Changes in repair of potentially lethal damage with culture age in EMT6 cells. We have investigated the changes in the amplitude of repair of potentially lethal damage (PLD) in EMT6 cells with increasing culture age and determined the delay necessary to achieve this repair. This experimental system presents all intermediaries between the exponential growth type and a plateau with a cell turnover nearly nil. The radiosensitivity was studied by the colony method. When the percentage of surviving cells was tested immediately after irradiation it was observed that their radiosensitivity increased with culture age. This percentage fell only slightly when the cells were tested for viability 6 hours after irradiation. Therefore, the amplitude of repair increases with culture age. Repair was found to terminate 1, 1.75, 3 and 6 hours after irradiation of cultures aged respectively 2, 4, 6 and 9 days. The delay and the amplitude of repair did not vary significantly for cultures of 9, 11 and 13 days.


Structure-activity relationships in the development of hypoxic cell radiosensitizers. I. Sensitization efficiency. The efficiency of 35 nitroaromatic and nitroheterocyclic compounds in radiosensitizing hypoxic Chinese Hamster cells in vitro was determined. The concentration C of the compound required to achieve an enhancement ratio of 1.6 was measured, and the redox and partition properties were quantified as the one-electron reduction potential at pH 7, E, and the octanol: water partition coefficient, P, respectively. Most of the compounds studied were 2-nitroimidazoles, but some 4- and 5-nitromidazoles, 5-nitrofurans and nitrobenzenes were investigated for comparison. Together with data for nine nitroimidazoles previously reported, the results were fitted to a structure-activity relationship of the form -log C = b0 + b1E + b2 log P + b3 (log P)2 using multiple linear regression analysis. Statistical tests showed that the coefficients b2 and b3 were not significantly different from zero and the simpler equation, obtained by omitting the terms in log P, explained 85 per cent of the variance in log C. Earlier reports that the radiosensitization efficiency of nitro compounds in vitro largely depends on the reduction potential were confirmed. The conclusive demonstration that P is unimportant in vitro is valuable in interpreting the results of experiments in vivo, where P is expected to have a much greater influence on biological response.


Structure-activity relationships in the development of hypoxic cell radiosensitizers. II. Cytotoxicity and therapeutic ratio. This paper describes measurements of the aerobic cytotoxicity of 42 nitroaromatic and nitroheterocyclic compounds towards Chinese Hamster cells in vitro. The results of acute and chronic exposure were quantified, and the concentration C required to achieve a standard response estimated. Fitting the data to an equation of the form - log C = b0 + b1E, where E is the one-electron reduction potential, explained 47 and 71 per cent of the variance in the acute and chronic aerobic cytotoxicity respectively. The addition of further terms to the equation, quantifying partition properties, was not statistically significant. The coefficient b1 was similar for both acute and chronic exposure; the dependence of both cytotoxicity and radiosensitization efficiency on reduction potential was also similar. A therapeutic ratio derived from these in vitro measurements showed no dependence on redox or partition properties. The insensitivity of cytotoxicity and radiosensitization properties to variations in molecular structure, other than those which influence redox behaviour, offers exceptional flexibility in developing compounds of improved therapeutic ratio.


Radiation effects on erythrocyte membrane structure studied by the intrinsic fluorescence. The changes in the intrinsic fluorescence, primarily from tryptophan residues, of sheep erythrocyte membranes following X-irradiation (0--4000 R) were investigated. The experiments showed that there was (1) a decrease in the intensity of fluorescence with increasing dose of X-rays, (2) a small shift of fluorescence emission to longer wavelengths, (3) a decrease in the fluorescence polarization, and that (4) treatment of membranes with a perturbing solvent, 2-chloroethanol, can eliminate the effects of X-rays. The amount of tryptophan in the membranes was not altered after X-irradiation. It was also shown that sulphydryl reagents, N-ethylmaleimide and 2,2'-dithiodipyridine, induced similar fluorescence changes. From these results it was concluded that the fluorescence changes could result from a change in the environment surrounding tryptophan residues, from being relatively non-polar to being more polar, implying that conformational changes of membrane proteins are brought about by low doses of X-rays.


1.5 MHz ultrasound irradiation of human lymphocytes. Human lymphocyte cultures are exposed to 1.5 MHz continuous wave ultrasound, and it is demonstrated that cell death, as monitored by pyknosis, follows immediately on sonication at intensities within the usual therapeutic range (less than 1.7 W/cm2,spatial average). The number of cells affected is determined by the ultrasound intensity only, but the rate at which they proceed through their pyknosis cycle is modified by both the intensity and the duration of exposure. There is a clear indication of an intensity threshold for the effect approximately 1.1 W/cm2. Pulsed 1.5 MHz ultrasound (70 mus, 1 :1 pulses, 1.7 W/cm2 space-time average) results in a 15-20 hour delay in the measurable response to sonication. It is shown that the intracellular presence of a lysosomal-enzyme inhibitor strongly modifies the course of the ultrasound action. Evidence is presented to suggest that the basic interaction mechanism is via a cavitation process, but there are some difficulties with this interpretation, which are also discussed.


The effect of ionizing radiation on the fatty acid composition of natural fats and on lipid peroxide formation. The effects of irradiation doses of 200-1000 krad on the fatty acid compositions of saturated and unsaturated natural food fats have been studied. Lard, coconut oil, corn oil, methyl linoleate and herring oil have been analysed before and after irradiation for lipid peroxide content and fatty acid composition. The effects of storage under varied conditions after irradiation have also been investigated. Irradiation doses of 200-1000 krad had little effect on the fatty acid compositions of saturated fats (lard and coconut oil) or of fats with a high antioxidant content (corn oil) but caused destruction of 98 per cent of the highly unsaturated acids (18: 4,20 :5,22 : 6) and 46 per cent of the diene acids (18:2) in herring oil. The destruction of the polyunsaturated fatty acids increased with increasing storage temperature and storage time. The destruction of polyunsaturated fatty acids is accompanied by an increase in lipid peroxide formation. It is considered that changes in fatty acid composition in natural foods after irradiation are important in consideration of the use of irradiation for food preservation.


Protection of liposomal lipids against radiation induced oxidative damage. Liposomes were prepared from phospholipids extracted from biological membranes. A comparison was made between the peroxidation rate in handshake liposomes and in sonicated liposomes. The smaller sonicated liposomes were more vulnerable to peroxidation, probably because of the smaller radius of curvature, which results in a less dense packing of lipid molecules in the bilayer and a facilitated action of water radicals produced by the X-irradiation. High oxygen enhancement ratios were obtained, especially at low dose rates, suggesting the operation of slowly progressing chain reactions initiated by ionizing radiation. Three compounds were tested for their ability to protect the liposomal membranes against lipid peroxidation. The naturally occurring compounds reduced glutathione (GSH) and vitamin E(alpha-T) and the powerful radiation protector cysteamine (MEA). All three molecules could protect the liposomes against peroxidation. The membrane-soluble compound vitamin E was by far the most powerful. About 50 per cent protection was achieved by using 5 X 10(-6) M alpha-T, 10(-4) M GSH and 5 X 10(-4) M MEA. The fatty acid composition of the lipids altered drastically as a result of the irradiation. Arachidonic acid and docosahexanoic acid were the most vulnerable of the fatty acids. Very efficient protection of these polyunsaturated fatty acids could be obtained with relatively low concentrations of vitamin E built into the membranes.


Characterization of feline whole-blood cultures and determination of the frequency of radiation-induced dicentrics in human and feline lymphocytes. The Ham's F-10 and PHA culture system was applied to whole feline blood and cell-cycle characteristics such as DNA synthesis and mitotic indices were studied. The results are comparable to those obtained from human whole-blood cultures. The yields of dicentrics were also determined in lymphocytes from X-irradiated human and feline blood. The ratio between the experimental yields of dicentrics in human as compared to feline lymphocytes was 1:0.27.


Modification by timolol of catecholamine stimulation of chloride transport in isolated corneas. In the isolated frog cornea, 10(-5)M timolol completely blocked the stimulation of chloride transport by 10(-6)M isoproterenol. In this preraration, timolol inconsistently modified the response to epinephrine. In some experiments, epinephrine added to the bathing medium after timolol caused a smaller than normal increase in chloride transport, but in other instances, epinephrine caused a decrease in chloride transport. In the isolated rabbit cornea, 10(-5)M timolol totally blocked the stimulation of chloride transport by 10(-6)M epinephrine. In vivo, topical treatment of rabbit eyes with 0.5% timolol resulted in corneas from these eyes having an inhibited response to epinephrine when incubated in vitro. This inability to stimulate chloride transport persisted for several days following termination of topical treatment with timolol.


Similar molecular properties of granulocyte-macrophage colony-stimulating factors produced by different mouse organs in vitro and in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was partially purified from post-endotoxin serum and conditioned media produced by organs from both normal and endotoxin-injected C57BL mice. The organs used to condition medium were heart, thigh muscle, salivary gland, thymus, spleen, kidney, brain, and femur shaft. The charge properties, molecular weights, and concanavalin A binding profiles of these GM-CSFs were analyzed and compared to purified mouse lung GM-CSF. All the GM-CSFs examined were shown to be gycoproteins since a proportion of the activity (80 to 100%) bound to concanavalin A-Sepharose. The organ-conditioned medium GM-CSFs were purified (3- to 13-fold) by absorption to calcium phosphate gel and chromatography on DEAE-Sepharose (further 2- to 10-fold). Analysis of the DEAE-Sepharose elution profiles indicated that there were two major charge species of GM-CSF eluting at conductivities of 10 and 14 mmho. These partially purified GM-CSFs showed considerable differences in their apparent molecular weights on Sephacryl S-200 (37,000 to 200,000). However, these differences could be eliminated by treating the GM-CSFs with neuraminidase and performing molecular sizing experiments under dissociating conditions (Sepharose CL-6B, 6 M guanidine hydrochloride). Although some of the GM-CSFs showed anomalously high molecular weights (40,000) on gel filtration columns, even under dissociating conditions, this appeared to be due to properties of the sialic acid residues. After neuraminidase treatment all of the conditioned medium GM-CSFs eluted from DEAE-Sepharose as a single peak of biological activity at a conductivity of 10 mmho and from gel filtration columns in the presence of 6 M guanidine hydrochloride as a single molecular weight species of approximately 23,000. GM-CSF from post-endotoxin serum (produced in vivo) eluted from the gel filtration column with an apparent molecular weight of 39,000, but analysis using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that this GM-CSF also had an apparent molecular weight of 23,000.


Purification of a colony-stimulating factor from cultured pancreatic carcinoma cells. Serum-free conditioned medium prepared from an established line of human pancreatic carcinoma (MIA PaCa-2) provides a rich source of colony-stimulating factor (CSF). Two activities distinctly separable by isoelectrofocusing have been identified: a high molecular weight CSF exhibiting greater activity in mouse bone marrow and a low molecular weight CSF more active in human bone marrow. The high molecular weight CSF has been purified 1000-fold to apparent homogeneity by a two-step procedure including isoelectrofocusing and gel filtration chromatography. The purified CSF has a molecular weight of 50,000 and an isoelectric point of 3.7 to 4.6. It is a glycoprotein as shown by periodic acid-Schiff stain and exhibits greater activity in mouse marrow than in human marrow.


Epidermal growth factor stimulates secretion of human chorionic gonadotropin by cultured human choriocarcinoma cells. Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCG-alpha.


Metabolism of radiolabeled corticosterone in an adult with the 17 alpha-hydroxylase deficiency syndrome. [4-14C]Corticosterone was administered to a woman with the 17 alpha-hydroxylase deficiency syndrome and urine was collected for 72 h. Sixty-three percent of the radioactivity was eliminated on the first day, 10.3% on the second, and 3.8% on the third, making a total recovery of 77%. On the first day, 85% of the recovered radioactivity was in the glucuronide conjugates of corticosterone, 10.6% was in the sulfate form of this steroid, and 3.9% was in the free forms of the steroid. On the following 2 days, the proportion of labeled glucuronides and free steroids decreased and that of labeled sulfates increased. On the first day of collection, the major radiolabeled metabolites were 21-hydroxylated steroids (e.g. allo-tetrahydrocorticosterone and 5 alpha- and beta-pregnane-3 alpha,11 beta,20 alpha,21-tetrol), but by the third day, at least 75% of the excreted activity was associated with 21-deoxysteroids, such as 3 alpha,20 alpha-dihydroxy-5 alpha (and beta)-pregnan-11-one and 5 alpha- and beta-pregnane-3 alpha,11 beta,20 alpha-triol. Bacterial metabolism in the intestinal tract is responsible for the dehydroxylation. 6 alpha-Hydroxytetrahydrocorticosterone was tentatively identified among several new metabolites of corticosterone.


Immune function in systemic lupus erythematosus. Impairment of in vitro T-cell proliferation and in vivo antibody response to exogenous antigen. Peripheral blood mononuclear cells from 46 systemic lupus erythematosus (SLE) patients showed reduced ability to proliferate in vitro in response to the soluble antigen, tetanus toxoid, as compared with 96 normal controls. Special studies of 27 untreated SLE patients also revealed significantly decreased blastogenic responses to tetanus toxoid. In both the total and untreated SLE populations, decreased mean tetanus antibody titers also were found as compared with the control population. However, the reduction in antibody titer and blastogenic response was not strictly parallel. A limited immunization program was initiated in low-responding volunteers from the SLE and normal populations. Three out of four SLE patients did not develop a significant blastogenic response despite increases in anti-tetanus titers after immunization, whereas all normals showed significant increases in both blastogenic and antibody responses. The accumulated evidence indicated that the unresponsiveness was the result of a defect in T-cell function. Monocyte reactivity was demonstrated to be normal, and no evidence was found for the presence of suppressor cells, inhibition by immune complexes, or increased prostaglandins to explain the defect.


Studies of immune functions of patients with systemic lupus erythematosus. Complement-dependent immunoglobulin M anti-thymus-derived cell antibodies preferentially inactivate suppressor cells. Patients with systemic lupus erythematosus (SLE) produce excessive amounts of autoantibodies. It has also been demonstrated in several systems that such patients have a relative loss of suppressor thymus-derived (T) cells that inhibit the immune response. This loss of suppressor cells has been suggested as one of the causes of the excessive production of antibodies in patients with SLE. In the present report we have tested the hypothesis that anti-T-cell antibodies found in the plasma of some patients with SLE preferentially kill suppressor cells. T cells from normal individuals can be activated by concanavalin A to develop suppressor cell activity. We therefore cultured normal T cells together with concanavalin A in the presence of plasma or plasma fractions from patients with SLE. We found that plasma from patients with active SLE, in which anti-T-cell antibodies were present, inhibited the development of suppressor activity in such cultures. In contrast, plasma from other active patients and patients with inactive SLE, in which no anti-T-cell antibodies could be detected, failed to block the development of such suppressor activity. Absorption of the plasma that contained anti-T-cell antibodies with T cell, but not non-T cells, could eliminate the suppressor-inhibiting activity of the SLE plasma that contained anti-T-cell antibodies. The immunoglobulin (Ig)M, but not the IgG, fraction of the plasma was shown to possess the inhibiting property and complement was found to be necessary for the effect of such anti-T-cell antibodies. We also demonstrated that exposure of normal T cells to such anti-T-cell antibodies and complement did not affect another population of T cells that could proliferate in response to mitogens.Thus, certain patients with SLE have in their plasma an antibody of the IgM class that can selectively eliminate a population of T cells capable of developing suppressor function. The loss of suppressor T cells in patients with SLE may be the result of the effects of such antibody activity in vivo.


Evidence that increased circulating 1 alpha, 25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis. Mean plasma 1(alpha),25-dihydroxyvitamin D[1(alpha),25(OH)(2)D] was significantly increased and serum parathyroid hormone was suppressed in three patients with sarcoidosis and hypercalcemia. Prednisone lowered the mean plasma 1(alpha),25(OH)(2)D to normal range and corrected the hypercalcemia. To elucidate the mechanism for the increased sensitivity to vitamin D in this disorder, the effects of orally-administered vitamin D(2) were determined in seven normal subjects, four patients with sarcoidosis and normal calcium metabolism and three patients with sarcoidosis and a history of hypercalcemia who were normocalcemic when studied. Serum and urinary calcium, serum 25-hydroxyvitamin D (25-OHD), plasma 1(alpha),25(OH)(2)D and, in some studies, calcium balance were measured. Vitamin D(2), 250 mug a day for 12 d, produced little, if any, change in mean plasma 1(alpha),25(OH)(2)D and in urinary calcium in the normals and in the patients with normal calcium metabolism. In contrast, vitamin D(2) produced increases in plasma 1(alpha),25(OH)(2)D from concentrations which were within the normal range (20-55 pg/ml) to abnormal values and increased urinary calcium in two patients with abnormal calcium metabolism. In an abbreviated study in the third patient, vitamin D(2), 250 mug a day for 4 d, also increased plasma 1(alpha),25(OH)(2)D abnormally from a normal value. There was a highly significant correlation between plasma 1(alpha),25(OH)(2)D and urinary calcium. Serum 25-OHD and serum calcium remained within the normal range in all subjects and patients. These findings provide evidence that the defect in calcium metabolism in sarcoidosis probably results from impaired regulation of the production and(or) degradation of 1(alpha),25(OH)(2)D. Prednisone may act to correct the abnormal calcium metabolism by reducing circulating 1(alpha),25(OH)(2)D.


T lymphocytes and blood eosinophils in early infancy in relation to heredity for allergy and type of feeding. T lymphocytes in 50 one-month-old infants were determined and correlated to heredity for allergy, blood eosinophil counts, and type of feeding. It was found that infants with heredity for atopy had significantly lower relative numbers of T lymphocytes than infants without heredity (p less than 0.05). This difference was particularly obvious in heredity for asthma on the paternal side (p equals 0.001). There was an inverse correlation between T lymphocyte counts and blood eosinophil counts in infants who were cow's milk-fed; i.e., low T lymphocyte counts were associated with high blood eosinophil counts (r = -0.59, p less than 0.01). T lymphocyte counts were not correlated to the type of feeding, whereas blood eosinophils were significantly higher in the cow's milk-fed than in the breast-fed group (p less than 0.01). The results suggest that atopic allergy may be associated with a genetically determined lymphocyte defect.


Neutropenia in three patients with rheumatic disorders. Suppression of granulopoiesis by control-sensitive thymus-dependent lymphocytes. A man with polymyalgia rheumatica (patient 1) and two patients (2 and 3) with Felty's syndrome had neutropenia at the time of diagnosis. Bone marrow samples in each patient were cellular but showed an "arrest" of granulocyte maturation at the myelocyte stage. Agar colony growth of marrow cells from each patient was subnormal but increased after removal of sheep erythrocytes rosette-forming cells (thymus-dependent [T] cells) from marrow cell suspensions before culture. Preincubation of marrow cells with cortisol also enhanced colony growth. Maximum enhancement with cortisol occurred at 1 mM (patient 1), 1 microM (patient 2), and 10 nM (patient 3). Cortisol failed to enhance colony growth after removal of T cells from marrow cell suspensions. Peripheral blood lymphocytes (PBL) and PBL-conditioned medium from all three patients inhibited colony growth of normal human marrow cells. Cortisol treatment of PBL or T depletion from PBL abrogated the inhibition in coculture and with conditioned medium. Prednisone therapy resulted in the disappearance of suppressor T-cell function concomitant with hematologic improvement in patients 2 and 3, but suppressor T cells persisted in patient 1, who did not respond to prednisone. We conclude that cortisol-sensitive T lymphocytes inhibited granulopoiesis in vitro probably by elaboration of a soluble factor or factors. Our results suggest (a) that neutropenia in these patients resulted, at least in part, from T-cell suppression of granulopoiesis, (b) that the effectiveness of prednisone therapy was a result of its inhibition of suppressor T cells, and (c) that responses to glucocorticoid therapy may be predicted in such patients with the agar culture technique and cortisol dose response in vitro.


Selective cell surface expression of thymus leukemia antigen during S phase of the cell cycle. The expression of the thymus leukemia antigen (TL) was studied on a murine leukemia cell line (ASL-1W) grown in vitro and separated into cell cycle phases by velocity sedimentation or growing synchronously in culture. The expression of TL was determined qualitatively by direct cytotoxicity and quantitatively by a modified inhibition of cytotoxicity assay. TL expression was found to vary with DNA synthesis. The hypothesis that expression is coordinately regulated with DNA synthesis, and the relationship of this to the restricted expression of TL on rapidly dividing cells is disucssed.


Inhibition of lymphocyte blastogenesis by C3c and C3d. The C3 cleavage products C3c and C3d were tested for their ability to alter the immunoproliferative response of human peripheral mononuclear cells to the antigens SLO and SK-SD, and to the mitogens PHA and PWM. It was found that both C3c (30 to 120 micrograms/ml) and C3d (10 to 40 micrograms/ml) inhibited lymphocyte blastogenesis in the presence on antigens but not mitogens, when cells were cultured in either autologous plasma or FCS. Similarly, the response to antigens of cell populations enriched for T lymphocytes was inhibited, whereas the response to optimal or suboptimal doses of mitogens was unaffected. When nonadherent (NA) cells were reconstituted with increasing numbers of adherent (AD) cells to potentiate the proliferative response of NA cells to the antigen SLO, the addition of either C3c or C3d abolished the potentiation of the response at low levels of reconstitution. However, at given dose of C3c or C3d, addition of excess AD cells could restore the proliferative response. These results suggest that both C3c and C3d can inhibit T cell proliferation in response to antigen and that they may act at the level of the monocyte-T lymphocyte interaction to modulate cellular immune responses.


Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages. The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.


Synergistic cytotoxicity. II. In vitro arming of monocytes and T cells by a heat labile fraction of human plasma. In a previous paper we demonstrated that freshly obtained human plasma contain a heat labile nonantibody factor that induced human mononuclear cells to become nonspecifically cytotoxic toward xenogeneic but not allogeneic RBC targets. We now present evidence that this factor has a loose affinity for human monocytes and human T cells and can arm then to kill xenogeneic RBC targets. Furthermore, proteolytic enzymes markedly enhance this arming effect. This ability to be armed by a heat labile component found in fresh human plasma and the fact that proteolytic enzymes markedly enhance cytotoxicity clearly dissociate this model of nonspecific cytotoxicity for previously reported NK models.


Suppression of cytotoxic response to histoincompatible cells. I. Evidence for two types of T lymphocyte-derived suppressors acting at different stages in the induction of a cytotoxic response. Spleen and thymus cell populations from normal or allograft tolerant mice have been cultured for 5 days with specific alloantigens and examined for their reactivity in three assay systems. No consistent correlation was observed between the production of cytotoxic T cells (CTL) in these cultures and the ability of such cultured cells to inhibit specifically a CML response from fresh normal spleen cells directed to the priming alloantigens. Furthermore, suppressor cells measured in this latter assay were apparently distinct from those able to inhibit the production of cytotoxic lymphocyte precursors (CTLp) from bone marrow stem cells in lethally irradiated bone marrow protected mice. Velocity sedimentation experiments confirmed that both the precursor and effector cells for the two suppressor systems were physically separable, and were distinct from CTLp or CTL, respectively. Precursor cells for the two suppressor systems investigated belong to the short-lived cortical thymus cell population.


Suppression of cytotoxic response to histoincompatible cells. II. Analysis of the role of two independent T suppressor pools in maintenance of neonatally induced allograft tolerance in mice. The kinetics of appearance of the precursors of SuppA cells (capable of inhibiting CTLp leads to CTL) or SuppB cells (capable of inhibiting (stem cells leads to CTLp) in neonatal mice, as well as the appearance of SuppA/SuppB cells in mice given neonatal innoculations of semiallogeneic spleen cells has been investigated. The data obtained are consistent with the idea that SuppA cells have a natural role to play in the induction of neonatal tolerance, whereas SuppB cells may be more important for the maintenance of the tolerant state. Unlike the level of SuppB cells, the level of SuppA cells in tolerant mice seems to be modulated by the presence of the tolerizing determinants. Data are provided to show that SuppB cells, once induced in tolerant mice, can adoptively transfer specific allograft unresponsiveness to newborn syngeneic mice in the absence of added tolerizing antigen, whereas SuppA cells are not able to do so. These data fit the notion that SuppB cells may be responsible for the phenotype of clonal deletion.


Activation of membrane phospholipase A by guinea pig lymphotoxin (GLT). The effect of GLT on target cells (L.P3 cells) labeled with radioactive fatty acids was examined. After incubation for 1 or 2 hr, no change in the survival ratio was observed, but radioactive fatty acid was released from phospholipids of the cells. When incubation was continued for 4 or 5 hr, the survival ratio was decreased with a concomitant increase of the radioactivity of triglyceride of the target cells and enhancement of calcium uptake into the target cells. These results could be explained by activation of membrane-bound phospholipase A after the binding of GLT to cell surface receptors.


Susceptibility of Friend virus antigen-modulated erythroleukemic cells to lysis by T lymphocytes from mice with dormant Friend virus infections. Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.


Activated T cells in the synovial fluid of arthritic patients: characterization and comparison with in vitro activated human and murine T cells in cooperation with monocytes in cytotoxicity. Lymphocytes were separated from the synovial fluid of 34 arthritic patients. The majority of lymphocytes rosetted with SRBC and were thus of T origin. A proportion of these cells were activated as indicated by their high 3H-thymidine incorporation in vitro, the formation of "stable" E rosettes, and their ability to attach to autologous monocytes and to human cells. Such attached activated T cells enhanced the cytotoxic activity of monocytes. Human and murine-activated T cells generated in mixed lymphocyte cultures also attached to cell lines of the same species, and after their attachment they enhanced the cytotoxic activity of monocytes and natural killer cells. It is suggested that one of the possible roles of activated T cells in immunologically damaged tissues is the attraction of circulating nonspecific killer cells to the site of the response.


Failure of NZB spleen to respond to prethymic bone marrow suppressor cells. Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.


Cyclical changes in susceptibility of a myeloma tumor (LPC-1) to immune destruction. I. Changes in reactivity with cytotoxic T lymphocytes and anti-H-2d sera. One of six transplantable ascites tumors of BALB/c mice was found to become periodically resistant to cytotoxic T lymphocytes (CTL). About 12 days after LPC-1, a myeloma tumor, was transplanted it became resistant to lysis by allogenic CTL (anti-H-2d) and by CTL directed to trinitrophenyl groups or minor histocompatibility antigens. Susceptibility to lysis by all of these CTL was regained within 2 to 4 days after transfer of resistant cells to a fresh BALB/c host. These changes were recurrent: in each transplantation cycle the early LPC-1 cells were susceptible and the late cells were resistant to CTL. Analyses with antisera (B10 anti-B10.D2) showed that the serologically recognized products of the H-2d haplotype were reduced about 10-fold on the LPC-1 cells that were resistant to CTL.


Three suppressor systems in human blood that modulate lymphoproliferation. Three suppressor system of T lymphocyte proliferation found in normal blood were characterized. The adherent cell suppressor system (ACSS) is effected by a steroid and radioresistant monocyte that survives well in culture. The prostaglandin-related suppressor system (PgSS) is effected by a similar cell but is distinct from the ACSS in terms of magnitude of suppression, effective monocyte concentration, and carrageenan or indomethacin sensitivity. The induced suppressor system (ISS) is effected by a T lymphocyte that is partially radiosensitive and loses activity after 24 hr in culture. Although the ISS is unaffected by age, ACSS and PgSS activity is, overall, higher among elderly than among young adult subjects. Activation of these three suppressor systems does not require cell replication. They are not restricted by histocompatibility barriers.


Subpopulations of human T lymphocytes. X. Alterations in T, B, third population cells, and T cells with receptors for immunoglobulin M (Tmu) or G (Tgamma) in aging humans. Peripheral blood from 120 healthy subjects, of whom 59 were young (35.5 +/- 9.6 years) and 61 aging (69.2 +/- 4.2 years), was examined for the proportions and numbers of lymphocyte populations, with a battery of surface markers. Absolute lymphocyte count and T,B and third population cells were comparable in both groups. Tgamma cell proportions were significantly (p less than 0.001) increased in aging subjects when compared with the young subjects. However, this difference was more significant (p less than 0.001) when aged females were compared with the young females as compared to aged males vs young males (p less than 0.05). When data were anlayzed for absolute numbers of Tmu and Tgamma cells, a similar significant decrease in Tmu, and increase in Tgamma cells were observed. Interestingly, when these data were anlayzed according to gender, significant differences in Tmu and Tgamma cell number were observed between young and old females but not between young and old men. Implications of these results are discussed.


In vitro differentiation of B lymphocytes from pre-B cells. Adult bone marrow contains both B lymphocytes and their immediate precursors, pre-B cells. These two cells differ in size and can be separated by velocity sedimentation; B cells are enriched in the subpopulation of cells sedimenting at between 2.0 and 3.5 mm/hr and pre-B cells in the subpopulation between 5.0 and 7.0 mm/hr. Incubation of pre-B cells in vitro for 4 or 5 days leads to their differentiation into functional B lymphocytes. The transition form pre-B to B appears to occur in two steps. The first step gives mitogen responsive B cells with an intermediate sedimentation velocity and the second step produces typical small, slowly sedimenting B cells. Pre-B cells can be quantified by using a limiting dilution assay and occur at a frequency of 1/60 in the subpopulation of rapidly sedimenting bone marrow cells.


The induction of TNP-altered, self-reactive human cytotoxic T cells by soluble factors: the role of Ia antigens. This report demonstrates that human peripheral blood T lymphocytes, triggered by allogeneic cells or soluble antigens, elaborate helper factor(s) that promotes the in vitro differentiation of TNP altered-self reactive human CTL. Helper factor(s) alone is not sufficient for the generation of these killer cells, but requires the presence of TNP-derivatized autologous stimulators during sensitization. Additional experiments were performed with antisera to a human Ia-like antigen, p23, 30. These studies indicate that human Ia-like antigens play an important role in both the induction of helper factor(s) and in the functional activity of preformed helper factor(s) molecules.


Helper T cell-replacing factors secreted by thymus-derived cells and macrophages: cellular requirements for B cell activation and synergistic properties. The biologic activities of helper T cell-replacing factors derived from concanavalin A-stimulated murine T cells (TRF-T) and from lipopolysaccharide-activated macrophages (TFR-M) have been compared. TRF-T stimulates immune responses to heterologous erythrocyte antigens (SRBC and BRBC) in T cell-depleted spleen cultures but not in macrophage-depleted spleen cultures. TRF-M stimulates immune responses in both T cell-depleted and macrophage-depleted spleen cultures. Under conditions where LPS stimulates the release of TRF-M from cultures of activated macrophages, TRF-t has no effect on TFR-M production. Thus. TRF-T does not appear to function by stimulating the release of TRF-M from macrophages. In macrophage-depleted spleen cultures, saturating concentrations of TRF-T and TRF-M when mixed together exhibit striking synergistic effects on the induction of immune responses to erythrocyte antigens. The kinetics of the synergistic effects of TRF-M and TRF-T are consistent with an effect of TRF-M on the production of TRF-T sensitive B cells.


Immune responses to weakly immunogenic virally induced tumors. III. Genetically unrestricted cytolysis of allogeneic tumor target cells. Splenocytes from A mice injected with YAC-1 or RBL5 could generate, after in vitro culture with or without stimulation, a genetically nonrestricted cytotoxic response against the allogenic tumor RBL5. YAC-1 tumor is an in vitro carried tumor induced in A mice (H-2a) by Moloney virus. RBL5 tumor is a Rauscher virus-induced tumor of C57BL/6 mice (H-2b). These tumors cross-react serologically. The effector cells that were generated after the in vitro cultivation recognized tumor-associated antigens on the target cells. H-2 alloantigens were not recognized by the effector cells. The effector cells that killed RBL5 tumor in a genetically nonrestricted manner were identified as T cells. The in vivo carried tumor YAC, in contrast to the in vitro carried tumor YAC-1, could not induce anti-RBL5 reactive cells in A mice. Instead, YAC tumor induced suppressor cells in A mice, which could abrogate the anti-RBL5 cytotoxic response of RBL5-primed splenocytes, but not that of YAC-1 primed splenocytes.


T100: a new murine cell surface glycoprotein detected by anti-Lyt-2.1 serum. A cell surface glycoprotein (designated T100) of apparent m.w. 100,000 by SDS-PAGE under reducing and nonreducing conditions was precipitated from NP-40 extracts of surface radiolabeled thymocytes from a variety of inbred strains of mice by the standard noncongenic Lyt-2.1-typing serum. The inbred stain distribution, trypsin sensitivity on intact cells, and apparent m.w. of T100 suggest that it is different from Lyt-2.1. Inheritance and expression of T100 suggest that it is determined by an allele at a single locus, and testing of CXB recombinant inbred strains and B6.C minor histocompatibility congenic strains suggest that this locus is linked to H-25. Antiserum absorption experiments, two-stage cytotoxicity assays, and results of immunoprecipitations performed after prebinding antibody to radiolabeled thymocytes suggest that some T100 is accessible to antibody on the intact cell surface. However, for unknown reasons the number of cells required to absorb anti-T100 precipitating activity from antiserum was much higher than for removal of anti-Lyt-2.1 activity. A molecule with properties of T100 was also detected on lymph node cells and on the AKTB-1 lymphoma.


Characterization of the immunosuppressive state during Schistosoma mansoni infection. Analysis of a murine model of schistosomiasis revealed that both the thymus (T)- and bursa (B)-derived compartments of the immune system are modified during acute infection. The functional capacity of T and B lymphocytes to respond to mitogenic stimuli and the humoral response to thymus-dependent (SRBC) and thymus-independent (DNP-Ficoll) antigens are severely depressed. In addition, it was found that suppressor cells capable of inhibiting the response of normal lymphocytes to SRBC arise during acute infection. Although the splenic frequency of T (theta) and B (Ig+) cells remained constant during chronic infection, quantitative changes were detected in each population. In the T cell pool there was a decrease in the percentage of Ly-1+ cells and a concomitant increase in Ly-1+, 2+, 3+, cells, whereas the B cell pool showed a progressive loss of complement receptor-bearing lymphocytes, which apparently was the result of inactivation of surface complement receptor by a serum factor specifically found in infected mice. Characterization of the serum factor strongly suggests it is an immune complex. Thus, it appears that both suppressor cells and immune complexes contribute to changes noted in the immune system during acute schistosomiasis. Additional studies carried out in mice after unisexual infection revealed that egg production is not a necessary prerequisite for several of the immunologic phenomena associated with acute schistosomiasis.


Immune responses during human schistosomiasis mansoni. VI. In vitro nonspecific suppression of phytohemagglutinin responsiveness induced by exposure to certain schistosomal preparations. Simultaneous in vitro exposure of human peripheral blood mononuclear cells to phytohemagglutinin-P (PHA) and either soluble schistosomal egg antigenic preparation (SEA) or soluble cercarial antigenic preparation (CAP) obtained from Schistosoma mansoni resulted in decreased responsiveness as compared to exposure to PHA alone. The addition of a soluble adult worm antigenic preparation (SWAP) did not predictably alter PHA responses in this system. The suppression due to in vitro exposure to either SEA or CAP was expressed whether the lymphocyte donors were S. mansoni patients (early infection, chronic, or treated), or uninfected subjects. The degree of suppression was related to the concentration of SEA used, and the timing of exposure. Preexposure to SEA for 3 days before the addition of PHA resulted in more potent suppression. However, a delay in the time of the addition of SEA of 6 and 24 hr after PHA exposure decreased and eliminated, respectively, its suppressive capacity. SEA and CAP were not directly toxic to responding cells, and appeared to exert their nonspecific suppressive influences through T lymphocyte-related mechanisms. It was observed that although these suppressive events could be induced and observed in vitro, the responsiveness of S. mansoni patient lymphocytes to PHA was equal with that of uninfected controls.


A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen. Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.


In vivo generation of hapten-specific killer T cells without elimination of suppressor cells. We have provided evidence to demonstrate that hapten-specific killer cells can be generated in vivo toward hapten-conjugated syngeneic splenic cells. The critical aspect is to provide an auxillary cellular antigenic stimulus in addition to the hapten-conjugated syngeneic cells. In our experiments this stimulus was CBA/J splenic cells that possess MIs disparate but H-2 compatible antigens with C3H/HeN hosts.  The Mls antigen has been shown by others to activate helper T cells in vitro to synthesize KAF, a signal that prekiller cells require besides target antigen. Killer cells (shown to be T cells) as well as helper T cells were found to be derived from the C3H host. Induction in the host animal of partial tolerance to the auxiliary cells possessing Mls antigen abrogated the response. This system, besides providing a better understanding of the control mechanisms involved in the development of T killer cells in vivo, points to a way in which the latter may be generated-ng suppressor cells.  The latter principle may prove highly useful in certain clinical situations.


Study on the turnover of the receptor for the third component of complement on human lymphoid cells. The in vitro turnover of the receptor for the third component of complement (C3) was studied in normal peripheral blood lymphocytes (PBL) and in lymphoblastoid cells from established cell cultures of both "normal" and "malignant" origin. The turnover was evaluated by studying i) the disappearance rate of the C3-receptor in cells in which the protein synthesis was blocked by cycloheximide and puromycin, ii) the reexpression rate of the C3-receptor after treatment of the cells with either rabbit antiserum against B lymphocytes or mouse C activated through the alternative pathway by inulin. The results show that the C3-receptor of all the lymphoid cells has roughly a half-life of about 3 to 4 hr. However, the cultured lymphoblastoid cells were less sensitive than normal PBL to inhibition by cycloheximide and showed a faster reexpression rate of the C3-receptor. A spontaneous release of the receptor was found to occur, since a receptor-like activity was detected in the spent culture medium of long-term cultured lymphoid cells.


A physicochemical and immunologic comparison of the cell growth inhibitory activity of human lymphotoxins and interferons in vitro. The physicochemical, immunologic, and biologic relationships between humam lymphotoxins (LT) and interferons (IF) present in supernatant fluids from lectin-stimulated peripheral blood lymphocytes (PBL) and a continuous B-lymphoblastoid cell line (PGLC-33h) were analyzed. LT activity obtained from lectin-activated PBL could not be resolved from IF activity by gel filtration chromatography. LT activity eluted in multiple peaks of activity at 70 to 90,000, and 40 to 50,000 m.w., characteristic of alpha and beta LT, respectively. IF activity in these supernatant fluids eluted as a broad band between 35 and 80,000 m.w., also suggestive of molecular heterogeneity. In contrast, this m.w. heterogeneity was not observed in LT and IF activities obtained from the PGLC-33h cell line. LT and IF eluted as separate peaks of activity at 90,000 and 25,000 m.w., respectively. In addition, acid and heat lability of PGLC-33h IF suggested similarity to type II IF. Immunologic studies, with a rabbit anti-alpha class serum that neutralized LT activity from both PBL and PGLC-33h, did not affect IF activity from either of these sources. Supernatant fluids from PGLC-33h cultures were also capable of inhibiting the proliferation of HeLa cells in vitro. The growth inhibitory activity was attributed to LT- and IF-like molecules. This evidence suggests that although cytotoxic and anti-viral activities were due to separate molecules, LT and IF have overlapping biologic activities in their ability to inhibit the proliferation of cells in vitro.


Macrophage cell lines produce a cytotoxin. Cytotoxic activity, measured by release of tritium from 3H-(methyl)-thymidine-labeled 7943 cells, was found in culture fluids from four of five macrophage cell lines. In the P388D1 cell line, cytotoxic activity was highest after 7 days of culture, 2 days after the cells reached confluence. The activity was not lost upon dialysis or lyophilization. When culture fluids were chromatographed on Sephadex G-200, two activity peaks were obtained: one in the void volume and the other corresponding to a m.w. of 40,000 to 50,000.


Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation. In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.


Carboxymethyl cellulose, a nonimmunogenic hapten carrier with tolerogenic properties. This communication reports on the tolerogenic properties of carboxymethyl cellulose (CMC) as a nonimmunogenic carrier for 2.4 dinitrophenyl (DNP) and the benzylpenicilloyl determinant (BPO). Either normal or primed mice, given an optimal dose of 250 micrograms per animal of DNP CMC, when challenged with an immunogenic form of the hapten as early as 30 min or as late as 21 days thereafter were completely and specifically unresponsive to it. Experimental evidence suggests that this unresponsiveness is not due to suppressor cells. Furthermore, DNP CMC induces tolerance in vivo but fails to do so in vitro under conditions at which other tolerogenic carbohydrate hapten conjugates such as DNP-dextran do. This together with comparative studies of tolerance induction kinetics by DNP CMC and DNP-dextran in vivo led us to conclude that molecular properties other than the epitope density must be attributed to CMC's tolerogenic potential. CMC may also be used as a tolerogenic carrier for BPO with respect to IgE antibody production. Thus, normal or primed mice injected with the BPO CMC conjugate were found specifically unresponsive to a challenge with an immunogenic form of penicillin.


Studies of congenitally immunologic mutant New Zealand mice. II. Absence of T cell progenitor populations and B cell defects of congenitally athymic (nude) New Zealand Black (NZB) mice. Congenitally athymic (nude) mice on an NZB, NZW, and BALB/c background were produced by repetitive selective backcrossing. F'12 generation nude mice of these three strains were compared to their littermate nu/+ controls with respect to survival, histology, blood counts, splenic surface markers, response to mitogens, spontaneous plaque-forming cells, and appearance of naturally occurring thymocytotoxic antibodies (NTA). Under specific pathogen-free conditions, NZB nude mice survive less than 3 weeks, dying of a runting-like disease with infection by local normally noninvasive organisms. A contributing factor to his premature death is the relative absence of T cell progenitor populations in the NZB nude vs NZW nude or BALB/c nude groups. Furthermore, NZB nude mice have a significantly earlier appearance of NTA than nu/+ littermates and likewise appear to have heightened spontaneous polyclonal B cell responses against the haptens dansyl, nitroiodophenyl, trinitrophenyl,2,4 dinitrophenyl, and sulfonate. It is suggested that NZB mice have several critical immunologic defects, including abnormalities of thymic epithelial cells, T cell differentiation pathways, and chronically polyclonal activated B cell populations. These defects interact to produce the clinical expression of autoimmunity.


Genetic and cellular requirements for primary antibody immune response to Thy-1 antigens in mice. An effective anti-Thy-1 response requires at least three conditions to be simultaneously fulfilled--1) The responder has to share an H-2 haplotype, or at least K and D alleles, with the donor of the Thy-1 disparate thymocytes, 2) The responder has to differ from the donor by some non-H-2 cell-surface antigens, and 3) The responder has to possess functionally capable T cells. All these requirements are consistent with the hypothesis that Thy-1 antigens and the non-H-2 antigen(s), the latter serving as carrier, form a complex that is viewed as a variant of self and then recognized in the context of the H-2 molecules by the responder's T cells. An attractive feature of the proposed hypothesis is that it could be applicable to a variety of cell-surface and also exogenous antigens.


PPD-induced immunoglobulin production in human peripheral blood lymphocytes. I. Necessary conditions for inducing the response. The ability of PPD to induce Ig production in human PBL was investigated. PPD proved to be a good B cell activator for inducing polyclonal Ig production in PBL from healthy Japanese. Comparative studies of this response with PWM-induced Ig production showed that the cellular mechanisms involved in the two responses were different. First, PBL from an atypical individual with a deficient IgM production to PWM responded normally to PPD with IgM production as well as IgG production. Secondly, in IgG production, the effects of the two mitogens (PPDand PWM) were additive. An analysis of the cellular requirements in PPD-induced Ig production clearly demonstrated that T cells played a role in this response as well as in the PWM-induced response. However, the head-to-head comparative study on the titration curves of helper T cells in the two responses showed that PWM-induced helper activity was 2 to 5 times more effective than PPD-induced helper activity. Moreover, PPD-induced helper activity was shown to be more sensitive to ionizing radiation than was PWM-induced helper activity. Thus, this system of PPD-induced Ig production may provide a useful tool for understanding the human antibody production system as well as the PWM-induced response.


Physicochemical characterization of lymphocyte-activating factor (LAF). The LAF produced by the mouse macrophage cell line, P388D1, is a single polypeptide chain of m.w. 12,000 to 16,000 daltons. Native LAF was destroyed by Streptomyces griseus protease, but not by trypsin, chymotrypsin, and papain, although in the presence of 8 M urea, papain completely destroyed LAF activity. LAF did not bind to concanavalin A-Sepharose, suggesting that LAF does not contain significant amounts of mannosyl or glycosyl residues. Since LAF activity was not inactivated by a treatment of reduction and alkylation the active conformation of LAF does not appear to be dependent on disulfide linkages. LAF was not irreversibly denatured by 8 M urea or 0.1 to 0.5% SDS. On SDS-polyacrylamide gels, the m.w. of LAF was 12,000 daltons, as compared to a value of 16,000 daltons, as determined by gel filtration. The isoelectric point of LAF was 5.0 to 5.4 as determined on 7.5% acrylamide gels (pH 3 to 10). On the basis of these results it appears that the P388D1 cell line-derived LAF is a relatively stable molecule that shares several physicochemical properties with normal human and mouse macrophage-derived LAF.


Natural thymocytotoxic autoantibodies in autoimmune and normal mice. Natural thymocytotoxic autoantibodies (NTA) were found in all mouse strains. Among those strains that show autoimmune syndromes resembling human systemic lupus erythematosus (SLE), the NZB and NZBxNZW had high levels of NTA, the BXSB had moderate levels, and the MRL/1 and MRL/n had very low levels. In addition, some normal strains had high levels, sometimes even higher than the autoimmune strains. The NTA were mostly IgM and were present, but not concentrated, in the cryoprecipitates of teh autoimmune mouse strains. In most strains, they were directed toward an antigen shared by thymocytes and brain. The failure to find high levels of NTA in all autoimmune mouse strains, as well as the finding of very high levels in some normal strains, make it unlikely that such auto-antibodies are a fundametnal etiologic factor in all murine SLE.


Lyb-7, a new B cell alloantigen controlled by genes linked to the IgCH locus. Anti-Lyb-5.1 serum contains antibodies against two different B cell surface antigenic determinants, Lyb-5.1 and Lyb-7.1, which are defined in cytotoxicity and functional tests, respectively. The antibody against Lyb-7.1 is identified by its ability to specifically inhibit in vitro primary antibody responses to TNP-Ficoll. Anti-Lyb-5.1 serum can be made monospecific for anti-Lyb-7.1 activity by absorption with spleen cells from AL/N mice which have been typed as Lyb-5.1+, Lyb-7.1-. Lyb-5.1 and Lyb-7.1 are each under control of one or one set of closely linked genes. The loci specifying Lyb-5.1 and Lby-7.1 are not linked to each other nor to M1s and H-2 loci. However, the gene controlling the expression of Lyb-7.1 is linked to the genes coding for the constant region of immunoglobulin heavy chains.


Genetic and biochemical evidence for the involvement of a bacterial component in the mitogenic properties of polyribonucleotides on murine B lymphocytes. The mitogenic response of C3H/HeJ mice to the B cell mitogens, poly C and poly I, is approximately one-half the response measured in various LPS-responder strains. C3H/HeJ mice respond normally to poly I:C, the heteroduplex polymer. The low responder phenotype of C3H/HeJ mice to poly C and poly I is shown by an analysis of (C3H/HeJ x C57BL/6J-By-Ps)F1 X C3H/HeJ backcross progeny to result from a gene locus that is closely linked or identical to the defective LPS response locus expressed by the C3H/HeJ strain. The entire mitogenic activity in poly C preparations and most of the mitogenic activity in poly I preparations is insensitive to ribonuclease degradation. Hot aqueous phenol extraction of the polynucleotides separates the majority of the mitogenic activity that is soluble in the combined interface and phenol phase fraction from the aqueous soluble polynucleotides. The ribonuclease-insensitive, phenolsoluble contaminant elicits a reduced response in C3H/HeJ mice as compared to an LPS responder  strain. We conclude that 1) poly C has no inherent mitogenic activity; 2) poly I preparations contain both ribonucleasesensitive and insensitive mitogenic activities; 3) the ribonuclease-resistant mitogenic activity in polynucleotide preparations has properties unlike those of LPS or lipid A; and 4) the product of LPS response gene has an effect upon the mitogenic stimulation of spleen cells by the contaminant.


Characteristics of in vitro production of antibodies to DNA in normal and autoimmune mice. The in vitro production of antibodies to dsDNA was studied with spleen cells from normal and autoimmune mice. After culture for 4 days, the binding of dsDNA in the culture supernatant was measured by a radioimmunoprecipitation assay. The production of antibodies to dsDNA by spleen cells appeared at 15 hr after culture and reached a plateau at 24 hr. No antibodies were produced by thymus cells or splenic T cells. The specificity for dsDNA was shown by competitive inhibition with nonradioactive nucleic acids. Autoimmune strains of mice (NZB/NZW, BXSB, MRL/1) produced more antibodies to dsDNA than did several control strains. Young B/W mice and control strain mice produced mainly IgM antibodies, whereas older B/W mice produced predominantly IgG antibodies to dsDNA. The in vitro production of antibodies to dsDNA by aged B/W spleen cells was macrophage and T cell dependent.


In vitro studies on lymphocyte differentiation. II. Generation of terminal deoxynucleotidyl transferase-positive cells in long-term cultures of mouse bone marrow. The enzyme TdT is the earliest known marker of lymphocytic differentiation in rodents. Cells containing this enzyme were demonstrated in suspension cultures of mouse bone marrow cells that had been maintained in vitro for periods of 7 to 45 days. The cells were detected by immunofluorescence using purified antibodies to homogeneous TdT. Between 0.04 and 2.0% of cultured bone marrow cells from a variety of mouse strains were positive. More than 40% of the TdT-positive cells incorporated 3H-thymidine during a 20-min pulse. Surface Ig and Thy-1 antigens were not detected on the TdT-positive cells. The prevalence of TdT-positive cells was decreased 10-fold in cultures that had been treated with 10(-6) M hydrocortisone 24 hr before  harvesting. The results indicate that lymphoid progenitor cells can be generated in vitro.


Induction of secretion of IgM from cells of the B cell line 38c-13 by somatic cell hybridization. Cells of the murine B cell line 38C-13 possess immunoglobulins of the IgM class on their surface but do not secrete them. Upon hybridization of 38C-13 cells with murine myeloma cells, hybridoma clones were obtained that secreted both pentameric IgM of 38C-13 origin and the myeloma protein. All hybridoma clones synthesized and secreted large amounts of homogeneous IgM with a half disappearance time of about 2 hr, typical of mature plasma cells. Concomitantly with the induction of IgM secretion, the hybridoma cells lost their surface IgM. The possibility of separate pathways for the synthesis of membrane and secreted IgM is discussed.


Genetic control of immune response to sperm whale myoglobin in mice. II. T lymphocyte proliferative response to the synthetic antigenic sites. The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one "nonsense" peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the "nonsense" peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.


The effect of nucleosides and deoxycoformycin on adenosine and deoxyadenosine inhibition of human lymphocyte activation. Micromolar deoxyadenosine inhibits leucine uptake during the 1st day of proliferation in mitogen-stimulated lymphocytes if adenosine deaminase is inhibited. This inhibition occurs before DNA synthesis begins, suggesting that deoxyadenosine can affect mitogenesis by mechanisms that do not involve ribonucleotide reductase inhibition. If deoxyadenosine addition to mitogen-stimulated lymphocytes is delayed to the 2nd or 3rd day post-stimulation, inhibition of proliferation is markedly reduced. Although the time dependence of deoxyadenosine toxicity resembles that of adenosine, these compounds appear to inhibit early protein synthesis by different mechanisms: 1) deoxycoformycin markedly potentiates deoxyadenosine but not adenosine; 2) deoxycytidine and thymidine reverse deoxyadenosine toxicity but do not alter adenosine toxicity.


Release of immature cells from the thymus during solid tumor growth: identification by assay of TdT activity. In vivo anti-tumor activity of spleen cells from C3H/eb mice bearing a syngeneic fibrosarcoma was shown previously to decline to an undetectable level and be replaced by tumor-enhancing activity as tumor growth proceeds. In the light of our findings that thymocytes in the early stages of thymic processing can bring about tumor enhancement, we postulated that premature release of thymocytes and their accumulation in the spleen might account for the loss of the anti-tumor response. In the present experiments an injection of thymocytes did in fact cancel the anti-tumor response of reactive splenocytes from tumor-bearing mice. In order to determine whether premature thymocyte release occurs naturally in the tumor-bearing animals, we assayed activity of the enzyme TdT (as a marker for thymus cells) in the spleens of these mice during progressive tumor growth. Cells with TdT activity were clearly evident in the spleens of the tumor-bearing animals, were derived from the thymus, and accumulated in parallel to the loss of anti-tumour reactivity.


Lymphocytes bearing Fc receptors for IgE. II. Induction of Fcepsilon-receptor bearing rat lymphocytes by IgE. The proportion of lymphocytes bearing receptors for IgE (FcepsilonR) markedly increased after infection of rats with Nippostrongylus brasiliensis (Nb). The FcepsilonR-bearing lymphocytes from the infected animals bound more IgE-coated erythrocytes in rosette assay than FcepsilonR-bearing cells from normal rats, suggesting that the number of FcepsilonR per cell may also increase following the infection. In contrast, the number of IgE-receptors on peritoneal mast cells did not change after Nb infection. The increase in the proportion of FcepsilonR-bearing lymphocytes in Nb-infected rats is probably due to an increased concentration of IgE in the environment. The proportion of FcepsilonR-bearing cells in normal rat lymphocyte suspensions increased by culture of the cells with rat IgE of 1 microgram/ml or higher concentration. Other immunoglobulins such as rat IgG, human IgE, or rabbit IgG failed to induce either FcepsilonR-bearing cells or FcgammaR-bearing cells. It was also found that induction of Fc receptors by rat IgE is confined to FcepsilonR. Kinetic studies on the induction of FcepsilonR-bearing lymphocytes in vitro showed that the proportion of these cells in lymphocyte suspensions increased within 8 hr incubation with rat IgE but not within 4 hr. Evidence was obtained that both RNA synthesis and protein synthesis, but no DNA synthesis, are required for the induction of FcepsilonR-bearing cells or the expression of the receptors on the cell surface.


The induction of cytolytic T lymphocytes with syngeneic trinitrophenyl-coupled membranes. Evidence is presented that trinitrophenyl-coupled tumor membranes are able to induce cytolytic T lymphocytes (CTL) when co-cultured with syngeneic spleen cells. These haptenated membranes stimulate spleen cells from naive and immune mice. The specificity of these CTL is determined by the H-2 antigens of the membranes used for stimulation.


Aging increases expression of LPS-induced autoantibody-secreting B cells. Escherichia coli lipopolysaccharide (LPS), a polyclonal B cell activator, has been employed to achieve in vitro stimulation of autoantibody-secreting B cells in young adult and aged mice of long-lived strains as assayed in a hemolytic plaque technique to syngeneic mouse erythrocytes. Aged 21- to 24-month-old C57BL/6J and (C57BL/10Sn x C3H/HeDiSn)F1 mice were found to express 3 to 4 times as many LPS-induced plaque-forming cells (PFC) to autologous erythrocytes than did younger 6-month-old animals. With the use of cyclophosphamide (CY), a significant enhancement of auto-PFC production in young mice occurred, approaching levels found in non-CY-treated old mice. Thus, autoreactive clones of lymphocytes exist in the spleens of young adult mice, but under normal circumstances produce little autoantibody. The situation in aged members of these strains, therefore, does not seem to involve an actual increase in numbers of autoreactive B cells, but may possibly involve some form of deregulation, permitting increased age-related expression of autoreactive lymphocyte clones.


Radioimmune double PEG precipitation technique for detecting complexed IgE. The presence of IgE in immune complex-enriched fractions prepared by 3% polyethylene glycol (PEG) precipitation was studied. The dissolved PEG-insoluble fraction was incubated with [125I]anti-IgE antibodies an then precipitated again with 3% PEG. Radioactivity in the washed pellets was taken as indicating IgE present in immune complex form. A calibration curve was constructed using artificially prepared IgE-anti-IgE complexes. The method developed may be useful to detect other components present in immune complex fractions.


Separation of rat T and B lymphocytes by density gradient electrophoresis. Density gradient electrophoresis has been employed for the preparative separation of T and B lymphocytes from rat spleen and peripheral blood. The high mobility cells were found to be predominantly T lymphocytes, as determined by their response to phytohemagglutinin and the relative absence of immunoglobulin-positive cells. The low mobility cells were predominantly B cells, as determined by the high percentage of immunoglobulin-positive cells and the total lack of response to PHA, an exclusive T cell mitogen. A better separation of peripheral blood T and B lymphocytes was achieved than with spleen T and B cells.


Normal T-lymphocyte function in psoriatic patients undergoing methoxsalen photochemotherapy. Peripheral blood lymphocytes from psoriatic patients undergoing photochemotherapy using oral 8-methoxypsoralen and high-intensity long-wave ultraviolet radiation, were isolated and their ability to respond to phytohemagglutinin and to form spontaneous rosettes with sheep red blood cells was measured in vitro. Seventeen patients were investigated who had received 200 to 3700 Joules/cm2 of ultraviolet radiation over a 6- to 33-month period. No significant defects in lymphocyte blastogenesis or E-rosette formation were detected in these patients.


The thymus: clock for immunologic aging? A brief overview of the effects of age on T cell function is presented. Normal immune functions can begin to decline shortly after an individual reaches sexual maturity. Foremost among the cellular changes are those in the stem cells as reflected in their growth properties and the availability of precursor T cells, and in the T cells, in which a shift in subpopulations may be occurring. Present evidence indicates that thymic involution precedes, and therefore may be responsible for, the age-dependent decline in the ability of the immune system to generate functional T cells. It now appears that the primary effect of thymic involution is on a T cell differentiation pathway; the more mature T cells are affected first, the less mature T cells only later. Thus, the thymus may be the aging clock for the immune system. Current studies are centered on processes regulating growth and atrophy of the thymus, and methods for restoring the impaired immune function of elderly individuals.


Penetration of cephapirin and cephalothin into the right atrial appendage and pericardial fluid of patients undergoing open-heart surgery. To prevent infection in 27 patients who underwent coronary artery bypass or cardiac valve replacement surgery, each patient received a single 2-g dose of either cephalothin or cephapirin intravenously before the operation (prior to opening of the chest cavity). Samples of the right atrial appendage, pericardial fluid, and serum were obtained at various intervals after injection of the antibiotic and were assayed for cephalosporin concentrations. Cephapirin consistently reached higher levels than cephalothin in the right atrial appendage and pericardial fluid; both cephalosporins, however, reach concentrations in these sites well above their minimal inhibitory concentrations (MICs) for penicillin-resistant staphylococci. Of particular interest was the brevity of the period (about 100 min) during which levels of both antibiotics were maintained above the MIC in the right atrial appendage. This finding emphasizes the need for administration of these antibiotics shortly before surgery.


Cyclophosphamide eliminates suppressor T cells in age-associated central regulation of delayed hypersensitivity in mice. Effect of treatment of mice with cyclophosphamide (CY) on the delayed hypersensitivity (DH) response was investigated in C57BL/6 mice. DH to methylated human serum albumin (MHSA) could be enhanced with CY in young mice but not in aged ones. DH enhancement with CY appeared to be due to elimination of suppressor T cells involved in DH. Effector T cells were also sensitive to CY, the damaging effect of CY on these latter cells was, however, transient suggesting the rapid recovery of effector T cells. The overshooting recovery of the effector T cells required the presence of the thymus. It is more probably that there are at least two distinct subpopulations of T cells in DH, effector T cells, and suppressor T cells. The distinction is already apparent in the thymus stage. The suppressor T cells, categorized as a central regulator, seem to be antigen nonspecific and regulate the more effectively the DH in young mice, thus physiological role of these cells in age-associated immune alterations is implicated.


Mechanisms of regulation of cell-mediated immunity. III. The characterization of azobenzenearsonate-specific suppressor T-cell-derived-suppressor factors. Delayed type hypersensitivity to the hapten azobenzenearsonate (ABA) can be induced and suppressed by the administration of hapten-coupled syngeneic spleen cells by the appropriate route. Suppressor T cells stimulated by the intravenous administration of ABA-coupled spleen cells have been shown to produce a discrete subcellular factor(s) which is capable of suppressing delayed type hypersensitivity to azobenzenearsonate in the mouse. Such suppressor factors may be produced by the mechanical disruption of suppressor cells or by placing such suppressor cells in culture for 24 h. The suppressor factor(s) (SF) derived from ABA-specific suppressor cells exhibit biological specificity for the suppression of ABA delayed type hypersensitivity (DTH), but not trinitro-phenyl DTH, as well as the capacity to bind to ABA immunoadsorbents. Passage of suppressor factor(s) over reverse immunoadsorbents utilizing a rabbit anti-mouse F(ab')2 antiserum demonstrated that the antigen-specific T-cell derived SF does not bear conventional immunoglobulin markers. The suppressor factor(s) are not immunoglobulin molecules was further demonstrated by the inability of anti-ABA antibodies to suppress ABA DTH. Gel filtration of ABA suppressor factor(s) showed that the majority of the suppressive activity was present in a fraction with molecular weight ranging between 6.8 x 10(4) and 3.3 x 10(4) daltons. We also analyzed for the presence of determinants encoded by the H-2 major histocompatibility complex (MHC) and found that immunoadsorbents prepared utilizing antisera capable of interacting with gene products of the whole or selected gene regions of H-2 MHC, i.e., B10.D2 anti-B10.A and B10 anti-B10.A immunoadsorbents, retained the suppressive activity of ABA-SF. Elution of such columns with glycine HCl buffers (pH 2.8) permitted recovery of specific suppressive activity. Taken collectively such data supports the notion that suppressor T-cell-derived ABA suppressor factors have antigen-binding specificity as well as determinants controlled by the K end of the H-2 MHC. The distribution of strains capable of making SF has also been analyzed. The relationship of the antigen-binding specificity to VH gene products is discussed in this and the companion paper.


Localization of H-2 antigens on mouse trophoblast cells. The presence of H-2 antigens of the paternal and maternal haplotypes on mouse trophoblast cells was examined at several stages of pregnancy by using a sensitive immunolabeling technique followed by quantitative radioautography. Results revealed the presence of H-2 antigens (determined by the K or D loci) of both parental haplotypes on the F1 trophoblast cells. At 14-16 d of gestation, the antigen density was equivalent to that on adult thymocytes and there was a further 50% increase on day 18. H-2 antigens of both parental haplotypes are also found to be expressed on 11-13 d trophoblast cells.


Endothelial injury in scleroderma.

Endothelial injury in scleroderma. Functional and structural vascular lesions have been observed in the organs involved in scleroderma. The etiology of these vascular changes is poorly understood. The ability to isolate, characterize, and maintain endothelial cells in vitro provides a target cell population to study endothelial damage in scleroderma. The present report describes the effect of scleroderma serum on endothelial, smooth muscle, and fibroblast cell types. Sera from patients with scleroderma (31/52) and Raynaud's syndrome (11/19) contain cytotoxic activity, specific for endothelial cells, which is nondialyzable, heat-stable, and elutes with albumin on gel-filtration chromatography.


Rosette formation between murine lymphocytes and erythrocytes. A new locus in the H-2 region. More than 5% of murine splenic lymphocytes form rosettes with syngeneic erythrocytes. This property was maximally expressed when the lymphocytes were cultured for 24 h before rosetting. About 70% of the rosetting lymphocytes were B cells and 30% were T cells on the basis of surface immunoglobulin and the Thy-1-antigen. Capping surface immunoglobulin had no effect on the capacity of lymphocytes to form rosettes, indicating that the receptor in question was not immunoglobulin. The capacity of lymphocytes to form rosettes with erythrocytes from other strains of mice was H-2 restricted. Extensive pairings of congenic and recombinant strains as donors of lymphocytes and erythrocytes showed that none of the known loci within the H-2 region-controlled rosetting. The involvement of regions on chromosome 17, telomeric or centromeric to H-2, was also excluded. The data were only compatible with the conclusion that this form of self-recognition is associated with a new locus (or loci) mapping between H-2G and H-2D.


Adaptive differentiation of murine lymphocytes. II. The thymic microenvironment does not restrict the cooperative partner cell preference of helper T cells differentiating in F1 leads to F1 thymic chimeras. The cooperating preference of helper T cells originating from F1 bone marrow, but differentiating in adult thymectomized, lethally irradiated F1 recipients reconstituted with either f1 or homozygous parental thymus grafts was investigated. Cooperating preference was assayed by determining the levels of helper activity provided by antigen-primed T cells derived from such thymic chimeras for hapten-primed B lymphocytes obtained from conventional F1 or parental donors in adoptive secondary antibody responses in vivo. The results of these analyses revealed a tendency of helper T cells derived from parental thymic chimeras to provide better help for B cells of the same parental type corresponding to the origin of the thymus graft than for the opposite parent. Such preference was, however, only marginal and rarely were differences in levels of helper activity provided to the respective parental types statistically significant. Moreover, this marginal preference, when observed, pertained only to responses of the IgG class; no concordant preference in providing helper activity for IgE antibody responses was observed even with the same populations of thymic chimera helper T cells. Finally, in no instance was there any evidence of restriction in the classical sense of presence versus absence of help as we have routinely observed in all of our previous studies concerning genetic restrictions of T-B-cell cooperative interactions. Although the basis for differences in the studies reported here when compared to observations made in cytotoxic T-lymphocyte systems is unclear, and could reflect genuine mechanistic requirements concerning what directs H-2 restrictions in helper T cells and cytotoxic T lymphocytes, respectively, it is also possible that we are placing too much faith in our interpretations of data obtained in bone marrow chimera systems than is perhaps justified by the potentially great fragility of such systems.


Isolation and immunological characterization of a major surface glycoprotein (gp54) preferentially expressed on certain human B cells. A major membrane glycoprotein with mol wt of approximately 54,000 has been isolated from membrane preparations of B-type lymphoid cell lines. Antiserum prepared against the isolated material specifically precipitated this glycoprotein from membranes labeled by surface radioiodination or by metabolic labeling. This antiserum was shown by complement-mediated cytotoxicity assay, membrane immunofluorescent staining, and by quantitative absorption analysis to react preferentially with certain B-lymphoblastoid cell lines, with a minor population of peripheral blood B lymphocytes, and a major population of tonsillar B lymphocytes. Certain B-cell leukemias also expressed the antigen, whereas others did not. Considerable variability was observed among positive B cells in the intensity of fluorescent staining even among the leukemic cells from a single individual. Although T cells, including T cells, were negative by direct immunofluorescent and cytotoxicity assay, evidence for low levels of the antigen on the cells of T cell lines was obtained. The whole specific antiserum and its F(ab')2 fragments stimulated B lymphocytes to proliferate. This proliferation did not produce differentiation to plasma cells and was T-cell independent. The monovalent Fab fragments had no effect. None of these preparations timulated T cells. The possibility that this antigen, termed gp54, may play some role in B-cell activation is discussed.


Age-dependent production of IgA and IgM autoantibodies against IgG2a in a colony of 129/Sv mice. Although much of the basic immunological work has been done with mice, little is known about anti-IgG autoantibodies in this species. Dresser (1, 2) has reported the occurrence, in CBA mice, of anti-IgG antibody (Ab)(1) detected by a hemolytic-plaque assay after stimulation with endotoxin or immunization against sheep erythrocytes. IgM rheumatoid factor has also been described in various strains of mice with a systemic lupus erythematosus-like disease (3). Recently, we have tried to induce anti-IgG in mice of the 129/Sv strain by inoculating autologous IgG. To our surprise, we found that the sera of all the animals had, before any inoculation, anti-IgG detectable by agglutination of particles coated with autologous IgG. The possibilities to investigate the mechanism of production and the biological role of this kind of Ab prompted us to undertake a study of the nature and specificity of the mouse anti-IgG.


Specificity and function of a human autologous reactive T cell. Normal human peripheral blood contains a population of T cells (autologous reactive cells [ARC]) capable of proliferating in response to signals from autologous B cells and monocytes. Selective suicide of proliferating ARC with 5-bromo-2-deoxyuridine and light demonstrated that this ARC was responsive to signals coded for by genes more closely linked to the HLA-DR, than to the HLA-A, or HLA-B, loci. Density-gradient fractionation of T cells indicated that populations enriched in ARC reactivity were also enriched for helper influences required for Ig synthesis by autologous B cells. In contrast, populations negatively selected for proliferating ARC were deficient in helper activity. These studies indicate that the ARC is responsive, at least in part, to products of genes closely linked to the HLA-DR locus and can function as a helper cell.


Qat-4 and Qat-5, new murine T-cell antigens governed by the Tla region and identified by monoclonal antibodies. Two new lymphocyte antigens, provisionally designated Qat-4 and Qat-5 have been identified with two different hybridoma-derived, monoclonal AKR antiC57BL/6 antibodies. These antigens are governed by genes located to the right (distal) end of the H-2 complex, within the Qa-2,3 region. Qat-4 and Qat-5 antigens which do not seem to be identical with Qa-2,3 or TL antigens are absent from Ig/ lymphocytes and thymocytes. They are only present on a fraction of peripheral T cells. Thus, Qat-4 is expressed on 70%, and Qat-5 on 30% of splenic and lymph node T cells, Qat-4 is also found on the majority of Ig- cells from athymic nude mice. These findings illustrate the complexity of the chromosome segment between the H-2D and Tla loci and they emphasize the role of major histocompatibility complex-associated genes for the differentiation of T cells into different subpopulations with possibly distinct immunologic functions.


Production of auto-anti-idiotypic antibody during the normal immune response to TNP-ficoll. I. Occurrence in AKR/J and BALB/c mice of hapten-augmentable, anti-TNP plaque-forming cells and their accelerated appearance in recipients of immune spleen cells. Attempts were made to elucidate the cause of the downward regulation of the splenic plaque-forming cell (PFC) response in AKR/J and BALB/c mice between days 4 and 7 after a single intravenous injection of 2,4,6,trinitrophenyl- lys-Ficoll(TNP-F). AKR/J spleen cells, taken 7 d after injection of TNP-F, were transferred, together with TNP-F, into normal AKR/J mice. The day-3 or - 4 PFC response of the recipients was much lower than that of recipients of normal cells. However, the suppression was only apparent because the presence of 10(-8)-10(-7) M 2,4,6-trinitrophenyl-epsilon-amino-n-caproic acid (TNP- EACA) (or 10(-7)-10(-6) M 2,4,-dinitrophenyl-epsilon-amino-n-caproic acid) in the PFC assay caused a dramatic increase in observed PFC, averaging 298 percent on day 3 and 122 percent on day 4. Recipients of normal cells showed no such hapten-augmentable PFC. T-depleted immune spleen cells did not cause any apparent suppression of the response to TNP-F, but hapten-augmentable PFC in recipient spleens were again prevalent. Suppression of the PFC response, as well as hapten-augmentable PFC, were seen after transfer of immune serum. It was postulated that hapten augmentation of PFC was caused by displacement of auto-anti-idiotypic antibody from the surface of blocked antibody- synthesizing cells. Further studies showed that such hapten-augmentable PFC occurred in the spleens of a large percentage of both AKR/J and BALB/c mice examined after day 4 of the primary response to TNP-F. Thus, it was hypothesized that the downward regulation of the magnitude and, possibly, also of the heterogeneity of the splenic-PFC response was due to an auto-antibody response to one or more major idiotypes of the anti-TNP response.


Production of auto-anti-idiotypic antibody during the normal immune response to TNP-Ficoll. II. Hapten-reversible inhibition of anti-TNP plaque-forming cells by immune serum as an assay for auto-anti-idiotypic antibody. Sera taken from AKR/J mice 7 d after the intravenous injection of 2,4,6-trinitrophenyl-lys-Ficoll (TNP-F) caused a specific inhibition of anti- trinitrophenol (TNP) plaque-forming cells (PFC) in vitro. This inhibition was reversed by the incorporation of 10(-8)-10(-7) M 2,4,6-trinitrophenyl- epsilon-amino-n-caproic acid (TNP-EACA) into the agar during the PFC assay. The factor responsible for the hapten-reversible PFC inhibition was removed from serum by passage through an anti-immunoglobulin column or through a 2,4,-dinitrophenyl-human-serum-albumin-bromoacetylcellulose plus anti-TNP- antibody column, but not by DNP-HSA-BAC alone. It was concluded that this immunoglobulin-like substance, lacking anti-TNP activity but reacting with anti-TNP antibody of AKR/J origin, was most likely an auto-anti-idiotypie antibody that had been produced during the normal course of the response of AKR/J mice to TNP-F. Pools of anti-idiotypic-antibody-containing antisera inhibited anti-TNP plaque formation to varying degrees when tested on d-4 PFC from different mice of the same inbred strain, suggesting a variability in idiotype expression. 4 d after transfer of immune (7 d after 10 mug TNP-F, administered intravenously) AKR/J spleen cells plus 10 mug TNP-F into syngeneic mice, the number of PFC detectable in the recipients' spleens could be markedly augmented by the inclusion of TNP-EACA in the agar during the PFC assay. Incubation of spleen cells containing such hapten-augmentable PFC with TNP- EACA yielded a factor in the supernate that caused a specific, in vitro, hapten-reversible inhibition of anti-TNP PFC. Studies with immunoadsorbents indicated that this PFC-inhibiting factor was antigenically immunoglobulin- like, lacked anti-TNP-antibody activity, but reacted with anti-TNP antibody of AKR/J origin. The results are consistent with the view that this PFC inhibitor is auto-anti-idiotypic antibody that is involved in the normal regulation of the immune response. It is proposed that hapten-reversible inhibition of plaque formation can be employed as an assay for anti-idiotypic antibody and the conditions for such an assay are described. It is further proposed that the detection of hapten-augmentable PFC suggests the presence of auto-anti-idiotypic antibody.


B-cell influences on the induction of allotype suppressor T cells. Allotype suppressor T-cell (Ts) populations that persist for the life of the animal arise in (BALB/c x SJL)F(1) hybrids exposed perinatally to antibody to the paternal (Ig-1b) allotype on IgG(2a)-isotype immunoglobulin H chains. These Ts suppress Ig-lb production by depleting the supply of allotype- specific helper T cells (Th) required, in addition to carrier-specific Th, for the latter stages of Ig-1b memory B-cell differentiation. In this publication, we show that specific Ig-1 allotype Ts are induced by perinatal exposure to antisera which interfere with normal B-cell maturation, i.e., by antibodies reactive with surface IgM on immature precursors of IgG(2a), memory cells. Antibodies to IgM (Ig-6) allotypes carried on precursors induce specific suppression for the IgG2, allotype produced by progeny of the target precursor. Anti-Ig-6a and anti-Ig-6b induce Ts that specifically suppress Ig-1a and Ig-1b, respectively. Heterologous (goat) anti-IgM induces suppression for both IgG(2a) immunoglobulins (Ig-1a and Ig-1b). Ts activity in these antiprecursor-Ig-suppressed mice is expressed in adoptive transfer assays and, as with anti-Ig-1b-induced Ts, is rendered ineffective by cotransfer of adequate numbers of T cells but not B cells from nonsuppressed mice. The Ts induction, in contrast with Ts expression, is reversed by the introduction of appropriate adult B-cell populations from nonsuppressed donors. Taken together, these data suggest that the development of mature B cells plays a central role in the early establishment of the balance between helper cells and suppressor cells that determines whether Ts or Th will dominate in regulating Ig-1b production in adult animals.


Idiotypic regulation of the immune system. Common idiotypic specificities between idiotypes and antibodies raised against anti-idiotypic antibodies in rabbits. Anti-idiotypic antibodies (Ab2) were raised in allotype-matched rabbits against anti-carbohydrate or anti-tobacco mosaic virus antibodies (Ab1). Several Ab2 were purified and injected into a third series of rabbits III which synthesized antiantiidiotypic antibodies (Ab3). Antigen was then given for the first time in those rabbits who had synthesized Ab3. The specific antibody synthesized in rabbits III was called Ab1'. Anti-idiotypic antibodies were raised against purified Ab3 antibodies (Ab4). In most cases, Ab1' antibodies are sharing idiotypic specificities with Ab1. Ab3 did not react with antigen but shared idiotopes with Ab1 and Ab1' because Ab4 antibodies, which are anti-idiotypes to Ab3 do recognize specifically Ab1 and Ab1' antibodies belonging to the same chain of immunization. It seems therefore that Ab3 looks idiotypically like Ab1 and Ab4 displays the same behaviour as Ab2. A general view of the functioning of the immune system is presented.


Primary generation of cytolytic T lymphocytes in the absence of DNA synthesis. The requirement for DNA synthesis during the primary differentiation of cytolytic T lymphocytes (CTL) had been investigated. CTL were induced polyclonally in vitro by stimulation of normal C57BL/6 spleen cells with concanavalin A (Con A)and their cytolytic activity was tested against 51Cr-labeled target cells in the presence of Bacto Phytohemagglutinin M. With this system, CTL activity could first be detected 48 h after exposure of spleen cells to Con A. Addition of cytosine arabinoside at concentrations sufficient to reduce DNA synthesis by 95-98% in Con A-stimulated cultures did not significantly inhibit the generation of cytolytic activity on a cell-to-cell basis. These results demonstrate that derepression of the genetic information required for the expression of CTL function can occur in the absence of detectable DNA synthesis.


CBA/N X-linked B-cell defect prevents NZB B-cell hyperactivity in F1 mice. NZB mice and their F1 hybrids produce excessive polyclonal IgM and autoantibodies of both IgM and IgG classes. CBA/N mice and CBA/N-mothered F1 males fail to make antibody to many T-independent antigens and have low levels of serum IgM; further, these mice lack a population of splenic B cells characterized by a low-to-intermediate density of surface IgM. We have studied male CBA/N, NZB, CBA/N X NZB, NZB X CBA/N, and CBA/J mice; female CBA/N X NZB mice; and males of several control crosses of NZB and CBA/N mice. We have found that the CBA/N X-linked defect of T-independent immune response is completely expressed in CBA/N X NZB mice. In marked contrast to NZB mice and to NZB mice and to NZB F1 hybrids bearing at least one normal X chromosome, the CBA/N X NZB males failed to respond to two T-independent antigens, had small numbers of splenic IgM-producing cells, barely detectable splenic IgM production, and splenic B-cell surface-Ig patterns resembling those of CBA/N mice. These data suggest that the NZB B-cell abnormality resulting in excessive IgM production occurs almost exclusively in that population of B cells affected by the CBA/N X chromome-linked defect. Preliminary studies suggest that CBA/N X chromosome retards the spontaneous development of anti-erythrocyte autoantibodies in CBA/N X NZB males. Castration, known to accelerate autoimmune disease in certain NZB F1 males, appears to have no influence on the immune functions examined in this study.