82(OH)22 n nanoparticles reported previously.
Previously, we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3⁺ cells present in ...CD25high, CD25low and even CD25⁻ subsets of CD4⁺ cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4⁺CD25⁺ Treg included all of the FOXP3⁺ cells present in the CD4⁺CD25high subset as well as a substantial proportion of the FOXP3⁺ cells present in the CD4⁺CD25low subset. Flow cytometric analysis of PB identified five-fold more Treg, determined by FOXP3 expression, in the CD4⁺CD25⁺TNFR2⁺ subset than in the CD4⁺CD25high subset. In addition, similar levels of FOXP3⁺ cells were identified in both the CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁺CD127low/⁻ subsets. Furthermore, the CD4⁺CD25⁺TNFR2⁺ subset expressed high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4⁺CD25⁺TNFR2⁺ cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T-responder cells. In contrast, CD4⁺CD25⁺TNFR2⁻ and CD4⁺CD25⁻TNFR2⁺ T cells did not show inhibitory activity. As some non-Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.
Defensins and cathelicidins are the two major families of mammalian anti‐microbial proteins. They contribute to host, innate, anti‐microbial defense by disrupting the integrity of the bacterial cell ...membrane. However, several members of the mammalian anti‐microbial proteins including defensins and cathelicidins have been shown recently to have chemotactic effects on host cells. Human neutrophil α‐defensins are chemotactic for resting, naïve CD45RA/CD4 T cells, CD8 T cells, and immature dendritic cells. Human β‐defensins are also chemotactic for immature dendritic cells but induce the migration of memory CD45RO/CD4 T cells. In contrast, cathelicidin/LL‐37 is chemotactic for neutrophils, monocytes, and T cells but not for dendritic cells. Thus, these anti‐microbial peptides have distinct, host‐target cell spectra. The chemotactic activities of human β‐defensins and cathelicidin/LL‐37 are mediated by human CC chemokine receptor 6 and formyl peptide receptor‐like 1, respectively. The capacities of defensins and cathelicidins to mobilize various types of phagocytic leukocytes, immature dendritic cells, and lymphocytes, together with their other effects such as stimulating IL‐8 production and mast cell degranulation, provide evidence for their participation in alerting, mobilizing, and amplifying innate and adaptive anti‐microbial immunity of the host.
Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not ...trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.
Display omitted
•N4-acetylcytidine (ac4C) incorporated into synthetic eGFP messenger RNAs (mRNA)•Synthetic ac4C mRNAs produce comparable eGFP as modified/unmodified mRNAs•Synthetic ac4C mRNAs are less inflammatory in immune cells than cytidine mRNAs•Cytidine acetylation alters the protein interaction landscape of synthetic mRNAs
Messenger RNA (mRNA) therapeutic platforms benefit from nucleotide components that enable protein translation while evading immune activation. Here, Nance et al. report the exploration of N4-acetylcytidine (ac4C) as a medicinal chemistry element in a synthetic mRNA.
PDF
![[Gd@C82(OH)22] n Nanopartic...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/1936-0851_1936-086X/small "[Gd@C82(OH)22] n Nanoparticles Induce Dendritic Cell Maturation and Activate Th1 Immune Responses")
