Regulatory factors produced by lymphocytes. I. The occurrence of multiple alpha-lymphotoxins associated with ribonuclease activity.

Supernatant fluids of mitogen-activated human tonsil lymphocytes contain large amounts of a factor toxic to mouse L cells. This substance, with a m.w. of 80,000 +/- 5,000 daltons, is called alpha-lymphotoxin (alpha-LT), to differentiate it from another toxin elaborated by mitogen activated human blood lymphocytes, called beta-lymphotoxin (beta-LT), which differs from alpha-LT in size (45,000 +/- 5,000 daltons), antigenicity, and stability. Further purification of alpha-LT by sequential phosphocellulose and DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) identifies a series of cytotoxins differing in ion exchange characteristics and electrophoretic mobilities. The three PAGE fractions (PAGE Ia, Ib and II), recovered in 2, 4.6, and 21% yield from the starting serum-free culture supernatant, represent purifications of 24-, 24- and 1851-fold, respectively. Each cytotoxic fraction has a ribonuclease activity. Comparison of RNase and mouse L cell cytotoxic activities of the three alpha-LT fractions shows that both activities for all three fractions have a similar temperature stability pattern and that both are similarly inhibited by DNA, single strand forms better than double strands, by glycerol in 5 to 20% concentration, and by protein denaturing reagents. These observations suggest, but do not prove, that mouse L cell toxicity and RNase activity are mediated by the same substance, which appears to occur in multiple or isozymic forms.     

The human LT serum. X. The initial form released by T-enriched lymphocytes is 150,000 m.w., associated with small nonlytic components, and can dissociate into the smaller alpha, beta, and gamma m.w. classes

The present studies examine the various lymphotoxin (LT) forms released in vitro by phytohemagglutinin- (PHA) activated T-enriched (Te) human peripheral blood lymphocytes. It is clear that Te cells rapidly released (24 to 48 hr) these molecules in vitro. The 1st cell-lytic form detected in these supernatants is a 140-160,000 m.w. molecule(s) termed precursor alpha heavy (P alpha H). This form does not express alpha-LT antigenic determinants but is neutralized by antisera from animals injected with serum-free PHA-activated unseparated lymphocyte supernatants (anti-WS). The P alpha H is converted into alpha H, which expresses alpha determinants, by passage through molecular sieving columns or by treatment with low levels of Nonidet P-40 or urea. These treatments dissociate a small nontoxic 10-20,000 m.w. molecule(s), termed precursor factor (Pf), which masks the alpha-LT determinant on the P alpha H molecule. The dissociation of Pf is reversible, since alpha H from the molecular sieving columns will reassociate with the Pf. The alpha H LT class can further dissociate into the smaller alpha, beta, and gamma LT forms upon chromatography on a molecular sieving column, and a certain small percentage of the alpha H forms appear capable of associating to form the high m.w. complex (Cx) LT class. These findings suggest P alpha H may represent an intermediate that requires additional processing in order to proceed down 1 of 2 pathways: a) formation of complexes that are highly cell-lytic, or b) degradation by dissociation into the smaller weakly cell-lytic molecules identified as LT forms.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Production of alpha-lymphotoxin by human T-cell subsets

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG-M-). When restimulated with alloantigen of the same derivation, TG+, TM+, and TG-M- cells yielded almost equal amounts of cytotoxin. Anti-alpha-lymphotoxin serum neutralized most of this cytotoxic activity indicating that alpha-lymphotoxin (alpha-LT) constituted most of this activity. Although TG-M- cells function as effectors in allogeneic cytotoxicity, TG+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by alpha-LT. Whether TM+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, TG+, TM+, and TG-M- cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-alpha-LT. The TG+ cells incorporated less thymidine than the TM+ or TG-M- cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in TM+ cells, where positive and negative results have been reported.     

Regulatory factors produced by lymphocytes. II. Inhibition of cellular DNA synthesis associated with a factor inhibiting DNA polymerase alpha activity.

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.     

Cytotoxic activity of lymphocytes. VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes.

Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M_r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N-ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.     

The human LT system. XII. Purification and functional studies of LT and "TNF-like" LT forms from a continuous human T cell line

A clone of the continuous human T cell line HUT-102, termed YM 1.2, can spontaneously release alpha-LT in vitro. However, when stimulated with phorbol myristic acetate, these cells release other LT forms. These LT forms were purified to homogeneity by DEAE chromatography, column isoelectric focusing, and polyacrylamide gel electrophoresis. One LT form, termed LT-2, is a 79,000 m.w. component in aqueous solution and composed of 21,000 m.w. subunits. This form is immunologically related to macrophage-derived TNF and has a lytic capacity in vitro on K-562, Molt-4F, and Raji cells similar to that described for cytotoxins derived from NK effector cells, termed NK-CF. A second LT form, termed LT-3, is a single 69,000 m.w. peptide which could not be reduced into the smaller subunits. This form expresses antigens in common with both alpha-LT and TNF, because both anti-LT and anti-TNF were required to completely neutralize cell lytic activity in vitro. Functional testing revealed that the LT-3 form is lytic on all continuous cells tested in vitro, including NK-resistant target cells. The LT-3 component appears similar by immunologic, biochemical, and functional criteria to the LT form derived from primary human cytolytic T cells in vitro. At the levels tested, none of these LT-TNF forms had measurable effects on primary fibroblasts in vitro.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Mouse lymphotoxin.

The addition of PHA to C3H mouse spleen cells in tissue culture led to the production of lymphotoxin (LT). Cytotoxicity was assayed by addition of the culture fluids to syngeneic target cells labeled with tritiated thymidine; after an incubation period of 72 hr the amount of radioactivity released into the supernatant was measured. The LT activity in unfractionated culture fluids survived lyophilization, remained unchanged for many weeks at 4 degrees C, and progressively decreased on heating at 56 degrees C for periods from 1 to 9 hr. Based on the G-200 Sephadex distribution coefficients for several preparations, the m.w. of mouse lymphotoxin was about 41,000 daltons. Lymphotoxin from three different spleen cell production runs was recovered from isoelectric focusing columns in sharply focused peaks, the pH of which ranged from 4.4 to 4.8.     

Granulocyte colony stimulating activity from lymphocytes: separation from lymphokines by cytochalasin B.

Lymphocytes from murine spleens released granulocyte colony stimulating activity (CSA), macrophage migration inhibition factor and lymphotoxin from 24–96 hr after stimulation with phytohemagglutinin (PHA). Cytochalasin B at 5 μg/ml completely inhibited the release of migration inhibition factor and lymphotoxin but significantly increased the release of CSA.     

Mechanisms of lymphocyte-mediated cytotoxicity. I. The effects of anti-human lymphotoxin antisera on the cytolysis of allogeneic B cell lines by MLC-sensitized human lymphocytes in vitro

Goat and rabbit anti-human lymphotoxin sera, IgG and F(ab')2 reagents were investigated for their capacity to effect a specific alloimmune lymphocyte-mediated cytotoxic reaction. The cytotoxic reaction employed human peripheral blood or adenoid lymphocytes sensitized in MLC to allogeneic B lymphocyte cell lines and lysis was measured in a short-term 51Cr-release assay. A polyspecific anti-LT sera (anti-WS), made against unfractionated whole supernatants from lectin-activated lymphocytes and its IgG and F(ab')2 fragments, was found to be a potent inhibitor of this reaction when the anti-WS reagent was present throughout the assay period. Absorption studies indicated the anti-WS was inhibiting cytolysis at the level of effector cell or its products. Two broadly defined antibody specificities were involved in the cytolytic-inhibitory activity of the polyspecific anti-LT; i) antigens present on the normal lymphocyte cell surface; and ii) lymphocyte surface antigens associated with activated cells. These results correlate with the previously defined antigenic structure of the LT Cx and alpha H classes. Anti-LT sera reactive with the smaller m.w. alpha and beta classes and subclasses were not inhibitory, although the anti-beta sera showed a moderate enhancing activity. The results indicated that several anti-LT antibody specificities may be required to inhibit alloimmune cytolysis. The results suggest LT molecules may mediate lymphocyte-induced alloimmune cytolysis as a multi-component toxin system, rather than as an individual toxin.     

Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells

Adenylate cyclase (AC) toxins produced by Bacillus anthracis and Bordetella pertussis were compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 degrees C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069). Treatment of target Chinese hamster ovary cells with trypsin or cycloheximide reduced anthrax AC toxin-induced cAMP accumulation by greater than 90%, but was without effect on pertussis AC toxin. In contrast, incubation of the AC toxins with gangliosides prior to addition to target cells inhibited cAMP accumulation by pertussis AC toxin, but not anthrax AC toxin. To evaluate the role of lipids in the interaction of pertussis AC toxin with membranes, multicompartmental liposomes were loaded with a fluorescent marker and exposed to toxin. Pertussis AC toxin elicited marker release in a time- and concentration-dependent manner and required a minimal calcium concentration of 0.2 mM. These data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.     

The thiazole derivative reduces transmembrane currents via ionic channels formed by alpha-latrotoxin and sea anemone toxin in the bilayer lipid membranes

It was shown that the thiazole derivative 3-decyloxycarbonylmethyl-4-methyl- 5-(2-hydroxyethyl)thiazole chloride (DMHT) (0.1 mM) reversibly reduced the transmembrane current in solutions of 10 mM CaCl2 and 100 mM KCl via ionic channels produced by alpha-latrotoxin from black widow spider (alpha-LT) and sea anemone toxin (RTX) in the bilayer lipid membranes (BLM). Introduction of DMHT from the cis-side of BLM inhibited transmembrane current by 31.6 +/- 3% and by 61.8 +/- 3% from the trans-side of BLM for alpha-LT channels. Application of DMHT to the cis-side BLM decreased the inward current through the RTX channels by 50 +/- 5%. Addition of Cd(2+) (0.1 mM) to the cis- or trans-side of a membrane after the DMHT induced depression of transmembrane current across the alpha-LT channels caused its further decrease by 85 +/- 5% that coincides completely with the intensity of Cd(2+)-inhibition in the control experiments without DMHT. These data suggest that DMHT may exert its inhibitory action on alpha-LT channels without considerable influence on the ionogenic groups inside the channel cavity. The comparative analysis of effective radii measured for alpha-LT and RTX channels on the cis- (0.9 nm and 0.55 nm, respectively) and the trans-side of BLM (< 0.467 nm for alpha-LT) allowed to propose the blocking action of DMHT for alpha-LT and RTX channels to result from direct penetration into the channel, achieved due to similar hydrodynamic size of blocking molecules and the size of toxin pores.     

The effect of antilymphocyte globulin (ALG) on human lymphocytes in vitro: inhibition of lymphotoxin production as a sensitive indicator for ALG activity.

The effect of antilymphocyte globulin (ALG) on lymphocyte function in the presence or absence of complement was investigated by comparing (i) ALG-induced blast-cell transformation, (ii) lymphotoxin production and (iii) its effect on PHA-induced blast-cell transformation, and on (iv) lymphotoxin production and (v) its cytolytic effect on lymphocytes. The most striking observation was that even extremely low doses of ALG were able to suppress significantly the lymphotoxin production without affecting other lymphocyte functions or cell viability; this effect was found to be independent of complement. From the comparisons of the mitogen-induced lymphocyte function in the presence of varying doses of ALG and PHA it can be concluded that ALG exerts its inhibitory effect on lymphokine production most likely by interfering with mitogen recognition rather than lymphokine secretion.     

Blocking of the induction and expression of immunologically functional T lymphocytes by rat antiactivated T-cell serum

A hybridoma, F133, that produces macrophage activation factor (MAF) after mitogen stimulation was developed by fusing the AKR-derived BW5147 thymoma with alloantigen-stimulated C3H/HeJ splenocytes. F133 supernatants were shown to contain MAF, migration inhibition factor, and a factor capable of suppressing the plaque-forming response to sheep erythrocytes but not lymphotoxin, interleukin II, or interferon. Both concanavalin A (Con A) and phytohemagglutinin (PHA) induced MAF production by F133. Time course and dose-response experiments showed that maximal concentrations of MAF were present 48 hr after stimulation with either 1.5 μg/ml Con A or 6 μg/ml PHA. F133 and normal splenocyte MAF preparations shared physicochemical properties in that heating at 100 °C for 30 min abolished MAF activity while 56 °C for 30 min or 100 °C for 2 min had little effect. In addition, both MAF preparations were dependent on the presence of lipopolysaccharide for macrophage activation and each was inactivated by pH 4.0 or pH 10 treatment while pH 6.0 and pH 8.0 had little effect. Also, pretreatment of both MAF preparations with either trypsin or chymotrypsin inactivated MAF activity.     

Elaboration of lymphotoxin by freshly isolated human T lymphocytes and continuous lymphoid-cell lines.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.     

In vitro stimulation of human lymphocytes by alpha, beta and delta toxins and toxoids of Staphylococcus aureus.

Alpha, beta and delta toxins of Staphylococcus aureus stimulate human peripheral blood lymphocytes to blastic transformation and formation of IgM, IgG and IgA. The toxins are efficient at concentrations that are not toxic for the cells in culture. A dose of a toxin suitable for stimulation is 100 ng/ml but a stimulation can be observed also at 10 ng/ml, in the case of Ig formation even at a concentration of 1 ng/ml. Toxoids are approximately as effective to elicit blastic transformation as the toxins themselves, their efficiency to stimulate Ig formation being somewhat lower but significant. Alpha and delta toxins and toxoids at the appropriate concentration appear to act as medium-strength polyclonal activators of lymphocytes. Beta toxin and its toxoid are weak polyclonal activators.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.