Selective proliferation of T cells in the mixed lymphocyte interaction

The contribution of avian thymus-derived (T) and bursa-derived (B₂) cells to the proliferating cell population in mixed lymphocyte cultures (MLC) was evaluated. When spleen cells of chickens containing chromosomally marked T and B₂ cells were stimulated in a one-way MLC by mitomycin C blocked allogeneic spleen cells, only T cells proliferated during a 4–9 day culture period. No evidence for significant recruitment of B₂ cells, expressed as proliferation of B₂ cells, was found. The initial viability and proliferative potential of B₂ cells was shown by a substantial and selective B₂ cell response to anti-immunoglobulin serum.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

Cyclosporin A inhibits the proliferative response and the generation of helper, suppressor and cytotoxic T-cell functions in the autologous mixed lymphocyte reaction

Chemical oxidation or reduction of lymphocyte cell surface thiol or disulfide groups, respectively, has been shown to alter the proliferative activity of murine T cells. S-2-(3-aminopropylamino)ethylphosphothioic acid, a compound containing no free thiol group until it is intracellularly dephosphorylated, did not enhance Con A-induced proliferation which suggested that thiols did not mediate proliferative enhancement via an intracellular mechanism. Glutathione, an impermeant thiol, enhanced T-cell proliferation 68% as effectively as 2-mercaptoethanol (2-ME), which suggested that the thiol-sensitive site was at the cell surface. A battery of structural analogs to 2-ME was employed to elucidate the chemical requirements for the biological activity of the thiols. The necessity for a hydrogen-binding moiety on the thiol reagent was determined by the use of non-hydrogen-binding analogs and by competitive inhibition of the thiol-enhancing activity of 2-ME by non-thiol-containing hydrogen-binding analogs. Pretreatment of cells with the copper:phenanthroline complex (CuP), an impermeant oxidant of thiol groups, reduced the Con A-induced response >79%; however, the presence of 2-ME in culture completely reversed the inhibitory effect of CuP pretreatment. Oxidation of T cells by high oxygen tension (17% O₂) also ablated the Con A response but did not alter the response to Con A + 2-ME. Protection from oxidative inhibition also was afforded T cells by sequential reduction and blockage of cell surface thiol groups. Finally, a model which correlates the chemical study of cell surface residues with T-lymphocyte responsiveness is presented.     

The assay, properties, and kinetics of human T cells forming clusters with sheep erythrocytes (CFC) in stimulated cultures.

This study describes a new method of detecting the in vitro stimulation of lymphocytes based on the appearance of cells having the property to cluster several layers of SRBC around themselves (CFC). The formation of multilayer rosettes is temperature dependent and requires trypan-blue-excluding, metabolically active blastoid cells. Non-separated cells as well as purified T cells stimulated with allogeneic leucocytes (MLR), specific antigens, or polyclonal mitogens (PWM, Con A) gave rise to CFC. Multilayer rosettes were not formed by separate B cells. In the MLR, CFC were detected 48 hr after the beginning of cultures and increased in number thereafter just like thymidine incorporation. The response to Con A and PWM was detected earlier and gave rise to two or three peaks, the first rise in the number of CFC coinciding with the peak of thymidine incorporation but the maximum increase occuring two or three days later. Treatment of blastoid cells with a serum specific for T cells, but not with an anti-immunoglobulin serum, abolished their ability to form ordinary or multilayer rosettes. When separated, however, on a nylon wool column, CFC were more adherent than the bulk of T cells. It is suggested that CFC form a subpopulation of T cells with distinct characteristics, allowing a direct assessment of membrane changes which take place when T lymphocytes are activated in vitro.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Inhibition of the lymphocyte blastogenic response to antigen by serum-free culture supernatants of leukemic B cells

Suppressive factors were detected in culture supernatants of the guinea pig B-cell L₂C leukemia. Dialyzed culture supernatants (DCS) inhibited the blastogenic response of sensitized lymph node cells (LNC) to a wide dose range of the sensitizing antigen (ovalbumin or PPD) but failed to inhibit the proliferative response to PHA or Con A. In addition, DCS inhibited the response of blast cells to preformed T-cell growth factor (TCGF). The inhibitor(s) in DCS was noncytotoxic, heat stable (30 min at 80 °C), resistant to treatment with trypsin, and exerted its effect subsequent to activation of sensitized LNC by antigen. Washing of DCS-treated cells restored normal reactivity to a subsequent antigen challenge. The target cell for the inhibitor may be cells responding to amplification signals produced by activated T cells. KCl (3 M) extracts of L₂C cells behaved like DCS in inhibiting only antigen responses. Both undialyzed culture supernatants (UCS) and leukemic sera inhibited mitogen, allogeneic, and antigen-stimulated proliferative responses by greater than 80%. These soluble factors may participate in the depression of cell-mediated immunity associated with lymphoid malignancies.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

Stimulant-free preculture in heterologous serum-supplemented medium induces unresponsiveness of T cells to subsequent mitogenic stimulation

Quite often freshly isolated lymphocytes are kept in culture before experimentation for 1 or more days without any stimulus. Most of the time, culture is supplemented with fetal bovine serum (FBS) which is heterologous to all species except bovine. In the present study, we found that freshly isolated murine T cells show a good proliferative response to concanavalin A (Con A) and phorbol ester (PMA)/ionomycin in FBS medium, without any detectable background proliferation. However, the cells kept in the same culture without any stimulus for prolonged period of time (referred to as preculture in this report) showed reduced response to Con A and PMA/ionomycin in a time-dependent manner. Almost a complete loss of response to Con A was observed within 1 day of preculture. However, loss of response to PMA/ionomycin was observed only after 2 days of preculture. Interestingly, similar preculture in autologous mouse serum-supplemented media did not cause any loss of the response to these mitogens. The loss of responsiveness of T cells during preculture in heterologous serum was irreversible. The heterologous serum-induced unresponsiveness of T cells to these mitogens was also prevented by adding Calphostin C, a specific protein kinase C (PKC) inhibitor, during preculture in heterologous serum. These results showed that prolonged stimulant-free preculture in heterologous serum induces irreversible unresponsiveness of T cells to mitogens through the down regulation of T cell receptor signaling pathway, which can be prevented by autologous serum or a PKC inhibitor.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.     

Suppressor cells present in the spleens of Trypanosoma cruzi-infected mice.

Infection with Trypanosoma cruzi decreases the ability of spleen cells from mice to respond to either T cell, concanavalin A (Con A), or B cell, lipopolysaccharide (LPS), mitogens. The effect of infection on the mitogenic response depends on the elapsed time between the day of infection and the time of mitogen presentation. Responses early in infection are normal, whereas later responses to either mitogen are depressed. Spleen cells from late trypanosome-infected mice inhibit the ability of normal spleen cells to respond to Con A or LPS. The cell in the T. cruzi-infected spleen cells responsible for this effect is nonadherent, sensitive to treatment with anti-mouse thymus serum plus complement, but insensitive to treatment with anti-immunoglobulin plus complement. These data indicate that infection with T. cruzi elicits over time the generation of T cells suppressive to T and B cell mitogenic responses.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Synergistic effect of concanavalin A and Bu-WSA on DNA synthesis in human peripheral blood lymphocytes

Butanol-extracted water soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was not mitogenic for human peripheral blood mononuclear cells (PBM) but was capable of enhancing (3H) thymidine uptake of T cells stimulated by concanavalin A (Con A) in the presence of B cells or macrophages (M phi) in vitro. The mechanisms of the synergy of Con A and Bu-WSA were studied by using separated cell populations from PBM. Both subfractioned OKT4+ and OKT8+ cells were responsive to co-stimulation by Con A and Bu-WSA in the presence of an accessory cell population. Allogeneic B cells and M phi as well as autologous cells had helper function as accessory cells. Heavy irradiation with gamma-rays did not affect the function of the accessory cells, but previous treatment of B cells with anti-Ig serum plus complement (C) or treatment of M phi with anti-M phi serum plus C deprived them of their function. The treatment of accessory cells with anti-HLA-DR serum, regardless of the presence or absence of C, resulted in loss of their helper function. Cultures in Marbrook-type vessels showed that a mixed cell population of T cells and accessory cells in the lower chamber produced some active factor(s) after co-stimulation with Con A and Bu-WSA, and by passing through the membrane filter separating the chambers, the factor(s) enhanced the proliferation of the Con A-activated T cell population in the upper chamber. The factor(s) was presumed to be interleukin 2 (IL 2), because it supported the growth of IL 2-dependent CTLL cells. These results indicate that the synergy of Con A and Bu-WSA on the proliferative response of human PBM is due to the elevation of growth factor production from T cells stimulated by those mitogens.     

The regulatory role of adenosine-activated T-lymphocyte subset on the immune response in humans: I. Mitogenic response and production of mediators

Human T lymphocytes, rerosetted with sheep erythrocytes in the presence of adenosine, yield two subpopulations: a major one (E_R), still capable of forming E rosettes; and a minor nonrosetting (E_S) one. The two subpopulations differed in their proliferative responses to various mitogens. E_R cells responded well to galactose oxidase (GO), soybean agglutinin (SBA), and phytohemagglutinin (PHA) but responded poorly to concanavalin A (Con A). The response of E_S cells was poor to GO and SBA, intermediate to PHA, and significantly high to Con A. The different response of E_R and E_S subsets to Con A was not greatly affected by adherent cells, but an enhancing effect on the proliferation of E_S cells to Con A was observed when prostaglandin synthesis was inhibited by indomethacin. Addition of E_S cells to E_R cells in a ratio of 1:5 resulted in an enhanced synergistic effect of Con A-induced proliferation. A soluble mitogenic factor released from Con A-activated T cells appeared involved in this enhanced proliferation. This factor (E_SF) was produced only by the minor T-cell subpopulation which is sensitive to adenosine (E_S). The induction of E_SF was not dependent on the addition of adherent cells and required 72 hr of incubation for its production. E_SF was mitogenic to nonactivated and Con A-activated PBL as well as to T, E_R, and E_S subpopulations. Following incubation of E_R cells with E_SF, a suppressor factor (E_RSF) was produced which abolished the mitogenic activity of E_SF. Differences between these factors and a known mediator like Interleukin-2 (IL-2) and suppressor factors are discussed.     

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/ mass spectrometry

Human alveolar macrophages, obtained during diagnostic bronchoscoy, were maintained in monolayer culture. Challenge of these cells (>95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B₄ into the medium, 7.28 ± 5.99 ng/mg cell protein at 2 h mean ± S.D⁴, n = 4). Leukotriene B₄ was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B₂, prostaglandins D₂, E₂, F_2α and the lysosomal enzyme N-acetyl-β-D- glucosaminidase was also measured. Thromboxane B₂ was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 ± 14.8 ng/mg cell protein 2 h after the addition of zymosanA, n = 4), and the synthesis of thromboxane B₂ was inhibited by >90% in 1 μM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B₄ synthesis.     

Fluorescence-activated cell sorting of human T and B lymphocytes. II. Identification of the cell type responsible for interferon production and cell proliferation in response to mitogens

We have employed the fluorescence-activated cell sorter to separate pure viable preparations of human T and enriched B lymphocytes. Using such preparations, we have demonstrated that both human T and B cells can respond to PHA and PWM in vitro in the presence of macrophages with proliferation and the production of interferon, a mediator of cellular immunity. However, selective T cell interferon production and proliferative response can be assessed at 3 days in culture; B cell interferon production and proliferative response is delayed to 5 and 7 days. T cells or T cell products are ineffective in inducing or accelerating B cell interferon or proliferative response at 3 days. The use of 3-day T cell interferon production as a new technique for the assessment of T cell effector function and competence is suggested.     

Modulation of IL-4 production in murine spleen cells by prostaglandins

Recently, it has been reported that IL-4 production by murine Th2 cell lines is insensitive to inhibition by E-type prostaglandins. In the present study, IL-4 production in vitro by freshly isolated concanavalin A (Con A)-stimulated murine spleen cells was readily suppressed by PGE₂ with an I₅₀ of 2 nM. Comparable suppression by PGE₂ was seen after priming by anti-CD3? antibody instead of Con A or with other changes in the culture conditions. PGE₂ was an effective inhibitor after elimination of Ly2.2⁺ T cells, consistent with a direct effect on Th2 cells. In the absence of added prostaglandins, IL-4 production was enhanced 1.5- to 7.0-fold by 0.2–2.0 μM indomethacin, indicating that endogenous arachidonate metabolites such as PGE₂ and PGI₂ regulate IL-4 production in our usual culture system. The inhibition of Th2 cell secretion by PGE₂in vitro may have physiologic and pharmacologic implications for the regulation of Th2 cell function and IgE production in vivo.