BackgroundThe eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8+ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8+ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.Methodology/Principal FindingsHere we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8+ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8+ T cells is dependent on CD4+ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.Conclusions/SignificanceB cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.
The pleiotropic cytokine interleukin-6 (IL-6) interacts with the specific ligand binding subunit (IL-6R alpha) of the IL-6 receptor, and this complex associates with the signal-transducing subunit gp130 (IL-6R beta). Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a region (residues 43-55) within the human IL-6 protein, which is important for the interaction with gp130. Subdividing this region shows that mainly residues 50-55 of the human IL-6 are necessary for this interaction. Recently, another human IL-6 double mutant (Q159E and T162P) showed reduced affinity to gp130 but residual activity on the human myeloma cell line XG-1. Into this IL-6 mutant we introduced the murine residues 43-49 or 50-55 together with two point mutations, F170L and S176A, which had been reported to increase the affinity of IL-6 to the IL-6R alpha. The resulting IL-6 molecule, which contained the murine residues 50-55, was inactive on human myeloma cells and in addition completely inhibited wild type IL-6 activity on these cells. Such an antagonist may be used as a specific inhibitor of IL-6 activity in vivo.
IL-17-producing CD4 IntroductionIL-17 producing CD4 ϩ T helper (Th17) cells are important in immunity against extracellular pathogens, in particular at the mucosa, and are implicated in a variety of immune-mediated inflammatory disorders. Similar to other effector T cell types, Th17 cells develop from naive CD4 ϩ T cells in response to antigen presenting cell (APC)-derived signals. Whereas the cytokines IL-1, IL-6, IL-23, and TGF- have been identified to support Th17 differentiation in both mice and human, there is less consensus on the costimulatory signals that drive the development of human Th17 cells. [1][2][3][4] A basic principle of T-cell activation is that proper stimulation of naive T cells requires costimulation via CD28 for survival and expansion, which licenses their subsequent development into distinct effector Th subsets driven by appropriate signal 3 factors. Surprisingly, 2 recent studies reported that Th17 development is selectively inhibited by CD28 costimulation. 5,6 One of these studies suggested that human Th17 development is alternatively mediated via ligation of the inducible costimulator (ICOS). 6 Among the other molecules that may alternatively costimulate CD4 ϩ T cells are the lymphocyte receptor CD5 and CD6. 7,8 CD5 and CD6 are both group B members of the Scavenger Receptor Cystein-Rich domains superfamily (SRCR-SF). CD5 and CD6 share important structural and functional properties, and have probably arisen from a common ancestral gene. During interaction between the APC and T cell, CD5 and CD6 form part of the immunologic synapse. 9,10 This localization makes them well positioned to modulate the signals that follow antigen-specific T-cell receptor (TCR) ligation. For CD5, this concept has been most clearly demonstrated. CD5 plays a role in the late events of synapse-mediated signal transduction, whereby the large cytoplasmatic domain of CD5 can recruit both positive and negative regulators of T-cell signaling. Consequently, CD5 is a modulator of T-cell responses with both stimulatory and inhibitory activities. 8,11 In the thymus, CD5 plays a role in regulating TCR-mediated expansion and survival during T-cell ontogeny. 7,8,12 In peripheral resting CD4 ϩ T cells, CD5 costimulation results in proliferation levels as high as obtained with classic CD28-mediated costimulation. A possible role of CD5-mediated costimulation of T cells in Th17 cell development is favored by the finding that mice deficient in signaling between CD5 and CK-2, a prosurvival serine/threonine kinase that associates with CD5, show diminished populations of IL-17-expressing T cells in the central nervous system, in a model of experimental autoimmune encephalomyelitis. 13 In the present study, we show that costimulation via CD5 or CD6 is superior to classic CD28 costimulation in driving Th17 cell development from human naive CD4 ϩ T cells. CD5 induces high and consistent levels of IL-17, indicating the induction of stable terminal differentiation. Indeed, CD5 promotes elevated expression of various intracellular fact...
Stlmmal~The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)-transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R)ot complex with the signaltransducing "B" chain, gp130, but leave the binding of IL-6 to IL-6Rot intact. The resulting mutant protein (with substitutions of residues Gin160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL6Rot was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL-6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.
The pleiotropic cytokine interleukin-6 (IL-6) has been predicted to be a protein with four antiparallel alpha-helices. On target cells, IL-6 interacts with a specific ligand binding receptor subunit (IL-6R), and this complex associates with the signal-transducing subunit gp130. Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of chimeric human/murine IL-6 proteins has allowed us to define a region (residues 77-95, region 2c) within the human IL-6 protein that is important for IL-6R binding and a region (residues 50-55, region 2a2) that is important for IL-6R dependent gp130 interaction. Guided by sequence alignment and molecular modeling, we have constructed several IL-6 variants with point mutations in these regions and have tested them for receptor binding and signal initiation. Within region 2c, phenylalanine 78 was involved in receptor binding, whereas lysine 54 within region 2a2 participated in gp130 activation. Furthermore, some IL-6 variants with lysine 54 replacements could be used to construct muteins that retained receptor binding but failed to activate gp130. Such IL-6 muteins were efficient IL-6 receptor antagonists.
A model of the tertiary structure of human IL-6, derived from the crystal-structure of granulocyte-colony stimulating factor, reveals a 5th helical region in the loop between the first and second alpha-helix. To investigate the importance of this region for biological activity of IL-6, residues Glu-52, Ser-53, Ser-54, Lys-55, Glu-56, Leu-58, and Glu-60 were individually replaced by alanine. IL-6.Leu-58Ala displayed a 5-fold reduced biological activity on the IL-6 responsive human cell lines XG-1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL-6 receptor-alpha-chain-dependent binding to the IL-6 signal transducer, gp130.
interaction (respectively termed a-and 13-sites) have been Abstract We have previously shown that L58 in the putative 5th helical region of human interleuidn-6 (IL-6) is important for identified and are dispersed throughout the putative tertiary activation of the IL-6 signal transducer gpl30 Ide Hon et al.structure (reviewed in [9]). The a-site is spatially separated (1995) FEBS Lett. 369, 187-1911. To further explore the from the I~-sites (l~l-~a), and is comprised of residues in the importance of individual residues in this region for gpl30A-and D-helix and in the loop between helices A and B. The activation we have now combined Ala substitutions of residues 131-site comprises residues Q153-H165 at the end of the C-D E52, $53, $54, K55, E56, L58 and E60 with other substitutions loop and top of helix D, the 132-site residues K42-A57 coverin IL-6, known to affect gpl30 activation (QI60E and T163P).ing the loop created by the first cysteine bridge and part of the The combination mutant protein with L58A completely lost the putative 5th helical region in the A-B loop, and the I~a-site capacity to induce the proliferation of XG-I myeloma cells, and residues Y32, G36, S119 and V122, located in the A-and Ccould effectively antagonize wild type IL-6 activity on these cells, helix [8,10]. The a-and 133-site of IL-6 correspond to binding Moreover, the data suggest that besides L58, $54 particularly, but also E52, $53, K55 and E56 contribute to gpl30 activation, sites 1 and 2 of human growth hormone respectively [10,11]. In the hexameric IL-6-receptor complex, regions 131 and 132 are
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