Transplantation tolerance at the T-lymphocyte receptor level. II. Interaction of T-cell receptors with alloantigen and with anti-receptor antiserum.

CBA T lymphocytes deprived temporarily of receptors for alloantigens A[RS(A)] cultivated in vitro for 30 h with anti-receptor antibody-forming (AxCBA)F1 spleen cells were capable of resynthesizing RS(A) if primed F1 cells exceeded parental T cells by a factor of 25 or less, but not if the excess was 50-fold or more. This indicated that resynthesis of CBA T-cell RS(A) was successful if primed F1 cells formed insufficient amounts of anti-CBA T-cell RS(A) antibody. Abortive or successful receptor resynthesis was measured by two parameters, (a) reappearing RS(A) formed PAR together with A alloantigens of (AxCBA)F1 spleen cells and (b) budding receptors bound anti-receptor antibody. CBA B lymphocutes did not interfere with these reactions. A search for putative T suppressor cells in the F1 cell population was unsuccessful. PAR formation and anti--RS antibody consumption by reappearing receptors differed temporally: receptors forming PAR were present after a delay lasting 8 h; receptor structures fixed anti-RS antibody as early as 5 h after being cultivated. With due caution, these results might reflect processes operating in maintenance of transplantation tolerance, suggesting that this condition is a serum-mediated suppression of long duration. The suppression would encompass continued neutralisation of receptors for the alloantigen to be tolerated by anti-T-cell receptor antibody formed by the F1 chimeric cells within an animal with acquired transplantation tolerance.     

Lipopolysaccharide receptors on lymphocytes. I. Lack of immunologic recognition of a putative LPS receptor on LPS-responder lymphocytes by LPS-nonresponder mice

We have examined the potential immunogenicity of viable lymphocytes from C3HeB/FeJ responder mice adoptively transferred into congenic nonirradiated C3H/HeJ nonresponder mice. Immunologic rejection or acceptance of donor cells was employed as indirect evidence for the presence or absence of an antigenically distinct "LPS receptor" present on donor lymphocytes. Immunogenicity was evaluated by in vitro assessment of the subsequent proliferative response of recipient splenocytes to protein-free LPS after multiple i.p. injections of responder lymphocytes. Control experiments have made use of syngeneic donor lymphocytes differing immunologically by the presence of the H-Y minor histocompatibility antigen. The results of these experiments provide evidence for the concept that if the phenotypic difference between LPS responder and nonresponder mice is also expressed antigenically in the form of an LPS receptor, that antigenic difference is significantly less immunogenic than a minor histocompatibility antigen.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. V. Evidence for b-cell deletion in vitro with serum from tolerant mice.

The serum from mice that had been rendered specifically tolerant (TolS) to the trinitrophenyl (TNP) hapten by the injection of trinitrobenzenesulfonic acid (TNBS) is effective in the in vitro induction of immunological unresponsiveness in murine spleen cells. This tolerance system was investigated with particular emphasis upon the mode of induction. The observed inhibition by TolS of responses to the thymic-independent (TI) antigen TNP-lipopolysaccharide (TNP-LPS) was stable following adoptive transfer to lethally irradiated recipients and was due neither to the delay of in vitro responsiveness nor to effector cell blockade at the level of the antibody-forming cell. Neither suppressor cells nor cell-bound tolerogen carry-over were responsible for the tolerance induced by TolS. TNP-LPS doses, including a wide range of polyclonal activating concentrations, were ineffective in reversing the unresponsive state induced by cocultivation with TolS. Additionally, unconjugated LPS in either fetal calf serum (FCS)-containing or FCS-free cultures did not break tolerance. This failure of polyclonal activating substances to reverse the unresponsive state suggests that blockade of TNP-specific receptors is not the mechanism of tolerogenesis, since such compounds trigger cells polyclonally through nonimmunoglobulin receptors. Tolerance induced by incubation of spleen cells with TolS for 24 hr followed by extensive washing was stable whether the immunogenic stimulus was the TI antigen TNP-LPS or the thymic-dependent (TD) form of the hapten, TNP-sheep erythrocytes (TNP-SRC). Washing spleen cells at elevated temperatures after preculturing with TolS to avoid possible reassociation of surface Ig (sIg)-bound TNP conjugates did not lead to escape from tolerance. Antigen-free incubation for 24 hr following cultivation with TolS was equally unsuccessful in reversing the unresponsive state. Thus, extensive washing following tolerance induction and antigen-free cultivation where unblocking or turnover and resynthesis of sIg receptors should have taken place provided no support for receptor blockade as the mode of in vitro induction and maintenance of tolerance by TolS. Treatment with the proteolytic enzyme pronase with the intention of removing potential tolerogen from the cell surface revealed a stable tolerant state. Incubation with anti-Ig or anti-TNP antisera under conditions designed to allow capping and removal of sIg-bound tolerogen or surface-bound TNP conjugates also failed to reverse the tolerance induced by incubation with TolS. The results presented here and previously lend no support to active or passive suppression or blockade of reactive cells as the mechanism of tolerance induction in vitro by TolS. The data are consistent with the hypothesis that TolS-induced unresponsiveness is due to a functional deletion of TNP-specific B lymphocytes. Furthermore, the similarities observed between the induction of tolerance by TNBS injection and TolS-induced unresponsiveness are consistent with the suggestion that TNBS-induced tolerance in vivo is mediated by a component of TolS which is active as a tolerogen in vitro.     

T-cell specific rearrangement of T-cell receptor beta transgenes in mice.

To study rearrangement of T cell receptor (TCR) genes, transgenic mice were generated with a TCR beta minilocus in germline configuration, containing three V beta, two D beta, fourteen J beta and two C beta gene segments and the TCR beta enhancer. Using the polymerase chain reaction as an analytical tool both partial DJ as well as complete VDJ rearrangements were seen, indicating that the minilocus contained all sequence elements required for rearrangment. Rearrangements of minilocus gene segments were restricted to T cells in the thymus and the periphery and did not occur in B cells. V beta 8.3 and V beta 5 sequences encoded by the minilocus were expressed on the surface of peripheral T cells at high frequencies. Transgenic mice with TCR minilocus genes will be a useful system to identify DNA sequence elements required for regulation of rearrangement in vivo.     

Specific suppression of the immune response by HGG tolerant spleen cells. I. Parameters affecting the level of suppression.

After adoptive transfer, the spleen cells from mice made tolerant to human gamma-globulin (HGG) specifically suppress the immune response of normal spleen cells. However, this suppressive activity in the spleen cells of tolerant mice is only present for a brief period after treatment with tolerogen. Spleen cells from animals injected 10 days earlier with tolerogen reduce the immune response of an equal number of normal spleen cells by 75%. Spleen cells from mice made tolerant 40 days previously are, however, no longer suppressive, even though they remain completely unresponsive. These data suggest that active suppression of antigen-reactive cells is not the mechanism responsible for maintaining tolerance to HGG, but rather is only transiently associated with the tolerant state. Further evidence in favor of the separation of the tolerant state from suppressive activity is that complete suppression of the normal spleen cell response requires either a high ratio of tolerant to immune competent cells or a delay in the antigenic challenge of the reconstituted recipients. By contrast, such manipulations are not required to demonstrate the complete unresponsiveness of the tolerant cells after adoptive transfer.     

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.     

Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes

T lymphocytes are activated by the engagement of their antigen receptors (TCRs) with complexes of peptide and major histocompatibility complex (MHC) molecules displayed on the cell surface of antigen-presenting cells (APCs) [1]. An unresolved question of antigen recognition by T cells is how TCR triggering actually occurs at the cell-cell contact area. We visualized T-cell-APC contact sites using confocal microscopy and three-dimensional reconstruction of z-sections. We show the rapid formation of a specialized signaling domain at the T-cell-APC contact site that is characterized by a broad and sustained area of tyrosine phosphorylation. The T-lymphocyte cell-surface molecule CD2 is rapidly recruited into this signaling domain, whereas TCRs progressively percolate from the entire T-cell surface into the phosphorylation area. Remarkably, the highly expressed phosphatase CD45 is excluded from the signaling domain. Our results indicate that physiological TCR triggering at the T-cell-APC contact site is the result of a localized alteration in the balance between cellular kinases and phosphatases. We therefore provide experimental evidence to support current models of T-cell activation based on CD45 exclusion from the TCR signaling area [2] [3] [4].     

Rabbit lymphocytes with receptors for IgM. I. The development of receptors on lymphocytes following intravenous injection of antigen.

Under conditions optimal for detection of receptors for IgM (RFcμ) on human lymphocytes, RFcμ⁺ lymphocytes were not found in any of the tissues of nonimmune rabbits. However, 1 day after iv immunization with alum-precipitated keyhole limpet hemocyanin (KLH) or polymerized human IgG (pol-Ig), rabbit peripheral blood (PB) and spleen (S) demonstrated a significant percentage of lymphocytes (10%) with RFcμ. At 3 days after immunization there was a further increase in RFcμ⁺ lymphocytes (10–13%). By 6 days, however, the percentage of lymphocytes bearing receptors for IgM had returned nearly to zero. Pronase treatment was found to increase expression of RFcμ at 1 and 3 days in pol-Ig-primed tissues (up to 25%) but not in KLH-primed tissues. The percentage of RFcμ⁺ lymphocytes in tissues remained low at 6 days even after Pronase treatment. This time sequence of development of RFcμ⁺ cells after immunization seems interesting in the light of the findings of others that human lymphocytes with receptors for IgM are responsible for help in the PWM-induced differentiation of B cells to plasma cells.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors.

The role of muscarinic receptors in the down-regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10(-6) M) and atropine (10(-8) to 10(-6) M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10(-6) M) caused a slight depression in the amplitude of responses to ACh pressure-applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA-immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10(-6) M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10(-6) M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10(-6) M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse.     

Resistance to viral infection by intraepithelial lymphocytes in HIV-1 P18-I10-specific T-cell receptor transgenic mice

For the analysis of mucosal immunity to HIV-1, we have recently established a line of transgenic (Tg) mice expressing the TCRalpha and TCRbeta genes of the murine CTL clone RT1 specific for P18-I10 (RGPGRAFVTI), an immunodominant gp160 envelope-derived epitope of IIIB isolate, restricted by the H-2D(d) MHC-I molecule. Here we examine those cells bearing specific TCR among the intraepithelial lymphocytes (IELs), with flow cytometric analysis using H-2D(d)/P18-I10 tetramers. We observed three distinct CD3(+), tetramer positive populations among the IELs: extra-thymic CD8alphabeta(+), alphabetaTCR T-cells; CD8 alphaalpha+, gammadeltaTCR T-cells; and thymus-derived CD8alphabeta+, alphabetaTCR T-cells. Challenge of these Tg mice with P18-I10 encoded by a vaccinia virus vector, either intrarectally (i.r.) or intraperitoneally (i.p.), revealed that the intraepithelial compartment seems to be a major site for prevention of the spread of viral infection. Such immunity appears due to the thymus-derived, CD8alphabeta+ antigen-specific CTLs together with CD8alphaalpha+ gammadelta cells, which regulate virus spread. This model system for studying CTL based immunity at mucosal sites should prove helpful in developing rational approaches for HIV control.     

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.