Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

Nonspecific inhibitor of contact sensitivity made by T-acceptor cells: triggering of T cells armed with antigen-specific T-suppressor factor (TsF) requires both occupancy of the major histocompatibility complex recognition site by soluble I-J product and cross-linking of the antigen recognition sites of the TsF

The phenomenon of associative recognition, i.e., the recognition of antigen together with major histocompatibility complex products (MHC) was studied in a model system. T-acceptor cells armed with antigen-specific T-suppressor factor (TsF) released a nonspecific inhibitor of the transfer of contact sensitivity when exposed to antigen together with MHC. The MHC product occurred in a KCl extract of cells and behaved genetically and serologically as I-J. Cells armed with anti-picryl or anti-"oxazolone" TsF could be triggered by the corresponding "bis-picryl-L-lysine" and "bis-oxazolone-L-lysine" together with MHC. This suggested that cross-linking of antigen recognition sites on separate molecules of TsF might be required. To investigate this possibility the bifunctional "mixed" hapten "N alpha-picryl-N epsilon-oxazolone-L-lysine," which is univalent with respect to the picryl and oxazolone haptenic groups, was synthesized. This triggered cells armed with a mixture of anti-picryl and anti-oxazolone TsF but not cells armed with either TsF alone. It was concluded that both occupancy of the I-J recognition site and the cross-linking of separate molecules of TsF was required for triggering. Moreover the hapten and the KCl extract could be given sequentially and in either order. This finding suggested that the triggering of the release of nonspecific inhibitor was due to the separate recognition of I-J and antigen and not to new antigenic determinants produced by their interaction.     

Suppression of delayed-type hypersensitivity to third party "bystander" alloantigens by antigen-specific suppressor T cells

Delayed-type hypersensitivity (DTH) against alloantigens can be induced by sc immunization with allogeneic cells. The induction of DTH can be suppressed by iv preimmunization of the mice with similar allogeneic spleen cells, provided the cells are irradiated before injection. This suppression is mediated by T cells. The suppressor activity can be induced not only by H-2-and non-H-2-coded antigens, but also by H-2 subregion-coded antigens. Suppression induced by K, I, or D subregion-coded antigens is specific for that particular subregion as well as for its haplotype. I-J-coded alloantigens were found to not be necessary for the induction of antigen-specific suppressor T cells. After restimulation of suppressor T cells by the "specific" alloantigens, the DTH to simultaneously administered third-party alloantigens becomes suppressed as well. This nonspecific suppression of DTH to third party "bystander" alloantigens also occurs when the specific and the third-party antigens are presented on separate cells, provided that both cell types are administered together at the same site. The simultaneous presentation of both sets of alloantigens during the induction phase of DTH only is sufficient to prevent the normal development of DTH to the third-party antigens.     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Characterization of two suppressor cells that together prevent in vivo development of cytolytic T cells to hapten-altered self

Two suppressor cell populations that interact to down-regulate in vivo development of the cytolytic T-cell (CTL) response to trinitrophenyl-modified syngeneic spleen cells (TNP-SC) have been further characterized. Suppressor cells induced by the iv injection of trinitrophenyl-modified syngeneic spleen cells possess Thy 1.2 antigen. Their precursors are insensitive to pretreatment of host animals with cyclophosphamide (CY). Suppressor cells that arise after dermal sensitization with trinitrochlorobenzene are also Thy 1.2 antigen positive but their precursors are sensitive to pretreatment with CY. These characteristics of the two suppressor T cells (Ts) are identical to those of the two Ts that are generated by similar methodologies and that together suppress contact sensitivity (CS) to picryl chloride. Neither the CS nor CTL response was suppressed when host animals possessed only one set of Ts. In contrast to suppression of CS at the efferent phase, development of CTL was suppressed only when the two Ts were present early during sensitization (afferent phase). Since the results point to several similarities between the two sets of Ts that are active in the down-regulation of the CS and CTL responses, it is suggested that the two dissimilar immune responses directed to the same hapten, namely CS and CTL, may be controlled by the same suppressor cells. Since it appears that the two sets of Ts interact to affect different phases of the CS and CTL responses, down-regulation of each must be accomplished through different mechanisms.     

Effects of pertussis toxin on delayed-type hypersensitivity responses and on the activity of suppressor T cells on the responses

Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 micrograms/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH.     

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with ¹²⁵I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for ¹²⁵I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.     

Partial characterization of a prostaglandin-induced suppressor factor

Glass adherent splenic T cells, cultured in the presence of prostaglandin E₂ (10^?5M), were found to elicit a factor capable of nonspecifically suppressing PHA- and LPS-induced mitogenesis. Cells from C57B1/6J, Balb/C, and C3H/He mice were all capable of producing this suppressor factor, although some degree of variability in the response of cells from C3H mice to the factor was observed. The suppressor (designated prostaglandin-induced T-cell derived suppressor, PITS) was characterized biochemically and it was found that the activity was resistant to boiling, and treatment with RNase and DNase, yet was sensitive to treatment with proteinase K, trypsin, and Pronase. Further, PITS supernatants were found to contain at least two suppressors with approximate molecular weights of 35,000 (PITS_α) and 5000 (PITS_β). Results from experiments with cycloheximide-treated glass-adherent T cells indicate that prostaglandin E₂ may function by inducing the release rather than de novo synthesis of the PITS. These results indicate that the reported overall suppressive effect of prostaglandin E₂ on lymphocytes may in part be due to the release by certain T cells of a suppressive factor.     

Identification of a new marker (Ly RL male 1) for cells of the T lineage by an auto-antithymocyte serum

The sera of mice surviving challenge with a Thy-1-negative variant of the thymoma RL male 1 contain antibodies which identify a new cell surface antigen (Ly RL male 1) present on cells of the T lineage. This antigen appears early in the development of the lineage and it can be detected on most thymocyte precursors. Its presence on prothymocytes can serve to distinguish these cells from their multipotential precursors. The antigen is present on many thymocytes, and dividing thymocytes are more susceptible to its cytotoxic activity than is the total population. Ly RL male 1-antigen-positive cells can be detected in peripheral lymphoid tissues by both functional assays and absorption. Treatment of peripheral lymphoid cells with the antiserum leads to significant reduction in MLR and helper activity but does not alter mitogen reactivity or lymphocytotoxicity. Animals with significant serum levels of anti-RL male 1 are deficient in their ability to produce IgG antibody to sheep erythrocytes.     

Phosphate uptake by kidney epithelial (LLC-PK1) cells

Phosphate uptake by the cultured kidney epithelial cell (LLC-PK1) was studied. The uptake was Na+ dependent, saturable with respect to phosphate and Na+, and energy dependent. The characteristics of the cell uptake system resembled the properties of phosphate transport in the kidney. Parathyroid hormone, dibutyryl cyclic AMP, and forskolin decreased Na+-dependent phosphate uptake. These agonists did not affect Na+-dependent alpha-methylglucoside uptake. Vasopressin and isoproterenol, which do not affect renal phosphate transport, did not inhibit phosphate uptake by the cell. These findings suggest that the cultured cell system may be a useful experimental model for studies of renal phosphate transport and its regulation.     

Affinity-purified antigen-specific products produced by T cells share epitopes recognized by heterologous antisera raised against several different antigen-specific products from T cells

Heterologous antisera to murine or rat T-cell antigen-binding molecules (T-ABM) were raised in rabbits or sheep. The T-ABM used for immunization were purified by affinity for antigen and did not bear known immunoglobulin isotypes. T-ABM and anti-T-ABM were raised in three separate laboratories. Antisera to T-ABM were exchanged and tested for binding to T-ABM in three separate laboratories. Thus antisera to at least three distinct T-ABM were tested directly for binding to T-ABM or by adsorption of biological activity. Rabbit antisera to murine trinitrophenol (TNP)-specific T-ABM or rat AgB-specific T-ABM bound both murine or rat T-ABM, indicating evolutionary conservation of T-ABM. Similar results were found with sheep antisera to murine T-ABM. In addition, all heterologous anti-T-ABM antisera used bound murine T-ABM specific for TNP, 4-hydroxy-3-nitrophenyl acetate (NP), SRBC, or T-cell membrane proteins with similar structure. Thus, there is a commonality of antigenic determinants between various T-ABM and T-cell membrane homologues which may be T-cell surface receptors for foreign antigen.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

Binding and reactivity at the "glucose" site of galactosyl-beta-galactosidase (Escherichia coli)

A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase. Two classes of compounds were found to bind well to the galactosyl form of the enzyme. One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols. On the other hand, several factors seemed to control k4. Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars. Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule. In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low. Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site. More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small. Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors.     

Methylated cycloamyloses (cyclodextrins) and their inclusion properties

2,3,6-Tri-O-methyl and 2,6-di-O-methyl derivatives of cyclohexa- and cyclo-hepta-amylose (2,3,6-tri-O-methyl- and 2,6-di-O-methyl-α- and -β-cyclodextrin) have been prepared and shown to be versatile complexing agents. Their complexes in aqueous solution are usually more stable than the corresponding complexes of un-substituted cycloamyloses. The methylated cycloamyloses also form crystalline complexes, the stability of which depends on the size and shape of the guest molecule. The decomposition temperature of the crystalline complexes with homologous n-alkanes, which is relatively high increases with increasing chain-length of the hydro-carbon. The shift of the i.r. carbonyl band of oleic acid in its solid complex with methylated α-cyclodextrin probably reflects inclusion of the fatty acid in the mono-meric form. The methylated cycloamyloses, when used as the stationary phases for g.l.c. or dissolved in a conventional stationary phase, affect the retention times of organic compounds in a manner which suggests that inclusion phenomena are operative.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Effect of atrial natriuretic factor (ANF)-related peptides on aldosterone secretion by adrenal glomerulosa cells: critical role of the intramolecular disulphide bond

We previously demonstrated that synthetic 48-73 atrial natriuretic factor (ANF) (previously called 8-33 ANF) blocked the response of rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. We have now investigated the effects of natural 43-73 ANF, oxidised synthetic 48-73 ANF and the natural 1-73 ANF on aldosterone output by rat glomerulosa cells. The natural 43-73 ANF and the natural 1-73 ANF were equipotent to 48-73 ANF in inhibiting the stimulation of aldosterone secretion produced by angiotensin II with an IC50 of 2 X 10(-9)M. Similar results were obtained with ACTH and potassium. After oxidation with performic acid, 48-73 ANF was completely devoid of activity on the response of aldosterone to angiotensin II, ACTH and potassium. We conclude that the intramolecular disulphide bond in 48-73 ANF is critical for maintaining the active conformation of ANF.     

Stimulation of splenic prostaglandin levels by DNP-protein antigens.

Secondary challenge of DNP-BGG primed mice with either DNP-BSA or DNP-BGG stimulates significant increases in splenic prostaglandin levels. This secondary increase appears to be dependent on the amount of total DNP groups present on the challenging antigen, and is independent of the contribution by the secondary response to the carrier itself. Mice primed with BGG or BSA do not show any significant early increases in prostaglandin in response to DNP-BSA and DNP-BGG. The effects of DNP-BGG hyperimmune serum transfer (as passive antibody) on the primary responses to DNP-BSA and DNP-BGG as compared to effects of the same serum depleted of antibody suggest that the interaction of the challenging antigen and the existing anti-DNP antibody is of prime importance in the antigenic stimulation of prostaglandin levels.     

Inhibitory effect of a low dose of prednisone on PHA-induced Ia antigen expression by human T cells and on proliferation of T cells stimulated with autologous PHA-T cells

Administration of a small dose of prednisone markedly reduced (1) the PHA-induced expression of Ia antigens by T cells, (2) the stimulatory activity of Ia antigen-bearing T cells in autologous and allogeneic mixed lymphocyte reactions (MLRs), and (3) the proliferative response of T cells stimulated with autologous PHA-activated T cells or autologous or allogeneic non-T cells. The inhibitory effects of prednisone are reversible and are not detectable on T cells isolated from blood drawn 24 hr following prednisone administration. The kinetics of the prednisone-mediated inhibition of MLRs with autologous PHA-T cells is different from that of MLRs with autologous non-T cells. These data in conjunction with the information available in the literature suggest that the mechanisms underlying these two types of autologous MLRs are different.