Migration inhibitory factor (MIF) production by skin fibroblast cultures from patients with severe combined immunodeficiency

The supernatants of fibroblast cultures derived from skin biopsies of each of two patients with severe combined immunodeficiency were studied for the presence of migration inhibitory activity (MIF). The supernatants of both of these fibroblast cultures were found to contain inhibitory activity for the migration of cultured human lymphoid cells (PGLC-33H). This MIF activity was found to share chromatographic similarities with the MIF contained in the supernatants of a lymphoid cell line (PGLC-33H) and phytohemagglutinin (PHA) and tuberculin (PPD) stimulated human peripheral lymphocytes. These data suggest that MIF is not solely a lymphoid product and that severe combined immunodeficiency does not represent a gene deletion for MIF production.     

Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.     

Inhibitory activity of medium-dialyzed transfer factor on lymphocyte blastogenesis.

Human transfer factor was prepared from normal donors with marked skin reactivity against common microbial antigens by dialysis of lymphocyte extracts versus tissue culture medium (TFmd). Several batches of TFmd were tested for their ability to specifically increase the DNA synthesis of unsensitized lymphocytes in vitro in the presence of the corresponding antigens (PPD, SKSD, Candida, Thistoplasmin). No transfer or immunologic reactivity by TFmd was observed. There was, however, a significant unspecific inhibition by TFmd of lymphocyte cultures stimulated by antigens, PHA or allogeneic cells.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Reversal of cell surface abnormalities of T lymphocytes in hodgkin's disease after in vitro incubation in fetal sera.

The capacity of periphal blood lymphocytes from patients with untreated Hodgkin's disease to form E rosettes with sheep erythrocytes and to respond in vitro to PHA stimulation were found to be profoundly impaired. In 49% of the patients, the percentage of E rosette-forming cells (E-RFC) was more than two standard deviations below the mean for normal donors. Overnight incubation of the peripheral blood lymhocytes from these patients in culture media containing 20% fetal calf serum was followed by restoration of the percentage of E-RFC up to normal levels. Similar results have been observed after incubation in fetal human serum, but not in adult human AB serum or adult bovine serum. Incubation of peripheral blood lymphocytes from untreated patients in20% fetal calf serum also resulted in a remarkable restoration of their capacity to respond normally to PHA. Possible mechanisms involved in these reversible cell surface and in vitro lymphocyte function abnormalities in Hodgkin's disease are discussed.     

Lymphocyte reactivity in pregnant women and newborn infants.

The mitotic response to phytohaemagglutinin (PHA) was determined in lymphocytes of mothers and their newborn infants obtained at delivery and seven days later by measuring the rate of 125 I-idoxuridine uptake into DNA in lymphocytes cultured in their own plasma and after washing and resuspension in fetal bovine serum. There was no difference in the unstimulated counts of maternal lymphocytes taken at delivery, whether unwashed or washed, compared with those from nonpregnant controls. With PHA stimulation the mitotic response of the maternal lymphocytes cultured in their own plasma was reduced compared with that of the control lymphocytes but washed maternal cells showed a similar response to the controls. These findings suggest that the reduced lymphocyte mitotic response to PHA in pregnancy is due to a plasma inhibitory factor This inhibition was not evident in maternal blood taken seven days after delivery. DNA synthesis in unstimulated cultures from newborn infants at birth and seven days after birth was greater than that in adult control cultures. With PHA stimulation the mitotic response of cord-blood lymphocytes cultured in their own plasma paralleled that of control lymphocytes but washed newborn cells showed a greater response. Thus plasma suppression similar to that observed in the mother seems also to affect infants at birth. This inhibition was not demonstrable in blood taken from infants of 7 days.     

Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses.

Leukocytes from the various lymphoid tissues of rainbow trout (RBT) were tested for their capacity to respond to the lymphocyte mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and purified protein derivative of tuberculin (PPD). Thymocytes responded to Con A but not to LPS or PPD. In contrast, leukocytes from anterior kidney were stimulated with LPS but not with Con A or PPD. Cells from spleen and peripheral blood were stimulated by each mitogen. However, the degree of stimulation at optimally stimulatory concentrations of each mitogen was distinctive. The finding that the patterns of mitogenic responses of cells from each tissue were significantly different suggested that there is lymphoid heterogeneity in the RBT with a unique tissue distribution. The species source of serum utilized as a medium supplement appeared to be capable of markedly affecting mitogenesis. Thus, LPS and PPD stimulation occurred in medium supplemented with rainbow trout serum (RBTS). On the other hand, LPS and PPD stimulation was not observed in medium supplemented with fetal bovine serum (FBS), with the exception of peripheral blood leukocytes which were stimulated by LPS in culture medium supplemented with FBS. Con A stimulated leukocytes from each lymphoid tissue in medium supplemented with RBTS and, with the exception of cells from anterior kidney, also stimulated cells from each tissue in medium supplemented with FBS. The kinetic profiles of the responses of peripheral blood leukocytes to Con A, LPS, and PPD suggested that the extent as well as the time required for maximal stimulation was dependent on the dose of mitogen.     

Maintenance of lymphocyte surface Ig by mitogen stimulation in vitro.

In 4 to 24 hr cultures of rabbit lymphoid cells in medium supplemented with autologous serum, most B cells lost their surface Ig as assayed by rosette formation with anti-Ig antibody-coated erythrocytes. This loss was prevented by adding selected mitogens such as streptococcal mitogen (SM), lipopolysaccharide, and concanavalin A or by supplementing the medium with fetal calf serum. When SM was added at various times to the cultures (1, 2, 3, and 4 hr), it was effective in maintaining the approximate level of Ig-bearing cells present at the time of its addition but was ineffective in restoring the level of Ig-bearing cells present at the time the cultures were intiated. Very small, submitogenic doses of SM were sufficient to maintain the level of Ig-bearing cells. The data suggest that lymphocytes require continuous stimulation to maintain their surface receptors.     

Effect of interferons on protein synthesis in human lymphocytes: enhanced synthesis of eight specific peptides in T cells and activation-dependent inhibition of overall protein synthesis

Lymphoblastoid and fibroblast IFN inhibited PHA stimulation of overall protein synthesis in human lymphocytes by ca. 30%. Inhibition occurred within the first 6 hr of PHA treatment and was not progressive. DNA synthesis at 48 hr was inhibited to the same extent. Overall protein synthesis in resting lymphocytes was not detectably inhibited by IFN concentrations up to 1000 U/ml. Thus, inhibition of protein synthesis and subsequent reduction of cell proliferation by IFN require certain early events in mitogen activation. Resting lymphocytes were not unresponsive to IFN treatment, however. Two-dimensional electrophoretic analysis of newly synthesized proteins after IFN treatment showed enhanced synthesis of a specific set of eight peptides (I-peptides), which were shown to be synthesized in T lymphocytes. This enhancement was produced by both IFN-alpha and IFN-beta after 4 to 6 hr of exposure and was identical for all lymphocyte donors studied. After growth stimulation, IFN treatment produced no enhancements of additional peptides, although the original eight I-peptides were enhanced as usual. It is concluded that the biochemical activities of the I-peptides, which remain to be determined, cannot inhibit protein synthesis in resting lymphocytes, but may do so after mitogen activation, when the major physiologic restriction of lymphocyte protein synthesis is released. Alternatively, the I-peptides may be unrelated to regulation of protein synthesis but may be involved in viral protection or enhancement of NK activity.     

Production of IL 2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro

T cells from patients acutely infected with malaria exhibit a disease-related stimulation of DNA synthesis in response to Plasmodium falciparum antigen in vitro. This response is weak and short-lived, suggestive of induction of suppressor mechanisms. Exogenous T cell growth factor (IL 2) that was added to antigen-stimulated T cell cultures enhanced proliferation in antigen-responsive cultures, indicating that the lymphocytes expressed IL 2 receptors. In contrast, the addition of IL 2 to cultures that did not respond to antigen had no effect. Antigen-responsive cultures contained endogenous IL 2 as well, and the antigen-induced lymphocyte proliferation was correlated with IL 2 production. However, the results suggested that IL 2 production by the patients' T cells was insufficient or actively shut off, and that this was responsible for the premature cessation of their DNA synthesis. Supernatants from 60% of the T cell cultures treated with malaria antigen and from 30% treated with RBC ghost antigen contained interferon-gamma (IFN-gamma), as determined by a cytopathic effect inhibition assay combined with acid treatment and antibody neutralization or by an IFN-gamma-specific ELISA. There was no obvious correlation between antigen-induced lymphocyte proliferation and the presence of IFN-gamma in the culture supernatants. A high IFN-gamma activity was also seen in antigen-treated cultures from P. falciparum-immune donors living in highly endemic malaria areas. In contrast, no IFN-gamma was found in supernatants of antigen-treated T cells from healthy donors or patients with Plasmodium vivax malaria. Thus, the IFN-gamma activity of these cultures appears to reflect the presence of antigen-reactive T cells and may be useful as a sensitive indicator of cellular immunity in P. falciparum malaria.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Presence of a reversible inhibitor for human lymphoid cell DNA synthesisin the extracts of human peripheral lymphocytes.

An endogenous inhibitor of human lymphocyte DNA synthesis contained in extracts of purified human peripheral lymphocytes is described. It was found that the peripheral lymphocyte extract inhibits the DNA synthesis of phytohemagglutinin (PHA) stimulated human peripheral lymphocytes, lymphocytes in mixed lymphocyte culture, and human lymphoid cells in a long-term culture (PGLC-33H). This extract did not inhibit the DNA synthesis of nonlymphoid cells including HeLa and human embryonic lung. The effects of the inhibitor were reversible and noncytotoxic. Initial characterization showed the inhibitor to be thermolabile, DNase resistant, trypsin sensitive, and stable in a pH range 5.4–8.4. It appears that the inhibitor contained in the purified human peripheral lymphocyte extract is similar to a previously described inhibitor extracted from a human lymphoid cell line (PGLC-33H). Quantitation of the inhibitor in various lymphoid cell populations showed the amount of inhibitor per cell to be higher in resting peripheral lymphocytes than in PHA stimulated peripheral lymphocytes or human lymphoid cells in long-term culture (PGLC-33H). This data suggest that the inhibitor described may play a regulatory role in lymphocyte metabolism.     

Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures

Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained from Hodgkin's disease, have many similarities to the murine as well as human dendritic cells.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Interferon and the cellular immune response: separation of interferon-producing cells from DNA-synthetic cells

Spleen cells from mice were separated by albumin discontinuous density gradient centrifugation into six fractions. Cells from each fraction were cultured with a variety of general mitogens and a specific antigen PPD. The cultures then were assayed for mitogenesis and interferon production. The fraction of cells producing interferon were found to belong to a different population of cells from those undergoing a DNA synthetic response. Treatment of the interferon-producing fraction of cells with anti-theta serum and complement eliminated the interferon-producing capabilities of the remaining cells after exposure to PHA, Con A, and PPD but not after exposure to PWM. The results suggest at least a two-cell requirement for the production of interferon in response to PHA, Con A or PPD stimulation. They also suggest that the interferon-producing cells do not belong to the thymus-dependent lymphocyte subpopulation.     

Role of the thymus in generation of lymphocyte functions: II. Serum-mediated inhibition of development of mitogen-reactive lymphocytes in embryonic mouse thymus organ cultures

The thymuses of 14-day-old mouse embryos could be grown in serum-free organ cultures for at least 14 days with development of relatively large numbers of lymphocytes. These also acquired a strong reactivity to the mitogens concanavalin A (Con A) and leucoagglutinin (LA). Supplementing the organ culture medium with serum from calf (CS), fetal calf (FCS), mouse (MS), or fetal mouse (FMS) gave a serum concentration-dependent inhibition of development of mitogen reactivity, without clearly altering the quantitative lymphoid development in the organ cultures. Adult sera were more suppressive than fetal sera. All of nine tested FCS lots were inhibitory and the inhibiting activity was mainly found in the albumin fraction upon Sephadex G-200 gel filtration. Although FCS prevented development of mitogen-reactive cells in organ cultures of thymuses of 14-day-old embryos, it had much less effect on cultures of 15-day-old thymuses. FCS present during the entire organ culture period most efficiently inhibited generation of mitogen reactivity. If present only during the first or second half of the 14-day culture period, the inhibition was still marked but less complete.     

Inhibition of normal allogeneic lymphocyte mitogenesis by a factor released from human tumor cells in culture

Summary Human tumor and normal cell lines in culture were examined for the release of factors capable of inhibiting lymphocyte blastogenesis. Supernatants from tumor cell cultures of melanoma, carcinoma (lung, colon, breast), sarcoma, and normal fibroblasts inhibited normal lymphocyte response to PHA. Only supernatants from the tumor cell lines C1 (colon carcinoma), 734B (breast carcinoma), and 231 (breast carcinoma) were found to inhibit both PHA and ConA responses significantly. The two breast carcinoma cell lines, 734B and 231, which also were capable of inhibiting lymphocyte responses to PPD and alloantigens, were investigated further. The inhibition of lymphocyte blastogenesis caused by the supernatant of these two cell lines could not be overcome by the addition of added mitogen. Further experiments showed that the inhibition was not due to nutrient deficiencies and the supernatants were not directly toxic to the lymphocyte cultures as judged by trypan blue exclusion.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

The role of mitogens and lymphokines in the induction of ornithine decarboxylase (ODC) in T lymphocytes

Polyamine synthesis occurs early in lymphocyte activation after stimulation with antigen or mitogen. Ornithine decarboxylase (ODC) is the primary enzyme in the polyamine cascade. We have examined the induction of ODC by mitogens and/or lymphokines in human peripheral blood T lymphocytes. When isolated populations of monocytes and T lymphocytes were stimulated with phytohemagglutinin (PHA) there was little or no change in ODC activity. The combination of T lymphocytes and monocytes enhanced mitogen-induced ODC activity 10-fold. Several interleukin 1 (IL 1)-containing supernatants and fractionated human IL 1 were capable of substituting for monocytes in supporting PHA induction of ODC in T lymphocytes. Interleukin 2 (IL 2) and IL 2-containing supernatants were also capable of increasing ODC activity in T lymphocytes in the absence of monocytes. Lymphokines alone in the absence of PHA could not induce ODC. We conclude that both mitogens and monocytes are required for the induction of polyamine synthesis in T lymphocytes, and that supernatants containing IL 1 or IL 1 and IL 2 can substitute for monocytes in the induction of ODC in mitogen-stimulated T lymphocytes.