Low-dose X-irradiation and 4-hydroperoxycyclophosphamide distinguishes three Lyt-1+ lymph node T-cell subtypes that respond to specific antigen in vitro

We investigated whether preculture exposure to low-dose X-irradiation and culture treatment with 4-hydroperoxycyclophosphamide (4-HPCY), a synthetic derivative of cyclophosphamide (CY), might distinguish subtypes of thymic-dependent (T) lymphocytes that respond to specific antigen in vitro. Lymph node (LN) cells were obtained from mice pretreated with CY and immunized with aggregated (A) human IgG (HGG) in Freund's complete adjuvant (CFA), and proliferation was assessed by incorporation of tritiated thymidine. Primed LN cells were untreated or exposed to low-dose irradiation before being cultured in medium alone and in medium containing 4-HPCY. The results show that these agents (irradiation and 4-HPCY) distinguished, in a dose-dependent manner, subtypes of T-cells which contribute to the specific antigen-stimulated proliferative response in vitro. For LN T-cells and LN Lyt-1+ T-cells, 20-25 rads and 1.0 microM 4-HPCY inactivated non-overlapping cell subtypes that respectively accounted for 26% and 28% of the response to HGG. The remaining 46% of HGG-responding cells were not affected by either agent. Although similar cell subtypes were discerned in unseparated LN cells, it required use of higher agent-doses. Cell cycle analysis revealed that treatment with irradiation, 4-HPCY, and the combination (both agents) caused S-phase arrest of 29%, 30%, and 55% of HGG-responding cells, respectively. Thus, identification of these cell subtypes could not be attributed to agent-mediated inactivation of HGG-responding cells that might be in exclusively different phases of the cell cycle.     

Evidence that the spontaneous blastogenesis of lymphocytes from bovine leukemia virus-infected cattle is viral antigen specific.

Cattle lymphocytes cultured for 3 days were found to spontaneously incorporate thymidine (3STI). Under optimal conditions of culture, the median magnitude of 3STI activity in lymphocytes from bovine leukemia virus (BLV)-infected cattle was higher than that of BLV-free cattle, but the ranges of the values overlapped. However, the 3STI activity of most BLV-infected cattle was specifically inhibited by serum containing BLV antibodies, whereas the 3STI activity of BLV-free cattle was not. The 3STI inhibitor copurified with immunoglobulin, and its activity could be absorbed with BLV. Rabbit anti-BLV serum inhibited 3STI, but rabbit anti-BLV p25 did not. These results indicate that BLV infection induces or expands a BLV-specific lymphocyte population. Spontaneous blastogenesis may be indicative of an immune response which controls virus spread.     

B lymphocytes that migrate to tuberculous pleural fluid via the SDF-1/CXCR4 axis actively respond to antigens specific for Mycobacterium tuberculosis

B-cell biology has been largely uncharacterized in the field of tuberculosis (TB). In this study, we investigated the immunophenotypical and functional characteristics of B cells obtained from the pleural fluid (PF) and peripheral blood of patients with tuberculous pleuritis (TP). Our results indicated that the total numbers of B cells, CD27(+) memory B cells and plasmablasts were clearly lower in the PF than in peripheral blood. Furthermore, we found significantly higher expression of CXCR4 on B cells in the PF, and a chemotaxis assay showed that B cells in the PF were more responsive to stromal cell-derived factor-1 (SDF-1) than B cells from peripheral blood. In addition, SDF-1 levels in PF were remarkably high compared with SDF-1 levels in plasma, suggesting that the SDF-1/CXCR4 axis might facilitate the migration of circulating B cells into tuberculous pleural space. Importantly, we observed that significantly more antibodies were produced by B cells in the PF following stimulation with BCG, early secretory antigenic target (ESAT-6)/culture filtrate protein-10 (CFP-10) or ESAT-6 protein. Collectively, these data demonstrate that Mycobacterium tuberculosis-specific B cells exist at local sites of infection in TP patients and this localization might influence the immune response to M. tuberculosis.     

Selection of DMA aptamer that specific binding human carcinoembryonic antigen in vitro

Objective:To select the specific aptamer of carcinoembryonic antigen (CEA), one of the most attractive molecule for cancer target therapy and imaging. Methods: Seven rounds in vitro selection were performed against the purified CEA protein. Ligand-mediated target purification and Co-immunoprecipitation were adopted to verify the specific binding of the aptamer to the purified and native protein separately. Results:The CEA-specific aptamer which can bind both the purified and native protein with the high specificity was obtained. Conclusion:This is the first time the CEA specific apatmer was produced. The results in this study provides the preliminary evidence for further investigation and application of CEA-aptamer in the future.     

Evidence that blood group A antigen on lymphocytes is derived from the plasma

Serum from group O volunteers, who had been injected with porcine A blood group substance, was used in lymphocytotoxicity tests. Positive reactions were obtained only with lymphocytes of group A secretors; the strongest reactors were Le(a--b--). The same group O sera reacted with group O lymphocytes which had been exposed to a glycosphingolipid fraction prepared from the plasma of A,Le(a--b--) secretors. These reactions were specifically inhibited by A substance. It is suggested that, unlike the A antigen on red cells, the A antigen detected in lymphocytotoxicity tests is entirely derived from the plasma.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ α β T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153 – 166 and p108 – 122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193 – 204 in the context of DRB1*1401 and for p153 – 165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193 – 204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Dose-response effects of 4-hydroperoxycyclophosphamide on human T and B cell function in vitro

4-Hydroperoxycyclophosphamide (4-OOH-CYP) is spontaneously converted in aqueous solution to 4-hydroxycyclophosphamide (4-OH-CYP), the major active metabolic of cyclophosphamide. We studied the dose related effects of in vitro treatment with 4-OOH-CYP on human T- and B cell-mediated immune responses. T-cell proliferation to mitogens and alloantigens was only partially inhibited even relatively high-doses of 4-OOH-CYP (greater than 6-12 micrograms/ml). In contrast cytotoxic functions of activated T-cells and natural killer (NK) cells were inhibited at lower doses (3-6 micrograms/ml). PWM induced in vitro synthesis of IgG by B-cells was inhibited at less than 3 micrograms/ml of 4-OOH-CYP. These data indicate that 4-HOO-CYP has selective, dose-dependent effects on human T and B cells in vitro.     

The participation of thymus-derived and of bone marrow-derived lymphocytes of sensitized mice, in the proliferative response to specific antigen, in vitro

Spleen cells of mice primed with sheep red cells (SRC) responded with increased thymidine uptake when stimulated with SRC in vitro. Depletion of bone marrow-derived (B) cells by filtering the spleen cells through columns of plastic beads coated with anti-mouse immunoglobulin serum resulted in a drastic loss of the proliferative response. A similar loss occurred on depleting the thymus-derived (T) cells by treatment with anti-Θ serum and complement. Addition of peritoneal exudate cells failed to restore the reactivity of the purified T cells to SRC. The proliferative response could be restored to a level exceeding the sum of the individual responses, by mixing the purified T with the purified B cells. A synergistic interaction between T and B cells is suggested.     

T lymphocytes respond to solid-phase antigen: a novel approach to the molecular analysis of cellular immunity.

Using cloned human T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminus of the HA-1 molecule of influenza haemagglutinin (HA), we have investigated the ability of solid-phase antigen to induce antigen-specific T-cell proliferation. The activation by nitrocellulose-bound virus and p20 was accessory-cell dependent and was not caused by immobilized antigen directly cross-linking the specific receptors. Furthermore, we report that separation of complex antigen mixtures such as influenza virus and HA by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) followed by transfer to a nitrocellulose membrane can be used to allow direct screening of individual polypeptides in T-cell proliferation assays. With this immunoblotting procedure the antigenic site recognized by HA-reactive T cells was confirmed to reside in the HA-1 molecule of influenza virus of only the appropriate subtype. The general application of this approach is discussed in the case of infections and autoimmune diseases in which the immune response is predominantly T-cell mediated and where antibody studies may fail to identify key antigenic determinants involved in the activation of T cells.     

Drosophila melanogaster prophenoloxidases respond inconsistently to Cu2+ and have different activity in vitro

Dipteran insects, like mosquitoes, possess more than two prophenoloxidase (PPO) genes, but it is unclear whether their gene products differ in biochemical properties and physiological functions. Here, we used three Drosophila melanogaster PPOs as models to study their properties through expression in S2 cells. Our data revealed that the PPOs were expressed in the ethanol-activatable conformation: rPPO1 and rPPO2 needed additional Cu²⁺ in the medium, but rPPO3 did not. rPPO1 bound Cu²⁺ within minutes; rPPO2 did that in hours when Cu²⁺ were present at a higher concentration. Thus, rPPO1 and rPPO2 were expressed as apo-rPPO and became holo-PPO upon Cu²⁺ binding; rPPO3 was holo-PPO immediately after expression. Surprisingly, in the absence of ethanol, the apparently intact rPPO3 catalyzed dopamine oxidation and melanization. The successful method for rPPO expression in S2 cells described in this paper will provide us with an opportunity to study the properties of a specific PPO gene in a small insect like mosquitoes in the future.     

Responses of alloantigen-primed lymphocytes in vitro Quantitative analysis of the relative frequency of reactive lymphocytes in primed populations which respond to allogeneic stimulating cells

Populations of lymphocytes primed to alloantigens in vitro are stimulated by allogeneic cells which are unrelated to the specific stimulator cells used for priming. The number of lymphocytes in the primed population which responded to unrelated stimulators could be estimated in relation to the number of primed lymphocytes responding to the specific stimulator. This was done by measuring the responses of decreasing numbers of primed lymphocytes to various stimulator cell populations. Similar estimates were obtained on several occasions using the same combination of lymphocytes which were primed and retested at different times. There was a close correlation between the estimated frequency of lymphocytes in a primed population which responded to unrelated stimulators and the number of responder lymphocytes required to obtain a measurable response. Stimulators which activated a high frequency of primed cells required fewer responder lymphocytes than those which activated lower frequencies of primed cells. The specific stimulator was capable of initiating a response using fewer responder lymphocytes than were most unrelated stimulator cells. This confirms that primed populations are predominantly enriched for lymphocytes responding to the specific stimulator. At least some lymphocytes in a primed population responded to both the specific stimulator and unrelated stimulator cells. This was demonstrated in two types of experiments. In the first, it was possible to enrich lymphocytes in a primed population which responded to unrelated stimulator cells without depleting lymphocytes which also responded to the specific stimulator. In the second, primed lymphocytes from donor A, which showed strong reactivity to stimulators from donor D after priming with B stimulator cells, also showed significant reactivity to B stimulators after priming with D cells. These results strongly suggest that primed populations contain several subpopulations (clones) of lymphocytes whose specificities differ as exhibited by their responsiveness to unrelated stimulator cells.     

B lymphocytes from individuals with common variable immunodeficiency respond to B lymphocyte stimulator (BLyS protein) in vitro

B lymphocyte stimulator (BLyS protein) is a member of the human TNF family of ligands. BLyS induces B-lymphocyte proliferation and Ig secretion in vitro and in vivo. These qualities suggest that it may be useful as a therapeutic in the treatment of immunodeficiencies characterized by low or absent serum immunoglobulin, such as common variable immunodeficiency (CVID). CVID is characterized by the inability to generate adequate serum Ig despite normal or slightly depressed peripheral B, T, and myeloid cell populations. We tested the ability of BLyS to stimulate B lymphocytes obtained from CVID patients. Among five patients studied, 60% (three of five) produced normal quantities of IgM when cultured in the presence of BLyS. B-cell proliferation among patients was comparable, with 60% (three of five) responding to BLyS stimulation. These results suggest that BLyS induces proliferative and Ig-secretory responses in B lymphocytes isolated from some CVID patients and lend support to its potential use in therapy of this disorder.