Differentiation-associated alteration in human monocyte-macrophage accessory cell function

Human monocyte (Mo) to macrophage (Mx) differentiation is associated with marked and well studied changes in morphology, biochemical parameters, and effector cell function. Nevertheless, the comparative accessory cell (AC) function of blood Mo and differentiated Mx has not been carefully studied. We, therefore, examined the kinetics and mechanisms of change in AC function during in vitro Mo to Mx differentiation. The system utilized has two distinctive features: blood Mo and resultant cultured Mx represent a cohort of cells derived from the bone marrow within a 12-hr period. Moreover, the in vitro derived Mx utilized herein have been characterized extensively and are functionally and biochemically similar to pulmonary macrophages (PMx). In the experiments reported, AC functions of blood Mo, Mx derived from Mo after 1 to 6 days of culture, and PMx was compared. AC were cultured with nylon wool column-purified autologous T cells and were stimulated with concanavalin A (Con A) or streptokinase-streptodornase (SKSD). Blood T cell proliferation to Con A or SKSD was inhibited greater than 90% by the removal of Mo and was reconstituted by 20% Mo. Mx derived from Mo by culture for 1 to 3 days exhibited the same (or better) AC function as Mo when T cells were stimulated with either SKSD or Con A. In marked contrast, Mx derived from 6-day cultures exhibited less than or equal to 15% of Mo (i.e., control) capacity to support T cell proliferative response to SKSD. Six-day Mx support T cell proliferation to Con A was somewhat variable. Similar to 6-day cultured Mx, PMx failed to function as AC. The mechanism of loss of AC function was examined: a) cultured Mx maintained Ia antigen positivity for greater than 8 days; b) mixing experiments with Mo + 6-day cultured Mx or Mo + PMx demonstrated no T cell suppression; c) the normal capacity of most 6-day cultured Mx to support Con A but not SKSD induced T cell proliferation, apparently ruled out the loss of the ability to deliver a nonspecific "second signal" as the involved mechanism; d) inhibition of Mo to Mx differentiation by dexamethasone preserved AC activity. Thus, human culture-derived Mx and PMx exhibit deficit AC function through loss of an undefined mechanism. However, loss of AC antigen processing or presentation may occur.     

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.     

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.     

A spleen-derived maturational factor allows immature thymocytes, prepared as cells bearing low amounts of surface sialic acid, to become cytotoxic T cells

A population of immature mouse thymocytes bears low levels of surface sialic acid and can be separated from the more mature high sialic acid-bearing thymocytes by selective agglutination with the sialic acid-specific lectin, lobster agglutinin 1. These immature thymocytes do not proliferate in response to concanavalin A (Con A). They do not produce interleukin 2 (IL-2), do not provide T cell help to B cells for an in vitro antibody response, and as shown here, do not become cytotoxic T lymphocytes when polyclonally stimulated with Con A + IL-2. We describe here a spleen-derived maturational factor which stimulates these immature thymocytes, in the presence of Con A and IL-2, to become cytotoxic T lymphocytes. The maturational factor is a protein secreted by Con A-stimulated mouse or rat spleen cells; it is apparently neither interleukin 1, IL-2, interleukin 3, gamma-interferon, nor combinations of these cytokines, because these materials do not replace the maturational factor. The active material in Con A-stimulated mouse spleen cell supernatant was recovered from a G-75 column in the 33,000-48,000 m.w. range. These experiments suggest that within the lobster agglutinin 1-negative thymocyte population there are cells which can mature under the influence of a spleen-derived factor. It is possible that these cells represent the small subpopulation of immature cells destined to become immunocompetent peripheral T cells. On the other hand, the factor may be rescuing cells destined to die in the thymus.     

Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of T-cell proliferation and enhancement of NK activity by supernatants from Con A-stimulated human peripheral blood mononuclear cells: a new lymphokine

Supernatants from human peripheral blood mononuclear cells activated by Con A contain a factor(s) that stimulates blastogenic activity of normal human peripheral blood mononuclear cells. This Con A supernatant (CAS) contains stimulatory activity for E-rosette positive lymphocytes (T cells) and requires adherent cells for stimulation of T-cell proliferation. CAS does not contain detectable amounts of IL-2 as determined by its inability to support CTLL cell growth. Nor does it contain IL-1 or interferon. Examination of functional activity of lymphocytes stimulated for 3 days by CAS revealed that NK activity is augmented. This supernate does not appear to have any direct effect on B-cell function, although it induces suppression of polyclonal PWM stimulation of immunoglobulins. Thus, CAS appears to contain a new cytokine with immunomodulating potential.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

The role of accessory molecules in T-helper activation induced by antigen, lectin, or CD3 antibodies

We examined the role of L3T4 and LFA-1 molecules in T-helper-cell activation, under conditions where the physical stability of T helper-accessory cell interactions was not an issue. T-helper hybridomas were activated by accessory cells coated either with concanavalin A (Con A) or with CD3 antibodies. Activation of the T helper cells was measured by microtubule-organizing center (MTOC) reorientation as an early activation event, and by interleukin-2 (IL-2) production as an indication of a fully matured response. Both parameters were strongly blocked by L3T4 and LFA-1 antibodies in the case of Con A activation. In the case of stimulation with accessory cell-bound CD3 antibody, activation was blocked by LFA-1 but not L3T4 antibody. These results support the notion that L3T4 and LFA-1 molecules play more than a simple adhesion role in T-cell activation. The differential effect of L3T4 antibody in the case of Con A activation vs CD3 activation is consistent with the possibility that L3T4 and the alpha/beta portions of the T-cell receptor must interact during antigen- and lectin-stimulated T-cell activation.     

LFA-1/ICAM-1在ConA诱导的小鼠胸腺细胞活化中的作用

用抗LFA-1/ICAM-1粘附分子单克隆抗体和ConA联合刺激小鼠胸腺细胞,初步研究了该膜分子在经TCR/CD3介导的胸腺细胞活化信号传导以及胸腺细胞亚群选择中的作用。在ConA刺激系统中,抗ALFA-1/ICAM-1单抗均能抑制胸腺细胞的增殖应答,且以抗LFA-1单抗的作用更为显著;而在PMA加钙离子载体A23187刺激体系中,抗LFA-1单抗却表现出明显的促活化效应。当加入IL-2 时,抗LFA——1/ICAM-1单抗便不能抑制ConA刺激的胸腺细胞活化。此外,抗体对已活化的胸腺母细胞增殖也无影响。FACS分析的结果还显示,抗LFA-1单抗可明显降低CD4~-CD8~ 胸腺细胞亚群的比例,而抗ICAM-1单抗对此无影响。表明胸腺细胞表面粘附分子LFA-1具有直接参与TCR/CD3途径介导的跨膜信号传导的功能,并对CD4~-CD8~ 胸腺细胞亚群的功能分化与成熟可能起重要作用。     

Capacity of different cell types to prime in vivo for secondary in vitro cytotoxic T-cell responses against non-major-histocompatibility antigens

The capacity of several types of cell preparations to induce in vivo a state of memory for a secondary in vitro cytotoxic response against non-major-histocompatibility antigen was markedly reduced (on a per cell basis) by uv-irradiation. This indicated that memory induction requires metabolically active stimulator cells. An “adherent cell preparation” (AC) that was enriched for dendritic cells was among the most effective memory-inducing cell populations; but concanavalin A-activated nylon-wool-nonadherent spleen cells (Con A-NWT) or concanavalin A-activated unfractionated spleen cells (Con A-spl) were on the average equally effective. Normal unfractionated spleen cells (spl) or nonactivated nylon-wool-nonadherent cells (NWT) were markedly less effective on a per cell basis. This pattern of stimulatory activity was in line with the relative stimulatory activity of these cell types in primary cytotoxic responses in the presence of interleukin 2 (IL-2) and also in line with the relative capacity to induce IL-2-dependent proliferation in H-2D-incompatible T-cell populations (cf. W. Dröge et al., J. Immunol.132, 2749, 1984). These differences in the immunogenic potential and the requirement for metabolically active stimulator cells suggested that these cells stimulated the CTL system directly and not indirectly through antigen processing cells of the immunized host. Nevertheless, the secondary cytotoxic response after injection of low numbers of Con A-spl into H-2 heterozygous recipients, (BALB/c × BALB/b)F1, or into recipients with recombinant H-2 haplotype (A.J) was only preferentially but not exclusively restricted to the H-2 haplotype of the immunizing cell populations. Restriction was considerably more complete when AC were used for immunization.     

Relative roles of T-cell receptor ligands and interleukin-2 in driving T-cell proliferation

Stimulation of T cells by the T-cell receptor (TCR)/CD3 complex results in interleukin-2 (IL-2) synthesis and surface expression of the IL-2 receptor (IL-2R), which in turn drive T-cell proliferation. However, the significance of the requirement of IL-2 in driving T-cell proliferation, when TCR stimulation itself delivers potential mitogenic signals, is unclear. We show that blocking of IL-2 synthesis by Cyclosporin A (CsA) suppressed both the Concanavalin A (Con A)- and phorbol myristate acetate (PMA)/ionomycin-induced proliferation of T cells. The latter is also inhibited by anti-IL-2R. Kinetic studies showed that T-cell proliferation begins to become resistant to CsA inhibition by about 12 h and became largely resistant by 18 h of stimulation. PMA, the protein kinase C activator, enhanced Con A-induced T-cell proliferation if added only within first 12 h of stimulation, and not after that. Given the fact that, in the present study, TCR is downregulated within 2 h of Con A stimulation and T cells entered the S phase of cell cycle by about 18 h of stimulation, the above results suggest that TCR stimulation provides the initial trigger to the resting T cells, which allows the cells to traverse the first two third portions of G1 phase of cell cycle and become proliferation competent. IL-2 action begins afterward, delivering the actual proliferation signal(s), allowing the cells to traverse the rest of G1 phase and enter the S phase of the cell cycle.     

Phorbol myristic acetate enhances the production of Interleukin 2

Interleukin 2 is an antigen-nonspecific factor produced by Concanavalin A (Con A)-activated mouse spleen cells that has a number of biologic activities including the capacity to stimulate thymocyte proliferation, support the growth of continuous T cell lines, augment the antibody response of nude mouse spleen cells, and support the generation of antigen-specific cytotoxic T cells. In order to obtain increased amounts of Interleukin 2 for further purification and biologic studies, we have examined the use of Phorbol Myristic Acetate (PMA) as a costimulant. In this report we demonstrate that the addition of PMA to Con A-induced mouse spleen cell cultures results in a 5- to 20-fold increase in the production of Interleukin 2 activity under serum-containing and serum-free culture conditions.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.     

Growth of immature (double negative) rat thymocytes in the presence of IL-1

It is unclear which growth factors, if any, are involved in the growth and differentiation of immature T cells in the thymus. Because IL-1 has been previously implicated in thymocyte proliferation, we examined the effects of IL-1 on precursor thymocytes, the CD4-CD8- or double-negative (DN) cells. We show that IL-1 (together with Con A) is a growth factor for DN, TCR- alpha beta-cells in vitro. After 5 days of culture in IL-1 and Con A, a number of phenotypic changes were observed and two subsets of DN cells were distinguished. One subset expressed full length TCR-alpha and -beta mRNA and surface alpha beta TCR, the other expressed low levels of full length TCR-gamma together with high levels of full length TCR-delta mRNA. Thus, DN cells are induced by IL-1 and Con A to proliferate and express TCR without parallel acquisition of CD4 or CD8 markers. These data suggest that IL-1 drives early steps of intrathymic T cell differentiation.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

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.     

Interleukin 2 promotes expression of an interleukin 2 receptor and proliferation of T cells stimulated with non-mitogenic dose of concanavalin A

Interleukin 2 (IL-2) initiated proliferation of the cells of T-enriched population stimulated with non-mitogenic dose of concanavalin A (Con A), whereas it could not induce proliferation of the cells treated with non-mitogenic lectin wheat germ agglutinin (WGA). Use of monoclonal antibody (MAb) directed against interleukin 2 receptor (IL-2R) showed that IL-2 significantly increased expression of IL-2R on the cells of T-enriched population stimulated with Con A, whereas it could not cause any significant enhancement of expression of IL-2R on the cells treated with WGA. We concluded that IL-2 activated T cells in combination with non-mitogenic does of Con A, and induced both expression of IL-2R and proliferation.     

Effector activity of OKT4+ and OKT8+ T-cell subsets in lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.     

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.