Induction of peripheral tolerance in dual TCR T cells: an evidence for non-dominant signaling by one TCR.

Recently, the existence of T cells with dual T cell receptor (TCR) in the immune system is generally accepted, while it has been controversial whether signals through one TCR would affect the functions of the other. In this study T cells expressing two different TCR were obtained from cross-hybrids of LCMV and AND TCR transgenic mice specific for the gp33 and peptide fragment of PCC (fPCC), respectively. Peptide stimulation demonstrated that the dual TCR T cells functioned independently in an antigen-specific manner. To examine whether the tolerance targeted for the one TCR affects the responsiveness of the other, the cross-hybrids were treated with gp33. Although T cells from F1 mice were rendered anergenic to gp33, no functional changes to fPCC were observed in terms of cellular proliferation and IL-2 secretion, suggesting that the dual TCR T cells remained reactive to fPCC. We therefore propose that signaling through the TCR is receptor-specific and 'negative dominance' of one TCR by tolerance induction is not applicable in this dual TCR system.

Stereoselective synthesis of 3-hydroxymethyl-D-cyclopentenone, the versatile intermediate for the synthesis of carbocyclic nucleosides.

The preparative and stereoselective synthesis (45- 50% overall yields, >50 g scale) of the key carbasugars 7a-d was achieved from D-ribose via stereoselective Grignard reaction and oxidative rearrangement as key reactions.

Tetramethoxy hydroxyflavone p7F downregulates inflammatory mediators via the inhibition of nuclear factor kappaB.

Artemisia has been traditionally used in Korean herbal medicine to clear damp heat and to treat uteritis and jaundice. Flavonoids isolated from Artemisia are also known to possess anti-inflammatory activities. In this study, 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (p7F) was isolated from Artemisia absinthium. We examined in vitro and in vivo regulatory functions of p7F on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) as well as the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and collagen-induced arthritis. p7F inhibited the expression or production of proinflammatory mediators such as COX-2/PGE(2) and iNOS/NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. p7F also suppressed the serum level of TNF-alpha in mice treated with collagen and inhibited nuclear factor-kappaB (NF-kappaB) activation as well as NF-kappaB promoter activity in RAW 264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in hydrogen peroxide-stimulated RAW 264.7 cells. p7F has antioxidant activity and inhibits NF-kappaB activation. Taken together, these results suggest that p7F can be clinically applied to the treatment of inflammatory diseases.

Response of primed human PBMC to synthetic peptides derived from hepatitis B virus envelope proteins: a search for promiscuous epitopes.

This investigation was aimed at identifying effective T helper cell epitopes to the hepatitis B virus in humans. A panel of synthetic peptides that represent the hepatitis B virus whole envelope proteins was examined for their capability to stimulate peripheral blood mononuclear cells from human subjects infected with hepatitis B virus naturally. In addition, a large number of subjects were examined and their human leukocyte antigen (HLA) class II allele types were identified to determine whether the helper T cell epitope is specific for a particular HLA allele or 'promiscuous'. The peptides of the amino acid residues 52-67, 110-125, 190-205, and 228-243 appeared to be immunogenic, and particularly, the 52-67 residue was the most promiscuous epitope peptide. These results would contribute to the better understanding of the helper T cell responses to the hepatitis B virus and provide a useful way in designing epitope-based vaccines and future therapeutic strategies.

Modification of concanavalin A-dependent proliferation by phosphatidylcholines isolated from deer antler, Cervus elaphus.

OBJECTIVE: The immunomodulatory effect of deer antler, which is used as traditional medicine, has been known, but the active component of antlers from Cervus elaphus has not been identified. In this study, we identified the immunomodulator from C. elaphus and examined its biological activities on the immune system. METHODS: To identify an immunomodulator, we used bioassay-guided fractionation after silica gel column chromatography. Structural analysis was performed with one- and two-dimensional nuclear magnetic resonance techniques and tandem mass spectrometry coupled with fast atom bombardment. RESULTS: The subfraction, phosphatidylcholines, isolated 70% ethanol extract of C. elaphus induced the proliferation of spleen cells in synergy with concanavalin A. According to the structural analysis, phosphatidylcholines were classified as a family (1,2-alkyl-sn-glycerol-3-phosphocholines) containing arachidonyl (C20:4), stearoyl (C18:0), oleoyl (C18:1), linoleoyl (C18:2), palmitoyl (C16:0), and myristoyl (C14:0) chains in their fatty acyl chains. Because the unsaturated fatty acids showed an inhibitory effect on the immune system, dialkyl phosphatidylcholines with different chain lengths from C10:0 to C20:0 that stimulate the proliferation of spleen cells were examined extensively. Among other saturated phosphatidylcholines used, dimyristoyl phosphatidylcholine (C14:0) induced the proliferation of spleen cells more efficiently, whereas dimyristoleoyl phosphatidylcholine (C14:1) effected little change in the proliferation of spleen cells. CONCLUSIONS: These data collectively suggest that phosphatidylcholines with saturated fatty acyl chains are immunostimulating factors. They may modify the proliferation of known mitogens. Further, chain length and saturation of the fatty acids may play important roles in the proliferation of spleen cells.

Synthesis of halogenated 9-(dihydroxycyclopent-4'-enyl) adenines and their inhibitory activities against S-adenosylhomocysteine hydrolase.

Novel halovinyl analogues of neplanocin A without 4'-hydroxymethyl group were easily synthesized starting from D-ribose via cyclopentenone 5 as a key intermediate and their inhibitory activity against SAH hydrolase was assayed.

Antigen dose governs the shaping of CTL repertoires in vitro and in vivo.

Although it is well established that antigen dose plays an important role in determining the quality of T cells induced in vitro, it has not well been determined whether antigen dose also affects T cell repertoires induced in vivo. This study demonstrates that variation of antigen doses in vivo as well as in vitro induce structurally and functionally different T cell repertoires. CTLs generated in vitro with a low antigen dose showed much higher T cell responsiveness than CTLs generated with a high antigen dose, and the two CTL populations employed different TCR Vbeta chains. This is most likely due to repertoire selection based on TCR affinity. The secondary in vivo responses with a high or low dose of antigen following the primary response raised with the same dose resulted in a reversed dominance pattern of two particular TCR Vbeta phenotypes. TCR affinity of these two T cell populations appeared different, suggesting avidity selection based on antigen availability. Indeed, they required a distinct level of antigen for maximal cytolytic function, implying a different functional avidity. These results suggest that antigen-specific T cell repertoire is substantially affected by the antigen dose employed in vivo as well as in vitro.

Diversity and complexity of CD8+ T cell responses against a single epitope of adenovirus E1B.

This study describes the characteristics of the immune responses against adenovirus in C57BL/6 mice. CTL responses could be induced against E1Bp of adenovirus type 5, when whole viruses were immunized. A panel of E1Bp-specific CTL clones showed a wide range of T cell avidity. Recognition of the E1Bp peptide and a panel of variant peptides containing a single alanine substitution by CTL clones revealed that the fine specificity of the CTL response was quite diverse, rather than being limited to a certain clonal preference. Moreover, the variant peptides with a substitution at the TCR contact residue had antagonistic properties to some of the CTL clones, while being agonistic to others, reflecting the extensive diversity of the T cells. These results imply that the functional diversity of T cells to even a single epitope should be considered in manipulating immunity to viruses and in developing adoptive immunotherapy for immunocompromised individuals.

Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells.

The hepatitis B virus (HBV) transgenic mouse (Tg) 50-4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2-month-old and 5-month-old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5-month-old Tg, but not in the 2-month-old Tg. Furthermore, CD4+ T cells from 2-month-old mice, but not those from 5-month-old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro.

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.

A dipalmitoyl peptide that binds SH3 domain, disturbs intracellular signal transduction, and inhibits tumor growth in vivo.

The Src homology 3 (SH3) domain plays a crucial role in protein-protein interactions during intracellular signal transduction. Blocking the SH3-mediated protein binding may inhibit the corresponding signal transduction, and thus, block the cellular functions. In this study, a peptide that specifically binds to SH3 domain could be introduced into the intracellular region when the peptides were conjugated with dipalmitic acid and appeared to disturb intracellular signaling. The dipalmitoyl peptide appeared to inhibit the phosphorylation of ZAP-70, Lck, and T-cell antigen receptor zeta in Jurkat. Mobilization of the intracellular free calcium induced by anti-CD3 antibody was reduced after treatment with the dipalmitoyl peptide. It was also observed that the dipalmitoyl peptide inhibited cancer cell growth both in vitro and in vivo. These results suggest that the dipalmitoyl peptide that presumably disturbs SH3-mediated signal transduction may have a potent anti-proliferative activity, which would be useful as a potential anti-tumor agent.