Accumulation of eosinophils and monocytes in lymphoid organs of chick-embryos. II. Effect of mitogenic stimulation.

The mitogen effect on migration of eosinophils and monocytes was studied in embryonic chickens. On the 13th embryonic day, chickens were injected with mitogens, such as concanavalin A (Con A), phytohemagglutinin-P (PHA-P), and lipopolysaccharide (LPS), into the allantoic cavity, and the mitogenic effect was estimated from the relative frequencies of eosinophils and monocytes by enumerating the number of oxidase positive cells (OPC) in the spleen, thymus, and bursa of Fabricius. Splenic frequencies of OPC increased in the embryos treated with mitogens. Similar influences were also detected in the thymic OPC. Higher responses were seen on the 18th embryonic day in the number of splenic OPC when embryos were treated with Con A or PHA-P than with LPS. These findings suggest that Con A and PHA-P are preferential OPC accumulation promoters. However, bursal frequencies of OPC in the cortex were low after mitogenic stimulations when compared with controls, although appreciable responses were detected in the bursal medulla after LPS stimulation. These results suggest that the migration pattern in the population of eosinophils and monocytes is affected not only by T cell mitogens but is also derived from LPS stimulation.     

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.     

Activation of a human T cell subpopulation bearing receptors for autologous erythrocytes by concanavalin A.

Human lymphocytes from different lymphoid organs were examined for rosette formation with autologous erythrocytes. The autorosette-forming cells (A-RFC) were shown to belong to a T cell subset including less mature lymphocytes. When normal human peripheral blood lymphocytes were stimulated with low doses of the plant lectin concanavalin A (Con A), in the presence of autologous plasma, the A-RFC levels were strongly enhanced. This response gave rise to two peaks: the first one coincided with the peak of thymidine incorporation but the maximum increase occurred 5 or 6 days later when the proliferative response was impaired. Depletion of A-RFC before stimulation with Con A led to a clear-cut decrease in autorosette levels at both peaks of the response. It is concluded that Con A, generally used for polyclonal activation against heteroantigens, may also result, in terms of A-RFC marker, in expansion of an autoreactive T cell population.     

Enhanced response to Con A and production of TCGF by lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis

The mitogenic response to Con A and the production of T cell growth factor or interleukin 2 (IL 2) by splenic and peripheral blood lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis have been investigated. By using an optimized method with Con A-coated chicken erythrocytes (MRC), lymphocytes of OS chickens were found to exhibit significantly elevated mitogenic responses as compared with cells from either Normal White Leghorn chickens (NWL) or animals of the Cornell C-Strain (CS), from which the OS has originally been developed. This difference was observed throughout ontogeny up to 15 mo of age, and was associated with increased levels of IL 2 activity in the culture supernatants. The elevated responsiveness of OS T lymphocytes was also found to be manifested in the expression of receptors for IL 2, because Con A-stimulated lymphocytes of OS birds were significantly more effective than those from normal controls in absorbing IL 2 activity from conditioned media (CM) of stimulated spleen cells. High concentrations of CM were suppressive in IL 2 assays, signaling the presence of an inhibitory factor(s) in addition to IL 2. An additional indication for defective immunoregulation was that CM from OS lymphocyte cultures showed significantly less of this suppressive activity in comparison with CM of normal (NWL and CS) lymphocyte cultures. Finally, the spontaneous uptake of 125IUdR of embryonic and early post hatching OS spleen lymphocytes was consistently and significantly enhanced. This difference, however, in contrast to the one observed in Con A responses, was found to decrease with age. The data are discussed in view of the contradictory results concerning T cell functions reported for several autoimmune states in mammals.     

Electrophoretic separation of rat spleen and thymus leukocytes and their reactions to mitogens in vitro]

Electrophoretic mobility (EPM) of lymphocytes from the thymus and spleen of August and Wistar rats as well as capacity of lymphocytes with different surface hemagglutinin (PHA) and concanavalin A (Con A) were studied by the method of free flow electrophoresis. Lymphocytes of the rat spleen were shown, depending on the surface charge, to divide into two groups during cultivation: cells with high and low electrophoretic mobility. At separation the lymphocytes consisted of 8--10 fractions with different EPM. There was a relationship between the surface charge of the lymphocytes and their stimulation rate by mitogens. Increased thymidine-3H uptake was recorded at mitogenic exposure of lymphocytes from the spleen with high EPM. Low mobile lymphoid elements of the spleen did not respond to mitogenic stimulation. A subpopulation of thymocytes with low EPM was resistant to Con A stimulation. The thymocytes of rats did not virtually respond to PHA irrespective of EPM.     

Characterization of the recognition of target cells sensitive to or resistant to cytolysis by activated macrophages: II. Competitive inhibition of macrophage-dependent tumor cell killing by mitogen-induced,nonmalignant lymphoblasts

We have previously reported that tumoricidal rat macrophages can distinguish quiescent normal lymphocytes from concanavalin A (Con A)-stimulated lymphocytes and thymic lymphoma cells on the basis of their ability to compete for the macrophage-dependent cytolysis of a sensitive tumor cell line. The present study was undertaken to determine (a) whether recognition was related to the proliferative response induced by Con A stimulation and (b) whether the competition of cytolysis was dependent upon the binding of sensitive target cells to activated macrophages. These possibilities were tested by examining Con A-treated lymphocytes in different functional stages of the Con A response with respect to their ability to compete either for cytolysis or binding of a tumor cell line susceptible to both activities. The results show that the ability to compete for either function was acquired coincidentally with the Con A-induced proliferative response. This competitive activity was not due solely to the presence of Con A in the culture medium nor to culture of unstimulated lymphocytes but rather required a blastogenic response to the mitogen. Blast-transformed nonproliferative cells (96 hr post-Con A stimulation) were as competitive as cells which had been stimulated to reinitiate DNA replication by treatment with Interleukin 2. Thus, competition for cytolysis is a consequence of blastogenesis rather than proliferation per se and operates mechanistically by competing for the binding of target cells to activated macrophages, an event known to be a necessary prerequisite to cytolysis.     

Genetic control of T-lymphocyte mitogenesis in chickens

The in vitro mitogenic response to PHA and Con A was determined in three inbred lines of chickens. Lymphocytes from one line consistently showed a greater stimulation by PHA than the other two lines. Analysis of F₁ crosses and backcrosses indicated that this quantitative difference was controlled by more than one gene. More substantial differences in Con-A stimulation were also observed between the three lines, and the data indicated that separate genetic systems were controlling the variation in PHA and Con-A stimulation. Analysis of F₁ crosses, backcrosses and assortative matings between backcrosses revealed that the variation in Con-A stimulation was controlled by at least two major genes, one of which may be linked to the major histocompatibility complex. Surprisingly, one line appeared to be segregating for Con-A stimulation in spite of an inbreeding coefficient greater than 0.98.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Studies on the activation mechanism of human mononuclear leukocytes isolated by physical methods

Responder lymphocytes, accessory lymphocytes and monocytes were isolated using countercurrent centrifugal elutriation and free flow electrophoresis. The Concanavalin A (Con A) binding behaviour of the isolated cell populations was studied. Responder lymphocytes and accessory cells bound Con A by different mechanisms. Cells in all the isolated populations were able to interact directly with Con A. Con A binding to responder lymphocytes was inhibited by alpha-methylmannoside (alpha MM) and by a distinct plasma protein. Accessory lymphocytes and monocytes bound Con A even in the presence of 10% human plasma or 50 mM alpha MM. The plasma protein which inhibited interaction of responder lymphocytes with Con A also reduced lymphocyte proliferation, when resting monocytes were used as accessory cells. If, however, monocytes were used after activation by lipopolysaccharide no inhibition effect was observed. From the results we conclude that there is a distinct plasma protein which protects the organism against lymphocyte stimulation not controlled by accessory cells. This inhibitory plasma protein appears to be a euglobulin.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Receptor mobility and the binding of cells to lectin-coated fibers

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation     

The role of macrophages in thymocyte mitogenesis.

The thymic lymphocytes of CBA/J mice respond to the mitogen Concanavalin A (Con A) only in the presence of adherent cells of the monocyte-macrophage series. Depletion of adherent cells abrogates the response, and macrophage-rich population of cells restore it. The need for macrophages and mitogen is completely provided by irradiated splenic macrophages which have been exposed to Con A and washed free of the soluble mitogen. The mitogenmacrophage effect in this case is apparently not due to soluble factors. Even more striking than the effect of macrophages on fresh cultures of thymic lymphocytes is their ability to restimulate quiescent cells 72 hr after their first stimulation with Con A. The quiescent cells respond immediately and quantitatively to Con A in the presence of fresh macrophages. This stimulation, like that of fresh thymocytes, is also controlled by a lymphokine ("costimulator") produced by mixing macrophages, mitogen, ant T lymphocytes. Our data suggest a model in which two signals are required for mitogenesis. First, the interaction of macrophage, T cell, and mitogen elicits a soluble costimulator, which is itself not mitogenic. Secondly, in the presence of costimulator, the mitogen (either soluble, or, more efficiently, bound to macrophages) induces a proliferative response in the T cell.     

Growth Stimulation of Tumor-Specific Cytotoxic T Lymphocytes on Concanavalin A-Immobilized Carrier Beads

Human tumor-specific CD4⁺ cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.     

Cytochemical examination of peripheral blood lymphocytes in bursectomized chickens

The starting material were 86 day-old utility hybrid Tetra SL chicks. Beginning from their third week of life, 59 chickens were kept in two separate rooms "A" and "B" for 42-56 days. The values of temperature and cooling were somewhat different in rooms "A" and "B", however essentially not deviating from the accepted zoohygienic norms. The obtained results revealed a significant reduction of the activity of acid phosphatase in blood lymphocytes of 8-10 week-old chickens bursectomized in the neonatal period and kept in room "A". No such changes were found concerning ATP-ase and 5'-nucleotidase. Similar effect appeared in the lymphocytes of non-bursectomized chickens kept in room "B". Antigen stimulation (SRBC) of bursectomized and non-bursectomized chickens brought about an increased activity of all the three enzymes in blood lymphocytes. At the same time it should be emphasized that the increased activity of the enzymes tested was modulated by bursectomy and conditions of the medium.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

Con A-stimulated thymocytes produce interleukin 2 after removal of Lyt-1 positive cells

Mouse lymphocytes produce several lymphokines, including interleukin 2 (IL-2) and colony-stimulating factors (CSFs) following stimulation with T-cell mitogens. However, very little IL-2 is produced by thymocytes upon concanavalin A (Con A) stimulation. Strong selective inhibition of IL-2 production was observed when fresh spleen cells were mixed with Con A-activated thymocytes. Sorting of populations on the basis of antigenic phenotype showed that the cell mediating the blockage in IL-2 secretion is a large T cell expressing markers for both Lyt-1 and Lyt-2. This specific inhibition of IL-2 accumulation was not mediated by a soluble product, or by absorption on expressed IL-2 receptors on the activated thymocytes. Removal of the Lyt-1 positive cells from a thymocyte population renders it capable to produce IL-2 upon Con A stimulation, indicating a functional role of these cells.     

In vitro T cell hyperreactivity in Obese strain (OS) chickens is due to a defect in nonspecific suppressor mechanism(s)

Spontaneous autoimmune thyroiditis of OS chickens is associated with a marked hyperreactivity of the T cell system. The purpose of the present study was to investigate the underlying regulatory mechanisms. Co-cultivation experiments between Con A-stimulated OS and NWL lymphocytes in communicating cultures revealed soluble regulatory factors to be responsible for the observed functional differences: the high proliferative response to Con A and hyperproduction of IL 2 of OS cells was found to be due to a deficiency in the conditioned medium of dialyzable inhibitory factor(s) that regulate IL 2 secretion of NWL lymphocytes. Furthermore, sera of young NWL chickens were found to profoundly inhibit the IL 2-promoted lymphoblast proliferation. This IL 2 antagonizing activity is lost with age (3 to 6 yr) and was found to be significantly diminished in OS birds throughout ontogeny, thus pointing to possible parallels between immune regulatory dysfunction in autoimmunity and in physiologic aging. Both enhanced T cell response and the defect in serum suppressor were inherited by (OS X CB)F1 animals, indicating that these two aberrations may be related to each other.     

Exogenous interleukin-2 abrogates differences in the proliferative responses to T cell mitogens among inbred strains of mice.

The proliferative response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, concanavalin A (Con A), was two or more times stronger than that of cells from C57BL/10SnSc (B10) mice. In contrast, the cells from B10 mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). The differences in the proliferative response to Con A stimulation were not associated with the function of macrophages nor did they depend on IL-1. Spleen cells from BALB/c and B10 mice synthesized comparable amounts of mRNA for IL-1 alpha, and the production of biologically active IL-1 was even higher in the B10 strain. Indomethacin, an inhibitor of prostaglandin synthesis, had no effect on the differences in reactivity between the cells from BALB/c and B10 mice. In addition, no differences in the synthesis of mRNA for the inducible 55-kDa interleukin-2 (IL-2) receptors were found between the spleen cells from BALB/c and B10 mice. However, Con A-stimulated spleen cells from B10 mice produced a significantly lower amount of biologically active IL-2 than similarly stimulated cells from BALB/c mice. In the presence of exogenous IL-2, these low responder spleen cells from the B10 mice responded by proliferation to Con A stimulation to the same extent as cells from the BALB/c mice. These results thus show that a low proliferative response to Con A stimulation in B10 mice was a consequence of a lower production of IL-2 and possibly abrogated the proliferative hyporeactivity produced by exogenous IL-2. We suggest that the differences in the ability to produce IL-2 could be a reason for the discrepancies observed in the immunological responsiveness between BALB/c and B10 mice.     

The role of insulin in the response of murine T lymphocytes to mitogenic stimulation in vitro

The ability of insulin to influence the responsiveness of murine T lymphocytes in a culture system containing a serum substitute was documented. The presence of insulin was found to enhance the concanavalin A (Con A) reactivity of the lymphocytes. Once the cells were activated by a short-term exposure to Con A, insulin was capable of replacing Con A for the continued stimulation of the cells. This was true both for lymphocyte proliferation and for the generation of nonspecific cytotoxic T lymphocytes. The presence or absence of insulin was not found to influence the phytohemagglutinin responsiveness of the T lymphocytes. Possible reasons for the observed results are discussed in relation to a proposed model for lymphocyte activation.