Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Cross-reactivity of T-cell clones specific for altered peptide ligands of myelin basic protein

We have determined that certain altered peptide ligands (APLs) can induce T-cells specific for the native peptide myelin basic protein (MBP) p85-99 to secrete Th2-type cytokines such as IL-4 and IL-5 in the absence of significant Th1-type cytokines. However, it is not known whether stimulation with APLs will activate autoreactive T cells or a distinct population of cells. In the present study, 18 T-cell clones that reacted with either MBP p85-99 or one of three APLs of the peptide substituted at TCR contact residues were generated. T-cells were tested functionally for their reactivity to the original stimulating peptide as well as to the MBP APLs. In addition, the T-cell receptor (TCR) alpha and beta chains of each of these clones were sequenced. In a series of T-cell clones isolated from a multiple sclerosis patient, stimulation of T-cells with the APL 93A, which has an alanine for lysine substitution at the TCR contact residue 93, did not induce substantial proliferation of MBPp85-99-specific T-cell clones, indicating that a distinct set of T-cell clones was induced. However, this was not the case for another set of T-cell clones from a different individual in which the 93A peptide induced clonal expansion of T-cells highly reactive with the native MBPp85-99 antigen. Thus, the potential beneficial effect of using APLs to induce downregulatory cytokines appears to depend on the specific T-cell repertoire of the individual patient.     

Establishment of cytotoxic T-cell clones specific for cells infected with mouse hepatitis virus.

Mouse hepatitis virus (MHV)-specific T-lymphocyte clones were established from MHV-infected BALB/c mice. They expressed Thy1 and Lyt2 antigens but lacked L3T4 and NK1 antigens. The clones killed MHV-infected but not uninfected or influenza virus-infected J774.1 cells. The specificity was further defined by a cold-target competition test.     

Monoclonal antibody differentiates chicken A system alloantigens

A monoclonal antibody (ISU-cA) was produced that recognized certain alloantigens of the chicken A blood group locus. Antigens produced by alleles A3, A4 and A8 were positive, and those produced by A2 and A5 were negative, by haemagglutination. The specificity of ISU-cA for chicken A blood group antigens was demonstrated by serologic analyses, genetic crosses and competitive inhibition of binding by anti-A alloantisera. To our knowledge, this is the first reported monoclonal antibody against a chicken blood group alloantigen system other than the B complex.     

T-cell clones in immunoparasitology

Cell-mediated immunity (CMI) is important in many parasitic infections. Stimulation by parasite-specific antigens can induce clonal expansion of parasite-specific T-cells which may act by direct cytotoxic action or by indirect effects on other cells such as natural killer cells or antibody-producing B-cells (Box 1). In many cases however, the precise effector functions and the identity of antigens that elicit protective responses in parasitic infections are poorly defined. Analysis of proliferative and cytotoxic activities of subcloned cultures of T-cells stimulated with parasite antigens can provide some clues about the importance of CMI, but, as this review shows, much more precise information can be obtained by analysis of the response of cloned T-cell cultures.     

N-acetyl-D-glucosamine specific hemagglutinin receptor of Vibrio cholerae O1 in chicken erythrocyte membranes

N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V. cholerae O1 cells. This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V. cholerae O1 strain from chicken erythrocyte membranes. The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V. cholerae O1 strain with chicken erythrocytes.     

The cell-surface distribution of alloantigens on the bovine erythrocyte

The topographical distribution of genetically defined alloantigens of the bovine erythrocyte was investigated by a serological absorption procedure known as the blocking test. Blocking was observed between the E′₁ and Y₂ antigens in theB system when these occurred in the same phenogroup. E′₁ was also blocked by G and Q′ in the same phenogroup and Y₂ was blocked by G. No blocking was observed between specificities residing either in different phenogroups or in different systems. We conclude that the several specificities in a givenB-system phenogroup are carried on a single molecule having multiple antigenic determinants, and that this arrangement is entirely consistent with the hypothesis that these factors are controlled by multiple alleles at a single locus.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans

Substantial progress in understanding the biology of regulatory T cells and their roles in health and disease has been achieved in the past 10 years. This has led to an increasing interest in the possibility of using regulatory T cells as a biological therapy to preserve and restore tolerance to self antigens and alloantigens. Immunotherapy by the adoptive transfer of regulatory T cells may have several advantages over conventional treatments. However, several hurdles have to be overcome before such a therapy can enter clinical practice. This Review summarizes our current knowledge of regulatory T cells, illustrates the ongoing regulatory T-cell-based clinical trials, analyses the strengths and pitfalls of this new therapeutic approach, and highlights the future perspectives.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

Sphingomyelinase of chicken erythrocyte membranes.

Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [³H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg²⁺ and Mn²⁺ activate the enzyme while Ca²⁺ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% ($\text{w}\text{v}$) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.     

Properties of chicken erythrocyte delta-aminolevulinate synthase

A procedure is described for preparing a fraction highly enriched for chicken blood delta-aminolevulinate synthase (ALA-S) using animals recovering from acetylphenylhydrazine-induced anemia. 1. Blood cells collected from chickens recovering from anemia were disrupted by nitrogen cavitation, and the mitochondrial fraction was prepared from the cell homogenates. ALA-S was released then from mitochondria by sonication and isolated by a procedure involving gel filtration chromatography on Sephadex G-150, fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephacel, and preparative isoelectric focusing. 2. Electrophoretic analyses under denaturing conditions indicated that the final ALA-S preparation was particularly enriched from a 62,200 Da polypeptide. The enzyme eluted from Sephadex G-200 with an equivalent molecular weight of 115,000; this suggested that active ALA-S was a dimer. 3. ALA-S was most active in the pH range of 7.0-8.0, with an apparent KM of 13 microM for succinyl-CoA and of 4.0 mM for glycine. The activity was inhibited 50% by 30 microM hemin.     

Advanced lymphoblastic clones detection in T-cell leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients.     

Octamer reconstitution from acid-extracted chicken erythrocyte histones

Histone octamers have been reconstituted from acid-extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido-ester cross-linking, the reconstituted octamers have a structure identical to that of salt-extracted octamers.     

Molecular characterization of different ataxia telangiectasia T-cell clones

Summary Using in situ chromosomal hybridization we have mapped the gene for the T-cell receptor -chain in three different non-malignant T-cell clones occurring in ataxia telangiectasia. The constant region was translocated in each of the three clones. The variable region remained in its original position in two cases and was deleted in one clone which lost the derivative chromosome 14. We have therefore demonstrated that the T-cell receptor -gene is split in at least two of these translocations. To our knowledge, this is the first direct evidence of the involvement of a gene from the immunoglobulin superfamily in chromosomal rearrangements in ataxia telangiectasia.