Abstract
The nature, function and pathways used to produce peptides for presentation by non-classical MHC class I molecules is poorly understood. Here we show that cells from mice lacking the ER ...aminopeptidase associated with antigen processing (ERAAP) present an immunologically distinct peptide-MHC class I repertoire. Wild-type mice immunized with ERAAP-deficient cells mount robust CD8 T cell responses specific for peptides presented by classical as well as non-classical MHC I molecules. These CD8 T cells produce pro-inflammatory cytokines and effectively kill ERAAP-deficient target cells in vitro as well as in vivo. Further, peptides presented by non-classical MHC class I molecules are expressed in a tissue-specific manner and elicit CD8 T cell responses both by direct and cross-priming. Thus, ERAAP trims the final peptides presented by classical as well as non-classical MHC class I molecules, and may be involved in trimming peptides for cross-presentation.
This research was supported by grants from the NIH (AI060040) to N.S.; N.A.N. is supported by the Irvington Institute Fellowship Program of the Cancer Research Institute.
Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient ...presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3–deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3–deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II–reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.
Abstract
Invariant (i)NKT cells influence the response to viral infections, although the mechanisms are poorly defined. Here we show that these innate-like lymphocytes secrete IFN-γ upon culture with ...CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but does not require CD1d expression. iNKT cells also produce IFN-γ in response to mouse CMV (MCMV) infection. Their mechanism of MCMV detection is quite similar to that of CpG, requiring bothTLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an antigen-independent manner, suggesting they may play a broad role in anti-pathogen defenses despite their limited TCR repertoire.
Invariant (
i
)NKT cells influence the response to viral infections, although the mechanisms are poorly defined. Here we show that these innate-like lymphocytes secrete IFN-γ upon culture with CpG ...oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but does not require CD1d expression.
i
NKT cells also produce IFN-γ in response to mouse CMV (MCMV) infection. Their mechanism of MCMV detection is quite similar to that of CpG, requiring bothTLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently,
i
NKT cells have the ability to respond to a variety of microbes, including viruses, in an antigen-independent manner, suggesting they may play a broad role in anti-pathogen defenses despite their limited TCR repertoire.
Certain glycolipid antigens (Ags) for Vα14
i
NKT cells can direct the overall cytokine balance of the immune response. T
H
2-biasing OCH has a lower TCR avidity than the most potent agonist known, ...αGalCer. Although the CD1d-exposed portions of OCH and αGalCer are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. T
H
1 biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFNγ, accounting for its promotion of T
H
1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer
in vivo
. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating
i
NKT cell responses.
Certain glycolipid Ags for Valpha14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, ...alpha-galactosylceramide. Although the CD1d-exposed portions of OCH and alpha-galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-gamma, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.
Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient ...presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3-deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3-deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II-reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.
Carbapenem resistant Enterobacteriaceae (CRE) infection has been widely treated with last resort antibiotics like colistin. Resistance to colistin has further jeopardized the situation. We have ...previously reported a combination of MarR inhibitor – salicylate (Sal) and an efflux pump inhibitor (BC1) that successfully restored colistin (Col) sensitivity in multidrug and colistin resistant clinical isolate of E. coli U3790. Since synthetic compounds usually fail during drug development initiatives, we attempted to replace synthetic efflux pump inhibitor (BC1) with plant metabolite as efflux pump inhibitor to restore colistin sensitivity in CRE. Screening 13 plant metabolites, we narrowed on curcumin (CUR) to effectively inhibit efflux in both colistin resistant E. coli U3790 and K. pneumoniae BC936. Combination of Col + CUR showed a remarkable reversal in colistin MIC by 128 fold and 32 fold in E. coli U3790 and K. pneumoniae BC936 respectively. Studies with knock out mutant strains of AcrAB-TolC pump components show that curcumin's efflux inhibition is partly mediated by acrB. Thus, curcumin reduced colistin MIC well below the CLSI breakpoint (<2 μg/ml). Curcumin also exhibited synergy with colistin against most of the clinical isolates of Enterobacteriaceae tested. Efficiency of Col + Sal + CUR was evident in time kill curve analysis, which displayed a 6 log and a 4 log decline in CFU/ml by 24 h in U3790 and BC936 strains respectively. In vivo intramuscular fish infection studies showed that the triad combination reduced the bacterial bioburden of E. coli U3790 by 2.6 log and that of K. pneumoniae BC936 by 1.6 log. Hence, our study shows the efficacy of inhibiting MarR by salicylate and inhibiting efflux pump with curcumin restores colistin sensitivity in colistin resistant Enterobacteriaceae in vitro and in vivo.
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•Curcumin (CUR) enhanced ethidium bromide accumulation in colistin (Col) resistant Enterobacteriaceae.•Curcumin synergised with colistin and reduced colistin's MIC by 128 fold and 32 fold in resistant Enterobacteriaceae.•Curcumin along with MarR inhibitor Salicylate (Sal) augmented the bactericidal efficacy of colistin in vitro and in vivo.•Curcumin would enhance therapeutic utility of colistin and salicylate which are already in therapeutic use.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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