Anchorage and lymphocyte function. Antibodies as adhesion and spreading factors for human T lymphocytes

When attached to a solid surface coated with protein A various antibodies reacting with lymphocyte membrane antigens (anti-beta 2m, OKT3, OKT8, Leu2, 3, 4 and certain patient sera) catalyse the formation of peripheral lamellar activity, i.e. an active spreading process in human T lymphocytes. In contrast, binding only of the same antibodies to the cells or allowing antibody-coated cells to settle and bind to a protein A-coated surface did not induce spreading although the number of cells attached to the solid surface was virtually the same as in the former case. The peripheral lamellar activity markedly facilitated short-range lymphocyte interactions and appeared to constitute the region of the lymphocyte that actively contacts other cells. These results show that antibodies can act as spreading factors, and indicate that this function is critically dependent on the presentation of the inducing ligand. The asymmetry in the induction of active cell edges may influence functional lymphocyte interactions with environmental surfaces.     

Activation of human peripheral blood lymphocytes by concanavalin A dependence of monocytes.

The activation of human peripheral blood lymphocytes or isolated T lymphocytes by concanavalin A (Con A) is hightly potentiated by the presence of autologous, mitomycin C-treated monocytes. The optimal lymphocyte: monocyte ratio within a broad dose range is 1:1 when the incorporation of [14C]thymidine is expressed as total incorporation per culture tube and 1:10 when expressed per lymphocyte. A five-to-ten-fold increase of total DNA synthesis is noted in the presence of 10-90% monocytes. The data may help to explain the wide variations in Con A responsiveness of human peripheral lymphocytes which may be partly related to differences in purification which give rise to cell preparations containing varying amounts of monocytes.     

Anchorage and lymphocyte function: collagen and the maintenance of motile shape in T cells.

When cultured on a collagen matrix at a density of 7 X 10(4) cells/cm2 for 48 hr, 80 +/- 10% of unfractionated and 70 +/- 14% of T-enriched human blood lymphocytes from eight healthy individuals developed a motile morphology defined as the presence of lamellar surface activity and cytoplasmic flattening. During culture on plastic for 48 hr, 32 +/- 5% of unfractionated and 38 +/- 6% of T-enriched lymphocytes from the same individuals developed a motile morphology. The motile morphology was not a rigid state but a series of oscillations in cell shape. The conversion from a spherical into a motile morphology was independent of cell density. After preculture on plastic at 'high' density (1.5 X 10(6) cells/cm2), and subsequent transfer to a plastic surface, 54 +/- 12% of the cells from separate individuals exhibited a motile morphology within 2 hr. These motile forms were, however, transient and approximately 50% disappeared within 12 hr. Collagen augmented the motile behaviour of unfractionated and T-enriched lymphocytes by two to four times when fresh from the blood compared with glass or plastic. During culture on plastic, the lymphocytes lost this prompt responsiveness to collagen contact. Thus, during culture on plastic, the ratio between percentage motile lymphocytes after subsequent transfer to collagen and plastic, respectively decreased from values between 2 and 4 immediately after purification to close to 1 within 2 days. However, when retransferred to collagen, the majority of the lymphocytes within another 2-day period acquired responsiveness to collagen measured as potentiation of motile cell shape on this substrate compared with on plastic. These data suggest that the variation in motile behaviour in the T lymphocyte reflects a labile property which is enhanced by contact with a collagen matrix.     

Anchorage and lymphocyte function. Spreading-capacity distinguishes common thymocytes and peripheral T lymphocytes

Contact of T-enriched human blood lymphocytes with an adhesive surface in the presence of Concanavalin A (Con A) almost immediately induced a sequence of motile changes in virtually all cells. The initial event in this spreading process was the formation of filopodia distinct from the microvilli of lymphocytes in suspension. The filopodia were accompanied by lamellipodia, ruffles and flattening of the nucleus. Contact with a nonadhesive substratum in the presence of Con A did not trigger this sequence of changes. Cytochalasin B and D or low temperature inhibited the contact-induced changes. With the exception of a small number of cells (5-15%), T-enriched lymphocytes that were allowed to settle in the absence of Con A showed a radius of action (area occupied by the cells/translational movement per hr) of 39 micrometers 2/ less than 1 micrometer. The small 'motile' population showed a radius of action of 74 micrometers 2/8 micrometers. The Con-A-mediated spreading-process yielded a radius of action of the lymphocytes of 117 micrometers 2/6 micrometers. This augmented radius of action markedly facilitated cell-cell interaction in a high frequency of the cells and appeared to be a prerequisite for such interactions at 'low' cell density. Thymocytes reactive with OKT 6 antibodies or belonging to the 'high-density' fraction of cells attached to a Con-A-coated surface to the same extent as peripheral OKT 3 positive lymphocytes, but did not exhibit the morphological changes characteristic of a spreading-process. In contrast, OKT 6 negative thymocytes or thymocytes with a relatively low density showed spreading indistinguishable from that of OKT 3 positive peripheral lymphocytes. These results characterize the spreading-process in human T lymphocytes and demonstrate its functional importance for interactions with the environment. Spreading-capacity appears to reflect the stage of maturation of T cells.     

Anchorage and lymphocyte function. Contact-induced augmentation of T-cell activation.

Beads of polyacrylamide, latex or DEAE-Sephadex markedly augmented the stimulation of unfractionated or T-enriched lymphocytes by concanavalin A (Con A) or phytohaemagglutinin (PHA). The beads were not mitogenic in the absence of Con A or PHA. A prerequisite for the bead-induced augmentation was that the stimulated lymphocytes had been depleted of phagocytic and/or adherent accessory cells. The enhancing effect of beads was most pronounced during the initial 12 h after the beginning of lymphocyte stimulation, but not limited to this early phase of the growth period. The stimulation of lymphocytes in petri dishes of adhesive tissue culture plastic and non-adhesive bacterial plastic were compared. The magnitude of the stimulation on the non-adhesive surface was 10--50% lower than on the adhesive one, this difference being most pronounced at hyperoptimal mitogen concentrations. These results indicate that contact between some cell type and a solid surface can improve lymphocyte stimulation under experimental conditions when the number of phagocytic and adherent accessory cells is a limiting factor. The fact that cultivation on bacterial plastic, where adhesion and spreading were abolished, produced substantial stimulation (albeit reduced) demonstrates that substrate contact may be important, but is not a prerequisite, for lymphocyte activation.     

Amphotericin B modifications of peripheral blood lymphocyte and tonsil lymphocyte responses to concanavalin A

Peripheral blood lymphocytes (PBL) were shown previously to be activated to incorporate ³H-thymidine by concanavalin A (con A). Amphotericin B (Am B) was reported to be both immuno-enhancing and suppressive. We investigated the response of PBL and tonsil lymphocytes (TL) to con A in culture, and the effect of Am B on these responses. PBL were activated by con A as expected, and the magnitude of the response diminished as cell density increased. The responses of PBL usually were significantly enhanced by Am B at 2.5 or 5 μg/ml, but 10 μg Am B/ml significantly inhibited the con A response. TL had relatively high rates of spontaneous proliferation, but Am B was a potent inhibitor of this. TL responded to con A just as well as PBL, and Am B strongly inhibited these responses too. When TL were cultured with various doses of con A in the presence of 5 or 10 μg Am B/ml, the shape of the dose curves resembled those of PBL stimulated in the absence of Am B except that the incorporation of ³H-thymidine increased with increasing cell density. It was concluded that Am B at 2.5–5.0 μg/ml enhanced the response of PBL to con A and suppressed the response at 10 μg/ml. All doses of Am B inhibited both spontaneous proliferation and con A-induced proliferation in TL. In spite of this inhibition, the TL population responded to various doses of con A in the presence of Am B.     

Human peripheral blood lymphocyte transformation by concanavalin A in a chemically defined medium

Human small lymphocytes from peripheral blood were cultured for 72 hours in a chemically defined medium consisting of NCTC 109 supplemented with methyl cellulose, glucosamine, and hormones. About two thirds of the cells died during the first 24 hour period, but thereafter the counts were fairly stable. The addition of concanavalin A stimulated the production of transformed cells, although during the 72 hour observation period none of these was seen in mitosis. As the amount of concanavalin A added to 3 ml. cultures was increased from 5 to 40 μg, the number of small lymphocytes in the defined medium that underwent transformation increased from 34 per thousand to 61 per thousand.     

Modulation of concanavalin A-induced lymphocyte stimulation by human low-density lipoproteins

Spleen lymphocytes and T cells were stimulated by concanavalin A in the presence of various concentrations of human low-density lipoproteins (LDL). The proliferative responses were measured by [¹⁴C]thymidine incorporation. Low LDL doses (5–50 μg/ml) significantly enhanced the stimulation of splenic lymphocytes and T cells. Further, LDL had the capacity to partially relieve the suppression produced by supraoptimal doses of concanavalin A.     

Isolation of human spontaneous killer lymphocytes by bacterial adherence.

Human lymphocyte subpopulations (B, T1, T2, T3, and T4 our denomination) have been identified previously by bacterial adherence and differences between them in mitogen responses and specific cytotoxic activity have been found. In this study another aspect has been investigated in order to find functions associated with these subpopulations, namely the spontaneous killing (SK) ability. Freshly isolated human peripheral blood lymphocytes (PBL) were separated into adherent and non-adherent cells following centrifugation against various bact:rial monolayers. The PBL and the resulting subpopulations of PBL were tested alone or in combination as effector cells in a 4 hr cytotoxicity assay against human lymphoblastoid cel- lines of B or T cell origin. The T3 + T4 cells or T4 cells alone showed a significantly higher SK activity against both B and T target cell lines when compared with unseparated PBL, T1 + T2, or T3 cells alone. Whe Fc portion of IgG, contain the lymphocytes responsible for SK activity and that SK cells can be purified by negative selection using bacterial adherence.     

Changes in concanavalin A capping of human lymphocytes with age

Cap formation of peripheral blood lymphocytes from human donors of different ages was compared using fluorescein-labeled concanavalin A as the ligand. The mean percentage capping in donors aged 20–40 years (21.6 ± 0.7%) was significantly higher than values found for donors aged 40–60 years (18.4 ± 1.1%) and for donors aged > 60 years (14.5 ± 1.3%). Regression analysis of the entire age range studied (3–95 years) indicated that capping decreases in a linear fashion with age, although in the old age group some individuals showed capping values comparable to those of young donors. Preincubation of lymphocytes with colchicine increased capping in both young and old donors. Cells from both young and old donors with capping values of more than 20% showed a marked increase in capping following colchicine treatment. Capping values after colchicine in these two groups were significantly greater than values after colchicine treatment of old donors' cells with low pretreatment capping values. This finding suggests that abnormalities of microtubules do not totally account for the age-related reduction of concanavalin A capping unless colchicine interacts differently with lymphocyte microtubules in the aged. This study provides further evidence for altered cell membrane function with age.     

Activation of human T and non-T lymphocytes by sepharose-bound concanavalin A and the differential effect of macrophages.

The mitogenic effect of insoluble concanavalin A (Con A) bound to Sepharose beads on human peripheral blood mononuclear cells has been investigated. Sepharose-Con A, with a maximum activity at 100-400 micrograms/ml and an optimal incubation time of 3 or 4 days, stimulated the mononuclear cells with a high mitogenic response. Sepharose-Con A also stimulated the monocyte-depleted lymphocytes and their T- and non T-enriched fractions with a definite mitogenic response, although the levels of response were much lower than those of unpurified mononuclear cells. Addition of the adherent monocytes strongly augmented the Sepharose-Con A responses of monocyte-depleted lymphocytes and their T-enriched fractions, whereas it gave no effect on those of non-T-enriched fractions. Culture fluids of adherent monocytes also augmented the Sepharose-Con A responses of monocyte-depleted lymphocytes. These results indicate that, although insoluble Sepharose-Con A activates both human T and B cells, the response of T cells but not B cells may be dependent in part on the presence of monocytes or a monocyte-derived soluble factor(s). Thus, it is concluded that Sepharose-Con A may be a useful mitogen to elucidate the role of macrophages or their soluble factors in activation of human T and B cells.     

Effects of concanavalin A and Fc fragments of IgG on human monocyte cAMP content: modulation of monocyte secretory function by cAMP

Exposure of human monocytes in monolayer culture to concanavalin A (Con A) or Fc fragments of IgG results in decreased levels of lysozyme, acid phosphatase and the second component of complement (C2), but increased levels of prostaglandin E₂ (PGE₂) in the culture medium. Indomethacin blocked the effect of Con A and Fc fragments on PGE₂ levels but did not alter the effects on the other secretory products. The addition of Con A to the monocyte cultures resulted in increased cAMP levels in short (<20 min) and in long term incubations (24 hr). Long term exposure to the Fc fragments of IgG also resulted in elevated levels of cAMP. In long term cultures, elevation of cAMP levels by Fc fragments and Con A may in part be mediated by stimulation of endogenous prostaglandin production, since addition of indomethacin resulted in a return of cAMP towards basal levels. The addition of D-L isoproterenol, IBMX, or exogenous PGE₂, reagents that increase cAMP content, or addition of Dibutyryl 3′, 5′ adenosine monophosphate (DBcAMP) to cultures resulted in a dose-dependent inhibition of secretion of acid phosphatase, lysozyme and C2.

These findings suggest that certain secretory responses of human monocytes are reduced by agents which increase cAMP levels. These changes may be related to direct effects on adenylate cyclase stimulation or indirect effects on the release of other products which effect cAMP generation. These changes in cAMP content may in part be responsible for the reduced secretory response.

     

Function and distribution pattern of human T lymphocytes. I. Production of anti-T lymphocyte specific sera as estimated by cytotoxicity and elimination of function lymphocytes

Antisera were prepared in rabbits against lymphoid cells from peripheral blood of a patient with Bruton-type agammaglobulinaemia. Such antisera could be shown to display a two plateau level of cytotoxicity against human peripheral lymphocytes in the presence of complement. This suggested the presence of species- as well as subgroup-specific antibodies in these antisera.

The subgroup-activity of the antisera could be shown to be directed against human T lymphocytes on the basis of the following results. When lymphocytes are filtered through columns coated with anti-immunoglobulin antibodies lymphocytes with high surface concentrations of immunoglobulin are retained. The filtered cells are highly enriched in cells sensitive to the subgroup-specific antibodies. A close to complete inactivation of mixed leucocyte reactivity or stimulability with soluble PHA was induced by preincubating with the antisera and complement. Using identical conditions only marginal inhibition of immunoglobulin production of peripheral lymphocytes in vitro was induced.

In conclusion, we believe these antisera to selectively kill human T lymphocytes at serum concentrations, which will not kill or inhibit the function of human B lymphocytes.

     

Activation of B lymphocytes by locally concentrated concanavalin A

B lymphocytes are not stimulated by soluble concanavalin A (Con A). However, Con A cross-linked at the bottom of tissue culture petri dishes could activate DNA synthesis in B cells. Addition of free Con A to such cultures inhibited the proliferative response to insoluble Con A. T lymphocytes were not activated by locally concentrated Con A, although they responded to soluble Con A. In the presence of both soluble and cross-linked Con A there was a shift in the dose response curve to soluble Con A, the magnitude of which varied with the concentration of cross-linked Con A.     

Regulation of lymphocyte proliferation by soluble products released by stimulated human lymphocytes.

Human lymphocytes pulsed with phytothaemagglutinin (PHA) release mitogenic factors and other lymphokines upon subsequent incubation in fresh medium. Such active supernatants (SUPs) were assayed for their capacity to modify the DNA synthetic response of human lymphocytes exposed to various mitogenic stimuli. Both non-fractionated peripheral lymphocytes and preparations of fractionated T cells were stimulated by active SUPs to DNA synthesis. In contrast, active SUPs reduced the responses of lymphocytes to allogeneic cells, PPD tuberculin and PHA. These reductions were most evident at the end of the culture periods. The results suggest that soluble products released by stimulated lymphocytes activate suppressor lymphocytes which inhibit proliferative responses of lymphocytes.     

Alcohol and immune regulation. I. In vivo effects of ethanol on concanavalin A sensitive thymic lymphocyte function

Alcohol is known to suppress the immune response, but the underlying mechanism to account for this immune suppression is still not clearly elucidated. In an attempt to clarify such mechanisms, experimental rats were fed for 50 days on a 36% ethanol, Lieber diet (LED) while control (LCD) rats were fed a similar diet supplying the same amount of calories but lacking ethanol. It was found that both LCD and LED animals grew at a linear rate (LCD: r = 0.981, LED: r = 0.961) but that LCD animals grew more rapidly. While thymic weights in the LED group were significantly smaller (P less than 0.05) than in the LCD group, the ratios of thymic weight/body weight between these groups were not significantly different. To identify the effects of ethanol on immune response, thymic (Th) or splenic (S) cells were prepared and incubated in culture with the mitogen, Con A and rat serum prepared from LCD or LED groups. It was found that lymphocytes prepared from thymus of LED animals appeared to be depressed in mitogen-driven blastogenic transformation when incubated in LCD serum but not LED serum. Furthermore, lymphocytes prepared from the spleen of LED animals appeared to be depressed in mitogen driven blastogenic transformation when incubated in LED serum but not LCD serum. Since lymphocytes of the thymus and spleen are undergoing maturation and replication this implies that ethanol may alter these processes.     

Studies on the accessory requirement for T lymphocyte activation by concanavalin A.

In this study we have examined the interactions between accessory cells (AC) and T cells in response to Con A. Highly purified peripheral blood T cells and AC exposed to a variety of treatments were used. We found that untreated AC provided optimal help for T cell proliferation and this was not mediated by soluble factors since whole cells could not be replaced with supernatants from activated AC. Furthermore, cycloheximide-treated AC were able to supply the accessory signal although unable to elaborate soluble activation factors. To find out more about the accessory signal, we examined the ability of monocytes mildly fixed with glutaraldehyde to supply help. These cells were completely unable to perform as AC, although they were viable and had unaltered surface antigen expression. They could not secrete activation factors, but this alone could not explain their inability to supply help because this function was not restored with the addition of soluble activation factors. This indicated that AC-T cell contact was of prime importance to accessory function. To investigate the possibility that AC work by cross-linking structures on the lymphocyte surface, we attempted to substitute for the soluble Con A plus AC with Con A bound to the surface of erythrocytes. Comparable stimulation was observed, suggesting that the cross-linking of Con A-bound structures on the lymphocyte surface generates the accessory signal.