In vitro studies of the rabbit immune system. V. Suppressor T cells activated by concanavalin A block the proliferation, not the induction of antierythrocyte plaque-forming cells.

The late B-cell proliferative phase of the in vitro antibody response by rabbit spleen cells is highly susceptible to suppression by activated T cells. The in vitro antisheep erythrocyte plaque-forming cell (PFC) response by spleen cells from normal or primed rabbits can be suppressed by adding concanavalin A (Con A), Con A-prestimulated peripheral blood or spleen lymphocytes, or supernates from Con A-prestimulated peripheral blood lymphocytes. The suppression is not mediated by a direct interaction of Con A with responding cells as shown by the effectiveness of prestimulated cells. Primed spleen cultures remain sensitive to Con A suppression as late as 72 h after initiation, and the addition of Con A after 24-72 h rapidly stops the increase in the number of PFC. T cells are required for Con A addition to be effective but the suppression can be induced at a time when T-helper cells are no longer necessary. Further, the suppressive effect of Con A addition is abrogated by specific antisera to rabbit T cells. We propose that Con A activates suppressor T cells which then exert their effects on proliferating PFC or their immediate precursor B cells. The early inductive or recruitment phase of the response is probably not blocked by suppressor cells. Also, there is an apparent relationship between the number of proliferating B cells and the number of suppressor cells required. Finally, the difficulties in inducing a stimulatory effect by Con A and the prolonged period that Con A addition is suppressive suggests that the rabbit has relatively more and/or longer-lived suppressor cells than the mouse and may be a particularly useful species for studying suppressive phenomena and their mechanisms.

Flow cytometric method for quantifying viable Mycoplasma agassizii, an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii).

AIMS: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. METHODS AND RESULTS: Here, we demonstrate that the nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml(-1) can be labelled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. CONCLUSIONS: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.

Active tumor cell resistance to human natural killer lymphocyte attack.

Inhibition of protein synthesis by cycloheximide, puromycin, or emetine increased tumor lysis mediated by human natural killer (NK) cells to "slow" but not "fast" tumor targets. Human T24 bladder carcinoma cells were used as slow targets which are killed after a approximately 3-hr lag period, and K562 cells derived from a patient with myelogenous leukemia were used as fast targets which are killed without a lag period. This enhancement of killing exceeded that which would have been expected simply from a reduction of tumor cell growth during the time of assay. Pretreatment of the NK effector cells and the tumor cells separately showed that the effect was on the tumor cells and not due to enhancement of NK cell activity. These observations imply that some tumor cells can actively resist NK attack. The discovery that some but not all human tumor cells actively resist cellular immune attack, perhaps by repair mechanisms dependent upon protein synthesis, provides a new model for evaluation of tumor cells in their resistance to host defenses.

In vitro studies of the rabbit immune system. VIII. The production of rabbit T cell growth factor (TCGF) and its relationship to mouse and human TCGF.

Rabbit spleen cells (1 X 10(6)/ml) stimulated with Con A (2.5 micrograms/ml) begin producing T cell growth factor (TCGF) activity within 2-3 h at 37 degrees C and reach a plateau after 20-24 h. Lymphocytes from the mesenteric lymph nodes (MLN) also produce TCGF activity but require higher lectin and/or cell concentrations for optimal production. TCGF generation by MLN can be improved by including a small proportion of spleen cells (10-40%). Rabbit lymphocytes also produce TCGF in serum-free, protein-free medium, but the Con A concentration must be reduced to 0.5-1.25 micrograms/ml. Rabbit TCGF activity eluted from gel filtration columns at a volume equivalent to approximately 15,000 molecular weight, similar to human TCGF activity. However, when rabbit, mouse and human TCGFs were compared for their ability to support proliferation by activated cells from the 3 species, rabbit and mouse TCGFs appeared more similar. In all cases, the homologous combinations of cells and TCGF were most efficient. Human TCGF would support some proliferative activity in all 3 cell types but rabbit and mouse TCGFs failed to support the growth of human cells.

In vitro studies of the rabbit immune system. IX. Extended culture of rabbit cytotoxic cells and the generation of TCGF-dependent T lymphocyte lines.

Rabbit T lymphocytes with cytotoxic activity for mouse target cells can be maintained in culture for several weeks by periodically restimulating with fresh mouse cells or by culturing with a source of TCGF. The former procedure greatly enriches the cultured cells for cytotoxic activity but has the disadvantage of introducing cellular debris. Cell growth and cytotoxic activity waned after 4-6 weeks using either procedure. However, T lymphocyte lines (TLL) could be isolated from limiting dilution cultures containing mitomycin C-treated mouse 'feeder' cells and a source of TCGF. These cells grew out with a frequency of one in 50-150 and approximately 15% of these growing cells had cytotoxic activity. Some cytotoxic cells lost their lytic activity after 4-5 months but others have persisted for over 7 months. Some TLL have now been growing for over 20 months.

In vitro studies of the rabbit immune system. VII. The generation of rabbit anti-mouse cytotoxic T lymphocytes.

Lymphocytes from normal or immunized rabbits generated cell-mediated cytotoxic activity (CML) after culture with mitomycin C-treated mouse stimulator cells for 4--7 days. CML activity was detected in short term (4--6 h) isotope-release assays using 51Cr-labeled tumor cells or mitogen stimulated blast cells as targets. Rabbit CML effectors could distinguish between different mouse strains including congenics differing only at H-2. It was previously shown that rabbit CML precursors and effectors expressed antigens recognized by a specific anti-T cell serum (ATS). The current results demonstrate that T-enriched fractions from nylon wool columns were enriched for CTL precursors and were sufficient to generate CML responses. These data were most consistent with the xenogeneic CML being mediated by rabbit cytotoxic T-lymphocytes (CTL) analogous to murine CTL. Rabbit lymphocytes also produced a strong mixed lymphocyte (MLC) response when tested in microculture with mouse stimulator cells. However, optimal CML and MLC responses did not occur in lymphocytes from the same organ sources or subpopulation. These data implied that CML activity could be generated with little, if any, proliferation necessary.

Lysis by RNK-16 cytotoxic lymphocyte granules. Rate assays and conditions to study control of cytolysis.

Dense subcellular granules of cytolytic lymphocytes can mediate rapid lysis of erythrocytes or nucleated cells. The granules contain several different proteases and proteoglycans that regulate cytolysis. We describe a rate assay that we have already used to demonstrate the requirement for serine proteases in granule-mediated lysis. In this assay, 51Cr-labeled erythrocytes are lysed by limiting concentrations of granules from RNK-16 tumor cells. Cytolysis is initiated by the addition of calcium (1 mM final concentration) and stopped at 0.5-1 min intervals by acidification to pH 6.0. The effects of the granule protein concentration, temperature, the concentration of erythrocytes, pH, and the concentration of calcium on the rate of lysis are reported. A preliminary mathematical approach is described and suggested as a means to differentiate 'lag' or activation times from the initial burst of lysis. With this rate assay, we have found four classes of protease inhibitors that block granule-mediated lysis (Hudig et al. (1987) Biochem. Biophys. Res. Commun. 149, 882). The utility of the rate assays is underscored by the observation that reversible protease inhibitors only showed rates of cytolysis whereas irreversible protease inhibitors stopped cytolysis completely. Rate assays are essential for future analyses of the complex physiological regulation of granule-mediated cytotoxicity by proteases, endogenous protease inhibitors and proteoglycans.

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

Antigen-specific proliferative responses to vesicular stomatitis virus following immunization of cattle with inactive virus.

Peripheral blood mononuclear cells (PBM) from four normal cows with no known exposure to vesicular stomatitis virus (VSV) were cultured with a New Jersey (NJ) serotype (Ogden) VSV that had been UV-irradiated and inactivated. PBM from these animals produced no detectable proliferative response when incubated with varying concentrations of VSV-NJ (Ogden) ranging from 10 ng to 10 micrograms protein/ml. Two of these cows were immunized with an experimental VSV-NJ vaccine and their PBM were tested at various intervals after immunization. PBM tested 14 days after the initial immunization produced readily detectable antigen-specific proliferative responses when cultured with UV-irradiated strains of VSV-NJ. Following a second immunization, lower concentrations of antigen were sufficient to stimulate the proliferative response and the magnitude of the proliferative response was increased. The responsiveness persisted for at least 6 months after these two immunizations. The specificity of the proliferative response was examined by comparing the responses stimulated by one VSV-Ind and four VSV-NJ serotype strains. The PBM from the immunized cows produced proliferative responses that were essentially specific for the VSV-NJ serotype antigens. In dose titrations, the VSV-NJ antigens were 300-1000-fold more effective than was the VSV-Ind antigen. Thus, persistent antigen-specific proliferative responsiveness that is serotype specific can be stimulated by immunizing cattle with an inactivated VSV vaccine.

Experimental vesicular stomatitis virus infection of swine: extent of infection and immunological response.

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3-5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.

Characterization of Tritrichomonas foetus antigens, using bovine antiserum.

Tritrichomonas foetus antigens were identified, using the serum of an Angus heifer that had been repeatedly immunized with suspensions of 1 X 10(8) organisms in Freund's complete adjuvant. Antibody activity against T foetus was determined by dot-blot analysis, using horse-radish peroxidase-conjugated anti-bovine immunoglobulin to detect bound antibody. The antiserum contained antibodies against surface and flagellar components of live or fixed T foetus, as determined by use of immunofluorescence. The antiserum reacted with approximately 38 proteins in a pool of 55 to 60 components resolvable by polyacrylamide-gel electrophoresis of T foetus extracts.

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation.

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.

Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25).

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+-sensitive dye Indo-1 showed that concentrations of CsA sufficient to inhibit 90-100% of tritiated thymidine incorporation had no effect on the PHA-stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37 degrees C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin-2, IL-2), as measured by monoclonal antibodies to CD25 after 16-24-hr incubation. In like manner, CsA also prevented the increase in the expression of the E-rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40-72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of "activation antigens."

The induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis.

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.

The mechanism of cell-mediated cytotoxicity. IV. K-76 COONa, which inhibits the activity of Factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation.

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.

The mechanism of cell-mediated cytotoxicity. III. Protease-specific inhibitors preferentially block later events in cytotoxic T lymphocyte-mediated lysis than do inhibitors of methylation or thiol-reactive agents.

Highly active mouse cytotoxic T lymphocytes (CTL) generated in secondary mixed-lymphocyte responses were used to examine the manner in which adenosine derivatives, thiol-specific reagents, or protease-specific probes affected CTL-mediated lysis (CML). The adenosine deaminase inhibitor deoxycoformycin (dCF) enhanced inhibition by adenosine (AR) or by deoxyadenosine (AdR), but not by 7-deazaadenosine (tubercidin). L-Homocysteinethiolactone (L-Hcy) acted synergistically with AR, but not with AdR or tubercidin, to block CML. Thus, AR derivatives may act both by affecting cellular methylation reactions, as demonstrated by the synergism between AR and L-Hcy, and by inhibiting other events required for CML. Conditions were then established to determine whether these reagents preferentially affected either the Ca2+-independent initial stage of cytolysis or the subsequent Ca2+-dependent events. Methylation inhibitors blocked lysis most effectively if added before effector-target binding. Similarly, the nonpenetrating thiol-specific reagent quaternary ammonium monobromobimane (qBBr) was more inhibitory when added prior to the Ca2+-dependent stage. Protease inhibitors such as alpha-1-antichymotrypsin and protease substrates such as acetyltyrosine ethyl ester (ATEE) or tyrosine ethyl ester (TEE) also inhibited CML. But, in contrast to qBBr or methylation inhibitors, neither TEE nor ATEE was more effective when added prior to the initial effector-target interaction. Furthermore, TEE did not appreciably affect CTL binding to target cells at concentrations that nearly abrogated CML. Thus, the implicated protease step is unique in that it does not appear to participate in recognition or binding.