Synthesis and biological activity of ester and ether analogues of α-galactosylceramide (KRN7000)

α-Galactosylceramide (αGalCer, KRN7000) has been identified as a modulator of immunological processes through its capacity to bind iNKT cells mediated by CD1d molecules. Some analogues in while the amide group in αGalCer is replaced with ester or ether groups were synthesized from d-arabinitol or l-ribose to evaluate their ability to activate iNKT cells. Ester analogues 30a, 31a, and 61 showed activity for IFNγ and IL-4 production of iNKT cells, while ether (31b) and 4-methoxy ester (76) analogues of α-galactosylceramide were not active for iNKT cells.     

Evidence for a positive role of PtdIns5P in T-cell signal transduction pathways

Phosphatidylinositol 5-phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4, 5)P₂ 4-phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a new PtdIns5P interacting domain blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis.     

Serine Residues in the Cytosolic Tail of the T-cell Antigen Receptor α-Chain Mediate Ubiquitination and Endoplasmic Reticulum-associated Degradation of the Unassembled Protein

The T-cell antigen receptor (TCR) α-chain (TCRα) is a type I integral membrane protein that becomes ubiquitinated and targeted to the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway when it fails to assemble into the heteromeric TCR complex. Remarkably, TCRα has a cytosolic tail of only five amino acid residues (i.e. RLWSS), none of which is the conventional ubiquitin acceptor, lysine. Herein we report that substitution of two conserved serine residues in the cytosolic tail of TCRα to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e. cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCRα for ERAD. We also found that this ubiquitination was mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicate that serine-dependent, HRD1-mediated ubiquitination targets TCRα to the ERAD pathway.     

Molecular characterization,tissue distribution and expression analysis of Interleukin-12 receptor β2 chain in sheep

Ovine β2 subunit of the interleukin (IL)-12 receptor (IL-12Rβ2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rβ2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rβ2. The ovine IL-12 Rβ2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rβ2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rβ2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rβ2 mRNA expression was detected at all the detected tissues with the exception of thymus.     

An increased number of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis

Abstract There is evidence that γ/δ TCR + T cells are specialized in recognizing different antigens, but their immunologic role as a second TCR is still unclear. The aim of this study was to investigate the percentage and absolute numbers of circulating γ/δ TCR + T cells in patients with chronic viral hepatitis (CVH) and to compare with HBsAg⁺, HCV healthy carriers and healthy subjects. Forty nine patients with CVH-24 with chronic active (CAH) and 25 with chronic persistent hepatitis (CPH)-, 21 HBsAg⁺, 20 HCV asymptomatic carriers and 20 healthy subjects were enrolled in the study. Lymphocyte subsets were determined after incubation with monoclonal antibodies to T total (CD5) and T γ/δ cells (γ/δ-1) using immunofluorescence microscopy. An increased number of circulating γ/δ TCR + T cells was found in patients with CVH in comparison with asymptomatic carriers and normal controls: this increase was more profound in patients with CAH, compared to CPH patients. These results indicate a correlation between circulating γ/δ TCR + T cells in CVH patients and activity and chronicity of the disease.     

Cytotoxic effector T cells elicited by the killed tumor vaccine differ significantly from the effectors generated during active growth of a murine B-cell lymphoma

Active specific immunotherapy of neoplastic diseases is an elusive goal. Using a murine B lymphoma 2C3, we showed that vaccination with the killed tumor cells effectively induces protective immunity and a cytotoxic T cell (CTL) response. Similar protection, however, is rarely observed in mice bearing live tumor cells. These animals usually succumb to the progressively growing tumor. In this study, we inquired whether the splenic CTL induced during tumor progression in mice differ from those evoked by the killed tumor cells. Here we demonstrate that the CTL generated following vaccination are significantly different from those induced in the tumor-bearing hosts. Adding to the complexity, the CTL from the early tumor bearers also differ significantly from those induced at the late stages. These differences are based on their cytotoxic activity, MHC allele specificity, mitogen responsiveness, cytokine secretion profile and T cell receptor Vβ gene expression. The results clearly indicate that passive immunization with killed tumor is most effective, possibly because the CTL induced are not subject to the same regulatory pressure as those induced during active tumor growth. This decreasing effectiveness of CTL could be due to greater variability in antigenic stimulus, less involvement of innate immunity, changes in cytokine milieu and/or costimulatory factors. Received: 5 February 1999 / Accepted: 22 June 1999     

TCR基因免疫治疗中优化转TCR基因配对的研究进展

TCR基因修饰T细胞的过继性免疫治疗是指将识别肿瘤抗原的特异性TCR基因转导至外周血T细胞,经大量扩增后回输给患者,从而发挥抗肿瘤效应的一种治疗技术。目前TCR基因治疗所面临的关键问题之一是如何改造修饰转TCR基因使得转TCR α链和β链在T细胞表面优先配对以提高转T细胞的功能,并避免off-target反应毒性的产生。最近,各种基因修饰策略被用于优化转TCR基因配对和减少错配。介绍了近年来针对TCR基因进行修饰改造的各种策略及TCR基因治疗的临床试验。     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.